Method of Reducing Acetylation in Plants to Improve Biofuel Production
The invention provides methods for engineering plants to have reduced levels of acetylation by decreasing expression of one or more Cas1L genes. Such plants can be used, e.g., to increase yield for biofuel production.
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This application claims priority benefit of U.S. provisional patent application No. 61/153,202, filed Feb. 17, 2009, which application is incorporated by reference herein.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENTThe invention described and claimed herein was made using funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.
BACKGROUND OF THE INVENTIONBiomass for biofuel production contains large amounts of xylan as well as smaller amounts of other matrix polysaccharides, such as xyloglucan, mannans, pectins, and the like. Efficient biofuel production requires efficient degradation of these polysaccharides.
Enzymatic degradation into monosaccharides is hindered, however, by acetyl ester substitutions on the polysaccharide backbone. The inhibition of degradation by acetyl esters can be significant, as substitution can be at a high level, e.g., typically 25-50% of the xylose residues of grass xylan are acetylated. Moreover, the acetic acid that is released during enzymatic or chemical degradation is inhibitory to organisms, such as yeast, that are used for fermentation. The acetic acid contained in a biomass mixture for fermentation can easily be in the order of 0.1 M or 6 g/l, which is a highly inhibitory level. Reduction in the level of acetic acid would therefore be highly beneficial for fermentation. Accordingly, there is a need for improvement to biofuel production to reduce acetic acid levels.
A pathogenic fungus, Cryptococcus neoformans, has a polysaccharide coat consisting of O-acetylated glucuronoxylomannans. A protein encoded by the Cas1p gene has been identified as being essential for acetylation of the coat polysaccharide (Janbon et al., Molec. Microbiol. 42:453-467, 2001). Although the gene has been putatively annotated as an acetyltranserase, its biochemical activity was not confirmed. Homologs of this gene have been identified in various plants.
The present invention is based, in part, on the discovery that mutations in homologs to Cas1p reduce polysaccharide acetylation. Plants having reduced polysaccharide acetylation in accordance with the present invention can be used, for example, to provide plant mass that produces lower levels of acetic acid during fermentation.
BRIEF SUMMARY OF THE INVENTIONThe invention provides, in part, methods of engineering plants to reduce the level of acetylation in plants, plants that have been engineered in accordance with the methods and methods of using such plants, e.g., to improve the yield for biofuel production. Thus, in one aspect, the invention provides a method of improve the yield of a fermentation reaction from an enzymatic or chemical degradation of acetylated products in a reaction comprising plant material, e.g., cell wall material, the method comprising providing a plant that has been engineered to decrease the activity of at least one CAS1L gene, e.g., a CAS1L1 or CAS1L2 gene, in the plant; and incubating the plant material in the reaction. The plants can be engineered to decrease the activity of at least two CAS1L genes, or to decrease the activity of at least three CAS1L genes. In some embodiments, a plant can be engineered to reduce the activity of four CAS1L genes.
In some embodiments, improvement in yield can be an improvement of the final yield from, or efficiency of, a fermentation reaction, wherein the method comprises: enzymatically or chemically degrading plant material from a mutant CAS1L plant that has decreased activity of at least one CAS1L gene to obtain degradation products; and fermenting the degradation products in a fermentation reaction, wherein the final yield, or efficiency of, the fermentation reaction is increased relative to the final yield, or efficiency, of a fermentation reaction using corresponding plant material from a wildtype CAS1L plant. In some embodiment, the steps of degrading the plant material and fermenting the degradation products occur in the same reaction mixture. Alternatively, the steps of degrading the plant material and fermenting the degradation products can occur in separate reaction mixtures. In some embodiments, the improvement of the efficiency of the fermentation reaction is an increase in the amount of product obtained per unit enzyme over a period of time compared to the amount of product obtained using corresponding plant material from a wildtype CAS1L plant per unit enzyme over the same period of time. In some embodiments, the mutant CAS1L plant has been engineered to decrease the activity of at least one CAS1L gene, e.g., a CAS1L1 or a CAS1L2 gene. In some embodiments, the plants are engineered to decrease the activity of at least two CAS1L genes, or to decrease the activity of at least three CAS1L genes. In some embodiments, a plant can be engineered to reduce the activity of four CAS1L genes.
In some embodiments, the invention provides a method of chemically degrading or enzymatically degrading plant material from a mutant CAS1L plant that has decreased activity of at least one CAS1L gene to obtain degradation products for a fermentation reaction, wherein the amount of degradation product obtained and/or the efficiency of the degradation reaction is improved compared to a corresponding reaction in which wildtype CAS1L plant material is employed. In some embodiments, the degradation reaction is an enzymatic reaction and the efficiency (e.g., amount of product obtained per unit enzyme per unit of time) of the degradation reaction is improved. In some embodiments, the mutant CAS1L plant has been engineered to decrease the activity of at least one CAS1L gene, e.g., a CAS1L1 or a CAS1L2 gene. In some embodiments, the plants are engineered to decrease the activity of at least two CAS1L genes, or to decrease the activity of at least three CAS1L genes. In some embodiments, a plant can be engineered to reduce the activity of four CAS1L genes.
In some embodiments, the plant is a transgenic plant that comprises a vector comprising a nucleic acid sequence that encodes an RNAi, e.g., an artificial microRNA (miRNA) or other nucleic acid that encodes an RNAi, that inhibits expression of at least one CAS1L gene.
In some embodiments, the plants that is engineered in accordance with the invention is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
In an additional aspect, the invention provides a plant comprising a vector that comprises an RNAi that inhibits a CAS1L gene. In some embodiments, the plants is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
The invention also provides an isolated mutant CAS1L plant in which at least two CAS1L genes are inhibited. In some embodiments, three or four CAS1L genes are inhibited.
In some embodiments, the plant is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
In some embodiments, the invention also provides a method of producing a plant that has reduced acetylation in the cell wall material, the method comprising inhibiting expression of at least one CAS1L gene in the plant using RNAi. In some embodiments, the method further comprises determining the level of acetylation in the cell wall of the plant.
In another aspect, the invention provides bulk harvested material comprising material from at least two engineered plants, e.g., grass plants, that have decreased expression of at least one CAS1L gene. In some embodiments, the bulk harvested material is present in a fermentation reaction.
In an additional aspect, the invention provides a plant, e.g., a grass, engineered to have reduced expression of at least one CAS1L gene, wherein the plant has at least a 10% reduction in cell wall acetylation in comparison to a plant that has not been engineered.
The invention further provides a polysaccharide or polysaccharide fraction, e.g., homogalacturonan or pectin, isolated from a plant that has at least a 10% reduction in cell wall acetylation in comparison to a wildtype plant. In some embodiment, the invention provides a food ingredient containing such a polysaccharide.
The term “biomass” in the context of this application refers to plant material that is processed to provide a product, e.g., a biofuel such as ethanol, or livestock feed. Such plant material can include whole plants, or parts of plants, e.g., stems, leaves, branches, shoots, roots, tubers, and the like.
The term “acetate” as used herein is used to refer to acetyl esters bound to polysaccharides and glycan structures on glycoproteins and proteoglycans. The acetyl ester can be bound to different OH-groups on sugars, and individual sugar residues can contain more than one acetyl ester group. Many different plant polysaccharides are known to be acetylated, including xylan, mannan, xyloglucan, and pectin.
A “fermentation reaction” as used herein is used to refer to the conversion of a substrate into a product, typically by an enzymatic reaction. Such reactions in the context of this invention can be anaerobic or aerobic. Typically, a fermentation reaction used in this invention is an anaerobic reaction in which yeast or bacteria convert polysaccharides, oligosaccharides and/or sugars to alcohols, acids, hydrocarbons and/or esters.
In the context of this invention, the term “yield”, when referring to an enzymatic or chemical reaction for the conversion of a substrate into a product, refers to efficiency as well as overall amount of the product. Thus, the term “improved yield” not only refers to increases in overall, or final, yield of a reaction (i.e., the amount of product obtained per amount of starting material), but also includes a faster reaction rate that increases the amount of product produced over a shorter period when using biomass from a mutant CAS1L plant in comparison to corresponding biomass from the wildtype plant. In some embodiments in which CAS1L mutant plant material is degraded in an enzymatic reaction, “improved yield” includes an increase in the amount of product obtained per enzyme unit over a defined unit of time in comparison to the yield obtained using wildtype CAS1L plant material.
In the context of this invention “corresponding plant material from a wildtype CAS1L plant” refers to plant material that is from the same part of the plant as the CAS1L mutant plant material. As understood in the art, improved yield is based upon comparisons of the same amount of corresponding plant material.
A plant that has a “mutant CAS1L gene” in the context of this invention refers to a plant in which at least the CAS1L gene is inactivated, or mutated to reduce expression, relative to a plant with a wildtype CAS1L gene.
A plant that has been “engineered to have decreased expression of a CAS1L gene” refers to a plant that has been modified by mutagenesis, and/or genetic engineering, e.g., engineering to express RNAi that targets one or more CAS1L genes, to exhibit decreased expression of the targeted CAS1L gene or genes.
A “CAS1L gene” refers generally to a nucleic acid encoding a polypeptide that is a member of the CAS1L gene family. Examples of members of the CAS1L gene family include CAS1L1, CAS1L2, CAS1L3, and CAS1L4. In the context of this invention, a CAS1L polypeptide that is encoded by a CAS1L gene has substantial identity to a polypeptide comprising the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; or to a CAS1L gene sequence set forth in
The terms “decreased expression”, “reduced expression”, or “inhibited expression” of a CAS1L gene refers to a reduction in the level of expression of the CAS1L gene in an engineered plant compared to the level of expression in a wildtype plant. Thus, decreased expression can be a reduction in expression of a CAS1L gene of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater. Decreased expression can be assessed by measuring decreases in the level of RNA encoded by the gene and/or decreases in the level of CAS1L protein or protein activity. CAS1L protein/protein activity can be assessed directly or indirectly, e.g., by measuring an endpoint such as the acetate content of a part of plant in which the CAS1L gene is expressed.
The phrase “substantially identical,” in the context of two nucleic acids or polypeptides, refers to a sequence or subsequence that has at least 50% identity, typically at least 60% sequence identity, to a reference sequence. Percent identity can be any integer from 50% to 100%. In some embodiments, a sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical when compared to a reference sequence.
In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be perfectly identical and may be “substantially identical” to a sequence of the gene from which it was derived. In the case of polynucleotides used to inhibit expression of an endogenous gene, the introduced sequence need not be perfectly identical to a sequence of the target endogenous gene. The introduced polynucleotide sequence will typically be at least substantially identical (as determined below) to the target endogenous sequence. Thus, an introduced “polynucleotide sequence from” a CAS1L gene may not be identical to the target CAS1L gene to be suppressed, but is functional in that it is capable of inhibiting expression of the target CAS1L gene. As explained below, these variants are specifically covered by this term.
The phrase “at least one CAS1L gene, or “at least two CAS1L genes”, or “at least three CAS1L genes” is used to refer to members of the CAS1L gene family in a plant that has multiple CAS1L genes. For example, in the alignment in
Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term “complementary to” is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needle man and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. In some embodiments, percent identity is determined using the BLAST2 algorithm set at the default settings.
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. A “comparison window” may be, e.g., 20, 50, 100, 400, or more nucleotides ore amino acids in length; or may be the entire length of the sequences being compared.
Proteins that are substantially identical include those that have conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C. For example, stringent conditions for hybridization, such as RNA-DNA hybridizations in a blotting technique are those which include at least one wash in 0.2×SSC at 55° C. for 20 minutes, or equivalent conditions.
A CAS1L polynucleotide sequence for use in the invention can also be amplified using PCR techniques, e.g., primers that are designed to amplify nucleic acid sequences that encode CAS1L protein sequences set forth in SEQ ID NOs: 1, 2, 3, 4, or 5.
The term “plant”, as used herein, can refer to a whole plant or parts plant parts e.g., cuttings, tubers, pollen, leaves, stems, flowers, roots, fruits, branches, and the like. The term also encompasses individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof. The tetin includes plants of a variety of a ploidy levels, including polyploid, diploid and haploid.
The term “progeny” refers generally to the offspring of a cross, and includes direct F1 progeny, as well as later generations of F2, F3, etc.
A polynucleotide sequence is “heterologous to” a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively.
The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest.
The term “bulk harvested material” refers to combined plant material harvested from at least three plants, preferably at least 5, 10, 25, 50, 100, 500, or 1000 or more plants. The plant material may be whole plants, or parts of the plants, e.g., leaves or stems harvested from the plants. In some embodiments, the plant material present in the bulk harvested material is crushed or milled to a desired particle size, e.g., a size that is useful for producing biofuel.
IntroductionThis invention is based, in part, on the discovery that plants can be engineered to suppress CAS1L gene expression to reduce the amount of acetate, i.e., polymer-bound acetyl esters, present in the plant material, thereby enhancing yield in an enzymatic reaction, e.g., a fermentation reaction, to obtain a desired product. Often, the product is a biofuel, such as ethanol. In typical embodiments, the fermentation reaction employs a microorganism, such as a yeast or bacteria, that is sensitive to acetic acid levels in the reaction.
In some embodiments, a plant such as a sugar beet or potato, that is engineered to reduce the amount of acetate in the plant material can be used as a source of other products, such as a pectin, that can be employed as a food ingredient.
The invention therefore provides methods of engineering plants to reduce acetate by suppressing expression of at least one, often two or three, CAS1L genes in the plant. The invention further provides plants that have been thusly engineered, as well as methods of using such plants, e.g., to enhance biofuel yield from plant material.
In the current invention, the yield of a fermentation reaction to obtain a desired product is increased due to the reduced acetylation in CAS1L mutant plants. This can be accomplished that by having less acetate in the products that are used in the fermentation. Either an enzymatic or chemical degradation can be used.
In some embodiments, an enzymatic degradation is employed. In such embodiments, the enzymatic degradation reaction can itself be improved due to the lowered acetate content in CAS1L mutant plants. Typically, this results in increased yield in a fermentation reaction (i.e., either in the rate of fermentation and/or the total amount of fermentation product generated).
In some embodiments, improved degradation can also be advantageous without an effect on the final yield in fermentation. For example, in some embodiments, a reaction may employ less enzyme in order to degrade the biomass obtained from CAS1L mutant plants compared to the amount of enzyme required to degrade the biomass from wildtype CAS1L plants. Accordingly, in some embodiments, improved yield from biomass from CAS1L mutant plants results from an increase in the amount of degradation product generated per enzyme unit per unit of time relative to the yield from corresponding biomass from a wildtype CAS1L plant.
In the current invention, the degradation and fermentation of the biomass from the plant can be performed in one reaction mixture or using separate reaction mixtures. Thus, plant material from a CAS1L mutant plant, e.g., cell wall material from shoots, stems, etc., can be degraded either enzymatically or chemically in one reaction and the degradation products then fermented in a separate reaction mixture. In other embodiments, the degradation reaction and the fermentation reaction are conducted in the same reaction mixture such that the degradation products generated from enzymatic or chemical degradation of the plant biomass is fermented in the same mixture in which the biomass is degraded.
An “improved yield” from a fermentation reaction can thus arise from an improvement in the overall amount of product obtained or from an increased efficiency of the overall reaction.
Plants that Can be Engineered in Accordance with the Invention
Various kind of plants can be engineered to reduce CAS1L gene expression as described herein to reduce acetate levels. The plant may be a monocotyledonous plant or a dicotyledonous plant. In certain embodiments of the invention, plants are green field plants.
In other embodiments, plants are grown specifically for “biomass energy”. For example, suitable plants include corn, switchgrass, sorghum, miscanthus, sugarcane, poplar, pine, wheat, rice, soy, cotton, barley, turf grass, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus. In further embodiments, the plant is switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×giganteus, Miscanthus sp., sericea lespedeza (Lespedeza cuneata), millet, ryegrass (Lolium multiflorum, Lolium sp.), timothy, Kochia (Kochia scoparia), forage soybeans, alfalfa, clover, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.), Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, or Kentucky bluegrass among others.
Inhibition of CALM Gene ExpressionThe invention employs various routine recombinant nucleic acid techniques. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Many manuals that provide direction for performing recombinant DNA manipulations are available, e.g., Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994-1999, updated through 2008).
Acetyl-related protein has been identified in Cryptococcus neoformans, where mutation in the Cas1p gene leads to lack of acetylation of the coat polysaccharide (Janbon et al. Molecular Microbiology 42: 453-467, 2001). The protein therefore plays a role in acetylation and has been described as a putative acetyl transferase, although no transferase activity has been demonstrated. In the Genbank database, several homologs of the Cryptococcus protein are annotated as ‘acetyl transferase’, ‘acetyl transferase-related’, ‘putative acetyl transferase’ etc. These annotations are based on the sequence similarity with the Cryptococcus protein.
A CAS1L nucleic acid that is targeted for suppression (inhibition) in this invention encodes a CAS1L protein that is substantially similar to SEQ ID NO:1, 2, 3, 4, or 5, or a fragment thereof. CAS1L proteins that are substantially similar to SEQ ID NO:1, 2, 3, 4, or 5, or a fragment thereof, have one or more conserved domains. For example,
This region is at least 78% conserved (i.e., in the context of this invention 78% identical to) Arabidopsis CAS1L2 in CAS1L sequences in
Accordingly, a CAS1L nucleic acid that is targeted for inhibition as described herein typically encodes a protein that contains a region that has at least 60%, 65%, 70%, or 75% or great identity to this sequence.
Another motif that is conserved is the sequence LHEWHFRSGLDRYIWI (SEQ ID NO:13). Accordingly, a CAS1L nucleic acid sequence that is targeted for inhibition typically encodes a protein that contains this motif.
Furthermore, a CAS1L nucleic acid typically encodes a protein that has substantial identity, e.g., at least 60%, 65%, 70%, or 75% identity to a central region, residues 116-350, shown with reference to an Arabidopsis CasL3 protein sequence (SEQ ID NO:14):
A plant may express multiple CAS1L family members. For example, Arabidopsis has multiple CAS1L family members, which have a high degree of amino acid sequence identity. For example, CAS1L1, CAS1L3, and CAS1L4 have at least 68%, 69%, and 74% amino acid sequence identity to CAS1L2.
In the present invention, at least one CAS1L gene sequence is inhibited in a plant. For example, in some embodiments, one CAS1L gene is inhibited. Such a gene may encode a protein that has at least 50%, 55%, 60%, 65%, 70%, or 75% identity, or greater, to a CAS1L1 or CAS1L2 reference sequence, e.g., SEQ ID NO:1 or 2. In some embodiments, plants are generated in which at least two CAS1L genes are inhibited. Plants may also be engineered in which at least three CAS1L genes or four CAS1L genes are inhibited. As understood in the art, the number of CAS1L genes that are inhibited will depend on such factors as effects on plant growth, plant general health and the like.
In some embodiments, a CAS1L gene that is inhibited has a coding nucleic acid sequence that is substantially identical, e.g., at least 50%, 60%, 65%, 70%, or 75% identical, to the CAS1L nucleic acid sequences SEQ ID NO:6, 7, 8, 9, or 10. Of the CAS1L nucleic acid sequences shown in SEQ ID NOs. 6-10, the sequences have the following percent identity as determined by Blast using standard parameters:
The CAS1L gene can be suppressed using any number of techniques well known in the art. For example, one method of suppression is sense suppression (also known as co-suppression). Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496 (1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity can exert a more effective repression of expression of the endogenous sequences. In some embodiments, sequences with substantially greater identity are used, e.g., at least about 80, at least about 95%, or 100% identity are used. As with antisense regulation, further discussed below, the effect can be designed and tested to apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. In some embodiments, a sequence of the size ranges noted above for antisense regulation is used, i.e., 30-40, or at least about 20, 50, 100, 200, 500 or more nucleotides.
Endogenous gene expression may also be suppressed by means of RNA interference (RNAi) (and indeed co-suppression can be considered a type of RNAi), which uses a double-stranded RNA having a sequence identical or similar to the sequence of the target gene. As used herein RNAi, includes the use of micro RNA, such as artificial miRNA to suppress expression of a gene.
RNAi is the phenomenon in which when a double-stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA. Although complete details of the mechanism of RNAi are still unknown, it is considered that the introduced double-stranded RNA is initially cleaved into small fragments, which then serve as indexes of the target gene in some manner, thereby degrading the target gene. RNAi is known to be also effective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc. Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl. Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431 (1998); Matthew, Comp Funct Genom 5: 240-244 (2004); Lu, et al., Nucleic Acids Res. 32(21):e171 (2004)).
Thus, in some embodiments, inhibition of a CAS1L gene is achieved using RNAi techniques. For example, to achieve suppression of the expression of a DNA encoding a protein using RNAi, a double-stranded RNA having the sequence of a DNA encoding the protein, or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a plant of interest. As used herein, RNAi and dsRNA both refer to gene-specific silencing that is induced by the introduction of a double-stranded RNA molecule, see e.g., U.S. Pat. Nos. 6,506,559 and 6,573,099, and includes reference to a molecule that has a region that is double-stranded, e.g., a short hairpin RNA molecule. The resulting plants may then be screened for a phenotype associated with the target CAS1L protein, e.g., reduced acetate, and/or by monitoring steady-state RNA levels for transcripts encoding the protein. Although the genes used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%, 95% or more identical to the target gene sequence. See, e.g., U.S. Patent Publication No. 2004/0029283. The constructs encoding an RNA molecule with a stem-loop structure that is unrelated to the target gene and that is positioned distally to a sequence specific for the gene of interest may also be used to inhibit target gene expression. See, e.g., U.S. Patent Publication No. 2003/0221211.
The RNAi polynucleotides may encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, or 500, nucleotides corresponding to the target sequence. In addition, in some embodiments, these fragments are at least, e.g., 50, 100, 150, 200, or more nucleotides in length. Interfering RNAs may be designed based on short duplexes (i.e., short regions of double-stranded sequences). Typically, the short duplex is at least about 15, 20, or 25-50 nucleotides in length (e.g., each complementary sequence of the double stranded RNA is 15-50 nucleotides in length), often about 20-30 nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, fragments for use in RNAi will be at least substantially similar to regions of a target protein that do not occur in other proteins in the organism or may be selected to have as little similarity to other organism transcripts as possible, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases. Thus, RNAi fragments may be selected for similarity or identity with conserved domains of CAS1L sequences, such as those described herein, that lacking significant homology to sequences in the databases).
In some embodiments, an RNAi is introduced into a cell as part of a larger DNA construct. Often, such constructs allow stable expression of the RNAi in cells after introduction, e.g., by integration of the construct into the host genome. Thus, expression vectors that continually express iRNA in cells transfected with the vectors may be employed for this invention. For example, vectors that express small hairpin or stem-loop structure RNAs, or precursors to microRNA, which get processed in vivo into small RNAi molecules capable of carrying out gene-specific silencing (Brummelkamp et al., Science 296:550-553 (2002), and Paddison, et al., Genes & Dev. 16:948-958 (2002)) can be used. Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001), Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395: 854 (1998).
Methods for selection and design of sequences that generate RNAi are well known in the art (e.g. Reynolds, 2004; see also U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; and U.S. Pat. No. 6,489,127).
One of skill in the art will recognize that using technology based on specific nucleotide sequences (e.g., antisense or sense suppression technology), families of homologous genes can be suppressed with a single sense or antisense, discussed below, transcript. For instance, if a sense or antisense transcript is designed to have a sequence that is conserved among a family of genes, then multiple members of a gene family can be suppressed. Conversely, if the goal is to only suppress one member of a homologous gene family, then the sense or antisense transcript should be targeted to sequences with the most variation between family members.
The term “target RNA molecule”, e.g., in this invention a target CAS1L RNA, refers to an RNA molecule to which an RNAi molecule is homologous or complementary.
One or more CAS1L genes can be inhibited using the same interfering RNA. For example, all of the CAS1L genes in a plant may be targeted by using an RNAi that is designed to a conserved region of the CAS1L gene. In other embodiments, individual CAS1L gene family members may be targeted by using an RNAi that is specific that CAS1L gene.
Antisense and Ribozyme SuppressionA reduction of CAS1L gene expression in a plant to reduce polysaccharide acetylation may be obtained by introducing into plants antisense constructs based on the CAS1L polynucleotide sequences. For antisense suppression, a CAS1L sequence is arranged in reverse orientation relative to the promoter sequence in the expression vector. The introduced sequence need not be a full length CAS1L cDNA or gene, and need not be identical to the CASL cDNA or a gene found in the plant variety to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native CASL sequence is used to achieve for effective antisense suppression. Preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous cspl gene. Suppression of endogenous CAS1L gene expression can also be achieved using a ribozyme. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No. 5,543,508 to Haselhoff.
MutagenesisAlternatively, random mutagenesis approaches may be used to disrupt or “knock-out” the expression of a CAS1L gene using either chemical or insertional mutagenesis, or irradiation. One method of mutagenesis and mutant identification is known as TILLING (for targeting induced local lesions in genomes). In this method, mutations are induced in the seed of a plant of interest, for example, using EMS treatment. The resulting plants are grown and self-fertilized, and the progeny are assessed. For example, the plants may be assed using PCR to identify whether a mutated plant has a CAS1L mutation, e.g., that reduces expression of a CAS1L gene, or by evaluating whether the plant has reduced levels of acetate in a part of the plant that expressed the CAS1L gene. TILLING can identify mutations that may alter the expression of specific genes or the activity of proteins encoded by these genes (see Colbert et al (2001) Plant Physiol 126:480-484; McCallum et al (2000) Nature Biotechnology 18:455-457).
Another method for abolishing or decreasing the expression of a CAS1L gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a CAS1L gene. Mutants containing a single mutation event at the desired gene may be crossed to generate homozygous plants for the mutation (Koncz et al. (1992) Methods in Arabidopsis Research. World Scientific).
Another method to disrupt a CAS1L gene is by use of the cre-lox system (for example, as described in U.S. Pat. No. 5,658,772).
Plants Having where Multiple CAS1L Genes are Inhibited
In some embodiments expression of two or more CAS1L genes is inhibited in a plant in accordance with the invention. As explained above, such plants can be generated by performing a molecular manipulation that targets all of the CAS1L gene family members in a plant, e.g., using an RNAi to a conserved region to inactivate all of the CAS1L genes. Such plants can also be obtained by breeding plants that each have individual mutations that inactivate different CAS1L genes to obtain progeny plants that are inactivated in all of the desired CAS1L genes. For example, to obtain a rice plant in which three CAS1L genes are inactivated, one of skill can target the genes using RNAi developed to a region that is conserved in all three of the rice CAS1L genes, or target the genes individually and breed the resulting mutant plants.
Expression of CAS1L Gene InhibitorsExpression cassettes comprising polynucleotides that encodes CAS1L gene expression inhibitors, e.g., an antisense or siRNA, can be constructed using methods well known in the art. Constructs include regulatory elements, including promoters and other sequences for expression and selection of cells that express the construct. Typically, plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
Examples of constitutive plant promoters which may be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al., (1985) Nature 313:810); the nopaline synthase promoter (An et al., (1988) Plant Physiol. 88:547); and the octopine synthase promoter (Fromm et al., (1989) Plant Cell 1:977).
Additional constitutive regulatory elements including those for efficient expression in monocots also are known in the art, for example, the pEmu promoter and promoters based on the rice Actin-1 5′ region (Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al., Mol. Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell 2:163 (1990)). Chimeric regulatory elements, which combine elements from different genes, also can be useful for ectopically expressing a nucleic acid molecule encoding an IND 1 polynucleotide (Comai et al., Plant Mol. Biol. 15:373 (1990)).
Other examples of constitutive promoters include the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens (see, e.g., Mengiste (1997) supra; O'Grady (1995) Plant Mol. Biol. 29:99-108); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang (1997) Plant Mol. Biol. 1997 33:125-139); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant Mol. Biol. 31:897-904); ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf Plant Mol. Biol. 29:637-646 (1995).
A variety of plant gene promoters that regulate gene expression in response to various environmental, hormonal, chemical, developmental signals, and in a tissue-active manner are known in the art. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Examples of environmental promoters include drought-inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897 909). Plant promoters that are inducible upon exposure to plant hormones, such as auxins, may also be employed. For example, the invention can use the auxin response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397 407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955 966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906 913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933 937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900 1902).
Plant promoters which are inducible upon exposure to chemicals reagents that can be applied to the plant, such as herbicides or antibiotics, may also be used in vectors as described herein. For example, the maize In2 2 promoter, activated by benzenesulfonamide herbicide safeners, can be used; application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Other promoters, e.g., a tetracycline inducible promoter; a salicylic acid responsive element promoter, promoters comprising copper-inducible regulatory elements; promoters comprising ecdysone inducible regulatory elements; heat shock inducible promoters, a nitrate-inducible promoter, or a light-inducible promoter may also be used.
In some embodiments, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters), such as a leaf or a stem. Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Examples of tissue-specific promoters include promoters that initiate transcription primarily in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Other examples are promoters that direct expression specifically to cells and tissues with secondary cell wall deposition, such as xylem and fibers.
Plant expression vectors may also include RNA processing signals that may be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors may include additional regulatory sequences from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.
Plant expression vectors routinely also include dominant selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin), herbicide resistance genes (e.g., phosphinothricin acetyltransferase), and genes encoding positive selection enzymes (e.g. mannose isomerase).
Once an expression cassette comprising a polynucleotide encoding an inhibitor of the expression of a CAS1L gene, e.g., an antisense or siRNA, has been constructed, standard techniques may be used to introduce the polynucleotide into a plant in order to modify CAS1L activity and accordingly, the level of acetylation in the plant or plant part in which the CAS1L target nucleic acid is expressed. See protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.
Transformation and regeneration of plants is known in the art, and the selection of the most appropriate transformation technique will be determined by the practitioner. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence. Examples of these methods in various plants include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.
Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants or the ability to grow on a specific substrate, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic, herbicide, or substrate.
Evaluation of Plants Engineered to Reduce CAS1L Expression.After transformed plants are selected parts of the plants may be evaluated to determine the level of CAS1L gene expression in a part of the plant that expresses the CAS1L gene, e.g., by evaluating the level of RNA or protein, or determining the levels of acetate in the plants. These analyses can be performed using any number of methods known in the art.
In some embodiments, acetyl esters in plant cell wall material can be measured. For example, cell walls are prepared from plant material. Several methods are known. In the simplest method, the plant material is ground and extracted repeatedly with 96% and 70% ethanol. The resulting ‘alcohol insoluble residue’ is highly enriched in cell wall material. The sample is dried and resuspended in buffer at neutral pH. An aliquot of the sample is saponified by treatment with 0.1 M NaOH at 4° C. overnight or at room temperature for several hours. Following saponification, the sample is neutralized by adding 1 M HCl. Acetic acid in the saponified and neutralized sample can be determined in several different ways, e.g. by gas chromatography or HPLC on an appropriate column. A convenient method is to use an acetic acid determination kit, e.g., such as the kit by R-Biopharm, Germany, according to the manufacturers instructions. The principle of the kit is that acetate is enzymatically consumed in a series of reactions leading to the formation of
NADPH, which can be determined spectrophotometrically at 340 nm. The kit allows for correction for interference in the determination.
The procedure above can be used to determine total acetyl esters in the alcohol insoluble residue. To determine acetate esters in specific polymers, the alcohol insoluble residue can be sequentially extracted and/or digested with specific enzymes. The extracts and digests can be analyzed by the methods described above or by mass spectrometry.
As appreciated by one in the art double, triple, or quadruple CAS1L mutant plants can be generated using techniques well known in the art, including breeding techniques as well as introducing mutations into a plant that already has a mutation in a CAS1L gene.
Plants that exhibit reduced CAS1L gene expression have at least 5% reduction in acetylation, e.g., as assessed by evaluating cell walls, typically at least 10% reduction in acetylation, or more often at least 15%, 20%, 30%, 40%, or 50% or more reduction in acetylation in comparison to a plant that has not been engineered to decrease expression of the CAS1L gene. In some embodiments, such a reduction in acetylation is observed when one CAS1L gene is reduced in expression in an engineered plant; in some embodiments, such a reduction in acetylation is observed when two CAS1L genes in the engineered plant have reduced expression, and in some embodiments, such a reduction in acetylation is observed when three or four CAS1L genes are engineered to decrease expression in the plant. As understood in the art, CAS1L-mediated reduction in acetylation may occur in one or more parts of the plants, e.g,. acetylation may be reduced in a leaf and/or a stem.
Plants that exhibit reduced CAS1L gene expression can be used in a variety of methods. The plants selected for reduced acetate levels may further be evaluated to further confirm that the plants provide for improved yield. For example, plant material from the plants with reduced acetate content is ground or milled to defined particle size. The plant material can be compared to ground or milled plant material from normal plants that have not been engineered to reduce CAS1L gene expression.
The milled plant material is subjected to a saccharification procedure. Many different procedures are currently used experimentally, e.g. dilute acid treatment, steam explosion or ionic liquid treatments. As the beneficial effect of reduced acetate content will differ depending on the exact procedure used several different pretreatment methods can be evaluated. For example, a dilute acid treatment method can be used. The pretreated plant material is then subjected to enzymatic hydrolysis using a mixture of cell wall degrading enzymes.
Procedures for cell wall pretreatment and enzymatic digestion are well known to those skilled in the art. The yield or efficiency of the procedure can be readily determined by measuring the amount of reducing sugar released, using a standard method for sugar detection, e.g. the dinitrosalicylic acid method well known to those skilled in the art. Plants engineered in accordance with the invention provide a higher sugar yield.
CAS1L-inhibited plants may also be evaluated in comparison to non-engineered plants to test for the effect of acetates and acetate-derived compounds on subsequent fermentation. For example, hydrolyzed biomass is subjected to fermentation using an organism such as yeast or E. coli that can convert the biomass into compounds such as ethanol, butanol, alkanes, lipids, etc. In the simplest test, the yield of ethanol obtained with a given amount of starting plant material and a standard yeast fermentation can be determined. Yield can be determined not only with organisms that can ferment glucose, but also with organisms that have the ability to ferment pentoses and or other sugars derived from the biomass. In addition to determining the yield of product, e.g. ethanol, one can determine the growth rate of the organism. The plants of the invention that are engineered to reduce CAS1L activity and accordingly, to have reduced acetate, will exhibit a reduced inhibitory effect due to acetate and acetate-derived compounds in comparison to corresponding plants that have not be engineered to reduce CAS1L activity. The decreased inhibitory effect may result in higher final yields of a fermentation reaction, or in faster fermentation, or both.
Plants having reduced CAS1L activity can be used in a variety of reactions, including fermentation reactions. Such reactions are well known in the art. For example, fermentation reactions noted above, e.g., a yeast or bacterial fermentation reaction, may employ CAS1L mutants, to obtain ethanol, butanol, lipids, and the like. For example the plants may be used in industrial bioprocessing reactions that include fermentative bacteria, yeast, or filamentous fungi such as Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp., Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonas sp., Serratia sp., Rhizobium sp., and Streptomyces sp., Zymomonas mobilis, acetic acid bacteria, methylotrophic bacteria, Propionibacterium, Acetobacter, Arthrobacter, Ralstonia, Gluconobacter, Propionibacterium, and Rhodococcus.
Evaluation for Increased Resistance to FungusCAS1L mutant plants, e.g., plants that have mutations in CAS1L2, also exhibit increased resistance to certain fungi in comparison to plants that have wildtype CAS1L2 genes. CAS1L-inhibited plants may also be evaluated for susceptibility to infection, e.g., fungus infection. In some embodiments, such an assay may also be used to identify mutant CAS1L plants. An example of an assay to evaluate fungus resistance is provided in Example 3. CAS1L mutant plants may thus be more readily propagated, e.g., the plants could grown using reduced amounts of fungicide.
EXAMPLES Example 1 Acetate Levels in CAS1L Mutant PlantsArabidopsis mutants generated by random insertion of T-DNA were obtained from the Arabidopsis Biological Resource Center, Ohio, or constructed. Mutants where the T-DNA had been inserted in the coding region of the genes of interest, e.g., At3g06550, At2g34410, and At1g29890 were identified by searching the website of www.arabidopsis.org. The website contains sequence information for flanking regions of T-DNA for all the mutants deposited. Mutant seeds were first screened to confirm the T-DNA insertion and identify homozygous plants. Seeds were germinated and leaf samples were used to prepare DNA and carry out PCR amplification using primers suggested by ABRC. The principle behind this PCR screening is that a primer complementary to the T-DNA together with a primer complementary to the plant genome near the insertion will yield a PCR product indicative of the insertion. Likewise, two primers complementary to the plant genome and placed on different sides of the site of insertion will only yield a PCR band if there is no insert, thus indicating the wild type allele. By using all three primers for PCR reactions, plants that were homozygous for the insertion could be identified by the presence of the insert-specific PCR product and the absence of the wild-type-specific PCR product. The homozygous individuals were grown to maturity and the seeds harvested. Homozygosity of the offspring was confirmed by PCR.
Acetate levels were initially evaluated in leaf samples from a small number of Arabidopsis plants that were generated. Cell walls were prepared from plant material. An aliquot of the sample was saponified using h 0.1 M NaOH. Following saponification, the sample was neutralized by adding 1 M HCl. Acetic acid in the saponified and neutralized sample was then determined with a colorimetric assay essentially according to Beutler (1984, in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) 3rd ed., vol. VI, pp. 639-645, Verlag Chemie, Weinheim, Deerfield Beach/Florida, Basel).
The results are shown in
An initial analysis also indicated that Arabidopsis plants in which CAS1L1 expression is inhibited exhibited reduced acetate levels in the stems.
Crosses of mutant CAS1L1, CAS1L2, CAS1L3, and CAS1L4 plants were also obtained in all combinations, i.e., all the single, double, triple, and quadruple mutants, were generated. The quadruple mutants and some of the triple mutant plants exhibited reduced plant growth.
Example 2 Enzymatic Digestion of Wild Type Arabadopsis and Mutant rwa2 to Determine the Effect of Acetylation on Saccharification Hydrolysis KineticsThis example demonstrates that plant material from CAS1L mutant plants is more easily saccharified into sugars.
Cell wall material was isolated as described (Harholt et al., Plant Physiol.140:49-58, 2006). Rosette leaves from mature plants were ground to a powder in a ball mill and extracted with 96% and 70% ethanol.
Enzyme digestions were performed using Novozyme cellulase NS50013 and beta-glucosidase NS50010, with enzyme loadings of 10% wt enzyme/wt glucan and 1% wt enzyme/wt glucan, respectively. The percentage glucan was assumed to be 30% (dw) in all samples, and a 1% glucan loading was used for all reactions. All reactions were performed as duplicate technical replicates and two or three biological replicates.
Reactions were performed in 2 ml screwcap microcentrifuge tubes. Aproximately 10 mg of cell wall material was measured into the vials. A master mix containing 10% and 1% of NS50013 and NS50010 was prepared in 50 mM sodium acetate buffer, pH 4.8. Enzyme mixture was added to tubes on ice. Samples were then incubated for up to 72 hours in an ATR multitron shaker at 50 C and 1000 RPM.
At various time points, samples were removed from the shaker and centrifuged at 14000×g for 10 min. Supernatants were collected and diluted 1:100 for analysis. Monosaccharide concentrations were determined using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) on a Dionex DX600 equipped with a Dionex Carbopac PA-20 analytical column (3×150 mm) and a Carbopac PA-20 guard column (3×30 mm) (Dionex, Sunnyvale, Calif.). Eluent flow rate was 0.4 mL min-1 and the temperature was 30° C. A gradient consisting of a 12 min elution with 14 mM NaOH followed by a 5 min ramp to 450 mM NaOH for 20 min, then a return to the original NaOH concentration of 14 mM for 10 min prior to the next injection. Product concentrations were determined using an external standard.
In one experiment, a much higher amount of sugar was released from the leaf cell walls from the rwa2-1 mutant (see
These experiments thus indicate that there is a benefit of reducing cell wall acetate on saccharification of biomass. The combined effect of a 20% reduction in hemicellulose acetylation on saccharification and on yeast fermentation has been simulated in a lignocellulose biorefinery model (Klein-Marchushamer et al., Techno-Economic Modeling of Cellulosic Biorefineries. Presented at DOE Genomic Science Awardee Workshop, Feb. 7-10, 2010, Crystal City, Va.). This simulation showed that an expected result would be a 10% decrease in cost per gallon of produced ethanol. The main effect is on the improved fermentation.
Example 3 RWA Mutant Plants have Decreased Susceptibility to FungusRWA muant plants also demonstrated increased resistant to fungus.
Botrytis cinerea IK2018 was isolated from strawberry fruit and obtained from Dr. Birgit Jensen, Dept. of Plant Biology and Biotechnology, University of Copenhagen, Denmark. B. cinerea was maintained on potato dextrose agar (Difco, USA). Spores were collected in 3 ml of 12 g/L potato dextrose broth (PDB; Difco, USA) by gentle rubbing and filtered through miracloth (Calbiochem/Merck, Germany) to remove mycelium. The number of spores was counted using a haemocytometer, and the suspension was adjusted to 5×105 conidiospores ml−1 in PDB for infection of leaves.
Rosette leaves from 4-week-old soil-grown Arabidopsis plants (wild type Col-0 and the knock out mutants rwa2-1 and rwa2-3) were placed in Petri dishes containing 0.6% agar, with the petiole embedded in the medium. Inoculation was performed by placing 5 μl of a suspension of 5×105 conidiospores ml−1 in 12 g l−1 potato dextrose broth (PDB; Difco, Detroit, USA) on each side of the middle vein. The plates were incubated at 22° C. with a 12-h photoperiod. High humidity was maintained by covering the plates with a clear plastic lid. Lesion diameter in centimeters was obtained by hand analysis of high-resolution digital images of infected leaves using ImageJ (Abramoff et al., Biophotonics International 11:36-42, 2004). Included scale objects allowed standardization of measurements across images.
The results showed that rwa2 mutant plants with decreased wall acetylation exhibited a surprising tolerance to the necrotrophic fungal pathogen Botrytis cinerea. The experiment was carried out twice with essentially the same results. The result of one of the experiments is shown in
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
All publications, accession numbers, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.
Examples of CAS1L1 (RWA) Polypeptide and Nucleic Acid Sequences
Claims
1. A method of improving the final yield from, or efficiency of, a fermentation reaction comprising plant material that is acetylated, the method comprising:
- enzymatically or chemically degrading plant material from a mutant CAS1L plant that has decreased activity of at least one CAS1L gene to obtain degradation products; and fermenting the degradation products in a fermentation reaction, wherein the final yield, or efficiency of, the fermentation reaction is increased relative to the final yield, or efficiency, of a fermentation reaction using corresponding plant material from a wildtype CAS1L plant.
2. The method of claim 1, wherein the steps of degrading the plant material and fermenting the degradation products occur in the same reaction mixture.
3. The method of claim 1, wherein the steps of degrading the plant material and fermenting the degradation products occur in separate reaction mixtures.
4. The method of claim 1, wherein the improvement of the efficiency of the fermentation reaction is an increase in the amount of enzymatically degraded degradation product obtained per unit enzyme over a period of time compared to the amount of product obtained using corresponding plant material from a wildtype CAS1L plant per unit enzyme over the same period of time.
5. The method of claim 1, wherein the mutant Cas1L plant has been engineered to decrease the activity of at least two Cas1L genes.
6. The method of claim 1, wherein the mutant Cas1L_plan has been engineered to decrease the activity of at least three Cas1L genes.
7. The method of claim 1, wherein the plant is a transgenic plant that comprises a vector comprising a nucleic acid sequence that encodes an RNAi that inhibits expression of at least one Cas1L gene.
8. The method of claim 1, wherein the plant is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
9. A plant comprising a vector that comprises an RNAi that inhibits a Cas1L gene.
10. The plant of claim 9, wherein the plant is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
11. An isolated mutant Cas1L plant in which expression of at least two Cas1L genes is inhibited.
12. The plant of claim 11, wherein the plant is selected from the group consisting of corn, sorghum, millet, miscanthus, sugarcane, poplar, pine, eucalyptus, wheat, rice, soy, cotton, barley, switchgrass, turfgrass, ryegrass, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, Indian grass, fescue, Dactylis sp. Brachypodium, smooth bromegrass, orchardgrass, Kentucky bluegrass, timothy, Kochia, forage soybeans, alfalfa, clover, hemp, kenaf, tobacco, potato, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
13. A method of engineering a plant to reduce acetylation in the cell wall of a plant, the method comprising inhibiting expression of at least one Cas1L gene in the plant using RNAi.
14. The method of claim 13, further comprising determining the level of acetylation in the cell wall of the plant.
15. Bulk harvested material comprising material from at least two engineered plants that have decreased expression of at least one Cas1L gene.
16. The bulk harvested material of claim 15, wherein the at least two engineered plants are grass plants.
17. The bulk harvested material of claim 15, wherein the material is present in a fermentation reaction.
18. A plant engineered to have reduced expression of at least one Cas1L gene, wherein the plant has at least a 10% reduction in cell wall acetylation in comparison to a plant that has not been engineered.
19. The plant of claim 18, wherein the plant is a grass.
20. A polysaccharide or polysaccharide fraction isolated from a plant of claim 18.
21. A polysaccharide of claim 20, where the polysaccharide is homogalacturonan or pectin.
22. A food ingredient containing the polysaccharide of claim 20.
23. A food product containing an ingredient according to claim 22.
Type: Application
Filed: Feb 17, 2010
Publication Date: Feb 23, 2012
Applicant: The Regents of the University of California (Oakland, CA)
Inventor: Henrik Vibe Scheller (Millbrae, CA)
Application Number: 13/201,851
International Classification: C12P 1/00 (20060101); A01H 3/04 (20060101); C08B 37/06 (20060101); A01H 5/00 (20060101); C08B 37/00 (20060101);
