ISOPRENE PRODUCTION USING THE DXP AND MVA PATHWAY
The invention provides for methods for producing isoprene from cultured cells using various components of the DXP pathway and MVA pathway, or components associated with the DXP pathway and MVA pathway, iron-sulfur cluster-interacting redox polypeptides, and isoprene synthase. The invention also provides compositions that include these cultured cells.
Latest The Goodyear Tire & Rubber Company Patents:
This application is a continuation of U.S. patent application Ser. No. 12/817,134, filed Jun. 16, 2010, which claims priority to U.S. Provisional Application No. 61/187,941, filed Jun. 17, 2009; U.S. Provisional Application No. 61/187,930, filed Jun. 17, 2009; U.S. Provisional Application No. 61/314,985, filed Mar. 17, 2010; U.S. Provisional Application No. 61/314,979, filed Mar. 17, 2010; the disclosures of which are herein incorporated by reference in their entirety.
INCORPORATION BY REFERENCEThe content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 643842001101_Sequence_Listing.txt, date created: Jul. 8, 2013, size: 395,413 bytes).
FIELD OF THE INVENTIONThe present invention relates generally to compositions and methods for improving the production of isoprene from cultured cells using the DXP pathway and MVA pathway.
BACKGROUND OF THE INVENTIONIsoprene (2-methyl-1,3-butadiene) is the critical starting material for a variety of synthetic polymers, most notably synthetic rubbers. Isoprene is naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway (
Currently, the tire and rubber industry is based on the use of natural and synthetic rubber. Natural rubber is obtained from the milky juice of rubber trees or plants found in the rainforests of Africa. Synthetic rubber is based primarily on butadiene polymers. For these polymers, butadiene is obtained as a co-product from ethylene and propylene manufacture.
While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only about 15% isoprene. Thus, more economical methods for producing isoprene are needed. In particular, methods that produce isoprene at rates, titers, and purity that are sufficient to meet the demands of a robust commercial process are desirable. Also desired are systems for producing isoprene from inexpensive starting materials.
BRIEF SUMMARY OF THE INVENTIONThe invention provides, inter alia, compositions and methods for the production of isoprene in increased amounts using various DXP pathway genes and polypeptides and various MVA pathway genes and polypeptides, iron-sulfur cluster-interacting redox genes and polypeptides, isoprene synthase, and optionally, various genes and polypeptides associated with the DXP pathway, various genes and polypeptides associated with the MVA pathway, and IDI genes and polypeptides. In one aspect, the invention features cells or cells in culture which have been engineered for producing isoprene in increased amounts by using a combination of various DXP pathway genes and polypeptides, various MVA pathway genes and polypeptides, iron-sulfur cluster-interacting redox genes and polypeptides, isoprene synthase genes and polypeptides, and optionally, DXP pathway associated genes and polypeptides, MVA pathway associated genes and polypeptides, and IDI genes and polypeptides.
In some embodiments, the cells or cells in culture comprise (i) a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide and/or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide. In some embodiments, the cells or cells in culture comprise (i) one or more copies of heterologous or endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, (ii) one or more copies of heterologous or endogenous nucleic acid encoding a DXP pathway polypeptide and/or a MVA pathway polypeptide, and (iii) one or more copies of heterologous or endogenous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the iron-sulfur cluster-interacting redox polypeptide, the DXP pathway polypeptide, a MVA pathway polypeptide, and isoprene synthase polypeptide are operably linked to a promoter.
In some embodiments, the DXP pathway polypeptide is selected from the group consisting of DXS (1-deoxy-D-xylulose-5-phosphate synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT (4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some embodiments, the DXP pathway polypeptide is DXS, HDS, or HDR. In some embodiments, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
In some embodiments, the MVA pathway polypeptide is selected from the group consisting acetyl-CoA acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase (MVD), phosphomevalonate decarboxylase (PMDC) and isopentenyl phosphate kinase (IPK). In some embodiments, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
In one embodiment, both the DXP and MVA pathways can be present in any ratio to produce isoprene from each pathway in any proportion in cells or cells in culture. In another embodiment, about 10% to 50% of the isoprene is produced utilizing the DXP pathway and the remainder is produced utilizing the MVA pathway. In another embodiment, at least about 50% of the isoprene is produced utilizing the DXP pathway and the remainder is produced utilizing the MVA pathway.
In some embodiments, the invention provides cells or cells in culture that produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) of isoprene. In some embodiments, the cells or cells in culture convert more than about 0.002% of the carbon in a cell culture medium into isoprene.
In some embodiments, the invention provides cells or cells in culture where the level of HMBPP and DMAPP are maintained below 1 mM for the duration of the fermentation run. In other embodiments, the invention provides cells in culture where the level of HMBPP and DMAPP are maintained below 1 mM during the exponential phase of the fermentation. In other embodiments, the invention provides cells or cells in culture in which late DXP pathway enzymes, particularly IspG and IspH are maintained at levels consistent with minimizing phosphorylation level of Dxr.
In some embodiments of any of the aspects of the invention, the iron-sulfur cluster-interacting redox polypeptide comprises flavodoxin (e.g., flavodoxin I), flavodoxin reductase, ferredoxin (e.g., ferredoxin I), ferredoxin-NADP+ oxidoreductase, and genes or polypeptides encoding thereof (e.g., fpr and fldA).
In some embodiments, the cells or cells in culture comprise (i) a heterologous nucleic acid encoding a ferredoxin polypeptide, a ferredoxin-NADP+ oxidoreductase polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide and/or (ii) a duplicate copy of an endogenous nucleic acid encoding a ferredoxin polypeptide, a ferredoxin-NADP+ oxidoreductase polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide. In some embodiments, the cells or cells in culture comprise IspG and fldA. In another embodiment, the cells or cells in culture comprise IspG, fldA, and IspH. In some embodiments, the ferredoxin polypeptide, the ferredoxin-NADP+ oxidoreductase, the DXP pathway polypeptide, and isoprene synthase polypeptide are operably linked to a promoter. In some embodiments, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI polypeptide.
In some embodiments, the cells in culture comprise (i) a heterologous nucleic acid encoding a flavodoxin polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide and/or (ii) a duplicate copy of an endogenous nucleic acid encoding a flavodoxin polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide. In some embodiments, the flavodoxin polypeptide, the DXP pathway polypeptide, MVA pathway polypeptide, and isoprene synthase polypeptide are operably linked to a promoter. In some embodiments, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI polypeptide.
In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.
In other aspects, the invention provides for methods of producing isoprene, the method comprising (a) culturing cells comprising (i) a heterologous nucleic acid encoding a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, and (b) producing isoprene. In one embodiment, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide. In other embodiments, the cells in culture produce greater than about 400 nmole/gwcm/hr of isoprene. In other embodiments, more than about 0.02 molar percent of the carbon that the cells consume from a cell culture medium is converted into isoprene.
In one aspect, the invention features methods of producing isoprene, such as methods of using any of the cells described herein to produce isoprene. In some embodiments, the method involves culturing cells comprising (i) a heterologous nucleic acid encoding a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide, and/or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide. In some embodiments, the cells are cultured under suitable culture conditions for the production of isoprene, and isoprene is produced. In some embodiments, the iron-sulfur cluster-interacting redox polypeptide, isoprene synthase polypeptide, and DXP pathway polypeptide are operably linked to a promoter. In some embodiments, the DXP pathway polypeptide is selected from the group consisting of DXS (1-deoxy-D-xylulose-5-phosphate synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT (4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some embodiments, the DXP pathway polypeptide is DXS, HDS, or HDR. In some embodiments, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide. In some embodiments, the method involves culturing cells under conditions sufficient to produce greater than about 400 nmole/gwcm/hr of isoprene. In some embodiments, the method involves culturing cells under conditions sufficient to convert more than about 0.002% (mol/mol) of the carbon in a cell culture medium into isoprene.
In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2 or more times the amount of isoprene produced during the growth phase for the same length of time. In some embodiments, the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In particular embodiments, (i) the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit, and (ii) the cells produce greater than about 400 nmole/gwcm/hr of isoprene.
In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time.
In one aspect, the invention features compositions and systems that comprise isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene (w/w) of the volatile organic fraction of the composition is isoprene.
In some embodiments, the composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the composition has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.
In some embodiments, the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also has greater than about 2 mg of isoprene.
In some embodiments, the composition comprises (i) a gas phase that comprises isoprene and (ii) cells in culture that produce greater than about 400 nmole/gwcm/hr of isoprene. In some embodiments, the composition comprises a closed system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ug/L of isoprene when normalized to 1 mL of 1 OD600 cultured for 1 hour. In some embodiments, the composition comprises an open system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ug/L of isoprene when sparged at a rate of 1 vvm. In some embodiments, the volatile organic fraction of the gas phase comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.
In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene.
In some embodiments of any of the compositions of the invention, at least a portion of the isoprene is in a gas phase. In some embodiments, at least a portion of the isoprene is in a liquid phase (such as a condensate). In some embodiments, at least a portion of the isoprene is in a solid phase. In some embodiments, at least a portion of the isoprene is adsorbed to a solid support, such as a support that includes silica and/or activated carbon. In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol. In some embodiments, the composition includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.
In some embodiments, the invention also features systems that include any of the cells and/or compositions described herein. In some embodiments, the system includes a reactor that chamber comprises cells in culture that produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene. In some embodiments, the system is not a closed system. In some embodiments, at least a portion of the isoprene is removed from the system. In some embodiments, the system includes a gas phase comprising isoprene. In various embodiments, the gas phase comprises any of the compositions described herein.
In one aspect, the invention provides a tire comprising polyisoprene. In some embodiments, the polyisoprene is produced by (i) polymerizing isoprene in any of the compositions described herein or (ii) polymerizing isoprene recovered from any of the compositions described herein. In some embodiments, the polyisoprene comprises cis-1,4-polyisoprene.
In some embodiments of any of the compositions, systems, and methods of the invention, a nonflammable concentration of isoprene in the gas phase is produced. In some embodiments, the gas phase comprises less than about 9.5% (volume) oxygen. In some embodiments, the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 100% (volume) oxygen, such as between about 10% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume) CO2.
In some embodiments of any of the aspects of the invention, the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding a DXP pathway associated polypeptide.
In some embodiments of any of the aspects of the invention, the cells in culture produce isoprene at greater than or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene. In some embodiments of any of the aspects of the invention, the cells in culture convert greater than or about 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbon in the cell culture medium into isoprene. In some embodiments of any of the aspects of the invention, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/gwcm/h). In some embodiments of any of the aspects of the invention, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium). Other exemplary rates of isoprene production and total amounts of isoprene production are disclosed herein.
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by using a mutant DXP pathway polypeptide and nucleic acid derived from thereof. In some embodiments, the mutant DXP pathway polypeptide is a HDR polypeptide with the iron-sulfur cluster regulator (iscR) removed. In some embodiments, the mutant DXP pathway polypeptide is a mutant HDR polypeptide that produces solely DMAPP or a majority of DMAPP relative to IPP.
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by increasing the carbon flux through the DXP pathway and/or MVA pathway. In some embodiments, the carbon flux can be increased by avoiding any feedback inhibition of DXS activity by metabolites downstream the DXP pathway or/and intermediates of other pathways that use a DXP pathway polypeptide as a substrate. In some embodiments, the other pathway that uses DXP pathway polypeptide as a substrate (e.g., DXP) is the thiamine (Vitamin B1) or pyridoxal (Vitamin B6) pathway. In some embodiments, the carbon flux can be increased by expressing a DXP pathway polypeptide from a different organism that is not subject to inhibition by downstream products of the DXP pathway. In some embodiments, the carbon flux can be increased by deregulating glucose uptake. In other embodiments, the carbon flux can be increased by maximizing the balance between the precursors required for the DXP pathway and/or MVA pathway. In some embodiments, the balance of the DXP pathway precursors, pyruvate and glyceraldehydes-3-phosphate (G-3-P), can be achieved by redirecting the carbon flux with the effect of elevating or lowering pyruvate or G-3-P separately. In some embodiments, the carbon flux can be increased by using a CRP (cAMP Receptor Protein)-deleted mutant.
In some embodiments, the carbon flux can be increased by using a strain (containing one or more DXP pathway genes or one or more both DXP pathway and MVA pathway genes) containing a pyruvate dehydrogenase E1 subunit variant. In some embodiments, the pyruvate dehydrogenase (PDH) E1 subunit variant has an E636Q point mutation.
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by utilizing the downstream genes or polypeptides of the DXP pathway by introducing a heterologous terpene synthase nucleic acid or a duplicate copy of an endogenous terpene synthase nucleic acid into the cells, which includes, but is not limited to ocimene synthase, farnesene synthase, and artemesinin synthase.
In some embodiments of any of the aspects of the invention, in some embodiments, the vector comprises a selective marker, such as an antibiotic resistance nucleic acid.
In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects of the invention, the heterologous isoprene synthase nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase nucleic acid integrates into a chromosome of the cells without a selective marker.
In some embodiments, iron-sulfur cluster-interacting redox nucleic acid, any one or more of the nucleic acids in the DXP pathway, MVA pathway, and isoprene synthase nucleic acid are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. In one embodiment, IDI nucleic acid is also included for IDI expression to produce a higher amount of isoprene than when IDI is not used. For example, one or more iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, IDI nucleic acid, or isoprene synthase nucleic acid may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, MVA pathway nucleic acid, IDI nucleic acid, or isoprene synthase nucleic acid are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.
In some embodiments of any of the aspects of the invention, cells expressing iron-sulfur cluster-interacting redox polypeptide, isoprene synthase polypeptide, and DXP pathway polypeptide are grown under non-inducing conditions. In some embodiments of any of the aspects of the invention, cells expressing iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, IDI polypeptide, and isoprene synthase polypeptide are grown under non-inducing conditions. For example, the non-inducing condition is that IPTG-induced expression from the Trc promoter regulated gene constructs is not performed.
In some embodiments of any of the aspects of the invention, the cells express a second DXP pathway polypeptide, in addition to the first DXP pathway polypeptide, including DXS (1-deoxy-D-xylulose-5-phosphate synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT (4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some embodiments of any of the aspects of the invention, the cells express two or more DXP pathway polypeptides, in addition to the first DXP pathway polypeptide as described above. In some embodiments of any of the aspects of the invention, the cells express 2, 3, 4, 5, 6, or 7 DXP pathway polypeptides, in addition to the first DXP pathway polypeptide as described above.
In some embodiments of any of the aspects of the invention, the cells express a second MVA pathway polypeptide, in addition to the first MVA pathway polypeptide, including acetyl-CoA acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase (MVD), phosphomevalonate decarboxylase (PMDC) and isopentenyl phosphate kinase (IPK). In some embodiments of any of the aspects of the invention, the cells express two or more MVA pathway polypeptides, in addition to the first MVA pathway polypeptide as described above. In some embodiments of any of the aspects of the invention, the cells express 2, 3, 4, 5, 6, or 7 MVA pathway polypeptides, in addition to the first MVA pathway polypeptide as described above.
In some embodiments of any of the aspects of the invention, at least a portion of the cells maintain the heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, and isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, at least a portion of the cells maintain the heterologous iron-sulfur cluster-interacting redox nucleic acid, IDI nucleic acid, DXP pathway nucleic acid, and isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, at least a portion of the cells maintain the heterologous isoprene synthase nucleic acid, DXS nucleic acid, IDI nucleic acid, and iron-sulfur cluster-interacting redox nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, the nucleic acid comprising the iron-sulfur cluster-interacting redox nucleic acid, isoprene synthase nucleic acid, DXP pathway nucleic acid, and/or IDI nucleic acid also comprises a selective marker, such as an antibiotic resistance nucleic acid.
In some embodiments of any of the aspects of the invention, the isoprene synthase polypeptide is a polypeptide from a plant such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, or the hybrid, Populus alba×Populus tremula).
In some embodiments of any of the aspects of the invention, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus cells) or cyanobacterial cells (e.g., Thermosynechococcus cells such as Thermosynechococcus elongates cells). In some embodiments of any of the aspects of the invention, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells or Pantoea cells such as Pantoea citrea cells) or cyanobacterial cells (e.g., Thermosynechococcus cells such as Thermosynechococcus elongates cells). In some embodiments of any of the aspects of the invention, the cells are fungal, cells such as filamentous fungal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger), or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells).
In some embodiments of any of the aspects of the invention, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects of the invention, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
In one aspect, the invention features a product produced by any of the compositions or methods of the invention.
FIGS. 18A1-18A2 shows a schematic outlining construction of vectors pYLA 1, pYL1 and pYL2 (SEQ ID NOS: 44, 45, 46, 47, 48 and 50).
The invention provides, inter alia, compositions and methods for the production of isoprene in increased amounts using various DXP pathway genes and polypeptides, various MVA pathway genes and polypeptides, iron-sulfur cluster-interacting redox genes and polypeptides, isoprene synthase genes and polypeptides, and optionally, IDI genes and polypeptides and various genes and polypeptides associated with the DXP pathway and/or MVA pathway.
Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of skill in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this invention. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. One of skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.
As used herein, the term “isoprene” or “2-methyl-1,3-butadiene” (CAS#78-79-5) refers to the direct and final volatile C5 hydrocarbon product from the elimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate (DMAPP), and does not involve the linking or polymerization of one or more isopentenyl diphosphate (IPP) molecules to one or more DMAPP molecules. The term “isoprene” is not generally intended to be limited to its method of production.
As used herein, the phrase, “various genes and polypeptides associated with the DXP pathway,” or “DXP pathway associated nucleic acid(s) or polypeptide(s)” refers to any nucleic acid or polypeptide that interacts with DXP pathway polypeptides or nucleic acids, including, but not limited to, a terpene synthase (e.g., ocimene synthase, farnesene synthase, and artemesinin synthase), either directly or indirectly.
For use herein, unless clearly indicated otherwise, use of the terms “a”, “an,” and the like refers to one or more.
Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.
It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.
The present invention is based in part on the surprising discovery that an increased amount of an iron-sulfur cluster-interacting redox polypeptide increases the activity demonstrated by the DXP pathway polypeptides (such as HDS (GcpE or IspG) or HDR polypeptide (IspH or LytB). While not intending to be bound to a particular theory, it is believed that the increased expression of one or more endogenous or heterologous iron-sulfur interacting redox nucleic acids or polypeptides improve the rate of formation and the amount of DXP pathway polypeptides containing an iron sulfur cluster (such as HDS or HDR), and/or stabilize DXP pathway polypeptides containing an iron sulfur cluster (such as HDS or HDR). This in turn increases the carbon flux to isoprene synthesis in cells by increasing the synthesis of HMBPP and/or DMAPP and decreasing the cMEPP and HMBPP pools in the DXP pathway. For example, overexpression of an iron-sulfur cluster-interacting redox polypeptide (flavodoxin I) in cells overexpressing a DXP pathway polypeptide (DXS), isoprene synthase polypeptide, and IDI polypeptide resulted in increased production of isoprene by about 1- to 2-fold in comparison to cells overexpressing DXP pathway polypeptide, isoprene synthase polypeptide, and IDI polypeptide only. See Example 8. Overexpression of one or more iron-sulfur cluster-interacting redox polypeptide (ferredoxin and ferredoxin-NADP+ oxidoreductase), one or more DXP pathway polypeptide, isoprene synthase polypeptide, and IDI polypeptide resulted in increased production of isoprene. See Example 9.
Accordingly, in one aspect of the invention, cells in culture comprise (i) a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a heterologous nucleic acid encoding DXP pathway polypeptide, and an heterologous nucleic acid encoding isoprene synthase and/or (ii) a duplicate copies of endogenous nucleic acids encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide. In some embodiments, the cells in culture comprise (i) one or more copies of heterologous or endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, (ii) one or more copies of heterologous or endogenous nucleic acid encoding a DXP pathway polypeptide, and (iii) one or more copies of heterologous or endogenous nucleic acid encoding an isoprene synthase polypeptide.
In another aspect of the invention, provided are methods of producing isoprene. In one embodiments, the method comprises (a) culturing cells comprising (i) a heterologous nucleic acid encoding a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide and/or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, and (b) producing isoprene.
As used herein, iron-sulfur cluster-interacting redox polypeptide is a polypeptide that is capable of transferring electrons to a polypeptide containing an iron-sulfur cluster. An iron-sulfur cluster-interacting redox polypeptide includes, but is not limited to, flavodoxin (e.g., flavodoxin I), flavodoxin reductase, ferredoxin (e.g., ferredoxin I), ferredoxin-NADP+ oxidoreductase, and genes or polypeptides encoding thereof (e.g., fpr or fldA). For example, DXP pathway polypeptide HDS (GcpE) is a metallo-enzyme possessing a [4Fe-4S]2+ center and catalyzes the reduction of cMEPP into HMBPP via two successive one-electron transfers mediated by the reduction of [4Fe-4S]2+ center in the presence of flavodoxin/flavodoxin reductase (see, Wolff et al., FEBS Letters, 541:115-120 (2003)), which is hereby incorporated by reference in its entirety). Similarly, DXP pathway polypeptide HDR (LytB) is also a Fe/S protein catalyzing the reduction of HMBPP into IPP or DMAPP via two successive one-electron transfers in the presence of flavodoxin/flavodoxin reductase/NADPH system. See, for example, Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339 (2002); Wolff, M. et al., FEBS Letters, 541: 115-120 (2003), which are each hereby incorporated by reference in their entirety, particularly with respect to the description of GcpE, LytB, and flavodoxin/flavodoxin reductase/NADPH system).
As used herein, flavodoxin is a protein that is capable of transferring electrons and contains the prosthetic group flavin mononucleotide. In Escherichia coli (E. coli), flavodoxin is encoded by the fldA gene and reduced by the FAD-containing protein NADPH:ferredoxin oxidoreductase, and plays an essential role in the DXP pathway for isoprenoid biosynthesis (see, example, Kia-Joo, P. et al. FEBS Letters, 579: 3802-3806, 2005, which is hereby incorporated by reference in its entirety).
As used herein, ferredoxin is a protein that is capable of transferring electron and contains iron and labile sulfur in equal amounts and plays an essential role in the DXP pathway for isoprenoid biosynthesis. For example, HDS from plants and cyanobacteria have been shown to be ferredoxin, rather than flavodoxin-dependent, enzymes (Seemann et al., FEBS Lett., 580(6):1547-52 (2006), which is hereby incorporated by reference in its entirety).
As used herein, Fpr encodes flavodoxin/ferredoxin NADPH-oxidoreductase and provides the necessary electron derived from NADPH via FldA for HDS and HDR to perform their catalytic functions (reviewed in report by L. A. Furgerson, The Mevalonate-Independent Pathway to Isoprenoid Compounds: Discovery, Elucidation, and Reaction Mechanisms, published Feb. 13, 2006, which is hereby incorporated by reference in its entirety).
As used herein, the encoded DXS, DXR, MCT, CMK, MCS, HDS, and HDR polypeptides are part of the DXP pathway for the biosynthesis of isoprene (
DXS polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate (DXP). While not intending to be bound by any particular theory, it is believed that increasing the amount of DXS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
DXR polypeptides convert 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). While not intending to be bound by any particular theory, it is believed that increasing the amount of DXS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate (MEP) into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-Me). While not intending to be bound by any particular theory, it is believed that increasing the amount of MCT polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
CMK polypeptides convert 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP). While not intending to be bound by any particular theory, it is believed that increasing the amount of CMK polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
MCS polypeptides convert 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into 2-C-methyl-D-erythritol 2,4-cyclodiphoshphate (ME-CPP or cMEPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of MCS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
HDS polypeptides convert 2-C-methyl-D-erythritol 2,4-cyclodiphoshphate (ME-CPP or cMEPP) into (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate (HMBPP or HDMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of HDS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate (HMBPP) into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of HDR polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
Isoprene synthase polypeptides convert dimethylallyl diphosphate (DMAPP) into isoprene.
Heterologous iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, and isoprene synthase polypeptide can be expressed in a variety of host cells, such as Escherichia coli (E. coli), Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica, and Trichoderma reesei. All of these cells produced more isoprene than the naturally occurring DXP pathway alone.
As discussed further below, isoprene production by cells can be enhanced by increasing the amount of expression of an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, and an isoprene synthase polypeptide. The DXP pathway polypeptides include DXS, DXR, MCT, CMK, MCS, HDS, and HDR. For example, one or more DXP pathway nucleic acids can be introduced into the cells, which includes DXS, DXR, MCT, CMK, MCS, HDS, and HDR. The DXS, DXR, MCT, CMK, MCS, HDS, or HDR nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. Similarly, the iron-sulfur cluster-interacting redox nucleic acid may be a heterologous nucleic acid or duplicate copy of an endogenous nucleic acid. Similarly, the isoprene synthase nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. In some embodiments, the amount of one or more iron-sulfur cluster-interacting redox polypeptide, one or more of DXS, DXR, MCT, CMK, MCS, HDS, or HDR polypeptide, and isoprene synthase polypeptide are increased by replacing one or more endogenous iron-sulfur cluster-interacting redox promoters or regulatory regions, one or more of the endogenous DXS, DXR, MCT, CMK, MCS, HDS, or HDR promoters or regulatory regions, and isoprene synthase promoter or regulatory region with other promoters and/or regulatory regions that result in greater transcription of iron-sulfur cluster-interacting redox nucleic acids, one or more of DXS, DXR, MCT, CMK, MCS, HDS, or HDR nucleic acids, and isoprene synthase nucleic acid.
In some embodiments, the presence of heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, and isoprene synthase nucleic acid cause cells to grow more reproducibly and/or remain viable for longer compared to the corresponding cell with only one or two of these heterologous or extra endogenous nucleic acids. While not intending to be bound to a particular theory, it is believed that the overexpressing an iron sulfur cluster-interacting redox polypeptide can increase the rate of formation or the amount of one or more DXP pathway polypeptides (e.g., GcpE and/or LytB) or stabilizes one or more DXP pathway polypeptides (e.g., GcpE and/or LytB), so that one or more DXP pathway polypeptides are active for a longer period of time, which in turn cause cells containing heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, and isoprene synthase nucleic acid to grow more reproducibly and/or remain viable for longer compared to the corresponding cell with only one or two of these heterologous or extra endogenous nucleic acids. For example, cells containing heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, and isoprene synthase nucleic acid grow better than cells with only a DXP pathway nucleic acid, with only a heterologous iron-sulfur cluster-interacting redox nucleic acid, with a heterologous iron-sulfur cluster-interacting redox nucleic acid and DXP pathway nucleic acid, iron-sulfur cluster-interacting redox nucleic acid and isoprene synthase nucleic acid, or DXP pathway nucleic acid and isoprene synthase nucleic acid. Also, large amounts of iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, and isoprene synthase polypeptide can be expressed in the cells without causing an excessive amount of toxicity to the cells.
In some embodiments of any of the aspects of the invention, the cells express a second DXP pathway polypeptide, in addition to the first DXP pathway polypeptide, including DXS (1-deoxy-D-xylulose-5-phosphate synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT (4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some embodiments of any of the aspects of the invention, the cells express two or more DXP pathway polypeptides, in addition to the first DXP pathway polypeptide as described above. In some embodiments of any of the aspects of the invention, the cells express 2, 3, 4, 5, 6, or 7 DXP pathway polypeptides, in addition to the first DXP pathway polypeptide as described above.
Additionally, isoprene production by cells that contain a heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid (e.g., DXS, DXR, MCT, CMK, MCS, HDS, or HDR), and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells.
In some embodiments, isoprene production by cells that contain a heterologous iron-sulfur cluster-interacting redox nucleic acid, DXS nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells. In other embodiments, isoprene production by cells that contain a heterologous iron-sulfur cluster-interacting redox nucleic acid, HDS (IspG or GcpE), and isoprene synthase nucleic acids can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells. In some embodiments, the cells comprise IspG and fldA. In another embodiment, the cells comprise IspG, fldA, and IspH.
In some embodiments, isoprene production by cells that contain a heterologous flavodoxin nucleic acid, DXS nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells. In other embodiments, isoprene production by cells that contain a heterologous flavodoxin nucleic acid, HDS (IspG or GcpE) nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells.
In some embodiments, isoprene production by cells that contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+ oxidoreductase nucleic acid, DXS nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells. In other embodiments, isoprene production by cells that contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+ oxidoreductase nucleic acid, HDS (IspG or GcpE) nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI polypeptide expressed by the cells.
In some embodiments, isoprene production by cells that contain a heterologous iron-sulfur cluster-interacting redox nucleic acid, HDR (IspH or LytB) nucleic acid, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide expressed by the cells. In some embodiments, the cells comprise IspG and fldA. In another embodiment, the cells comprise IspG, fldA, and IspH.
In some embodiments, isoprene production by cells that contain a heterologous flavodoxin, HDR (IspH or LytB), and isoprene synthase nucleic acids can be enhanced by increasing the amount of an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide expressed by the cells.
In some embodiments, isoprene production by cells that contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+ oxidoreductase nucleic acid, HDR (IspH or LytB) nucleic acid, and isoprene synthase nucleic acids can be enhanced by increasing the amount of an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide expressed by the cells.
In some embodiments, isoprene production by cells that contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+ oxidoreductase nucleic acid, HDS and HDR nucleic acids, and isoprene synthase nucleic acid can be enhanced by increasing the amount of an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide expressed by the cells.
IDI polypeptides catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of IDI polypeptide in cells increases the amount (and conversion rate) of IPP that is converted into DMAPP, which in turn is converted into isoprene.
The IDI nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. In some embodiments, the amount of iron-sulfur cluster-interacting redox polypeptide, one or more of DXP pathway polypeptide (e.g., DXS, DXR, MCT, CMK, MCS, HDS, or HDR), isoprene synthase polypeptide, and IDI polypeptide are increased by replacing endogenous iron-sulfur cluster-interacting redox promoter or regulatory region, one or more of the endogenous DXP pathway promoter or regulatory region, and IDI promoters or regulatory region with other promoters and/or regulatory regions that result in greater transcription of iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, isoprene synthase nucleic acid, and IDI nucleic acid.
Heterologous IDI polypeptides can also be expressed in a variety of host cells in the presence of isoprene synthase, such as Escherichia coli (E. coli), Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica, and Trichoderma reesei. All of these cells produced more isoprene than when IDI is not used.
Additionally, isoprene production by cells that contain a heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid (e.g., DXS, DXR, MCT, CMK, MCS, HDS, or HDR), isoprene synthase nucleic acid, and optionally IDI nucleic acid, can be enhanced by increasing the amount of a DXP pathway associated polypeptide expressed by the cells
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by using a mutant DXP pathway polypeptide or nucleic acid derived from thereof. In some embodiments, the mutant DXP pathway polypeptide is a HDR polypeptide with the iron-sulfur cluster regulator (iscR) removed. In some embodiments, the mutant DXP pathway polypeptide is a mutant HDR polypeptide that produces solely DMAPP or a majority of DMAPP relative to IPP. For example, the use of the LytBG120D in a DXP pathway-mediated isoprene production strain allows the unique generation of an isoprenoid product that is derived almost entirely from DMAPP. See Example 18.
As used herein, iscR is encoded by an ORF located immediately upstream of genes coding for the E. coli Fe—S cluster assembly proteins. In the DXP pathway, the implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an E. coli strain engineered for HDR protein anaerobically purified from this strain by a factor of at least 200. (Gräwert et al., J Am Chem Soc. 126(40):12847-55 (2004); Schwartz et al., PNAS, 98(26):14751-3 (2001); Akhtar and Jones, Appl. Microbiol. Biotechnol. 78(5):853-62 (2008), which are each hereby incorporated by reference in their entireties).
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by increasing the carbon flux through the DXP pathway. In some embodiments, the carbon flux can be increased by avoiding any feedback inhibition of DXS activity by metabolites downstream the DXP pathway or/and intermediates of other pathways that use a DXP pathway polypeptide as a substrate (e.g., DXR). In some embodiments, the feedback inhibition by some DXP pathway polypeptides (e.g., DXR) can be alleviated by rebalancing pathyway enzymes and maintaining levels of HMBPP and DMAPP at concentrations below 1 to 2 mM DMAPP and 1 to 2 mM HMBPP. In some embodiments, the level of HMBPP and DMAPP are maintained below 1 mM for the duration of the fermentation run. In other embodiments, the level of HMBPP and DMAPP are maintained below 1 mM during the exponential phase of the fermentation. In other embodiments, late DXP pathway enzymes, particularly IspG and IspH, are maintained at levels consistent with minimizing phosphorylation level of Dxr.
In some embodiments, the other pathway that uses DXP pathway polypeptide as a substrate (e.g., DXP) is the thiamine (Vitamin B1) or pyridoxal (Vitamin B6) pathway. In some embodiments, the carbon flux can be increased by expressing a DXP pathway polypeptide from a different organism that is not subject to inhibition by downstream products of the DXP pathway. In some embodiments, the carbon flux can be increased by deregulating glucose uptake. In other embodiments, the carbon flux can be increased by maximizing the balance between the precursors required for the DXP pathway. In some embodiments, the balance of the DXP pathway precursors, pyruvate and glyceraldehydes-3-phosphate (G-3-P) can be achieved by redirecting the carbon flux with the effect of elevating or lowering pyruvate or G-3-P separately. In some embodiments, the carbon flux can be increased by using a strain (containing one or more DXP pathway genes or one or more both DXP pathway and MVA pathway genes) containing a pyruvate dehydrogenase E1 subunit variant. In some embodiments, the pyruvate dehydrogenase (PDH) E1 subunit variant has an E636Q point mutation. In some embodiments, the carbon flux can be increased by using a CRP-deleted mutant. As used herein, CRP (cAMP Receptor Protein) is a positive regulator protein activated by cyclic AMP. It is required for RNA polymerase to initiate transcription of certain (catabolite-sensitive) operons of E. coli.
In some embodiments of any of the aspects of the invention, isoprene production can be further increased by utilizing the downstream genes or polypeptides of the DXP pathway by introducing a heterologous terpene synthase nucleic acid or a duplicate copy of an endogenous terpene synthase nucleic acid into the cells, which includes, but is not limited to ocimene synthase, farnesene synthase, and artemesinin synthase.
In some embodiments, a renewable carbon source is used for the production of isoprene. In some embodiments, the concentrations of isoprene and any oxidants are within the nonflammable ranges to reduce or eliminate the risk that a fire may occur during production or recovery of isoprene. See for example, U.S. Appl. No. 61/133,947, which is hereby incorporated by reference in its entirety, particularly with respect to flammability modeling and testing of isoprene in Example 13 and WO2010/003007. The compositions and methods of the present invention are desirable because they allow high isoprene yield per cell, high carbon yield, high isoprene purity, high productivity, low energy usage, low production cost and investment, and minimal side reactions. This efficient, large scale, biosynthetic process for isoprene production provides an isoprene source for synthetic isoprene-based rubber and provides a desirable, low-cost alternative to using natural rubber.
In some embodiments, at least a portion of the cells maintain the heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, and isoprene synthase nucleic acid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments, at least a portion of the cells maintain the heterologous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and IDI nucleic acid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects of the invention, the nucleic acid comprising the heterologous or duplicate copy of an endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid and DXP pathway associated nucleic acid also comprises a selective marker, such as a kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol antibiotic resistance nucleic acid.
The amount of isoprene produced can be further increased by adding yeast extract to the cell culture medium. For example, the amount of isoprene produced that are linearly proportional to the amount of yeast extract in the cell medium for the concentrations are tested. Increasing the amount of yeast extract in the presence of glucose can result in more isoprene being produced than increasing the amount of glucose in the presence of yeast extract. Also, increasing the amount of yeast extract can allow the cells to produce a high level of isoprene for a longer length of time and improved the health of the cells.
Isoprene production can also be demonstrated using three types of hydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as the carbon source. If desired, any other biomass carbon source can be used in the compositions and methods of the invention. Biomass carbon sources are desirable because they are cheaper than many conventional cell mediums, thereby facilitating the economical production of isoprene.
In some embodiments, an oil is included in the cell medium. See, for example, U.S. 61/134,094, which is hereby incorporated by reference in its entirety, particularly with respect to oils included in the cell medium In some embodiments, more than one oil (such as 2, 3, 4, 5, or more oils) is included in the cell medium. While not intending to be bound to any particular theory, it is believed that (i) the oil may increase the amount of carbon in the cells that is available for conversion to isoprene, (ii) the oil may increase the amount of glyceraldehyde 3-phosphate and/or pyruvate in the cells, thereby increasing the carbon flow through the DXP pathway, and/or (ii) the oil may provide extra nutrients to the cells, which is desirable since a lot of the carbon in the cells is converted to isoprene rather than other products. In some embodiments, cells that are cultured in a cell medium containing oil naturally use the DXP pathway to produce isoprene or are genetically modified to contain nucleic acids for the entire DXP pathway. In some embodiments, the oil is partially or completely hydrolyzed before being added to the cell culture medium to facilitate the use of the oil by the host cells.
Exemplary Polypeptides and Nucleic AcidsVarious iron-sulfur cluster-interacting redox polypeptides and nucleic acids, DXP pathway polypeptides and nucleic acids, DXP pathway associated polypeptides and nucleic acids, isoprene synthase polypeptides and nucleic acids, and IDI polypeptides and nucleic acids can be used in the compositions and methods of the invention.
As used herein, “polypeptides” includes polypeptides, proteins, peptides, fragments of polypeptides, and fusion polypeptides. In some embodiments, the fusion polypeptide includes part or all of a first polypeptide (e.g., an iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and IDI polypeptide, or catalytically active fragment thereof) and may optionally include part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, the fusion polypeptide has an activity of two or more DXP pathway polypeptides.
In various embodiments, a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In some embodiments, the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of a corresponding full-length polypeptide. In particular embodiments, the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, or IDI polypeptide. In some embodiments, the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, or IDI polypeptide.
In some embodiments, the polypeptide is an isolated polypeptide. As used herein, an “isolated polypeptide” is not part of a library of polypeptides, such as a library of 2, 5, 10, 20, 50 or more different polypeptides and is separated from at least one component with which it occurs in nature. An isolated polypeptide can be obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide.
In some embodiments, the polypeptide is a heterologous polypeptide. By “heterologous polypeptide” is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host cell. In particular, a heterologous polypeptide is not identical to a wild-type nucleic acid that is found in the same host cell in nature.
As used herein, a “nucleic acid” refers to two or more deoxyribonucleotides and/or ribonucleotides in either single or double-stranded form. In some embodiments, the nucleic acid is a recombinant nucleic acid. By “recombinant nucleic acid” means a nucleic acid of interest that is free of one or more nucleic acids (e.g., genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest. The term therefore includes, for example, a recombinant DNA which is hereby incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In various embodiments, a nucleic acid is a recombinant nucleic acid. In some embodiments, an iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, part or all of a recombinant nucleic acid is chemically synthesized. It is to be understood that mutations, including single nucleotide mutations, can occur within a nucleic acid as defined herein.
In some embodiments, the nucleic acid is a heterologous nucleic acid. By “heterologous nucleic acid” is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell.
In particular embodiments, the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic, or IDI nucleic acid. In some embodiments, the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic, or IDI nucleic acid. In some embodiments, the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid. In some embodiments, the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid. In some embodiments, the nucleic acid is a degenerate variant of any nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, or IDI polypeptide.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid for improved expression in a host cell, it is desirable in some embodiments to design the nucleic acid such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The accession numbers of exemplary isoprene synthase and DXP pathway polypeptides and nucleic acids are listed in Appendix 1 (the accession numbers of Appendix 1 and their corresponding sequences are herein incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase and/or DXP pathway polypeptides and nucleic acids). The Kegg database also contains the amino acid and nucleic acid sequences of numerous exemplary isoprene synthase and/or DXP pathway polypeptides and nucleic acids (see, for example, the world-wide web at “genome.jp/kegg/pathway/map/map00100.html” and the sequences therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase and/or DXP pathway polypeptides and nucleic acids). In some embodiments, one or more of the isoprene synthase and/or DXP pathway polypeptides and/or nucleic acids have a sequence identical to a sequence publicly available on Dec. 12, 2007 or Sep. 14, 2008 such as any of the sequences that correspond to any of the accession numbers in Appendix 1 or any of the sequences present in the Kegg database. Additional exemplary isoprene synthase and/or DXP pathway polypeptides and nucleic acids are described further below.
Exemplary Isoprene Synthase Polypeptides and Nucleic AcidsAs noted above, isoprene synthase polypeptides convert dimethylallyl diphosphate (DMAPP) into isoprene. Exemplary isoprene synthase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an isoprene synthase polypeptide. Standard methods can be used to determine whether a polypeptide has isoprene synthase polypeptide activity by measuring the ability of the polypeptide to convert DMAPP into isoprene in vitro, in a cell extract, or in vivo. In an exemplary assay, cell extracts are prepared by growing a strain (e.g., the E. coli/pTrcKudzu strain described herein) in the shake flask method as described in Example 1. After induction is complete, approximately 10 mL of cells are pelleted by centrifugation at 7000×g for 10 minutes and resuspended in 5 ml of PEB without glycerol. The cells are lysed using a French Pressure cell using standard procedures. Alternatively the cells are treated with lysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thaw at −80 C.
Isoprene synthase polypeptide activity in the cell extract can be measured, for example, as described in Silver et al., J. Biol. Chem. 270:13010-13016, 1995 and references therein, which are each hereby incorporated by reference in their entireties, particularly with respect to assays for isoprene synthase polypeptide activity. DMAPP (Sigma) is evaporated to dryness under a stream of nitrogen and rehydrated to a concentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 and stored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl2, 1 mM (250 μg/ml) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 5% glycerol, and 2 mM DTT) is added to 25 μL of cell extract in a 20 ml Headspace vial with a metal screw cap and teflon coated silicon septum (Agilent Technologies) and cultured at 37° C. for 15 minutes with shaking. The reaction is quenched by adding 200 μL of 250 mM EDTA and quantified by GC/MS as described in Example 1, part II.
Exemplary isoprene synthase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an isoprene synthase polypeptide. Exemplary isoprene synthase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
In some embodiments, the isoprene synthase polypeptide or nucleic acid is from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the isoprene synthase polypeptide or nucleic acid is a polypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (such as Populus alba, Populus nigra, Populus trichocarpa, or Populus alba×tremula (CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen (such as Populus tremuloides) Silver et al., JBC 270(22): 13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO 98/02550), which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene synthase nucleic acids and the expression of isoprene synthase polypeptides. Suitable isoprene synthases include, but are not limited to, those identified by Genbank Accession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241, which are each hereby incorporated by reference in their entireties, particularly with respect to sequences of isoprene synthase nucleic acids and polypeptides. In some embodiments, the isoprene synthase polypeptide or nucleic acid is not a naturally-occurring polypeptide or nucleic acid from Quercus robur (i.e., the isoprene synthase polypeptide or nucleic acid is an isoprene synthase polypeptide or nucleic acid other than a naturally-occurring polypeptide or nucleic acid from Quercus robur). In some embodiments, the isoprene synthase nucleic acid or polypeptide is a naturally-occurring polypeptide or nucleic acid from poplar. In some embodiments, the isoprene synthase nucleic acid or polypeptide is not a naturally-occurring polypeptide or nucleic acid from poplar.
Exemplary DXP Pathway Polypeptides and Nucleic AcidsExemplary DXP pathways polypeptides include, but are not limited to any of the following polypeptides: DXS polypeptides, DXR polypeptides, MCT polypeptides, CMK polypeptides, MCS polypeptides, HDS polypeptides, HDR polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of one, two, or more of the DXP pathway polypeptides. In particular, DXP pathway polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of a DXP pathway polypeptide. Exemplary DXP pathway nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a DXP pathway polypeptide. Exemplary DXP pathway polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
In particular, DXS polypeptides convert pyruvate and D-glyceraldehyde 3-phosphate into 1-deoxy-d-xylulose 5-phosphate (DXP). Standard methods can be used to determine whether a polypeptide has DXS polypeptide activity by measuring the ability of the polypeptide to convert pyruvate and D-glyceraldehyde 3-phosphate in vitro, in a cell extract, or in vivo.
DXR polypeptides convert 1-deoxy-d-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). Standard methods can be used to determine whether a polypeptide has DXR polypeptides activity by measuring the ability of the polypeptide to convert DXP in vitro, in a cell extract, or in vivo.
MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate (MEP) into 4-(cytidine 5′-diphospho)-2-methyl-D-erythritol (CDP-ME). Standard methods can be used to determine whether a polypeptide has MCT polypeptides activity by measuring the ability of the polypeptide to convert MEP in vitro, in a cell extract, or in vivo.
CMK polypeptides convert 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP). Standard methods can be used to determine whether a polypeptide has CMK polypeptides activity by measuring the ability of the polypeptide to convert CDP-ME in vitro, in a cell extract, or in vivo.
MCS polypeptides convert 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP or cMEPP). Standard methods can be used to determine whether a polypeptide has MCS polypeptides activity by measuring the ability of the polypeptide to convert CDP-MEP in vitro, in a cell extract, or in vivo.
HDS polypeptides convert 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP or HDMAPP). Standard methods can be used to determine whether a polypeptide has HDS polypeptides activity by measuring the ability of the polypeptide to convert ME-CPP in vitro, in a cell extract, or in vivo.
HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Standard methods can be used to determine whether a polypeptide has HDR polypeptides activity by measuring the ability of the polypeptide to convert HMBPP in vitro, in a cell extract, or in vivo.
IDI polypeptides convert isopentenyl diphosphate into dimethylallyl diphosphate. Standard methods can be used to determine whether a polypeptide has IDI polypeptides activity by measuring the ability of the polypeptide to convert isopentenyl diphosphate in vitro, in a cell extract, or in vivo.
Exemplary MVA Pathway Polypeptides and Nucleic AcidsIn some aspects of the invention, the cells described in any of the compositions or methods described herein comprise a nucleic acid encoding an MVA pathway polypeptide. In some embodiments, the MVA pathway polypeptide is an endogenous polypeptide. In some embodiments, the cells comprise one or more additional copies of an endogenous nucleic acid encoding an MVA pathway polypeptide. In some embodiments, the endogenous nucleic acid encoding an MVA pathway polypeptide operably linked to a constitutive promoter. In some embodiments, the endogenous nucleic acid encoding an MVA pathway polypeptide operably linked to a constitutive promoter. In some embodiments, the endogenous nucleic acid encoding an MVA pathway polypeptide is operably linked to a strong promoter. In a particular embodiment, the cells are engineered to over-express the endogenous MVA pathway polypeptide relative to wild-type cells.
In some embodiments, the MVA pathway polypeptide is a heterologous polypeptide. In some embodiments, the cells comprise more than one copy of a heterologous nucleic acid encoding an MVA pathway polypeptide. In some embodiments, the heterologous nucleic acid encoding an MVA pathway polypeptide is operably linked to a constitutive promoter. In some embodiments, the heterologous nucleic acid encoding an MVA pathway polypeptide is operably linked to a strong promoter.
Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA pathway polypeptides. In particular, MVA pathway polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein. In addition, variants of MVA pathway polypeptide that confer the result of better isoprene production can also be used as well.
Types of MVA pathway polypeptides and/or DXP pathway polypeptides which can be used and methods of making microorganisms (e.g., facultative anaerobes such as E. coli) encoding MVA pathway polypeptides and/or DXP pathway polypeptides are also described in International Patent Application Publication No. WO2009/076676; U.S. patent application Ser. Nos. 12/496,573, 12/560,390, 12/560,317, 12/560,370, 12/560,305, and 12/560,366; and U.S. Provisional Patent Application Nos. 61/187,930, 61/187,934, and 61/187,959.
One of skill in the art can readily select and/or use suitable promoters to optimize the expression of isoprene synthase or and one or more MVA pathway polypeptides and/or one or more DXP pathway polypeptides. Similarly, one of skill in the art can readily select and/or use suitable vectors (or transfer vehicle) to optimize the expression of isoprene synthase or and one or more MVA pathway polypeptides and/or one or more DXP pathway polypeptides. In some embodiments, the vector contains a selective marker. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the host cell. In some embodiments, an isoprene synthase or MVA pathway nucleic acid integrates into a chromosome of the cells without a selective marker.
Exemplary Iron-Sulfur Cluster-Interacting Redox Polypeptides and Nucleic AcidsAs noted above, the iron-sulfur cluster-interacting redox polypeptide plays an essential role in the DXP pathway for isoprenoid biosynthesis. Exemplary iron-sulfur cluster-interacting redox polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of a iron-sulfur cluster-interacting redox polypeptide. Standard methods can be used to determine whether a polypeptide has iron-sulfur cluster-interacting redox polypeptide activity by using a hydrogenase-linked assay measuring the rate of metronidazole[1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] reduction (Chen and Blanchard, Analytical Biochem, 93:216-222 (1979)), which is hereby incorporated by reference in its entirety, especially with respect to the hydrogenase-linked assay for ferredoxin and flavodoxin).
Exemplary iron-sulfur cluster-interacting redox polypeptide nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an iron-sulfur cluster-interacting redox polypeptide. Exemplary iron-sulfur cluster-interacting redox polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.
Exemplary Methods for Isolating Nucleic AcidsIron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic, or IDI nucleic acid can be isolated using standard methods. Methods of obtaining desired nucleic acids from a source organism of interest (such as a bacterial genome) are common and well known in the art of molecular biology (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the isolation of nucleic acids of interest). For example, if the sequence of the nucleic acid is known (such as any of the known nucleic acids described herein), suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired nucleic acid sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202, which is hereby incorporated by reference in its entirety, particularly with respect to PCR methods) to obtain amounts of DNA suitable for transformation using appropriate vectors.
Alternatively, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic, and/or IDI nucleic acid (such as any isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, and/or IDI nucleic acid with a known nucleic acid sequence) can be chemically synthesized using standard methods.
Additional iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic, and/or IDI nucleic acid which may be suitable for use in the compositions and methods described herein can be identified using standard methods. For example, cosmid libraries of the chromosomal DNA of organisms known to produce isoprene naturally can be constructed in organisms such as E. coli, and then screened for isoprene production. In particular, cosmid libraries may be created where large segments of genomic DNA (35-45 kb) are packaged into vectors and used to transform appropriate hosts. Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the heterologous DNA. In addition to the cos sequence, these vectors also contain an origin of replication such as ColEI and drug resistance markers such as a nucleic acid resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.
Typically to clone cosmids, heterologous DNA is isolated using the appropriate restriction endonucleases and ligated adjacent to the cos region of the cosmid vector using the appropriate ligases. Cosmid vectors containing the linearized heterologous DNA are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process, the cos sites are cleaved and the heterologous DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli. Once injected into the cell, the heterologous DNA circularizes under the influence of the cos sticky ends. In this manner, large segments of heterologous DNA can be introduced and expressed in host cells.
Additional methods for obtaining iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid include screening a metagenomic library by assay (such as the headspace assay (see for example, in U.S. application Ser. No. 12/335,071 and PCT/US2008/086809, which are hereby incorporated by reference in their entireties, particularly with respect to headspace assay for isoprene production in Example 1 and 7) or by PCR using primers directed against nucleotides encoding for a length of conserved amino acids (for example, at least 3 conserved amino acids). Conserved amino acids can be identified by aligning amino acid sequences of known iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide. Conserved amino acids for isoprene synthase polypeptides can be identified based on aligned sequences of known isoprene synthase polypeptides. An organism found to produce isoprene naturally can be subjected to standard protein purification methods (which are well known in the art) and the resulting purified polypeptide can be sequenced using standard methods. Other methods are found in the literature (see, for example, Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007; Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which are each hereby incorporated by reference in their entireties, particularly with respect to identification of nucleic acids involved in the synthesis of isoprene).
Additionally, standard sequence alignment and/or structure prediction programs can be used to identify additional DXP pathway polypeptides and nucleic acids based on the similarity of their primary and/or predicted polypeptide secondary structure with that of known DXP pathway polypeptides and nucleic acids. Standard databases such as the swissprot-trembl database (world-wide web at “expasy.org”, Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue Michel Servet CH-1211 Geneva 4, Switzerland) can also be used to identify isoprene synthase, flavodoxin I, DXP pathway, and/or IDI polypeptides and nucleic acids. The secondary and/or tertiary structure of an iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide can be predicted using the default settings of standard structure prediction programs, such as PredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA). Alternatively, the actual secondary and/or tertiary structure of an iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide can be determined using standard methods. Additional iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid can also be identified by hybridization to probes generated from known iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid.
Exemplary Promoters and VectorsAny of the iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid described herein can be included in one or more vectors. Accordingly, the invention also features vectors with one more nucleic acids encoding any of the iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide that are described herein. As used herein, a “vector” means a construct that is capable of delivering, and desirably expressing one or more nucleic acids of interest in a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, DNA or RNA expression vectors, cosmids, and phage vectors. In some embodiments, the vector contains a nucleic acid under the control of an expression control sequence.
As used herein, an “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid of interest. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. An “inducible promoter” is a promoter that is active under environmental or developmental regulation. The expression control sequence is operably linked to the nucleic acid segment to be transcribed.
In some embodiments, the vector contains a selective marker. The term “selective marker” refers to a nucleic acid capable of expression in a host cell that allows for ease of selection of those host cells containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the host cell. Exemplary nutritional selective markers include those markers known in the art as amdS, argB, and pyr4. Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6 in Biotechnology of Filamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann, Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, the selective marker is the amdS nucleic acid, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of an A. nidulans amdS nucleic acid as a selective marker is described in Kelley et al., EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987 (which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, an isoprene synthase, flavodoxin I, DXP pathway, or IDI nucleic acid integrates into a chromosome of the cells without a selective marker.
Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to the use of vectors).
Promoters are well known in the art. Any promoter that functions in the host cell can be used for expression of an iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid in the host cell. Initiation control regions or promoters, which are useful to drive expression of iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid in various host cells are numerous and familiar to those skilled in the art (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors for the expression of nucleic acids of interest). Virtually any promoter capable of driving these nucleic acids is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, PL, PR, T7, tac, and trc (useful for expression in E. coli).
In some embodiments, a glucose isomerase promoter is used (see, for example, U.S. Pat. No. 7,132,527 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect promoters and plasmid systems for expressing polypeptides of interest). Reported glucose isomerase promoter mutants can be used to vary the level of expression of the polypeptide encoded by a nucleic acid operably linked to the glucose isomerase promoter (U.S. Pat. No. 7,132,527). In various embodiments, the glucose isomerase promoter is contained in a low, medium, or high copy plasmid (U.S. Pat. No. 7,132,527).
In various embodiments, an iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is contained in a low copy plasmid (e.g., a plasmid that is maintained at about 1 to about 4 copies per cell), medium copy plasmid (e.g., a plasmid that is maintained at about 10 to about 15 copies per cell), or high copy plasmid (e.g., a plasmid that is maintained at about 50 or more copies per cell). In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to a T7 promoter. In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid operably linked to a T7 promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to a Trc promoter. In some embodiments, the heterologous or extra endogenous is iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid operably linked to a Trc promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to a Lac promoter. In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid operably linked to a Lac promoter is contained in a low copy plasmid. In some embodiments, the heterologous or extra endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to an endogenous promoter, such as an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces, Trichoderma or Thermosynechococcus promoter or an endogenous alkaline serine protease iron-sulfur cluster-interacting redox promoter, DXP pathway promoter, DXP pathway associated promoter, isoprene synthase promoter, or IDI promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, and/or IDI nucleic acid operably linked to an endogenous promoter is contained in a high copy plasmid. In some embodiments, the vector is a replicating plasmid that does not integrate into a chromosome in the cells. In some embodiments, part or all of the vector integrates into a chromosome in the cells.
In some embodiments, the vector is any vector which when introduced into a fungal host cell is integrated into the host cell genome and is replicated. Reference is made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, the world-wide web at “fgsc.net” and the references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors) for a list of vectors. Additional examples of suitable expression and/or integration vectors are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18); van den Hondel et al. in Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp. 396-428, 1991; and U.S. Pat. No. 5,874,276, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors. Particularly useful vectors include pFB6, pBR322, PUC18, pUC100, and pENTR/D.
In some embodiments, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is operably linked to a suitable promoter that shows transcriptional activity in a fungal host cell. The promoter may be derived from one or more nucleic acids encoding a polypeptide that is either endogenous or heterologous to the host cell. In some embodiments, the promoter is useful in a Trichoderma host. Suitable non-limiting examples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, and amy. In some embodiments, the promoter is one that is native to the host cell. For example, in some embodiments when T. reesei is the host, the promoter is a native T. reesei promoter. In some embodiments, the promoter is T. reesei cbh1, which is an inducible promoter and has been deposited in GenBank under Accession No. D86235, which is hereby incorporated by reference in its entirety, particularly with respect to promoters. In some embodiments, the promoter is one that is heterologous to the fungal host cell. Other examples of useful promoters include promoters from the genes of A. awamori and A. niger glucoamylase (glaA) (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984, which are each hereby incorporated by reference in their entireties, particularly with respect to promoters); Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1, and the T. reesei cellobiohydrolase 1 (EP 137280, which is hereby incorporated by reference in its entirety, particularly with respect to promoters).
In some embodiments, the expression vector also includes a termination sequence. Termination control regions may also be derived from various genes native to the host cell. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is endogenous to the host cell. A particularly suitable terminator sequence is cbh1 derived from a Trichoderma strain (such as T. reesei). Other useful fungal terminators include the terminator from an A. niger or A. awamori glucoamylase nucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated by reference in their entireties, particularly with respect to fungal terminators). Optionally, a termination site may be included. For effective expression of the polypeptides, DNA encoding the polypeptide are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.
In some embodiments, the promoter, coding, region, and terminator all originate from the iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid to be expressed. In some embodiments, the coding region for iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is inserted into a general-purpose expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences. In some embodiments, genes or part thereof are inserted downstream of the strong cbh1 promoter.
An iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors). Methods used to ligate the DNA construct comprising a nucleic acid of interest (such as an iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid), a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art. For example, restriction enzymes can be used to cleave the iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid and the vector. Then, the compatible ends of the cleaved iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid and the cleaved vector can be ligated. Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide linkers are used in accordance with conventional practice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to oligonucleotide linkers). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).
In some embodiments, it may be desirable to over-express iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid at levels far higher than currently found in naturally-occurring cells. This result may be accomplished by the selective cloning of the nucleic acids encoding those polypeptides into multicopy plasmids or placing those nucleic acids under a strong inducible or constitutive promoter. Methods for over-expressing desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning techniques.
The following resources include descriptions of additional general methodology useful in accordance with the invention: Kreigler, Gene Transfer and Expression; A Laboratory Manual, 1990 and Ausubel et al., Eds. Current Protocols in Molecular Biology, 1994, which are each hereby incorporated by reference in their entireties, particularly with respect to molecular biology and cloning techniques.
Exemplary Source OrganismsIron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid (and their encoded polypeptides) can be obtained from any organism that naturally contains iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, and/or IDI nucleic acid. As noted above, isoprene is formed naturally by a variety of organisms, such as bacteria, yeast, plants, and animals. Organisms contain the MVA pathway, DXP pathway, or both the MVA and DXP pathways for producing isoprene (
In some embodiments, the nucleic acid sequence of the iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid is identical to the sequence of a nucleic acid that is produced by any of the following organisms in nature. In some embodiments, the amino acid sequence of iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, or IDI polypeptide is identical to the sequence of a polypeptide that is produced by any of the following organisms in nature. In some embodiments, the iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid or its encoded polypeptide is a mutant nucleic acid or polypeptide derived from any of the organisms described herein. As used herein, “derived from” refers to the source of the nucleic acid or polypeptide into which one or more mutations is introduced. For example, a polypeptide that is “derived from a plant polypeptide” refers to polypeptide of interest that results from introducing one or more mutations into the sequence of a wild-type (i.e., a sequence occurring in nature) plant polypeptide.
In some embodiments, the source organism is a fungus, examples of which are species of Aspergillus such as A. oryzae and A. niger, species of Saccharomyces such as S. cerevisiae, species of Schizosaccharomyces such as S. pombe, and species of Trichoderma such as T. reesei. In some embodiments, the source organism is a filamentous fungal cell. The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. The filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, (e.g., Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea); Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or “Trichoderma spp.” refer to any fungal genus previously or currently classified as Trichoderma.
In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Aspergillus strains are disclosed in Ward et al., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur et al., Curr Gene 41:89-98, 2002, which are each hereby incorporated by reference in their entireties, particularly with respect to fungi. In particular embodiments, the fungus is a strain of Trichoderma, such as a strain of T. reesei. Strains of T. reesei are known and non-limiting examples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No. 56765, ATCC No. 56767, and NRRL 15709, which are each hereby incorporated by reference in their entireties, particularly with respect to strains of T. reesei. In some embodiments, the host strain is a derivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl. Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated by reference in its entirety, particularly with respect to strains of T. reesei.
In some embodiments, the source organism is a yeast, such as Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.
In some embodiments, the source organism is a bacterium, such as strains of Bacillus such as B. lichenformis or B. subtilis, strains of Pantoea such as P. citrea, strains of Pseudomonas such as P. alcaligenes, strains of Streptomyces such as S. lividans or S. rubiginosus, strains of Thermosynechococcus such as T. elongatus, strains of Sinorhizobium such as S. meliloti, strains of Helicobacter such as H. pylori, strains of Agrobacterium such as A. tumefaciens, strains of Deinococcus such as D. radiodurans, strains of Listeria such as L. monocytogenes, strains of Lactobacillus such as L. spp, or strains of Escherichia such as E. coli.
As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thennobacillus, Ureibacillus, and Virgibacillus.
In some embodiments, the source organism is a gram-positive bacterium. Non-limiting examples include strains of Streptomyces (e.g., S. lividans, S. coelicolor, or S. griseus), Bacillus, Listeria (e.g., L. monocytogenes) or Lactobacillus (e.g., L. spp). In some embodiments, the source organism is a gram-negative bacterium, such as E. coli, Pseudomonas sp, or H. pylori.
In some embodiments, the source organism is a plant, such as a plant from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the source organism is kudzu, poplar (such as Populus alba×tremula CAC35696), aspen (such as Populus tremuloides), Quercus robur, Arabidopsis (such as A. thaliana), or Zea (such as Z. mays).
In some embodiments, the source organism is an algae, such as a green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.
In some embodiments, the source organism is a cyanobacterium, such as cyanobacteria classified into any of the following groups based on morphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, or Stigonematales. In some embodiments, the cyanobacterium is Thermosynechococcus elongates.
Exemplary Host CellsA variety of host cells can be used to express iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, MVA pathway polypeptide, MVA pathway associated polypeptide, isoprene synthase polypeptide, or IDI polypeptide and to produce isoprene in the methods of the claimed invention. Exemplary host cells include cells from any of the organisms listed in the prior section under the heading “Exemplary Source Organisms.” The host cell may be a cell that naturally produces isoprene or a cell that does not naturally produce isoprene. In some embodiments, the host cell naturally produces isoprene using the DXP pathway and an isoprene synthase, and one or more DXP pathway polypeptide and iron-sulfur cluster-interacting redox polypeptides are added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the DXP pathway and isoprene synthase, and one or more DXP pathway nucleic acids, one or more iron-sulfur cluster-interacting redox nucleic acids, and IDI are added to enhance production of isoprene using this pathway.
Exemplary Transformation Methodsiron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid or its vectors containing them can be inserted into a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a bacterial cell described herein) using standard techniques for expression of the encoded iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide. Introduction of a DNA construct or vector into a host cell can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet. 16:53-56, 1989, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods). The expression of heterologous polypeptide in Trichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat. No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes,” in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation and expression methods). Reference is also made to Cao et al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods) for transformation of Aspergillus strains. The introduced nucleic acids may be integrated into chromosomal DNA or maintained as extrachromosomal replicating sequences.
Any method known in the art may be used to select transformants. In one non-limiting example, stable transformants including an amdS marker are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium containing acetamide. Additionally, in some cases a further test of stability is conducted by growing the transformants on a solid non-selective medium (e.g., a medium that lacks acetamide), harvesting spores from this culture medium, and determining the percentage of these spores which subsequently germinate and grow on selective medium containing acetamide.
In some embodiments, fungal cells are transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a known manner. In one specific embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods). In some embodiments, the mycelia are obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is desirable to use about a 1.2 M solution of sorbitol in the suspension medium.
Uptake of DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration. Generally, between about 10 mM CaCl2 and 50 mM CaCl2 is used in an uptake solution. In addition to the calcium ion in the uptake solution, other compounds generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). While not intending to be bound to any particular theory, it is believed that the polyethylene glycol acts to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA to be transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.
Usually a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 105 to 107/mL (such as 2×106/mL) are used in the transformation. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. In some embodiments, about 0.25 volumes are added to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like may also be added to the uptake solution and aid in transformation. Similar procedures are available for other fungal host cells (see, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods).
Generally, the mixture is then cultured at approximately 0° C. for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired nucleic acid sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is desirably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then cultured either at room temperature or on ice before the addition of a sorbitol and CaCl2 solution. The protoplast suspension is then further added to molten aliquots of a growth medium. When the growth medium includes a growth selection (e.g., acetamide or an antibiotic) it permits the growth of transformants only.
The transformation of bacterial cells may be performed according to conventional methods, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.
Exemplary Cell Culture MediaThe invention also includes a cell or a population of cells in culture that produce isoprene. By “cells in culture” is meant two or more cells in a solution (e.g., a cell medium) that allows the cells to undergo one or more cell divisions. “Cells in culture” do not include plant cells that are part of a living, multicellular plant containing cells that have differentiated into plant tissues. In various embodiments, the cell culture includes at least or about 10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or more cells.
Any carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a host cell or organism. For example, the cell medium used to cultivate the host cells may include any carbon source suitable for maintaining the viability or growing the host cells.
In some embodiments, the carbon source is a carbohydrate (such as monosaccharide, disaccharide, oligosaccharide, or polysaccharids), invert sugar (e.g., enzymatically treated sucrose syrup), glycerol, glycerine (e.g., a glycerine byproduct of a biodiesel or soap-making process), dihydroxyacetone, one-carbon source, oil (e.g., a plant or vegetable oil such as corn, palm, or soybean oil), animal fat, animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbial or plant protein or peptide), renewable carbon source (e.g., a biomass carbon source such as a hydrolyzed biomass carbon source), yeast extract, component from a yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, or any combination of two or more of the foregoing. In some embodiments, the carbon source is a product of photosynthesis, including, but not limited to, glucose.
Exemplary monosaccharides include glucose and fructose; exemplary oligosaccharides include lactose and sucrose, and exemplary polysaccharides include starch and cellulose. Exemplary carbohydrates include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). In some embodiments, the cell medium includes a carbohydrate as well as a carbon source other than a carbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, or a component from a yeast extract). In some embodiments, the cell medium includes a carbohydrate as well as a polypeptide (e.g., a microbial or plant protein or peptide). In some embodiments, the microbial polypeptide is a polypeptide from yeast or bacteria. In some embodiments, the plant polypeptide is a polypeptide from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
In some embodiments, the concentration of the carbohydrate is at least or about 5 grams per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the carbohydrate is between about 50 and about 400 g/L, such as between about 100 and about 360 g/L, between about 120 and about 360 g/L, or between about 200 and about 300 g/L. In some embodiments, this concentration of carbohydrate includes the total amount of carbohydrate that is added before and/or during the culturing of the host cells.
In some embodiments, the cells are cultured under limited glucose conditions. By “limited glucose conditions” is meant that the amount of glucose that is added is less than or about 105% (such as about 100%) of the amount of glucose that is consumed by the cells. In particular embodiments, the amount of glucose that is added to the culture medium is approximately the same as the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added glucose such that the cells grow at the rate that can be supported by the amount of glucose in the cell medium. In some embodiments, glucose does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited glucose conditions may allow more favorable regulation of the cells.
In some embodiments, the cells are cultured in the presence of an excess of glucose. In particular embodiments, the amount of glucose that is added is greater than about 105% (such as about or greater than 110, 120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, glucose accumulates during the time the cells are cultured.
Exemplary lipids are any substance containing one or more fatty acids that are C4 and above fatty acids that are saturated, unsaturated, or branched.
Exemplary oils are lipids that are liquid at room temperature. In some embodiments, the lipid contains one or more C4 or above fatty acids (e.g., contains one or more saturated, unsaturated, or branched fatty acid with four or more carbons). In some embodiments, the oil is obtained from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, linseed, oleagineous microbial cells, Chinese tallow, or any combination of two or more of the foregoing.
Exemplary fatty acids include compounds of the formula RCOOH, where “R” is a hydrocarbon. Exemplary unsaturated fatty acids include compounds where “R” includes at least one carbon-carbon double bond. Exemplary unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid. Exemplary polyunsaturated fatty acids include compounds where “R” includes a plurality of carbon-carbon double bonds. Exemplary saturated fatty acids include compounds where “R” is a saturated aliphatic group. In some embodiments, the carbon source includes one or more C12-C22 fatty acids, such as a C12 saturated fatty acid, a C14 saturated fatty acid, a C16 saturated fatty acid, a C18 saturated fatty acid, a C20 saturated fatty acid, or a C22 saturated fatty acid. In an exemplary embodiment, the fatty acid is palmitic acid. In some embodiments, the carbon source is a salt of a fatty acid (e.g., an unsaturated fatty acid), a derivative of a fatty acid (e.g., an unsaturated fatty acid), or a salt of a derivative of fatty acid (e.g., an unsaturated fatty acid). Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like. Di- and triglycerols are fatty acid esters of glycerol.
In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is at least or about 1 gram per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 10 and about 400 g/L, such as between about 25 and about 300 g/L, between about 60 and about 180 g/L, or between about 75 and about 150 g/L. In some embodiments, the concentration includes the total amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both (i) a lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride and (ii) a carbohydrate, such as glucose. In some embodiments, the ratio of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e., one carbon in the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride per carbohydrate carbon). In particular embodiments, the amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 60 and 180 g/L, and the amount of the carbohydrate is between about 120 and 360 g/L.
Exemplary microbial polypeptide carbon sources include one or more polypeptides from yeast or bacteria. Exemplary plant polypeptide carbon sources include one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.
Exemplary renewable carbon sources include cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt, and components from any of the foregoing. Exemplary renewable carbon sources also include glucose, hexose, pentose and xylose present in biomass, such as corn, switchgrass, sugar cane, cell waste of fermentation processes, and protein by-product from the milling of soy, corn, or wheat. In some embodiments, the biomass carbon source is a lignocellulosic, hemicellulosic, or cellulosic material such as, but are not limited to, a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, corn stover, switch grass, rice hull product, or a by-product from wet or dry milling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat, and/or distillers grains). Exemplary cellulosic materials include wood, paper and pulp waste, herbaceous plants, and fruit pulp. In some embodiments, the carbon source includes any plant part, such as stems, grains, roots, or tubers. In some embodiments, all or part of any of the following plants are used as a carbon source: corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassava, legumes, such as beans and peas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. In some embodiments, the carbon source is a biomass hydrolysate, such as a biomass hydrolysate that includes both xylose and glucose or that includes both sucrose and glucose.
In some embodiments, the renewable carbon source (such as biomass) is pretreated before it is added to the cell culture medium. In some embodiments, the pretreatment includes enzymatic pretreatment, chemical pretreatment, or a combination of both enzymatic and chemical pretreatment (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No. 6,106,888; which are each hereby incorporated by reference in their entireties, particularly with respect to the pretreatment of renewable carbon sources). In some embodiments, the renewable carbon source is partially or completely hydrolyzed before it is added to the cell culture medium.
In some embodiments, the renewable carbon source (such as corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment before it is added to the cell culture medium (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005). During AFEX pretreatment, a renewable carbon source is treated with liquid anhydrous ammonia at moderate temperatures (such as about 60 to about 100° C.) and high pressure (such as about 250 to about 300 psi) for about 5 minutes. Then, the pressure is rapidly released. In this process, the combined chemical and physical effects of lignin solubilization, hemicellulose hydrolysis, cellulose decrystallization, and increased surface area enables near complete enzymatic conversion of cellulose and hemicellulose to fermentable sugars. AFEX pretreatment has the advantage that nearly all of the ammonia can be recovered and reused, while the remaining serves as nitrogen source for microbes in downstream processes. Also, a wash stream is not required for AFEX pretreatment. Thus, dry matter recovery following the AFEX treatment is essentially 100%. AFEX is basically a dry to dry process. The treated renewable carbon source is stable for long periods and can be fed at very high solid loadings in enzymatic hydrolysis or fermentation processes. Cellulose and hemicellulose are well preserved in the AFEX process, with little or no degradation. There is no need for neutralization prior to the enzymatic hydrolysis of a renewable carbon source that has undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sources produces clean sugar streams for subsequent fermentation use.
In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to at least or about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalent amount of glucose can be determined by using standard HPLC methods with glucose as a reference to measure the amount of glucose generated from the carbon source. In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to between about 0.1 and about 20% glucose, such as between about 0.1 and about 10% glucose, between about 0.5 and about 10% glucose, between about 1 and about 10% glucose, between about 1 and about 5% glucose, or between about 1 and about 2% glucose.
In some embodiments, the carbon source includes yeast extract or one or more components of yeast extract. In some embodiments, the concentration of yeast extract is at least 1 gram of yeast extract per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In some embodiments, the concentration of yeast extract is between about 1 and about 300 g/L, such as between about 1 and about 200 g/L, between about 5 and about 200 g/L, between about 5 and about 100 g/L, or between about 5 and about 60 g/L. In some embodiments, the concentration includes the total amount of yeast extract that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both yeast extract (or one or more components thereof) and another carbon source, such as glucose. In some embodiments, the ratio of yeast extract to the other carbon source is about 1:5, about 1:10, or about 1:20 (w/w).
Additionally the carbon source may also be one-carbon substrates such as carbon dioxide, or methanol. Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources) and in bacteria (Hunter et. al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose-momophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York, 1986, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form a six carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate.
In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th ed., 415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK, 1993, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). Similarly, various species of Candida metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153(5), 485-9, 1990, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources).
In some embodiments, cells are cultured in a standard medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ. Microbiol. 63:1298-1306, 1997, which are each hereby incorporated by reference in their entireties, particularly with respect to cell medias). Exemplary growth media are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of particular host cells are known by someone skilled in the art of microbiology or fermentation science.
In addition to an appropriate carbon source, the cell medium desirably contains suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art suitable for the growth of the cultures or the enhancement of isoprene production (see, for example, WO 2004/033646 and references cited therein and WO 96/35796 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect cell medias and cell culture conditions). In some embodiments where an isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic acid is under the control of an inducible promoter, the inducing agent (e.g., a sugar, metal salt or antimicrobial), is desirably added to the medium at a concentration effective to induce expression of an isoprene synthase polypeptide, iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, or IDI polypeptide. In some embodiments, cell medium has an antibiotic (such as kanamycin) that corresponds to the antibiotic resistance nucleic acid (such as a kanamycin resistance nucleic acid) on a vector that has one or more isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic acid.
Exemplary Cell Culture ConditionsMaterials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Exemplary techniques may be found in Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture techniques. In some embodiments, the cells are cultured in a culture medium under conditions permitting the expression of one or more isoprene synthase polypeptide, iron-sulfur cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP pathway associated polypeptide, or IDI polypeptide encoded by a nucleic acid inserted into the host cells.
Standard cell culture conditions can be used to culture the cells (see, for example, WO 2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture and fermentation conditions). Cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 to about 37° C., at about 6% to about 84% CO2, and at a pH between about 5 to about 9). In some embodiments, cells are grown at 35° C. in an appropriate cell medium. In some embodiments, e.g., cultures are cultured at approximately 28° C. in appropriate medium in shake cultures or fermentors until desired amount of isoprene production is achieved. In some embodiments, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Reactions may be performed under aerobic, anoxic, or anaerobic conditions based on the requirements of the host cells. Exemplary culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center.
In various embodiments, the cells are grown using any known mode of fermentation, such as batch, fed-batch, or continuous processes. In some embodiments, a batch method of fermentation is used. Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the cell medium is inoculated with the desired host cells and fermentation is permitted to occur adding nothing to the system. Typically, however, “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly until the time the fermentation is stopped. Within batch cultures, cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. In some embodiments, cells in log phase are responsible for the bulk of the isoprene production. In some embodiments, cells in stationary phase produce isoprene.
In some embodiments, a variation on the standard batch system is used, such as the Fed-Batch system. Fed-Batch fermentation processes comprise a typical batch system with the exception that the carbon source is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of carbon source in the cell medium. Fed-batch fermentations may be performed with the carbon source (e.g., glucose) in a limited or excess amount. Measurement of the actual carbon source concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.
In some embodiments, continuous fermentation methods are used. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or isoprene production. For example, one method maintains a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allows all other parameters to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration (e.g., the concentration measured by media turbidity) is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, the cell loss due to media being drawn off is balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.
In some embodiments, cells are immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isoprene production.
In some embodiments, bottles of liquid culture are placed in shakers in order to introduce oxygen to the liquid and maintain the uniformity of the culture. In some embodiments, an incubator is used to control the temperature, humidity, shake speed, and/or other conditions in which a culture is grown. The simplest incubators are insulated boxes with an adjustable heater, typically going up to ˜65° C. More elaborate incubators can also include the ability to lower the temperature (via refrigeration), or the ability to control humidity or CO2 levels. Most incubators include a timer; some can also be programmed to cycle through different temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the size of small rooms.
If desired, a portion or all of the cell medium can be changed to replenish nutrients and/or avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifuging or filtering the suspension culture and then resuspending the cells in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced. In some embodiments, the cell medium allows at least a portion of the cells to divide for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution).
In some embodiments, a constitutive or leaky promoter (such as a Trc promoter) is used and a compound (such as IPTG) is not added to induce expression of the isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic acid, operably linked to the promoter. In some embodiments, a compound (such as IPTG) is added to induce expression of the isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic acid operably linked to the promoter.
Exemplary Methods for Decoupling Isoprene Production from Cell Growth.
The invention provides, inter alia, compositions and methods for increasing the production of isoprene from cultured cells. When feedstock is used, it is desirable for the carbon from the feedstock to be converted to isoprene rather than to the growth and maintenance of the cells. In some embodiments, the cells are grown to a low to medium OD600, then production of isoprene is started or increased. This strategy permits a large portion of the carbon to be converted to isoprene.
In some embodiments, cells reach an optical density such that they no longer divide or divide extremely slowly, but continue to make isoprene for several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or more hours). In some cases, the optical density at 550 nm decreases over time (such as a decrease in the optical density after the cells are no longer in an exponential growth phase due to cell lysis), and the cells continue to produce a substantial amount of isoprene. In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 nmole/gwcm/hr, such as between about 2 to about 100 nmole/gwcm/hr, about 100 to about 500 nmole/gwcm/hr, about 150 to about 500 nmole/gwcm/hr, about 500 to about 1,000 nmole/gwcm/hr, about 1,000 to about 2,000 nmole/gwcm/hr, or about 2,000 to about 5,000 nmole/gwcm/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/gwcm/hr, about 100 to about 5,000 nmole/gwcm/hr, about 200 to about 2,000 nmole/gwcm/hr, about 200 to about 1,000 nmole/gwcm/hr, about 300 to about 1,000 nmole/gwcm/hr, or about 400 to about 1,000 nmole/gwcm/hr.
In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/Lbroth, such as between about 2 to about 100 mg/Lbroth, about 100 to about 500 mg/Lbroth, about 500 to about 1,000 mg/Lbroth, about 1,000 to about 2,000 mg/Lbroth, or about 2,000 to about 5,000 mg/Lbroth. In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/Lbroth, about 100 to about 5,000 mg/Lbroth, about 200 to about 2,000 mg/Lbroth, about 200 to about 1,000 mg/Lbroth, about 300 to about 1,000 mg/Lbroth, or about 400 to about 1,000 mg/Lbroth.
In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene during this time period. In some embodiments, the percent conversion of carbon into isoprene is between such as about 0.002 to about 4.0%, about 0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 to about 0.3%.
In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells are in stationary phase. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells divide slowly or not at all such that the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is only produced in the growth phase.
In some embodiments, one or more isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, and/or IDI nucleic acid are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, and/or IDI nucleic acid may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more isoprene synthase nucleic acid, iron-sulfur cluster-interacting redox nucleic acid, DXP pathway nucleic acid, DXP pathway associated nucleic acid, and/or IDI nucleic acid are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.
Production of Isoprene within Safe Operating Ranges
The invention provides, inter alia, compositions and methods for increasing the production of isoprene from cultured cells. The production of isoprene within safe operating levels according to its flammability characteristics simplifies the design and construction of commercial facilities, vastly improves the ability to operate safely, and limits the potential for fires to occur. In particular, the optimal ranges for the production of isoprene are within the safe zone, i.e., the nonflammable range of isoprene concentrations. In one such aspect, the invention features a method for the production of isoprene within the nonflammable range of isoprene concentrations (outside the flammability envelope of isoprene).
Thus, computer modeling and experimental testing were used to determine the flammability limits of isoprene (such as isoprene in the presence of O2, N2, CO2, or any combination of two or more of the foregoing gases) in order to ensure process safety. The flammability envelope is characterized by the lower flammability limit (LFL), the upper flammability limit (UFL), the limiting oxygen concentration (LOC), and the limiting temperature. For a system to be flammable, a minimum amount of fuel (such as isoprene) must be in the presence of a minimum amount of oxidant, typically oxygen. The LFL is the minimum amount of isoprene that must be present to sustain burning, while the UFL is the maximum amount of isoprene that can be present. Above this limit, the mixture is fuel rich and the fraction of oxygen is too low to have a flammable mixture. The LOC indicates the minimum fraction of oxygen that must also be present to have a flammable mixture. The limiting temperature is based on the flash point of isoprene and is that lowest temperature at which combustion of isoprene can propagate. These limits are specific to the concentration of isoprene, type and concentration of oxidant, inerts present in the system, temperature, and pressure of the system. Compositions that fall within the limits of the flammability envelope propagate combustion and require additional safety precautions in both the design and operation of process equipment.
The following conditions were tested using computer simulation and mathematical analysis and experimental testing. If desired, other conditions (such as other temperature, pressure, and permanent gas compositions) may be tested using the methods described herein to determine the LFL, UFL, and LOC concentrations.
(1) Computer Simulation and Mathematical Analysis Test Suite 1:isoprene: 0 wt %-14 wt %
O2: 6 wt %-21 wt % N2: 79 wt %-94 wt % Test Suite 2:isoprene: 0 wt %-14 wt %
O2: 6 wt %-21 wt % N2: 79 wt %-94 wt %Saturated with H2O
Test Suite 3:isoprene: 0 wt %-14 wt %
O2: 6 wt %-21 wt % N2: 79 wt %-94 wt % CO2: 5 wt %-30 wt % (2) Experimental Testing for Final Determination of Flammability Limits Test Suite 1:isoprene: 0 wt %-14 wt %
O2: 6 wt %-21 wt % N2: 79 wt %-94 wt % Test Suite 2:isoprene: 0 wt %-14 wt %
O2: 6 wt %-21 wt % N2: 79 wt %-94 wt %Saturated with H2O
Simulation software was used to give an estimate of the flammability characteristics of the system for several different testing conditions. CO2 showed no significant affect on the system's flammability limits. Test suites 1 and 2 were confirmed by experimental testing. The modeling results were in-line with the experimental test results. Only slight variations were found with the addition of water.
The LOC was determined to be 9.5 vol % for an isoprene, O2, N2, and CO2 mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO2 did not significantly affect the flammability characteristics of an isoprene, O2, and N2 mixture. Only slight variations in flammability characteristics were shown between a dry and water saturated isoprene, O2, and N2 system. The limiting temperature is about −54° C. Temperatures below about −54° C. are too low to propagate combustion of isoprene.
In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % to about 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % to about 12.0 vol. %, depending on the amount of oxygen in the system. In some embodiments, the LOC is about 9.5 vol % oxygen. In some embodiments, the LFL of isoprene is between about 1.5 vol. % to about 2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0 vol. %, and the LOC is about 9.5 vol % oxygen when the temperature is between about 25° C. to about 55° C. (such as about 40° C.) and the pressure is between about 1 atmosphere and 3 atmospheres.
In some embodiments, isoprene is produced in the presence of less than about 9.5 vol % oxygen (that is, below the LOC required to have a flammable mixture of isoprene). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is below the LFL (such as below about 1.5 vol. %). For example, the amount of isoprene can be kept below the LFL by diluting the isoprene composition with an inert gas (e.g., by continuously or periodically adding an inert gas such as nitrogen to keep the isoprene composition below the LFL). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is above the UFL (such as above about 12 vol. %). For example, the amount of isoprene can be kept above the UFL by using a system (such as any of the cell culture systems described herein) that produces isoprene at a concentration above the UFL. If desired, a relatively low level of oxygen can be used so that the UFL is also relatively low. In this case, a lower isoprene concentration is needed to remain above the UFL.
In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol % oxygen, the isoprene concentration is within the flammability envelope (such as between the LFL and the UFL). In some embodiments when the isoprene concentration may fall within the flammability envelope, one or more steps are performed to reduce the probability of a fire or explosion. For example, one or more sources of ignition (such as any materials that may generate a spark) can be avoided. In some embodiments, one or more steps are performed to reduce the amount of time that the concentration of isoprene remains within the flammability envelope. In some embodiments, a sensor is used to detect when the concentration of isoprene is close to or within the flammability envelope. If desired, the concentration of isoprene can be measured at one or more time points during the culturing of cells, and the cell culture conditions and/or the amount of inert gas can be adjusted using standard methods if the concentration of isoprene is close to or within the flammability envelope. In particular embodiments, the cell culture conditions (such as fermentation conditions) are adjusted to either decrease the concentration of isoprene below the LFL or increase the concentration of isoprene above the UFL. In some embodiments, the amount of isoprene is kept below the LFL by diluting the isoprene composition with an inert gas (such as by continuously or periodically adding an inert gas to keep the isoprene composition below the LFL).
In some embodiments, the amount of flammable volatiles other than isoprene (such as one or more sugars) is at least about 2, 5, 10, 50, 75, or 100-fold less than the amount of isoprene produced. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 100% (volume) oxygen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 99% (volume) nitrogen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 99% (volume) nitrogen.
In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 1% to about 50% (volume) CO2, such as between about 1% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to about 50% (volume) CO2.
In some embodiments, an isoprene composition also contains ethanol. For example, ethanol may be used for extractive distillation of isoprene, resulting in compositions (such as intermediate product streams) that include both ethanol and isoprene. Desirably, the amount of ethanol is outside the flammability envelope for ethanol. The LOC of ethanol is about 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standard conditions, such as about 1 atmosphere and about 60° F. (NFPA 69 Standard on Explosion Prevention Systems, 2008 edition, which is hereby incorporated by reference in its entirety, particularly with respect to LOC, LFL, and UFL values). In some embodiments, compositions that include isoprene and ethanol are produced in the presence of less than the LOC required to have a flammable mixture of ethanol (such as less than about 8.7% vol %). In some embodiments in which compositions that include isoprene and ethanol are produced in the presence of greater than or about the LOC required to have a flammable mixture of ethanol, the ethanol concentration is below the LFL (such as less than about 3.3 vol. %).
In various embodiments, the amount of oxidant (such as oxygen) is below the LOC of any fuel in the system (such as isoprene or ethanol). In various embodiments, the amount of oxidant (such as oxygen) is less than about 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. In various embodiments, the amount of oxidant (such as oxygen) is less than the LOC of isoprene or ethanol by at least 2, 4, 5, or more absolute percentage points (vol %). In particular embodiments, the amount of oxygen is at least 2 absolute percentage points (vol %) less than the LOC of isoprene or ethanol (such as an oxygen concentration of less than 7.5 vol % when the LOC of isoprene is 9.5 vol %). In various embodiments, the amount of fuel (such as isoprene or ethanol) is less than or about 25, 20, 15, 10, or 5% of the LFL for that fuel.
Exemplary Production of IsopreneThe invention provides, inter alia, compositions and methods for increasing the production of isoprene from cultured cells using various DXP pathway enzymes in combination with iron-sulfur cluster-interacting redox genes or polypeptides and isoprene synthase genes or polypeptides, optionally with IDI and DXP pathway associated genes and polypeptides. In some embodiments, the cells are cultured in a culture medium under conditions permitting the production of isoprene by the cells. By “peak absolute productivity” is meant the maximum absolute amount of isoprene in the off-gas during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak absolute productivity time point” is meant the time point during a fermentation run when the absolute amount of isoprene in the off-gas is at a maximum during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the isoprene amount is measured at the peak absolute productivity time point. In some embodiments, the peak absolute productivity for the cells is about any of the isoprene amounts disclosed herein.
By “peak specific productivity” is meant the maximum amount of isoprene produced per cell during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By “peak specific productivity time point” is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. The specific productivity is determined by dividing the total productivity by the amount of cells, as determined by optical density at 600 nm (OD600). In some embodiments, the isoprene amount is measured at the peak specific productivity time point. In some embodiments, the peak specific productivity for the cells is about any of the isoprene amounts per cell disclosed herein.
By “cumulative total productivity” is meant the cumulative, total amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the cumulative, total amount of isoprene is measured. In some embodiments, the cumulative total productivity for the cells is about any of the isoprene amounts disclosed herein.
By “relative detector response” refers to the ratio between the detector response (such as the GC/MS area) for one compound (such as isoprene) to the detector response (such as the GC/MS area) of one or more compounds (such as all C5 hydrocarbons). The detector response may be measured as described herein, such as the GC/MS analysis performed with an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm film thickness). If desired, the relative detector response can be converted to a weight percentage using the response factors for each of the compounds. This response factor is a measure of how much signal is generated for a given amount of a particular compound (that is, how sensitive the detector is to a particular compound). This response factor can be used as a correction factor to convert the relative detector response to a weight percentage when the detector has different sensitivities to the compounds being compared. Alternatively, the weight percentage can be approximated by assuming that the response factors are the same for the compounds being compared. Thus, the weight percentage can be assumed to be approximately the same as the relative detector response.
In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr). In some embodiments, the amount of isoprene is between about 2 to about 5,000 nmole/gwcm/hr, such as between about 2 to about 100 nmole/gwcm/hr, about 100 to about 500 nmole/gwcm/hr, about 150 to about 500 nmole/gwcm/hr, about 500 to about 1,000 nmole/gwcm/hr, about 1,000 to about 2,000 nmole/gwcm/hr, or about 2,000 to about 5,000 nmole/gwcm/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/gwcm/hr, about 100 to about 5,000 nmole/gwcm/hr, about 200 to about 2,000 nmole/gwcm/hr, about 200 to about 1,000 nmole/gwcm/hr, about 300 to about 1,000 nmole/gwcm/hr, or about 400 to about 1,000 nmole/gwcm/hr.
The amount of isoprene in units of nmole/gwcm/hr can be measured as disclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of isoprene production. For example, two mL of headspace (e.g., headspace from a culture such as 2 mL of culture cultured in sealed vials at 320 C with shaking at 200 rpm for approximately 3 hours) are analyzed for isoprene using a standard gas chromatography system, such as a system operated isothermally (850 C) with an n-octane/porasil C column (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2 mercuric oxide reduction gas detector (Trace Analytical, Menlo Park, Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A: 2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of isoprene production). The gas chromatography area units are converted to nmol isoprene via a standard isoprene concentration calibration curve. In some embodiments, the value for the grams of cells for the wet weight of the cells is calculated by obtaining the A600 value for a sample of the cell culture, and then converting the A600 value to grams of cells based on a calibration curve of wet weights for cell cultures with a known A600 value. In some embodiments, the grams of the cells is estimated by assuming that one liter of broth (including cell medium and cells) with an A600 value of 1 has a wet cell weight of 1 gram. The value is also divided by the number of hours the culture has been incubating for, such as three hours.
In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/gwcm/h). In some embodiments, the amount of isoprene is between about 2 to about 5,000 ng/gwcm/h, such as between about 2 to about 100 ng/gwcm/h, about 100 to about 500 ng/gwcm/h, about 500 to about 1,000 ng/gwcm/h, about 1,000 to about 2,000 ng/gwcm/h, or about 2,000 to about 5,000 ng/gwcm/h. In some embodiments, the amount of isoprene is between about 20 to about 5,000 ng/gwcm/h, about 100 to about 5,000 ng/gwcm/h, about 200 to about 2,000 ng/gwcm/h, about 200 to about 1,000 ng/gwcm/h, about 300 to about 1,000 ng/gwcm/h, or about 400 to about 1,000 ng/gwcm/h. The amount of isoprene in ng/gwcm/h can be calculated by multiplying the value for isoprene production in the units of nmole/gwcm/hr discussed above by 68.1 (as described in Equation 5 below).
In some embodiments, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/Lbroth, such as between about 2 to about 100 mg/Lbroth, about 100 to about 500 mg/Lbroth, about 500 to about 1,000 mg/Lbroth, about 1,000 to about 2,000 mg/Lbroth, or about 2,000 to about 5,000 mg/Lbroth. In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/Lbroth, about 100 to about 5,000 mg/Lbroth, about 200 to about 2,000 mg/Lbroth, about 200 to about 1,000 mg/Lbroth, about 300 to about 1,000 mg/Lbroth, or about 400 to about 1,000 mg/Lbroth.
In some embodiments, the cells in culture produce at least about 2 g/Lbroth, at least about 2.1 g/Lbroth, at least about 2.2 g/Lbroth, at least about 2.3 g/Lbroth, at least about 2.4 g/Lbroth, at least about 2.5 g/Lbroth, at least about 2.6 g/Lbroth, at least about 2.7 g/Lbroth, at least about 2.8 g/Lbroth, at least about 2.9 g/Lbroth, at least about 3.0 g/Lbroth, at least about 3.2 g/Lbroth, at least about 3.5 g/Lbroth, at least about 3.7 g/Lbroth, or at least about 4.0 g/Lbroth.
The specific productivity of isoprene in mg of isoprene/L of headspace from shake flask or similar cultures can be measured by taking a 1 ml sample from the cell culture at an OD600 value of approximately 1.0, putting it in a 20 mL vial, incubating for 30 minutes, and then measuring the amount of isoprene in the headspace (as described, for example, in Example I, part II). If the OD600 value is not 1.0, then the measurement can be normalized to an OD600 value of 1.0 by dividing by the OD600 value. The value of mg isoprene/L headspace can be converted to mg/Lbroth/hr/OD600 of culture broth by multiplying by a factor of 38. The value in units of mg/Lbroth/hr/OD600 can be multiplied by the number of hours and the OD600 value to obtain the cumulative titer in units of mg of isoprene/L of broth.
The instantaneous isoprene production rate in mg/Lbroth/hr in a fermentor can be measured by taking a sample of the fermentor off-gas, analyzing it for the amount of isoprene (in units such as mg of isoprene per Lgas) as described, for example, in Example I, part II and multiplying this value by the rate at which off-gas is passed though each liter of broth (e.g., at 1 vvm (volume of air/volume of broth/minute) this is 60 Lgas per hour). Thus, an off-gas level of 1 mg/Lgas corresponds to an instantaneous production rate of 60 mg/Lbroth/hr at air flow of 1 vvm. If desired, the value in the units mg/Lbroth/hr can be divided by the OD600 value to obtain the specific rate in units of mg/Lbroth/hr/OD. The average value of mg isoprene/Lgas can be converted to the total product productivity (grams of isoprene per liter of fermentation broth, mg/Lbroth) by multiplying this average off-gas isoprene concentration by the total amount of off-gas sparged per liter of fermentation broth during the fermentation. Thus, an average off-gas isoprene concentration of 0.5 mg/Lbroth/hr over 10 hours at 1 vvm corresponds to a total product concentration of 300 mg isoprene/Lbroth.
In some embodiments, the cells in culture convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene. In some embodiments, the percent conversion of carbon into isoprene is between such as about 0.002 to about 4.0%, about 0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 to about 0.3%.
The percent conversion of carbon into isoprene (also referred to as “% carbon yield”) can be measured by dividing the moles carbon in the isoprene produced by the moles carbon in the carbon source (such as the moles of carbon in batched and fed glucose and yeast extract). This number is multiplied by 100% to give a percentage value (as indicated in Equation 1).
% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon in carbon source)*100 Equation 1
For this calculation, yeast extract can be assumed to contain 50% w/w carbon. As an example, for the 500 liter described in Example 7, part VIII, the percent conversion of carbon into isoprene can be calculated as shown in Equation 2.
% Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 g glucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12 mol/g)]*100=0.042% Equation 2
For the two 500 liter fermentations described herein (Example 7, parts VII and VIII), the percent conversion of carbon into isoprene was between 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14 liter systems as described herein.
One skilled in the art can readily convert the rates of isoprene production or amount of isoprene produced into any other units. Exemplary equations are listed below for interconverting between units.
Units for Rate of Isoprene production (total and specific)
1 g isoprene/Lbroth/hr=14.7 mmol isoprene/Lbroth/hr (total volumetric rate) Equation 3
1 nmol isoprene/gwcm/hr=1 nmol isoprene/Lbroth/hr/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a wet cell weight of 1 gram.) Equation 4
1 nmol isoprene/gwcm/hr=68.1 ng isoprene/gwcm/hr (given the molecular weight of isoprene) Equation 5
1 nmol isoprene/Lgas O2/hr=90 nmol isoprene/Lbroth/hr (at an O2 flow rate of 90 L/hr per L of culture broth) Equation 6
1 ug isoprene/Lgas isoprene in off-gas=60 ug isoprene/Lbroth/hr at a flow rate of 60 Lgas per Lbroth (1 vvm) Equation 7
Units for Titer (total and specific)
1 nmol isoprene/mg cell protein=150 nmol isoprene/Lbroth/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a total cell protein of approximately 150 mg)(specific productivity) Equation 8
1 g isoprene/Lbroth=14.7 mmol isoprene/Lbroth (total titer) Equation 9
If desired, Equation 10 can be used to convert any of the units that include the wet weight of the cells into the corresponding units that include the dry weight of the cells.
Dry weight of cells=(wet weight of cells)/3.3 Equation 10
If desired, Equation 11 can be used to convert between units of ppm and ug/L. In particular, “ppm” means parts per million defined in terms of ug/g (w/w). Concentrations of gases can also be expressed on a volumetric basis using “ppmv” (parts per million by volume), defined in terms of uL/L (vol/vol). Conversion of ug/L to ppm (e.g., ug of analyte per g of gas) can be performed by determining the mass per L of off-gas (i.e., the density of the gas). For example, a liter of air at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K) has a density of approximately 1.29 g/L. Thus, a concentration of 1 ppm (ug/g) equals 1.29 ug/L at STP (equation 11). The conversion of ppm (ug/g) to ug/L is a function of both pressure, temperature, and overall composition of the off-gas.
1 ppm (ug/g) equals 1.29 ug/L at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K). Equation 11
Conversion of ug/L to ppmv (e.g., uL of analyte per L of gas) can be performed using the Universal Gas Law (equation 12). For example, an off-gas concentration of 1000 ug/Lgas corresponds to 14.7 umol/Lgas. The universal gas constant is 0.082057 L.atm K−1mol−1, so using equation 12, the volume occupied by 14.7 umol of HG at STP is equal to 0.329 mL. Therefore, the concentration of 1000 ug/L HG is equal to 329 ppmv or 0.0329% (v/v) at STP.
PV=nRT, where “P” is pressure, “V” is volume, “n” is moles of gas, “R” is the Universal gas constant, and “T” is temperature in Kelvin. Equation 12
The amount of impurities in isoprene compositions are typically measured herein on a weight per volume (w/v) basis in units such as ug/L. If desired, measurements in units of ug/L can be converted to units of mg/m3 using equation 13.
1 ug/L=1 mg/m3 Equation 13
In some embodiments encompassed by the invention, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acid encoding the isoprene synthase polypeptide.
In some embodiments encompassed by the invention, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide and one or more heterologous nucleic acids encoding a DXP pathway polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acids.
In some embodiments, the isoprene composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.
In some embodiments, the isoprene composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition.
In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 ug/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a hydrocarbon other than isoprene (such as 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 ug/L of a hydrocarbon other than isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a protein or fatty acid (such as a protein or fatty acid that is naturally associated with natural rubber).
In some embodiments, the isoprene composition comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the isoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, the isoprene composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as pentyne-1, butyne-2, 2MB1-3yne, and 1-pentyne-4yne). In some embodiments, the isoprene composition comprises less than or about 2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g., cyclic C10 compounds derived from the dimerization of two isoprene units).
In some embodiments, the composition comprises greater than about 2 mg of isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the amount of isoprene in the composition is between about 2 to about 5,000 mg, such as between about 2 to about 100 mg, about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount of isoprene in the composition is between about 20 to about 5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about 1,000 mg. In some embodiments, greater than or about 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organic fraction of the composition is isoprene.
In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol. In some embodiments in which the composition includes ethanol, the composition also includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.
Exemplary Isoprene Purification MethodsIn some embodiments, any of the methods described herein further include recovering the isoprene. For example, the isoprene produced using the compositions and methods of the invention can be recovered using standard techniques, such as gas stripping, membrane enhanced separation, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods). In one embodiment, the isoprene is recovered by absorption stripping (see, e.g., U.S. Appl. 61/288,142). In particular, embodiments, extractive distillation with an alcohol (such as ethanol, methanol, propanol, or a combination thereof) is used to recover the isoprene. In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation (such as condensation due to exposure to a condensation coil or do to an increase in pressure). In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene. In some embodiments, the isoprene is compressed and condensed.
The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent. In one embodiment, the isoprene is recovered by using absorption stripping as described in U.S. Provisional Appl. No. 61/288,142.
In some embodiments, any of the methods described herein further include purifying the isoprene. For example, the isoprene produced using the compositions and methods of the invention can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid. Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography. As used herein, “purified isoprene” means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC analysis, or GC-MS analysis.
In some embodiments, at least a portion of the gas phase remaining after one or more recovery steps for the removal of isoprene is recycled by introducing the gas phase into a cell culture system (such as a fermentor) for the production of isoprene.
In some embodiments, any of the methods described herein further include polymerizing the isoprene. For example, standard methods can be used to polymerize the purified isoprene to form cis-polyisoprene or other down stream products using standard methods. Accordingly, the invention also features a tire comprising polyisoprene, such as cis-1,4-polyisoprene and/or trans-1,4-polyisoprene made from any of the isoprene compositions disclosed herein.
EXAMPLESThe examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Example 1 Production of Isoprene in E. coli Expressing Recombinant Kudzu Isoprene SynthaseI. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in E. coli
The protein sequence for the kudzu (Pueraria montana) isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprene synthase gene, optimized for E. coli codon usage, was purchased from DNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen) that had been digested with NcoI/PstI. The construct was designed such that the stop codon in the isoprene synthase gene 5′ to the PstI site. As a result, when the construct was expressed the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid, pTrcKudzu, was verified by sequencing (
The isoprene synthase gene was also cloned into pET16b (Novagen). In this case, the isoprene synthase gene was inserted into pET16b such that the recombinant isoprene synthase protein contained the N-terminal His tag. The isoprene synthase gene was amplified from pTrcKudzu by PCR using the primer set pET-His-Kudzu-2F: 5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) and pET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). These primers added an NdeI site at the 5′-end and a BamH1 site at the 3′ end of the gene respectively. The plasmid pTrcKudzu, described above, was used as template DNA, Herculase polymerase (Stratagene) was used according to manufacture's directions, and primers were added at a concentration of 10 pMols. The PCR was carried out in a total volume of 25 μl. The PCR product was digested with NdeI/BamH1 and cloned into pET16b digested with the same enzymes. The ligation mix was transformed into E. coli Top10 (Invitrogen) and the correct clone selected by sequencing. The resulting plasmid, in which the kudzu isoprene synthase gene was expressed from the T7 promoter, was designated pETNHisKudzu (
The kudzu isoprene synthase gene was also cloned into the low copy number plasmid pCL1920. Primers were used to amplify the kudzu isoprene synthase gene from pTrcKudzu described above. The forward primer added a HindIII site and an E. coli consensus RBS to the 5′ end. The PstI cloning site was already present in pTrcKudzu just 3′ of the stop codon so the reverse primer was constructed such that the final PCR product includes the PstI site. The sequences of the primers were: HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQ ID NO:6) and BamH1-Kudzu R:
5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The PCR product was amplified using Herculase polymerase with primers at a concentration of 10 pmol and with 1 ng of template DNA (pTrcKudzu). The amplification protocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1 minute, 72° C. for 2 minutes). The product was digested with HindIII and PstI and ligated into pCL1920 which had also been digested with HindIII and PstI. The ligation mix was transformed into E. coli Top10. Several transformants were checked by sequencing. The resulting plasmid was designated pCL-lac-Kudzu (
For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed as described below (see Table 1 for some experimental values from this assay).
In cases where isoprene production in fermentors was determined, samples were taken from the off-gas of the fermentor and analyzed directly as described below (see Table 2 for some experimental values from this assay).
The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/min. The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 2000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method.
III. Production of Isoprene in Shake Flasks Containing E. coli Cells Expressing Recombinant Isoprene Synthase
The vectors described above were introduced to E. coli strain BL21 (Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu and BL21/pETHisKudzu. The strains were spread for isolation onto LA (Luria agar)+carbenicillin (50 μg/ml) and incubated overnight at 37° C. Single colonies were inoculated into 250 ml baffled shake flasks containing 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures were grown overnight at 20° C. with shaking at 200 rpm. The OD600 of the overnight cultures were measured and the cultures were diluted into a 250 ml baffled shake flask containing 30 ml MagicMedia (Invitrogen)+carbenicillin (100 μg/ml) to an OD600˜0.05. The culture was incubated at 30° C. with shaking at 200 rpm. When the OD600˜0.5-0.8, 400 μM IPTG was added and the cells were incubated for a further 6 hours at 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after induction with IPTG, 1 ml aliquots of the cultures were collected, the OD600 was determined and the amount of isoprene produced was measured as described above. Results are shown in
IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation
Large scale production of isoprene from E. coli containing the recombinant kudzu isoprene synthase gene was determined from a fed-batch culture. The recipe for the fermentation media (TM2) per liter of fermentation medium was as follows: K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2SO4 3.2 g, yeast extract 5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) and q.s. to volume. The final product was filter sterilized with 0.22μ filter (only, do not autoclave). The recipe for 1000× Modified Trace Metal Solution was as follows: Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with a 0.22μ filter.
This experiment was carried out in 14 L bioreactor to monitor isoprene formation from glucose at the desired fermentation, pH 6.7 and temperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD550=0.6, two 600 ml flasks were centrifuged and the contents resuspended in 70 ml supernatant to transfer the cell pellet (70 ml of OD 3.1 material) to the bioreactor. At various times after inoculation, samples were removed and the amount of isoprene produced was determined as described above. Results are shown in
The protein sequence for the poplar (Populus alba×Populus tremula) isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786) was obtained from GenBank (CAC35696). A gene, codon optimized for E. coli, was purchased from DNA2.0 (p9796-poplar,
The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 were electroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformants were selected on LA containing carbenicillin (200 μg/ml) or spectinomycin (50 μg/ml) respectively. Production of isoprene from shake flasks and determination of the amount of isoprene produced was performed as described in Example 1 for E. coli strains expressing recombinant kudzu isoprene synthase. Results are shown in
I. Construction of a B. subtilis Replicating Plasmid for the Expression of Kudzu Isoprene Synthase
The kudzu isoprene synthase gene was expressed in Bacillus subtilis aprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid (pBS19 with a chloramphenicol resistance cassette) under control of the aprE promoter. The isoprene synthase gene, the aprE promoter and the transcription terminator were amplified separately and fused using PCR. The construct was then cloned into pBS19 and transformed into B. subtilis.
a) Amplification of the aprE Promoter
The aprE promoter was amplified from chromosomal DNA from Bacillus subtilis using the following primers:
The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu (SEQ ID NO:2). The gene had been codon optimized for E. coli and synthesized by DNA 2.0. The following primers were used:
The terminator from the alkaline serine protease of Bacillus amyliquefaciens was amplified from a previously sequenced plasmid pJHPms382 using the following primers:
The kudzu fragment was fused to the terminator fragment using PCR with the following primers:
The kudzu-terminator fragment was fused to the promoter fragment using PCR with the following primers:
The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes MfeI and BamHI. This digested DNA fragment was gel purified using a Qiagen kit and ligated to a vector known as pBS19, which had been digested with EcoRI and BamHI and gel purified.
The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA+50 carbenicillin plates. A total of six colonies were chosen and grown overnight in LB+50 carbenicillin and then plasmids were isolated using a Qiagen kit. The plasmids were digested with EcoRI and BamHI to check for inserts and three of the correct plasmids were sent in for sequencing with the following primers:
The plasmid designated pBS Kudzu #2 (
II. Production of Isoprene in Shake Flasks Containing B. subtilis Cells Expressing Recombinant Isoprene Synthase
Overnight cultures were inoculated with a single colony of CF 443 from a LA+Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB+Cm at 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) were used to inoculate 250 ml baffled shake flasks containing 25 ml Grants II media and chloramphenicol at a final concentration of 25 μg/ml. Grants II Media recipe was 10 g soytone, 3 ml 1M K2HPO4, 75 g glucose, 3.6 g urea, 100 ml 10×MOPS, q.s. to 1 L with H2O, pH 7.2; 10×MOPS recipe was 83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K2SO4 solution, 10 ml 0.528M MgCl2 solution, 29.22 g NaCl, 100 ml 100×micronutrients, q.s. to 1 L with H2O; and 100× micronutrients recipe was 1.47 g CaCl2*2H2O, 0.4 g FeSO4*7H2O, 0.1 g MnSO4*H2O, 0.1 g ZnSO4*H2O, 0.05 g CuCl2*2H2O, 0.1 g CoCl2*6H2O, 0.1 g Na2MoO4*2H2O, q.s. to 1 L with H2O. Shake flasks were incubated at 37° C. and samples were taken at 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and the control strain were sampled. This represented 18 hours of accumulation of isoprene. The amount of isoprene was determined by gas chromatography as described in Example 1. Production of isoprene was enhanced significantly by expressing recombinant isoprene synthase (
Large scale production of isoprene from B. subtilis containing the recombinant kudzu isoprene synthase gene on a replication plasmid was determined from a fed-batch culture. Bacillus strain CF 443, expressing a kudzu isoprene synthase gene, or control stain which does not express a kudzu isoprene synthase gene were cultivated by conventional fed-batch fermentation in a nutrient medium containing soy meal (Cargill), sodium and potassium phosphate, magnesium sulfate and a solution of citric acid, ferric chloride and manganese chloride. Prior to fermentation the media is macerated for 90 minutes using a mixture of enzymes including cellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batch fermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADM Versadex greens or Danisco invert sugar) and 99% wt/wt oil (Western Family soy oil, where the 99% wt/wt is the concentration of oil before it was added to the cell culture medium). Feed was started when glucose in the batch was non-detectable. The feed rate was ramped over several hours and was adjusted to add oil on an equal carbon basis. The pH was controlled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case of foaming, antifoam agent was added to the media. The fermentation temperature was controlled at 37° C. and the fermentation culture was agitated at 750 rpm. Various other parameters such as pH, D0%, airflow, and pressure were monitored throughout the entire process. The DO % is maintained above 20. Samples were taken over the time course of 36 hours and analyzed for cell growth (OD550) and isoprene production. Results of these experiments are presented in
IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.
The kudzu isoprene synthase gene was cloned in an integrating plasmid (pJH101-cmpR) under the control of the aprE promoter. Under the conditions tested, no isoprene was detected.
Example 5 Production of Isoprene in TrichodermaI. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Trichoderma reesei
The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized by DNA 2.0 (SEQ ID NO:8) (
The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kit following manufacturer's protocol: 1 μl PCR reaction, 1 μl Salt solution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a total reaction volume of 6 μl. The reaction was incubated at room temperature for 5 minutes. One microliter of TOPO reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA+50 μg/ml kanamycin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB+50 μg/ml kanamycin and the cultures grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit, following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.
A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimized kudzu isoprene synthase gene, was used for Gateway Cloning into a custom-made pTrex3g vector. Construction of pTrex3g is described in WO 2005/001036 A2. The reaction was performed following manufacturer's protocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μl Y. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPO donor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 in a total reaction volume of 8 μl. The reaction was incubated at room temperature for 1 hour and then 1 μl proteinase K solution was added and the incubation continued at 37° C. for 10 minutes. Then 1 μl of reaction was transformed into TOP10 chemically competent E. coli cells. The transformants were selected on LA+50 μg/ml carbenicillin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB+50 μlg/ml carbenicillin and the cultures were grown overnight at 37° C. with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen, Inc.), following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA sequence was correct.
Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprene synthase pTrex3g plasmid (
II. Production of Isoprene in Recombinant Strains of T. reesei
One ml of 15 and 36 hour old cultures of isoprene synthase transformants described above were transferred to head space vials. The vials were sealed and incubated for 5 hours at 30° C. Head space gas was measured and isoprene was identified by the method described in Example 1. Two of the transformants showed traces of isoprene. The amount of isoprene could be increased by a 14 hour incubation. The two positive samples showed isoprene at levels of about 0.5 μg/L for the 14 hour incubation. The untransformed control showed no detectable levels of isoprene. This experiment shows that T. reesei is capable of producing isoprene from endogenous precursor when supplied with an exogenous isoprene synthase.
Example 6 Production of Isoprene in YarrowiaI. Construction of Vectors for Expression of the Kudzu Isoprene Synthase in Yarrowia lipolytica.
The starting point for the construction of vectors for the expression of the kudzu isoprene synthase gene in Yarrowia lipolytica was the vector pSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) is shown in
The following fragments were amplified by PCR using chromosomal DNA of a Y. lipolytica strain GICC 120285 as the template: a promotorless form of the URA3 gene, a fragment of 18S ribosomal RNA gene, a transcription terminator of the Y. lipolytica XPR2 gene and two DNA fragments containing the promoters of XPR2 and ICL1 genes. The following PCR primers were used:
For PCR amplification the PfuUltraII polymerase (Stratagene), supplier-provided buffer and dNTPs, 2.5 μM primers and the indicated template DNA were used as per the manufacturer's instructions. The amplification was done using the following cycle: 95° C. for 1 min; 34× (95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 min at 72° C. followed by a 4° C. incubation.
Synthetic DNA molecules encoding the kudzu isoprene synthase gene, codon-optimized for expression in Yarrowia, was obtained from DNA 2.0 (
A similar cloning procedure can be used to express a poplar (Populus alba×Populus tremula) isoprene synthase gene. The sequence of the poplar isoprene is described in Miller B. et al. (2001) Planta 213, 483-487 and shown in
II. Production of Isoprene by Recombinant Strains of Y. lipolytica.
Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digested with SacII and used to transform the strain Y. lipolytica CLIB 122 by a standard lithium acetate/polyethylene glycol procedure to uridine prototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) overnight, were collected by centrifugation (4000 rpm, 10 min), washed once with sterile water and suspended in 0.1 M lithium acetate, pH 6.0. Two hundred μl aliquots of the cell suspension were mixed with linearized plasmid DNA solution (10-20 μg), incubated for 10 minutes at room temperature and mixed with 1 ml of 50% PEG 4000 in the same buffer. The suspensions were further incubated for 1 hour at room temperature followed by a 2 minutes heat shock at 42° C. Cells were then plated on SC his leu plates (0.67% yeast nitrogen base, 2% glucose, 100 mg/L each of leucine and histidine). Transformants appeared after 3-4 days of incubation at 30° C.
Three isolates from the pYLA(KZ1) transformation, three isolates from the pYLI(KZ1) transformation, two isolates from the pYLA(MAP29) transformation and two isolates from the pYLI(MAP29) transformation were grown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0) at 30° C. with shaking. Cells from 10 ml of culture were collected by centrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 ml screw cap vials. The vials were incubated overnight at room temperature with gentle (60 rpm) shaking. Isoprene content in the headspace of these vials was analyzed by gas chromatography using mass-spectrometric detector as described in Example 1. All transformants obtained with pYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene (0.5 μg/L to 1 μg/L,
I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, or dxs, or idi and dxs for the Production of Isoprene in E. coli
i) Construction of pTrcKudzuKan
The bla gene of pTrcKudzu (described in Example 1) was replaced with the gene conferring kanamycin resistance. To remove the bla gene, pTrcKudzu was digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP), heat killed at 65° C., then end-filled with Klenow fragment and dNTPs. The 5 kbp large fragment was purified from an agarose gel and ligated to the kanr gene which had been PCR amplified from pCR-Blunt-II-TOPO using primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) and MCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested with HindIII and PvuI, and end-filled. A transformant carrying a plasmid conferring kanamycin resistance (pTrcKudzuKan) was selected on LA containing kanamycin 50 μg/ml.
ii) Construction of pTrcKudzu yIDI Kan
pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding idi from S. cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F 5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1 R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S. cerevisiae genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The ligation mixture was transformed into chemically competent TOP10 cells and selected on LA containing 50 μg/ml kanamycin. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-yIDI(kan) (
iii) Construction of pTrcKudzu DXS Kan
Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding dxs from E. coli with a synthetic RBS. The primers for PCR were MCM13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG (SEQ ID NO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); and the template was E. coli genomic DNA. The PCR product was digested with NsiI and PstI and gel purified prior to ligation. The resulting transformation reaction was transformed into TOP10 cells and selected on LA with kanamycin 50 μg/ml. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (
iv) Construction of pTrcKudzu-yIDI-Dxs (Kan)
pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding E. coli dxs with a synthetic RBS (primers MCM 13 5′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG (SEQ ID NO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); template TOP10 cells) which had been digested with NsiI and PstI and gel purified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (
A fragment of DNA containing the promoter, structural gene and terminator from Example 1 above was digested from pTrcKudzu using SspI and gel purified. It was ligated to pCL1920 which had been digested with PvuII, treated with SAP and heat killed. The resulting ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and two were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insert in opposite orientations (
vi) Construction of pCL PtrcKudzu yIDI
The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) above was ligated into pCL PtrcKudzu which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu yIDI (
vii) Construction of pCL PtrcKudzu DXS
The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) above was ligated into pCL PtrcKudzu (A3) which had been digested with PstI, treated with SAP, and heat killed. The ligation mixture was transformed into TOP10 cells and selected in LA containing spectinomycin 50 μg/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu DXS (
II. Measurement of Isoprene in Headspace from Cultures Expressing Kudzu Isoprene synthase, idi, and/or dxs at Different Copy Numbers.
Cultures of E. coli BL21(λDE3) previously transformed with plasmids pTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C), pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Cultures of pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCL PtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures were induced with 400 μM IPTG at time 0 (OD600 approximately 0.5) and samples taken for isoprene headspace measurement (see Example 1). Results are shown in
Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21 by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS was grown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and used to inoculate shake flasks of TM3 (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) containing 1% glucose. Flasks were incubated at 30° C. until an OD600 of 0.8 was reached, and then induced with 400 μM IPTG. Samples were taken at various times after induction and the amount of isoprene in the head space was measured as described in Example 1. Results are shown in
III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS
The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generate isoprene from three types of biomass; bagasse, corn stover and soft wood pulp with glucose as a control. Hydrolysates of the biomass were prepared by enzymatic hydrolysis (Brown, L and Torget, R., 1996, NREL standard assay method Lap-009 “Enzymatic Saccharification of Lignocellulosic Biomass”) and used at a dilution based upon glucose equivalents. In this example, glucose equivalents were equal to 1% glucose. A single colony from a plate freshly transformed cells of BL21 (DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB plus kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. The feedstock was corn stover, bagasse, or softwood pulp. Glucose was used as a positive control and no glucose was used as a negative control. Cultures were incubated at 30° C. with shaking at 180 rpm. The culture was monitored for OD600 and when it reached an OD600 of ˜0.8, cultures were analyzed at 1 and 3 hours for isoprene production as described in Example 1. Cultures are not induced. All cultures containing added feedstock produce isoprene equivalent to those of the glucose positive control. Experiments were done in duplicate and are shown in
IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS
A single colony from a plate freshly transformed cells of BL21 (λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB+kanamycin (50 μg/ml). The culture was incubated overnight at 25° C. with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. Feedstock was glucose, inverted glucose or corn stover. The invert sugar feedstock (Danisco Invert Sugar) was prepared by enzymatically treating sucrose syrup. AFEX corn stover was prepared as described below (Part V). The cells were grown at 30° C. and the first sample was measured when the cultures reached an OD600˜0.8-1.0 (0 hour). The cultures were analyzed for growth as measured by OD600 and for isoprene production as in Example 1 at 0, 1 and 3 hours. Results are shown in
V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover
AFEX pretreated corn stover was obtained from Michigan Biotechnology Institute. The pretreatment conditions were 60% moisture, 1:1 ammonia loading, and 90° C. for 30 minutes, then air dried. The moisture content in the AFEX pretreated corn stover was 21.27%. The contents of glucan and xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (dry basis), respectively. The saccharification process was as follows; 20 g of AFEX pretreated corn stover was added into a 500 ml flask with 5 ml of 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121 (Danisco xylanase product from Aspergillus niger for bread-making industry), and 72.65 ml of DI water. The flask was put in an orbital shaker and incubated at 50° C. for 96 hours. One sample was taken from the shaker and analyzed using HPLC. The hydrolysate contained 38.5 g/1 of glucose, 21.8 g/1 of xylose, and 10.3 g/1 of oligomers of glucose and/or xylose.
VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grown in Fed-Batch Culture
Fermentation was performed at the 14-L scale as previously described with E. coli cells containing the pTrcKudzu yIDI DXS plasmid described above. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed at an exponential rate. The total amount of yeast extract delivered to the fermentor was varied between 70-830 g during the 40 hour fermentation. Optical density of the fermentation broth was measured at a wavelength of 550 nm. The final optical density within the fermentors was proportional to the amount of yeast extract added (
VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI
A 500 liter fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21 (λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels of isoprene varied from 50 to 300 μg/L over a time period of 15 hours. On the basis of the average isoprene concentrations, the average flow through the device and the extent of isoprene breakthrough, the amount of isoprene collected was calculated to be approximately 17 g.
VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown in Fed-Batch Culture
Medium Recipe (Per Liter Fermentation Medium):K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium gas (NH3) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotic were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution:Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component is dissolved one at a time in DI H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.
Fermentation was performed in a 500-L bioreactor with E. coli cells containing the pTrcKudzu yIDI DXS plasmid. This experiment was carried out to monitor isoprene formation from glucose and yeast extract at the desired fermentation pH 7.0 and temperature 30° C. An inoculum of E. coli strain taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD 0.15, measured at 550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-L soytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at 30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.
Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose were fed at exponential rates. The total amount of glucose and yeast extract delivered to the bioreactor during the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively. The optical density within the bioreactor over time is shown in
BL21 (DE3) strain harboring pTrcKudzuDXSyIDI produced more isoprene under non-inducing conditions compared to IPTG induction conditions, and was observed to accumulate HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate), the substrate of HDS (GcpE or IspG). Using the BL21 (DE3) strain harboring pTrcKudzuDXSyIDI as the parental host strain, the introduction of an additional plasmid-born copy of the Kudzu isoprene synthase gene alone and in combination with the fldA gene encoding flavodoxin I were assessed for the effects on isoprene production by the strains under non-inducing conditions relative to the empty vector control strain.
These experiments investigated whether an additional copy of the isoprene synthase improves isoprene production under non-inducing conditions in the BL21 (DE3) strain harboring the pTrcKudzuDXSyIDI construct. Under the non-inducing conditions, isoprene synthase may be limiting and an additional copy of the Kudzu enzyme may be able to improve the specific productivity of isoprene generation by the strain. The experiments also investigated whether other factor(s) contributed to the modest level of isoprene produced by the strain and whether a plasmid-born copy of fldA could increase isoprene production by the BL21 (DE3) strain that harbors the pTrcKudzuDXSyIDI construct under non-inducing conditions. The flavodoxin I encoded by fldA was intended to be expressed ectopically from the pTrcHgSfldA/pBAD33 construct at a level surpassing that generated from the endogenous fldA locus. An increased amount of flavodoxin I may increase the activity demonstrated by the DXP pathway enzymes GcpE (HDS or IspG) and LytB (HDR or IspH) in vivo, as was previously seen in vitro (Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339, 2002; Wolff, M. et al. FEBS Letters, 541: 115-120, 2003), and possibly improve carbon flux to isoprene synthesis in the strain of interest over that of the comparable pTrcKudzuDXSyIDI-containing BL21 (DE3) control strain.
Bacterial transformation and molecular biology techniques were performed using standard protocols (Sambrook et al), which is hereby incorporated by reference in its entirety, particularly with respect to bacterial transformation. The E. coli strains BL21 (DE3) and TOP10 were obtained from Invitrogen. TOP10 cells were used during the preparation of the pTrcHgS/pBAD33 and pTrcHgSfldA/pBAD33 constructs described below. Vector constructs were moved via chemical transformation into the BL21 (DE3) strain for the subsequent assessment of isoprene production.
Constructs
Primers were purchased from Integrated DNA Technologies (Coralville, Iowa). PCR reactions were performed with Herculase II Fusion (Stratagene) according to manufacturer's specifications.
Primers were purchased from Integrated DNA Technologies (Coralville, Iowa). PCR reactions were performed with Herculase II Fusion (Stratagene) according to manufacturer's specifications.
E. coli 12 MG1655 (world wide web at genome.wisc.edu/resources/strains.htm) was the source of genomic template used to amplify the fldA locus; cells were added directly to the PCR reaction using a sterile toothpick.
The fldA PCR product was cleaned utilizing the MinElute PCR Purification Kit (Qiagen). pBAD33 is described, for example, in Luz-Maria, G. et al., J. Bacteriology, 77: 4121-4130, 1995, which is hereby incorporated by reference in its entirety, particularly with respect to pBAD33. pTrcKudzu, and pTrcKudzuDXSyIDI kan, were described, for example, in U.S. application Ser. No. 12/335,071 and PCT/US2008/086809, which are hereby incorporated by reference in their entireties, particularly with respect to Examples 1 and 7.
pTrcHgS/pBAD33 was constructed here by cloning the SspI-PstI (1934 bp) fragment containing the Trc promoter region, rbs, and the coding sequence of the Kudzu isoprene synthase derived from pTrcKudzu into the SmaI-PstI sites of pBAD33.
pTrcHgSfldA/pBAD33 was constructed here. The NsiI-PstI (1471 bp) digested PCR amplified fldA fragment encompassing 22 bp upstream of the fldA start, including the endogenous rbs, through the stop codon of the fldA gene was cloned into the PstI site located just downstream of the isoprene synthase open reading frame in pTrcHgS/pBAD33.
Constructs were verified by sequencing that was performed by Sequetech (Mountain View, Calif.).
Culture ConditionsBacteria were grown at 25° C. and 30° C. on LB 1.5% agar plates and in TM3 liquid media (see description of TM3, for example, U.S. application Ser. No. 12/335,071 and PCT/US2008/086809, which are hereby incorporated by reference in their entireties, particularly with respect to TM3 liquid media) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose. When appropriate, kanamycin (Kan) and/or chloramphenicol (Cmp) were added to the growth media at 50 μg/ml and 10 μg/ml, respectively; pTrc-based constructs encode KanR and pBAD33-based constructs encode CmpR. Bacterial growth was monitored by optical density measured at 600 nm.
Assessment of Isoprene ProductionHeadspace assay for isoprene production was described in Example 1. The specific productivity of each strain was reported as μg/L·OD·hour; note ratio of 1900 μl headspace:100 μl broth in assay vials. Graphs depicting the growth rate and specific productivity of each strain were generated using Microsoft Office Excel 2003 software.
Construction of BL21 (DE3) Strains and Assessment of the Isoprene ProductionThe following BL21 (DE3) strains were constructed and assessed for the production of isoprene relative to one another: BL21 (DE3) harboring the pTrcKudzuDXSyIDI vector and either 1) empty pBAD33 vector (also referred to as “empty vector”); 2) pTrcHgS/pBAD33 construct (also referred to as “HgS”), or 3) pTrcHgSfldA/pBAD33 construct (as referred to as “HgS-FldA”).
All three BL21 (DE3) test strains harbor KanR and CmpR and were grown under appropriate selection for both plasmid constructs. The empty vector strain represented the parental control strain; the HgS strain represented the parental strain harboring an addition plasmid-born copy of the Kudzu isoprene synthase gene; the HgS-FldA strain represented the parental strain harboring the addition plasmid-born copies of flavodoxin I and isoprene synthase genes.
The bacteria strains were grown overnight shaking (250 rpm) at 25° C. in 10 ml of supplemented TM3 media containing antibiotics; here and for the following experiments 50 μg/ml of kanamycin and 10 μg/ml of chloramphenicol were present in the growth media. The cultures were then diluted into fresh supplemented TM3 media containing antibiotics to an optical density at 600 nm of approximately 0.05 and allowed to grow shaking (250 rpm) at 30° C. in 12.5-25 ml of supplemented TM3 media containing antibiotics in 250 ml Erlenmeyer flasks. Strains were typically assessed for isoprene production once the optical density at 600 nm of the culture reached 0.4. In the most densely sampled experiments, once isoprene measurements commenced the isoprene production for each culture was monitored in 45 min. intervals. The results from two independent experiments depicting growth rate and specific productivity of isoprene generation for the empty vector (control), HgS, and HgS-FldA strains are shown in the
Under the non-inducing conditions tested, the results obtained from the isoprene headspace assays performed on the empty vector, HgS, and HgS-FldA strains indicate that an additional copy of fldA present on the pTrcHgSfldA/pBAD33 construct substantially increases isoprene production in the HgS-FldA strain over that produced by both the HgS and empty vector control strains. The HgS-FldA strain was observed to exhibit increased specific productivity of isoprene generation ranging from 1.5- to 1.9-fold and 1.3- to 1.8-fold higher than the control strain over a 3.75-hour and 2.5-hour time course, respectively, during two independent experiments. The observed effect on isoprene production appears to be specific to the presence of the fldA-containing construct, as the HgS strain produces comparable levels of isoprene under the non-inducing conditions to that produced by the empty vector control strain.
Example 9 Expression of Alternative ispG (gcpE or HDS) and ispH (lytB or HDR) and their Corresponding Reducing Shuttle System, from Thermosynechococcus elongatus BP-1 in an Isoprene-Producing E. coli to Improve Isoprene ProductionIn this example, we demonstrated that the ferredoxin/ferredoxin-NADP oxidoreductase/NADPH reducing system together with the GcpE and LytB enzymes from T. elongates improve isoprene production in E. coli BL21(DE3).
T. elongatus, like E. coli, synthesizes isoprenoids via the DXP pathway, but does not harbor any genes coding for a flavodoxin protein. It was previously shown that the plant GcpE enzyme is a ferredoxin-dependent enzyme, and that flavodoxin could not support the enzymatic conversion of cMEPP (ME-CPP) into HDMAPP (HMBPP) by this enzyme (see Seemann et al., FEBS Lett., 580(6):1547-52 (2006), which is hereby incorporated by reference in its entirety). It was also demonstrated in vitro that GcpE of T. elongatus together with PetF (ferredoxin), Pet H (ferredoxin-NADP+ oxidoreductase), and NADPH could convert cMEPP into HDMAPP (Okada and Hase, J Biol Chem, 280(21):20627-9 (2005)), which is hereby incorporated by reference in its entirety). With the lack of other small electron carrier proteins besides ferredoxin in the genome, it is likely that LytB of T. elongatus also utilizes the same reducing shuttle system as GcpE.
Demonstration of increased isoprene production and elevated cMEPP levels in REM23-26 by overexpression of GcpE, PetF, and PetH from T. elongatus BP-1
We have previously demonstrated that increased expression of dxs increases flux through the DXP pathway in E. coli. Isoprene-producing strains (REM19-22) harboring increased and varied levels of dxs expression were constructed by integrating the GI 1.X-promoter series immediately upstream of the dxs locus within the E. coli BL21(DE3) genome. Subsequently, the test set of strains, REM23-26 were created by transformation with plasmids expressing the T. elongatus GcpE and its corresponding reducing shuttle system encoded by petF and petH. The parental and test strains were evaluated for growth, isoprene production, and the presence of DXP pathway metabolites. The results are presented in
The GI 1.X-promoter insertions and subsequent loopout of the antibiotic resistance markers described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21(DE3) (Invitrogen) was used.
degenerate N base: A base yields GI 1.6-, T base yields GI 1.5-, G base yields GI1.2-, and C base yields GI 1.0-promoter.
The strategy for inserting the GI1.X-promoter series in front of dxs is shown in
To amplify the GB-NeoR cassette for inserting the GI 1.X-promoters immediately upstream of the dxs locus the following PCR reactions were set up:
1 ul (100 ng GB-NeoR) 10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) MCM319
1.25 ul primer (10 uM) MCM320
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 minutes, [95° C.×20 seconds, 55° C.×20 seconds, 72° C.×50 seconds]×30 cycles; 72° C.×3 minutes, 4° C. until cool (BioRadPCR machine).
The resulting PCR fragments were separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stock was GB-NeoR-GI 1.X-dxs fragment.
Integration of GB-NeoR-GI 1.X-Dxs PCR Product into BL21(DE3)/pRed-ET Strain
The pRed-ET vector (Gene Bridges kit) was transformed into BL21(DE3) by electroporation resulting in strain MCM327 (BL21(DE3)/pRed-ET). Approximately 500 ng of the GB-NeoR-GI 1.x-dxs PCR fragment was electroporated into MCM327. The transformants were recovered in L Broth for 1 hour with shaking at 200 rpm at 37° C. and then plated on L agar containing kanamycin (10 ug/ml). Kanamycin resistant colonies were analyzed by PCR for the presence of the GB-NeoR cassette and the GI 1.X-promoters using primers GB-DW/MCM327. The PCR fragments from a number of transformants (MCM617-625) were sequenced using the MCM327 and GB-DW primers (Quintara; Berkeley, Calif.) and the various GI 1.X-dxs strains of interest identified. The correct strains were designated MCM617 (FRT-neo-FRT-GI 1.0-dxs), MCM618 (FRT-neo-FRT-GI 1.5-dxs), MCM623 (FRT-neo-FRT-GI 1.2-dxs), and MCM625 (FRT-neo-FRT-GI 1.6-dxs). The kanamycin resistance cassette was looped out of the strains using pCP20 from the RED/ET kit according to the manufacturer's instructions. Transformants were verified by loss of resistance to kanamycin (10 ug/ml) and PCR demonstrating loopout of the GB-NeoR cassette. The resulting strains were designated MCM638 (BL21(DE3) GI 1.0-dxs), MCM639 (BL21(DE3) GI 1.5-dxs), MCM640 (BL21(DE3) GI 1.2-dxs) and MCM641 (BL21(DE3) GI 1.6-dxs).
Construction of the Parental Strains REM19-22 from MCM638, MCM640, MCM639, and MCM641, Respectively
The construction of the T7-MEARR alba/pBBR1MCS-5 described in this example was carried out using standard molecular biology techniques (Sambrook et al., 1989, which is hereby incorporated by reference in its entirety). The pBBR1MCS-5 plasmid has been previously described (Kovach et al., Biotechniques, 16(5):800-2 (1994), which is hereby incorporated by reference in its entirety, particularly with respect to cloning of the pBBR1MCS). A picture illustrating the resulting plasmid construct is shown in
Electroporation of T7-MEARR alba/pBBR1MCS-5 into strains MCM638-641. The vector construct harboring the T7 polymerase governed MEARR alba allele, MD09-173 (BL21(DE3)pLysS, pET24a-P. alba (MEA) Untagged (pDu39)), was used as the PCR template.
Amplification of the T7-MEARR alba Fragment
To amplify the T7-MEARR alba fragment for cloning into the pBBR1MCS-5 plasmid the following PCR reaction was performed:
1 ul (approx. 120 ng MDO9-173)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ KpnI to lad MEARR T7 frag
1.25 ul primer (10 uM) 3′ SpeI to T7 terminator MEARR T7 frag
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene.
95° C.×2 minutes, [95° C.×30 seconds, 63° C.×30 seconds, 72° C.×3 minutes]×29 cycles; 72° C.×5 minutes,
4° C. until cool (Biometra T3000 Combi Thermocycler).
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions. The resulting stock was T7-MEARR alba fragment.
Cloning of the T7-MEARR alba Fragment into pBBR1MCS-5
Approximately 600 ng of the T7-MEARR alba fragment and 200 ng of the pBBR1MCS-5 plasmid were digested with KpnI and SpeI (Roche) according to the manufacturer's specifications. The digests were subsequently combined and cleaned using the Qiagen QiaQuick Gel Extraction Kit. Approximately a fourth to a third of the cleaned cut DNA was ligated using T4 DNA Ligase (New England Biolabs) according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) was transformed with the ligation reaction using a standard heat-shock protocol (See, e.g., Sambrook et al., 1989, which is hereby incorporated by reference in its entirety), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing gentamycin (10 ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40 ug/ml; Sigma). White, gentamycin resistant colonies were selected, grown overnight in L broth containing gentamycin (10 ug/ml), and harvested for plasmid preparation the following day. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit and first analyzed by restriction enzyme digestion and electrophoresis (as described above) for the putative presence of the T7-MEARR alba fragment. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers M13 Forward (−20), EL-1000, A-rev, and A-rev2, and the correct T7-MEARR alba/pBBR1MCS-5 clone identified.
Transformation of T7-MEARR alba/pBBR1MCS-5 into MCM638-641
To build the isoprene-producing strains REM19-22 the T7-MEARR alba/pBBR1MCS-5 plasmid was transformed by electroporation into MCM638-641. Transformants were recovered in L broth and plated on L agar containing gentamycin (10 ug/ml). The resulting strains were designated as such: REM19 (MCM638/T7-MEARR alba/pBBR1MCS-5), REM20 (MCM640/T7-MEARR alba/pBBR1MCS-5), REM21 (MCM639/T7-MEARR alba/pBBR1MCS-5), and REM22 (MCM641/T7-MEARR alba/pBBR1MCS-5).
Construction of the Test Strains REM23-26REM23-26 were constructed by transformation of the Ptac-gcpE-petF-petH/pK184 construct into MCM638, MCM640, MCM639, and MCM641. The plasmid Ptac-gcpE-petF-petH/pK184 described in this example was synthesized by Gene Oracle, Inc. (Mountain View, Calif.) with codon optimization of gcpE, petF, and petH for expression in E. coli. The Ptac promoter and aspA terminator sequences have been previously described (Genbank accession # E02927 and CP001164, respectively). The pK184 cloning vector has been described, for example, by Jobling and Holmes, Nucleic Acids Res. 18(17):5315-6 (1990), which is hereby incorporated by reference in its entirety, particularly with respect to the pK184 cloning vector. A picture illustrating the resulting plasmid construct is shown in
Electroporation of Ptac-gcpE-petF-petH/pK184 into strains REM19-22. A plasmid preparation of Ptac-gcpE-petF-petH/pK184 was provided by Gene Oracle, Inc.
Transformation of Ptac-gcpE-petF-petH/pK184 into REM19-22
To build the isoprene-producing test strains, REM23-26, the Ptac-gcpE-petF-petH/pK184 plasmid was transformed by electroporation into REM19-22. Transformants were recovered in L broth and plated on L agar containing kanamycin (10 ug/ml) and gentamycin (10 ug/ml). The resulting strains were designated as such: REM23 (REM19/Ptac-gcpE-petF-petH/pK184), REM24 (REM20/Ptac-gcpE-petF-petH/pK184), REM25 (REM21/Ptac-gcpE-petF-petH/pK184), and REM26 (REM22/Ptac-gcpE-petF-petH/pK184).
Analysis of REM19-26 for Growth, Isoprene Production, and DXP Metabolite GenerationThe parental strains REM19-22 were compared against the test strains REM23-26 in a shake flask isoprene headspace assay as well as in a DXP metabolite determination study. The benefits of expressing the T. elongatus GcpE enzyme on DXP metabolite generation and isoprene production from the E. coli host is illustrated in
Strains REM19-26 were grown at 30° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including kanamycin (10 ug/ml) and gentamycin (10 ug/ml). Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production was analyzed using a headspace assay. For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 200 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isoprene ug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental and test strains, REM19-22 and REM23-26, respectively, described above and depicted in
Cell metabolism was rapidly inactivated by withdrawing 3.5 mL of the culture into a tube filled with 3.5 mL of dry ice-cold methanol. Cell debris was pelleted by centrifugation and the supernatant was loaded onto Strata-X-AW anion exchange column (Phenomenex) containing 30 mg of sorbent. The pellet was re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM NH4HCO3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1 mM NH4HCO3 buffer (pH=7.0). After each extraction, cell debris was pelleted by centrifugation and the supernatants were consecutively loaded onto the same anion exchange column. During the extraction and centrifugation steps the samples were kept at below +4° C. Prior to metabolite elution, the anion exchange columns were washed with water and methanol (1 mL of each) and the analytes were eluted by adding 0.35 mL of concentrated NH4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated NH4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant was neutralized with 30 μL of glacial acetic acid and cleared by centrifugation in a microcentrifuge.
Metabolite QuantificationMetabolites were analyzed using a Thermo Scientific TSQ Quantum Access mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). All system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a chiral Nucleodex β-OH 5 μM HPLC column (100×2 mm, Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH guard cartridge was eluted with a mobile phase gradient shown in Table 1 (flow rate of 0.4 mL/min). The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of metabolites were determined based on the integrated intensities of peaks generated by PO3− product ion (m/z=79.0). Calibration curves obtained by injection of corresponding standards purchased from Echelon Biosciences Inc. Intracellular concentrations of metabolites were calculated based on the assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Demonstration of Increased Isoprene Production in REM31 and REM29 by Overexpression of GcpE, LytB PetF and PetH of T. elongatus BP-1
We have demonstrated that increased expression of dxs permits increased flux through the DXP pathway within E. coli, while the additional overexpression of an idi gene increases the production of downstream isoprenoids significantly. To demonstrate the benefits of expressing the non-flavodoxin-dependent GcpE and LytB enzymes on carbon flux through the endogenous E. coli DXP pathway to isoprene synthesis, E. coli BL21(DE3) isoprene-producing strains with constitutive expression of dxs and the yeast IDI enzyme were constructed. The BL21(DE3) GI 1.6-dxs strain MCM641 is described above. The construction of the vector construct harboring the yeast IDI enzyme, pDU9-pET-16b rev-yIDI, is described herein. The T7-(−3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 P. alba isoprene synthase-containing constructs are described below. A set of parental isoprene-producing, IDI-overexpressing strains derived from MCM641 were created (REM H76 and REMH86) to compare to the newly generated test set of strains (REM31 and REM29) which harbor the T. elongatus GcpE, LytB, and their corresponding reducing shuttle system (described below). The parental and test strains were evaluated for growth, isoprene production, and the presence of DXP pathway metabolites. The results are depicted in
Construction of pDU-9
The IPP isomerase from Saccharomyces cerevisiae (yIDI) was cloned into the vector pET16b (Invitrogen). The primer set Hg-yIDI-R2/Hg-yIDI-F2 was used for PCR with the template DNA pTrcKudzu yIDI Kan. The PCR cycle conditions:
PCR Reaction
-
- 1 ul of template (pMVK1—Fernando's template)
- 5 ul of 10× PfuII Ultra buffer
- 1 ul of dNTP
- 1 ul of primer (50 uM) Hg-MVK-F2-NdeI
1 ul of primer (50 uM) Hg-yIDI-R2
40 ul of DiH2O
+1 ul of Pfu UltraII Fusion DNA Polymerase from Stratagene
Cycle Parameter: (95° C. 2 min., 95° C. 20 sec., 55° C. 20 sec., 72° C. 21 sec., 29×, 72 C 3 min.,4° C. until cool, use Eppendorf Mastercycler Gradient Machine)
The PCR product was purified using the QiaQuick PCR purification kit according to the manufacturer's suggestion. An aliquot of 5 uL purified of the PCR product was ligated to Invitrogen pET-16b Vector that was previously digested with NdeI-SAP (Shrimp Alkaline Phosphatase) treated using T4 ligase enzyme (NEB). The ligation was carried out overnight at 16° C.
5 uL of overnight ligation mixture was introduced into Invitrogen TOP10 cells and transformants were selected on L agar containing Carbenicillin (50 ug/ml) incubated at 37° C. Plasmids from transformants were isolated using QiaQuick spin miniprep kit. The insert is sequenced with T7 promoter and T7 terminator (Use Quintara Bio Sequencing Service). The resulting plasmid r is called pDu-9.
Once the sequence is verified, 1 ul of plasmid pDu-9 was transformed into BL21(DE3) pLysS hst strain according to manufacturer's protocol. Transformants are selected on L agar containing Carbenicillin (50 ug/ml) plate and incubated at 37° C.
Construction of REMD76 (MCM641/pDU9-pET-16b Rev-yIDI), REMH76 and REMH86 (REMD76/T7-(−3) alba/pBBR1MCS-5 and REMD76/T7-MTE alba/pBBR1MCS-5, Respectively)
pDU9-pET-16b rev-yIDI was electroporated into MCM641.
Transformation of pDU9-pET-16b rev-yIDI into MCM641
To build the BL21(DE3) GI 1.6-dxs yIDI-overexpressing strain REMD76, the pDU9-pET-16b rev-yIDI plasmid expressing a yeast IDI (yIDI) allele was transformed by electroporation into MCM641. Transformants were recovered in L broth and plated on L agar containing carbinicillin (50 ug/ml). A carbinicillin resistant colony was selected and designated REMD76.
Generation of the Parental Strains REMH76 and REMH86 (REMD76/T7-(−3) alba/pBBR1MCS-5 and REMD76/T7-MTE alba/pBBR1MCS-5, Respectively
The construction of the T7-(−3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 constructs described in this example were carried out using standard molecular biology techniques (ee, e.g., Sambrook et al., 1989). The pBBR1MCS-5 plasmid has been previously described (see, Kovach et al., Biotechniques, 16(5):800-2 (1994), which is hereby incorporated by reference in its entirety, particularly with respect to the pBBR1MCS-5 plasmid). The pictures illustrating the resulting plasmid constructs are shown in
Electroporation of T7-(−3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 into strain REMD76. The vector constructs harboring the T7 polymerase governed (−3) and MTE alba alleles, pDU47-3-pET24a-P. alba (−3) and pDU42 pET24a-P. alba-MTE untagged, were used as the PCR templates.
Amplification of the T7-(−3) and T7-MTE alba Fragments
To amplify the T7-(−3) and T7-MTE alba fragments for cloning into the pBBR1MCS-5 plasmid the following PCR reactions were performed: 1 ul (approx. 100 ng pDU47-3-pET24a-P. alba (−3) or pDU42 pET24a-P. alba-MTE untagged)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ KpnI to lad MEARR T7 frag
1.25 ul primer (10 uM) 3′ SpeI to T7 terminator MEARR T7 frag
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 minutes, [95° C.×30 seconds, 63° C.×30 seconds, 72° C.×3 minutes.]×29 cycles; 72° C.×5 minutes,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragments were separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stocks were T7-(−3) alba fragment and T7-MTE alba fragment.
Cloning of the T7-(−3) alba and T7-MTE alba Fragments into pBBR1MCS-5
Approximately 600 ng of the T7-(−3) alba fragment or T7-MTE alba fragment and 200 ng of the pBBR1MCS-5 plasmid were digested with KpnI and SpeI from Roche according to the manufacturer's specifications. The digests were subsequently combined and cleaned using the Qiagen QiaQuick Gel Extraction Kit. Approximately a fourth to a third of the cleaned cut DNA was ligated using T4 DNA Ligase from New England Biolabs according to the manufacturer's suggested protocol.
Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989, which is hereby incorporated by reference in its entirety), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing gentamycin (10 ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40 ug/ml; Sigma). White gentamycin resistant colonies were selected, grown overnight in L broth containing gentamycin (10 ug/ml), and harvested for plasmid preparation the following day. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit and first analyzed by restriction digest and electrophoresis (as described above) for the putative presence of the T7-(−3) alba fragment or T7-MTE alba fragment. Plasmid preparations of interest identified were sequenced (Sequetech; Mountain View, Calif.) using primers M13 Forward (−20), EL-1000, A-rev, and A-rev2, and the correct T7-(−3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 clones identified.
Construction of the Test Strains REM31 and REM29To create strains REM31 and 29 the plasmid Ptac-gcpE-lytB-petF-petH/pK184 was transformed into REMH76 and REMH86. The synthesis and codon optimization for E. coli of the Ptac-gcpE-lytB-petF-petH/pK184 described in this example was performed by Gene Oracle, Inc. (Mopuntain View, Calif.). The Ptac promoter and aspA terminator sequences have been previously described (Genbank accession # E02927 and CP001164, respectively) and were also constructed synthetically. The pK184 cloning vector has been described previously (see, Jobling and Holmes, Nucleic Acids Res. 18(17):5315-6 (1990), which is hereby incorporated by reference in its entirety, particularly with respect to the pK184 cloning vector). A picture illustrating the resulting plasmid construct is shown in
Electroporation of Ptac-gcpE-lytB-petF-petH/pK184 into strains REMH76 and REMH86 strains: A plasmid preparation of Ptac-gcpE-lytB-petF-petH/pK184 was provided by Gene Oracle, Inc.
Transformation of Ptac-gcpE-lytB-petF-petH/pK184 into REMH76 and REMH86
To build the isoprene-producing test strains (REM31 and REM29) which harbor the T. elognatus GcpE and LytB enzymes to assess against the parental strains (REMH76 and REMH86) for benefits in DXP pathway flux and isoprene production, the Ptac-gcpE-lytB-petF-petH/pK184 plasmid was transformed by electroporation into REMH76 and REMH86. Transformants were recovered in L broth and plated on L agar containing carbinicillin (50 ug/ml), kanamycin (10 ug/ml), and gentamycin (10 ug/ml). The resulting strains are designated as such: REM31 (REMH76/Ptac-gcpE-lytB-petF-petH/pK184) and REM29 (REMH86/Ptac-gcpE-lytB-petF-petH/pK184).
Comparing REMH76 and REMH86 to REM31 and REM29 for Growth and Isoprene ProductionThe parental strains REMH76 and REMH86 were compared against the test strains REM31 and REM29, respectively, in a shake flask isoprene headspace assay as well as in a DXP metabolite determination study. The benefit of expressing the T. elongatus GcpE and LytB enzymes on isoprene production from the E. coli host is illustrated in
Parental strains REMH76 and REMH86 and test strains REM31 and REM29 were grown at 30° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including carbinicillin, (50 ug/ml) kanamycin (10 ug/ml) and gentamycin (10 ug/ml). Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production was analyzed using a headspace assay. For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 200 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method.
The specific productivity of each strain was reported as ug/L OD Hr. Ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation resulted in the following conversion of isopreneug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental (REMH76 and REMH86) and test strains (REM31 and REM29) described above were isolated and quantified as described below. The resulting data is discussed in the legend to
Cell metabolism was rapidly inactivated by withdrawing 3.5 mL of the culture into a tube filled with 3.5 mL of dry ice-cold methanol. Cell debris was pelleted by centrifugation and the supernatant was loaded onto Strata-X-AW anion exchange column (Phenomenex) containing 30 mg of sorbent. The pellet was re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM NH4HCO3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1 mM NH4HCO3 buffer (pH=7.0). After each extraction, cell debris was pelleted by centrifugation and the supernatants were consecutively loaded onto the same anion exchange column. During the extraction and centrifugation steps the samples were kept at below +4° C. Prior to metabolite elution, the anion exchange columns were washed with water and methanol (1 mL of each) and the analytes were eluted by adding 0.35 mL of concentrated NH4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated NH4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant was neutralized with 30 μL of glacial acetic acid and cleared by centrifugation in a microcentrifuge.
Metabolite QuantificationMetabolites were analyzed using a Thermo Scientific TSQ Quantum Access mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). All system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a chiral Nucleodex β-OH 5 μM HPLC column (100×2 mm, Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH guard cartridge was eluted with a mobile phase gradient shown in Table 1 (flow rate of 0.4 mL/min). The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of metabolites were determined based on the integrated intensities of peaks generated by PO3− product ion (m/z=79.0). Calibration curves obtained by injection of corresponding standards purchased from Echelon Biosciences Inc. Intracellular concentrations of metabolites were calculated based on the assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Example 10 Deletion of iscR in E. coli BL21(DE3) Genotype to Improve Isoprene ProductionPrevious studies suggest that repair of damaged Fe—S centers and the turnover or regeneration of active 4Fe-4S centers within GcpE is partially contributable to the perceived bottleneck in DXP-mediated isoprenoid biosynthesis at the catalytic step carried out by GcpE. Increased levels of the related enzyme LytB have been obtained from E. coli engineered to overexpress the isc operon (Gräwert et al., J Am Chem Soc. 126(40):12847-55 (2004), which is hereby incorporated by reference in its entirety). The enzymes encoded by the E. coli isc operon have been shown to play a role in Fe—S cluster biogenesis and maintenance (Tokumoto and Takahashi, J. Biochem., 130: 63-71 (2001); Djaman et al., J. of Biol. Chem., 279(43):44590-44599 (2004), which are each hereby incorporated by reference in their entireties). An alternative approach to overexpressing the isc operon in E. coli to generate increased levels of active 4Fe-4S cluster containing enzymes such as GcpE and LytB is to remove the IscR transcriptional repressor that inhibits expression of the isc operon (Schwartz et al., PNAS, 98(26):14751-3 (2001), which is hereby incorporated by reference in its entirety). Such an approach was recently proved successful for a group expressing Clostridial hydrogenase in E. coli BL21(DE3) (Akhtar and Jones, Appl. Microbiol. Biotechnol. 78(5):853-62 (2008), which is hereby incorporated by reference in its entirety).
In this example, we demonstrated that the removal of iscR from the E. coli BL21(DE3) genome significantly improves isoprene production over that produced from the corresponding wild-type strain.
Deletion of iscR from BL21(DE3)/pRed/ET
The gene deletions and subsequent loopout of the antibiotic resistance markers described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21(DE3) (Invitrogen) was used.
Primer Sequences Used
The strategy for the deletion of iscR is shown in
To amplify the GB-CmR cassette for deletion of iscR the following PCR reactions were set up:
1 ul (100 ng GB-CmR) 10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) top iscR deletion
1.25 ul primer (10 uM) bottom iscR deletion
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 63° C.×30 sec., 72° C.×3 min]×29 cycles; 72° C.×5 min,
4° C. until cool (Biometra T3000 Combi Thermocycler).
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) to verify successful amplification, and purified using QIAquick PCR Purification kit according to manufacturer's instructions. The resulting stock was designated GB-CmR-iscR fragment.
Integration of GB-CmR-iscR Product into the BL21(DE3) Genome
The pRed-ET vector (Gene Bridges) was transformed into electrocompetent BL21(DE3) (Invitrogen) by electroporation resulting in strain BL21(DE3)/pRed-ET. Approximately 500 ng of GB-CmR-iscR PCR fragment was electroporated into BL21(DE3)/pRed-ET. The transformants were recovered in L Broth for 1 hour at 37° C. and then plated on L agar containing chloramphenical (10 ug/ml). Chloramphenicol resistant colonies were analyzed by PCR for the replacement of the iscR by the GB-CmR-iscR fragment using primers 5′ screen up of up iscR/3′ screen down of down iscR. The correct strain was designated REM14::CMP. The chloramphenicol resistance cassette was looped out of the strain using pCP20 from the RED/ET kit according to the manufacturer's instructions. Transformants were verified by loss of resistance to chloramphenicol (10 ug/ml) and PCR demonstrating loopout of the GB-CmR cassette. The resulting strain was designated REM14.
Creation of Strains REM65-1 and REM4, the Parental Strains to REM12 and REM13The wild-type BL21(DE3) (Invitrogen) and ΔiscR strain REM14 were transformed with the T7-MEARR alba/pBBR1MCS-5 construct to create the isoprene-producing strains REM65-1 and REM4 strains, respectively. A picture of the isoprene synthase containing vector, T7-MEARR alba/pBBR1MCS-5, is shown in
Transformation of T7-MEARR alba/pBBR1MCS-5 into BL21(DE3) and REM14
To build the isoprene-producing strains REM65-1 and REM4 strains, the T7-MEARR alba/pBBR1MCS-5 plasmid was transformed by electroporation into BL21(DE3) (Invitrogen) and REM14. Transformants were recovered in L broth and plated on L agar containing gentamycin (10 ug/ml). The resulting strains are designated as such: REM65-1 (BL21(DE3)/T7-MEARR alba/pBBR1MCS-5 and REM4 (REM14/T7-MEARR alba/pBBR1MCS-5).
Construction of the Test Strains REM12 and REM13The entire DXP pathway from E. coli was synthesized by DNA2.0 (Menlo Park, Calif.) and cloned into pET24a (see
To build the higher flux DXP pathway isoprene-producing REM12 and REM13 strains, the DXP operon pET24a plasmid was transformed by electroporation into REM65-1 and REM4. A picture of the DXP pathway enzyme containing vector, DXP operon pET24a plasmid, is shown in
Transformation of DXP Operon pET24a into REM65-1 and REM4
To build the test strains REM12 and REM13 strains, the DXP operon pET24a plasmid was transformed by electroporation into REM65-1 and REM4. Transformants were recovered in L broth and plated on L agar containing gentamycin (10 ug/ml) and kanamycin (10 ug/ml). The resulting strains are designated as such: REM12 (REM65-1/DXP operon pET24a) and REM13 (REM4//DXP operon pET24a).
Analysis of REM12 and REM13 for Growth and Isoprene ProductionThe wild-type strain REM12 and otherwise isogenic ΔiscR strain REM13 were compared in a shake flask isoprene headspace assay. The benefits on isoprene production and effect on growth rate the loss of iscR causes in the E. coli host are illustrated in
Strains REM12 and REM13 were grown at 30° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including kanamycin (10 ug/ml) and gentamycin (10 ug/ml). Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf). 50 uM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cultures to induce expression of the isoprene synthase and DXP enzymes harbored by the strains at time zero, as indicated in the legend to
Isoprene production was analyzed using a headspace assay. For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 200 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isopreneug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
Example 11 Evaluation of Alternative ispG (gcpE) and ispH (lytB) Alleles from Different Organisms by Complementation of dispG and/or dispH Strains of BL21(DE3)PL.2 mKKDyI::FRTWe constructed an E. coli strain expressing the lower mevalonic acid pathway (mevalonate kinase, phosphomevalonate kinase, diphosphomevalonte decarboxylase and IPP isomerase from yeast) as a base strain for testing the functionality of DXP pathway enzymes from heterologous organisms. This strain produces IPP and DMAPP from the lower mevalonate pathway if it is grown in the presence of mevalonate. Deletions of enzymes of the DXP pathway can be rescued by growing the stain in the presence of mevalonate. Therefore, functionality of heterologous DXP pathway genes can be expressed in the E. coli containing the lower MVA pathway and looking for growth in the absence of mevalonate.
Construction of MD09-170 (BL21(DE3)PL.2 mKKDyI::FRT
A P1 phage lysate was generated from MCM521 (BL21 neo-PL.2-mKKDyI) and transduced into BL21(DE3) (according to Procedure 12-Genetic Transduction Using P1vir protocol). The transductants were selected on L agar plates containing kanamycin (20 ug/ml), with incubation at 37° C. overnight. Four colonies were verified by PCR to be correct transductants. One of these colonies was selected and designated MD09-169 (BL21(DE3)PL.2 mKKDyI::Kan). The kanamycin resistance marker was looped out of this strain using pCP20 from the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The correct loopout was confirmed by testing for sensitivity to kanamycin (20 ug/ml) and then loss of the kanamycin resistance cassette was verified by PCR. The correct strain was designated MD09-170 (BL21(DE3)PL.2 mKKDyI::FRT).
Deletion of ispG and ispH from MD09-170
The gene deletions and subsequent loopout of the antibiotic resistance markers described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain MD09-170 was used.
Primer Sequences Used
The strategy for the deletion of ispG and ispH is shown in
To amplify the GB-CmR cassette for deletion of ispG or ispH the following PCR reactions were set up:
2 ul (100 ng GB-CmR) 10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) MQ09-18F/19F
1.25 ul primer (10 uM) MQ09-18R/19R
32 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×20 sec., 55° C.×20 sec., 72° C.×50 sec]×29 cycles; 72° C.×3 min,
4° C. until cool (Eppendorf Mastercycler PCR machine)
The resulting PCR fragments were separated on a 1.2% E-gel (Invitrogen), and purified using the Qiagen QiaQuick Gel Extraction and QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stocks were: GB-CmR-ispG fragment (1.593 kb)-180 ng/ul, and GB-CmR-ispH fragment (1.593 kb)-165 ng/ul.
Integration of GB-CmR-ispG or GB-CmR-ispH PCR Products into MD09-170/pRed-ET Strain
The pRed-ET vector (Gene Bridges kit) was transformed into MD09-170 by electroporation resulting in strain MD09-170/pRed-ET. Approximately 300-500 ng of GB-CmR-ispG or GB-CmR-ispH PCR fragments were electroporated into MD09-170/pRed-ET. The transformants were recovered in L Broth containing 500 uM mevalonic acid (Sigma) for 1 hour at 37° C. and then plated on L agar containing chloramphenical (5 ug/ml) and mevalonic acid (MVA) (500 uM). Chloramphenicol resistant colonies were analyzed by PCR for the presence of the GB-CmR cassette and the absence of the ispG or ispH genes using primers MQ09-20F/MQ09-20R or MQ09-21F/MQ09-21R respectively. The correct strains were designated MD09-209(BL21(DE3)PL.2 mKKDyI::FRT-ΔispG::Cm) and MD09-210 (BL21(DE3)PL.2 mKKDyI::FRT-ΔispH::Cm). The chloramphenicol resistance cassette was looped out of both strains using pCP20 from the RED/ET kit according to the manufacturer's instructions. Transformants were verified by loss of resistance to chloramphenicol (5 ug/ml) and PCR demonstrating loopout of the GB-CmR cassette.
The resulting strains were designated MD09-219 (BL21(DE3)PL.2 mKKDyI::FRT-ΔispG::FRT) and MD09-220 (BL21(DE3)PL.2 mKKDyI::FRT-ΔispH::FRT).
Complementation of MD09-219 and MD09-220 with Alleles from Thermosynechococcus elongatus BP-1
To test the functionality of the gcpE and lytB genes (annotated) from T. elongates, the following plasmids expressing these constructs or gcpE and lytB from E. coli were transformed by electroporation into MD09-219 and MD09-220:
-
- 1. E. coli: GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO (Kan) (positive control)
- 2. T. elong: Ptac-gcpE-petF-petH/pK184 (Kan)
- 3. T. elong: Ptac-gcpE-lytB-petF-petH/pK184 (Kan)
Transformants from 1. (E. coli) were recovered in L broth containing MVA (500 uM) and plated on L agar containing kanamycin (50 ug/ml). The resulting strain is designated MD09-219/GI1.6-gcpE-lytB-yidi/pCRII-TOPO (Kan).
Transformants from 2. (T. elong) or 3. (T. elong) were recovered in L broth containing MVA (500 uM) and IPTG (200 uM) and then plated on L agar containing on kanamycin (50 ug/ml) and IPTG (200 uM). The resulting strains were designated MD09-219/Ptac-gcpE-petF-petH/pK184 (Kan) and MD09-219/Ptac-gcpE-lytB-petF-petH/pK184 (Kan) respectively.
Several transformants were obtained on all of the plates suggesting that the T. elongatus gcpE and lytB were functional in E. coli. To confirm this, transformants were grown in L broth containing kanamycin (50 ug/ml) with and without IPTG (200 uM).
Construction of GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO
The construction of the GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO described in this example was carried out using standard molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning techniques). A picture illustrating the resulting plasmid construct is shown in
degenerate N base: A base yields GI 1.6-, T base yields GI 1.5-, G base yields GI1.2-, and C base yields GI 1.0-promoter
Strategy for Constructing GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO
The vector construct harboring the T7 polymerase governed synthetic DXP operon, DXP operon pET24a, was used as the PCR template.
Amplification of the GI 1.6-gcpE-lytB-yidi Fragment
To amplify the GI 1.6-gcpE-lytB-yidi fragment (among the other GI 1.X-possibilities) for cloning into the pCR-Blunt II-TOPO vector the following PCR reaction was performed:
1 ul (approx. 100 ng DXP operon pET24a)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ EcoRI-GI 1.X-BamHI gcpE DXP oper
1.25 ul primer (10 uM) 3′ PstI idi DXP operon
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 63° C.×30 sec., 72° C.×3.5 min.]×29 cycles; 72° C.×5 min,
4° C. until cool (Biometra T3000 Combi Thermocycler).
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions. The resulting stock was GI 1.X-gcpE-lytB-yidi fragments.
Cloning of the GI 1.6-gcpE-lytB-yidi Fragment into pCR-Blunt II-TOPO
The GI 1.X-gcpE-lytB-yidi fragments were cloned into pCR-Blunt II-TOPO using Invitrogen's Zero Blunt® TOPO® PCR Cloning Kit using the suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with 2 ul of the ligation reaction using a standard heat-shock protocol, and recovered in L broth for 1 hour at 37° C. and then plated on L agar containing kanamycin (10 ug/ml). Resulting colonies were selected, grown overnight in L broth containing kanamycin (10 ug/ml), and harvested for plasmid preparation the following day. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. A number of plasmid preparations were sequenced (Quintara; Mountain View, Calif.) using primers M13 Forward (−20) and M13 Reverse (−27) and the correct GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO clone identified.
Example 12 Improving Isoprene Production in E. coli by Deregulating Glucose UptakeIn Escherichia coli, glucose is transported using the phosphoenolpyruvate transport system (PTSg1c), which consists of PtsHICRR and the transporter PtsG (see Tchieu et al., J. Mol. Microbiol. Biotechnol. 3(3):329-46 (2001), which is hereby incorporated by reference in its entirety). Glucose is phosphorylated as it is transported into the cell, with the phosphate originating from phosphoenol pyruvate. The resulting glucose-6-phosphate is metabolized via glycolysis regenerating the PEP. Glucose transport continues through exponential growth but is down-regulated as cells enter stationary phase. For commercial purposes it is desirable to maximize production time and yield of the desired molecule, which is difficult to achieve if the feedstock transporter is downregulated. To solve this problem, the PTSglc system is deleted by deleting ptsHIcrr, and in some embodiments, ptsG, and constitutively express galP and glk, encoding the galactose permease and glucokinase respectively. The galactose permease transports glucose without phosphorylation so it is necessary to express the glucokinase (see US Patent Application No. 20050079617, which is hereby incorporated by reference in its entirety).
The ptsHIcrr operon is deleted in BL21 using the Red/ET system from Gene Bridges. Electrocompetent BL21 (Invitrogen) are transformed with the pRed/ET plasmid and the resulting cells are made electrocompetent by washing 3-4× in ice cold dH2O. The GB-cmR cassette is amplified using forward and reverse primers have at least 50 bases of homology to the regions immediately upstream of ptsH or immediately downstream of crr. The resulting PCR product is used to transform BL21/pRED and transformants are plated on MacConkey agar containing glucose (1%) and chloramphenical (5 ug/ml). Transformants that grown and are white in color will be the correct genotype. The ptsHIcrr knockout is transduced into the desired isoprene-producing hosts using P1 transduction.
The Ptrc-galP-cat and Ptrc-glk-cat cassettes are amplified by PCR from strains KLpts::gal-trc::Cm or KLgalPglk-trc-cat S (see U.S. Patent Application No. 20050079617, which is hereby incorporated by reference in its entirety) with at least 50 base pairs (bp) of homology on the 5′ and 3′ ends to allow homologous recombination into BL21 with either the DXP or the MVA or both pathways and isoprene synthase (example ispS from P. alba or a variant thereof) expressed and the ptsHIcrr and/or ptsG deleted. The desired strain is made competent and transformed with the pRed/ET plasmid, and after being made competent, the new strain is transformed with the galP-trc-cat cassette. Transformants are selected on MacConkey agar containing 1% glucose and chloramphenicol (5 ug/ml). Colonies which are slightly pink have the correct genotype. The CAT markers in these cassettes are flanked by loxP sites and can be looped out by standard methods (Palmeros et al., Gene 18; 247(1-2):255-64 (2000)) which is hereby incorporated by reference in its entirety). The strain expressing galP from Ptrc is then transformed with the glk-trc-cat cassette and transformants are select on MacConkey agar containing 1% glucose and chloramphenicol (5 ug/ml). Colonies which are deep red in color are the correct colonies.
The resulting strains have the full MVA pathway, with or without the DXP pathway constitutively expressed, an isoprene synthase (example P. alba IspS or a variant thereof), a deletion of the ptsHIcrr and/or ptsG, and constitutive expression of the galactose permease and glucokinase. To demonstrate that isoprene production is enhanced and/or prolonged in these strains compared to the parent which transports glucose via the PTSglc system, the strains are tested in shake flask (TM3 containing 1% glucose, 0.1% yeast extract), microfermentor (TM3 containing 1% glucose, 0.1% yeast extract), and in 14-Liter fermentation. These strains are also tested using pretreated and saccharified biomass, for example corn fiber, corn stover, switch grass, forage sorghum, softwood pulp, hardwood pulp or other suitable biomass.
Isoprene production is enhanced and/or prolonged in the strains with ptsHIcrr and/or ptsG deletion and constitutive expression of the galactose permease and glucokinase compared to the compared to the parent strains without the deletion of ptsHIcrr and/or ptsG and constitutive expression of the galactose permease and glucokinase.
Example 13 Expression of Monoterpene and Sesquiterpene Synthases in Combination with the Expression of Isoprene Synthase Increases the Specific Productivity of Isoprene in E. coliIsopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) are biosynthesized by the DXP pathway (also called the non-mevalonate pathway and MEP pathway) in E. coli. IPP and DMAPP can be condensed to form geranyl pyrophosphate (GPP) and subsequently farnesyl pyrophosphate (FPP) by farnesene synthase (IspA). FPP can be converted to octaprenyl pyrophosphate (OPP) and undecaprenyl pyrophosphate (UPP) by extension of FPP with IPP. These products serve a variety of functions in E. coli including prenylation of tRNA (protein synthesis component) with DMAPP, formation of quinones (respiratory chain component) with OPP, and peptidoglycan formation (cell wall component) with UPP.
The products of the DXP pathway may be regulated by the production of IPP and DMAPP. Accordingly, the example shows that the introduction of a terpene synthase that utilizes downstream products of the DXP pathway in combination with isoprene synthase in E. coli results in increased flux through the DXP pathway and increased specific productivity of isoprene.
Methods Strain ConstructionThe following strains are constructed.
Ocimene synthase, farnesene synthase and artemesinin synthase are cloned into pTrchis2A plasmids to give pTrcFPP, pTrcAS, or pTrcOS. Isoprene synthase (for example IspS from P. alba or variants thereof) is cloned into pBBR under control of the Ptrc promoter to give pBBRPtrcalba.
Strain set 1) BL21GI1.6yIDI/pBBRPtrcalba itself or combined with PtrcFPP or pTrcAS or pTrcOS.
Strain set 2) BL21GI1.6yIDIGI1.6DXS/pBBRPtrcalba itself or combined with PtrcFPP or pTrcAS or pTrcOS.
The strains in strain set 1) or 2) are grown in shake flask or in the microfermentor in TM3 containing 0.1% yeast extract and 1% glucose. The specific productivity of isoprene is measured over time.
The specific productivity of isoprene from strains in strain set 1) are compared. The specific productivity of isoprene in the strains containing FPP, OS, or AS is higher than in the strain without FPP, OS, or AS.
The specific productivity of isoprene from strains in strain set 2) are compared. The specific productivity of isoprene in the strains containing FPP, OS, or AS is higher than in the strain without FPP, OS, or AS.
Example 14 Deletion or Reduction of Carbon into Thiamine and Pyridoxine Paths for Relief of Inhibition1-deoxy-D-xylulose-5-phosphate (DXP) is a substrate in three essential anabolic pathways in E. coli, namely isoprenoids, thiamine and pyridoxal synthesis. In order to avoid any feedback regulation from thiamine or pyridoxal pathways, which could then decrease the flux in the DXP pathway for isoprenoid production, we build strains mutated in the thiamine and/or pyridoxal pathways.
A: Construction of an E. coli Strain Deleted in the Thiamine Synthesis Pathway
Several enzymes are involved in the biosynthesis of thiamine from DXP. ThiG and ThiH combine to form a complex containing an iron-sulfur cluster (Leonardi et al. FEBS Lett. 539(1-3):95-9 (2003), PMID: 12650933, which is hereby incorporated by reference in its entirety). Together, they are required for the synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole phosphate, which is the rate-limiting step in thiamine synthesis (Leonardi et al. J Biol. Chem. 279(17):17054-62 (2004), PMID: 14757766; Vander et al., J. Bacteriol. 175(4):982-92 (1993), PMID: 8432721; which are hereby incorporated by reference in their entireties). Since it is in the rate-limiting step, and it is the first enzyme after 1-deoxy-D-xylulose-5-phosphate, thiG was chosen as the gene to be deleted.
A PCR product was obtained using primers GB400thiGF (caggagccagaacgcaactgc (SEQ ID NO:100) and GB400thiGR (CACTTTCGCCTGATGTTCACC (SEQ ID NO:101), and genomic DNA of strain JW5549 from the Keio collection (Baba et al., Mol. Syst. Biol. 2006.008 (2006), which is hereby incorporated by reference in its entirety). The PCR product contains a kanamycin cassette replacing most of the thiG gene and around 400 bp flanking regions of both sides of the thiG gene.
A BL21(DE3) thiG::Kan mutant is then obtained by Red/ET recombineering (Gene Bridges, Dresden, Germany) using the PCR product mentioned above. It is proven correct by amplification and sequencing. The strain is named CMP179.
B: Construction of an E. coli Strain Deleted in the Pyridoxal Synthesis Pathway
PdxJ catalyses the formation of pyridoxine-5-phosphate (precursor of pyridoxal-5-phosphate then pyridoxal) from 1-deoxy-D-xylulose-5-phosphate and 1-amino-propan-2-one-3-phosphate. The latter is produced by a sequence of reactions coming from erythrose-4-phsophate, the first one catalyzed by D-erythrose 4-phosphate dehydrogenase (epd). Thus both pdxJ and epd are good candidates for deleting the production of pyridoxal. However, epd has been reported not to be required for glycolysis or for synthesis of pyridoxal (Seta et al., J. Bacteriol. 179(16):5218-21 (1997), which is hereby incorporated by reference in its entirety). Thus, pdxJ is chosen as the target for mutation.
A PCR product is obtained using primers GB400pdxJF (CAT TCA GTC TCT TGC AGG GGT C (SEQ ID NO:102) and GB400pdxJR (gcatagtgccgctcatctgcc (SEQ ID NO:103)), and genomic DNA of strain JW2548 from the Keio collection (Baba et al. 2006). The PCR product contains a kanamycin cassette replacing most of the pdxJ gene and around 400 bp flanking regions of both sides of the pdxJ gene.
A BL21(DE3) pdxJ::Kan mutant is then obtained by Red/ET recombineering (Gene Bridges, Dresden, Germany) using the PCR product mentioned above. It is proven correct by amplification and sequencing. The strain is named CMP180.
C: Construction of an E. coli Strain Deleted in the Thiamine and Pyridoxal Synthesis Pathways
The kanamycin cassette is removed from CMP179 and/or CMP180 by Flp-mediated excision, using plasmid 706-Flp from Gene Bridges (Dresden, Germany). Then the PCR product described in section A is used to mutate BL21(DE3) pdxJ through Red/ET recombineering.
D: Production of Isoprene Via the DXP Pathway, in a thiG and/or a pdxJ Mutant
The effect of the thiG, pdxJ or thiG pdxJ mutations on the production of isoprene through the DXP pathway is assessed in different constructs enhancing DXP pathway flux and expressing IspS (isoprene synthase) from Populus alba, such as MCM597 (BL21(DE3)pLysS pET24(MEA)alba-DXS-yIDI) or MCM719 (BL21 gi1.6-yIDI gi1.6-dxs, pTrc(MEA)alba)).
Strains are grown overnight at 30° C., 200 RPM, in HM1 medium (Table 2) plus appropriate antibiotics. The morning after, they are resuspended to an OD=0.2 in fresh HM1 medium+appropriate antibiotics. Flasks are incubated at 30° C., 200 RPM, and regularly sampled for OD and isoprene productivity.
Specific productivity (ug isoprene/OD.h) is increased when strains MCM597 or MCM719 contains thiG, pdxJ, or thiG pdxJ mutations.
Example 15 Balancing Pyruvate and G-3-P (Glyceraldehyde-3-Phosphate) to Increase Isoprene ProductionFlux to the DXP pathway may be positively (more flux) effected to increase isoprene production by maximizing the balance between the two precursors required for the DXP pathway, pyruvate and G-3-P (glyceraldehyde-3-phosphate). Accordingly, adjusting the expression level of enzymes that determine flux into glycolysis, into the pentose phosphate pathway (PPP) and into the Entner-Doudoroff (ED) pathway (
Cells that have been constructed by procedures known to practitioners of the art and as exemplified in this application to overexpress the DXP pathway or wild type cells are fed with various carbon sources, but more specifically cells are fed glucose plus gluconic acid or gluconic acid alone. The culture is sampled and analyzed for improved evolution of isoprene. This analysis is accomplished by monitoring the head space of the culture with a mass spectrometer either continuously or at specific time points during the cultivation of cells with different concentrations of the carbon sources.
Section BCells in Section A harboring the overexpressed DXP pathway or wild type cells are genetically engineered to overexpress glucose-6-phosphate dehydrogenase to redirect flux to PPP and ED. Effect and benefit of these mutations can be assessed by measuring isoprene specific productivity.
Section CCells in Section A harboring the overexpressed DXP pathway or wild type cells are genetically engineered to limit expression of glucose-6-phosphate isomerase to redirect flux to PPP and ED. Effect and benefit of these mutations can be assessed by measuring isoprene specific productivity.
Section DCells in Section A harboring the overexpressed DXP pathway or wild type cells are genetically engineered to limit expression of Gluconate-6-phosphate dehydrogenase (gnd) to limit flux to pentose phosphate and maximize flux to ED. Effect and benefit of these mutations can be assessed by measuring isoprene specific productivity.
Section EIn this section, the DXP precursor pyruvate is adjusted by the level of expression of the mevalonic acid pathway for which pyruvate is the sole precursor. Cells are constructed to overexpress the DXP pathway enzymes as well as the mevalonic acid pathway enzymes and expression of both pathways is adjusted, by choosing the appropriate promoter strengths, such that pyruvate flux is balanced with G-3-P flux and neither precursor accumulates in the cell. Similar, approaches in the presence of zwf, gnd, and pgi mutations, singly or in all possible combination, have potential for improved performance.
Section FThe strains created in Sections B-E, are combined for potential additivity. Combination of zwf and gnd in a overexpressed DXP pathway strain is tested for improved performance of the strain. Similarly, the combination of pgi and gnd is envisaged to provide similar results.
Example 16 Improved Carbon Flux Through the DXP Pathway in Strains Containing PDH E1 E636Q Subunit VariantsThis example describes methods for the construction of E. coli BL21 strains containing pyruvate dehydrogenase E1 subunit (PDH) variants that increase carbon flux through the DXP pathway. In particular, these strains contain a mutant aceE gene, encoding for a PDH variant with an E636Q point mutation which possesses a reduced activity (26% of wild-type PDH activity) for the conversion of pyruvate to acetyl-CoA. In addition, the PDH E636Q variant is thought to have a dxs-like activity that results in the production of 1-deoxyxylulose-5-phosphate (DXP) from the aldol condensation of pyruvate and glyceraldehyde-3-phosphate. The carboligase activity of the pyruvate dehydrogenase E1 E636Q mutant has been reported by Nemeria et al. (J. Biol. Chem., 280(22), 21473-21482 (2005), which is hereby incorporated by reference in its entirety). The net effect is increased carbon flux into the DXP pathway, and reduced carbon flux to acetyl-CoA relative to strains containing wild-type PDH E1 activity.
The construction of E. coli BL21 strains containing the PDH E1 E636Q mutant was as described by Sauret-Giieto et al. (FEBS Lett., 580, 736-740 (2006)), which is hereby incorporated by reference in its entirety. Briefly, the chromosomal copy of the dxs gene is disrupted by the insertion of a chloramphenicol acetyl transferase (CAT) containing cassette into the dxs locus of an E. coli BL21 strain that contains one or more plasmids encoding a heterologous mevalonic acid pathway (MVA). The resulting E. coli BL21 MVA+(dxs::CAT) strain requires mevalonic acid for normal growth. When the strain is cultured in the absence of mevalonic acid, a suppressor mutation aceE gene arises at a low to moderate frequency that rescues the surviving clones from the otherwise lethal dxs-phenotype. Sequencing of the aceE gene and associated promoter region is performed in order to confirm the presence of the missense mutation that results in the PDH E636Q mutant.
The resulting E. coli BL21 dxs::CAT PDH E1 E636Q MVA+strain is complemented with one or more functional copies of the dxs gene derived from E. coli or from a heterologous source as described herein. The resulting strains exhibits improved flux into the DXP pathway relative to strains that do not possess the PDH E1 E636Q variant.
Additionally, the resulting E. coli BL21 dxs::CAT PDH E1 E636Q MVA+strain can be further complemented with one or more functional copies of a DXP pathway gene, a DXP pathway associated gene, an iron-sulfur cluster-interacting redox gene (e.g., fldA or fpr), and/or an IDI gene derived from E. coli or from a heterologous source as described herein.
The strains can also be transformed with one or more copies of genes encoding isoprene synthases, for example IspS from P. alba or variants thereof as described herein. These strains produce isoprene by both the DXP and MVA pathways where a greater proportion of isoprene is derived from the DXP pathway relative to the MVA pathway, as compared to strains that do not possess the PDH E636Q variant. The ratio the DXP to MVA carbon flux is determined using isotope-labeling techniques known to those skilled in the art.
The strains can be optionally cured of the MVA pathway encoding plasmids (e.g., CHL18 or any other MVA pathway strains as described in U.S. Patent Application Nos. 61/097,186, 61/097,189, and 61/125,336, which are each hereby incorporated by reference in their entireties) if desired using techniques known to those skilled in the art.
Example 17 Mutation of CRP Increases Flux to the DXP Pathway and Increases the Production of IsopreneCatabolite repression, in which the transcription of sensitive operons is reduced by certain carbon sources, could be a major restriction to flux in the DXP pathway, thereby reducing the amount of isoprene which could be produced.
A CRP (cAMP Receptor Protein)-delete mutant is available from the Keio collection and could easily be assessed for the production of isoprene through the DXP pathway. Impact of its global transcriptional regulation has been studied (Perrenoud and Sauer, J. Bact. 187:3171-3179 (2005). which is hereby incorporated by reference in its entirety). Other types of CRP mutants could also be beneficial to the process. One such example is the CRP mutant described by Eppler and Boos (Eppler and Boos, Mol. Microbiol. 33:1221-1231 (1999), which is hereby incorporated by reference in its entirety). CRP* is a cAMP-independent CRP variant.
A: Construction of an Isoprene-Producing Crp* Mutant of E. coli
CRP* mutation is introduced by P1 transduction (lysate prepared from E. coli strain ET25 (to be obtained from W. Boos)) in an isoprene-producing strain, such as MCM597 (BL21(DE3)pLysS pET24(MEA)alba-DXS-yIDI) or MCM719 (BL21 gi1.6-yIDI gi1.6-dxs, pTrc(MEA)alba)) to form strains CMP220 and CMP221 respectively.
B: Production of Isoprene in a Crp* Mutant of E. coli, Via the DXP Pathway
Strains CMP220 and CMP221, and strains MCM597 and MCM719, are grown overnight at 30 C, 200 RPM, in HM1 medium (Table 3) plus appropriate antibiotics+10 g/L glucose+1 g/L yeast extract. The morning after, they are resuspended to an OD=0.2 in fresh HM1 medium+appropriate antibiotics+5 g/L glucose+1 g/L yeast extract. Flasks are incubated at 30° C., 200 RPM, and regularly sampled for OD600 and isoprene productivity.
Specific productivity (ug isoprene/OD.h) is increased in strains CMP220 and CMP221 in comparison to strains MCM597 or MCM719.
C: Production of Isoprene in a Crp* Mutant of E. coli, Via the DXP Pathway, when the Strain is Grown on a Glucose/Xylose Mixture
Pretreated biomass samples contain a mixture of glucose, xylose and acetate as the main components. Xylose consumption by E. coli is usually prevented in the presence of glucose. The CRP* mutation should be helpful to enhance glucose and xylose coconsumption (Cirino et al. biotech. Bioeng. 95:1167-1176 (2006), which is hereby incorporated by reference in its entirety).
Strains CMP220 and CMP221, and strains MCM597 and MCM719, are grown overnight at 30° C., 200 RPM, in HM1 medium (Table 3) plus appropriate antibiotics+10 g/L glucose+1 g/L yeast extract. The morning after, they are resuspended to an OD600=0.2 in fresh HM1 medium+appropriate antibiotics+2.5 g/L xylose and 2.5 g/L glucose+1 g/L yeast extract. Flasks are incubated at 30° C., 200 RPM, and regularly sampled for Mao, isoprene productivity and carbohydrate concentration. Carbohydrate concentration is determined by HPLC (Ion exclusion column Aminex HPX-87H, 300 mm×7.8 mm, 0.005 M H2504, 0.6 mL/min as the mobile phase).
While strains MCM597 and MCM719 show a diauxic growth curve, co-consumption of xylose and glucose is increased in strains CMP220 and CMP221. This allows the fermentation to be completed in a shorter time.
Example 18 Increased Isoprene Production in an E. coli Strain with LytBG120D MutationThe primary issues of this concept involve the biochemical determination of the mutant DXP pathway enzyme LytBG120D and whether or not the anticipated function of the LytBG10D enzyme can help serve a relevant aspect of our target DXP pathway strain to be used for BioIsoprene production. In this example, the desired DXP pathway strain is to produce a majority (if not as close to all as possible) of isoprene via the dimethylallyl pyrophosphate (DMAPP) molecule derived directly from the LytBG120D catalysis of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP); as opposed to DMAPP generated via the IDI enzyme, which isomerizes isopentenyl pyrophosphate (IPP) into DMAPP.
The wild-type LytB of E. coli and the LytB enzyme common to a number of other organisms, including plants and algae as well as other bacteria, have been reported to produce both DMAPP and IPP in ratios typically ranging from 1:4 to 1:6 (DMAPP:IPP). The work by Kia-Joo Puan et al. (FEBS Letters, 579:3802-3806 (2005), which is hereby incorporated by reference in its entirety) provides in vivo data that supports the hypothesis that the LytBG120D mutant enzyme can produce DMAPP, but can not generate sufficient levels of IPP to support the viability of an E. coli deficient for IDI. No in vitro data supporting the suggested activity for LytBG120D has been introduced to the field yet.
Currently, isoprenoid production systems derive the majority of their products from IPP. If the LytBG120D is determined to solely generate DMAPP or a majority of DMAPP relative to IPP, then the use of the lytBG120D allele in a DXP pathway-mediated isoprene production strain may allow the unique generation of an isoprenoid product that is derived almost entirely from DMAPP.
The lytBG12D is generated via PCR-based methods using E. coli MG1655 as a template and cloned into an expression vector (pET-15b). For comparison, the wild-type lytB is cloned into the same pET-15b expression vector backbone. Each construct is moved into BL21(DE3), or a comparable expression host, once the sequence of the construct has been verified. From the expression strains, LytB and LytBG120D is produced and subsequently purified using standard affinity purification procedures. The protein may need to be reconstituted under anaerobic conditions prior to activity assessment (protocols exist in the literature) for robust enzymatic function to be determined. LytB is a 4Fe-4S cluster containing enzyme and is known to be sensitive to oxygen. Alternatively, LytB and LytBG120D may be able to be assayed directly from cell lysates prior to purification if sufficient activity of each enzyme can be supported under those conditions and if an absence of significant Idi activity can be achieved. Expression of each enzyme is determined and quantified by gel electrophoresis and/or immuno-blot. Activity assays are described in the literature, but briefly may include incubation of each enzyme (purified or contained within a cell extract) in a previously described buffer including the substrate HMBPP and in the absence of Idi activity. After a defined time(s) the ratio of DMAPP to IPP is determined using HPLC methods. The resulting data are the first in vitro results for LytBG120D available to us.
If LytBG120D is found to solely produce DMAPP, or at least produce DMAPP in vast abundance to IPP, then the use of the lytBG120D allele is incorporated in the DXP pathway isoprene production strains. Initially, this is accomplished by overexpressing the lytBG120D gene relative to the wild-type allele under isoprene-production phases within a host background that supports carbon flux through the DXP pathway to isoprene synthase. As a control to assess, any benefits specific to generating increased DMAPP levels relative to IPP that are expected to accompany the overexpression of lytBG120D, a similar strain overexpressing the wild-type lytB gene is also constructed and assessed. The levels of DMAPP and IPP generated by these strains, as well as isoprene and other downstream isoprenoids, are determined by HPLC and/or GC-MS methods.
Our past findings indicate that increased IPP levels are not tolerated well by E. coli. Further more, we have seen that increased IPP levels accompanied by a significantly active Idi result in the synthesis of larger downstream isoprenoid products, which also cause a significant decrease in viability. Because LytB produces a majority of IPP to DMAPP, and because the endogenous IdI activity of E. coli is minimal, and because DMAPP is the substrate for isoprene synthase, our current D×P system relies on the use of an IdI derived from yeast. The use of LytBG120 in a DXP production strain removes the dependence our current system has on the yeast IdI (if LytBG120D is determined to produce mostly DMAPP). The use of LytBG120 is also expected to reduce the levels of downstream isoprenoid synthesis since IPP, the major subunit of larger isoprenoids, is not abundantly available.
Example 19 Host Change for Relief of Endogenous Regulation of DXP PathwayThe DXP pathway, required for isoprenoids production in most Prokaryotes, is a strongly regulated pathway. Indeed, it is essential but also needed in small amount, as it diverts carbon from the central metabolism intermediates glyceraldehyde-3-P and pyruvate. As such, it might be difficult to escape regulation when working with endogenous genes.
A solution to this problem may be to express the whole DXP pathway from one organism into another host organism, the latter organism being close or far on the phylogenetic tree. These host organisms include, but not limited to industrial organisms, such as Escherichia coli, Pseudomonas fluorescens, Zymomonas mobilis, Bacillus sp., Saccharomyces cerevisiae, Clostridium sp., Corynebacterium glutamicum, and Saccharomyces cerevisiae. The fact that all the genes involved in the pathway are cloned from one organism guarantees that the enzymes produced by those genes can work together to produce the end product DMAPP.
A: Construction of a DXP Pathway-Expressing Plasmid by Cloning E. coli DXP Genes
A Ptrc promoter, PCR-amplified from plasmid pTrcHis2A (Invitrogen, Carlsbad, Calif.) is cloned into pBBR1-MCS4 plasmid (Kovach et al, Gene, 166:175-176 (1995), which is hereby incoporated by reference in its entirety) multiple cloning site, leaving a PstI site downstream of the promoter. This plasmid is named pBBR4Ptrc. E. coli genes yajP (dxs), ispC (dxr), ispD, ispE, ispF, gcpE, lytB and idi are amplified from genomic DNA of E. coli MG1655 with primers containing an NsiI site and a RBS on the upstream primer, and a PstI site on the downstream primer. Genes are added one by one to the plasmid. Restriction digestion is used to check and select clones with the right orientation. Alternatively, a terminator is introduced after ispF and a new promoter (e.g. Ptrc) has been introduced in front of an operon constituted from gcpE, lytB and idi. The plasmid thus generated is named pBBR4PtrcDXPc and pBBR4PtrcDXPc2.
B: Construction of a Codon-Optimized DXP Pathway-Expressing Plasmid by Synthetic DNA SynthesisA synthetic operon similar to the one described above is designed and ordered, codon-optimized for Pseudomonas fluorescens, from GeneArt (Regensburg, Germany). It is subcloned in plasmid pBBR4Ptrc to generate plasmid pBBR4PtrcDXPa.
C: Expression of E. coli DXP Pathway in Pseudomonas fluorescens, and its Effect on Isoprene Production
An ispS (isoprene synthase from Populus) gene codon optimized for Pseudomonas (see other Pseudomonas patent example) is cloned into plasmid pHRP309 (gentamycin resistant) (Parales and Harwood, Gene 133:23-30 (1993), which is hereby incorporated by reference in it entirety), and transformed by biparental mating into Pseudomonas fluorescens ATCC 13525. Plasmids pBBR4PtrcDXPc, PBBR4PtrcDXPc2 and pBBR4PtrcDXPa are transformed in E. coli S17-1 by electroporation and selection of transformants on LB+kanamycin 50 ug/ml. The plasmids are then transformed into Pseudomonas fluorescens with IspS-expressing pHRP309 by biparental mating and selection on M9 medium+16 mM sodium citrate+kanamycin 50 μg/ml+gentamycin 50 ug/ml, to form strain CMP222, CMP223 and CMP224 respectively.
When strains CMP222, CMP223 and CMP224 are grown in HM1 medium+10 g/L glucose, isoprene specific productivity is higher than for the Pseudomonas fluorescens strain devoid of the DXP pathway-expressing plasmids.
Example 20 Identification of Compounds Affecting Production of Isoprene Via the DXP PathwayIsoprene production and growth by a strain of E. coli that over-expresses DXP pathway enzymes and isoprene synthase was investigated using 96-well microtiter plates with a range of different carbon, nitrogen or phosphate sources. A number of compounds that affected production of isoprene to a significant degree either positively or negatively were surprisingly identified. Compounds positively or negatively affecting the specific productivity of isoprene may help identify metabolic pathways that affect isoprene production. Such pathways may be implicated directly in the production of isoprene or they may have regulatory roles. The identified compounds or metabolic pathways may be modified for example by genetic modification to optimize the production of isoprene. The identified carbon, nitrogen or phosphate sources may also be supplemented directly to the media for increased production of isoprene.
Experimental Procedure: TM3 Media Recipe (Per Liter Fermentation Media):K2HPO4 13.6 g, KH2PO4 13.6 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2SO4 3.2 g, 1000× Trace Metal Solution 1 ml. All of the components were dissolved sequentially in diH2O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought to volume. Media was filter sterilized with a 0.22 micron filter. Before use, MgSO4*7H2O 2 g, yeast extract 0.2 g was added to the media. Carbon source was added to a final concentration of 0.5% if needed. Required antibiotics were added after sterilization and pH adjustment.
1000× Trace Metal Solution (Per Liter Fermentation Media):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HCl/NaOH, and then brought to volume and filter sterilized with 0.22 micron filter.
Strain: MCM597(i) Construction of MCM597 (BL21(DE3) pLysS pet24(MEA)albadxsyIDI
Construction of pDU-39
A truncated version of the Populus alba isoprene synthase was created by amplifying the gene using the primer set Alba TRC(MEA)-NdeI-F/Alba FLTRC(−) TER-R and the template pET24 alba HGS (described in Example 10, U.S. patent application Ser. No. 12/335,071, which is hereby incorporated in its entirety). The PCR reaction was set up as follows:
1 ul (pET24a-P. alba)
5 ul 10× PfuUltraII Fusion buffer
1 ul dNTP's (10 mM)
1 ul primer (50 uM) Set #1 forward
1 ul primer (50 uM) Set #1 reverse
41 ul diH2O
+1 ul of PfuUltra II Fusion DNA Polymerase from Stratagene
95° C. 1 min. [95° C. 30 sec., 55° C. 20 sec., 72° C. 25 sec]×29 cycles, 72° C. 3 min,
4° C. until cool, (Eppendorf Mastercycler)
The PCR products were digested with NdeI-XhoI restriction endonucleases (Roche) and gel purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. An aliquot of 3 ul of the purified product was ligated using T4 ligase (New England BioLabs) to pET-24a vector (Invitrogen) that was previously digested with NdeI-XhoI, gel purified and treated with Shrimp Alkaline Phosphatase (SAP, Roche). The ligation was carried out overnight at 16° C.
An aliquot of 5 uL of the overnight ligation mixture was transformed into TOP10 cells (Invitrogen) and transformants were selected on L agar containing kanamycin (50 ug/ml) at 37° C. overnight.
Plasmids were isolated from a few of the transformants using the QiaQuick Spin Kit (Qiagen) according to the manufacturer's instructions. The insert was verified by digestion NdeI-XhoI restriction endonucleases and the clones were sequenced with the commercially available T7 promoter and T7 terminator (Quintara Bio Sequencing Service, Berkeley, Calif.).
The Correct Plasmid was Designated pDu-39 (
The DXS-yIDI genes were amplified by PCR using primers MCM270/MCM271 and the template pMCM72 (described in Example 7 U.S. patent application Ser. No. 12/335,071, which is hereby incorporated by reference in its entirety). Two identical PCR reactions were set up according to the manufacturer's protocol for Herculase II Fusion (Stratagene). 35 uL water, 10 uL buffer, 1.25 uL each primer, 0.5 uL dNTPs, 1 uL polymerase. Reactions were cycled: 95 C, 2:00; (95 C 0:15, 55 C 0:15, 72 C 1:45)×30; 72 C 3:00, 4 C until cold.
The resulting PCR fragment was digested with BamHI and NotI (Roche), and then ligated using Roche Rapid Ligation Kit into pDu39 that had been digested with the same restriction endonucleases. The ligation reaction was set up in 10 uL containing 5 uL Buffer 1, 1 uL vector, 3 uL insert and 1 uL ligase and incubated for 1 hour at room temperature. An aliquot of 5 uL was transformed into E. coli Top10 chemically competent cells (Invitrogen). Transformants were selected on L agar containing kanamycin (50 ug/ml) at 37° C. overnight. Plasmids were purified from a few transformants and screened for the presence of insert using Herculase II Fusion (Stratagene). 17.5 uL water, 5 uL buffer, 0.625 uL each primer, 0.25 uL dNTPs, 0.5 uL polymerase. Reactions were cycled: 95 C, 2:00; (95 C 0:15, 52 C 0:15, 72 C 0:45)×30; 72 C 3:00, 4 C until cold. Clones with a PCR product near 1.5 kbp were sequenced (Quintara Biosciences, Berkeley Calif.). A correct plasmid was designated MCM596. The plasmid was then transformed into electrocompetent BL21(DE3)pLysS cells (Invitrogen) and transformants were selected on L agar containing kanamycin (50 ug/ml) and chloramphenicol (35 ug/mL). One colony was selected and designated MCM597.
Experimental ProtocolAn inoculum of the E. coli strain MCM597 over-expressing the DXP pathway enzymes dxs from E. coli and idi from Saccharomyzes cereviciae and the isoprene synthase ispS from Populus alba was taken from a frozen vial and streaked onto an LB broth agar plate (with antibiotics) and incubated at 30° C. overnight. A single colony was inoculated into TM3 media containing glucose as the only carbon source and grown overnight at 30° C. The overnight cultures were washed by centrifugation and resuspended into fresh TM3 media containing no glucose or yeast extract. The bacteria were then diluted into 20 mL of TM3 media to reach an optical density of 0.05 measured at 600 nm. For experiments testing the effect of different nitrogen sources using the Biolog PM3B microtiter plates (Biolog, USA), the bacteria were diluted into media containing 0.5% glucose and no yeast extract. For experiments testing the effect of different carbon sources using the Biolog PM1 and PM2A microtiter plates (Biolog, USA), the bacteria were diluted into media containing 0.2% yeast extract and either no or 0.5% glucose. A total of 120 μL of culture was dispensed into each well of the Biolog plates and the plate was incubated on an orbital shaker (250 rpm) at 30° C. The optical density was measured in the wells at 600 nm using a 384 well microtiter plate reader (Molecular Devices, Spectramax Plus 384) in the beginning of the experiment and every hour thereafter to follow growth of the bacteria. None of the compounds in the biolog plates were found to interfere with the optical density measurement at 600 nm. After four to six hours of growth, the optical density was measured again and two times 50 μL was transferred to two 96 well quartz glass blocks (Zinsser, Germany) and sealed with Biomek aluminum foil tape lids (Beckman Coulter, USA). The glass blocks were shaken at 450 rpm at 30° C. for 30 minutes and then heat treated for 12 minutes at 70° C. The produced isoprene was measured using a GC-MS (GC 7889A and MSD 5975C, Agilent Technologies, USA). To account for differences from glass block to glass block, the isoprene measurement was normalized to the block average. The specific isoprene productivity was calculated by dividing the isoprene production with the optical density for each well. Each Biolog experiment was performed in duplicate. Statistical analysis (students T-test) was used to identify compounds in the microtiter plates that affected specific isoprene productivity with statistical significance (p<0.1).
Results Nitrogen Sources Affecting Isoprene Production:When E. coli harboring the DXP pathway and isoprene synthase was grown on 0.5% glucose as the sole carbon source in media lacking yeast extract, a number of nitrogen containing compounds were found to either positively or negatively affect the production of isoprene through the DXP pathway. The PM3B plates from Biolog were used for these experiments. Statistical analysis was used to identify compounds that most significantly affect isoprene production (Table 4). The addition of nitrite, nitrate, ammonia and urea did not significantly change the specific isoprene production, suggesting the bacteria were not directly lacking nitrogen in the fermentation media. Compounds increasing the specific production of isoprene surprisingly include L-glutamic acid, L-aspartic acid, the purines inosine and guanosine, L-threonine, L-serine, L-tryptophan and L-asparagine. Compounds negatively affecting specific isoprene productivity particularly include adenine, and L-methionine, and L-tyrosine among others. Some of these compounds are involved in purine and thiamine biosynthesis, which are related to the DXP pathway, and may as such play important roles in the regulation of the DXP pathway.
Carbon Sources Affecting Isoprene Production During Growth on Glucose:When E. coli harboring the DXP pathway and isoprene synthase was grown on 0.5% glucose in fermentation media containing 0.2% yeast extract, a range of carbon sources were found to affect the specific productivity of isoprene through the DXP pathway to a surprisingly high degree. The PM1 and PM2A carbon source plates from Biolog were used for these experiments. Statistical analysis was used to identify compounds that most significantly affect isoprene production (Table 5). Compounds most significantly increasing specific productivity of isoprene include, but are not limited to, phenylethylamine, propionic acid, D-galacturonic acid, inosine, L-galactonic acid-γ-lactone, D-psicose, glucuronamide, 2-aminoethanol, D-cellobiose, sucrose, mucic acid, L-malic acid, L-phenylalanine, 2,3-butanediol, L-ornithine, D-gluconic acid, D-glucosaminic acid, D-mannose. It is to be expected that the addition of these compounds to glucose fed fermentations would increase the specific productivity of isoprene. A range of other compounds were found to negatively affect specific productivity (Table 5). These effects may be caused by regulatory roles of the compounds or associated metabolic pathways, making these pathways interesting for genetic modification.
Identification of Carbon Sources Useful for the Production of Isoprene:When E. coli harboring the DXP pathway and isoprene synthase was grown in media containing 0.2% yeast extract in micro titer plates containing a range of different carbon sources, it was possibly to identify carbon sources that lead to the production of isoprene with a surprisingly high specific productivity. The PM1 carbon source plate from Biolog was used for these experiments. A range of compounds that lead to a very high specific isoprene productivity is shown in Table 6. Compounds most significantly increasing specific productivity of isoprene include, but is not limited to, D-galacturonic acid, D-trehalose, N-acetyl-D-glucosamine, D-mannitol, D-fructose, D-glucose-6-phosphate, α-D-glucose. The final optical densities of the cultures grown on the different compounds are shown in Table 6. Some of these carbon sources may be used for the production of isoprene.
In this example, we demonstrate an increase in activity of the GcpE and LytB enzymes of the DXP pathway by providing more of an essential auxiliary factor, Fpr, which has been shown to positively influence their in vitro and in vivo activities (Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339 (2002); Wolff, M. et al. FEBS Letters, 541: 115-120 (2003), which are hereby incorporated by reference in their entireties). Fpr provides the necessary electrons derived from NADPH via FldA for GcpE and LytB to perform their catalytic functions (reviewed in report by L. A. Furgerson, The mevalonate-independent Pathway to Isoprenoid Compounds: Discovery, Elucidation, and Reaction Mechanisms, published Feb. 13, 2006, which is hereby incorporated by reference in its entirety).
The expression of fpr (encoding flavodoxin/ferredoxin NADPH-oxidoreductase) is increased in an engineered, isoprene producing strain of E. coli. Our previously tested higher DXP flux strains produce only modest isoprene levels, and are observed to accumulate significant levels of both cMEPP, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, and HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. The cMEPP and HMBPP DXP intermediates are the substrates of GcpE and LytB, respectively. The increased amount of Fpr may increase the activity demonstrated by the DXP pathway enzymes GcpE and LytB resulting in improved carbon flux to isoprene synthesis in the strain of interest over that of the comparable BL21 (DE3) control strain producing only endogenous levels of Fpr. The improved flux is demonstrated by an increase in isoprene titer.
The flavodoxin/ferredoxin NADPH-oxidoreductase encoded by fpr is intended to be expressed at increased levels from the E. coli chromosome by incorporating a constitutive highly active GI 1.6-promoter in front of the fpr open-reading frame, while replacing the endogenous promoter sequence. Alternatively, fpr can be expressed ectopically from a multi-copy vector construct. For either method, our goal is to express and accumulate Fpr at a level surpassing that generated from the endogenous fldA locus. Our preliminary qRT-PCR results suggest GI 1.6 fpr generates more fpr-transcript than the endogenous locus, and will likely accumulate more Fpr than the control as a result of the increased level of fpr-message. This is confirmed by immuno-blot once we receive the antibodies to Fpr.
Using a BL21(DE3) high DXP flux strain as the parental host strain, the introduction of the up-regulated fpr locus is assessed for the effects on isoprene production relative to the control strains. In addition, metabolite studies on the DXP intermediates provides insight into the beneficial affects of increased Fpr levels on GcpE and LytB activities.
Initially, the following BL21 (DE3) test strain is constructed and assessed for growth and the production of isoprene relative to the control: BL21 (DE3) GI 1.6-dxs GI 1.6-fpr T7-MEARR alba/pBBR1MCS-5. This strain is compared to the parental control strain (BL21 (DE3) GI 1.6-dxs T7-MEARR alba/pBBR1MCS-5) for growth, isoprene production, and DXP metabolite accumulation.
GrowthStrains are grown at 30° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including the appropriate antibiotics. Growth is monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production is analyzed using a headspace assay. For the shake flask cultures, one ml of a culture is transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753; cap cat#5188 2759). The cap is screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials are removed from the incubator and analyzed. The analysis is performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μm film thickness) is used for separation of analytes. The sampler is set up to inject 500 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port is held at 250° C. with a split ratio of 50:1. The oven temperature is held at 37° C. for the 2 minute duration of the analysis. The Agilent 5793N mass selective detector is run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) is observed to elute at 1.78 minutes. A calibration table is used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 200 μg/L. The limit of detection is estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isopreneug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental and test strains will be isolated and quantified as follows:
Metabolite ExtractionCell metabolism is rapidly inactivated by withdrawing 3.5 mL of the culture into a tube filled with 3.5 mL of dry ice-cold methanol. Cell debris is pelleted by centrifugation and the supernatant is loaded onto Strata-X-AW anion exchange column (Phenomenex) containing 30 mg of sorbent. The pellet is re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM NH4HCO3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1 mM NH4HCO3 buffer (pH=7.0). After each extraction, cell debris is pelleted by centrifugation and the supernatants are consecutively loaded onto the same anion exchange column. During the extraction and centrifugation steps the samples are kept at below +4° C. Prior to metabolite elution, the anion exchange columns are washed with water and methanol (1 mL of each) and the analytes were eluted by adding 0.35 mL of concentrated NH4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated NH4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant is neutralized with 30 μL of glacial acetic acid and cleared by centrifugation in a microcentrifuge.
Metabolite QuantificationMetabolites are analyzed using a Thermo Scientific TSQ Quantum Access mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). All system control, data acquisition, and mass spectral data evaluation are performed using XCalibur and LCQuan software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a chiral Nucleodex β-OH 5 μM HPLC column (100×2 mm, Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH guard cartridge is eluted with a mobile phase gradient shown in Table 7 (flow rate of 0.4 mL/min). The sample injection volume was 10 μL.
Mass detection is carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions are selected to detect the metabolites of interest in SRM mode: 245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of metabolites are determined based on the integrated intensities of peaks generated by PO3− product ion (m/z=79.0). Calibration curves obtained by injection of corresponding standards purchased from Echelon Biosciences Inc. Intracellular concentrations of metabolites are calculated based on the assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL
Example 22 Improved Carbon Flux into the DXP Pathway Using a Heterologous DXSLiving organisms synthesize isoprenoids via two distinct pathways: the mevalonate (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. MEP pathway starts from 1-deoxy-D-xylulose 5-phosphate (DXP), which is synthesized by condensation of pyruvate and glyceraldehyde-3-phosphate. This reaction is catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS). In some bacteria, including E. coli, DXP serves not only as a precursor of isoprenoids but is also used for biosynthesis of two important cofactors: thiamine (vitamin B1) and pyridoxol phosphate (vitamin B6).
The rate of isoprenoid synthesis in E. coli is regulated at the level of DXS. One of the mechanisms of this regulation may involve feedback inhibition of DXS activity by metabolites downstream the MEP pathway or/and intermediates of vitamin B1/B6 biosynthesis. Accordingly, the overall flux into the MEP pathway may be increased in E. coli by expressing an enzyme from a different organism that is not subject to inhibition by downstream products. Heterologous DXS may also be superior to the native E. coli DXS due lower Km or higher Kcat values with respect to pyruvate or glyceraldehyde-3-phosphate. Earlier studies have shown that a single Y392F substitution in the DXS of E. coli results in two-fold increase in the activity of the enzyme in vitro, although catalytic properties of the modified enzyme have not been studied in detail.
The choice of the sources of DXS for heterologous expression in E. coli can be based on the following considerations (see Table 8). First, organisms which have genome coding for several dxs isogenes can be selected. These organisms include plants (different forms of DXS in plants are classified as DXS 1 and DXS2), and bacteria (e.g. species of Streptomyces) having two or more dxs isogenes. Second, bacteria in which isoprenoids are synthesized via both the MEP (or DXP) pathway and the MVA pathway can be selected. Third, bacteria, which synthesize isoprenoids via the MVA pathway but contain a copy of the dxs gene in their genome specifically needed to make the vitamin cofactors. The DXS sequence this group of microorganisms is characterized by a significantly shorter loop corresponding to the amino acids 203-242 of E. coli DXS sequence (
In one set of the experiment, DXS from a variety of organisms (examples are listed in Table 8) is introduced into E. coli cells over-expressing plant isoprene synthase and isopentenyl-diphosphate delta-isomerase (IDI). (IDI activity in E. coli is normally very low; therefore enhanced expression of this enzyme is necessary to provide efficient conversion of isopentenyl-diphosphate into dimethylallyl-diphosphate, the substrate of isoprene synthase.). The resulting strains are tested for isoprene production and accumulation of DXP pathway intermediates, including but not limited to DXP, MEP, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol, 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, and compared to the control strain containing native E. coli DXS expressed in the same context as in the tested mutants. Increased concentrations of DXP intermediates and/or elevated rate of isoprene evolution in mutants containing heterologous DXSs indicated that the enzyme from the particular organism has higher activity in E. coli and is not subject to feedback inhibition by accumulated products.
In another set of experiments, a set of mutants over-expressing either heterologous dxs genes or dxs from E. coli (the control) are introduced into the background E. coli strain containing plant isoprene synthase, IDI, and several enzymes of MVA pathway allowing that strain to synthesize excessive amounts of isoprenoids when grown in the media containing exogenous MVA. These strains are tested for the accumulation of the intermediates specific to the DXP pathway. As in the previous case, increased concentrations of DXP intermediates compared to the control showed that DXS from specific organisms have higher activity in E. coli than the native enzyme and is not subject to feedback inhibition by isopentenyl-diphosphate and/or downstream isoprenoid products. To verify that a particular mutant have an improved rate of the isoprene production specifically due to the modified DXS, isoprene production rate is measured in cells grown on 13C-uniformly labeled glucose in the presence of non-labeled MVA. In this case, 13C composition of isoprene analyzed by mass spectrometry unequivocally indicated that this compound is synthesized via the DXP pathway from the labeled glucose, not from exogenous non-labeled MVA.
In a third set of experiments, experiments are performed to demonstrate that substitution of the tyrosine at position 392 of E. coli DXS for phenylalanine results in higher flux rate into the DXP pathway compared to the wild type enzyme. For this experiment the wild-type and the mutated DXS are over-expressed in an E. coli strain containing plant isoprene synthase and IDI. The two strains are compared for isoprene production rate and accumulation of DXP pathway intermediates. Increased concentrations of DXP intermediates and/or elevated rate of isoprene evolution in the strain bearing the superior properties of the engineered enzyme demonstrated the superior attributes of the mutant enzyme.
Populations of cells with a high degree of genotypic diversity are generated to identify combinations of genes, gene expression or mutations that increase flux through the DXP pathway. Three different methods are used in this example. First, combinations of genes, either endogenous to E. coli or from heterologous organisms, are assembled using the Multisite Gateway (Invitrogen) procedure and introduced into the E. coli screening strain. Second, libraries of genomic DNA, either from E. coli or heterologous organisms, are generated and introduced in the E. coli screening strain. Third, transposons that can result in either gene disruption or activation due to an internal promoter that is directed towards the inverted repeat of the transposable element are introduced.
A. The Multisite Gateway (Invitrogen) Procedure for Generating Synthetic OperonsGenes either endogenous to E. coli or from heterologous organisms are assembled into synthetic operons that are subsequently screened for increased flux through the DXP pathway and resulting isoprene production. The Multisite Gateway (Invitrogen) kit provides for a maximum of four discrete DNA “elements” that can be assembled together into one operon. Four genes are individually cloned into pENTR vectors, according to the manufacturer's protocol. For example, the last two genes in the DXP pathway, ispG and ispH are amplified by PCR with appropriate att recombination sites (according to manufacturer's protocol) and variable RBS (see Yarchuk et al., J. of Mol. Biol., 226(3):581-596 (1992), which is hereby incorporated by reference in its entirety) to generate plasmid pools with varying expression levels of each gene. The same procedure is applied to the electron carrier genes fldA and fpr, and the four resulting plasmid pools are recombined together onto Gateway destination vectors (pDEST-14 (Invitrogen), pET54-DEST or pCOLA-2-DEST (Novagen)) according to the manufacturer's protocol. The resulting plasmids harbor four gene operons with varying expression levels of each ORF. The pooled destination vectors are then introduced into E. coli strains by selecting for antibiotic resistance markers (kanamycin or ampicillin) and resulting pools are screened by GC-MS (described below).
B. Generation of Genomic LibrariesGenomic DNA either endogenous to E. coli or from heterologous organisms is cloned into the pSMART LCKan vector (Lucigen) according to the manufacturer's recommended protocol (see Lynch et al., Nat. Methods, 4(1):87-93 (2007), which is hereby incorporated by reference in its entirety). DNA from E. coli BL21 and K12 strains, B. subtilis, Lb. plantarum, Lb. sakei, P. citrea, S. coelicolor, S. spheroides, L. monocytogenes, A. tumefaciens, S. meliloti, and C. jejuni is used to generate libraries. The genomic DNA inserts of up to 20 kb in size are then introduced into E. coli strains for screening. Positive transformants are selected for by introduction of antibiotic resistance (kanamycin), pooled, and screened by GC-MS.
C. Transposon Mutagenesis and Gene ActivationA transposon that can both inactive genes by disruption of the ORF and also drive expression of proximal genes due to an endogenous promoter in the transposable element is introduced into E. coli for screening. The custom transposon is generated by inserting either a constitutive or inducible promoter into the MCS of the EZ-Tn5 transposon construction vectors (Epicentre). Examples of internal promoters include PT7, Ptrc, Ptac, Pbad, Plac, PL (phage lambda), the gi series, and Ptet. These promoters are cloned into the transposable element, and the resulting custom transposon is introduced into E. coli. Strains harboring transposon insertions are identified by antibiotic resistance, pooled, and subjected to screening by GC-MS.
E. coli Strains and Screening
Plasmid pools or transposons are introduced into different E. coli strains for screening. Positive transformants are identified by antibiotic resistance markers (typically Kan or Amp) located on the plasmid or within the transposable element. Strains include: A strain harboring a plasmid carrying dxs, dxr, idi, and IspS (isoprene synthase) under control of the T7 promoter; a strain harboring integrated and constitutively expressed dxs, dxr, and idi with ispS also integrated or expressed from a plasmid; a strain expressing the entire DXP operon under the control of the T7 Promoter; any strain harboring the current best conformation of DXP pathway genes for isoprene production, yet still displays clear accumulation of DXP pathway metabolites (e.g. HDMAPP). Individual transformants are pooled (in groups of 100 to 1000 individuals per pool) and screened via GC-MS in a 96-well glass block. The analysis is performed (for the 2 mL and 96-well plate methods) using an Agilent 6890 GC/MS system interfaced with a 5973 MS Leap CTC CombiPAL autosampler operating in headspace mode. An Agilent HP-5 (5% Phenyl Methyl Siloxane (15 m×0.25 mm×0.25 uM)) column is used for separation of analytes. The sampler is set up to inject 100 μL of headspace gas. The GC/MS method utilizes helium as the carrier gas at a flow of 1 ml/min. The injection port is held at 250° C. with a split ratio of 50:1. The oven temperature is held at 37° C. for the 2 min duration of the analysis. The Agilent 5793N mass selective detector is run in single ion monitoring (SIM) mode on mass 67. The detector is switched off from 0.00 to 0.44 minutes to allow the elution of permanent gases and on 0.44 mins to 0.60 mins. Under these conditions isoprene (2-methyl-1,3-butadiene) is observed to elute at 0.49 minutes. A calibration table is used to quantify the absolute amount of isoprene and was found to be linear from 0 μg/L to 5600 mg/L (using calibration gas). Positive pools are then re-assayed to confirm any positive effect on isoprene production. The individual plasmids or constructs in strains or pools which display increased isoprene production are identified to determine the precise nature of positive influence on DXP pathway flux.
Genes of Organisms ExaminedGenes including, but not limiting to, the following organisms are examined: Arabidopsis thaliana, Zea mays, Campylobacter jejuni, Sinorhizobium meliloti, Helicobacter pylori Agrobacterium tumefaciens, Deinococcus radiodurans, Bacillus subtilis, Pantoea citrea, Listeria monocytogenes, Lactobacillus spp., and Streptomyces spp.
Materials Multisite Gateway kit (Invitrogen)Lucigen (Clonesmart Cloning Kits)—library construction
EZ-Tn5 System (EpiCentre)—gene disruption/activation
pET—PT7-driven full DXP pathway plasmid
pET—PT7 driven dxs, dxr. idi, ispS
pET—best conformation of DXP pathway genes for isoprene production
pBBR—PT7 or Ptrc ispS
pET-54-DEST, pCOLA-DEST vectors (Novagen)
pDEST14, pDEST15 (Invitrogen)
This example provides further demonstration of increased isoprene production in REMG39 by overexpression of GcpE, LytB PetF and PetH of T. elongatus BP-1 within CMP272, a BL21 derived host.
As described and shown infra, increased expression of both dxs and yeast idi allow increased flux through the endogenous DXP pathway of E. coli. Previous work by the field (see, for example, Chao et al., Biotechnol Prog., 18(2):394-400 (2002) and Zhang et al., Protein Expression and Purification, 29(1): 132-139 (May 2003)) has lead to the conclusion that T7-based expression systems are unstable and their behavior not entirely predictable when subjected to 14-L fermentation conditions. The CMP271 and subsequent CMP272 strain were constructed to: (1) replace our current T7-governed plasmid-based expression of yeast idi with expression originating from the chromosome; permitting the use of a non-T7 based expression strain for DXP-mediated isoprene production and/or (2) introduce the genomically encoded locus harboring the genes for the lower MVA pathway enzymes and yeast IDI to provide sufficient levels of yeast IDI for maximal flux to Isoprene Synthase.
The CMP271 strain was made into an isoprene generating strain by the addition of pDW33, harboring a P. alba isoprene synthase allele, via electroporation, and subsequently yielding strain CMP272.
The CMP272 strain serves as the baseline host in which isoprene production has been successfully improved by the addition of the T. elogatus IspG (GcpE) and IspH (LytB) encoding genes along with their putative reducing shuttle system (PetF and PetH). The construct harboring the T. elongatus genes, Ptac-gcpE-lytB-petF-petH/pK184, has been described infra in the example utilizing T. elongatus. The parental CMP272 and test strain REMG39 were evaluate for growth, isoprene production, metabolite profile, and product yield on carbon under 14-L fermentation conditions described below. The results are depicted in
The GI 1.X-promoter insertions and subsequent loopout of the antibiotic resistance markers described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21 (Novagene) was used. P1 lysate preparations and transductions were performed as previously described (Thomason et al., 2007).
The strategy for inserting the GI1.X-yidi series into the E. coli idi locus using the Gene Bridges GmbH methods is illustrated in
0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) MQ09-10F
1.25 ul primer (10 uM) MQ09-10R
32 ul diH2O
+1 ul of HerculaseII Fusion from Stratagene
0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) MQ09-11F
1.25 ul primer (10 uM) MQ09-11R
32 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) MQ09-10F″
1.25 ul primer (10 uM) MQ09-11R
32 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
(95° C. 2 min., 95° C. 20 sec., 55° C. 20 sec., 72° C. 1 min., 29×, 72° C. 3 min,
4° C. until cool, use Eppendorf Mastercycler)
(95° C. 2 min., 95° C. 20 sec., 55° C. 20 sec., 72° C. 35 sec., 29×, 72° C. 3 min,
4° C. until cool, use Eppendorf Mastercycler)
(95° C. 2 min., 95° C. 20 sec., 55° C. 20 sec., 72° C. 1.2 min., 29×, 72° C. 3 min,
4° C. until cool, use Eppendorf Mastercycler)
The resulting PCR fragments Frag A, B, and A+B were separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The purifed stocks of Frag A and Frag B were used in the Frag A+B PCR reaction described above. The resulting purified stock of Frag A+B is referred to as GB-CMP-GI1.X-yidi.
Amplification of the galM Locus of CMP263
One colony of CMP263 was stirred in 30 uL H2O and then heated to 95° C. for 5 min. The resulting solution was spun down to pellet debris and 2 uL of the supernatant was used as the template in the following PCR reaction:
2 ul colony in H2O (see above)
5 ul Herculase Buffer1 ul dNTP's (100 mM)
1 ul galMF primer (10 uM)
1 ul galMR primer (10 uM)
+0.5 ul of Herculase Enhanced DNA polymerase from Stratagene
Cycle Parameter:95° C.×2 min., [95° C.×30 sec., 52° C.×30 sec., 72° C.×60 sec]×30 cycles; 72° C.×7 min,
4° C. until cool (PCRExpress Thermocycler from ThermoHybaid).
The size of the resulting PCR fragment was determined on a 0.8% E-gel (Invitrogen), using DNA Molecular Weight X (Roche) as a ladder; a corresponding PCR product was not obtained from BL21 cells, as expected for the negative control.
Integration of GB-CMP GI 1.X-yidi PCR Product into BL21/pRed-ET Strain
The pRed-ET vector (Gene Bridges kit) was transformed into BL21 by electroporation using the BIO RAD Gene Pulser system and a transformation protocol suggested by the manufacturer (BIO RAD) resulting in strain MD08-114 (BL21/pRed-ET). Approximately 400 ug of the purified GB-CMP GI 1.X-yidi PCR fragment was electroporated into MD08-114. The transformants were recovered in L Broth and then plated on L agar containing chloramphenicol (5 ug/ml). Chloramphenicol resistant colonies were analyzed by PCR for the presence of the GB-CMP GI 1.X-yidi sequence at the desired locus using the top Gb's CMP and MQ09-12R primers. The PCR fragments from a number of transformants were sequenced using the MQ09-12R and top GB's CMP primers (Quintara; Albany, Calif.) and the various GI1.X-yidi strains of interest identified. One chloramphenicol resistant clone harboring the GI1.6-yidi locus (BL21 FRT-CmR-FRT GI1.6(A)-yidi) was chosen and designated MD09-211.
B. Strategy for Creating the CMP271 StrainThe GI1.6-dxs::kan locus of strain MCM625, described in Example 9, was introduced into MD09-211 via P1-mediated transduction and the resulting kanamycin and chloramphenicol resistant strain named MD09-221. The antibiotic resistance markers of strain MD09-221 were looped out using pCP20 from the pRed-ET kit according to the manufacturer's instructions (GeneBridges). Transformants of interest were verified by the loss of resistance to chloramphenicol (5 ug/ml) and kanamycin (50 ug/ml); one chloramphenicol and kanamycin sensitive clone was chosen and designated MCM710. The FRT-Neo-FRT PL.2 mKKDyI locus (harboring an additional copy of the yeast idigene) of strain MCM521, described in US Appl. No. 61/289,959, was moved into MCM710 by P1-mediated transduction. One kanamycin resistant clone was chosen and designated MCM783. MCM783 was transduced with a P1 lysate of E. coli K-12 MG1655, and selected on M9 medium (Na2HPO4 6 g/L, KH2PO4 3 g/L, NaCl 0.5 g/L, NH4Cl 0.5 g/L, 0.1 mM CaCl2, 2 mM MgSO4)+0.4% w/v galactose. One galactose ultizing clone was chosen and designated CMP263. The presence of the galM locus within the 17,257 bp of MG1655 that is not endogenous to BL21, but was now harbored by CMP263, was verified by PCR using the primer set galMF/galMR; this PCR reaction is described above. The kanmycin resistance marker within strain CMP263 was looped out using Gene Bridges GmbH methods. One kanamycin sensitive clone was chosen and designated CMP271.
C. Strategy for Creating the CMP272 StrainElectroporation of pDW33 into strain CMP271 was done using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The vector construct harbors the PTrc-governed MEARR P. alba allele encoding a truncated form of Isoprene Synthase. The template for pDW33 construction, EWL230, has been described in US. Publ. No. 2009/0203102 and WO 2009/076676. A picture of the pDW33 vector map is presented in
Construction of pDW33
pDW33 was constructed in order to generate an isoprene producing Escherichia coli strain harboring the truncated version of P. alba isoprene synthase (the MEA variant) under control of the Ptrc promoter.
Construction of Strain DW194:The plasmid harboring truncated P. alba isoprene synthase (IspS) was constructed by Quikchange PCR mutagenesis (Stratagene—see Table below for primer sequences) upon the template EWL230 (aka pTrc-P. alba). PCR reaction and cycling parameters are described below. The PCR product was visualized by gel electrophoresis (E-gel, Invitrogen), and then treated with 1 μl DpnI restriction endonuclease (Roche) for three hours at 37° C. Ten μl of the PCR product was then de-salted using a microdialysis membrane (MilliPore) and transformed into electrocompetent E. coli strain MCM531 (previously described) using standard molecular biology techniques. Cells were recovered in one ml of LB medium for 1.5 hours at 30° C., plated onto LB solid agar plates containing 50 μg/ml carbenicillin and 5 mM mevalonic acid, and then incubated overnight at 37° C. The next day, positive colonies (of strain DW194, see below) were selected for growth and plasmid purification (Qiagen), and ultimately confirmed by DNA sequencing (Quintara) with the primers listed below. The final plasmid, pDW33, carries the open reading frame encoding the truncated version (MEA) of IspS.
1 ul plasmid EWL230 (aka pTrc P. alba)
5 ul 10× PfuUltra HF buffer
1 ul dNTPs (100 mM)
39 ul diH2O
1 ul PfuUltra HF Polymerase (Stratagene) PCR Cycling Parameters: 1. 95° C. 1 min. 2. 95° C. 30 sec. 3. 55° C. 1 min. 4. 68° C. 6 min.5. Go to step 2-18 cycles
6. 4° C.
Transformation of pDW33 into CMP271
This step was done to build the isoprene-producing strain CMP272 the pDW33 plasmid was transformed by electroporation into CMP271. Transformants were recovered in L broth and plated on L agar containing carbenicillin (50 ug/ml). The resulting strain was designated as CMP272.
D. Strategy for Creating the REMG39 StrainElectroporation of Ptac-gcpE-lytB-petF-petH/pK184 into strain CMP272 was performed using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). A plasmid preparation of Ptac-gcpE-lytB-petF-petH/pK184 was provided by Gene Oracle, Inc. Ptac-gcpE-lytB-petF-petH/pK184 has been described infra (see, e.g., Example 11).
Transformation of Ptac-gcpE-lytB-petF-petH/pK184 into CMP272
To build the REMG39 test strain, Ptac-gcpE-lytB-petF-petH/pK184 was transformed by electroporation into CMP272. Transformants were recovered in L broth and plated on L agar containing carbenicillin (50 ug/ml) and kanamycin (50 ug/ml). The resulting strain was designated as REMG39.
E. Comparing CMP272 to REMG39 for Growth, Isoprene Production, DXP Metabolite Profile, and Product Yield on Carbon During 14-L FermentationThe parental strain CMP272 was compared to the test strain REMG39 under 14-L fermentation conditions. The benefit of the T. elongatus IspG (GcpE) and IspH (LytB) activities on isoprene production and overall flux through the otherwise endogenous DXP pathway of E. coli is illustrated in
Isoprene production from E. coli expressing genes from the DXP pathway and isoprene synthase, grown in fed-batch culture at the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in Di H2O. This solution was heat sterilized (123° C. for 20 minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g, Mercury Vitamin Solution 8 mL, and antibiotics were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
Mercury Vitamin Solution (Per Liter):Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0 g. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100% Foamblast 10 g. All components were mixed together and autoclaved. Mercury Vitamin Solution 6.7 mL was added after the solution had cooled to 25° C.
Fermentation was performed in a 15-L bioreactor with E. coli BL21 cells overexpressing the first enzyme in the dxp pathway (GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6y-IDI), the lower MVA pathway (PL.2-mKKDyI) and truncated isoprene synthase from P. alba (pDW33) and containing a restored 17,257 bp chromosomal galM-containing region derived from MG1655 (strain name CMP272). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium for the bioreactor. After the inoculum grew to optical density 1.0, measured at 550 nm (OD550), 500 mL was used to inoculate a 15-L bioreactor and bring the initial tank volume to 5 L.
The feed solution was fed at an exponential rate until a top feed rate of 4.8 g/min was reached. After this time, the glucose feed was fed to meet metabolic demands at rates less than or equal to 4.8 g/min. The total amount of glucose delivered to the bioreactor during the 45 hr fermentation was 5.6 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). A single shot of IPTG was added to the tank to bring the concentration to 200 uM when the cells were at an OD of 8.
The isoprene level in the off-gas from the bioreactors was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 0.97 g/L at 45 hr.
Equation for calculating Isoprene Titer: ∫(Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 45 hrs [=] g/L broth
Equation for calculating Specific Productivity levels: (mg isoprenet−mg isopreneto)/[(OD550t*L brotht−OD550to*L brothto)/(2.7 OD*L/g cell)]/(t−t0) [=] mg isoprene/g cell/hr
Isoprene production from E. coli expressing genes from the DXP pathway and isoprene synthase, grown in fed-batch culture at the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in Di H2O. This solution was heat sterilized (123° C. for 20 minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g, Mercury Vitamin Solution 8 mL, and antibiotics were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
Mercury Vitamin Solution (Per Liter):Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0 g. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100% Foamblast 10 g. All components were mixed together and autoclaved. Macro Salt Solution 3.4 mL, 1000× Modified Trace Metal Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added after the solution had cooled to 25° C.
Macro Salt Solution (Per Liter):MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate 49.6 g. All components were dissolved in water, q.s. to volume and filter sterilized with 0.22 micron filter.
Fermentation was performed in a 15-L bioreactor with E. coli BL21 cells overexpressing the first enzyme in the dxp pathway (GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), and truncated isoprene synthase from P. alba (pDW33) and containing a restored 17,257 bp chromosomal galM-containing region derived from MG1655 (strain name REMG39). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium for the bioreactor. After the inoculum grew to optical density 1.0, measured at 550 nm (OD550), 500 mL was used to inoculate a 15-L bioreactor and bring the initial tank volume to 5 L.
The feed solution was fed at an exponential rate until a top feed rate of 4.8 g/min was reached. After this time, the glucose feed was fed to meet metabolic demands at rates less than or equal to 4.8 g/min. The total amount of glucose delivered to the bioreactor during the 56 hr fermentation was 7.0 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). A single shot of IPTG was added to the tank to bring the concentration to 300 uM when the cells were at an OD of 5. After a run time of 36 h, whole broth, including cell mass, was drawn off periodically to prevent overflow of the bioreactor.
The isoprene level in the off-gas from the bioreactors was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 2.7 g/L at 56 hr.
Equation for calculating Isoprene Titer: ∫(Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 56 hrs [=] g/L broth
Equation for calculating Specific Productivity levels: (mg isoprenet−mg isopreneto)/[(OD550t*L brotht−OD550to*L brothto)/(2.7 OD*L/g cell)]/(t−t0) [=] mg isoprene/g cell/hr
Cell metabolism was rapidly inactivated by withdrawing several milliliters of the fermentor culture into a pre-weighed tube filled with 9.0 mL of dry ice-cold methanol. The resulting sample was weighed again to calculate the amount of withdrawn cell culture and then put to −80° C. for storage until further analysis. In order to extract metabolites, 500 μL of methanol-quenched fermentation sample was spun down by centrifugation for 4 min at 4500×g, at −9° C. The pellet was then re-extracted twice, first with 350 μL of 85% methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and then with 350 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and all three supernatants were pooled together for further analysis.
Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Example 26 Result of Increased Activity of IspGThis example demonstrates that increased activity of IspG can be detrimental to isoprene production when it occurs in the absence of increased FldA expression.
Data obtained using 14-L REMG39 indicates that despite the increased production of isoprene in REMG39, the strain is still limited for IspG activity; this is suggested by the approx. 19 mM cMEPP level the REMG39 strain maintains across the majority of the fermentation (see
A. Construction of test strains REM C9—12, REM D6—12, and REM E7—12
The construction of GI1.6 fldA/pCL, GI1.6 fldA-ispG/pCL, and GI1.6 ispG/pCL were done using standard molecular biology techniques (Sambrook et al., 1989). The pCL1920 (pCL) cloning vector has been described in publications, see, e.g., Lerner, C. G. et al., Nucleic Acids Research, Vol. 18: 4631(1990).
Chromosomal DNA from strain REM I6—4 was used as a PCR template for the generation of the PCR fragment harboring GI1.6 fldA, which was used to create GI1.6 fldA/pCL. Generation of strain REM I6—4 is described below. The DNA ultimately derived from the DXP operon pET24a plasmid (see, e.g., Example 11) was used as the PCR template for both the generation of the PCR fragments harboring ispG and GI1.6 ispG, which were used to create GI1.6 fldA-ispG/pCL and GI1.6 ispG/pCL, respectively. The DXP operon pET24a plasmid and GI1.6 gcpE-lytB-yidi pCR Blunt II TOPO vector PCR templates utilized have been described previously (see, e.g., Example 11).
B. The Generation of REM I6 4, the Precursor to GI1.6 fldA/pCL
The GI 1.X-promoter insertions and subsequent loopout of the antibiotic resistance markers described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21(DE3) (Invitrogen) was used. The BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD) was used for the electroporations described.
The GI1.X promoters introduced upstream of the endogenous fldA coding region using the Gene Bridges GmbH methods are illustrated in
To amplify the GB-CmpR cassette for inserting the GI 1.X-promoters immediately upstream of the fldA locus the following PCR reaction was set up:
1 ul template (100 ng GB-CmpR)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) fldA cmpGI1.X-F
1.25 ul primer (10 uM) fldA cmpGI1.X-R
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., (95° C.×30 sec., 63° C.×30 sec., 72° C.×2 min.)×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragments were separated on a 0.8% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions. The resulting stock was GB-CmpR-GI 1.X-fldA fragment.
Integration of GB-CmpR-GI 1.X fldA PCR Product into BL21(DE3)/pRed-ET Strain
The pRed-ET vector (Gene Bridges kit) was transformed into BL21(DE3) by electroporation resulting in strain DW30 (BL21(DE3)/pRed-ET). The purified GB-CmpR-GI 1.X-fldA PCR fragment was electroporated into DW30. The transformants were recovered in L Broth and then plated on L agar containing chloramphenicol (10 ug/ml). Chloramphenicol resistant colonies were analyzed by PCR for the presence of the GB-CmpR cassette and the GI 1.X-promoters using primers fldA confirm-F, fldA confirm-R, top GB's CMP, and bottom Pgb2. The PCR fragments from a number of transformants were sequenced using the fldA confirm-R and top GB's CMP primers (Sequetech; Mountain View, Calif.) and the various GI 1.X fldA strains of interest identified. The chloramphenicol resistant strain, BL21(DE3) CMP::GI1.6fldA, was designated REM I6—4.
C. Strategy for Creating REM C9—12Electroporation of GI1.6 fldA/pCL into CMP272 was performed using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). Cells of the strain REM I6—4 encoding GI1.6 fldA were used as the PCR template for vector construction.
Amplification of the GI1.6 fldA Fragment
To amplify the GI1.6 fldA fragment for inserting the GI1.6 fldA fragment into pCL the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6—4 cells)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ SalI GI1.X
1.25 ul primer (10 uM) 3′ NruI/SacII fldA stop
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×3 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stock was GI 1.6-fldA fragment.
Cloning of the GI1.6 fldA Fragment into pCL
Approximately 600 ng of the GI1.6 fldA fragment was digested with SalI (Roche) according to the manufacturer's specifications and approx. 200 ng of the pCL plasmid was digested with SalI and SmaI (Roche) according to the manufacturer's specifications. The digests were subsequently combined and cleaned using the Qiagen QiaQuick Gel Extraction Kit. Approximately one half of the cleaned cut DNA was ligated using T4 DNA Ligase from New England Biolabs according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing spectinomycin (50 ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40 ug/ml; Sigma). White, spectinomycin resistant colonies were selected, grown overnight in L broth containing spectinomycin (50 ug/ml), and harvested for subsequent plasmid preparation. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers M13 (−20) and M13 Reverse, and the correct GI1.6 fldA/pCL clone identified, which has been designated as strain REM A1—11 (TOP10 w/GI1.6 fldA/pCL; 5′ Sal I-3′ SacII/NruI uncut (blunt 3′) end PCR fragment into 5′ SalI-3′Sma I of pCL). A picture of the GI1.6 fldA/pCL vector map is presented in
Transformation of GI1.6 fldA/pCL into CMP271
To build the isoprene producing test strain REM C9—12, the GI1.6 fldA/pCL plasmid was transformed by electroporation into CMP272. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). The resulting strain was designated REM C9—12.
Strategy for Creating REM D6—12Electroporation of GI1.6 fldA-ispG/pCL into CMP272 was performed using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The DXP operon pET24a plasmid was used as the PCR template for vector construction.
Primers Sequences
Amplification of they′ SacII-ispG-3′ NruI Fragment
To amplify the ispG fragment for inserting into GI1.6 fldA/pCL the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6—4 cells)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ SacII Ec ispG w/rbs
1.25 ul primer (10 uM) 3′ NruI Ec ispG stop
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×2 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stock was 5′ SacII-ispG-3′ NruI fragment.
Cloning of the GI1.6 fldA Fragment into pCL
Approximately 600 ng of the 5′ SacII-ispG-3′ NruI fragment was digested with Sac II (New England BioLabs) according to the manufacturer's specifications and approx. 200 ng of the GI1.6 fldA/CL plasmid was digested with SacII and NruI (New England BioLabs) according to the manufacturer's specifications. The digests were subsequently combined and cleaned using the Qiagen QiaQuick Gel Extraction Kit. Approximately one half of the cleaned cut DNA was ligated using T4 DNA Ligase (New England Biolabs) according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing spectinomycin (50 ug/ml). Some spectinomycin resistant colonies were selected, grown overnight in L broth containing spectinomycin (50 ug/ml), and harvested for subsequent plasmid preparation. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers 5′ SacII Ec ispG, 3′ NruI Ec ispG stop, M13For, 5′ BamHI Ec ispG w/rbs, 3′ SacII Ec ispG w/stop, pRA42, and pRA43 and the correct GI1.6fldA-ispG/pCL clone identified, which has been designated as strain REM D9—11 (TOP10 w/GI1.6 fldA-ispG/pCL; 5′ Sac II-3′ NruI uncut (blunt 3′ end) PCR fragment into 5′ SacII-3′NruI of pCL). A picture of the GI1.6 fldA-ispG/pCL vector map is presented in
Transformation of GI1.6 fldA-ispG/pCL into CMP271
To build the isoprene producing test strain REM D6—12, the GI1.6 fldA-ispG/pCL plasmid was transformed by electroporation into CMP272. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). The resulting strain was designated REM D6—12.
E. Strategy for Creating REM E7 12Electroporation of GI1.6 ispG/pCL into CMP272 was performed using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The GI1.6 gcpE-lytB-yidi pCR Blunt II TOPO vector was used as the PCR template for vector construction.
Amplification of the GI1.6 ispG Fragment
To amplify the GI1.6 ispG fragment for inserting into pCL the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6—4 cells)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ SalI GI1.X
1.25 ul primer (10 uM) 3′ SacI Ec ispG w/stop
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×2 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits according to manufacturer's instructions. The resulting stock was GI1.6 ispG fragment.
Cloning of the G11.6 ispG Fragment into pCL
Approximately 600 ng of the GI1.6 ispG fragment and 200 ng of the pCL vector were digested with SalI and SacI (Roche) according to the manufacturer's specifications. The digests were subsequently combined and cleaned using the Qiagen QiaQuick Gel Extraction Kit. Approximately one half of the cleaned cut DNA was ligated using T4 DNA Ligase (New England Biolabs) according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing spectinomycin (50 ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40 ug/ml; Sigma). White spectinomycin resistant colonies were selected, grown overnight in L broth containing spectinomycin (50 ug/ml), and harvested for subsequent plasmid preparation. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers 3′ SacI Ec ispG w/stop, M13For, and M13 Rev and the correct GI1.6 ispG/pCL clone identified, which has been designated as strain REM H5—11 (TOP10 w/GI1.6 ispG/pCL; 5′ SalI-3′ SacI PCR fragment into 5′ SalI-3′SacI of pCL). A picture of the GI1.6 ispG/pCL vector map is presented in
Transformation of GI1.6 ispG/pCL into CMP271
To build the isoprene producing test strain REM E7—12, the GI1.6 ispG/pCL plasmid was transformed by electroporation into CMP272. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). The resulting strain was designated REM E7—12.
F. Analysis of Test Strains REM C9—12, REM D6—12, and REM E7—12 and the Parental Strain CMP272 for Growth, Isoprene Production, and DXP Metabolite Accumulation.The parental strain CMP272 was compared against the test strains (REM C9—12, REM D6—12, and REM E7—12) in a shake flask assay as well as in a DXP metabolite determination study
Strains CMP272, REM C9—12, REM D6—12, and REM E7—12 were grown as 2-5 ml cultures at 30° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). Induction of LacI-regulated gene expression was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a concentration of 600 uM. Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production was anlayzed using a headspace assay. For the shake flask cultures, 200 ul of a culture was transferred from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (15 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 100 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for 0.6 minute, the duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 0 to 0.42 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at approx. 0.49 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 5000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isoprene ug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental and test strains, CMP272 and REM C9—12, REM D6—12, and REM E7—12, respectively, that are described above and depicted in
Metabolite Extraction: Processing Samples from Small-Scale Experiments.
To measure accumulation of metabolites in small-scale experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min at 7500×g, at −9° C. Immediately after centrifugation the supernatant was aspirated to a clean tube for analysis of excreted metabolites and 100 μL of dry ice-cold methanol was added to pelleted cells. The resulting samples were then stored at −80° C. until further processing.
To determine concentrations of excreted metabolites, 500 μL of methanol was added to 300 μL of the supernatant and the resulting mixture was centrifuged for 10 min at 20000×g at 4° C. to remove insoluble material before the LCMS analysis.
For metabolites extraction from the pellet (further referred as intracellular metabolites), 10 μL of water was added to methanol-containing samples, the pellet was resuspended in the resulting methanol/water mix and cell debris were spun down by 4-min centrifugation at 4500×g. The pellet was re-extracted two more times, first with 100 μL of 75% methanol buffered with 1 mM ammonium acetate in water (pH=8.0), then with 90 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and the supernatants from all three extractions were combined and analyzed by LCMS. During the extraction procedure, samples were kept on ice or in a refrigerated centrifuge whenever possible to minimize metabolites degradation.
Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Example 27 Effects of Increased Activity of IspGThis example demonstrates that increased activity of IspG can be detrimental to isoprene production as a result of insufficient IspH activity within strain REM G4—11.
As described in the example above, increased expression of fldA alone or in combination with ispG within the CMP272 strain background improved isoprene production (
To further improve the IspG activity generated by the REMG39 strain background, the vector constructs GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL were introduced into the strain, subsequently generating the test strains REM G2—11 and REM G4—11, respectively.
B. Strategy for Creating REM G2—11 and REM G4—11Electroporation of GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL into REMG39 was performed using the BIO RAD Gene Pulser system and a transformation protocol suggested by the manufacturer (BIO RAD). Plasmid preparations of GI1.6 fldA/pCL, generated from strain REM A1—11, and GI1.6 fldA-ispG/pCL, generated from strain REM D9—11, were used; these strains and constructs are described above.
Transformation of GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL into CMP271
To build the isoprene producing test strains REM G2—11 and REM G4—11, the GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL plasmids were transformed, separately, by electroporation into REMG39. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml), kanamycin (50 ug/ml), and carbenicillin (50 ug/ml). The resulting strains were designated REM G2—11 and REM G4—11, respectively.
C. Analysis of Test Strains REM G2—11, REM G4—11, and the Parental Strain REMG39 for Growth, Isoprene Production, and DXP Metabolite Accumulation.The parental strain REMG39 was compared against the test strains (REM G2—11, REM and REM G4—11) in a shake flask assay as well as in a DXP metabolite determination study. The increase in isoprene production provided by the presence of GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL within the REMG39 background is depicted in
Strains REMG39, REM G2—11, and REM G4—11 were grown at 30° C. as 2-5 ml cultures in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). Induction of LacI-regulated gene expression was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a concentration of 400 uM. Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production was anlayzed using a headspace assay. For the shake flask cultures, 200 ul of a culture was transferred from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (15 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 100 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for 0.6 minute, the duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 0 to 0.42 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at approx. 0.49 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 5000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isoprene ug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental and test strains, REMG39 and REM G2—11 and REM G4—11, respectively, that are described above and depicted in
Metabolite Extraction: Processing Samples from Small-Scale Experiments.
To measure accumulation of metabolites in small-scale experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min at 7500×g, at −9° C. Immediately after centrifugation the supernatant was aspirated to a clean tube for analysis of excreted metabolites and 100 μL of dry ice-cold methanol was added to pelleted cells. The resulting samples were then stored at −80° C. until further processing.
To determine concentrations of excreted metabolites, 500 μL of methanol was added to 300 μL of the supernatant and the resulting mixture was centrifuged for 10 min at 20000×g at 4° C. to remove insoluble material before the LCMS analysis.
For metabolites extraction from the pellet (further referred as intracellular metabolites), 10 μL of water was added to methanol-containing samples, the pellet was resuspended in the resulting methanol/water mix and cell debris were spun down by 4-min centrifugation at 4500×g. The pellet was re-extracted two more times, first with 100 μL of 75% methanol buffered with 1 mM ammonium acetate in water (pH=8.0), then with 90 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and the supernatants from all three extractions were combined and analyzed by LCMS. During the extraction procedure, samples were kept on ice or in a refrigerated centrifuge whenever possible to minimize metabolites degradation.
Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
D. Analysis of Test Strain REM G4—11 for Growth, Isoprene Production, and DXP Metabolite Accumulation at Large Scale.The increased HDMAPP present in the REM G4—11 cells was higher than the parental control strain; however, the averaged 0.63 mM HDMAPP intracellular concentration measured in the REM G4—11 cells was significantly less than the >10 mM intracellular HDMAPP level that has been correlated with poor cell growth and reduced isoprene production (see
-
- 1) IspG activity has been improved within the REM G4—11 strain.
- 2) a bottleneck in DXP flux now occurs at the point of IspH activity in the REM G4—11 strain.
The large scale fermentation of the parental strain REMG39 is described above. Isoprene production from E. coli expressing genes from the DXP pathway and isoprene synthase, grown in fed-batch culture at the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in Di H2O. This solution was heat sterilized (123° C. for 20 minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g, Mercury Vitamin Solution 8 mL, and antibiotics were added after sterilization and pH adjustment.
1000× Modified Trace Metal Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
Mercury Vitamin Solution (Per Liter):Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0 g. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100% Foamblast 10 g. All components were mixed together and autoclaved. Macro Salt Solution 3.4 mL, 1000× Modified Trace Metal Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added after the solution had cooled to 25° C.
Macro Salt Solution (Per Liter):MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate 49.6 g. All components were dissolved in water, q.s. to volume and filter sterilized with 0.22 micron filter.
Fermentation was performed in a 15-L bioreactor with E. coli BL21 cells overexpressing the first enzyme in the dxp pathway (GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), the E. coli ispG and fldA genes (GI1.6 fldA-ispG/pCL), and truncated isoprene synthase from P. alba (pDW33) and containing a restored 17,257 bp chromosomal galM-containing region derived from MG1655 (strain name REM G4—11). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium for the bioreactor. After the inoculum grew to optical density 1.0, measured at 550 nm (OD550), 500 mL was used to inoculate a 15-L bioreactor and bring the initial tank volume to 5 L.
The feed solution was fed at an exponential rate until a top feed rate of 4.9 g/min was reached. After this time the glucose feed was fed to meet metabolic demands a rates less than or equal to 4.9 g/min. The total amount of glucose delivered to the bioreactor during the 44 hr fermentation was 3.0 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The inital IPTG concentration when the tank was first inoculated was 50 uM. Shots of 50 uM were added over the next five hours to bring the IPTG concentration to 350 uM when the cells were at an OD550 of 10.
The isoprene level in the off-gas from the bioreactors was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 0.98 g/L at 44 hours.
Equation for calculating Isoprene Titer: ∫(Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 44 hrs [=] g/L broth
Equation for calculating Specific Productivity levels: (mg isoprenet−mg isopreneto)/[(OD550t*L brotht−OD550to*L brothto)/(2.7 OD*L/g cell)]/(t−t0) [=] mg isoprene/g cell/hr
Cell metabolism was rapidly inactivated by withdrawing several milliliters of the fermentor culture into a pre-weighted tube filled with 9.0 mL of dry ice-cold methanol. The resulting sample was weighted again to calculate the amount of withdrawn cell culture and then put to −80° C. for storage until further analysis. In order to extract metabolites, 500 μL of methanol-quenched fermentation sample was spun down by centrifugation for 4 min at 4500×g, at −9° C. The pellet was then re-extracted twice, first with 350 μL of 85% methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and then with 350 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and all three supernatants were pooled together for further analysis.
B. Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Example 29 Increased Isoprene Production by Expression of the IspH Enzyme and Coincident Demonstration of Maintained Accumulation of Higher DXP Metabolite LevelsThis example demonstrates increased isoprene production by expression of the IspH enzyme from Anabaena sp. PCC7120 in strain REM H8—12 and coincident demonstration of maintained accumulation of higher DXP metabolite levels in the REM H8—12 strain exhibiting increased IspG activity.
Data in the above example(s) generated with test strain REM G4—11 indicates that increased IspG activity within an enhanced DXP fluxing strain needs to be balanced by sufficient IspH activity in order to avoid high levels of HDMAPP accumulation during 14-L fermentation. Intracellular levels of HDMAPP, the substrate for IspH, in excess of 10 mM have been correlated in both small scale and large scale experiments with poor cell growth, reduced flux through the DXP pathway, and subsequently reduced isoprene generation from isoprene production strains. Therefore, increased IspH activity within an enhanced DXP pathway strain (REM I7—11; described below) was achieved by over-expressing the ispH allele of Anabaena sp. PCC7120, generating test strain REM H8—12. Demonstrated in
REM I7—11 and REM H8—12 are derivatives of WW119. This strain was constructed by electoporation of Strain WW103 with plasmid pDW33 (see Example 30 for construction of WW119). WW119 exhibits improved DXP-flux, but generates similar isoprene levels to that of the previous parental strain CMP272; this is potentially due to a bottleneck in flux at the point of IspG. WW119 harbors two improvements over the CMP272 strain. These beneficial modifications include increased dxs expression and increased dxr expression and are described infra. REM I7—11 was generated by introducing GI1.6 fldA-ispG/pCL into WW119 and REM H8—12 was made by moving Ptac Anabaena ispH aspA term/pEWL454 into REM I7—11; both plasmids were incorporated into their corresponding host strain via electroporation transformation methods.
To verify/amplify the pgl locus of REM I1—9 the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I1—9 cells)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ Asel F-pgl pET-15b
1.25 ul primer (10 uM) 3′ BamHI R-pgl pET-15b
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 55° C.×30 sec., 72° C.×2 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification. A pgl+ verified clone was selected as REM I1—9
Amplification of the pEWL454 Fragment
To generate pEWL454 the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of pK184 w/aspA term vector (Gene Oracle, Inc.))
5 ul 10×Pfu Ultra II Fusion DNA polymerase
2.5 ul dNTP's (10 mM)
1.0 primer (10 uM) EL-1098
1.0 primer (10 uM) EL-1099
39.5 ul diH2O
+1 ul of Pfu Ultra II Fusion DNA polymerase from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×52 sec.]×29 cycles; 72° C.×3 min.,
4° C. until cool (MJ Research PTC-200 Peltier Thermal Cycler)
The resulting PCR fragment was separated on a 1.2% E-gel (Invitrogen) for verification of successful amplification.
Strain REM I1—9 description
The strain REM I1—9 was used to clone the Anabaena sp. PCC7120 ispH allele, which had been codon optimized for expression in E. coli (provided by Gene Oracle, Inc.). Surprisingly Gene Oracle, Inc. was unable to provide an E. coli strain harboring the desired clone. Therefore, strain REM I1—9 was used as a host to obtain the Ptac Anabaena ispH aspA term/pEWL454 clone of interest using a survival based strategy.
Strain REM I1—9 is derived from MD09-220 (BL21(DE3)PL.2 mKKDyI::FRT-ΔispH::FRT) and has been described previously. The FRT-neo-FRT-GI1.6-dxs locus of strain MCM625 was transduced into the genome of MD09-220 via standard P1 lystate/P1 transduction protocol (Thomason et al., 2007) and the resulting kanamycin resistant strain named REM C5—9. Using Gene Bridge's GmbH methods the antibiotic marker was looped out, generating strain REM H5—9. Subsequently, the pgl and galP region of MG1655 was transduced into strain REM H5—9 using standard P1 lystate/P1 transduction protocol (Thomason et al., 2007), and the cells selected for growth on M9 agar (Na2HPO4 6 g/L, KH2PO4 3 g/L, NaCl 0.5 g/L, NH4Cl 0.5 g/L, 0.1 mM CaCl2, 2 mM MgSO4, 1.5% agar) containing 0.4% w/v galactose and 500 uM mevalonic acid. The presence of the pgl locus in the galactose-utlizing, mevalonic acid-dependent, kanamycin sensitive cells was verified by PCR (see above) and one clone selected as REM I1—9 (BL21(DE3) PL.2 mKKDy/::FRT-ΔispH::FRT pgl FRT::GI1.6-dxs).
Cloning of the Anabaena sp. PCC7120 ispH Allele into pEWL454
Approximately 90 ng of a precut 5′ BamHI-3′ PstI purified DNA fragment harboring the Anabaena sp. PCC7120 ispH allele codon optimized for expression in E. coli (provided by Gene Oracle, Inc.) was ligated to precut 5′ BamHI-3′ PstI purified DNA vector backbone pEWL454 (provided by Gene Oracle, Inc.), harboring the tac promoter and aspA terminator sequences separated by a multiple cloning site (MCS) within a pK184 (Jobling and Holmes, 1990) derived plasmid, using T4 DNA Ligase (New England Biolabs) according to the manufacturer's suggested protocol. The aspA terminator sequences present in pEWL454 were synthesized by Gene Oracle, Inc. Using the PCR method outlined above, the lac promoter sequence present in pK184 was removed and the tac promoter and MCS harbored within pEWL454 was inserted using the oligos detailed above (Integrated DNA Technologies). The resulting PCR fragment was ligated using T4 DNA Ligase from New England Biolabs according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing kanamycin (50 ug/ml). A kanamycin resistant clone was selected, grown overnight in L broth containing kanamycin (50 ug/ml), and harvested for subsequent plasmid preparation. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Quintara; Albany, Calif.) using primers EL-1100, EL-1101, EL-1102, EL-1103, and EL-1104 and the correct pEWL454 clone identified, which has been designated as strain EWL454 (TOP10 w/pEWL454; pK184-derived cloning vector harboring Ptac-RBS-NdeI-SacI-MluI-NotI-XhoI-PstI-aspA terminator). A picture illustrating pEWL454 is shown in
Water-washed REM I1—9 cells were transformed with the ligation reaction via electroporation using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The cells were recovered in L broth plus 500 uM mevalonic acid (available commercially, for example, Sigma-Aldrich) for 1 hour at 37° C. and then plated on L agar containing kanamycin (50 ug/ml). Kanamycin resistant colonies that grew in the absence of mevalonic acid were selected, grown overnight in L broth containing kanamycin (50 ug/ml), and harvested for subsequent plasmid preparation; the presence of the Anabaena ispH allele relieved the cell's dependence on mevalonic acid for growth. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers EL-1105 and Neo.21 and the correct Ptac Anabaena ispH aspA term/pEWL454 clone identified, which has been designated as strain REM F5—12 (REM I1—9 w/Ptac Anabaena ispH aspA term/pEWL454; 5′ BamHI-3′ PstI synthetic fragment into 5′ BamHI-3′ PstI of pEWL454). A picture of the resulting Ptac Anabaena ispH aspA term/pEWL454 construct is shown in
REM I7—11 was constructed by transformation of GI1.6 fldA-ispG/pCL into WW119. The transformation was performed by electroporation using a BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). A plasmid preparation of GI1.6 fldA-ispG/pCL, generated from strain REM D9—11, was used; this strain and corresponding plasmid construct are described infra.
REM H8—12 was constructed by transformation of Ptac Anabaena ispH aspA term/pEWL454. The transformation was performed by electroporation using a BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). A plasmid preparation of Ptac Anabaena ispH aspA term/pEWL454 was made from strain REM F5—12.
Transformation of GI1.6 fldA-ispG/pCL into WW119 and Ptac Anabaena ispH aspA Term/pEWL454 into REM I7—11
To build the isoprene producing parental strain, REM I7—11, from which the test strain REM H8—12 is derived, the GI1.6 fldA-ispG/pCL plasmid was transformed by electroporation into WW119. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml). The resulting strain was designated REM I7—11.
REM I7—11 was then transformed by electroporation with Ptac Anabaena ispH aspA term/pEWL454. Transformants were recovered in L broth and plated on L agar containing spectinomycin (50 ug/ml), kanamycin (50 ug/ml), and carbenicillin (50 ug/ml). The resulting strain was designated REM H8—12.
C. Analysis of Test Strain REM H8—12 and the Parental Strain REM I7—11 for Growth, Isoprene Production, and DXP Metabolite Accumulation at Small Scale.The parental strain REM I7—11 was compared against the test strain REM H8—12 in a shake flask assay as well as in a DXP metabolite determination study. The increased benefit on isoprene production of the REM H8—12 strain harboring the Ptac Anabaena ispH aspA term/pEWL454 construct over the parental control strain REM 17—11 is depicted in
Strains REM I7—11 and REM H8—12 were grown at 30° C. in 2-5 ml cultures of TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 0.2 g yeast extract, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with 0.1% yeast extract and 1.0% glucose and including spectinomycin (50 ug/ml) and carbenicillin (50 ug/ml).). Induction of LacI-regulated gene expression was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a concentration of 500 uM. Growth was monitored periodically by recording each of the culture's optical density measured at 600 nm using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene ProductionIsoprene production was analyzed using a headspace assay. For the shake flask cultures, 200 ul of a culture was transferred from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (15 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 100 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minutes The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for 0.6 minute, the duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67. The detector was switched off from 0 to 0.42 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at approx. 0.49 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 5000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in assay vials for 30 min. incubation results in the following conversion of isoprene ug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(38)/(OD 600 nm of the culture).
DXP Metabolite AccumulationThe DXP metabolites of the isoprene-producing parental strain REM I7—11 and test strain REM H8—12 that are described above and depicted in
Metabolite Extraction: Processing Samples from Small-Scale Experiments.
To measure accumulation of metabolites in small-scale experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min at 7500×g, at −9° C. Immediately after centrifugation the supernatant was aspirated to a clean tube for analysis of excreted metabolites and 100 μL of dry ice-cold methanol was added to pelleted cells. The resulting samples were then stored at −80° C. until further processing.
To determine concentrations of excreted metabolites, 500 μL of methanol was added to 300 μL of the supernatant and the resulting mixture was centrifuged for 10 min at 20000×g at 4° C. to remove insoluble material before the LCMS analysis.
For metabolites extraction from the pellet (further referred as intracellular metabolites), 10 μL of water was added to methanol-containing samples, the pellet was resuspended in the resulting methanol/water mix and cell debris were spun down by 4-min centrifugation at 4500×g. The pellet was re-extracted two more times, first with 100 μL of 75% methanol buffered with 1 mM ammonium acetate in water (pH=8.0), then with 90 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and the supernatants from all three extractions were combined and analyzed by LCMS. During the extraction procedure, samples were kept on ice or in a refrigerated centrifuge whenever possible to minimize metabolites degradation.
Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
D. Analysis of Test Strain REM H8—12 for Growth, Isoprene Production, and DXP Metabolite Accumulation at 14-L Fermentation Scale.REM_H8—12 produced 2.6 g/L isoprene in 14-L fermentation (
Isoprene production from E. coli expressing genes from the DXP pathway and isoprene synthase, grown in fed-batch culture at the 15-L scale.
1000× Modified Trace Metal Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
Mercury Vitamin Solution (Per Liter):Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0 g. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100% Foamblast 10 g. All components were mixed together and autoclaved. Macro Salt Solution 3.4 mL, 1000× Modified Trace Metal Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added after the solution had cooled to 25° C.
Macro Salt Solution (Per Liter):MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate 49.6 g. All components were dissolved in water, q.s. to volume and filter sterilized with 0.22 micron filter.
Fermentation was performed in a 15-L bioreactor with E. coli BL21 cells overexpressing the first enzyme in the dxp pathway (GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), the E. coli ispG and fldA genes (GI1.6 fldA-ispG/pCL), and truncated isoprene synthase from P. alba (pDW33) and containing a restored 17,257 bp chromosomal galM-containing region derived from MG1655 (strain name REM H8—12). This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 34° C. A frozen vial of the E. coli strain was thawed and inoculated into tryptone-yeast extract medium for the bioreactor. After the inoculum grew to optical density 1.0, measured at 550 nm (OD550), 500 mL was used to inoculate a 15-L bioreactor and bring the initial tank volume to 5 L.
The feed solution was fed at an exponential rate until a top feed rate of 5.8 g/min was reached. After this time, the glucose feed was fed to meet metabolic demands at rates less than or equal to 5.8 g/min. The total amount of glucose delivered to the bioreactor during the 44 hr fermentation was 4.4 kg. Induction was achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). A single shot of IPTG was added to the tank to bring the concentration to 300 uM when the cells were at an OD550 of 7.
The isoprene level in the off-gas from the bioreactors was determined using a Hiden mass spectrometer. The isoprene titer increased over the course of the fermentation to a maximum value of 2.6 g/L at 44 hr.
Equation for calculating Isoprene Titer: ∫(Instantaneous isoprene production rate, g/L/hr)dt from t=0 to 84 hrs [=] g/L broth
F. DXP Metabolite Determination Metabolite Extraction: Processing 14-L Fermentor Samples.Cell metabolism was rapidly inactivated by withdrawing several milliliters of the fermentor culture into a pre-weighted tube filled with 9.0 mL of dry ice-cold methanol. The resulting sample was weighted again to calculate the amount of withdrawn cell culture and then put to −80° C. for storage until further analysis. In order to extract metabolites, 500 μL of methanol-quenched fermentation sample was spun down by centrifugation for 4 min at 4500×g, at −9° C. The pellet was then re-extracted twice, first with 350 μL of 85% methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and then with 350 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris was pelleted by centrifugation and all three supernatants were pooled together for further analysis.
Metabolite QuantitationExtracted metabolites were analyzed by LC-ESI-MS/MS on a Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.). The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following m/z values for precursor ions were selected to detect the metabolites of interest in SRM mode: 213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0 for CDP-MEP. Concentrations of metabolites were determined based on integrated intensities of peaks generated by PO3− product ion (m/z=79.0) using calibration curves obtained by injection of corresponding standards (Echelon Biosciences Inc). The concentration of CDP-MEP was expressed in arbitrary units because of the unavailability of commercial standard. Intracellular concentrations of metabolites were calculated based on a standard assumption that in 1 mL of the culture at OD=200 the integrated volume of all cells is 50 μL.
Example 30 Discovery of Apparent Biochemical Feedback Inhibition of Dxr and Alleviation of Negative Effects ThereofWe made the surprising observation that in a DXP strain production of isoprene was shut off while cells were still in a vigorous growth phase. In addition these cells also accumulate1-deoxyxylulose-5-phosphate, the substrate for Dxr. Without being bound by theory, one possible hypothesis to explain this observation is that the pathway is subject to regulation either at the genetic level or at the biochemical level. Jawaid et. al., PLoS One, 4(12):e8288 (2009) reported that a fraction of Dxr protein from Francisella tularensis was phosphorylated at ser177 when overexpressed in E. coli. This phosphorylation was presumed to inactivate the protein based on the observation that the mutations S177D and S177E led to inactive protein. We subsequently showed that purified Dxr from E. coli is inactivated when incubated with dimethylallyl diphosphate (DMAPP) or 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP). Further, an E. coli strain with a genetically modified deoxyxylulose phosphate (DXP) pathway was shown to accumulate DMAPP and/or HMBPP to levels higher than that observed in wild type. Without being bound by theory, based on the result of in vitro inactivation of Dxr and in vivo metabolite accumulation observed in the engineered DXP pathway strain, we postulate that the shut down of the pathway and the accumulation of 1-deoxyxylulose-5-phosphate is due to the in vivo inactivation of Dxr in the engineered strain. We discovered that shut down of the pathway in engineered strains is prevented by rebalancing pathway enzymes and maintaining levels of HDMAPP and DMAPP at concentrations below 1 to 2 mM DMAPP and 1 to 2 mM HDMAPP. These observations are exemplified in
REM I7—11—This strain arose from the modification of CMP271 detailed infra. CMP271 was transduced with P1 lysate MCM754, obtained as described below, harboring a modified PL.6 promoter (DNA seq.#1) replacing the native promoter in front of the dxs gene.
FRT-neo-FRT PL.x(Trimmed) Integrated at dxs.gb DNA Seq.#1 Sequence Includes Upstream FRT to and Including ATG of dxs
Looping out the associated antibiotic marker according to Gene Bridges instructions yielded strain WW102. This strain was additionally transduced with P1 lysate MCM755 harboring a promoter named gi1.6 (DNA seq.#2).
FRT-neo-FRT-gi1.x-d×r Region BL21.gb DNA seq#2; Sequence Includes Upstream FRT to and Including ATG of dxr
This promoter was targeted to replace the native promoter of the dxr gene. Looping out the antibiotic marker according to Gene Bridges instructions yielded strain WW103. Strain WW103 was transformed by electroporation with plasmid pDW33 (Example 24 Part C) providing ispS, the isoprene synthase expression cassette and the resultant strain is designated WW119.
B. Detailed Strain Construction Protocols Construction of Strain CMP271 Construction of StrainConstruction of P1 lysates MCM754 and MCM755 are detailed below:
PL.6(trim)-dxs
PCR reactions were carried out in quadruplicate using the Herculase II Fusion Kit (Stratagene).
35 uL ddH2O
10 uL 5× buffer
1.25 uL 10 uM primer MCM320, (gel purified)
1.25 uL 10 uM primer MCM347, (gel purified)
0.5 uL dNTPs
1 uL polymerase
1 uL FRT-PGK-gb2-neo-FRT template DNA, GeneBridges Cat. No. K006
Reactions were cycled as follows:
95 C×2 min followed by (95 C×15 sec; 55 C×15 sec; 72 C×1 min)×30 cycles 72 C×3 min 30 sec 4 C until cold.
gi1.6-dxr
Four PCR reactions were carried out in using the Herculase II Fusion Kit (Stratagene). Reactions varied by the presence or absence of 2 uL DMSO and an annealing temperature of 55 C or 60 C.
35 uL ddH2O
10 uL 5× buffer
1.25 uL 10 uM primer MCM321, IDT (gel purified)
1.25 uL 10 uM primer MCM337, IDT (gel purified)
0.5 uL dNTPs
1 uL polymerase
1 uL FRT-PGK-gb2-neo-FRT template DNA, GeneBridges Cat. No. K006
Reactions were cycled as follows:
95 C×2 min followed by (95 C×20 sec; 55 C or 60 C×20 sec; 72 C×1 min)×30 cycles 72 C×3 min; 4 C until
For each amplicon, four reactions were pooled and purified using a QIAquick PCR Purification kit (Qiagen) PCR column, eluting in 30 uL EB.
D. Integration of Amplicon onto Chromosome
Strain MCM327 (BL21) carrying pRedET-carb (GeneBridges) was grown in L broth (LB) containing carbenicillin (50 ug/ml) at 30 C overnight and then diluted 1:100 into fresh LB+carb50 and cultured at 30 C for 2 hr. 130 uL of 10% arabinose was added and cells cultured at 37 C for approximately 2 hours. Cells were prepared for electroporation by washing 3× in one half culture volume iced ddH2O and resuspended in one tenth culture volume of the same. 100 uL of cell suspension was combined with 3 uL DNA amplicon in a 2 mm electroporation cuvette, electroporated at 25 uFD, 200 ohms, 2.5 kV, (Gene Pulser MXcell; BioRad) and immediately quenched with 500 uL LB. Cells were recovered shaking at 37 C for 1-3 hrs and then transformants selected overnight on L agar (LA) plates containing kanamycin (10 ug/ml) at 37 C.
Single colonies arising from transformations with each DNA amplicon were patched to LA+kan50 and grown overnight at 37 C. Clones were inoculated into 5 mL LB+kan10, grown to an OD600˜1 and then frozen by mixing 1 mL 50% glycerol and 0.5 mL culture, placing on dry ice until solid, and then storing at −80 C. These manipulations resulted in strain MCM754 [PL.6(trim) dxs] and strain MCM755 (gi1.6 dxr).
The integrated promoters were amplified for sequencing by colony PCR using the Herculase II Fusion kit (Stratagene).
35 uL ddH2O
10 uL 5× buffer
1.25 uL 10 uM primers GB-DW
1.25 uL 10 uM primer MCM327 (dxs) or MCM330 (dxr)
0.5 uL dNTPs
1 uL polymerase
Colony scraping
Reactions were cycled as follows:
95 C for 2 min; (95 C for 20 sec; 55 C for 20 sec; 72 C for 30 sec)×30 cycles; 72 C 3 min; 4 C until cold
PCR products were sequenced (Quintara Biosciences) following treatment by ExoSAP.
E. Preparation of P1 Lysates from Strains MCM754 and MCM755.
100 uL of respective overnight cultures (LB+kan 10) were diluted into 10 mL LB+0.2% glucose+5 mM CaCl2, and grown with shaking at 250 rpm, 37 C. After 30 min., 100 uL of a generic P1 lysate from MG1655 was added and the culture returned to the shaker for ˜3 hours. The lysed culture was transferred to a 15 mL tube, 200 uL chloroform added, and it was vortexed for 30 sec. The sample was centrifuged at 4500 g for 10 min and then the aqueous supernatant transferred to a fresh 15 mL tube. 200 uL chloroform was added and the lysate stored at 4 C.
F. Cloning and Purification of the Enzyme.Dxr from E. coli was cloned and purified by methods well known to those of skill in the art. The gene was inserted into the pET15b vector as described by the vendor to include a N-terminal His tag sequence (Invitrogen, Carlsbad, Calif.). A BL21(λDE3) E. coli culture harboring the plasmid and expressing the protein was harvested, the cell pellet lysed in a French pressure cell and protein was purified using a Ni-NTA column following the protocol recommended by the manufacturer (GE Healthcare, Pittsburgh, Pa.).
G. Dxr Inactivation by Incubation with DMAPP and HDMAPP.
The purified protein, 5 uM, was incubated at several concentration of DMAPP or HMBPP (Echelon Bioscience, Salt Lake City, Utah) in buffer consisting of 100 mM Tris, 100 mM NaCl pH 8, 5 mM MgCl2, 0.2 mM NADPH, 0.2 mM DXP, and 250 nM DXR. D at 37° C. for two hour in a total volume of 50 uL. Dxr activity was measured periodically according to standard assay, see, e.g., Koppisch et al, Biochemistry, 41:236-43 (2002) with a 20-fold dilution of the inactivation reaction mixture. Control incubations and assays of the enzyme were conducted under similar conditions in the absence of DMAPP or HMBPP in the inactivation reaction. Where appropriate additional control activity assays were conducted in the presence of a 20-fold diluted concentration of inactivators (DMAPP or HMBPP). A larger aliquot of enzyme (about 400 ug) was inactivated similarly with DMAPP for analysis by mass spectroscopy to verify the anticipated amino acid residue modification. As shown in
Comparison of the energetics and carbon utilization efficiency for the DXP pathway and the MVA pathway reveal that the DXP pathway is more efficient in carbon utilization but less efficient in redox balance than the MVA pathway. When glucose is the carbon source stoichiometric yield on carbon of the DXP pathway is about 85% (grams of isoprene produced per grams of glucose utilized). The energy balance of the DXP pathway is less efficient when compared to the MVA pathway. For DXP glucose to isoprene suffers a shortage of 3 moles of NAD(P)H per mole of isoprene formed and is minus 2 moles of ATP. For the similar comparison of glucose to isoprene via MVA this pathway produces an excess of 4 moles of NAD(P)H; ATP is balanced, however, the carbon utilization efficiency is only about 55%. Without being bound by theory, a more balanced and more efficient production host can be made by combining the two pathways in a single host to optimize redox chemistry and efficiency of carbon utilization.
In this example, we provide evidence consistent with that combination of the two pathways in a single host can be established in practice. Combination of the two pathways should lead to an improved process. A series of cultures comprising two strains, REM H8—12 and REM I7—11, described above, were set up in a 48-deep-well plate (cat# P-5ML-48-C-S Axygen Scientific, California, USA) with each well providing a 2 mL culture. The media, named TM3, is described below. The two strains were grown overnight at 30 degrees Celsius at 250 rpm in TM3 medium supplemented with 1% glucose and 0.1% yeast extract. In the morning, the two strains were inoculated into the 48-deep well block in replicate. The TM3 medium was supplemented with 1% [U-13C]-glucose and 0.1% yeast extract. The cultures were shaken at 30 degrees C. at 600 rpm (Shel-Lab Inc. Model SI6R Refrigerated Shaking Incubator; Oregon, USA). Culture OD was determined after two hours and then at timed intervals out to 4.25 hours. The cultures were induced at two hours of growth by the addition of 400 uM IPTG. After one hour of induction the cultures of each strain also received from 0 to 8 mM (R)-mevalonic acid [cat#; Sigma M4667]. At timed intervals a 100 uL aliquot of each culture was transferred to a 98-deep well glass block (cat#3600600 Zinsser; North America) which was immediately sealed with an impermeable adhesive aluminum film and incubated for 30 minutes with shaking at 450 rmp on an Eppendorf thermomixer (Eppendorf; North America.). The cultures were killed by heating at 70 degrees C. for 7 minutes on a second Eppendorf thermomixer. The glass block was transferred to an Agilent 6890 GC attached to an Agilent 5973 MS and outfitted with a LEAP CTC CombiPAL autosampler for head space analysis. The column was an Agilent HP-5 (5% Phenyl Methyl Siloxane (15 m×0.25 mm×0.25 um)). A 100 uL gas volume was injected on the column. Other conditions were as follows. Oven Temperature: 37 C (held isothermal for 0.6 mins); Carrier Gas: Helium (flow −1 mL/min), split ratio of 50:1 at 250° C. on the injection port; Single Ion Monitoring mode (SIM) on mass 67 or 73; Detector off: 0.00 min-0.42 mins; Dectector on: 0.42 mins-0.60 mins; elution time for Isoprene (2-methyl-1,3 butadiene) was ˜0.49 min for a total analysis time of 0.6 mins. Calibration of the instrument was performed by methods well known to those of skill in the art.
Isoprene head space measurements were normalized by culture OD600 to yield a measure of specific isoprene production in units of ug/L/H/OD. All reactions were followed for 4 hours.
K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 1.0 g, 1000× Modified Trace Metal stock solution 1 ml. All of the components were added together and dissolved in Di H2O. The pH is adjusted to 6.8 with NH4OH and the solution is filter sterilized over a 0.22 micron membrane. Antibiotics were added post-sterile as needed. U-13C-Glucose and [R]-mevalonic acid were added post sterile as indicated.
1000× Modified Trace Metal Stock Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
Example 32 Demonstration of Isoprene Generated by Strain REM A2—17 Via Dual Isoprenoid Biosynthetic PathwaysDescribed here is the construction of an isoprene producing E. coli strain that harbors both an exogenous MVA isoprenoid biosynthetic pathway and an enhanced DXP biosynthetic pathway. Data presented here indicates that isoprene produced by strain REM A2—17 is derived from both types of isoprenoid biosynthetic pathways simultaneously. For this particular example, roughly 3:2 to 1:1 MVA-flux:DXP-flux contributions to isoprene production were observed; see
The genomic insertions described in this example were carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21 (Novagene) was used. P1 lysate preparations and transductions were performed as previously described (Thomason et al., 2007). The pBBR1MCS-5 vector has been described (Kovach et al., 1994) as have vector constructs MCM82, pMCM296, pDW34, pDW33, GI1.6 fldA-ispG/pCL, and Ptac Anabaena ispH aspA term/pEWL454 (see, e.g., Example 29 above and WO 2009/076676). MCM82 contains the pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS). The Trc promoter, Trp promoter and aspA terminator sequences were obtained from the information provided by NCBI www.ncbi.nlm.nih.gov and EcoCyc www.ecocyc.org.
Construction of pDW15 (Ptrc-Upper MVA Pathway on pBBR1MCS-5)
To insert the upper MVA pathway onto the pBBR1MCS-5 vector, the entire expression cassette containing Ptrc, mvaE, mvaS, and the rrn terminator was amplified by PCR from MCM82 using the primers Upper5′XhoI and Upper3′XbaI. See below for PCR primer sequences (Table 9), reaction and cycling parameters. The approximately 4.2 kb PCR product was confirmed by gel electrophoresis (E-Gel, Invitrogen) and then purified using QiaQuick purification columns (Qiagen) according to the manufacturers recommended protocol. Purified PCR product and the pBBR1MCS-5 vector were then treated with XbaI and XhoI restriction endonucleases overnight at 37° C. See below for reaction conditions. The next day, reactions were heated to 65° C. to deactivate restriction enzymes prior to ligation. Ligation reactions (see below for conditions) were carried out at 4° C. overnight. Approximately 5 μl of the ligation reactions were transformed into chemically competent E. coli TOP10 cells (Invitrogen) according to the manufacturer's recommended protocol, recovered at 37° C. in LB for 1 hour, and then plated onto LB plates containing X-gal and Gentamicin at 10 μg/ml. Colonies displaying no β-galactosidase activity were selected for further analysis by PCR using primers M13 Reverse and MCM163 to confirm the presence of the insert. The plasmid from one of these colonies was purified (Qiagen) and completely sequenced (Quintara Biosciences, see Table 9 for primer sequences) to verify that it contained the complete upper MVA pathway expression cassette in the correct orientation. The sequence and map of pDW15 is listed below and in
-
- 1 μl MCM82 (approx. 30 ng)
- 10 μl 5× Herculase Buffer (Stratagene)
- 0.5 μl dNTPs (100 mM)
- 1 μl Upper5′XhoI (20 uM)
- 1 μl Upper3′XbaI (20 uM)
- 35.5 μl diH2O
- 1 μl Herculase DNA Polymerase (Stratagene)
1. 95° C. 4 min.
2. 95° C. 20 min, 52° C. 20 sec., 72° C. 4 min., 5×
3. 95° C. 20 min, 55° C. 20 sec., 72° C. 4 min., 25×
4. 72° C. 10 min,
5. 4° C. until cool
DNA Digestion:
-
- 6 μl diH2O
- 2 μl 10× Buffer H (Roche)
- 10 μl DNA (pBBR1MCS-5 or PCR insert)
- 1 μl XhoI (Roche)
- 1 ulXbaI (Roche)
1. 37° C. overnight
2. 65° C. 20 min (heat kill)
Ligation:
-
- 2 μl diH2O
- 1 μl 10× ligase buffer (NEB)
- 1 μl T4 DNA ligase (NEB)
- 2 μl vector (pBBR1MCS-5)
- 4 μl insert (upper MVA expression cassette)
1. 4° C. overnight
2. microdialyze (Millipore) and transform into competent E. coli (Invitrogen)
Construction of PTrp mMVK/pDW15
Amplification of the PTrp mMVK Fragment
PCR Reaction for PTrp mMVK
0.5 ul vector template pDW34
0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ phos Ptrp 5′ mMVK
1.25 ul primer (10 uM) 3′ phos aspA term 3′ mMVK
36 ul diH2O
+0.5 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×2 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 0.8% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions. The resulting purified stock is referred to as PTrp mMVK; note the primers used contained 5′ phosphorylated ends.
Cloning of the PTrp mMVK fragment into pDW15
Approximately 500 ng of the pDW15 plasmid was digested with SfoI (New England Biolabs) according to the manufacturer's specifications. The SfoI cut vector was then dephosphorylated using rAPpid Alkaline Phosphatase (Roche) according to the manufacturer's suggested protocol. The digested/dephosphorylated DNA was cleaned using the Qiagen QiaQuick Gel Extraction Kit prior to ligation. A portion of the PTrp mMVK fragment (5′ ends phosphorylated) was ligated to the cleaned/SfoI cut/dephosphorylated pDW15 plasmid using T4 DNA Ligase from New England Biolabs according to the manufacturer's suggested protocol. Chemically competent TOP10 cells (Invitrogen) were transformed with the ligation reaction using a standard heat-shock protocol (Sambrook et al., 1989), recovered in L broth for 1 hour at 37° C. and then plated on L agar containing gentamicin (10 ug/ml). Gentamicin resistant colonies were selected, grown overnight in L broth containing gentamicin (10 ug/ml), and harvested for subsequent plasmid preparation. Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were sequenced (Sequetech; Mountain View, Calif.) using primers 5′ mMVK seq prim, 3′ mMVK seq prim, EL1003, and MCM 177, and the correct PTrp mMVK/pDW15 clone identified; the resulting clone of interest has been designated as strain REM H9—14 (TOP10 w/PTrp mMVK/pDW15; SfoI site destroyed with PTrp mMVK inserted in the orientation as the Ptrc mvaE-mvaS operon present in the construct; see
Construction of Integration Construct pMCM900
The GI1.6 promoter and yeast MVK gene of pMCM296 were replaced with a chloramphenicol resistance cassette and Trc promoter. The cmR resistance cassette-Ptrc fragment was created by amplification from pMCM883 (GeneBridges cmR cassette) using primers MCM127 and MCM375. 2, 50 uL reactions were created according the manufacturer's protocol for Herculase II Fusion (Agilent #600679) containing 35 uL water, 10 uL buffer, 0.5 uL dNTPs, 1.25 uL each primer at 10 uM, 1 uL plasmid template, 1 uL polymerase. Reactions were cycled as follows: 95° C., 2:00; 30× (95° C., 0:20; 55° C., 0:20; 72° C., 1:00); 72° C., 3:00; 4° C. until cold.
The ˜1.6 kb amplicon and plasmid pMCM296 (described infra) were digested at 37° C. for 2 hour in 10 uL reactions containing 5 uL DNA, 1 uL EcoRV, 1 uL NotI (amplicon) or 1 uL StuI (pMCM296), 1 uL Roche Buffer H, and 2 uL ddH2O. Reactions were heat-killed at 65° C. for 2 hr then digested DNA was purified on Qiagen PCR columns and eluted in 30 uL EB. The eluted DNAs were ligated 1 hr at room temperature in a 10 uL Roche Rapid Ligation kit reaction containing 1 uL pMCM296, 3 uL cut amplicon, 5 uL buffer 3, and 1 uL ligase. Ligated DNA was transformed into Invitrogen Pir1 chemically competent cells, recovered for 1 hr at 37° C., plated on LB/cmp 25 ug/mL, then grown overnight at 37° C. The resulting plasmids were purified and sequenced across the promoter region. Clone four was frozen as pMCM900; see
Integration of cmR-Ptrc-KDyI into Host BL21 t pgl to Create MCM928
Strain MCM865 is an aliquot of strain MD253 (BL21 t pgl pRedET-carb). MCM865 was grown in LB+carb50 at 30° C. overnight and then diluted 1:100 into fresh LB+carb50 and cultured at 30° C. for 2 hr. 130 uL 10% arabinose was added and cells cultured at 37° C. for approximately 2 hours. Cells were prepared for electroporation by washing 3× in one half culture volume iced ddH2O and resuspended in one tenth culture volume of the same. 100 uL of cell suspension was combined with 1 uL pMCM900 DNA in a 2 mm electroporation cuvette, electroporated at 25 uFD, 200 ohms, 2.5 kV, and immediately quenched with 500 uL LB. Cells were recovered shaking at 37° C. for 1-3 hrs and then transformants selected overnight on LB cmp5 plates at 37° C.
After restreaking on LB cmp5, transformants were tested for growth on LB cmp5, LB kan10 and LB carb50. A cmpR/carbS/kanS clone was frozen as MCM928.
The chloramphenicol marked PTrc PMK-MVD-yIDI locus of strain MCM928, described above, was introduced into strain WW103 (see, e.g., Examples 29 and 30) via P1-mediated transduction. The resulting chloramphenicol resistant strain was named REM H4—15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, CMP::PTrc PMK-MVD-yIDI).
Strategy for Creating REM A2—17REM A2—17 was created by subsequent plasmid transformations of pDW33, PTrp mMVK/pDW15, Ptac Anabaena ispH aspA term/pEWL454, and lastly GI1.6 fldA-ispG/pCL initially into strain REM H4—15.
Water-washed REM H4—15 cells were transformed with pDW33 via electroporation using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The cells were recovered in L broth for 1 hour at 37° C. and then plated on L agar containing carbenicillin (50 ug/ml). One carbenicillin resistant colony was chosen, named REM A4—16, and subsequently transformed with PTrp mMVK/pDW15 via the method described; in this case L agar containing carbenicillin (50 ug/ml) and gentamicin (10 ug/ml) was used as a selection, resulting in the carbenicillin and gentamicin resistant strain REM I4—16. Similarly, REM I4—16 was transformed with Ptac Anabaena ispH aspA term/pEWL454 resulting in the carbenicillin (50 ug/ml), gentamicin (10 ug/ml) and kanamycin (50 ug/ml) resistant strain REM C5—16. Lastly, strain REM C5—16 was transformed with GI1.6 fldA-ispG/pCL resulting in the carbenicillin (50 ug/ml), gentamicin (10 ug/ml) kanamycin (50 ug/ml), and spectinomycin (50 ug/ml) resistant strain REM A2—17.
Example 33 Analysis of Strain REM A2—17 in the Presence and Absence of Fosmidomycin for Growth, Isoprene Production, and DXP and MVA Metabolite Accumulation Using Unlabeled, 1-13C Labeled, or 3-13C Labeled Glucose as the Sole Carbon SourceIt was previously determined that the addition of 1 mM fosmidomycin to the growth media of an E. coli BL21 strain harboring the GI1.6-dxr locus common to the REM A2—17 strain could inhibit isoprene production to an undetectable level. Fosmidomycin inhibits the activity of the DXR enzyme that performs the committed step of the endogenous E. coli DXP pathway (Kuzuyama et al., 1998). Furthermore, the addition of 1 mM fosmidomycin to the growth media of a dxr null E. coli BL21 strain that harbors the same heterologous MVA isoprenoid biosynthetic pathway enzymes present in REM A2—17 was found to maintain the same level of isoprene production as that grown in the absence of fosmidomycin. This data indicates that the DXR inhibitor (fosmidomycin) does not adversely affect in vivo flux through the MVA isoprenoid biosynthetic pathway.
Specific Productivity of Isoprene Generated by REM A2—17 Strain.2 mM fosmidomycin in combination with 1-13C (Isotec) or 3-13C glucose (Omicron Biochemicals, Inc) was used in small scale headspace assays and corresponding DXP and MVA metabolite determination studies to demonstrate the simultaneous flux to isoprene via the dual MVA and DXP isoprenoid biosynthetic pathways expressed within REM A2—17. See below for the rationale of using 1-13C glucose and 3-13C glucose to generate uniquely labeled isoprene derived from the DXP pathway that can be differentiated from the isoprene generated via the MVA pathway. Shown in
The dual flux of carbon to isoprene down the MVA and DXP isoprenoid biosynthetic pathways harbored by REM A2—17 was further support by use of tryptophan to repress expression of the MVK enzyme common to the MVA pathway (see
To demonstrate that in the REM A2—17 strain isoprene is produced by the DXP and MVA pathways operating simultaneously, the above strain was grown on glucose containing 13C isotope at specific positions. As illustrated in
The distribution of cumomers of isoprene produced exclusively via the MVA pathway by REM A2—17 cells grown on 1-13C or 3-13C glucose was estimated by measuring GC spectra of isoprene emitted in the presence of 2 mM fosmidomycin (
Strains REM A2—17 was grown at 34° C. in TM3 liquid media (13.6 g K2PO4, 13.6 g KH2PO4, 2.0 g MgSO4*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2SO4, 1.0 ml 1000× Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H2O, and filter sterilized) supplemented to a final concentration with either 1% unlabeled glucose and 0.1% yeast extract (
Isoprene production was analyzed using a headspace assay. For the headspace cultures, 100 uL to 200 ul of the cultures was transferred from the shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After approx. 30 min. to 1 hour the vials were removed from the incubator, heat killed at 70° C. for 7 min., and analyzed. The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS GC/MS column (15 m×0.25 mm; 0.25 μm film thickness) was used for separation of analytes. The sampler was set up to inject 100 μL of headspace gas. The GC/MS method utilized helium as the carrier gas at a flow of 1 ml/minute. The injection port was held at 250° C. with a split ratio of 50:1. The oven temperature was held at 37° C. for 0.6 minute, the duration of the analysis. The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on m/z 67 or in a full scan mode covering m/z from 25 to 80. The detector was switched off from 0 to 0.42 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) standard (SCOTTY® Analyzed Gases) was observed to elute at approx. 0.49 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 μg/L to 5000 μg/L. The limit of detection was estimated to be 50 to 100 ng/L using this method. The specific productivity of each strain is reported as ug/L OD Hr. Note, ratio of 1800 ul headspace:200 ul broth in assay vials for 1 hour incubation results in the following conversion of isoprene ug/L of culture to specific productivity: (isoprene/L determined by GC-MS)×(9)/(OD 600 nm of the culture). To quantify the amount of isoprene produced from 13C-labeled glucose, the concentration obtained based on the calibration curve with the non-labeled standard was multiplied by the conversion factor K to compensate for isotopic effects. The conversion factors were calculated as
K=(Σ(Ai)/A67)/(Σ(Pi)/P67), (Eq. 1)
where Ai are the measured intensities of GC peaks produced by 13C-enriched isoprene and Pi are the measured intensities of GC peaks produced by the isoprene standard (subscript indices i=60 . . . 72 indicate m/z values of corresponding peaks, which include peaks A67 and P67). For the experiments referred to in this document the conversion factors of 2.901 and 3.369 were applied to no fosmidomycin and to 2 mM fosmidomycin conditions, respectively, for cells grown on 1-13C glucose and the factors of 1.476 and 1.315 were applied to no fosmidomycin and to 2 mM fosmidomycin conditions, respectively, for cells grown on 3-13C glucose.
LC-MS/MS Analysis of Cellular MetabolitesFor metabolite analysis 1.5 to 5 mL of cell culture was spun down by centrifugation and 100 or 150 uL of dry ice-cold methanol was added to pelleted cells after the centrifugation. The resulting samples were then stored at −80° C. until further processing. To extract cellular metabolites, 10 or 15 μL of water was added to methanol-containing samples, the pellet was resuspended in the resulting methanol/water mix and then cell debris were spun down for 4-min at 4500×g. The pellet was re-extracted two more times, first with 90 μL of 75% methanol buffered with 1 mM ammonium acetate in water (pH=8.0), then with 100 μL of 50% methanol in the ammonium acetate buffer. After each extraction, cell debris were spun down by centrifugation and the supernatants from all three extractions were combined. During the extraction procedure, samples were kept on ice or in a refrigerated centrifuge whenever possible to minimize metabolites degradation.
The extract was analyzed by LC-MS/MS on a TSQ Quantum triple quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, Calif.) using electrospray ionization in the negative mode. The system control, data acquisition, and mass spectral data evaluation were performed using XCalibur and LCQuan software (Thermo Electron Corp). LC separation was done on a Synergi 45 μM Hydro-RP HPLC column (150×2 mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column temperature of 40° C. The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM tributylamine/15 mM acetic acid in water and solvent B was LCMS-grade methanol. The sample injection volume was 10 to 25 μL.
Mass detection was carried out using electrospray ionization in the negative mode. The following MS/MS transitions were chosen to detect the metabolites of interest: 213→79 for DXP, 215→79 for MEP, 245→79 for IPP and DMAPP, 261→79 for HDMAPP, 277→79 for cMEPP, 520.1→79 for CDP-ME, 227→79 for MVP, 307→79 for MVPP, and 147→59 for MVA. Other mass spec settings were optimized to obtain the highest sensitivity using corresponding standards purchased from Echelon Biosciences Inc. or synthesized in house. To quantify the absolute concentrations of cellular metabolites a calibration table was constructed by injecting the known amounts of these standards. Note that the LC-MS/MS method that was used for metabolite analysis does not discriminate between structurally similar IPP and DMAPP, therefore their amount in samples was determined as a sum of concentrations of the two compounds.
Cumomer distribution analysis for cMEPP was done by calculating relative intensities of peaks arising from 277→79, 278→79, 279→79, 280→79, 281→79 and 282→79 MS/MS transitions corresponding to M0, M+1, M+2, M+3, M+4, and M+5 cumomers of this metabolite. In a separate experiment it has been verified that at t z 14.3 min (the retention time of cMEPP) extracts from E. coli cells grown on a regular glucose do not generate detectable peaks with MS/MS transitions 272→79, 273→79, 274→79, 275→79, 276→79. These control measurements exclude the possibility that compounds potentially co-eluting with cMEPP but having slightly lower molecular weight can contribute to the MS/MS peaks generated by cMEPP when cells are grown on 13C-enriched glucose.
Cumomer Analysis of 13C Labeled IsopreneTo measure 13C enrichment of isoprene emitted by cells grown on 13C-glucose, GC spectra were monitored from m/z 58 to m/z 68, i.e. over the range of mass to charge ratios that can originate from five-carbon isoprene derivatives.
The data shown in
P60=1.00000*k60
P61=0.05561*k60+1.00000*k61
P62=0.01238*k60+0.05561*k61+1.00000*k62
P63=0.01238*k61+0.05561*k62+1.00000*k63
P64=0.01238*k62+0.05561*k63+1.00000*k64
P65=0.01238*k63+0.05561*k64+1.00000*k65
P66=0.01238*k64+0.05561*k65+1.00000*k66
P67=0.01238*k65+0.05561*k66+1.00000*k67
P68=0.01238*k66+0.05561*k67+1.00000*k68
P69=0.01238*k67+0.05561*k68+1.00000*k69
P70=0.01238*k68+0.05561*k69+1.00000*k70 (Eqs. 2),
where P60 . . . P70 are the measured intensities of GC peaks produced by the isoprene standard (subscript indices indicate m/z values of corresponding peaks), k60 . . . k70 are the calculated intensities of GC peaks that would be generated by all-12C5 isoprene (subscript indices indicate m/z values of corresponding peaks), and the coefficients 1.00000, 0.05561, and 0.05561 are the estimated relative abundances of three C5 cumomers containing zero, one or two 13C isotopes per molecule assuming that this C5 compound has natural abundance of 13C isotope equal to 1.1%. (Note that in our calculations of all-12C5 isoprene spectrum it was assumed that the natural abundance of deuterium is too small to affect the final results). The positive values of k60 . . . k70 were obtained using lsqlin solver (MATLAB 7.0, MathWorks). The calculated values of k69 and k70 were effectively zero indicating that GC spectrum of all-12C5 isoprene should not have any peaks with m/z=69 and higher.
Cumomer distribution analysis of labeled isoprene samples was done according to the following set of linear equations based on the values of k62 . . . k68 obtained as described above:
A67=k67*χM0+k66*χM+1+k65*χM+2+k64*χM+3+k63*χM+4+k62*χM+5
A68=k68*χM0+k67*χM+1+k66*χM+2+k65*χM+3+k64*χM+4+k63*χM+5
A69=k68*χM+1+k67*χM+2+k66*χM+3+k65*χM+4+k64*χM+5
A70=k68*χM+2+k67*χM+3+k66*χM+4+k65*χM+5
A70=k68*χM+3+k67*χM+4+k66*χM+5
A71=k68*χM+4+k67*χM+5
A72=k68*χM+5 (Eqs. 3),
where A67-A72 are the measured intensities of GC peaks produced by 13C-enriched isoprene (subscript indices indicate m/z values of corresponding peaks) and χM0 . . . χM+5 are the relative abundances of isoprene cumomers having from zero to five 13C atoms (χM0 corresponds to the isoprene molecules in which all carbons atoms are represented by the isotope 12C). The non-negative values of χM0 . . . χM+5 were obtained using the isqlin solver (MATLAB 7.0, MathWorks).
Determination of Relative Contribution of DXP and MVA Pathways to the Isoprene ProductionThe relative contribution of DXP and MVA pathways to the total isoprene production (φDXP and φMVA, respectively) was estimated by solving in MATLAB (MathWorks) the following overdetermined system of linear equations:
χMO,Isp−FM=φDXP*χMO,cMEPP+φMVA*χMO,Isp+FM
χM+1,Isp−FM=φDXP*χM+1,cMEPP+φMVA*χM+1,Isp+FM
χM+2,Isp−FM=φDXP*χM+2,cMEPP+φMVA*χM+2,Isp+FM
χM+3,Isp−FM=φDXP*χM+3,cMEPP+φMVA*χM+3,Isp+FM
χM+4,Isp−FM=φDXP*χM+4,cMEPP+φMVA*χM+4,Isp+FM (Eqs. 4),
where χM0,Isp−FM . . . χM+4,Isp−FM and χM0,Isp+FM . . . χM+4,Isp+FM are the relative abundances of isoprene cumomers containing from zero to four 13C atoms calculated according to Eqs. 3 (subscript indices “Isp+FM” and “Isp-FM” indicate that calculations were done for cells incubated with and without fosmidomycin, respectively), χM0,cMEPP . . . χM+4,cMEPP are the relative abundances of corresponding cMEPP cumomers measured by LC-MS/MS as described above.
Example 34 13C NMR Method for the Determination of Carbon Fluxes Through the MVA and MEP Pathways Leading to BioIsoprene™ ProductIn this Example, BioIsoprene™ refers to isoprene that is biologically produced, e.g., using the methods described herein. BioIsoprene™ was obtained from bioisoprene composition. The relative contributions of the two isoprenoid precursor pathways, the MVA and MEP (DXP) pathways, to isoprene production in a REM A2—17 dual pathway strain were determined by 13C NMR spectroscopy and the resulting information used to calculate the MVA/MEP carbon ratio. Similar techniques have been used to determine the respective contributions of the MVA and MEP pathways to the biosynthesis of polyisoprenoids (Skorupinska-Tudek, K. et al. (2008) J. Biol. Chem., 283(30), pp. 21024-21035.) and isoprene (Wagner, W. P., Helmig, D. and Fall, R. (2000) J. Nat. Prod., 63, pp. 37-40). The labeling patterns of isoprene derived from 13C enriched glucose labels differ according to which pathway was utilized to channel carbon from the substrate to product. These patterns are shown in
As can be seen from
In the case where carbon flux though the MVA and MEP pathways is equal (1:1 MVA/MEP ratio), the extent of labeling at C-1 relative to C-2 and C-4 is also equivalent in the Bioisoprene™ product, with a maximum enrichment of 25%. At a MVA/MEP ratio of 9:1, C-1 is only enriched to the extent of 5%, whereas C-2 and C-4 are enriched to a level of 45%.
A method for the small-scale generation, collection and analysis of 13C-labeled BioIsoprene™ product was developed in order to determine the relative contributions of the MVA and MEP (DXP) pathways to isoprene production in strain REM A2—17. The strain was grown in HM-1 media with [1-13C]-D-glucose (10 mg/mL) as the sole carbon source in a stirred bottle format and the resulting BioIsoprene™ product was adsorbed to a small carbon filter consisting of 200 mg activated carbon (Koby filters, MA) packed into a glass Pasteur pipette with cotton wool (Scheme xx-1). After overnight growth at 34° C., the carbon filter was removed and desorbed directly into a glass NMR tube with CDCl3 (1 mL). A reference spectrum of unlabeled isoprene was obtained by diluting an isoprene standard (5 uL) (Sigma-Aldrich) into 0.75 mL of deuterochloroform (CDCl3) and acquiring a 13C NMR spectrum.
Relative 13C-enrichment of isoprene at each carbon atom was determined by 13C nuclear magnetic resonance spectroscopy (13C-NMR) by determining the relative intensities of the signals corresponding to each carbon atom of isoprene and comparing these values to the relative intensities of the carbon signals from unlabeled (natural 13C abundance) isoprene. 13C NMR spectra were obtained on a Varian 500 MHz VNMRS system operating at 125.7 MHz. Acquisition parameters were sw=30487, at=1.3 sec, dl=1, nt=10000, do=H1, dm=yyy, dmm=w, dpr=42, dmf=12600. 13C signal intensity was determined by peak height and integrated peak area. The 13C-NMR spectrum of unlabeled isoprene (% 13C=1.1%) is shown in
The 13C NMR spectrum of the BioIsoprene™ product derived from dual pathway strain REM A2—17 is shown in
In this example, we show that when the promoter PL.6 replaced the native promoter of the operon fkpB-ispH in strain WW119 to create strain REM D8—15, isoprene production drops from ≈500 to 600 ug/L/H/OD seen in strain WW119 (see
Construction of Strains for this Example.
Generation of the PL.6-fkpB Locus
Within the E. coli BL21 genome the ispH gene is located immediately downstream of the fkpB gene which encodes a FKBP-type peptidyl-prolyl cis-trans isomerase. Interestingly, the structure of the E. coli IspH enzyme shows that the protein has 2 proline residues that are isomerized (Gräwert, T. et al., 2009). The idea that FkpB could be involved in IspH function may also be reflected by the fact that the fkpB and ispH orfs are separated by just one nucleotide and together have been shown to be transcribed as the last 2 genes of the ribF-ileS-lspA-fkpB-ispH 5-gene operon (see www.ecocyc.org).
Further more, BLAST analysis of the 125 bases separating the stop codon of lspA and the start codon of fkpB revealed a highly conserved sequence that occurs many times throughout the E. coli genome. This commonly found sequence is:
This sequence harbors characteristics of a transcriptional terminator, which includes the likely formation of a stem loop. The bases with potential of hybridizing together to form the stem loop are highlighted above in bold and underlined text (bold anneals to bold; underlined anneals to underlined). The location of this repeated sequence, in each instance observed, was always found just downstream of the 3′ end of a single gene or downstream of the 3′ ends of 2 genes transcribed toward one another. The repeated sequence was not found within the coding region of over 40 regions analyzed. Together, this information suggests that the sequence functions as a transcriptional terminator and hints at the possibility of fkpB and ispH being transcribed as an independent 2-gene operon.
Our in-house transcriptional analyses of BL21 14-L fermentations show the ispH transcript to be present at almost undetectable levels; a result inline with that previously reported in the field. Similarly, the level of IspH protein accumulates to low levels within these and small scale grown cells (for small scale result see
The PL.6-promoter insertion and subsequent loopout of the chloramphenicol resistance marker described in this example was carried out using the Red/ET system from Gene Bridges GmbH according to the manufacturer's instructions. The strain BL21 (Novagen) was used. P1 lysate preparations and transductions were performed as previously described (Thomason et al., 2007). The BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD) was used for the electroporations described.
The PL.6-promoter introduced upstream of the endogenous fkpB coding region using the Gene Bridges GmbH methods is illustrated in
Amplification of the CMP:: PL.6 fkpB Fragment
To amplify the GB-CmpR cassette for inserting the PL.6-promoter immediately upstream of the fkpB locus the following PCR reaction was set up:
1 ul template (100 ng GB-CmpR)
10 ul HerculaseII Buffer0.5 ul dNTP's (100 mM)
1.25 ul primer (10 uM) 5′ CMP::80 bp up of fkpB
1.25 ul primer (10 uM) 3′ CMP::PL.6-fkpB
35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×3 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 0.8% E-gel (Invitrogen) for verification of successful amplification, and purified using the QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions. The resulting stock was CMP::PL.6 fkpB fragment.
Integration of CMP::PL.6 fkpB Fragment PCR Product into BL21/pRed-ET Strain
The pRed-ET vector (Gene Bridges kit) was transformed into BL21 (Novagen) by electroporation resulting in strain REM F7—13 (BL21/pRed-ET). The purified CMP::PL.6 fkpB PCR fragment was electroporated into REM F7—13. The transformants were recovered in L Broth and then plated on L agar containing chloramphenicol (10 ug/ml). Chloramphenicol resistant colonies were analyzed by PCR for the presence of the GB-CmpR cassette and the PL.6-promoter upstream of fkpB using primers 5′ confirm CMP::80 bp up of fkpB and bottom Pgb2 as well as 3′ confirm CMP::PL.6-fkpB and top Gb's CMP. The PCR fragments from a number of transformants were sequenced using the 3′ confirm CMP::PL.6-fkpB and top GB's CMP primers (Sequetech; Mountain View, Calif.) and PL.6 fkpB strain of interest identified. The chloramphenicol resistant strain, BL21 CMP::PL.6 fkpB, was designated REM A4—14.
Strategy for Creating REM D1—14Verification of the Presence of PL.6 fkpB within REM D1—14
To verify the REM D1—14 strain harbored the PL.6 fkp locus the following PCR reaction was set up:
Approx. 0.5 ul cells from a colony
5 ul HerculaseII Buffer0.25 ul dNTP's (100 mM)
0.625 ul primer (10 uM) 5′ primer to PL.6
0.625 ul primer (10 uM) 3′ confirm CMP::PL.6-fkpB
17.5 ul diH2O
+0.5 ul of HerculaseII fusion from Stratagene
95° C.×2 min., [95° C.×30 sec., 60° C.×30 sec., 72° C.×2 min.]×29 cycles; 72° C.×5 min.,
4° C. until cool (Biometra T3000 Combi Thermocycler)
The resulting PCR fragment was separated on a 2% E-gel (Invitrogen) for verification of successful amplification.
The chloramphenicol marked PL.6 fkpB locus of strain REM A4—14, described above, was introduced into strain WW103 via P1-mediated transduction. The resulting chloramphenicol resistant strain was named REM A9—14. After Flipase-mediated excision of the antibiotic cassette the resulting chloramphenicol sensitive strain was designated REM D1—14 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, CMP::PL.6 fkpB). The presence of the PL.6-promoter upstream of fkpB within REM D1—14 was verified by PCR using primers 5′ primer to PL.6 and 3′ confirm CMP::PL.6-fkpB, which are described above.
Strategy for Creating REM A8—15The chloramphenicol marked ΔiscR locus of strain REM14::CMP, described previously, was introduced into strain WW103 via P1-mediated transduction. The resulting chloramphenicol resistant strain was named REM A5—15. After Flipase-mediated excision of the antibiotic cassette the resulting chloramphenicol sensitive strain was designated REM A8—15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, ΔiscR).
Strategy for Creating REM A7—15The chloramphenicol marked ΔiscR locus of strain REM14::CMP was introduced into strain REM D1—14 via P1-mediated transduction. The resulting chloramphenicol resistant strain was named REM A2—15. After Flipase-mediated excision of the antibiotic cassette the resulting chloramphenicol sensitive strain was designated REM A7—15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, CMP::PL.6 fkpB, ΔiscR).
Verification of Increased Accumulation of IspH within REM D1—14 and REM A7—15
Western Blot MethodREM D1—14, REM A7—15, REM A8—15, and WW103 cells were grown in TM3 medium (1% glucose, 0.1% yeast extract) to limiting OD and cells were harvested by centrifugation and pellets stored at −80 deg until analyzed. For analysis culture pellets were resuspended in 0.05 M sodium phosphate, 0.3 M sodium chloride, 0.02 M imidazole, pH 8 with 0.2 mg/ml DNaseI to 100 OD/ml. Cells were broken by repeated pass through the French Press. 8 ml of each lysate was then clarified by ultracentrifugation at 50,000 rpm for 30 minutes. Soluble material was removed and the insoluble pellet was resuspended in 8 ml of 0.05 M sodium phosphate, 0.3 M sodium chloride, 0.02 M imidazole, pH 8 buffer. Analysis for E. coli ispH expression was performed using Nitrocellulose western blot, following transfer and development techniques recommended by Invitrogen as described in iBlot® and WesternBreeze® user manuals. The western blot was probed using primary polyclonal antibody produced against purified E. coli ispH in rabbit by ProSci Inc. The detection used a fluorescent secondary antibody from Invitrogen, Alexa Fluor® 488 goat anti-rabbit IgG (H+L). The raw data is shown in
The increased expression of ispH driven by the PL.6-promoter located upstream of the fkpB-ispH 2 gene operon of strains REM D1—14 and REM A7—15 relative to strain REM A8—15 and WW103 was indirectly assessed by measuring the level of IspH accumulation via a Western blot method (see
Strains WW119, REM D8—15, REM D7—15, and REM D6—15 were created by transforming pDW33 into WW103, REM D1—14, REM A8—15, and REM A7—15, respectively (strains described above).
Water-washed REM D1—14, REM A8—15, and REM A7—15 cells were transformed with pDW33 via electroporation using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a transformation protocol suggested by the manufacturer (BIO RAD). The cells were recovered in L broth for 1 hour at 37° C. and then plated on L agar containing carbenicillin (50 ug/ml). One carbenicillin resistant colony was chosen for each strain. The resulting carbenicillin resistant strains were named as such:
REM D8—15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, PL.6 fkpB, and pDW33);
REM D7—15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, ΔiscR, and pDW33);
REM D6—15 15 (BL21 pgl+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, PL.6 fkpB, ΔiscR, and pDW33).
Method Section for Isoprene Production and Quantitation of ispH
Isoprene Measurements.
Cultures to measure isoprene production were set up in a 48-deep-well plate (cat# P-5ML-48-C-S Axygen Scientific, California, USA) with each well providing a 2 mL culture. The culture medium, named TM3, is described below. The strains to be compared were grown o/n at 30 degrees at 250 rpm in TM3 medium supplemented with 1% glucose and 0.1% yeast extract. In the morning the strains were inoculated at 1:100 in quadruplicate sets of wells in the 48-deep well block. The cultures were covered with a “Breath Easier”™ membrane (Electron Microscopy Sciences Cat#70536-10) and were continuously shaken at at 600 rpm and 30 deg C (Shel-Lab Inc. Model SI6R Refrigerated Shaking Incubator; Oregon, USA). Culture OD was determined after two hours and then at timed intervals out to 6 hours. Induction with IPTG was after two hours of growth by the addition of 50, 100, 200, and 400 uM IPTG to the quadruplicated sets of wells, one through four. At two hours post-induction and hourly thereafter out to six hours these cultures were samples for isoprene production assays as follow: A 100 uL aliquot of each culture was transferred to a 98-deep well glass block (cat#3600600 Zinsser; North America) which was immediately sealed with an impermeable adhesive aluminum film and incubated for 30 minutes with shaking at 450 rmp on an Eppendorf thermomixer (Eppendorf; North America.). The isoprene assay cultures were killed by heating at 70 deg C for 7 min on a second Eppendorf thermomixer. The glass block was transferred to an Agilent 6890 GC attached to an Agilent 5973 MS and outfitted with a LEAP CTC CombiPAL autosampler for head space analysis. The column was an Agilent HP-5 (5% Phenyl Methyl Siloxane (15 m×0.25 mm×0.25 um)). A 100 uL gas volume was injected on the column. Other conditions were as follows. Oven Temperature: 37 C (held isothermal for 0.6 mins); Carrier Gas: Helium (flow −1 mL/min), split ratio of 50:1 at 250° C. on the injection port; Single Ion Monitoring mode (SIM) on mass 67; Detector off: 0.00 min-0.42 mins; Dectector on: 0.42 mins-0.60 mins; elution time for Isoprene (2-methyl-1,3 butadiene) was ˜0.49 min for a total analysis time of 0.6 mins. Calibration of the instrument was performed by methods well known to those trained in the art.
Isoprene head space measurements were normalized by culture OD600 to yield a measure of specific isoprene production in units of ug/L/H/OD. All reactions were followed for 4 to 8 hours. The surprising results from this experiment is that when the ΔiscR mutation is combined with the chromosomal mutation of PL.6 fkpB-ispH isoprene activity is restored. This result is consistent with that iscR in a background of overexpressed ispH takes on a regulatory role or at least interferes with flux through the DXP pathway. For high flux ispH needs to be overexpressed and under these condition ΔiscR expected to be beneficial for the process.
Verification of Increased ispH Expression Level by Western Blot.
The substitution of the PL.6 promoter for the native promoter of the fkpB-ispH operon was expected to raise the level of ispH. This was confirmed in strain REM A7—15, REM D1—14 by comparison to control strains REM A8—15 and WW103 by western Blot with polyclonal antibody prepared against this enzyme as described; the promoter swap resulted in a 5-fold increase of soluble ispH. Cells were grown in TM3 medium (1% glucose, 0.1% yeast extract) to limiting OD and were harvested by centrifugation and the pellets were stored at −80 deg until the next day. For analysis pellets were resuspended in 0.05 M sodium phosphate, 0.3 M sodium chloride, 0.02 M imidazole, pH 8 with 0.2 mg/ml DNaseI to 100 OD/ml. Cells were broken by repeated passage through the French press. Eight ml of each lysate was clarified by ultracentrifugation at 100,000×g for 30 minutes. Supernatant was removed and the pellet was resuspended in 8 ml of buffer pH8, 0.05 M sodium phosphate, 0.3 M sodium chloride, 0.02 M imidazole. Western blot was performed as described in the users manuals iBlot® and WesternBreeze® (in Vitrogen). The primary polyclonal antibody was against purified E. coli IspH overexpressed in E. coli and raised in rabbit by ProSci Inc (Poway, Calif.). For detection a fluorescent secondary antibody from Invitrogen (Alexa Fluor® 488 goat anti-rabbit IgG H+L), was used. The raw data is shown in
K2HPO4 13.6 g, KH2PO4 13.6 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 1.0 g, 1000× Modified Trace Metal stock solution 1 ml. All of the components were added together and dissolved in Di H2O. The pH is adjusted to 6.8 with NH4OH and the solution is filter sterilized over a 0.22 micron membrane. Glucose was typically added at 1% and yeast extract was typically boosted to 0.1%. Antibiotics were added post-sterile as needed (TM3 medium was sometimes prepared w/o any MgSO4 as this Mg++ led to precipitation over time. In this case MgSO4 was added from a sterile 1M solution just prior to use).
1000× Modified Trace Metal Stock Solution (Per Liter):Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to volume and filter sterilized with a 0.22 micron filter.
APPENDIX 1 Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleic acids and polypeptides
AY182241
Claims
1. Cells comprising (i) a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide.
2. The cells of claim 1, wherein the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
3. The cells of claim 1, wherein the cells in culture produce greater than about 400 nmole/gwcm/hr of isoprene.
4. The cells of claim 1, wherein more than about 0.02 molar percent of the carbon that the cells consume from a cell culture medium is converted into isoprene.
5. A method of producing isoprene, the method comprising
- (a) culturing cells comprising (i) a heterologous nucleic acid encoding a heterologous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide or (ii) a duplicate copy of an endogenous nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, and
- (b) producing isoprene.
6. The method of claim 5, wherein the cells further comprise a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid encoding an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
7. The method of claim 5, wherein the cells in culture produce greater than about 400 nmole/gwcm/hr of isoprene.
8. The method of claim 5, wherein more than about 0.02 molar percent of the carbon that the cells consume from a cell culture medium is converted into isoprene.
Type: Application
Filed: Jul 11, 2013
Publication Date: Jun 12, 2014
Applicants: The Goodyear Tire & Rubber Company (Akron, OH), Danisco US Inc. (Palo Alto, CA)
Inventors: Gopal K. CHOTANI (Cupertino, CA), Joseph C. MCAULIFFE (Sunnyvale, CA), Michael C. MILLER (San Francisco, CA), Rachel E. MUIR (Redwood City, CA), Dmitrii V. VAVILINE (Palo Alto, CA), Walter WEYLER (San Francisco, CA)
Application Number: 13/940,092
International Classification: C12P 5/00 (20060101); C12N 15/70 (20060101);
