WO2011160050A2 - Systèmes de réduction du caractère récalcitrant de la biomasse cellulosique et d'augmentation des rendements des sucres fermentescibles - Google Patents

Systèmes de réduction du caractère récalcitrant de la biomasse cellulosique et d'augmentation des rendements des sucres fermentescibles Download PDF

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WO2011160050A2
WO2011160050A2 PCT/US2011/040928 US2011040928W WO2011160050A2 WO 2011160050 A2 WO2011160050 A2 WO 2011160050A2 US 2011040928 W US2011040928 W US 2011040928W WO 2011160050 A2 WO2011160050 A2 WO 2011160050A2
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plant
transgenic
transgenic plant
grain
cellulose
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WO2011160050A3 (fr
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Kirk Pappan
Michael Blaylock
David Lee
Bruce Ferguson
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Edenspace Systems Corporation
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis

Definitions

  • bio fuels such as ethanol as a fuel additive.
  • the potential market for the bio fuels in industries such as transportation fuel is one of the largest in the U.S. economy.
  • Lignocellulosic production would also help to increase the net energy balance of corn ethanol.
  • the conversion of lignocellulosic feedstocks into ethanol has advantages including ready availability of large amounts of feedstock, avoidance of burning or land filling the materials, and relatively easy conversion of glucose (produced by hydrolysis of cellulose) into ethanol.
  • Most studies show substantial energy advantages of using cellulosic feedstocks ⁇ e.g., Farrell et ah, Science, 2006, 311 : 506-508).
  • glucan i.e., cellulose
  • cellulose a series of enzymes known as cellulases.
  • cellulases can efficiently hydrolyze cellulose to simpler sugars, the surrounding matrix of hemicellulose, lignin, beta-glucans, homogalacturonans and
  • rhamnogalacturonans must be partially or completely removed to expose the cellulose.
  • Hemicellulose, lignin, and pectin are cell wall structural polymers that provide additional strength to cell wall by extensive network of cross-links with one another. Side chains of hemicellulose and pectin provide sites for covalent cross-linking and these side chains can also limit the accessibility of the polysaccharide backbone to enzymatic hydrolysis.
  • (l,3),(l,4)-beta-D-glucans also embed within cellulosic microfibrils and act as an additional barrier to cellulose.
  • the diversity of chemical components and bonds involved in the matrix surrounding cellulose necessitates the action of several distinct classes of enzymes for its breakdown.
  • stay-green characteristics of plants.
  • Corn hybrids have varying degrees of "stay-green” characteristic. Good stay-green means grain dries faster than stover. This is useful in a grain hybrid because as the grain dries, the stalk stays green and healthy, and is less likely to have broken stalks, stalk rot or to lodge in late season.
  • Some hybrids are designed only for use in silage and have less stay-green, so that the grain will have higher moisture relative to the whole plant. Lodging is less important in silage and having more moisture in the grain portion increases starch digestibility.
  • Hybrids with good stay-green ratings will have milk lines that are more advanced relative to whole plant moistures, while silage-only hybrids that have less stay-green characteristic will be ready to harvest at less advanced milk line.
  • Clostridia bacteria convert forage sugars and organic acids into butyric acid, carbon dioxide and ammonia. This silage will have high levels of foul-smelling butyric acid, with a higher pH, high dry matter losses, and poor feed quality, palatability and intake potential. Laboratory fermentation profile analysis is available to determine relative amounts of lactic, acetic, butyric and propionic acids, all of which affect quality. Seepage results in a loss of nutrients and can be harmful to the environment. Very wet or frozen silage can be difficult to unload in the winter.
  • the invention encompasses the recognition that several aspects of plant processing could be improved to increase ethanol yields and/or reduce costs.
  • ethanol in the United States is currently produced from the starch present in corn grain, which makes up over 70% of the weight of a kernel.
  • Approximately 9% of the mass of the kernel is composed of cellulose and hemicellulose, which are also sources of fermentable sugars.
  • an additional 500 million gallons of ethanol could be produced from the level corn currently processed for ethanol.
  • the invention describes methods of using enzymes to modify feruloyl ester linkages, xylans, xylan side chains, glucuronoarabinoxylans, xyloglucans, mixed-linkage glucans, pectins, pectates,
  • transgenic plants expressingcellulases and hemicellulasestargeted to fiber in corn kernels and methods of using such plants to increase the yield of fermentable sugars from processing corn grain.
  • lysine an essential amino acid for nutrition in most livestock, in fuel processing byproducts.
  • the primary byproduct of ethanol production from corn grain is Dried Distillers Grains with Solubles (DDGS), which is commonly used as animal feed. Nevertheless, corn grain is naturally low in lysine.
  • DDGS Dried Distillers Grains with Solubles
  • DDGS is supplemented with lysine before being fed to swine and poultry.
  • the present invention provides corn plants that have been engineered to
  • DDGS lysine-enhanced DDGS
  • methods comprise using corn hybrids that retain higher levels of moisture in their stalks.
  • the invention provides methods of using varieties of maize that retain moisture in stalks while grain is drying in the field. Such varieties retain higher levels of moisture in the stalks compared to conventional corn varieties; the increased moisture facilitates reducing the recalcitrance of the stover to pretreatment and hydrolysis, while concurrently preserving the storage and transportation advantages of using dried grain in biorefmeries that produce ethanol or other bioproducts.
  • the present invention provides transgenic plants and methods using same; provided transgenic plants express enzymes that attack the backbone and sidechains of hemicellulose and pectin and feruloyl ester cross-links as a means of releasing fermentable sugars from cellulose, hemicellulose and pectin, improving forage and silage digestibility for livestock and exposing cellulose to direct hydro lytic attack by cellulases.
  • the present invention provides isolated polypeptides that can be used as accessory enzymes to facilitate hydrolysis of lignocellulosic biomass.
  • Polypeptides comprising amino acid sequences SEQ ID NO: 1 through SEQ ID NO: 70; expression vectors for expressing such polypeptides, and plants transformed with such expression vectors, are provided.
  • the term “gene” refers to a discrete nucleic acid sequence responsible for a discrete cellular product and/or performing one or more intracellular or extracellular functions. More specifically, the term “gene” refers to a nucleic acid that includes a portion encoding a protein and optionally encompasses regulatory sequences, such as promoters, enhancers, terminators, and the like, which are involved in the regulation of expression of the protein encoded by the gene of interest.
  • the gene and regulatory sequences may be derived from the same natural source, or may be heterologous to one another.
  • the definition can also include nucleic acids that do not encode proteins but rather provide templates for transcription of functional RNA molecules such as tRNAs, rRNAs, etc.
  • a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids.
  • gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g. , mRNA, tRNA, rRNA, antisense RNA, ribozyme structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
  • Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, proteins post-translationally modified, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • transgenic or genetically modified organism is one that has a genetic background which is at least partially due to manipulation by the hand of man through the use of genetic engineering.
  • transgenic cell refers to a cell whose DNA contains an exogenous nucleic acid not originally present in the non-transgenic cell.
  • a transgenic cell may be derived or regenerated from a transformed cell or derived from a transgenic cell.
  • Exemplary transgenic cells in the context of the present invention include plant calli derived from a stably transformed plant cell and particular cells (such as leaf, root, stem, or reproductive cells) obtained from a transgenic plant.
  • a "transgenic planf is any plant in which one or more of the cells of the plant contain heterologous nucleic acid sequences introduced by way of human intervention.
  • Transgenic plants typically express DNA sequences, which confer the plants with characters different from that of native, non-transgenic plants of the same strain.
  • the progeny from such a plant or from crosses involving such a plant in the form of plants, seeds, tissue cultures and isolated tissue and cells, which carry at least part of the modification originally introduced by genetic engineering, are comprised by the definition.
  • nucleic acid construct refers to a polynucleotide or oligonucleotide comprising nucleic acid sequences not normally associated in nature.
  • a nucleic acid construct of the present invention is prepared, isolated, or manipulated by the hand of man.
  • the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” are used herein
  • DNA deoxyribonucleotide
  • RNA ribonucleotide
  • operably linked refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled by, regulated by or modulated by the other nucleic acid sequence.
  • a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such second sequence, although any effective three-dimensional association is acceptable.
  • a single nucleic acid sequence can be operably linked to multiple other sequences. For example, a single promoter can direct transcription of multiple RNA species.
  • plant can refer to a whole plant, plant parts ⁇ e.g., cuttings, tubers, pollen), plant organs ⁇ e.g., leaves, stems, flowers, roots, fruits, branches, etc.), individual plant cells, groups of plant cells ⁇ e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof.
  • the class of plants which can be used in the methods of the present invention is as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants, as well as certain lower plants such as algae.
  • plants of a variety of a ploidy levels including polyploid, diploid and haploid.
  • plants are green field plants.
  • plants are grown specifically for "biomass energy".
  • suitable plants include, but are not limited to, corn, switchgrass, sorghum, miscanthus, sugarcane, poplar, pine, wheat, rice, soy, cotton, barley, turf grass, tobacco, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
  • suitable plants include, but are not limited to, corn, switchgrass, sorghum, miscanthus, sugarcane, poplar, pine, wheat, rice, soy, cotton, barley, turf grass, tobacco, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
  • transformation methods genetically modified plants, plant cells, plant tissue, seeds, and the like can be obtained.
  • polypeptide generally has its art-recognized meaning of a polymer of at least three amino acids. However, the term is also used to refer to specific functional classes of polypeptides, such as, for example, lignocellulolytic enzyme polypeptides (including, for example, AcidothermuscellulolyticusEl endo-l,4- -glucanase polypeptide, AcidothermuscellulolyticusxylE polypeptide, Acidothermuscellulolyticusguxl polypeptide, AcidothermuscellulolyticusayiUI polypeptide, Talaromycesemersoniicb E polypeptide, and PyrococcusfuriosusfaoE (ferulic acid esterase) polypeptide).
  • lignocellulolytic enzyme polypeptides including, for example, AcidothermuscellulolyticusEl endo-l,4- -glucanase polypeptide, AcidothermuscellulolyticusxylE polypeptide, Acidothermuscellulolyticus
  • polypeptide is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides.
  • polypeptides generally tolerate some substitution without destroying activity.
  • polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%>, 60%>, 70%>, or 80%>, and further usually including at least one region of much higher identity, often greater than 90%> or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term "polypeptide" as used herein. Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides presented herein.
  • promoter refers to a polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to the promoter, and which effects expression of the selected polynucleotide sequence in cells.
  • plant promoter refers to a promoter that functions in a plant.
  • the promoter is a constitutive promoter, i.e., an unregulated promoter that allows continual expression of a gene associated with it.
  • a constitutive promoter may in some embodiments allow expression of an associated gene throughout the life of the plant. Examples of constitutive plant promoters include, but are not limited to, rice actl promoter, Cauliflower mosaic virus (CaMV) 35S promoter, and nopalinesynthase promoter from
  • the promoter is a tissue- specific promoter that selectively functions in a part of a plant body, such as a flower. In some embodiments of the invention, the promoter is a developmentally specific promoter. In some embodiments of the invention, the promoter is an inducible promoter. In some embodiments of the invention, the promoter is a senescence promoter, i.e., a promoter that allows transcription to be initiated upon a certain event relating to the age of the organism.
  • the term "stably transformed?', when applied to a plant cell, callus or protoplast refers to a cell, callus or protoplast in which an inserted exogenous nucleic acid molecule is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome.
  • the stability is demonstrated by the ability of the transformed cells to establish cell lines or clones comprised of a population of daughter cells containing the exogenous nucleic acid molecule.
  • the phrase "substantial homology,” when used to refer to polypeptide sequences, refers to at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%), 95%), 96%o, 97%), 98%>, 99%, or more sequence identity over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or more residues.
  • the term "transformation” refers to a process by which an exogenous nucleic acid molecule (e.g., a vector or recombinant DNA molecule) is introduced into a recipient cell, callus or protoplast.
  • the exogenous nucleic acid molecule may or may not be integrated into (i.e., covalently linked to) chromosomal DNA making up the genome of the host cell, callus or protoplast.
  • the exogenous polynucleotide may be maintained on an episomal element, such as a plasmid.
  • the exogenous polynucleotide may become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • Methods for transformation include, but are not limited to, electroporation, magnetoporation, Ca 2+ treatment, injection, particle bombardment, retroviral infection, and lipofection.
  • transgene refers to an exogenous gene which, when introduced into a host cell through the hand of man, for example, using a process such as transformation, electroporation, particle bombardment, and the like, is expressed by the host cell and integrated into the cell's DNA such that the trait or traits produced by the expression of the transgene is inherited by the progeny of the transformed cell.
  • a transgene may be partly or entirely heterologous (i.e., foreign to the cell into which it is introduced).
  • a transgene may be homologous to an endogenous gene of the cell into which it is introduced, but is designed to be inserted (or is inserted) into the cell's genome in such a way as to alter the genome of the cell (e.g. , it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can also be present in a cell in the form of an episome.
  • a transgene can include one or more transcriptional regulatory sequences and other nucleic acids, such as introns.
  • a transgene is one that is not naturally associated with the vector sequences with which it is associated according to the present invention.
  • the water content in biomass affects the degree of swelling (Browning, 1975), the crystallinity (Fan et al, 1981) and the digestibility (Focher et al, 1981) of cellulose microfibrils.
  • Air-drying of biomass samples is thought to collapse the capillary structure irreversibly (Esteghlalian et al, 2001; Weise, 1998), significantly decreasing internal surface area (Stone and Scallan, 1968; Browning, 1975).
  • Recent studies have demonstrated that drying dilute acid pre-treated corn stover decreases cellulose digestibility, suggesting that the efficiency of biomass hydrolysis could be improved with using biomass with a higher moisture content.
  • a number of plant mutants have been identified whose leaves remain green longer than wild type plants. These have been defined as 'stay-green' and examples have been identified in a number of crops, including maize (Gentinetta et al, 1986; Bekavac et al, 1995) and sorghum (Duncan et al., 1981; Tao et al., 2000). Most of these mutations are involved in the timing and rate of leaf senescence, and some of these alterations result in a phenotype that continues to photosynthesize for longer than normal (i.e. is 'functional stay green') and that might therefore be expected to result in a higher yield. Even in varieties that are cosmetic stay green plants and do not have increased photosynthetic competence (Thomas and Stoddart, 1975), the biomass retains greater levels of moisture as their cells do not senesce correctly.
  • the present invention encompasses the recognition that using stay-green varieties as biomass feedstocks for cellulosic biofuel production may be advantageous. Using varieties that continue to photosynthesize later in the growing season may produce greater quantities of biomass even as the grain on the plant is drying out. This type of variety, along with cosmetic stay green plants, will also contain higher moisture levels than wild type varieties when the grain is harvested. In some embodiments, altered harvesting processes are used.
  • the biomass will not have undergone hornification, the process in which adjacent cellulose fibers form hydrogen bond upon dessication, significantly increasing the recalcitrance of the biomass. Consequently, the cell walls in high moisture stay green biomass will be more readily accessible to hydrolytic enzymes during conversion. Additionally, the higher moisture content in stay green biomass will also be beneficial for ensilement, a process beneficial to both store biomass prior to biofuel conversion and to activate the plant produced enzymes.
  • Biomass for ensilement is usually harvested green, before the grain has dried down, because a certain level of moisture is necessary to avoid spontaneous combustion during ensilement. Use of stay green plants will allow the grain to be harvested dry and be available for conversion to grain ethanol, and the biomass to be more efficiently pretreated and hydrolyzed for biofuel production.
  • transgenic plants having a stay-green trait e.g., as conferred by an altered stay-green gene
  • a lignocellulo lytic polypeptide e.g., as expressed from a recombinant polynucleotide
  • such plants are used for cellulosic biofuel production.
  • corn plants were stably transformed to express cell wall- modifying enzyme polypeptides.
  • Stable transformation of corn was performed according to a protocol using immature embryos of the Hi-II corn genotype.
  • NPTII neomycin phosphotransferase II
  • infected embryos were moved to selection medium containing paromomycin (100 mg/L) and incubated in an incubator at 27 °C in the dark in a plant tissue cuture chamber.
  • Resistant Type II calli induced from immature embryos were selected for 8 weeks at 27 °C in the dark with 200 mg/L of paromomycin.
  • proliferated embryogenic calli were sub-cultured into somatic embryo maturation medium for two weeks at 27 °C in dark.
  • Matured somatic embryos were subcultured on regeneration medium for another two weeks under light at 27 °C (16 h/8 h light/dark cycle, Conviron TC26; tissue culture chamber). Green and elongated somatic embryos that emerged in 2-4 weeks were transferred to basic nutrient medium for further elongation and rooting in magenta boxes and grown at 27 °C under a 16 h/8 h light/dark photoperiod. Plantlets with well-established roots were transferred to soil and acclimatized in a plant growth chamber (Conviron, Adaptis A 1000). After molecular characterization for transgene integration plants were moved to green house and grown to maturity.
  • Corn plants were successfully transformed with expression vectors encoding cell wall- modifying enzyme polypeptides, including a feruloyl esterase polypeptide and an exoglucanase polypeptide.
  • Example 2 Characterization of corn plants stably transformed with a construct for expressing an exoglucanase
  • Corn plants were stably transformed with expression vectors as described in Example 1.
  • the present Example presents experimental results characterizing corn plants that had been transformed with expression vectors for an exoglucanase.
  • Corn plants were transformed with pEDEN122, an expression vector encoding CBH-E (an exoglucanase expressed by Talaromyces emorsonii) and selected for paromomycin resistance. Paromomycin-selected plants were screened by PCR for presence of CBH-E and npt II (the selectable marker) genes, using CBH-E and npt II primers. Plants for which positive signals for CBH-E and the selectable marker were detected by PCR were chosen for further study.
  • CBH-E an exoglucanase expressed by Talaromyces emorsonii
  • Extractive compounds were removed from exoglucanase-expressing and control corn stover composite samples using a standard ethanol-acetone extraction procedure and dried to completeness in a fume hood.
  • the tare weight of empty sample tubes was recorded and then ground material from exoglucanase-expressing and control biomass ( ⁇ 50 mg) was transferred to each tube. The dry weight of the sample plus the tube was recorded.
  • Samples were then treated according to their experimental group.
  • Half of the exoglucanase-expressing and control samples, the "Pretreated” group were reconstituted in 100 mM sulfuric acid and heated at 120 °C for 10 minutes followed by neutralization with 0.5 N sodium hydroxide.
  • the second half of the samples, the "Not Treated” group were kept in their dry state.
  • samples in the Pretreated group were centrifuged and the supernatant was discarded.
  • Samples in the Pretreated and Not Treated groups were reconstituted in buffer (sodium acetate pH 5.0, 5 mM CaC12, and 0.02% sodium azide) containing either 0.4 mg or 8 mg Novozymes Celluclast 1.5L/g of starting dry weight and 0.2 units of Novozymes 188 ⁇ -glucosidase.
  • the samples were incubated at 50 °C for 24 h after which time the solids rinsed extensively with water to remove hydrolyzed materials liberated during the 24 h hydrolysis period. Samples were dried to completeness in a dehydrator and the final dry weight of the sample plus tube recorded.
  • the amount of mass lost during the enzyme digestion was determined by subtracting the final sample weight from the starting weight.
  • the digestibility of a sample was determined by calculating percentage of mass lost during the in vitro dry matter digestibility (IVDMD) procedure. Data were graphed and analyzed by one-way Analysis of Variance (ANOVA) with post-hoc testing using the Tukey method.
  • Transgenic corn plants such as those described in examples 1 and 2, will be crossed with stay green corn plants to generate hybrid plants containing both the stay green trait and the cell wall hydrolyzing enzymes. Seeds will be collected from these hybrids, germinated and genotyped, and again back-crossed with the stay green corn parent line to produce genetically stable stay green corn plants expressing cell wall hydrolyzing enzymes.
  • Example 4 Processing of transgenic stay green corn for biofuel production
  • transgenic corn seed will be sown and grown using standard agronomic practices. Plants will be allowed to grow until they produce mature ears and the grain dries down to 15% moisture, at which point the stover will still be green. Plants will be harvested in a manner to separate the grain from the green biomass and the grain will be processed for ethanol using standard methods. Stover may be collected for ensilement onsite or at the processing facility and ensiled for an indefinite period of time until the facility is ready to process the biomass, or utilized immediately if desired.
  • the ensilement period also provides a time for the plant produced enzymes to act on the cell wall, likely reducing the recalcitrance of the biomass prior to pretreatment. Enzymes will have a greater impact on the biomass of stay green corn compared to standard varieties due to the increased moisture in the biomass.
  • the facility When the facility is ready to process the biomass, it will first perform an activation step to allow the enzymes to act on the biomass at the optimal temperature, pH, and other processing conditions. After activation, the biomass will be pretreated using dilute sulfuric acid, AFEX, or other common methods, saccharified, and fermented to produce biofuel.
  • AFEX dilute sulfuric acid
  • the stay green biomass retains significant levels of moisture compared to standard corn varieties, it is expected that water use during pretreatment and hydrolysis will be less than from conversion of standard corn stover.
  • Corn kernel has four main components: the pericarp, the endosperm, the germ and the tip cap.
  • the tip is the component that attaches the kernel to the cob.
  • the germ is a small portion of the kernel that can be seen on one surface.
  • the germ has oil, protein and enzymes that start the germination process for growth.
  • the outer fibrous layer is the pericarp, or bran, which protects the kernel.
  • the majority of the kernel is endosperm .
  • the endosperm contains approximately 98 percent of the starch in the kernel and is approximately 83 percent of the dry weight of the kernel.
  • starch is the constituent of the corn that is converted to alcohol.
  • An object of fractionation for ethanol plants is to separate the endosperm from the other components because it contains 98 percent of the starch.
  • the present invention encompasses the recognition that removing non-endosperm components (fiber and germ) of corn grain during the beginning stages of processing offers advantages for ethanol production.
  • biorefmeries can improve yields of ethanol production by fractionating the corn kernel prior to saccarification of the starch.
  • the fiber fraction also has many new opportunities, which include cattle feed, human fiber additive, corn fiber oil extraction, and on-site burning to reduce natural gas costs involved in saccarification and fermentation. Additionally, the fiber is a good feedstock for cellulosic biofuel since it contains much less lignin and hemicellulose compared to other lignocellulosic feedstocks.
  • cell wall hydrolyzing enzymes By fractionating the kernel, cell wall hydrolyzing enzymes will be isolated in the protein fraction of the DDGS and avoid going through the starch hydrolysis reaction and being denatured. Instead, they can be recovered and applied to cellulosic feedstocks such as the fiber in the bran fraction of the kernel.
  • Example 4 Fractionating of transgenic corn grain for improved yield of biofuel production
  • Transgenic corn plants expressing cell wall hydrolyzing enzymes can provide significant yield improvements in the production of grain ethanol.
  • the transgenic corn will be grown and harvested according to standard agronomic practices and the dried grain isolated for processing into ethanol.
  • the fractionation process will break the kernels into their basic components and separate the starch from the protein and fiber in the kernel.
  • the starch stream will be hydrolyzed with amylase enzymes using existing processes, and the fiber stream separated to process the cellulose into fermentable sugars.
  • the protein steam from the kernel will also be isolated to collect the recombinant enzyme produced in the corn.
  • the cell wall hydrolyzing enzyme such as cbhE
  • cbhE will then be added to the fiber fraction along with externally produced cellulase enzymes to efficiently convert the cellulose to glucose monomers.
  • the glucose will be recovered from the hydrolysis reaction and fermented to produce ethanol or other alcohol based biofuels, or as a feedstock for industrial chemicals such as poly-lactic acid (PLA). Se ⁇ K ace ⁇ Lisiing;
  • Organism Pyroeoccusf uriosits
  • Organism Bifidobacteriumlongum
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  • Sequence ID 6 Sequence Length: 540
  • Organism Solibacterusitatus
  • Organism Sulfolobussolfataricus
  • Organism Thermotoganeapolitana
  • Organism Erwiniacaroto vorum
  • Organism Bacillus sp.
  • Organism Bacillus subtilis MLK.NKKTWKRFFHLSSAALAAGLIFTSAAPAEAAFWGASNELLHDPTMIKEGSSWYALGTGLNEE RGLRVLKSSDAK.NWTVQKSIFSTPLSWWSNYVPNYEKNQWAPDIQYYNGKYWLYYSVSSFGNNTS AIGLASSTSISSGNWEDEGLVIRSTSSNNYNAIDPELTFDKDGNPWLAFGSFWSGIKLTKLDKST MKPTGSPYSIAARPNNNGALEAPTLTYQNGYYYLMVSFDKCCNGVNSTYKIAYGRSKSITGPYLD KSGKSMLDGGGTILDSGNDQWKGPGGQDIVNGNILVRHAYDANDNGTPKLLINDLNWSSGWPSY
  • Organism Erwiniacarotovorum
  • Organism Thermotogamaritima MVLMTKPGTSDFVWNGIPLSMELNLWNIKEYSGSVAMKFDGEKITFDADIQNLSPKEPERYVLGY PEFYYGYKPWENHTAEGSKLPVPVSSMKSFSVEVSFDIHHEPSLPLNFAMETWLTREKYQTEASI GDVEIMVWFYFNNLTPGGEKIEEFTIPFVLNGESVEGTWELWLAEWGWDYLAFRLKDPVKKGRVK FDVRHFLDAAGKALSSSARVKDFEDLYFTVWEIGTEFGSPETKSAQFGWKFENFSIDLEVRE
  • Organism Sinorhizobiummeliloti MTIDRYRRFARLAFIATLPLAGLATAAAAQEGANGKSFKDDFDTLDTRVWFVSDGWNNGGHQNCT WSKKQVKTVDGILELTFEEKKVKERNFACGEIQTRKRFGYGTYEARIKAADGSGLNSAFFTYIGP ADKKPHDEIDFEVLGKNTAKVQINQYVSAKGGNEFLADVPGGANQGFNDYAFVWEKNRIRYYVNG ELVHEVTDPAKIPVNAQKIFFSLWGTDTLTDWMGTFSYKEPTKLQVDRVAFTAAGDECQFAESVA CQLERAQSE
  • Organism Thermococcuss .
  • Organism Clostridium thermocellum
  • Organism Thermotogamaritima
  • Organism Vibriosp.
  • Organism Thermotogamaritima MNNTIPRWRGFNLLEAFSIKSTGNFKEEDFLWMAQWDFNFVRIPMCHLLWSDRGNPFIIREDFFE KIDRVIFWGEKYGIHICISLHRAPGYSVNKEVEEKTNLWKDETAQEAFIHHWSFIARRYKGISST HLSFNLINEPPFPDPQIMSVEDHNSLIKRTITEIRKIDPERLIIIDGLGYGNIPVDDLTIENTVQ SCRGYIPFSVTHYKAEWVDSKDFPVPEWPNGWHFGEYWNREKLLEHYLTWIKLRQKGIEVFCGEM GAYNKTPHDWLKWLEDLLEIFKTLNIGFALWNFRGPFGILDSERKDVEYEEWYGHKLDRK LEL LRKY
  • Organism Ruminococcusalbus MKQNGVNLYAISVQNEPDYAKDWTAWTPDETTDFIANYGDQITSTKLMSPESFQYGAYNNGKDYY SKILNNSKAYANCDIFGTHFYGTPRSK DFPALENCGKQLWMTEVYVPDSNVDSNIWPDNLKQAV SIHDSLWGGMQAYWWPLRRNYSILREDTHKISKRGYAFAQYSKFVRPGDVRVDVTEQPSSNVF VSAYKNNKNQVTIVAINNSSSGYSQQFSLNGKTIIDVDRWRTSGSENLAETDNLTIDNGTSFWAQ LPAQSVSTFVCTLSGGSSSGNNGSSNTELDSDGYYFHDTFEDDLTWQAHGGTELLKSGRTPYKGS ⁇ /TNRTSAWMGAERTLPSSWPGKTYSFSVNVTELDGEDTETFYLKLNYTDSSGTAHYPTIA EGVCPKGKYLQLSNTNYTIPSDAVDPVIYVE

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Abstract

Cette invention concerne des méthodes de réduction de la résistance de la paroi des cellules végétales à l'hydrolyse, des méthodes de stimulation de la conversion de biomasse végétale en sucres fermentescibles, des méthodes d'augmentation des rendements des sucres fermentescibles à partir de l'hydrolyse des grains de maïs, et des techniques de récolte des grains et des épis de maïs débarrassés des grains simultanément pour l'hydrolyse. Dans certains modes de réalisation, l'invention concerne des plantes transgéniques transformées par des vecteurs d'expression contenant une séquence d'ADN codant les enzymes de modification des parois cellulaires végétales à partir de sources microbiennes et animales. Dans certains modes de réalisation, l'invention concerne des méthodes d'amélioration des performances enzymatiques en créant des variants de l'enzyme parent. Dans certains modes de réalisation, l'invention concerne des méthodes d'utilisation de variétés « staygreen » (le maïs reste vert) comme charges de biomasse pour la production de biocarburants cellulosiques. Les utilisations de l'invention comprennent, sans s'y limiter, la stimulation de la conversion de biomasse, la conversion de biomasse lignocellulosique et la production de biocarburant efficaces, la réduction du caractère récalcitrant de la biomasse, l'amélioration de la digestibilité du fourrage, l'amélioration des qualités des fibres et l'amélioration des propriétés de traitement des fibres.
PCT/US2011/040928 2010-06-18 2011-06-17 Systèmes de réduction du caractère récalcitrant de la biomasse cellulosique et d'augmentation des rendements des sucres fermentescibles WO2011160050A2 (fr)

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WO2014077264A1 (fr) * 2012-11-16 2014-05-22 独立行政法人産業技術総合研究所 Composition pour la décomposition de biomasse et procédé de production d'un liquide sucré à l'aide de celle-ci
US8778641B1 (en) * 2013-02-12 2014-07-15 Novozymes Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2015002841A2 (fr) * 2013-07-02 2015-01-08 Board Of Trustees Of Michigan State University Digestibilité améliorée de biomasse végétale
WO2019161471A1 (fr) * 2018-02-23 2019-08-29 Cnpem - Centro Nacional De Pesquisa Em Energia E Materiais Composition pour l'hydrolyse enzymatique à activité arabinofuranosidase et procédé d'hydrolyse de biomasse végétale
US10745707B2 (en) 2013-07-02 2020-08-18 Board Of Trustees Of Michigan State University Digestibility of plant biomass

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CN106318957B (zh) * 2016-10-26 2019-05-07 南京林业大学 土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014077264A1 (fr) * 2012-11-16 2014-05-22 独立行政法人産業技術総合研究所 Composition pour la décomposition de biomasse et procédé de production d'un liquide sucré à l'aide de celle-ci
US8778641B1 (en) * 2013-02-12 2014-07-15 Novozymes Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2015002841A2 (fr) * 2013-07-02 2015-01-08 Board Of Trustees Of Michigan State University Digestibilité améliorée de biomasse végétale
WO2015002841A3 (fr) * 2013-07-02 2015-03-12 Board Of Trustees Of Michigan State University Digestibilité améliorée de biomasse végétale
US10202614B2 (en) 2013-07-02 2019-02-12 Board Of Trustees Of Michigan State University Digestibility of plant biomass
US10745707B2 (en) 2013-07-02 2020-08-18 Board Of Trustees Of Michigan State University Digestibility of plant biomass
WO2019161471A1 (fr) * 2018-02-23 2019-08-29 Cnpem - Centro Nacional De Pesquisa Em Energia E Materiais Composition pour l'hydrolyse enzymatique à activité arabinofuranosidase et procédé d'hydrolyse de biomasse végétale

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