SUPPLEMENTATION OF FATTY ACIDS FOR IMPROVING ALCOHOL PRODUCTIVITY
Fatty acids derived from biomass at a step in a fermentation process can be added to a fermentation medium comprising a recombinant microorganism that produces a product alcohol. At least one of growth rate and fermentable carbon consumption of the microorganism is greater in the presence of the fatty acids than the growth rate and the fermentable carbon consumption of the microorganism in the absence of the fatty acids. The addition of the fatty acids can increase glucose consumption, and can improve microorganism biomass production (cell growth/density) and growth rate, thereby reducing production time and increasing productivity of the fermentation process.
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This application claims the benefit of U.S. Provisional Application No. 61/356,290, filed on Jun. 18, 2010; U.S. Provisional Application No. 61/368,451, filed on Jul. 28, 2010; U.S. Provisional Application No. 61/368,436, filed on Jul. 28, 2010; U.S. Provisional Application No. 61/368,444, filed on Jul. 28, 2010; U.S. Provisional Application No. 61/368,429, filed on Jul. 28, 2010; U.S. Provisional Application No. 61/379,546, filed on Sep. 2, 2010; and U.S. Provisional Application No. 61/440,034, filed on Feb. 7, 2011; U.S. patent application Ser. No. 13/160,766, filed on Jun. 15, 2011; the entire contents of which are all herein incorporated by reference.
The Sequence Listing associated with this application is filed in electronic form via EFS-Web and hereby incorporated by reference into the specification in its entirety.
FIELD OF THE INVENTIONThe present invention relates to the production of fermentative alcohols, such as butanol, and in particular to alcohol fermentation processes for achieving improved alcohol productivity in which the fermentative growth of recombinant microorganisms is in the presence of fatty acids derived from biomass at a step in the fermentation process.
BACKGROUND OF THE INVENTIONAlcohols have a variety of applications in industry and science such as a beverage (i.e., ethanol), fuel, reagents, solvents, and antiseptics. For example, butanol is an alcohol that is an important industrial chemical with a variety of applications including use as a fuel additive, as a feedstock chemical in the plastics industry, and as a food-grade extractant in the food and flavor industry. Accordingly, there is a high demand for alcohols such as butanol, as well as for efficient and environmentally-friendly production methods.
Production of alcohol such as butanol utilizing fermentation by microorganisms is one environmentally friendly production method. Microorganisms such as yeasts have been used for the production of alcohol products where naturally produced pyruvate is used as a starting substrate in their biosynthetic pathways. Butanol can be produced biologically as a by-product of yeast fermentation, but the yield can be typically very low. To enhance production of desired products such as butanol, yeasts have been engineered to express enzymes that alter endogenous biosynthetic pathways or introduce new pathways, and/or by disrupting expression of endogenous enzymes to alter metabolite flow. Introduced pathways that use cellular pyruvate as a substrate include pathways for production of, for example, isomers of butanol. Disruption of pyruvate decarboxylase has been used to increase pyruvate availability for pathways that produce desired products such as butanol. Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (U.S. Patent Application Publication No. 2008/0182308A1), a 2-butanol biosynthetic pathway (U.S. Patent Application Publication Nos. 2007/0259410A1 and 2007/0292927), and an isobutanol biosynthetic pathway (U.S. Patent Application Publication No. 2007/0092957) have been described.
For example, Saccharomyces cerevisiae yeast can be metabolically engineered with disruptive mutations in the two primary pyruvate decarboxylase (PDC) genes. These genes, commonly referenced as PDC1 and PDC5, produce enzymes that are directly involved with ethanol production, and disruption of these genes has a negative impact on growth. Knock-out of pyruvate decarboxylase and alcohol dehydrogenase (ADH) alters the biosynthetic pathway resulting in the production of less fatty acid. Fatty acids are needed for cell wall formation and thus, necessary for cell growth. The importance of fatty acids for cell growth is demonstrated, for example, in Otoguro, et al., (J. Biochem. 89:523-529, 1981), which describes the effect of the antibiotic cerulenin, a known inhibitor of fatty acid synthesis, on cell growth. Cerulenin was added to a S. cerevisiae culture causing inhibition of the growth, but the growth was restored when oleic acid with certain saturated fatty acids (specifically, myristic acid, palmitic acid or pentadecanoic acid) was added.
Glucose metabolism in yeast generally follows a pathway of converting glucose to pyruvate to acetyl-CoA to cell mass. Correspondingly, there can be conversion of pyruvate to acetaldehyde to ethanol or a conversion of acetaldehyde to acetyl-CoA to fatty acid synthesis. With regard to recombinant microorganisms, a single PDC deletion reduces maximum growth but to a much lower extent. When only one PDC gene is disrupted, the other PDC gene is active enough to allow carbon flux to acetaldehyde and subsequently, ethanol and acetate. However, when butanol product is desired, ethanol production reduces butanol product yield on the substrate. The PDC genes are responsible for taking pyruvate to acetaldehyde, and the PDC1 and PDC5 double mutation prevents the production of acetaldehyde, altering the pathway to fatty acid biosynthesis and thereby inhibiting cell growth.
Thus, there exists a continuing need for methods for fermentative alcohol production using recombinant microorganisms in which growth rate and/or biomass production of the microorganisms can be improved despite reduction or elimination of fatty acid biosynthesis by the microorgansim. The present invention provides further related advantages, as will be made apparent by the description of the embodiments that follow.
BRIEF SUMMARY OF THE INVENTIONThe present invention is directed to a method comprising: (a) providing a fermentation broth comprising a recombinant microorganism that produces a product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity; (b) contacting the fermentation broth with a fermentable carbon source whereby the recombinant microorganism consumes the fermentable carbon source and produces the product alcohol; and (c) contacting the fermentation broth with fatty acids derived from biomass at a step in the fermentation process, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids. In a further embodiment, steps (b) and (c) occur substantially simultaneously. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof and in another embodiment, the biomass is derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. In a further embodiment, the fermentable carbon source is derived from the biomass. In one embodiment, the product alcohol is butanol and in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
The present invention is also directed to a method for producing a product alcohol comprising: (a) providing biomass comprising a fermentable carbon source and oil; (b) converting at least a portion of the oil into fatty acids to form a biomass comprising the fatty acids; (c) contacting the biomass with a fermentation broth comprising a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, and wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity; (d) contacting the fatty acids with the fermentation broth, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids. In a further embodiment, the step (b) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the portion of the oil into fatty acids. In one embodiment, the one or more substances comprise one or more enzymes and in another embodiment, the one or more enzymes comprise lipase enzymes. In a further embodiment, prior to step (c), the one or more enzymes may be inactivated after at least a portion of the oil is hydrolyzed. In one embodiment, one or more of steps (b), (c), and (d) occurs in the fermentation vessel and in another embodiment, one or more of steps (b), (c), and (d) occurs substantially simultaneously. In one embodiment, the method further comprises the step separating the oil from the biomass prior to the step (b) of converting at least a portion of the oil into fatty acids. In another embodiment, the method further comprises: liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides; and contacting the liquefied biomass with a saccharification enzyme capable of converting oligosaccharides into fermentable sugar to form a saccharified biomass, and wherein step (c) comprises contacting the saccharified biomass with the fermentation broth comprising a recombinant microorganism. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof and in another embodiment, the biomass is derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. In one embodiment, the method further comprises the step of fermenting the fermentable carbon source to produce product alcohol. In one embodiment, the product alcohol is butanol and in another embodiment, the fermentation broth further comprises ethanol. In another embodiment, the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
Another method of the present invention includes a method for producing a product alcohol comprising: (a) providing a feedstock; (b) liquefying said feedstock to create a feedstock slurry; (c) separating the feedstock slurry to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer, and (iii) a solids layer; (d) obtaining an oil from the oil layer and converting at least a portion of the oil into fatty acids; (e) feeding the aqueous layer of (c) to a fermentation vessel containing a fermentation broth comprising a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity; (f) fermenting the fermentable carbon source of the aqueous layer to produce the product alcohol; and (g) contacting the fermentation broth with the fatty acids, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids. In a further embodiment, the step (d) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the portion of the oil into fatty acids. In one embodiment, the one or more substances comprise one or more enzymes and in another embodiment, the one or more enzymes comprise lipase enzymes. In one embodiment, the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof and in another embodiment, the biomass is derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. In one embodiment, the product alcohol is butanol. In another embodiment, the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
The present invention is also directed to a composition comprising a recombinant microorganism comprising a reduction or elimination of pyruvate decarboxylase activity and fatty acids.
Fatty acids (e.g., oleic acid, palmitic acid, and mixtures thereof) derived from biomass at a step in a fermentation process, can be added to a fermentation medium comprising a recombinant microorganism that produces a product alcohol. The microorganism can be yeast or other alcohol-producing microorganism. Also, the microorganism can have one or more PDC gene deletions and/or have reduced or eliminated pyruvate decarboxylase activity. The addition of fatty acids can increase glucose consumption, and can improve microorganism biomass production (cell growth) and growth rate. Improving growth rate can reduce production time and thereby increase productivity of the alcohol fermentation process.
In some embodiments, a method for producing a product alcohol in a fermentation process includes (a) providing a fermentation broth including a recombinant microorganism that produces a product alcohol from a fermentable carbon source; (b) contacting the fermentation broth with a fermentable carbon source whereby the microorganism consumes the fermentable carbon source and produces the product alcohol; and (c) contacting the fermentation broth with fatty acids derived from biomass at a step in the fermentation process, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the microorganism in the absence of the fatty acids.
In some embodiments, the fatty acids are free fatty acids (FFA). In some embodiments, the fatty acids include oleic acid. In some embodiments, the fatty acids include saturated fatty acids. In some embodiments, the fatty acids include palmitic acid. In some embodiments, the fatty acids include myristic acid.
In some embodiments, the product alcohol is butanol.
In some embodiments, the fermentable carbon source is derived from the biomass. In some embodiments, the biomass includes corn and the fatty acids are corn oil fatty acids.
In some embodiments, the fermentation broth further includes ethanol. In some embodiments, the method further includes contacting the fermentation broth with ethanol.
In some embodiments, the step of contacting the fermentation broth with fatty acids includes contacting triglycerides derived from biomass with one or more enzymes capable of hydrolyzing triglycerides into free fatty acids, whereby the triglycerides are hydrolyzed into free fatty acids; and contacting the fermentation broth with the free fatty acids, wherein at least one of (i) the growth rate and (ii) the fermentable carbon consumption of the microorganism is greater in the presence of the free fatty acids than in the absence of the free fatty acids. In some embodiments, the one or more enzymes include lipase enzymes.
In some embodiments, the recombinant microorganism has a single pyruvate decarboxylase (PDC) gene deletion. In some embodiments, the recombinant microorganism has a double PDC gene deletion. In some embodiments, the recombinant microorganism has reduced or eliminated pyruvate decarboxylase activity.
In some embodiments, the concentration of the fatty acids in the fermentation broth is not greater than about 0.8 g/L.
In some embodiments, a method for producing a product alcohol from fermenting biomass includes (a) providing an aqueous biomass feedstream including water, fermentable carbon source, and an amount of oil, wherein the fermentable carbon source and the oil are both derived from said biomass; (b) hydrolyzing at least a portion of the oil into free fatty acids to form a biomass feedstream including the free fatty acids; (c) contacting a fermentation medium with the biomass feedstream in a fermentation vessel, the fermentation medium including a recombinant microorganism that produces a product alcohol; and (d) fermenting the fermentable carbon source in the fermentation vessel to produce said product alcohol, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism is greater in the presence of the free fatty acids than the growth rate and/or the fermentable carbon consumption of the microorganism in the absence of the free fatty acids.
In some embodiments, the step (b) of hydrolyzing at least a portion of the oil into free fatty acids includes contacting the oil with a composition including one or more enzymes capable of hydrolyzing the portion of the oil into free fatty acids. In some embodiments, the method further includes, prior to step (c), inactivating the one or more enzymes after at least a portion of the oil is hydrolyzed.
In some embodiments, the aqueous biomass feedstream is a liquefied mash formed from a milled, unfractionated grain. In some embodiments, the milled, unfractionated grain is corn and the oil is corn oil.
In some embodiments, a method of producing a product alcohol includes (a) providing biomass including glucose and oil including an amount of triglycerides; (b) contacting the oil with a composition including one or more substances capable of converting the triglycerides into free fatty acids, whereby at least a portion of the triglycerides in the oil are converted into free fatty acids; (c) contacting the biomass with a fermentation broth including a microorganism capable of converting the glucose to a product alcohol, whereby a product alcohol is produced; and (d) contacting the free fatty acids with the fermentation broth, wherein at least one of (i) growth rate and (ii) glucose consumption of the microorganism is greater in the presence of the free fatty acids than the growth rate and/or the glucose consumption of the microorganism in the absence of the free fatty acids.
In some embodiments, the method further includes separating the oil of (a) from the biomass prior to the step (b) of contacting the oil with the one or more substances.
In some embodiments, step (b) of contacting the oil with a composition including one or more substances includes contacting the oil with one or more catalysts capable of hydrolyzing triglycerides into free fatty acids.
In some embodiments, step (b) of contacting the oil with a composition including one or more substances includes contacting the oil with one or more reactants or solvents capable of chemically reacting the triglycerides to obtain a reaction product including the free fatty acids.
In some embodiments, a method for producing butanol includes (a) providing biomass including starch and oil, wherein the oil includes an amount of glycerides; (b) liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass includes oligosaccharides hydrolyzed from the starch; (c) contacting the biomass of step (a) or the liquefied biomass of step (b) with a composition including one or more enzymes capable of converting the glycerides into free fatty acids, whereby at least a portion of the glycerides in the oil are converted into free fatty acids; (d) contacting the liquefied biomass with a saccharification enzyme capable of converting oligosaccharides into fermentable sugar including monomeric glucose; (e) contacting the liquefied biomass with a recombinant microorganism capable of converting the fermentable sugar to butanol whereby butanol is produced; and (f) contacting the free fatty acids with the recombinant microorganism, wherein at least one of (i) growth rate and (ii) glucose consumption of the recombinant microorganism is greater in the presence of the free fatty acids than the growth rate and/or the glucose consumption of the recombinant microorganism in the absence of the free fatty acids.
In some embodiments, a fermentation process to produce a product alcohol from a feedstock includes: (a) liquefying said feedstock to create a feedstock slurry; (b) centrifuging the feedstock slurry to produce a centrifuge product including (i) an aqueous layer including glucose, (ii) an oil layer including glycerides, and (iii) a solids layer; (c) hydrolyzing at least a portion of the glycerides into free fatty acids; (d) feeding the aqueous layer of (b) to a fermentation vessel containing a fermentation broth including a recombinant microorganism capable of producing a product alcohol from glucose; (e) fermenting the glucose of the aqueous layer to produce the product alcohol; and (f) contacting the fermentation broth with the free fatty acids, wherein at least one of (i) growth rate and (ii) glucose consumption of the microorganism is greater in the presence of the free fatty acids than the growth rate and/or the glucose consumption of the microorganism in the absence of the free fatty acids.
In some embodiments, the process to produce a product alcohol from a feedstock further includes, prior to the step of hydrolyzing the glycerides, feeding the glycerides to the fermentation vessel.
In some embodiments, a fermentation process includes (a) providing a fermentation broth including a recombinant microorganism that produces a product alcohol from a fermentable carbon source, a fermentable carbon source, a product alcohol, and oil derived from biomass, wherein the oil includes glycerides; (b) contacting the fermentation broth with a first extractant to form a two-phase mixture including an aqueous phase and an organic phase, wherein the product alcohol and the oil partition into the organic phase to form a product alcohol-containing organic phase; (c) separating the product alcohol-containing organic phase from the aqueous phase; (d) separating the product alcohol from the organic phase to produce a lean organic phase; (e) contacting the lean organic phase with a composition including one or more catalysts capable of hydrolyzing the glycerides into free fatty acids to produce a second extractant including at least a portion of the first extractant and free fatty acids; and (f) repeating step (b) by contacting the fermentation broth with the second extractant of step (e), wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism is greater in the presence of the free fatty acids than the growth rate and/or the fermentable carbon consumption of the microorganism in the absence of the free fatty acids.
The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.
In order to further define this invention, the following terms and definitions are herein provided.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, that is, occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about,” the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.
“Biomass” as used herein refers to a natural product containing hydrolyzable polysaccharides that provide fermentable sugars including any sugars and starch derived from natural resources such as corn, cane, wheat, cellulosic or lignocellulosic material and materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and/or monosaccharides, and mixtures thereof. Biomass may also comprise additional components such as protein and/or lipids. Biomass may be derived from a single source or biomass can comprise a mixture derived from more than one source. For example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. For example, mash, juice, molasses, or hydrolysate may be formed from biomass by any processing known in the art for processing the biomass for purposes of fermentation such as by milling, treating, and/or liquefying and comprises fermentable sugar and may comprise water. For example, cellulosic and/or lignocellulosic biomass may be processed to obtain a hydrolysate containing fermentable sugars by any method known to one skilled in the art. A low ammonia pretreatment is disclosed in U.S. Patent Application Publication No. 2007/0031918A1, which is herein incorporated by reference. Enzymatic saccharification of cellulosic and/or lignocellulosic biomass typically makes use of an enzyme consortium for breaking down cellulose and hemicellulose to produce a hydrolysate containing sugars including glucose, xylose, and arabinose. (Saccharification enzymes suitable for cellulosic and/or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66:506-577, 2002).
Mash, juice, molasses, or hydrolysate may include feedstock 12 and feedstock slurry 16 as described herein. An aqueous feedstream may be derived or formed from biomass by any processing known in the art for processing the biomass for purposes of fermentation such as by milling, treating, and/or liquefying and comprises fermentable carbon substrate (e.g., sugar) and may comprise water. An aqueous feedstream may include feedstock 12 and feedstock slurry 16 as described herein.
“Biomass production” as used herein refers to microorganism biomass production (i.e., cell biomass production or cell growth) such as during cultivation of microorganisms pre-fermentation or during fermentative growth of microorganisms.
“Feedstock” as used herein means a feed in a fermentation process, the feed containing a fermentable carbon source with or without undissolved solids, and where applicable, the feed containing the fermentable carbon source before or after the fermentable carbon source has been liberated from starch or obtained from the break down of complex sugars by further processing such as by liquefaction, saccharification, or other process. Feedstock includes or is derived from a biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, cane, barley, cellulosic material, lignocellulosic material, or mixtures thereof.
“Fermentation broth” as used herein means the mixture of water, sugars, dissolved solids, optionally microorganisms producing alcohol, product alcohol, and all other constituents of the material held in the fermentation vessel in which product alcohol is being made by the reaction of sugars to alcohol, water, and carbon dioxide (CO2) by the microorganisms present. From time to time, as used herein the term “fermentation medium” and “fermented mixture” can be used synonymously with “fermentation broth.”
“Fermentable carbon source” or “fermentable carbon substrate” as used herein means a carbon source capable of being metabolized (or “consumed”) by the microorganisms disclosed herein for the production of fermentative alcohol. Suitable fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; C5 sugars such as xylose and arabinose; one carbon substrates including methane; and mixtures thereof. The term “consumed” as used herein includes processes by which compounds, for example, organic compounds such as glucose are broken down by the action of enzymes from a cell which results in the production of energy that may be used by the cell.
“Fermentable sugar” as used herein refers to one or more sugars capable of being metabolized (or “consumed”) by the microorganisms disclosed herein for the production of fermentative alcohol.
“Fermentation vessel” as used herein means the vessel in which the fermentation reaction is carried out whereby product alcohol such as butanol is made from sugars.
“Liquefaction vessel” as used herein means the vessel in which liquefaction is carried out. Liquefaction is the process in which oligosaccharides are liberated from the feedstock. In some embodiments where the feedstock is corn, oligosaccharides are liberated from the corn starch content during liquefaction.
“Saccharification vessel” as used herein means the vessel in which saccharification (i.e., the break down of oligosaccharides into monosaccharides) is carried out. Where fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentation vessel may be one in the same vessel.
“Sugar” as used herein refers to oligosaccharides, disaccharides, monosaccharides, and/or mixtures thereof. The term “saccharide” also includes carbohydrates including starches, dextrans, glycogens, cellulose, pentosans, as well as sugars.
As used herein, “saccharification enzyme” means one or more enzymes that are capable of hydrolyzing polysaccharides and/or oligosaccharides, for example, alpha-1,4-glucosidic bonds of glycogen, or starch. Saccharification enzymes may include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials as well.
“Undissolved solids” as used herein means non-fermentable portions of feedstock, for example, germ, fiber, and gluten.
“Product alcohol” as used herein refers to any alcohol that can be produced by a microorganism in a fermentation process that utilizes biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, C1 to C8 alkyl alcohols. In some embodiments, the product alcohols are C2 to C8 alkyl alcohols. In other embodiments, the product alcohols are C2 to C5 alkyl alcohols. It will be appreciated that C1 to C8 alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol. Likewise C2 to C8 alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, and pentanol. “Alcohol” is also used herein with reference to a product alcohol.
“Butanol” as used herein refers with specificity to the butanol isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol, and/or isobutanol (iBuOH or i-BuOH or I-BUOH, also known as 2-methyl-1-propanol), either individually or as mixtures thereof. From time to time, when referring to esters of butanol, the terms “butyl esters” and “butanol esters” may be used interchangeably.
“Propanol” as used herein refers to the propanol isomers isopropanol or 1-propanol.
“Pentanol” as used herein refers to the pentanol isomers 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.
The term “alcohol equivalent” as used herein refers to the weight of alcohol that would be obtained by a perfect hydrolysis of an alcohol ester and the subsequent recovery of the alcohol from an amount of alcohol ester.
The term “aqueous phase titer” as used herein refers to the concentration of a particular alcohol (e.g., butanol) in the fermentation broth.
The term “effective titer” as used herein refers to the total amount of a particular alcohol (e.g., butanol) produced by fermentation or alcohol equivalent of the alcohol ester produced by alcohol esterification per liter of fermentation medium. For example, the effective titer of butanol in a unit volume of a fermentation includes: (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) the amount of butanol recovered from the gas phase, if gas stripping is used; and (iv) the alcohol equivalent of the butyl ester in either the organic or aqueous phase.
“In Situ Product Removal (ISPR)” as used herein means the selective removal of a specific fermentation product from a biological process such as fermentation, to control the product concentration in the biological process as the product is produced.
“Extractant” or “ISPR extractant” as used herein means an organic solvent used to extract any product alcohol such as butanol or used to extract any product alcohol ester produced by a catalyst from a product alcohol and a carboxylic acid or lipid. From time to time, as used herein the term “solvent” may be used synonymously with “extractant.” For the processes described herein, extractants are water-immiscible.
The terms “water-immiscible” or “insoluble” refer to a chemical component such as an extractant or solvent, which is incapable of mixing with an aqueous solution such as a fermentation broth, in such a manner as to form one liquid phase.
The term “aqueous phase” as used herein refers to the aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant. In an embodiment of a process described herein that includes fermentative extraction, the term “fermentation broth” then specifically refers to the aqueous phase in biphasic fermentative extraction.
The term “organic phase” as used herein refers to the non-aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractant.
The term “fatty acid” as used herein refers to a carboxylic acid (e.g., aliphatic monocarboxylic acid) having C4 to C28 carbon atoms (most commonly C12 to C24 carbon atoms), which is either saturated or unsaturated. Fatty acids may also be branched or unbranched. Fatty acids may be derived from, or contained in esterified form, in an animal or vegetable fat, oil, or wax. Fatty acids may occur naturally in the form of glycerides in fats and fatty oils or may be obtained by hydrolysis of fats or by synthesis. The term fatty acid may describe a single chemical species or a mixture of fatty acids. In addition, the term fatty acid also encompasses free fatty acids.
The term “fatty alcohol” as used herein refers to an alcohol having an aliphatic chain of C4 to C22 carbon atoms, which is either saturated or unsaturated.
The term “fatty aldehyde” as used herein refers to an aldehyde having an aliphatic chain of C4 to C22 carbon atoms, which is either saturated or unsaturated.
The term “carboxylic acid” as used herein refers to any organic compound with the general chemical formula —COOH in which a carbon atom is bonded to an oxygen atom by a double bond to make a carbonyl group (—C═O) and to a hydroxyl group (—OH) by a single bond. A carboxylic acid may be in the form of the protonated carboxylic acid, in the form of a salt of a carboxylic acid (e.g., an ammonium, sodium, or potassium salt), or as a mixture of protonated carboxylic acid and salt of a carboxylic acid. The term carboxylic acid may describe a single chemical species (e.g., oleic acid) or a mixture of carboxylic acids as can be produced, for example, by the hydrolysis of biomass-derived fatty acid esters or triglycerides, diglycerides, monoglycerides, and phospholipids.
“Native oil” as used herein refers to lipids obtained from plants (e.g., biomass) or animals. “Plant-derived oil” as used herein refers to lipids obtain from plants in particular. From time to time, “lipids” may be used synonymously with “oil” and “acyl glycerides.” Native oils include, but are not limited to, tallow, corn, canola, capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya, sunflower, tung, jatropha, and vegetable oil blends.
The term “separation” as used herein is synonymous with “recovery” and refers to removing a chemical compound from an initial mixture to obtain the compound in greater purity or at a higher concentration than the purity or concentration of the compound in the initial mixture.
As used herein, “recombinant microorganism” refers to microorganisms such as bacteria or yeast, that are modified by use of recombinant DNA techniques, for example, by engineering a host cell to comprise a biosynthetic pathway such as a biosynthetic pathway to produce an alcohol such as butanol.
The present invention provides methods for producing product alcohol (e.g., fermentative alcohol) in which alcohol-producing microorganisms in a fermentation vessel are contacted with fatty acids which were derived from native oil such as biomass lipids at a step in a fermentation process. This fatty acid supplementation during the fermentative growth of the microorganism can increase the fermentable carbon consumption of the microorganisms, growth rate, and biomass production, particularly with regard to recombinant microorganisms that have reduced or eliminated pyruvate decarboxylase activity.
In some embodiments, glycerides in the oil can be chemically converted into fatty acids which are contacted with a fermentation broth including a recombinant microorganism that produces a product alcohol from fermentable carbon source. In other embodiments, the glycerides in the oil can be catalytically (e.g., enzymatically) hydrolyzed into fatty acids which are contacted with a fermentation broth including a recombinant microorganism. In some embodiments, the fatty acids can be obtained from hydrolysis of lipids found in the biomass which supplies the fermentable carbon source for fermentation. The fatty acids can also be used as an ISPR extractant to remove the product alcohol from the fermentation broth.
The fatty acids can be saturated, mono-unsaturated, poly-unsaturated, and mixtures thereof. For example, oil having a naturally-occurring fatty acid composition including a mixture of palmitic acid and oleic acid (e.g., corn oil) can be hydrolyzed to produce a mixture of free oleic acid and free palmitic acid which can be contacted with a fermentation broth in a fermentation vessel. In some embodiments, the concentration of the carboxylic acid (such as fatty acid) in the fermentation vessel exceeds the solubility limit in the aqueous phase and results in the production a two-phase fermentation mixture comprising an organic phase and an aqueous phase. In some embodiments, the concentration of carboxylic acids in the fermentation broth is typically not greater than about 0.8 g/L and is limited by the solubility of the carboxylic acid in the broth.
Growth rate and/or fermentable carbon consumption of the microorganism is greater in the presence of fatty acids than the growth rate and the fermentable carbon consumption of the microorganism in the absence of fatty acids. Correspondingly, fatty acid supplementation according to the methods of the present invention can achieve increased cell concentration and increased alcohol production than could be achieved in the absence of such fatty acid supplementation. In some embodiments, the microorganism may be a butanol-producing microorganism or other microorganism that typically requires supplementation of a 2-carbon substrate, for example, ethanol, to survive and grow. In such embodiments, the fatty acid supplementation according to methods of the present invention can allow such 2-carbon dependent microorganisms to survive and grow in the absence of ethanol supplementation. In some embodiments, the microorganisms can be deficient in production of acetyl-CoA from pyruvate. In some embodiments, the microorganism is metabolically engineered with disruptive mutations in one or more pyruvate decarboxylase (PDC) genes such that the pathway to fatty acid biosynthesis is modified. In some embodiments, the microorganism is metabolically engineered with disruptive mutations in two PDC genes such as genes PDC1 and PDC5, resulting in an altered pathway to fatty acid biosyntheses. Thus, the methods of the present invention can attain improved alcohol productivity by providing an optimal environment for fermentative growth of recombinant microorganisms.
The present invention will be described with reference to the Figures.
The process of liquefying feedstock 12 involves hydrolysis of polysaccharides in feedstock 12 into sugars including, for example, dextrins and oligosaccharides, and is a conventional process. Any known liquefying processes as well as the corresponding liquefaction vessel, normally utilized by the industry can be used including, but not limited to, the acid process, the acid-enzyme process, or the enzyme process. Such processes can be used alone or in combination. In some embodiments, the enzyme process can be utilized and an appropriate enzyme 14, for example, alpha-amylase, is introduced to an inlet in liquefaction vessel 10. Water can also be introduced to liquefaction vessel 10. In some embodiments, a saccharification enzyme, for example, glucoamylase, may also be introduced to liquefaction vessel 10. In additional embodiments, a lipase may also be introduced to liquefaction vessel 10 to catalyze the conversion of one or more components of the oil to free fatty acids.
Feedstock slurry 16 produced from liquefying feedstock 12 includes sugar, oil 26, and undissolved solids derived from the biomass from which feedstock 12 was formed. In some embodiments, the oil is in an amount of about 0 wt % to at least about 2 wt % of the fermentation broth composition. In some embodiments, the oil is in an amount of at least about 0.5 wt % of the feedstock. Feedstock slurry 16 can be discharged from an outlet of liquefaction vessel 10. In some embodiments, feedstock 12 is corn or corn kernels and therefore, feedstock slurry 16 is a corn mash slurry.
One or more substances 42 can be added to feedstock slurry 16. Substances 42 are capable of hydrolyzing glycerides in oil 26 to free fatty acids (FFA) 28. For example, when feedstock 12 is corn, then oil 26 is the feedstock's constituent corn oil and the free fatty acids 28 are corn oil fatty acids (COFA). Thus, after introduction of substances 42 to feedstock slurry 16, at least a portion of the glycerides in oil 26 are hydrolyzed to FFA 28 resulting in a feedstock slurry 18 having FFA 28.
In some embodiments, one or more substances 42 are one or more catalysts 42 capable of catalytically hydrolyzing glycerides in oil 26 to free fatty acids 28 (FFA). Thus, after introduction of catalyst 42 to feedstock slurry 16, at least a portion of the glycerides in oil 26 are hydrolyzed to FFA 28 resulting in a feedstock slurry 18 having FFA 28 and catalyst 42.
The resulting acid/oil composition from hydrolyzing oil 26 is typically at least about 17 wt % FFA. In some embodiments, the resulting acid/oil composition from hydrolyzing oil 26 is at least about 20 wt % FFA, at least about 25 wt % FFA, at least about 30 wt % FFA, at least about 35 wt % FFA, at least about 40 wt % FFA, at least about 45 wt % FFA, at least about 50 wt % FFA, at least about 55 wt % FFA, at least about 60 wt % FFA, at least about 65 wt % FFA, at least about 70 wt % FFA, at least about 75 wt % FFA, at least about 80 wt % FFA, at least about 85 wt % FFA, at least about 90 wt % FFA, at least about 95 wt % FFA, or at least about 99 wt % FFA.
Alternatively, in some embodiments, substance(s) 42 can alternatively constitute one or more reactants or solvents capable of chemically reacting oil 26 to FFA 28 for contacting with recombinant microorganism 32. For example, corn oil fatty acids can be synthesized from corn oil as oil 26 by base hydrolysis using NaOH and water as substances 42, as further described in co-pending, commonly owned U.S. Provisional Application Ser. No. 61/368,436, filed on Jul. 28, 2010, and incorporated herein in its entirety by reference thereto. Also, for example, corn oil triglycerides as oil 26 can be reacted with aqueous ammonium hydroxide as reactant 42 to obtain fatty acid (and fatty amide) as further described in Roe, et al., (Am. Oil Chem. Soc. 29:18-22, 1952), which is incorporated herein in its entirety by reference thereto. For purposes of the discussion herein with reference to the embodiments shown in the Figures, substance(s) 42 will often be described as constituting one or more catalysts as substance(s) 42 for the hydrolysis of biomass lipids to FFA 28 supplemented during fermentative growth of recombinant microorganism 32. However, it should be understood that the exemplary methods and systems described herein can be modified such that substance(s) 42 are reactant(s) and/or solvent(s) that are capable of chemically converting the biomass lipids into FFA 28.
In some embodiments, catalyst 42 can be one or more enzymes, for example, hydrolase enzymes such as lipase enzymes. Lipase enzymes used may be derived from any source including, for example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum, Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor, Nectria, Neurospora, Paecilomyces, Penicillium, Pseudomonas, Rhizoctonia, Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Sus, Sporobolomyces, Thermomyces, Thiarosporella, Trichoderma, Verticillium, and/or a strain of Yarrowia. In a preferred aspect, the source of the lipase is selected from the group consisting of Absidia blakesleena, Absidia corymbifera, Achromobacter iophagus, Alcaligenes sp., Alternaria brassiciola, Aspergillus flavus, Aspergillus niger, Aureobasidium pullulans, Bacillus pumilus, Bacillus strearothermophilus, Bacillus subtilis, Brochothrix thermosohata, Candida cylindracea (Candida rugosa), Candida paralipolytica, Candida Antarctica lipase A, Candida antartica lipase B, Candida ernobii, Candida deformans, Chromobacter viscosum, Coprinus cinerius, Fusarium oxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseum culmorum, Geotricum penicillatum, Hansenula anomala, Humicola brevispora, Humicola brevis var. thermoidea, Humicola insolens, Lactobacillus curvatus, Rhizopus oryzae, Penicillium cyclopium, Penicillium crustosum, Penicillium expansum, Penicillium sp. I, Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas wisconsinensis, Rhizoctonia solani, Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus, Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomyces cerevisiae, Sporobolomyces shibatanus, Sus scrofa, Thermomyces lanuginosus (formerly Humicola lanuginose), Thiarosporella phaseolina, Trichoderma harzianum, Trichoderma reesei, and Yarrowia lipolytica. In a further preferred aspect, the lipase is selected from the group consisting of Thermomcyces lanuginosus, Aspergillus sp. lipase, Aspergillus niger lipase, Candida antartica lipase B, Pseudomonas sp. lipase, Penicillium roqueforti lipase, Penicillium camembertii lipase, Mucor javanicus lipase, Burkholderia cepacia lipase, Alcaligenes sp. lipase, Candida rugosa lipase, Candida parapsilosis lipase, Candida deformans lipase, lipases A and B from Geotrichum candidum, Neurospora crassa lipase, Nectria haematococca lipase, Fusarium heterosporum lipase Rhizopus delemar lipase, Rhizomucor miehei lipase, Rhizopus arrhizus lipase, and Rhizopus oryzae lipase. Suitable commercial lipase preparations suitable as enzyme catalyst 42 include, but are not limited to, Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozym® CALA L, and Palatase 20000L, available from Novozymes, or from Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, hog pancreas, Candida cylindracea, Rhizopus niveus, Candida antarctica, Rhizopus arrhizus or Aspergillus available from SigmaAldrich.
After at least a portion of the glycerides are hydrolyzed, in some embodiments, catalyst 42 can be inactivated. Any method known in the art can be used to render catalyst 42 inactive. For example, in some embodiments, catalyst 42 can be inactivated by the application of heat, and/or by adjusting the pH of the reaction mass to a pH where catalyst 42 is irreversibly inactivated, and/or by adding a chemical or biochemical species capable of selectively inactivating the catalyst activity. As shown, for example, in the embodiment of
Inactivation of catalyst 42 is preferred when it is desirable to prevent catalyst 42 from esterifying alcohol with fatty acids 28 in the fermentation vessel. In some embodiments, production of an alcohol ester by esterification of product alcohol in a fermentation medium with an organic acid (e.g., fatty acid) and a catalyst (e.g., lipase) is desirable, as further described in co-pending, commonly owned U.S. Provisional Application Ser. No. 61/368,429, filed on Jul. 28, 2010; U.S. Provisional Application Ser. No. 61/379,546, filed on Sep. 2, 2010; and U.S. Provisional Application Ser. No. 61/440,034, filed on Feb. 7, 2011; all incorporated herein in its entirety by reference thereto. For example, for butanol production, active catalyst 42 in fermentation vessel (introduced via slurry 18) can catalyze the esterification of the butanol with fatty acids 28 (introduced via slurry 18) to form fatty acid butyl esters (FABE) in situ. In such embodiments in which alcohol esters of fatty acids are desirable, the methods described herein can be modified so as to omit inactivated of catalyst 42 prior to contacting a fermentation broth including product alcohol. Thus, with reference to the exemplary process flow diagrams of
Fermentation vessel 30 is configured to ferment slurry 18 to produce a product alcohol such as butanol. In particular, microorganism 32 metabolizes the fermentable sugar in slurry 18 and excretes a product alcohol. Microorganism 32 is selected from the group of bacteria, cyanobacteria, filamentous fungi, and yeast. In some embodiments, microorganism 32 can be a bacteria such as E. coli. In some embodiments, microorganism 32 can be a fermentative recombinant microorganism. The slurry can include sugar, for example, in the form of oligosaccharides and water, and can comprise less than about 20 g/L of monomeric glucose, more preferably less than about 10 g/L or less than about 5 g/L of monomeric glucose. Suitable methodology to determine the amount of monomeric glucose is well known in the art. Such suitable methods known in the art include HPLC.
In some embodiments, slurry 18 is subjected to a saccharification process in order to break the complex sugars (e.g., oligosaccharides) in slurry 18 into monosaccharides that can be readily metabolized by microorganism 32. Any known saccharification process that is routinely utilized by the industry, can be used including, but not limited to, the acid process, the acid-enzyme process, or the enzyme process. In some embodiments, simultaneous saccharification and fermentation (SSF) can occur inside fermentation vessel 30 as shown in
Optionally, ethanol 33 may be supplied to fermentation vessel 30 to be included in the fermentation broth. In some embodiments, when a recombinant microorganism having a butanol biosynthetic pathway is used as microorganism 32 for butanol production, microorganism 32 may require supplementation of a 2-carbon substrate (e.g., ethanol) to survive and grow. Thus, in some embodiments, ethanol 33 may be supplied to fermentation vessel 30.
However, it has been surprisingly found that methods of the present invention, in which free fatty acid (e.g., FFA 28) is present in the fermentation vessel, can allow reduction of the amount of ethanol 33 typically supplied for a given recombinant microorganism without detriment to the vitality of the recombinant microorganism. Further, in some embodiments, the methods of the present invention provide that the alcohol (e.g., butanol) production rate without ethanol supplementation to be comparable with the production rate that can be realized when ethanol 33 is supplemented. As further demonstrated by the comparative examples presented in the Examples 1-14 below, the butanol production rate when fatty acid but not ethanol is in the fermentation vessel can be greater than the butanol production rate when neither fatty acid nor ethanol is in the fermentation vessel. Thus, in some embodiments, the amount of ethanol 33 supplementation is reduced compared to conventional processes. For example, a typical amount of ethanol added to a fermentation vessel for microorganisms requiring supplementation of a 2-carbon substrate is about 5 g/L anhydrous ethanol (i.e., 5 g anhydrous ethanol per liter of fermentation medium). In some embodiments, the fermentation is not supplemented with any ethanol 33. In the latter case, the stream of ethanol 33 is entirely omitted from the fermentation vessel. Thus, in some embodiments of the present invention, it is possible to reduce or eliminate the cost associated with supplemental ethanol 33, as well as the inconvenience associated with storing vats of ethanol 33 and supplying it to the fermentation vessel during butanol fermentation. Moreover, regardless of ethanol supplementation, in some embodiments, the methods of the present invention can provide a higher rate of glucose uptake by microorganism 32 by virtue of the presence of fatty acids during the fermentation. According to the methods described herein, the fatty acids can be introduced into fermentation vessel 30 as FFA 28, hydrolyzed from feedstock oil 26 of slurry 16, or otherwise hydrolyzed from native oil such as biomass lipids at a step in the fermentation process. Fatty acids can also be introduced into fermentation vessel as an ISPR extractant 29.
In fermentation vessel 30, alcohol is produced by microorganism 32. In situ product removal (ISPR) can be utilized to remove the product alcohol from the fermentation broth. In some embodiments, ISPR includes liquid-liquid extraction. Liquid-liquid extraction can be performed according to the processes described in U.S. Patent Application Publication No. 2009/0305370, the disclosure of which is hereby incorporated in its entirety. U.S. Patent Application Publication No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water-immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C12 to C22 fatty alcohols, C12 to C22 fatty acids, esters of C12 to C22 fatty acids, C12 to C22 fatty aldehydes, and mixtures thereof, which contacts a fermentation broth and forms a two-phase mixture comprising an aqueous phase and an organic phase. The extractant may also be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C4 to C22 fatty alcohols, C4 to C28 fatty acids, esters of C4 to C28 fatty acids, C4 to C22 fatty aldehydes, and mixtures thereof, which contacts a fermentation broth and to form a two-phase mixture comprising an aqueous phase and an organic phase. Free fatty acids 28 from slurry 18 can also serve as an ISPR extractant 28. For example, when free fatty acids 28 are corn oil fatty acids (COFA), ISPR extractant 28 is COFA. ISPR extractant (FFA) 28 contacts the fermentation broth and forms a two-phase mixture comprising an aqueous phase 34 and an organic phase. The product alcohol present in the fermentation broth preferentially partitions into the organic phase to form an alcohol-containing organic phase 36. In some embodiments, fermentation vessel 30 has one or more inlets for receiving one or more additional ISPR extractants 29 which form a two-phase mixture comprising an aqueous phase and an organic phase, with the product alcohol partitioning into the organic phase.
The biphasic mixture can be removed from fermentation vessel 30 as stream 39 and introduced into a vessel 35, in which the alcohol-containing organic phase 36 is separated from the aqueous phase 34. The alcohol-containing organic phase 36 is separated from the aqueous phase 34 of the biphasic stream 39 using methods known in the art including, but not limited to, siphoning, decantation, aspiration, centrifugation, using a gravity settler, membrane-assisted phase splitting, and the like. All or part of the aqueous phase 34 can be recycled into fermentation vessel 30 as fermentation medium (as shown), or otherwise discarded and replaced with fresh medium, or treated for the removal of any remaining product alcohol and then recycled to fermentation vessel 30. Then, the alcohol-containing organic phase 36 is treated in a separator 50 to recover product alcohol 54, and the resulting alcohol-lean extractant 27 can then be recycled back into fermentation vessel 30, usually in combination with fresh FFA 28 from slurry 18 and/or with fresh extractant 29, for further extraction of the product alcohol. Alternatively, fresh FFA 28 (from slurry 18) and/or extractant 29 can be continuously added to the fermentation vessel to replace the ISPR extractant(s) removed in biphasic mixture stream 39.
In some embodiments, any additional ISPR extractant 29 can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. In some embodiments, ISPR extractant 29 can be a carboxylic acid or free fatty acid and in some embodiments, the carboxylic acid or free fatty acid have a chain of 4 to 28 carbons, 4 to 22 carbons in other embodiments, 8 to 22 carbons in other embodiments, 10 to 28 carbons in other embodiments, 7 to 22 carbons in other embodiments, 12 to 22 carbons in other embodiments, 4 to 18 carbons in other embodiments, 12 to 22 carbons in other embodiments, and 12 to 18 carbons in still other embodiments. In some embodiments, ISPR extractant 29 is one or more of the following fatty acids: azaleic, capric, caprylic, castor, coconut (i.e., as a naturally-occurring combination of fatty acids including lauric, myrisitic, palmitic, caprylic, capric, stearic, caproic, arachidic, oleic, and linoleic), dimer, isostearic, lauric, linseed, myristic, oleic, olive, palm oil, palmitic, palm kernel, peanut, pelargonic, ricinoleic, sebacic, soya, stearic acid, tall oil, tallow, #12 hydroxy stearic, or any seed oil. In some embodiments, ISPR extractant 29 is one or more of diacids, for example, azelaic acid and sebacic acid. Thus, in some embodiments, ISPR extractant 29 can be a mixture of two or more different fatty acids. In some embodiments, ISPR extractant 29 can be a free fatty acid derived from chemical or enzymatic hydrolysis of glycerides derived from native oil. For example, in some embodiments, ISPR extractant 29 can be free fatty acids 28′ obtained by enzymatic hydrolysis of native oil such as biomass lipids as later described with reference to the embodiment of
In situ extractive fermentation can be carried out in a batch mode or a continuous mode in fermentation vessel 30. For in situ extractive fermentation, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production, for example, the ISPR extractant can contact the fermentation medium at a time before the butanol concentration reaches a level which would be toxic to the microorganism. After contacting the fermentation medium with the ISPR extractant, the butanol product partitions into the extractant, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the production microorganism to the inhibitory butanol product.
The volume of the ISPR extractant to be used depends on a number of factors including the volume of the fermentation medium, the size of the fermentation vessel, the partition coefficient of the extractant for the butanol product, and the fermentation mode chosen, as described below. The volume of the extractant can be about 3% to about 60% of the fermentation vessel working volume. Depending on the efficiency of the extraction, the aqueous phase titer of butanol in the fermentation medium can be, for example, from about 1 g/L to about 85 g/L, from about 10 g/L to about 40 g/L, from about 10 g/L to about 20 g/L, from about 15 g/L to about 50 g/L or from about 20 g/L to about 60 g/L. In some embodiments, the resulting fermentation broth after alcohol esterification can comprise free (i.e., unesterified) alcohol and in some embodiments, the concentration of free alcohol in the fermentation broth after alcohol esterification is not greater than 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55, or 60 g/L when the product alcohol is butanol, or when the product alcohol is ethanol, the concentration of free alcohol in the fermentation broth after alcohol esterification is not greater than 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g/L. Without being held to theory, it is believed that higher butanol titer may obtained with the extractive fermentation method, in part, from the removal of the toxic butanol product from the fermentation medium, thereby keeping the level below that which is toxic to the microorganism.
In a batchwise mode of in situ extractive fermentation, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. This mode requires a larger volume of organic extractant to minimize the concentration of the inhibitory butanol product in the fermentation medium. Consequently, the volume of the fermentation medium is less and the amount of product produced is less than that obtained using the continuous mode. For example, the volume of the extractant in the batchwise mode can be 20% to about 60% of the fermentation vessel working volume in one embodiment, and about 30% to about 60% in another embodiment.
Gas stripping (not shown) can be used concurrently with the ISPR extractant to remove the product alcohol from the fermentation medium.
In the embodiment of
In a continuous mode of in situ extractive fermentation, the volume of the extractant can be about 3% to about 50% of the fermentation vessel working volume in one embodiment, about 3% to about 30% in another embodiment, 3% to about 20% in another embodiment; and 3% to about 10% in another embodiment. Because the product is continually removed from the reactor, a smaller volume of extractant is required enabling a larger volume of the fermentation medium to be used.
As an alternative to in situ extractive fermentation, the product alcohol can be extracted from the fermentation broth downstream of fermentation vessel 30. In such an instance, the fermentation broth can be removed from fermentation vessel 30 and introduced into vessel 35 for contacting with the ISPR extractant to obtain biphasic mixture 39 in vessel 35, which is then separated into the organic 36 and aqueous 34 phases. Alternatively, the ISPR extractant can be added to the fermentation broth in a separate vessel (not shown) prior to introduction to vessel 35.
As a non-limiting prophetic example, with reference to the embodiment of
In some embodiments, the system and processes of
In some embodiments, the system and processes of
For example, as shown in the embodiment of
Alternatively, in some embodiments, catalyst 42 can be added with saccharification enzyme 38 to simultaneously produce glucose and hydrolyze oil lipids 26 to free fatty acids 28. The addition of enzyme 38 and catalyst 42 can be stepwise (e.g., catalyst 42, then enzyme 38, or vice versa) or simultaneous. Alternatively, in some embodiments, slurry 62 can be introduced to fermentation vessel, with catalyst 42 being added directly to the fermentation vessel 30.
In the embodiment of
As described above with reference to
In still other embodiments, oil 26 derived from feedstock 12 can be catalytically hydrolyzed into FFA 28 either prior to or during liquefaction, such that feedstock slurry 18 having FFA 28 exits directly from liquefaction vessel 10 and can be fed to fermentation vessel 30. For example, feedstock 12 having oil 26 can be fed to liquefaction vessel 10 along with catalyst 42 for hydrolysis of at least a portion of the glycerides in oil 26 into FFA 28. Enzyme 14 (e.g., alpha-amylase) which hydrolyzes the starch in feedstock 12 can also be introduced to vessel 10 to produce a liquefied feedstock. The addition of enzyme 14 and catalyst 42 can be stepwise or simultaneous. For example, catalyst 42 can be introduced, and then enzyme 14 can be introduced after at least a portion of oil 26 has been hydrolyzed. Alternatively, enzyme 14 can be introduced, and then catalyst 42 can be introduced. The liquefaction process can involve the application of heat q. In such embodiments, catalyst 42 can be introduced prior to or during liquefaction when the process temperature is below that which inactivates catalyst 42, so that oil 26 can be hydrolyzed. Thereafter, application of heat q can provide a two-fold purpose of liquefaction and inactivation of catalyst 42, if inactivation is desired.
In some embodiments including any of the earlier described embodiments with respect to
In some embodiments, separator 20 removes oil 26 but not undissolved solids. Thus, aqueous stream 22 fed to fermentation vessel 30 includes undissolved solids. In some embodiments, separator 20 includes a tricanter centrifuge 20 that agitates or spins feedstock slurry 16 to produces a centrifuge product comprising an aqueous layer containing the sugar and water (i.e., stream 22), a solids layer containing the undissolved solids (i.e., wet cake 24), and an oil layer (i.e., oil stream 26). Methods and systems for removing undissolved solids from feedstock slurry 16 via centrifugation are described in detail in co-pending, commonly owned U.S. Provisional Application Ser. No. 61/356,290, filed Jun. 18, 2010, which is incorporated herein in its entirety by reference thereto.
In any case, catalyst 42 can be contacted with the removed oil 26 to produce a stream of FFA 28 including catalyst 42, as shown in
FFA 28 can serve as ISPR extractant 28 and forms a biphasic mixture in fermentation vessel 30. Product alcohol produced by SSF partitions into organic phase 36 constituted by FFA 28. In some embodiments, one or more additional ISPR extractants 29 can also be introduced into fermentation vessel 30. Thus, oil 26 (e.g., from feedstock) can be catalytically hydrolyzed to FFA 28, thereby decreasing the rate of build-up of lipids in an ISPR extractant while also producing an ISPR extractant. The organic phase 36 can be separated from the aqueous phase 34 of the biphasic mixture 39 at vessel 35. In some embodiments, separation of the biphasic mixture 39 can occur in the fermentation vessel, as shown in the embodiments of described in
When wet cake 24 is removed via centrifuge 20, in some embodiments, a portion of the oil from feedstock 12, such as corn oil when the feedstock is corn, remains in wet cake 24. Wet cake 24 can be washed with additional water in the centrifuge once aqueous solution 22 has been discharged from the centrifuge 20. Washing wet cake 24 will recover the sugar (e.g., oligosaccharides) present in the wet cake and the recovered sugar and water can be recycled to the liquefaction vessel 10. After washing, wet cake 20 can be dried to form Dried Distillers' Grains with Solubles (DDGS) through any suitable known process. The formation of the DDGS from wet cake 24 formed in centrifuge 20 has several benefits. Since the undissolved solids do not go to the fermentation vessel, DDGS does not have trapped extractant and/or product alcohol such as butanol, it is not subjected to the conditions of the fermentation vessel, and it does not contact the microorganisms present in the fermentation vessel. All these benefits make it easier to process and sell DDGS, for example, as animal feed. In some embodiments, oil 26 is not discharged separately from wet cake 24, but rather oil 26 is included as part of wet cake 24 and is ultimately present in the DDGS. In such instances, the oil can be separated from the DDGS and converted to an ISPR extractant 29 for subsequent use in the same or different alcohol fermentation process. Methods and systems for removing undissolved solids from feedstock 16 via centrifugation are described in detail in co-pending, commonly owned U.S. Patent Application No. 61/356,290, filed Jun. 18, 2010, which is incorporated herein in its entirety by reference thereto.
In still other embodiments (not shown), saccharification can occur in a separate saccharification vessel 60 (see
In still other embodiments as shown, for example, in the embodiment of
Alternatively, oil 26′ and catalyst 42 can be fed directly to fermentation vessel 30 in which oil 26′ is hydrolyzed to FFA 28′, rather than using vessel 40. Thereafter, active catalyst 42 can be subjected to heat q and inactivated while in the fermentation vessel before inoculation of microorganism 32. Alternatively, FFA 28′ and active catalyst 42 can be fed to fermentation vessel 30 from vessel 40, and active catalyst 42 can thereafter be subjected to heat q and inactivated while in the fermentation vessel before inoculation of microorganism 32. In such embodiments, feedstock slurry 16 including oil 26, rather than stream 22 in which oil 26 was removed, can be fed to fermentation vessel 30 and contacted with active catalyst 42. Active catalyst 42 can therefore be used to hydrolyze oil 26 into FFA 28, thereby reducing the loss and/or degradation of the partition coefficient of the extractant over time that is attributable to the presence of the oil in the fermentation vessel.
In some embodiments, the system and processes of
In some embodiments, native oil 26′ can be tallow, corn, canola, capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya, sunflower, tung, jatropha, vegetable oil blends, and mixtures thereof. In some embodiments, native oil 26′ is a mixture of two or more native oils, for example, a mixture of palm and soybean oils. In some embodiments, native oil 26′ is a plant-derived oil. In some embodiments, the plant-derived oil can be, though not necessarily, derived from biomass that can be used in a fermentation process. The biomass can be the same or different source from which feedstock 12 (shown in
In some embodiments of the present invention, biomass oil present in feedstock 12 can be converted to FFA 28 at a step following alcoholic fermentation. FFA 28 can then be introduced in the fermentation vessel, and contacted with fermentation broth for achieving improved growth rate and/or fermentable carbon consumption of the alcohol-producing microorganism. FFA 28 can as also serve as ISPR extractant 28. For example, in the embodiment of
ISPR extractant 29 is introduced to fermentation vessel 30 to form a biphasic mixture, and the product alcohol is removed by partitioning into the organic phase of the ISPR extractant 29. Oil 26 also partitions into the organic phase. Separation of the biphasic mixture occurs in fermentation vessel 30, whereby alcohol-containing organic phase stream 36 and aqueous phase stream 34 exit directly from fermentation vessel 30. Alternatively, separation of the biphasic mixture can be conducted in a separate vessel 35 as provided in the embodiments of
Thus,
From the above discussion and the Examples, one skilled in the art can ascertain essential characteristics of the present invention and can make various changes and modifications of the invention to adapt to various uses and conditions without departing from the present invention. For example, in some embodiments, fatty acid supplementation according to the present invention can be employed pre-fermentation, that is, during seed culturing of microorganisms 32 prior to fermentation in fermentation vessel 30. Typically, microorganisms 32 such as yeast can be grown from a seed culture to a desired cell concentration before being harvested and inoculated into fermentation vessel 30, as known in the art. Thus, according to some embodiments, the seed culture medium can be contacted with FFA 28 whereby improved growth rates and microorganism biomass production can be achieved, which can reduce the pre-fermentation time associated with the seed culturing phase of an alcohol fermentation process. Thus, it should be apparent that fatty acid supplementation according to the present invention can be employed at various stages in an alcohol fermentation process, for example, during pre-fermentation culturing and fermentation, for improving overall process efficiency without departing from the present invention.
In some embodiments, including any of the aforementioned embodiments described with reference to
Additionally, the carbon substrate may also be one-carbon substrates such as carbon dioxide or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. 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 yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion, et al., Microb. Growth C1-Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter, et al., Arch. Microbiol. 153:485-489, 1990). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable, in some embodiments, the carbon substrates are glucose, fructose, and sucrose, or mixtures of these substrates with C5 sugars such as xylose and/or arabinose for yeast modified to use C5 sugars. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described in, for example, U.S. Patent Application Publication No. 2007/0031918 A1, which is herein incorporated by reference. In addition to an appropriate carbon source (from aqueous stream 22), fermentation broth must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of an enzymatic pathway comprising a dihydroxyacid dehydratase (DHAD).
Recombinant microorganisms that produce butanol via a biosynthetic pathway can include a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In one embodiment, recombinant microorganisms can be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. In one embodiment, the recombinant microorganism is a crabtree-positive yeast selected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species of Candida. Species of crabtree-positive yeast include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata. For example, the production of butanol utilizing fermentation by a microorganism, as well as which microorganisms produce butanol, is known and is disclosed, for example, in U.S. Patent Application Publication No. 2009/0305370, herein incorporated by reference. In some embodiments, microorganisms comprise a butanol biosynthetic pathway. Suitable isobutanol biosynthetic pathways are known in the art (see, e.g., U.S. Patent Application Publication No. 2007/0092957, herein incorporated by reference). In some embodiments, at least one, at least two, at least three, or at least four polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the microorganism comprises a reduction or elimination of pyruvate decarboxylase activity. Microorganisms substantially free of pyruvate decarboxylase activity are described in U.S. Patent Application Publication No. 2009/0305363, herein incorporated by reference.
Construction of certain strains, including those used in the Examples, is provided herein.
Construction of Saccharomyces cerevisiae Strain BP1083 (“NGCI-070”)
The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6, and GPD2. BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1, described in U.S. Provisional Application Ser. No. 61/246,844) and pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1083, PNY1504).
Deletions, which completely removed the entire coding sequence, were created by homologous recombination with PCR fragments containing regions of homology upstream and downstream of the target gene and either a G418 resistance marker or URA3 gene for selection of transformants. The G418 resistance marker, flanked by loxP sites, was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion or if flanked by loxP sites, was removed using Cre recombinase.
The scarless deletion procedure was adapted from Akada, et al., (Yeast 23:399-405, 2006). In general, the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene). Fragments A and C, each 500 bp long, corresponded to the 500 bp immediately upstream of the target gene (Fragment A) and the 3′ 500 bp of the target gene (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target gene and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome. Using the PCR product ABUC cassette, the URA3 marker was first integrated into and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in the PCR cassette between fragments A and B.
URA3 DeletionTo delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxP cassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP sites to allow recombination with Cre recombinase and removal of the marker. PCR was done using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers BK505 and BK506 (SEQ ID NOs: 4 and 5). The URA3 portion of each primer was derived from the 5′ region upstream of the URA3 promoter and 3′ region downstream of the coding region such that integration of the loxP-kanMX-loxP marker resulted in replacement of the URA3 coding region. The PCR product was transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YPD containing G418 (100 μg/mL) at 30° C. Transformants were screened to verify correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs: 6 and 7) and designated CEN.PK 113-7D Δura3::kanMX.
HIS3 DeletionThe four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact, kit (Qiagen, Valencia, Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 14) and primer oBP453 (SEQ ID NO: 15) containing a 5′ tail with homology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO: 16) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 17) containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 18) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 19) containing a 5′ tail with homology to the 5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 20) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 21). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a his3 knockout were screened for by PCR with primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correct transformant was selected as strain CEN.PK 113-7D Δura3::kanMX Δhis3::URA3.
KanMX Marker Removal from the Δura3 Site and URA3 Marker Removal from the Δhis3 Site
The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMX Δhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 66, described in U.S. Provisional Application No. 61/290,639) using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation, Irvine, Calif.) and plating on synthetic complete medium lacking histidine and uracil supplemented with 2% glucose at 30° C. Transformants were grown in YP supplemented with 1% galactose at 30° C. for ˜6 hours to induce the Cre recombinase and KanMX marker excision and plated onto YPD (2% glucose) plates at 30° C. for recovery. An isolate was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) at 30° C. to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in and plated on YPD for removal of the pRS423::PGAL1-cre plasmid. Isolates were checked for loss of the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by assaying growth on YPD+G418 plates, synthetic complete medium lacking uracil plates, and synthetic complete medium lacking histidine plates. A correct isolate that was sensitive to G418 and auxotrophic for uracil and histidine was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletions and marker removal were confirmed by PCR and sequencing with primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) for Δura3 and primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23) for Δhis3 using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.).
PDC6 DeletionThe four fragments for the PCR cassette for the scarless PDC6 deletion were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B. PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 28), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment A, and primer oBP443 (SEQ ID NO: 29) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primer oBP444 (SEQ ID NO: 30) containing a 5′ tail with homology to the 3′ end of PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 31) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C was amplified with primer oBP446 (SEQ ID NO: 32) containing a 5′ tail with homology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO: 33). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). PDC6 Fragment AB was created by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 Fragment UC was created by overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C and amplifying with primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC6 ABUC cassette was created by overlapping PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made and transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc6 knockout were screened for by PCR with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1°)/0) at 30° C. to select for isolates that lost the URA3 marker. The deletion and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC6 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designated as BP891.
PDC1 Deletion ilvDSm Integration
The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC No. 700610. The A fragment followed by the ilvD coding region from Streptococcus mutans for the PCR cassette for the PDC1 deletion-ilvDSm integration was amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and NYLA83 (described herein and in U.S. Provisional Application No. 61/246,709) genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment A-ilvDSm (SEQ ID NO: 141) was amplified with primer oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO: 39) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment B. The B, U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSm integration were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment B was amplified with primer oBP516 (SEQ ID NO: 40) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 41) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO: 42) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B, and primer oBP519 (SEQ ID NO: 43) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primer oBP520 (SEQ ID NO: 44), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 45). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif. PDC1 Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41). PDC1 Fragment UC was created by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC1 A-ilvDSm-BUC cassette (SEQ ID NO: 142) was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc1 knockout ilvDSm integration were screened for by PCR with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC1 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC1, integration of ilvDSm, and marker removal were confirmed by PCR and sequencing with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm and designated as BP907.
PDC5 Deletion sadB Integration
The PDC5 gene was deleted and replaced with the sadB coding region from Achromobacter xylosoxidans. A segment of the PCR cassette for the PDC5 deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.
pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 gene from Saccaromyces cerevisiae situated within a multiple cloning site (MCS). pUC19 contains the pMB1 replicon and a gene coding for beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the sequences from upstream and downstream of this gene were included for expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.
The DNA encompassing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region from Saccaromyces cerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438 (SEQ ID NO: 12) containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 13) containing XbaI, PacI, and NotI restriction sites, using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.). Genomic DNA was prepared using a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The PCR product and pUC19 (SEQ ID NO: 144) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to create vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO: 10) and oBP265 (SEQ ID NO: 11).
The coding sequence of sadB and PDC5 Fragment B were cloned into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. The coding sequence of sadB was amplified using pLH468-sadB (SEQ ID NO: 67) as template with primer oBP530 (SEQ ID NO: 50) containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 51) containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B. PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 52) containing a 5′ tail with homology to the 3′ end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). sadB-PDC5 Fragment B was created by overlapping PCR by mixing the sadB and PDC5 Fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. The resulting plasmid was used as a template for amplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 54) and oBP546 (SEQ ID NO: 55) containing a 5′ tail with homology to the 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547 (SEQ ID NO: 56) containing a 5′ tail with homology to the 3′ end of PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 57). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). PDC5 sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C and amplifying with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 143) was created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ ID NO: 58) containing a 5′ tail with homology to the 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 57). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR cassette using a Frozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose) at 30° C. Transformants with a pdc5 knockout sadB integration were screened for by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC5 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 was grown overnight in YPE (1% ethanol) and plated on synthetic complete medium supplemented with ethanol (no glucose) and containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC5, integration of sadB, and marker removal were confirmed by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB and designated as BP913.
GPD2 DeletionTo delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxP cassette (SEQ ID NO: 145) was PCR-amplified using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. loxP-URA3-loxP contains the URA3 marker from (ATCC No. 77107) flanked by loxP recombinase sites. PCR was done using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5′ region upstream of the GPD2 coding region and 3′ region downstream of the coding region such that integration of the loxP-URA3-loxP marker resulted in replacement of the GPD2 coding region. The PCR product was transformed into BP913 and transformants were selected on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose). Transformants were screened to verify correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs: 63 and 64).
The URA3 marker was recycled by transformation with pRS423::PGAL1-cre (SEQ ID NO: 66) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and incubated at 30° C. to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid. The deletion and marker removal were confirmed by PCR with primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as PNY1503 (BP1064).
BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1083; PNY1504).
Construction of Strains NYLA74, NYLA83, and NYLA84Insertion-inactivation of endogenous PDC1 and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase is described as follows:
Construction of pRS425::GPM-sadB
A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 70) from Achromobacter xylosoxidans (disclosed in U.S. Patent Application Publication No. 2009/0269823) was cloned. The coding region of this gene called sadB for secondary alcohol dehydrogenase (SEQ ID NO: 69) was amplified using standard conditions from A. xylosoxidans genomic DNA, prepared using a Gentra® Puregene® kit (Qiagen, Valencia, Calif.) following the recommended protocol for gram negative organisms using forward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively. The PCR product was TOPO®-Blunt cloned into pCR®4 BLUNT (Invitrogen™, Carlsbad, Calif.) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-1 cells. Plasmid was subsequently isolated from four clones, and the sequence verified.
The sadB coding region was PCR amplified from pCR4Blunt::sadB. PCR primers contained additional 5′ sequences that would overlap with the yeast GPM1 promoter and the ADH1 terminator (N583 and N584, provided as SEQ ID NOs: 76 and 77). The PCR product was then cloned using “gap repair” methodology in Saccharomyces cerevisiae (Ma, et al., Gene 58:201-216, 1987) as follows. The yeast-E. coli shuttle vector pRS425::GPM::kivD::ADH which contains the GPM1 promoter (SEQ ID NO: 72), kivD coding region from Lactococcus lactis (SEQ ID NO: 71), and ADH1 terminator (SEQ ID NO: 73) (described in U.S. Patent Application Publication No. 2007/0092957 A1, Example 17) was digested with BbvCI and PacI restriction enzymes to release the kivD coding region. Approximately 1 μg of the remaining vector fragment was transformed into S. cerevisiae strain BY4741 along with 1 μg of sadB PCR product. Transformants were selected on synthetic complete medium lacking leucine. The proper recombination event, generating pRS425::GPM-sadB, was confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and 109).
Construction of pdc6:: PGPM1-sadB Integration Cassette and PDC6 Deletion:
A pdc6::PGPM1-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425::GPM-sadB (SEQ ID NO: 78) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:80) contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton, et al., Gene 77:61-68, 1989) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers 114117-11A through 114117-11D (SEQ ID NOs: 81, 82, 83, and 84), and 114117-13A and 114117-13B (SEQ ID NOs: 85 and 86).
The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′ ˜50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC No. 200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 87 and 88), and 112590-34F and 112590-49E (SEQ ID NOs: 89 and 90) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::PGPM1-sadB-ADH1t.
Construction of pdc1:: PPDC1-ilvD Integration Cassette and PDC1 Deletion:
A pdc1:: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO: 91) from pLH468 (SEQ ID NO: 2) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton, et al., Gene 77:61-68, 1989) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers 114117-27A through 114117-27D (SEQ ID NOs: 111, 112, 113, and 114).
The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′ ˜50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93), and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t.
HIS3 DeletionTo delete the endogenous HIS3 coding region, a his3::URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 95). URA3r2 contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers 114117-45A and 114117-45B (SEQ ID NOs: 96 and 97) which generated a ˜2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain, called NYLA73, has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3.
Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:
A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs: 98 and 99) which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/mL) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 100 and 101). The identified correct transformants have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 pdc5::kanMX4. The strain was named NYLA74.
Plasmid vectors pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB were transformed into NYLA74 to create a butanediol producing strain (NGCI-047).
Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were transformed into NYLA74 to create an isobutanol producing strain (NGCI-049).
Deletion of HXK2 (Hexokinase II):A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (described above) using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers 384 and 385 (SEQ ID NOs: 102 and 103) which generated a ˜2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5′ region upstream of the HXK2 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HXK2 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region using primers N869 and N871 (SEQ ID NOs: 104 and 105). The URA3r2 marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth, and by PCR to verify correct marker removal using primers N946 and N947 (SEQ ID NOs: 106 and 107). The resulting identified strain named NYLA83 has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.
Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:
A pdc5::kanMX4 cassette was PCR-amplified as described above. The PCR fragment was transformed into NYLA83, and transformants were selected and screened as described above. The identified correct transformants named NYLA84 have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4.
Plasmid vectors pLH468 and pLH532 were simultaneously transformed into strain NYLA84 (BY4700 pdc6::PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the resulting “butanologen NYLA84” was maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1 ethanol at 30° C.
Expression Vector pLH468
The pLH468 plasmid (SEQ ID NO:2) was constructed for expression of DHAD, KivD and HADH in yeast and is described in U.S. Patent Application Publication No. 2009/0305363, herein incorporated by reference. pLH486 was constructed to contain: a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nt position 3313-4849) expressed from the S. cerevisiae FBA1 promoter (nt 2109-3105) followed by the FBA1 terminator (nt 4858-5857) for expression of DHAD; a chimeric gene having the coding region of codon optimized horse liver alcohol dehydrogenase (nt 6286-7413) expressed from the S. cerevisiae GPM1 promoter (nt 7425-8181) followed by the ADH1 terminator (nt 5962-6277) for expression of ADH; and a chimeric gene having the coding region of the codon-optimized kivD gene from Lactococcus lactis (nt 9249-10895) expressed from the TDH3 promoter (nt 10896-11918) followed by the TDH3 terminator (nt 8237-9235) for expression of KivD.
Coding regions for Lactococcus lactis ketoisovalerate decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0, Inc. (Menlo Park, Calif.) based on codons that were optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 71 and 118, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs:117 and 119, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426::PTDH3-kivDy-TDH3t), vector pNY8 (SEQ ID NO: 121; also named pRS426.GPD-ald-GPDt, described in U.S. Patent Application Publication No. 2008/0182308, Example 17, which is herein incorporated by reference) was digested with AscI and SfiI enzymes, thus excising the GPD promoter and the ald coding region. A TDH3 promoter fragment (SEQ ID NO: 122) from pNY8 was PCR amplified to add an AscI site at the 5′ end and an SpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067 (SEQ ID NOs: 123 and 124). The AscI/SfiI digested pNY8 vector fragment was ligated with the TDH3 promoter PCR product digested with AscI and SpeI and the SpeI-SfiI fragment containing the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. The triple ligation generated vector pLH467 (pRS426::PTDH3-kivDy-TDH3t). pLH467 was verified by restriction mapping and sequencing.
pLH435 (pRS425::PGPM1-Hadhy-ADH1t) was derived from vector pRS425::GPM-sadB (SEQ ID NO: 78) which is described in U.S. Provisional Application Ser. No. 61/058,970, Example 3, which is herein incorporated by reference. pRS425::GPM-sadB is the pRS425 vector (ATCC No. 77106) with a chimeric gene containing the GPM1 promoter (SEQ ID NO:72), coding region from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB; DNA SEQ ID NO: 69; protein SEQ ID NO:70: disclosed in U.S. Patent Application Publication No. 2009/0269823), and ADH1 terminator (SEQ ID NO: 73). pRS425::GPMp-sadB contains BbvI and PacI sites at the 5′ and 3′ ends of the sadB coding region, respectively. A NheI site was added at the 5′ end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NOs: 126 and 127) to generate vector pRS425-GPMp-sadB-NheI, which was verified by sequencing. pRS425::PGPM1-sadB-NheI was digested with NheI and PacI to drop out the sadB coding region, and ligated with the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadhy-DNA2.0 to create pLH435.
To combine KivD and HADH expression cassettes in a single vector, yeast vector pRS411 (ATCC No. 87474) was digested with SacI and NotI, and ligated with the SacI-SalI fragment from pLH467 that contains the PTDH3-kivDy-TDH3t cassette together with the SalI-NotI fragment from pLH435 that contains the PGPM1-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the vector pRS411::PTDH3-kivDy-PGPM1-Hadhy (pLH441) which was verified by restriction mapping.
In order to generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy, and Hadhy, pRS423 FBA ilvD(Strep) (SEQ ID NO: 128) which is described in U.S. Patent Application Publication No. 2010/0081154 as the source of the IlvD gene, was used. This shuttle vector contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector has an FBA1 promoter (nt 2111 to 3108; SEQ ID NO: 120) and FBA terminator (nt 4861 to 5860; SEQ ID NO: 129). In addition, it carries the H is marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO: 115; protein SEQ ID NO: 116) from Streptococcus mutans UA159 (ATCC No. 700610) is between the FBA promoter and FBA terminator forming a chimeric gene for expression. In addition, there is a lumio tag fused to the ilvD coding region (nt 4829-4849).
The first step was to linearize pRS423 FBA ilvD(Strep) (also called pRS423-FBA(SpeI)-ilvD(Streptococcus mutans)-Lumio) with SacI and SacII (with SacII site blunt ended using T4 DNA polymerase), to give a vector with total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI site blunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment. This fragment was ligated with the 9,482 bp vector fragment from pRS423-FBA(SpeI)-ilvD(Streptococcus mutans)-Lumio. This generated vector pLH468 (pRS423::PFBA1-ilvD(Strep) Lumio-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t) which was confirmed by restriction mapping and sequencing.
pLH532 Construction
The pLH532 plasmid (SEQ ID NO: 130) was constructed for expression of ALS and KARI in yeast. pLH532 is a pHR81 vector (ATCC No. 87541) containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO: 139), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO: 137; protein SEQ ID NO: 138) and CYC1 terminator2 (SEQ ID NO: 133); 2) an ILV5 promoter (SEQ ID NO: 134), Pf5.IlvC coding region (SEQ ID NO: 132) and ILV5 terminator (SEQ ID NO: 135); and 3) the FBA1 promoter (SEQ ID NO: 136), S. cerevisiae KARI coding region (ILV5; SEQ ID NO: 131); and CYC1 terminator.
The Pf5.IlvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens that was described in U.S. Patent Application Publication No. 2009/0163376, which is herein incorporated by reference.
The Pf5.IlvC coding region was synthesized by DNA2.0, Inc. (Menlo Park, Calif.; SEQ ID NO: 132) based on codons that were optimized for expression in Saccharomyces cerevisiae.
pYZ090 Construction
pYZ090 (SEQ ID NO: 1) is based on the pHR81 (ATCC No. 87541) backbone and was constructed to contain a chimeric gene having the coding region of the alsS gene from Bacillus subtilis (nt position 457-2172) expressed from the yeast CUP1 promoter (nt 2-449) and followed by the CYC1 terminator (nt 2181-2430) for expression of ALS, and a chimeric gene having the coding region of the ilvC gene from Lactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4682-5304) for expression of KARI.
pYZ067 Construction
pYZ067 was constructed to contain the following chimeric genes: 1) the coding region of the ilvD gene from S. mutans UA159 (nt position 2260-3971) expressed from the yeast FBA1 promoter (nt 1161-2250) followed by the FBA terminator (nt 4005-4317) for expression of dihydroxy acid dehydratase (DHAD), 2) the coding region for horse liver ADH (nt 4680-5807) expressed from the yeast GPM promoter (nt 5819-6575) followed by the ADH1 terminator (nt 4356-4671) for expression of alcohol dehydrogenase, and 3) the coding region of the KivD gene from Lacrococcus lactis (nt 7175-8821) expressed from the yeast TDH3 promoter (nt 8830-9493) followed by the TDH3 terminator (nt 5682-7161) for expression of ketoisovalerate decarboxylase.
pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB and pLH475-Z4B8 Construction
Construction of pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB and pLH475-Z4B8 is described in U.S. Patent Application Publication No. 2009/0305363, incorporated herein by reference.
EXAMPLESThe following nonlimiting examples will further illustrate the invention. It should be understood that, while the following examples involve corn as feedstock and COFA as ISPR extractant obtained from enzymatic hydrolysis of corn lipids, other biomass sources can be used for feedstock and enzymatic hydrolysis of biomass oil, without departing from the present invention.
As used herein, the meaning of abbreviations used was as follows: “g” means gram(s), “kg” means kilogram(s), “L” means liter(s), “mL” means milliliter(s), “μL” means microliter(s), “mL/L” means milliliter(s) per liter, “mL/min” means milliliter(s) per min, “DI” means deionized, “uM” means micrometer(s), “nm” means nanometer(s), “w/v” means weight/volume, “OD” means optical density, “OD600” means optical density at a wavelength of 600 nM, “dcw” means dry cell weight, “rpm” means revolutions per minute, “° C.” means degree(s) Celsius, “° C./min” means degrees Celsius per minute, “slpm” means standard liter(s) per minute, “ppm” means part per million, “pdc” means pyruvate decarboxylase enzyme followed by the enzyme number.
General Methods Seed Flask GrowthA Saccharomyces cerevisiae strain that was engineered to produce isobutanol from a carbohydrate source, with pdc1 deleted, pdc5 deleted, and pdc6 deleted, was grown to 0.55-1.1 g/L dcw (OD600 1.3-2.6—Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Mass.) in seed flasks from a frozen culture. The culture was grown at 26° C. in an incubator rotating at 300 rpm. The frozen culture was previously stored at −80° C. The composition of the first seed flask medium was:
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- 3.0 g/L dextrose
- 3.0 g/L ethanol, anhydrous
- 3.7 g/L ForMedium™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: without HIS, without URA (Reference No. DSCK162CK)
- 6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
Twelve milliliters from the first seed flask culture was transferred to a 2 L flask and grown at 30° C. in an incubator rotating at 300 rpm. The second seed flask has 220 mL of the following medium:
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- 30.0 g/L dextrose
- 5.0 g/L ethanol, anhydrous
- 3.7 g/L ForMedium™ Synthetic Complete Amino Acid (Kaiser) Drop-Out: without HIS, without URA (Reference No. DSCK162CK)
- 6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
- 0.2 M MES Buffer titrated to pH 5.5-6.0
The culture was grown to 0.55-1.1 g/L dcw (OD600 1.3-2.6). An addition of 30 mL of a solution containing 200 g/L peptone and 100 g/L yeast extract was added at this cell concentration. Then, an addition of 300 mL of 0.2 uM filter sterilized Cognis, 90-95% oleyl alcohol was added to the flask. The culture continues to grow to >4 g/L dcw (OD600>10) before being harvested and added to the fermentation.
Fermentation Preparation Initial Fermentation Vessel PreparationA glass jacked, 2 L fermentation vessel (Sartorius AG, Goettingen, Germany) was charged with house water to 66% of the liquefaction weight. A pH probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bonaduz AG, Bonaduz, Switzerland) was calibrated through the Sartorius DCU-3 Control Tower Calibration menu. The zero was calibrated at pH=7. The span was calibrated at pH=4. The probe was then placed into the fermentation vessel through the stainless steel head plate. A dissolved oxygen probe (pO2 probe) was also placed into the fermentation vessel through the head plate. Tubing used for delivering nutrients, seed culture, extracting solvent, and base were attached to the head plate and the ends were foiled. The entire fermentation vessel was placed into a Steris (Steris Corporation, Mentor, Ohio) autoclave and sterilized in a liquid cycle for 30 minutes.
The fermentation vessel was removed from the autoclave and placed on a load cell. The jacket water supply and return line was connected to the house water and clean drain, respectively. The condenser cooling water in and water out lines were connected to a 6-L recirculating temperature bath running at 7° C. The vent line that transfers the gas from the fermentation vessel was connected to a transfer line that was connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Mass.). The sparger line was connected to the gas supply line. The tubing for adding nutrients, extract solvent, seed culture, and base was plumbed through pumps or clamped closed.
The fermentation vessel temperature was controlled at 55° C. with a thermocouple and house water circulation loop. Wet corn kernels (#2 yellow dent) were ground using a hammer mill with a 1.0 mm screen, and the resulting ground whole corn kernels were then added to the fermentation vessel at a charge that was 29-30% (dry corn solids weight) of the liquefaction reaction mass.
Lipase Treatment Pre-LiquefactionA lipase enzyme stock solution was added to the fermentation vessel to a final lipase concentration of 10 ppm. The fermentation vessel was held at 55° C., 300 rpm, and 0.3 slpm N2 overlay for >6 hrs. After the lipase treatment was complete, liquefaction was performed as described below (Liquefaction).
LiquefactionAn alpha-amylase was added to the fermentation vessel per its specification sheet while the fermentation vessel was mixing at 300-1200 rpm, with sterile, house N2 being added at 0.3 slpm through the sparger. The temperature set-point was changed from 55° C. to 85° C. When the temperature was >80° C., the liquefaction cook time was started and the liquefaction cycle was held at >80° C. for 90-120 minutes. The fermentation vessel temperature set-point was set to the fermentation temperature of 30° C. after the liquefaction cycle was complete. N2 was redirected from the sparger to the head space to prevent foaming without the addition of a chemical antifoaming agent.
Lipase Treatment Post-LiquefactionThe fermentation vessel temperature was set to 55° C. instead of 30° C. after the liquefaction cycle was complete (Liquefaction). The pH was manually controlled at pH=5.8 by making bolus additions of acid or base when needed. A lipase enzyme stock solution was added to the fermentation vessel to a final lipase concentration of 10 ppm. The fermentation vessel was held at 55° C., 300 rpm, and 0.3 slpm N2 overlay for >6 hrs. After the Lipase Treatment was complete, the fermentation vessel temperature was set to 30° C.
Lipase Heat Inactivation Treatment (Heat Kill Treatment Method)The fermentation vessel temperature was held at >80° C. for >15 minutes to inactivate the lipase. After the Heat Inactivation Treatment was complete, the fermentation vessel temperature was set to 30° C.
Nutrient Addition Prior to InoculationEthanol (6.36 mL/L, post-inoculation volume, 200 proof, anhydrous) was added to the fermentation vessel just prior to inoculation. Thiamine was added to a final concentration of 20 mg/L and 100 mg/L nicotinic acid was also added just prior to inoculation.
Oleyl Alcohol or Corn Oil Fatty Acids Addition Prior to InoculationAdded 1 L/L (post-inoculation volume) of oleyl alcohol or corn oil fatty acids immediately after inoculation.
Fermentation Vessel InoculationThe fermentation vessels pO2 probe was calibrated to zero while N2 was being added to the fermentation vessel. The fermentation vessels pO2 probe was calibrated to its span with sterile air sparging at 300 rpm. The fermentation vessel was inoculated after the second seed flask with >4 g/L dcw. The shake flask was removed from the incubator/shaker for 5 minutes allowing a phase separation of the oleyl alcohol phase and the aqueous phase. The aqueous phase (110 mL) was transferred to a sterile, inoculation bottle. The inoculum was pumped into the fermentation vessel through a peristaltic pump.
Fermentation Vessel Operating ConditionsThe fermentation vessel was operated at 30° C. for the entire growth and production stages. The pH was allowed to drop from a pH between 5.7-5.9 to a control set-point of 5.2 without adding any acid. The pH was controlled for the remainder of the growth and production stage at a pH=5.2 with ammonium hydroxide. Sterile air was added to the fermentation vessel, through the sparger, at 0.3 slpm for the remainder of the growth and production stages. The pO2 was set to be controlled at 3.0% by the Sartorius DCU-3 Control Box PID control loop, using stir control only, with the stirrer minimum being set to 300 rpm and the maximum being set to 2000 rpm. The glucose was supplied through simultaneous saccharification and fermentation of the liquified corn mash by adding a α-amylase (glucoamylase). The glucose was kept excess (1-50 g/L) for as long as starch was available for saccharification.
Analytical Gas AnalysisProcess air was analyzed on a Thermo Prima (Thermo Fisher Scientific Inc., Waltham, Mass.) mass spectrometer. This was the same process air that was sterilized and then added to each fermentation vessel. Each fermentation vessel's off-gas was analyzed on the same mass spectrometer. This Thermo Prima dB has a calibration check run every Monday morning at 6:00 am. The calibration check was scheduled through the Gas Works v1.0 (Thermo Fisher Scientific Inc., Waltham, Mass.) software associated with the mass spectrometer. The gas calibrated for were:
Carbon dioxide was checked at 5% and 15% during calibration cycle with other known bottled gases. Oxygen was checked at 15% with other known bottled gases. Based on the analysis of the off-gas of each fermentation vessel, the amount of isobutanol stripped, oxygen consumed, and carbon dioxide respired into the off-gas was measured by using the mass spectrometer's mole fraction analysis and gas flow rates (mass flow controller) into the fermentation vessel. Calculate the gassing rate per hour and then integrating that rate over the course of the fermentation.
Biomass MeasurementA 0.08% Trypan Blue solution was prepared from a 1:5 dilution of 0.4% Trypan Blue in NaCl (VWR BDH8721-0) with 1×PBS. A 1.0 mL sample was pulled from a fermentation vessel and placed in a 1.5 mL Eppendorf centrifuge tube and centrifuged in an Eppendorf, 5415C at 14,000 rpm for 5 minutes. After centrifugation, the top solvent layer was removed with an m200 Variable Channel BioHit pipette with 20-200 μL BioHit pipette tips. Care was made not to remove the layer between the solvent and aqueous layers. Once the solvent layer was removed, the sample was re-suspended using a Vortex-Genie® set at 2700 rpm.
A series of dilutions was required to prepare the ideal concentration for hemacytometer counts. If the OD was 10, a 1:20 dilution would be performed to achieve 0.5 OD which would give the ideal amount of cells to be counted per square, 20-30. In order to reduce inaccuracy in the dilution due to corn solids, multiple dilutions with cut 100-1000 μL BioHit pipette tips were required. Approximately, 1 cm was cut off the tips to increase the opening which prevented the tip from clogging. For a 1:20 final dilution, an initial 1:1 dilution of fermentation sample and 0.9% NaCl solution was prepared. Then, a 1:1 dilution of the previous solution (i.e., the initial 1:1 dilution) and 0.9% NaCl solution (the second dilution) was generated followed by a 1:5 dilution of the second dilution and Trypan Blue Solution. Samples were vortexed between each dilution and cut tips were rinsed into the 0.9% NaCl and Trypan Blue solutions.
The cover slip was carefully placed on top of the hemacytometer (Hausser Scientific Bright-Line 1492). An aliquot (10 μL) was drawn of the final Trypan Blue dilution with an m20 Variable Channel BioHit pipette with 2-20 μL BioHit pipette tips and injected into the hemacytometer. The hemacytometer was placed on the Zeis Axioskop 40 microscope at 40× magnification. The center quadrant was broken into 25 squares and the four corner and center squares in both chambers were then counted and recorded. After both chambers were counted, the average was taken and multiplied by the dilution factor (20), then by 25 for the number for squares in the quadrant in the hemacytometer, and then divided by 0.0001 mL which is the volume of the quadrant that was counted. The sum of this calculation is the number cells per mL.
LC Analysis of Fermentation Products in the Aqueous PhaseSamples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (about one hour). Approximately 300 μL of sample was transferred with a m1000 Variable Channel BioHit pipette with 100-1000 μL BioHit pipette tips into a 0.2 um centrifuge filter (Nanosep® MF modified nylon centrifuge filter), then centrifuged using a Eppendorf, 5415C for five minutes at 14,000 rpm. Approximately 200 μL of filtered sample was transferred into a 1.8 auto sampler vial with a 250 μL glass vial insert with polymer feet. A screw cap with PTFE septa was used to cap the vial before vortexing the sample with a Vortex-Genie® set at 2700 rpm.
Sample was then run on Agilent 1200 series LC equipped with binary, isocratic pumps, vacuum degasser, heated column compartment, sampler cooling system, UV DAD detector and R1 detector. The column used was an Aminex HPX-87H, 300×7.8 with a Bio-Rad Cation H refill, 30×4.6 guard column. Column temperature was 40° C., with a mobile phase of 0.01 N sulfuric acid at a flow rate of 0.6 mL/min for 40 minutes. Results are shown in Table 1.
Samples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (about one hour). Approximately 150 μL of sample was transferred using a m1000 Variable Channel BioHit pipette with 100-1000 μL BioHit pipette tips into a 1.8 auto sampler vial with a 250 μL glass vial insert with polymer feet. A screw cap with PTFE septa was used to cap the vial.
Sample was then run on Agilent 7890A GC with a 7683B injector and a G2614A auto sampler. The column was a HP-InnoWax column (30 m×0.32 mm ID, 0.25 μm film). The carrier gas was helium at a flow rate of 1.5 mL/min measured at 45° C. with constant head pressure; injector split was 1:50 at 225° C.; oven temperature was 45° C. for 1.5 minutes, 45° C. to 160° C. at 10° C./min for 0 minutes, then 230° C. at 35° C./min for 14 minutes for a run time of 29 minutes. Flame ionization detection was used at 260° C. with 40 mL/min helium makeup gas. Results are shown in Table 2.
Samples analyzed for fatty acid butyl esters were run on Agilent 6890 GC with a 7683B injector and a G2614A auto sampler. The column was a HP-DB-FFAP column (15 meters×0.53 mm ID (Megabore), 1-micron film thickness column (30 m×0.32 mm ID, 0.25 μm film). The carrier gas was helium at a flow rate of 3.7 mL/min measured at 45° C. with constant head pressure; injector split was 1:50 at 225° C.; oven temperature was 100° C. for 2.0 minutes, 100° C. to 250° C. at 10° C./min, then 250° C. for 9 minutes for a run time of 26 minutes. Flame ionization detection was used at 300° C. with 40 mL/min helium makeup gas. The following GC standards (Nu-Chek Prep; Elysian, Minn.) were used to confirm the identity of fatty acid isobutyl ester products: iso-butyl palmitate, iso-butyl stearate, iso-butyl oleate, iso-butyl linoleate, iso-butyl linolenate, iso-butyl arachidate.
Examples 1-14 describe various fermentation conditions that may be used for the claimed methods. As an example, some fermentations were subjected to Lipase Treatment pre-liquefaction and others were subjected to Lipase Treatment post-liquefaction. In other examples, the fermentation was subjected to Heat inactivation Treatment. Following fermentation, the effective isobutanol titer (Eff Iso Titer) was measured, that is, the total grams of isobutanol produced per liter aqueous volume. Results are shown in Table 3.
Example 1 ControlExperiment identifier 2010Y014 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 2Experiment identifier 2010Y015 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase Treatment Post-Liquefaction method, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 3Experiment identifier 2010Y016 included: Seed Flask Growth method, Initial Fermentation vessel Preparation method, Liquefaction method, Lipase Treatment Post-Liquefaction method, Nutrient Addition Prior to Inoculation method with the exception of the exclusion of ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 4Experiment identifier 2010Y017 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Heat Kill Treatment method Post-Liquefaction, Nutrient Addition Prior to Inoculation method with the exception of the exclusion of ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 5Experiment identifier 2010Y018 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase Treatment Post-Liquefaction method with the exception of only adding 7.2 ppm lipase after liquefaction, Heat Kill Treatment method post-liquefaction, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 6 ControlExperiment identifier 2010Y019 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Heat Kill Treatment method Post-Liquefaction, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 7 ControlExperiment identifier 2010Y021 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-Liquefaction method, the Liquefaction method, Heat Kill Treatment during liquefaction, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 8Experiment identifier 2010Y022 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 9Experiment identifier 2010Y023 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase Treatment Post-Liquefaction method, no Heat Kill Treatment, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Corn oil fatty acids made from crude corn oil was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 10Experiment identifier 2010Y024 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-Liquefaction method, Liquefaction method, Heat Kill Treatment during liquefaction, Nutrient Addition Prior to Inoculation method with the exception of there being no addition of ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 11Experiment identifier 2010Y029 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-Liquefaction method, Liquefaction method, Heat Kill Treatment during liquefaction, the Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Corn oil fatty acids made from crude corn oil was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 12Experiment identifier 2010Y030 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-Liquefaction method, Liquefaction method, Heat Kill Treatment during liquefaction, Nutrient Addition Prior to Inoculation method with the exception of there being no addition of ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Corn oil fatty acids made from crude corn oil was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 13 ControlExperiment identifier 2010Y031 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase Treatment Post Liquefaction method, no Heat Kill Treatment, Nutrient Addition Prior to Inoculation method with the exception of there being no addition of ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Corn oil fatty acids made from crude corn oil was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
Example 14Experiment identifier 2010Y032 included: Seed Flask Growth method, Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase Treatment Post-Liquefaction method, no Heat Kill Treatment, Nutrient Addition Prior to Inoculation method, Fermentation Vessel Inoculation method, Fermentation Vessel Operating Conditions method, and all of the Analytical methods. Corn oil fatty acids made from crude corn oil was added in a single batch between 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.
The experimental identifier was GLNOR432A. NGCI-047 (a butanediol producer) was grown in 25 mL medium in a 250 mL flask from a frozen vial to ˜1 OD. The pre-seed culture was transferred to a 2 L flask and grown to 1.7-1.8 OD. The medium for both flasks was:
3.0 g/L dextrose
3.0 g/L ethanol, anhydrous
6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
1.4 g/L Yeast Dropout Mix (Sigma Y2001)
10 mL/L 1% w/v L-Leucine stock solution
2 mL/L 1% w/v L-Tryptophan stock solution
A 1 L, Applikon fermentation vessel was inoculated with 60 mL of the seed flask. The fermentation vessel contained 700 mL of the following sterile medium:
20.0 g/L dextrose
8.0 mL/L ethanol, anhydrous
6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
2.8 g/L Yeast Dropout Mix (Sigma Y2001)
20 mL/L 1% w/v L-Leucine stock solution
4 mL/L 1% w/v L-Tryptophan stock solution
0.5 mL Sigma 204 Antifoam
0.8 mL/L 1% w/v Ergesterol solution in 1:1::Tween 80:Ethanol
The residual glucose was kept excess with a 50% w/w glucose solution. The dissolved oxygen concentration of the fermentation vessel was controlled at 30% with stir control. The pH was controlled at pH=5.5. The fermentation vessel was sparged with 0.3 slpm of sterile, house air. The temperature was controlled at 30° C.
Example 16The experimental identifier was GLNOR434A. This example is the same as example 15 with the exception of the addition of 3 g of oleic acid and the addition of 3 g of palmitic acid prior to inoculation. NGCI-047 (a butanediol producer) was the biocatalyst.
The experimental identifier was GLNOR435A. This example was the same as example 15 except it was inoculated with NGCI-049 (an isobutanol producer).
Example 18The experimental identifier was GLNOR437A. This example was the same as Example 16 except it was inoculated with NGCI-049 (an isobutanol producer).
The experimental identifier was 090420—3212. This example was run similarly to Example 15 except it was inoculated with butanologen NYLA84 (an isobutanol producer). This fermentation was run in a 1 L Sartorius fermentation vessel.
Example 20The experimental identifier was 2009Y047. This example was run similarly to Example 16 except it was inoculated with butanologen NYLA84 (an isobutanol producer). This fermentation was run in a 1 L Sartorius fermentation.
Samples of broth and oleyl alcohol taken from fermentations run as described above in Examples 1, 2, and 3 were analyzed for wt % lipid (derivatized as fatty acid methyl esters, FAME) and for wt % free fatty acid (FFA, derivatized as fatty acid methyl esters, FAME) according to the method described by E. G. Bligh and W. J. Dyer (Canadian Journal of Biochemistry and Physiology, 37:911-17, 1959, hereafter Reference 1). The liquefied corn mash that was prepared for each of the three fermentations was also analyzed for wt % lipid and for wt % FFA after treatment with Lipolase® 100 L (Novozymes) (10 ppm of Lipolase® total soluble protein (BCA protein analysis, Sigma Aldrich)) per kg of liquefaction reaction mass containing 30 wt % ground corn kernels). No lipase was added to the liquefied corn mash in Example 1 (control), and the fermentations described in Examples 2 and 3 containing liquefied corn mash treated with lipase (no heat inactivation of lipase) were identical except that no ethanol was added to the fermentation described in Example 3.
The % FFA in lipase-treated liquefied corn mash prepared for fermentations run as described in Examples 2 and 3 was 88% and 89%, respectively, compared to 31% without lipase treatment (Example 1). At 70 h (end of run (EOR)), the concentration of FFA in the OA phase of fermentations run as described in Examples 2 and 3 (containing active lipase) was 14% and 20%, respectively, and the corresponding increase in lipids (measured as corn oil fatty acid methyl ester derivatives) was determined by GC/MS to be due to the lipase-catalyzed esterification of COFA by OA, where COFA was first produced by lipase-catalyzed hydrolysis of corn oil in the liquefied corn mash. Results are shown in Table 5.
Tap water (918.4 g) was added to a jacketed 2-L resin kettle, then 474.6 g wet weight (417.6 g dry weight) of ground whole corn kernels (1.0 mm screen on hammer mill) was added with stirring. The mixture was heated to 55° C. with stirring at 300 rpm, and the pH adjusted to 5.8 with 2 N sulfuric acid. To the mixture was added 14.0 g of an aqueous solution containing 0.672 g of Spezyme®-FRED L (Genencor®, Palo Alto, Calif.), and the temperature of the mixture increased to 85° C. with stirring at 600 rpm and pH 5.8. After 120 minutes at 85° C., the mixture was cooled to 50° C. and 45.0 mL aliquots of the resulting liquefied corn mash were transferred to 50-mL polypropylene centrifuge tubes and stored frozen at −80° C.
In a first reaction, 50 g of liquefied corn mash prepared as described above was mixed with 10 ppm Lipolase® 100 L (Novozymes) for 6 h at 55° C. and with no inactivation of lipase at 85° C. for 1 h, the mixture was cooled to 30° C. In a second reaction, 50 g of liquefied corn mash was mixed with 10 ppm Lipolase® for 6 h at 55° C., then heated to 85° C. for 1 h (lipase inactivation), then cooled to 30° C. In a third reaction, 50 g of liquefied corn mash without added lipase was mixed for 6 h at 55° C., and with no heating at 85° C. for 1 h, the mixture was cooled to 30° C., 38 g of oleyl alcohol was added, and the resulting mixture stirred for 73 h at 30° C. In a fourth reaction, 50 g of liquefied corn mash without added lipase was mixed for 6 h at 55° C., then heated to 85° C. for 1 h, then cooled to 30° C. Each of the four reaction mixtures was sampled at 6 h, then 38 g of oleyl alcohol added, and the resulting mixtures stirred at 30° C. and sampled at 25 h and 73 h. Samples (both liquefied mash and oleyl alcohol (OA)) were analyzed for wt % lipid (derivatized as fatty acid methyl esters, FAME) and for wt % free fatty acid (FFA, derivatized as fatty acid methyl esters, FAME) according to the method described by Reference 1.
The % FFA in the OA phase of the second reaction run with heat inactivation of lipase prior to OA addition was 99% at 25 h and 95% at 73 h, compared to only 40% FFA and 21% FFA at 25 h and 73 h, respectively, when the lipase in lipase-treated liquefied corn mash was not heat inactivated (first reaction). No significant change in % FFA was observed in the two control reactions without added lipase. Results are shown in Table 6.
Three fermentations were run as described above in Examples 4, 5, and 6. No lipase was added to the liquefied corn mash in Examples 4 and 6 prior to fermentation, and the Lipase Treatment of the liquefied corn mash in the fermentation described in Example 5 (using 7.2 ppm of Lipolase® total soluble protein) was followed immediately by Heat Inactivation Treatment (to completely inactivate the lipase), and subsequently followed by Nutrient Addition Prior to Inoculation and fermentation. The % FFA in liquefied corn mash prepared without lipase treatment for fermentations run as described in Examples 4 and 6 was 31% and 34%, respectively, compared to 89% with lipase treatment (Example 5). Over the course of the fermentations listed in Table 10, the concentration of FFA in the OA phase did not decrease in any of the three fermentations, including that containing heat-inactivated lipase. The % FFA in the OA phase of the fermentation run according to Example 5 (with heat inactivation of lipase prior to fermentation) was 95% at 70 h (end of run (EOR)), compared to only 33% FFA for the remaining two fermentations (Examples 4 and 6) where liquefied corn mash was not treated with lipase. Results are shown in Table 7.
Tap water (1377.6 g) was added into each of two jacketed 2-L resin kettles, then 711.9 g wet weight (625.8 g dry weight) of ground whole corn kernels (1.0 mm screen on hammer mill) was added to each kettle with stirring. Each mixture was heated to 55° C. with stirring at 300 rpm, and the pH adjusted to 5.8 with 2 N sulfuric acid. To each mixture was added 21.0 g of an aqueous solution containing 1.008 g of Spezyme®-FRED L (Genencor®, Palo Alto, Calif.). To one mixture was then added 10.5 mL of aqueous solution of Lipolase® 100L Solution (21 mg total soluble protein, 10 ppm lipase final concentration) and to the second mixture was added 1.05 mL of aqueous solution of Lipolase® 100L Solution (2.1 mg total soluble protein, 1.0 ppm lipase final concentration). Samples were withdrawn from each reaction mixture at 1 h, 2 h, 4 h and 6 h at 55° C., then the temperature of the mixture was increased to 85° C. with stirring at 600 rpm and pH 5.8, and a sample was taken when the mixture first reached 85° C. After 120 minutes at 85° C., a sample was taken and the mixtures were cooled to 50° C. and final samples of the resulting liquefied corn mash were transferred to 50-mL polypropylene centrifuge tubes; all samples were stored frozen at −80° C.
In two separate reactions, a 50 g sample of the 10 ppm lipase-treated liquefied corn mash or a 55 g sample of the 1.0 ppm lipase-treated liquefied corn mash prepared as described above was mixed with oleyl alcohol (OA) (38 g) at 30° C. for 20 h, then the liquefied mash and OA in each reaction mixture were separated by centrifugation and each phase analyzed for wt % lipid (derivatized as fatty acid methyl esters, FAME) and for wt % free fatty acid (FFA, derivatized as fatty acid methyl esters, FAME) according to the method described by Reference 1. The % FFA in the OA phase of the liquefied mash/OA mixture prepared using heat inactivation of 10 ppm lipase during liquefaction was 98% at 20 h, compared to only 62% FFA in the OA phase of the liquefied mash/OA mixture prepared using heat inactivation of 1.0 ppm lipase during liquefaction. Results are shown in Table 8.
Seven reaction mixtures containing tap water (67.9 g) and ground whole corn kernels (35.1 g wet wt., ground with 1.0 mm screen using a hammer mill) at pH 5.8 were stirred at 55° C. in stoppered flasks. A 3-mL sample (t=0 h) was removed from each flask and the sample immediately frozen on dry ice, then ca. 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0) containing 1 mg total soluble protein (10 ppm final concentration in reaction mixture) of one of the following lipases (Novozymes) were added to one of each flask: Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozyme® CALA L, and Palatase 20000L; no lipase was added to the seventh flask. The resulting mixtures were stirred at 55° C. in stoppered flasks, and 3-mL samples were withdrawn from each reaction mixture at 1 h, 2 h, 4 h and 6 h and immediately frozen in dry ice until analyzed for wt % lipid (derivatized as fatty acid methyl esters, FAME) and for wt % free fatty acid (FFA, derivatized as fatty acid methyl esters, FAME) according to the method described by Reference 1, and the percent free fatty acid content was calculated relative to the total combined concentrations of lipid and free fatty acid was determined for each sample. Results are shown in Table 9.
Three fermentations were run as described above in Examples 7, 8, and 10. For fermentations run as described in Examples 7 and 10, lipase (10 ppm of Lipolase® total soluble protein) was added to the suspension of ground corn and heated at 55° C. for 6 h prior to Liquefaction to produce a liquefied corn mash containing heat-inactivated lipase. No lipase was added to the suspension of ground corn used to prepare liquefied corn mash for the fermentation described in Example 8, but the suspension was subjected to the same heating step at 55° C. prior to liquefaction. The % FFA in lipase-treated liquefied corn mash prepared for fermentations run as described in Examples 7 and 10 was 83% and 86%, respectively, compare to 41% without lipase treatment (Example 8). Over the course of the fermentations, the concentration of FFA did not decrease in any of the fermentations, including that containing heat-inactivated lipase. The % FFA in the OA phase of the fermentation run according to Examples 7 and 10 (with heat inactivation of lipase prior to fermentation) was 97% at 70 h (end of run (EOR)), compared to only 49% FFA for the fermentation run according to Example 8 where ground whole corn kernels had not been treated with lipase prior to liquefaction. Results are shown in Table 10.
Five fermentations were run as described above in Examples 9, 11, 12, 13, and 14. For the fermentations run as described in Examples 9, 13, and 14, lipase (10 ppm of Lipolase® total soluble protein) was added after Liquefaction and there was no heat-inactivation of lipase. Fermentations run as described in Examples 9 and 14 had 5 g/L of ethanol added prior to inoculation, whereas the fermentation run as described in Example 13 had no added ethanol. The fermentations run as described in Examples 11 and 12 employed the addition of 10 ppm Lipolase® total soluble protein to the suspension of ground corn prior to liquefaction, resulting in heat inactivation of lipase during liquefaction. The fermentation run as described in Example 11 had 5 g/L of ethanol added prior to inoculation, whereas the fermentation run as described in Example 12 had no added ethanol. The final total grams of isobutanol (i-BuOH) present in the COFA phase of the fermentations containing active lipase was significantly greater than the final total grams of i-BuOH present in the COFA phase of the fermentations containing inactive lipase. The final total grams of isobutanol (i-BuOH) present in the fermentation broths containing active lipase were only slightly less than the final total grams of i-BuOH present in the fermentation broths containing inactive lipase, such that the overall production of i-BuOH (as a combination of free i-BuOH and isobutyl esters of COFA (FABE)) was significantly greater in the presence of active lipase when compared to that obtained in the presence of heat-inactivated lipase. Results are shown in Tables 11 and 12.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Claims
1. A method comprising:
- (a) providing a fermentation broth comprising a recombinant microorganism that produces a product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity;
- (b) contacting the fermentation broth with a fermentable carbon source whereby the recombinant microorganism consumes the fermentable carbon source and produces the product alcohol; and
- (c) contacting the fermentation broth with fatty acids derived from biomass at a step in the fermentation process, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids.
2. The method of claim 1, wherein the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.
3. The method of claim 1, wherein biomass is derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
4. The method of claim 1, wherein the product alcohol is butanol.
5. The method of claim 1, wherein steps (b) and (c) occur substantially simultaneously.
6. The method of claim 1, wherein the fermentable carbon source is derived from the biomass.
7. The method of claim 1, wherein the fermentation broth further comprises ethanol.
8. The method of claim 1, wherein the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
9. A method for producing a product alcohol comprising:
- (a) providing biomass comprising a fermentable carbon source and oil;
- (b) converting at least a portion of the oil into fatty acids to form a biomass comprising the fatty acids;
- (c) contacting the biomass with a fermentation broth comprising a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, and wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity;
- (d) contacting the fatty acids with the fermentation broth, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids.
10. The method of claim 9, wherein the step (b) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the portion of the oil into fatty acids.
11. The method of claim 10, wherein the one or more substances comprise one or more enzymes.
12. The method of claim 11, wherein the one or more enzymes comprise lipase enzymes.
13. The method of claim 11, further comprising:
- prior to step (c), inactivating the one or more enzymes after at least a portion of the oil is hydrolyzed.
14. The method of claim 9, wherein one or more of steps (b), (c), and (d) occurs in the fermentation vessel.
15. The method of claim 9, wherein one or more of steps (b), (c), and (d) occurs substantially simultaneously.
16. The method of claim 9, wherein biomass is derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
17. The method of claim 9, wherein the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.
18. The method of claim 9, further comprising
- fermenting the fermentable carbon source to produce product alcohol.
19. The method of claim 18, wherein the product alcohol is butanol.
20. The method of claim 9, wherein the fermentation broth further comprises ethanol.
21. The method of claim 9, wherein the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
22. The method of claim 9, further comprising:
- separating the oil from the biomass prior to the step (b) of converting at least a portion of the oil into fatty acids.
23. The method of claim 9, further comprising:
- liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides; and
- contacting the liquefied biomass with a saccharification enzyme capable of converting oligosaccharides into fermentable sugar to form a saccharified biomass,
- and wherein step (c) comprises contacting the saccharified biomass with the fermentation broth comprising a recombinant microorganism.
24. A method for producing a product alcohol comprising:
- (a) providing a feedstock;
- (b) liquefying said feedstock to create a feedstock slurry;
- (c) separating the feedstock slurry to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer, and (iii) a solids layer;
- (d) obtaining an oil from the oil layer and converting at least a portion of the oil into fatty acids;
- (e) feeding the aqueous layer of (c) to a fermentation vessel containing a fermentation broth comprising a recombinant microorganism capable of producing a product alcohol from a fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity;
- (f) fermenting the fermentable carbon source of the aqueous layer to produce the product alcohol; and
- (g) contacting the fermentation broth with the fatty acids, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater in the presence of the fatty acids than the growth rate and/or the fermentable carbon consumption of the recombinant microorganism is in the absence of the fatty acids.
25. The method of claim 24, wherein the step (d) of converting at least a portion of the oil into fatty acids comprises contacting the oil with one or more substances capable of hydrolyzing the portion of the oil into fatty acids.
26. The method of claim 25, wherein the one or more substances comprises one or more enzymes.
27. The method of claim 26, wherein the one or more enzymes comprises lipase enzymes.
28. The method of claim 24, wherein feedstock comprises one or more fermentable sugars derived from corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sugar cane, soy, components obtained from milling of grains, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
29. The method of claim 24, wherein the fatty acids are selected from oleic acid, palmitic acid, myristic acid, and mixtures thereof.
30. The method of claim 18, wherein the product alcohol is butanol.
31. The method of claim 24, wherein the recombinant microorganism has a one or more pyruvate decarboxylase (PDC) gene deletions.
32. A composition comprising a recombinant microorganism comprising a reduction or elimination of pyruvate decarboxylase activity and fatty acids.
Type: Application
Filed: Jun 17, 2011
Publication Date: Dec 22, 2011
Applicant: BUTAMAX(TM) ADVANCED BIOFUELS LLC (Wilmington, DE)
Inventors: KEITH H. BURLEW (Middletown, DE), Robert Dicosimo (Chadds Ford, PA), Michael Charles Grady (Oaklyn, NJ), Ranjan Patnaik (Newark, DE)
Application Number: 13/162,631
International Classification: C12P 7/16 (20060101); C12N 1/21 (20060101); C12N 1/19 (20060101); C12P 7/04 (20060101);
