WO2011028623A2 - Organismes modifiés utilisés pour une meilleure saccharification de la biomasse - Google Patents

Organismes modifiés utilisés pour une meilleure saccharification de la biomasse Download PDF

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Publication number
WO2011028623A2
WO2011028623A2 PCT/US2010/046866 US2010046866W WO2011028623A2 WO 2011028623 A2 WO2011028623 A2 WO 2011028623A2 US 2010046866 W US2010046866 W US 2010046866W WO 2011028623 A2 WO2011028623 A2 WO 2011028623A2
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microorganism
another embodiment
product
fermentation
biomass
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PCT/US2010/046866
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WO2011028623A3 (fr
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Matthias Schmalisch
Glenn Richards
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Qteros, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Biomass is a renewable source of energy, which can be biologically fermented to produce an end-product such as a fuel (e.g. alcohol, ethanol, organic acid, acetic acid, lactic acid, methane, or hydrogen).
  • a fuel e.g. alcohol, ethanol, organic acid, acetic acid, lactic acid, methane, or hydrogen.
  • Biomass includes agricultural residues (corn stalks, grass, straw, grain hulls, bagasse, etc.), animal waste (manure from cattle, poultry, and hogs), woody materials (wood or bark, sawdust, timber slash, and mill scrap), municipal waste (waste paper, recycled toilet papers, yard clippings, etc.), and energy crops (poplars, willows, switch grass, alfalfa, prairie bluestem, algae etc.).
  • Lignocellulosic biomass has cellulose and hemicellulose as two major components. To obtain a high fermentation efficiency of lignocellulosic biomass to end- product (yield) it is important to provide an appropriate fermentation environment to enhance end-product yield. More complete saccharification of biomass and fermentation of the saccharification products results in higher fuel yields.
  • a composition for production of a biofuel comprising: a carbonaceous biomass, and a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein the microorganism is genetically modified to express at least one expansin and/or swollenin protein.
  • the microorganism is capable of direct fermentation of C5 and C6 carbohydrates.
  • the microorganism is a bacterium.
  • the microorganism is a species of Clostridia.
  • the microorganism is Clostridium phytofermentans .
  • the microorganism is Clostridium sp. Q.D.
  • the composition further comprises one or more expansin and/or swollenin enzymes.
  • the microorganism comprises one or more heterologous polynucleotides that enhance the activity of one or more cellulases.
  • the composition further comprises one or more cellulases, B-glucosidases, hemicellulases, xylanases, glucanases or xylanases.
  • the fermentation end-product is ethanol.
  • the fermentation end-product is lactic acid, acetic acid or formic acid.
  • an isolated microorganism is provided that is capable of directly fermenting a lignocellulosic biomass, wherein the microorganism is genetically modified to express at least one expansin and/or swollenin protein.
  • the microorganism is capable of direct fermentation of C5 and C6 carbohydrates.
  • the microorganism is a bacterium.
  • the microorganism is a species of Clostridia.
  • the microorganism is Clostridium
  • the microorganism is Clostridium sp. Q.D.
  • the microorganism comprises one or more heterologous polynucleotides that encode said at least one expansin and/or swollenin.
  • the microorganism comprises one or more heterologous polynucleotides that enhance the activity of one or more cellulases.
  • a product for production of a fermentation end-product comprising: (a) a fermentation vessel comprising a carbonaceous biomass; (b) a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein the microorganism is genetically modified to express at least one expansin and/or swollenin; and (c) a source of one or more proteins that is external to said microorganism, wherein the fermentation vessel is adapted to provide suitable conditions for fermentation of one or more carbohydrates into a fermentation end-product.
  • the microorganism is capable of direct fermentation of C5 and C6 carbohydrates.
  • the microorganism is a bacterium.
  • the microorganism is a species of Clostridia. In another embodiment, the microorganism is Clostridium phytofermentans. In another embodiment, the microorganism is Clostridium sp. Q.D. In another embodiment, the microorganism comprises one or more heterologous polynucleotides that encode said at least one expansin and/or swollenin. In another embodiment, the microorganism comprises one or more heterologous polynucleotides that enhance the activity of one or more cellulases. In another embodiment, the product further comprises one or more cellulases, B-glucosidases, hemicellulases, xylanases, glucanases or xylanases.
  • the biomass comprises one or more of corn steep solids, corn steep liquor, malt syrup, xylan, cellulose, hemicellulose, fructose, glucose, mannose, rhamnose, or xylose.
  • the fermentation end-products is ethanol.
  • the fermentation end-product is lactic acid, acetic acid or formic acid.
  • the external source of an enzyme comprises a cellulase mixture.
  • the cellulase mixture saccharifies cellulose to produce predominately saccharides with a polymerization higher than five.
  • the fermentation end-product is a fuel.
  • the fuel is ethanol.
  • the fermentation end-product is a chemical.
  • the fermentation end-product is an acid.
  • a fermentation end-product is produced by: (a) contacting a carbonaceous biomass with a microorganism of any of claims 3 or 15-21; and (b) allowing sufficient time for said hydrolysis and fermentation to produce the fermentation end- product.
  • the fermentation end-product is a fuel.
  • the fuel is ethanol.
  • the fermentation end-product is a chemical.
  • the fermentation end-product is an acid.
  • a process for producing a fermentation end-product comprising: contacting a carbonaceous biomass with: a microorganism that hydrolyses and ferments said biomass; an external source of at least one expansin or swollenin enzyme and at least one cellulase; and allowing sufficient time for said hydrolysis and fermentation to produce a fermentation end-product; wherein no external addition of xylanase, hemicellulase, glucanase or glucosidase is made.
  • Figure 1 illustrates a plasmid map for pIMPl .
  • Figure 2 illustrates a plasmid map for pIMPCphy.
  • Figure 3 illustrates a plasmid map for pCphyP3510.
  • Figure 4 illustrates a plasmid map for pCphyP3510-1163.
  • Figure 5 depicts a method for producing fermentation end products from biomass by first treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit.
  • Figure 6 depicts a method for producing fermentation end products from biomass by charging biomass to a fermentation vessel.
  • Figure 7 illustrates a pathway map for cellulose hydrolysis and fermentation.
  • Described herein are methods and compositions directed to saccharification and fermentation of various biomass substrates to desired products. Described methods and compositions utilize novel combinations of proteins, modified organisms, or processes to enhance saccharification of biomass. By described methods and compositions, fermentation end- products described herein are produced in a highly efficient manner that has not been previously known in the art. In one aspect, high efficiency is achieved by more complete or increased saccharification of biomass. In another aspect, high efficiency is achieved by more complete or increased fermentation of the saccharified products.
  • the term "about” means any integer and decimal point falling within plus or minus 10% of the referenced numeric indication.
  • about 10 refers to 9, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1 , 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 1 1.
  • the terms "increased” or “increasing” as used herein mean the ability of one or more recombinant microorganisms to produce a greater amount of a given product or molecule (e.g., commodity chemical, biofuel, or intermediate product thereof) as compared to a control microorganism, such as an unmodified microorganism or a differently modified microorganism.
  • An “increased” amount is typically a statistically significant amount, and may include an increase that is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (including all integers and decimal points in between, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by an unmodified microorganism or a differently modified microorganism.
  • gene refers to a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5' and 3' untranslated sequences).
  • the term "host cell” includes an individual cell which can be or has been a recipient of a recombinant vector(s) or isolated polynucleotide.
  • Host cells include progeny of a parent host cell, including progeny that are less than completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected, transformed, or infected in vivo or in vitro with a recombinant vector or a polynucleotide (e.g., dsDNA, ssDNA, dsR A or ssR A).
  • a host cell which comprises a recombinant vector can be referred to as a recombinant host cell, recombinant cell, or recombinant microorganism.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it in its native state.
  • isolated polynucleotide refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an "isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, i.e., it is not associated with in vivo substances.
  • external source as it relates to a quantity of an enzyme or enzymes provided to a product or a process means that the quantity of the enzyme or enzymes is not produced by a microorganism in the product or process.
  • An external source of an enzyme can include, but is not limited to an enzyme provided in purified form, cell extracts, culture medium or an enzyme obtained from a commercially available source.
  • operably linked means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the gene from which it is derived.
  • Constant promoters are typically active, i.e., promote transcription under most conditions.
  • “Inducible promoters” are typically active only under certain conditions, such as in the presence of a given molecule factor ⁇ e.g., IPTG) or a given environmental condition ⁇ e.g., C0 2 concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity.
  • polynucleotide variant and “variant” and the like refer to polynucleotides that display substantial sequence identity with any of the reference polynucleotide sequences or genes described herein, polynucleotides that hybridize with any polynucleotide reference sequence described herein or any polynucleotide coding sequence of any gene or protein referred to herein under low stringency, medium stringency, high stringency, or very high stringency conditions that are defined hereinafter and known in the art. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms
  • polynucleotide variant and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or has increased activity in relation to the reference polynucleotide (i.e., optimized).
  • Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with a reference polynucleotide described herein.
  • wild type and “naturally occurring” are used interchangeably to refer to biological entity that has the characteristics of a naturally occurring source. Where there are two or more naturally occurring sources, biological entities of both sources can be called as “wild type.” Alternatively, a most frequently occurring or observed source in a population can be called as “wild-type.”
  • biomass refers to plant or animal matter or other organic matter containing polysaccharides or polysaccharides in combination with other organic components.
  • polysaccharide comprises one or more carbohydrate polymers of sugars and sugar derivatives.
  • polysaccharide comprises derivatives of sugar polymers.
  • polysaccharide comprises polymeric materials that occur in plant and other organic matter.
  • Exemplary polysaccharides from plants and other organisms include lignin, cellulose, starch, pectin, hemicellulose, chitin, or sulfonated polysaccharides such as alginic acid, agarose, carrageenan, porphyran, furcelleran and funoran.
  • polysaccharide comprises two or more sugar units or derivatives of sugar units.
  • the sugar units are hexose units or pentose units, or combinations thereof.
  • the derivatives of sugar units are sugar alcohols, sugar acids, or amino sugars.
  • the polysaccharide is linear.
  • the polysaccharide is branched or cross-linked.
  • the polysaccharide is a mixture of linear and branched or cross-linked.
  • the polysaccharide is a mixture of different classes of polysaccharides.
  • polysaccharides, oligosaccharides, monosaccharides or other sugar components of biomass include, but are not limited to, alginate, agar, carrageenan, fucoidan, pectin, gluronate, mannuronate, mannitol, lyxose, cellulose, hemicellulose, glycerol, xylitol, glucose, mannose, galactose, xylose, xylan, mannan, arabinan, arabinose, glucuronate, galacturonate (including di- and tri-galacturonates), rhamnose, and the like.
  • a biomass comprises fermentable sugars.
  • a fermentable sugar is a sugar used by a microorganism as a carbon source.
  • a fermentable sugar is a monomer, dimer, or polymer of sugar.
  • a polymer is a trimer, quatrimer, pentamer, hexamer, septamer, octamer, nanomer, or decamer.
  • fermentable sugar is a product such as an intermediate metabolite of a polymer broken down by a microorganism.
  • a sugar is broken down by an enzymatic process such as hydrolysis.
  • Exemplary fermentable sugars include, but are not limited to glucose, xylose, arabinose, galactose, mannose, rhamnose, cellobiose, lactose, sucrose, maltose, and fructose.
  • a biomass is a carbonaceous biomass that can be converted to a fuel, biofuel, chemical or other fermentation end product.
  • a carbonaceous biomass comprises municipal waste, wood, plant material, plant extract, natural or synthetic polymers, or a combination thereof.
  • a biomass comprises plant matter.
  • plant matter include, but are not limited to, woody plant matter, non- woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, corn stover, sugar cane, grasses, switch grass, bamboo, and material derived from these.
  • Plant matter also includes algae and other autotrophic organisms, agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, distillers grains, peels, pits, fermentation waste, straw, lumber, sewage, garbage and food leftovers. These materials can come from farms, forestry, industrial sources, households, etc.
  • a biomass comprises animal matter, including, for example milk, meat, fat, animal processing waste, and animal waste.
  • a biomass comprises aquatic or marine biomass, fruit-based biomass such as fruit waste, or vegetable-based biomass such as vegetable waste.
  • aquatic or marine biomass include, but are not limited to, kelp, seaweed, algae, and marine microflora, microalgae, sea grass, salt marsh grasses such as Spartina sp. or Phragmites or the like.
  • fruit and/or vegetable biomass include, but are not limited to, any source of pectin such as plant peel and pomace including citrus, orange, grapefruit, potato, tomato, grape, mango, gooseberry, carrot, sugar-beet, or apple.
  • a biomass comprises one or more of corn steep solids, corn steep liquor, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), malt syrup, xylan, cellulose, hemicellulose, fructose, glucose, mannose, rhamnose, or xylose.
  • DDS Distillers Dried Solubles
  • DDG Distillers Dried Grains
  • CDS Condensed Distillers Solubles
  • DWG Distillers Wet Grains
  • DDGS Distillers Dried Grains with Solubles
  • malt syrup xylan, cellulose, hemicellulose, fructose, glucose, mannose, rhamnose, or xylose.
  • Microorganisms useful in compositions and methods described herein include but are not limited to bacteria, yeast or fungi. In one embodiment two or more different microorganisms can be utilized to produce a fermentation end-product. Microorganisms utilized herein can be recombinant, non-recombinant or wild type.
  • a microorganism utilized in a composition or method described herein is a Clostridium.
  • the Clostridium is C. phytofermentans ("C. phy").
  • C. phy is a microorganism that can simultaneously hydrolyze and ferment hexose (C6) and pentose (C5) polysaccharides.
  • C. phy is capable of acting directly on lignocellulosic biomass without any pretreatment of said biomass.
  • a Clostridium is used to enhance the yield of a fermentation end-product.
  • a genetically modified C. phy is used to enhance the yield of a fermentation end-product.
  • Clostridium sp. Q.D is described in U.S. serial No. 61/327,051, which is herein incorporated by reference in its entirety. Clostridium sp. Q.D forms moist, shiny, beige, opaque, irregular or undulate colonies. The cells are entire, small, short rods, diplo or chains, motile, and form subterminal endospores. Q.D is able to utilize crystalline cellulose as a carbon source, and can form ethanol and acetic acid as major fermentation end products. Clostridium sp. Q.D is a gram-positive bacterium, deposited under NRRL Accession No. NRRL B-50361 at the
  • Clostridium sp. U201 (99%>), and swine fecal bacterium strain RF3G-Cel2 (99%>).
  • Clostridium sp. Q.D can hydrolyze polysaccharides and higher saccharides that contain hexose sugar units, pentose sugar units, or that contain both, into lower saccharides and in some cases
  • a microorganism utilized for a process described herein is Clostridium sp. Q.D.
  • a microorganism described herein comprises one or more exogenous polynucleotides that encode an expansin or swollenin, and/or one or more cellulase proteins. Expansins or swollenins are part of a protein family derived from eukaryotes such as plants or fungi and also from some prokaryotes. They induce plant cell wall loosening and cellulose disruption without exerting cellulose hydrolysis.
  • a microorganism described herein comprises one or more exogenous polynucleotides that encode an expansin or swollenin, and/or one or more cellulase proteins. Expansins or swollenins are part of a protein family derived from eukaryotes such as plants or fungi and also from some prokaryotes. They induce plant cell wall loosening and cellulose disruption without exerting cellulose hydrolysis.
  • a protein family derived from eukaryotes such as plants or fungi and also from some
  • microorganism described herein does not contain any exogenous polynucleotides, but its genome is modified to change the function of one or more proteins.
  • the protein is associated with hydro lyzation of biomass.
  • the protein is associated with fermentation of a polysaccharide or monosaccharide, or both.
  • Selection of an optimal bacterial strain for fermentation depends on the composition of a particular biomass fermenting environment such as tolerance to ethanol, alkaline treatment, and other physical parameters of fermentation ⁇ e.g., pH, pressure, or temperature). Methods disclosed herein are applicable and readily adaptable to any bacterial strain that anaerobically ferment biomass.
  • a bacterial strain useful for the methods disclosed herein includes, but is not limited to those strains described below.
  • feedstock is saccharified by a microorganism that is a Clostridium strain, a Trichoderma strain, a Saccharomyces strain, a Zymomonas strain, or another microorganism suitable for fermentation of biomass.
  • feedstock is saccharified by a microorganism that is Clostridium phytofermentans, Clostridium sp.
  • lentocellum Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Clostridium beijerinckii, Clostridium bifermentans, Clostridium cellulose, Clostridium ljungdahlii, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum, Sacharophagus degradans, Thermoanaerobacterium saccharolyticum , Acetivibrio cellulosolvens, Acetivibrio
  • a microorganism is genetically modified to express one or more polypeptides, which can result in enhanced production of a fermentation end-product.
  • the enhanced production is enhanced yield or rate of production.
  • genetic modification enhances activity of one or more endogenous proteins.
  • genetic modification introduces one or more heterogeneous nucleic acid molecules into a host microorganism that encode a polypeptide not otherwise expressed in the host.
  • genetic modification modifies the physical or chemical conditions of a microorganism to enhance function of a protein (e.g. , modifying and/or maintaining a certain temperature, pH, nutrient concentration, temporal), or a combination of one or more such modifications.
  • one or more polynucleotides are introduced into a first embodiment.
  • the polynucleotide is a wild-type polynucleotide sequence found in the nature. In another embodiment, a
  • polynucleotide is a variant of a wild-type polynucleotide sequence. In one embodiment, a variant polynucleotide shares similarity to the wild-type polynucleotide sequence. In another
  • a variant polynucleotide is also found in the nature.
  • a variant polynucleotide is man-made.
  • a variant polynucleotide is chemically synthesized.
  • a variant polynucleotide is isolated from the nature.
  • a variant polynucleotide is a naturally-occurring allelic variant.
  • a variant polynucleotide is a homolog.
  • a variant polynucleotide is an ortholog.
  • a variant polynucleotide encodes a protein involved in the metabolism.
  • the protein is a swollenin variant.
  • the protein is an expansin variant.
  • a variant polynucleotide is a naturally-occurring mutant.
  • a mutant is different from that of the wild type by one nucleotide.
  • the mutated nucleotide changes one amino acid from the wild type polypeptide. In another embodiment, the mutated nucleotide does not change an amino acid it encodes. In another embodiment, the mutated nucleotide changes the rate of transcription. In another embodiment, the mutated nucleotide changes a start or stop codon.
  • a variant polynucleotide is made by mutagenesis.
  • mutagenesis is induced by introducing a mutagen to a cell.
  • a mutagen is a chemical.
  • a chemical is chemotherapeutic drug.
  • a mutagen is UV-light.
  • a mutagen is radiation.
  • mutagenesis occurs in vitro by inserting randomly selected nucleotide into or during the synthesis of a polynucleotide.
  • mutation introduced by mutagenesis occurs in coding-region. In another embodiment, mutation occurs in non-coding region.
  • mutagenesis is non-random, deliberate change of one or more nucleotides to make one or more pre-determined polynucleotide sequences.
  • a deliberate change is introduced to optimize the codon to a particular organism according to the principle of codon usage. It can be advantageous to modify a coding sequence to enhance its expression in a particular host.
  • the genetic code is redundant with 64 possible codons, but most organisms preferentially use a subset of these codons.
  • the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons (see, e.g., Zhang S P et al. (1991) Gene 105:61-72).
  • Codons can be substituted to reflect the preferred codon usage of the host, a process called "codon optimization” or "controlling for species codon bias.”
  • Optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S. cerevisiae and mammals are UAA and UGA respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer to use UAA as the stop codon (Dalphin M E et al. (1996) Nuc Acids Res 24: 216-218).
  • a variant polynucleotide is identified by a selection process designed to select for certain trait.
  • the trait is activity, stability, amount, intracellular location, extracellular location, or kinetics of a selected protein different to that of the wild type protein.
  • a trait is selected by monitoring phenotypic change.
  • a phenotypic change is at cellular level, i.e., a change of cellular phenotype.
  • a phenotypic change is at molecular level, i.e., a change of molecular phenotype.
  • a variant sequence has a sequence homology to a wild type sequence of about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %,
  • sequence homology is determined by counting the number of identical sequences between any two sequences.
  • an identical match is identified by the type of a nucleotide or an amino acid.
  • an identical match is identified by the type and location of a nucleotide or an amino acid.
  • a location is a relative position of a nucleotide or an amino acid within a polynucleotide or a polypeptide, respectively.
  • a pair of sequences are identical if both location and the type are matched.
  • a pair of sequences are identical if the type is matched.
  • a pair of sequences are identical if the type is matched and the location is closely matched.
  • a close match is within 1 sequence-distance.
  • two adjacent sequences i.e., two nucleotides (or amino acids) covalently linked to each other, are 1 sequence-distance apart from each other.
  • a unit of sequence-distance is nucleotide.
  • polypeptide a unit of sequence- distance is amino acid.
  • a close match is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, 97, 98, 99, or 100 sequence-distances.
  • a close match is within 1% of the distance of the polynucleotides or polypeptides in comparison. For example, if each of two compared sequences are 1000 base pair long, then a close match of 1% is a location within 10 nucleotides.
  • a close match is about 1%, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, or 49 %.
  • a close match of the location of a pair in comparison can be found between two sequences wherein one sequence contains an insertion, deletion,
  • one of the two sequences can contain an intron.
  • a close match can still be found for sequences that differ significantly in one section of the compared sequence, but are homologous in another section of the compared sequence.
  • a variant sequence is identified with a contiguous sequence found in the wild type.
  • a contiguous sequence is a conserved sequence that is generally found in naturally-occurring variants.
  • a contiguous sequence comprises about 6 nucleotides or amino acids.
  • a contiguous sequence comprises about 12 nucleotides or amino acids.
  • a contiguous sequence comprises 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, or 120 nucleotides or amino acids.
  • an optimal alignment of sequences for comparison is conducted by computerized implementations of one or more algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0).
  • the algorithm is a member of the BLAST family of programs as, for example, disclosed by Altschul et al, 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.
  • a wild-type, variant or a fragment thereof can be used to improve the efficiency of saccharification, fermentation and production of fermentation end-product.
  • a wild-type polynucleotide is introduced to a microorganism to improve the efficiency of saccharification, fermentation and production of fermentation end-product
  • a variant is introduced to a microorganism.
  • a fragment of a wild type polynucleotide or a variant polynucleotide is introduced to a
  • a variant polynucleotide encodes a protein with a biological activity equivalent to or exceeding the activity of the wild type protein.
  • a fragment of a wild-type polynucleotide or a variant polynucleotide has a biological activity equivalent to or exceeding the activity of the wild type protein.
  • the activity is increased target binding.
  • a target is cellulose.
  • a target is hemicellulose.
  • a target is lignocellulose.
  • a target is a cell wall.
  • a target is a cell wall polysaccharide.
  • a fragment is a fragment of swollenin.
  • a fragment is a fragment of expansin. In another embodiment, a fragment has the capacity to bind to carbohydrate. In another embodiment, a fragment is a carbohydrate binding domain of swollenin. In another embodiment, a fragment is a carbohydrate binding motif.
  • polynucleotide sequences of the present invention can be engineered in order to alter expansin or swollenin homologue coding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • alterations may be introduced using techniques which are well known in the art, e.g., PCR, to insert new restriction sites, to alter glycosylation patterns or other conjugation patterns, to change codon preference, to introduce splice sites, to introduce or remove introns.
  • a recombinant microorganism is produced to increase the yield of a fermentation end-product described herein. In another embodiment, a recombinant
  • microorganism comprises one or more exogenous nucleotide sequences.
  • an exogenous nucleotide sequence is a plasmid or vector.
  • an exogenous nucleotide sequence is a micro RNA.
  • an exogenous nucleotide sequence is a snRNA.
  • an exogenous nucleotide sequence is RNA capable of regulating host cell gene expression.
  • an exogenous sequence is a sequence not naturally associated with a wild-type host.
  • an exogenous sequence is a sequence naturally associated with a wild-type host but isolated from another wild- type host.
  • an exogenous sequence is a sequence substantially similar to sequence naturally associated with a wild-type host.
  • an exogenous sequence is a sequence introduced into the cell by molecular biological techniques.
  • a microorganism is modified to express a polypeptide, including but not limited to an expansin or swollenin. Examples of such modifications include modifying endogenous nucleic acid regulatory elements to increase expression of one or more proteins (e.g., operably linking a gene encoding a target protein to a strong promoter), introducing into a microorganism additional copies of nucleic acid molecules to provide enhanced activity of an protein, operably linking genes encoding one or more proteins to an inducible promoter or a combination thereof.
  • a nucleic acid sequence encoding a polypeptide is modified to include one or more signal sequences that facilitate transport of the polypeptide to the external environment relative to the microorganism in which the polypeptide is expressed.
  • a signal sequence is a sequence of amino acids bound to the N-terminal portion or C-terminal portion of a protein which facilitates the secretion of the mature form of the protein outside of the cell.
  • a mature form of the extracellular protein lacks the signal sequence.
  • a mature form of the extracellular protein retains the signal sequence.
  • the signal sequence is cleaved off during the secretion process.
  • a signal sequence translocates the expressed protein to a particular subcellular location.
  • the subcellular location is different than that of the location of the protein that does not contain the signal sequence.
  • an additional modification is expression of metabolic enzyme.
  • a metabolic enzyme is an enzyme in glycolytic pathway.
  • a metabolic enzyme is an enzyme in lipid-synthesis pathways.
  • a metabolic enzyme is an enzyme in nucleotide synthesis pathways.
  • metabolic enzyme is an enzyme in amino acid synthesis pathways.
  • a metabolic enzyme is an enzyme in complex sugar synthesis pathways.
  • a transcription regulatory sequence is operably linked to gene(s) of interest (e.g., in an expression construct).
  • the promoter may be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The selection of a particular promoter depends on what cell type is to be used to express the protein of interest. Generally, the most preferred transcription regulatory sequences are those from the host microorganism.
  • constitutive or inducible promoters are selected for use in a host cell. Depending on a host cell, there are multiple constitutive and inducible promoters which can be engineered to function in the host cell.
  • a variety of promoters e.g., constitutive promoters, inducible promoters, enhancers and regulators can be utilized to drive expression of the exogenous genes in a recombinant microorganism.
  • a promoter is selected based on its suitability for a host microorganism.
  • a promoter is a C. phytofermentans promoter. Promoters widely utilized in recombinant technology can also be used.
  • a promoter is E.
  • coli lac and trp operons the tac promoter, the bacteriophage p L promoter, bacteriophage T7 and SP6 promoters, beta-actin promoter, insulin promoter, baculoviral polyhedrin or plO promoter.
  • promoters can be used to drive expression of the heterologous genes in a recombinant host microorganism.
  • Promoter elements can be selected and mobilized in a vector (e.g., pIMPCphy).
  • a transcription regulatory sequence is operably linked to gene(s) of interest (e.g. , in a expression construct).
  • the promoter can be any array of DNA sequences that interact specifically with cellular transcription factors to regulate transcription of the downstream gene. The selection of a particular promoter depends on what cell type is to be used to express the protein of interest.
  • a transcription regulatory sequences can be derived from the host microorganism.
  • constitutive or inducible promoters are selected for use in a host cell. Depending on the host cell, there are potentially hundreds of constitutive and inducible promoters which are known and that can be engineered to function in the host cell.
  • Promoters typically used in recombinant technology such as E. coli lac and trp operons, the tac promoter, the bacteriophage pL promoter, bacteriophage T7 and SP6 promoters, beta- actin promoter, insulin promoter, baculoviral polyhedrin and plO promoter, can be used to initiate transcription.
  • a suitable constitutive promoter include, but are not limited to the int promoter of bacteriophage lambda, the bla promoter of the beta-lactamase gene sequence of pBR322, hydA or thlA in Clostridium, S. coelicolor hrdB or whiE, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, or the
  • Staphylococcal constitutive promoter blaZ Staphylococcal constitutive promoter blaZ.
  • an inducible promoter can be used that regulates the expression of downstream gene in a controlled manner, such as under a specific condition of a cell culture.
  • suitable inducible prokaryotic promoters include, but are not limited to, the major right and left promoters of bacteriophage, the trp, reca, lacZ, AraC and gal promoters of E. coli, the alpha-amylase (Ulmanen Ett at., J. Bacteriol. 162: 176-182, 1985, which is herein
  • a promoter that is constitutively active under certain culture conditions can be inactive in other conditions.
  • acetobutylicum is known to be regulated by the environmental pH.
  • a temperature- regulated promoter can be used to regulate transcription of a desired polynucleotide sequence.
  • a pH-regulatable promoters such as PI 70 functioning in lactic acid bacteria can be used to regulate transcription of a desired polynucleotide sequence, as disclosed in US Patent Application No. 20020137140, which is herein incorporated by reference in its entirety.
  • a pH-regulated or temperature- regulated promoter can be used with an expression construct to initiate transcription.
  • promoters can be used; e.g., the original promoter of the gene, promoters of antibiotic resistance genes such as for instance kanamycin resistant gene of Tn5, ampicillin resistant gene of pBR322, and promoters of lambda phage and any promoters which can be functional in the host cell.
  • antibiotic resistance genes such as for instance kanamycin resistant gene of Tn5, ampicillin resistant gene of pBR322, and promoters of lambda phage and any promoters which can be functional in the host cell.
  • promoters examples include those disclosed in the following patent documents: US20040171824, US 6410317, WO 2005/024019 , which are herein incorporated by reference in their entirety.
  • a Shine -Dalgarno (SD) sequence e.g., AGGAGG and so on including natural and synthetic sequences operable in the host cell
  • a transcriptional terminator inverted repeat structure including any natural and synthetic sequence
  • Repressors are protein molecules that bind specifically to particular operators. The structure formed by the repressor and operator blocks the productive interaction of the associated promoter with RNA polymerase, thereby preventing transcription. Other molecules, termed inducers, bind to repressors, thereby preventing the repressor from binding to its operator. Thus, the suppression of protein expression by repressor molecules may be reversed by reducing the concentration of repressor (depression) or by neutralizing the repressor with an inducer. Repressors are useful for controlling gene expression.
  • a repressor is the lac repressor molecule binds to the operator of the lac promoter-operator system.
  • a repressor is the cro repressor binds to the operator of the ApR promoter.
  • the lac promoter system is used to control the expression of exogenous expansin or swollenin.
  • the cro repressor system is used to control the expression of exogenous expansin or swollenin. Transcription under the control of the PH05 promoter is repressed by extracellular inorganic phosphate, and induced to a high level when phosphate is depleted. Detailed descriptions on PH05 promoter can be found in R. A. Kramer and N. Andersen, "Isolation of Yeast Genes With mRNA Levels Controlled By Phosphate Concentration", Proc. Nat.
  • PH05 repressor system is used to control the expression of exogenous expansin or swollenin.
  • CUP1 promoter which can be induced by Cu +2 ions.
  • the CUP1 promoter is regulated by a metallothionine protein.
  • the CUP1 repressor system is used to control the expression of exogenous expansin or swollenin.
  • Other combinations of repressor and operator known in the art can also be used for controlling gene expression in a recombinant microorganism described herein.
  • the repressor systems described herein are utilized to control the expression of metabolic enzymes or proteins regulating metabolic pathways.
  • a higher copy number plasmid can be utilized.
  • Copy number of a plasmid has been known to be regulated by the origin of replication (also known as "ori").
  • ori of pUC19-based plasmid produces high number of copies of the plasmid while ori of pBR322-based plasmid produces low number of copies.
  • a high copy number ori sequence is used. Examples of cellulase, hemicellulase or other enzymes are further described in U.S. Pat. Application Nos. 12/510,994 and 12/630,784, which are incorporated here in their entirety.
  • vectors comprising plasmid DNA Prior to transformation into C. phy, can be methylated to prevent restriction by Clostridial endonucleases. (Mermelstein and Papoutsakis. Appl. Environ. Microbiol. 59: 1077-1081 (1993)). In one embodiment, methylation can be accomplished by the phi3TI methyltransferase. In another embodiment, plasmid DNA can be transformed into ⁇ . E. coli containing vector pDHKM (Zhao, et al. Appl. Environ. Microbiol. 69: 2831-41 (2003)) carrying an active copy of the phi3TI methyltransferase gene.
  • a construct is prepared for chromosomal integration of the desired gene.
  • Chromosomal integration of one or more foreign gene can offer several advantages over plasmid-based constructions.
  • Ethanologenic genes have been integrated chromosomally in E. coli B; see Ohta et al. (1991) Appl. Environ. Microbiol. 57:893-900. Integration is accomplished by, for example, homologous recombination. Methods of preparing a plasmid for homologous recombination are known in the art. In general, a chromosomal sequence around the point of integration is obtained by genomic PCR.
  • a sequence upstream of the point of integration which is obtained by the PCR, is cloned into the 5 Of the exogenous sequence.
  • a sequence downstream of the point of integration is cloned into the 3' of the exogenous sequence.
  • a completed construct has the exogenous sequence flanked by the upstream and downstream regions of the PCR-isolated genomic DNA.
  • homologous recombination occurs between the vector and the corresponding homologous regions of genomic DNA, resulting in the swapping of the genomic DNA with the vector sequence.
  • a homologous recombination technique is used to integrate one or more copies of an exogenous gene.
  • two or more copies of an exogenous gene are integrated. In another embodiment, two or more copies of two, three, or four different exogenous genes are integrated. In another embodiment, two or more copies of an exogenous gene are cloned into single chromosomal integration vector and integrated into single point of insertion.
  • Viral integration is another method of chromosomal integration.
  • random integration via a viral vector such as a bacteriophage-based vector, is used to express exogenous genes than using the homologous recombination methods described herein.
  • a viral expression cassette containing swollenin, expansin, or other proteins described herein is prepared for chromosomal integration.
  • chromosomally integrated microorganisms are scored for the number of integration sites.
  • Microorganisms scored within the top 5 percentile are classified as high copy number integrators.
  • Microorganisms scored within the bottom 5 percentile are classified as low copy number integrators.
  • Each group is kept separately from each other and is subjected to a barrage of in vitro tests including amount of expression, kinetics, cellular location, protein binding, stability, and other molecular characteristics.
  • Strains containing exogenous genes with desired molecular traits are subjected to in vivo tests such as biomass digestion rate, fermentation end-product production rate, cell division rate, susceptibility to temperature, temperature fluctuation, shear force, hydrodynamic force, pressure, oxygen, carbon dioxide, depletion of nutrients, plant toxins, or the presence of other microorganisms and
  • Expansin and swollenin proteins have the ability to increase plant cell wall-loosening and cellulose disruption without exerting cellulose-hydrolytic activity. Coupled with cellulases, these proteins can increase the saccharification rates of biomass by making cellulose molecules and other cell wall components more accessible to hydrolytic enzymes. Recombinant cellulolytic organisms, such as C. phy and Clostridium sp. Q.D expressing an expansin or swollenin polypeptide can advantageously and more thoroughly increase saccharification in fermentation processes.
  • a microorganism is genetically modified to express one or more expansins, swollenins or both.
  • a genetically modified microorganism secretes one or more expansins, one or more swollenins or both.
  • a genetically modified microorganism produces fermentation conditions for cellulosic biomass material that are more amendable for cellulolytic proteins.
  • a genetically modified microorganism increases overall yield of a fermentation end product per a unit amount of biomass (e.g., 1 g, 1 kg, 1 ton, or any arbitrary unit used for testing the yield of a fermentation end-product per defined amount of biomass) by reducing process time for saccharification or increasing the amount of saccharification.
  • a genetically modified microorganism enhances overall yield of a fermentation end-product by reducing process time for fermentation.
  • expansin or swollenin is a protein or a polypeptide listed in a public database, such as Entrez or GeneBank, as an expansin, swollenin, or a fragment thereof.
  • a public database such as Entrez or GeneBank
  • an expansin, swollenin, or a fragment thereof is isolated from a microorganism.
  • an expansin, swollenin, or a fragment thereof is chemically synthesized either by using an enzyme or without an enzyme.
  • a sequence listed in a public database as a variant of an expansin, swollenin, or a fragment thereof is isolated either from a microorganism or synthesized.
  • an expansin-like or swollenin-like protein or fragment thereof capable of weakening cellulosic fiber association without using cellulo lytic activity, (e.g., without using catalytic activity involving the breakage of individual cellulose strands into smaller monomers or oligomers).
  • an expansin-like or swollenin-like protein has an ability to facilitate the weakening of the association of cellulosic fibers.
  • an expansin-like or swollenin-like protein has ability to directly enhance the expansion of cellulosic fibers.
  • an expansin-like or swollenin-like protein is capable of expanding cell walls and cell wall loosening.
  • an expansin-like or swollenin-like protein is capable of increasing the rate of expansion of cellulosic fibers in biomass.
  • expansin or expansin-like molecule is selected by measuring its ability to enlarge plant cells. In another embodiment, expansin or expansin-like molecule is selected by measuring its ability to loosen plant cell walls.
  • an expansin, swollenin, or a fragment thereof can be isolated or derived from a microorganism.
  • a microorganism is a fungus, a yeast, or a bacterium.
  • a microorganism is a strain of Trichoderma spp.
  • a microorganism is a strain of Trichoderma reesei.
  • Non-limiting examples of a fungus include, but are not limited to, Absidia spp.; Acremonium spp.; Agaricus spp.;
  • Anaeromyces spp. including auculeatus, A. awamori, A.flavus, A.foetidus, A. fumaricus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus or A. versicolor;
  • Neocallimastix spp. spp.
  • Orpinomyces spp. Penicillium spp; Phanerochaete spp.; Phlebia spp.; Piromyces spp.; Pseudomonas spp.; Rhizopus spp.; Schizophyllum spp.; Trametes spp.; T. viride; or
  • Non-limiting examples of cellulolytic bacteria include, but are not limited to, Bacillus spp.; Cellulomonas spp.; Clostridium spp.; Myceliophthora spp.; Thermomonospora spp.; Streptomyces spp., including S. olivochromogenes; or fiber degrading ruminal bacteria such as Firobacter succihogenes.
  • Non-limiting examples of yeast include, but are not limited to, Candida torresii; C. parapsUosis; C. sake; C. zeylanoides; Pichia minuta; Rhodotorula glutinis; R. mucilaginosa; or Sporobolomyces holsaticus.
  • a swollenin is isolated or purified.
  • purification is performed by techniques known in the art including, but is not limited to, ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or separation an a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or proteins undesired in the final composition.
  • purified swollenin is lyophilized.
  • purified swollenin is packaged into a stable storage solution in a container.
  • swollenin containing composition is formulated by adding activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other proteins such as cellulase.
  • a swollenin composition or purified swollenin is added to a biomass. Swollenins are described in U.S. Patent No. 6,967,246, and U.S. Patent Application Nos US20090061484 and US20080201123.
  • an expansin is a protein about 26 kDa comprising 2 domains: Dl has a fold similar to that of family 45 glycosyl hydrolases (GH45), and D2 has a ⁇ -sandwich fold.
  • an expansin is a polypeptide comprising 225 amino acids having the Dl and the D2 domains.
  • an expansin is a member of a-expansin family protein.
  • an expansin is a member of ⁇ -expansins. Examples of an expansin are described by Kerff et al.
  • one or more expansins described herein are produced in a strain of Clostridium phytofermentans.
  • the Clostridium phytofermentans is genetically modified.
  • a modified C. phytofermentans secretes one or more expansins into an extracellular space.
  • a modified C. phytofermentans produces a variant of one or more expansins.
  • a modified C. phytofermentans produces a fragment of an expansin described herein.
  • a modified C. phytofermentans produces a fragment of the variant of an expansin.
  • an optimized sequence is used to express expansin, its variant or its fragment in a C. phytofermentans.
  • Table 1 depicts expansins and corresponding codon optimized expansin sequences for expression in C. phytofermentans (SEQ ID NO: 1-4).
  • the codon-optimized sequences are fused to C. phytofermentans signal peptide.
  • a vector optimized for C. phytofermentans expression is used. For example, restriction endonuclease site Sacl (GAGCTC) and Xmal (CCCGGG) are placed on the N-terminal and C-terminal, respectively. The synthesized is then cloned into Sacl and Xmal cut vector, pCPHY-P3510-3368 to form pCPHY-P3510-Exp. This plasmid is used to transform a C. phytofermentans.
  • a modified C. phytofermentans is utilized to enhance the yield of fermentation end-product.
  • a modified C. phytofermentans is utilized to reduce the production time for producing fermentation end- product.
  • a modified C. phytofermentans is utilized as a cost-saving measure in the production of a fermentation end-product.
  • any of the known expansin genes can be used to improve the production of fermentation end product.
  • any of the 26 different a expansin genes or 6 different ⁇ expansin genes identified in Arabidopsis thaliana or their homologs can be used.
  • a structural analog to plant expansin that bears no sequence similarity can also be used.
  • a B. subtilis YOAJ protein can be used.
  • one or more swollenins described herein are produced in a strain of Clostridium.
  • the strain of Clostridium is Clostridium phytofermentans .
  • a genetically modified C. phytofermentans secretes one or more swollenins into the extracellular space.
  • a genetically modified C. phytofermentans produces a variant of one or more swollenins.
  • a genetically modified C. phytofermentans produces a fragment of swollenins described herein.
  • a genetically modified C. phytofermentans produces a fragment of the variant.
  • an optimized sequence is used to express swollenin, its variant or its fragment in a C. phytofermentans. For example, Table 2 depicts swollenins that can be expressed in C.
  • phytofermentans SEQ ID NO: 5-14.
  • a swollenin or its homo log is modified to enhance expression ⁇ e.g., stability, biological activity) in a host microorganism described herein.
  • FKYGCPLP AD SIHLDL SDI AMGRLQ GNGSLTNG VIPTRYRRV
  • a microorganism can be further genetically modified to enhance an activity of one or more cellulases, or enzymes associated with cellulose processing (e.g., Figure 7).
  • a microorganism is genetically modified to express one or more expansins and/or one or more swollenins and a heterologous or additional endogenous cellulase.
  • the classification of cellulases is usually based on grouping enzymes together that forms a family with similar or identical activity, but not necessary the same substrate specificity.
  • CAZy Carbohydrate- Active enZymes
  • GH Glycoside Hydrolases
  • cellulase enzymes whose function can be enhanced for expression endogenously or for expression heterologously in a microorganism include one or more of the genes disclosed in Table 3.
  • a polynucleotide comprises codons in its protein coding sequence that are optimized to increase the thermostability of an mRNA transcribed from the
  • a polynucleotide comprises codons in its protein coding sequence that are optimized to increase translation efficiency of an mRNA from the polynucleotide in a host cell. In one embodiment this optimization does not change the amino acid sequence encoded by the polynucleotide.
  • a polynucleotide has been optimized to remove one or more restriction enzyme recognition sites.
  • a polynucleotide has been optimized to remove one or more restriction enzyme recognition sites so that the polynucleotide is not digested when a host cell is transformed or transfected with the polynucleotide.
  • the host cell is a Clostridium cell.
  • the Clostridium is C. phytofermentans.
  • the Clostridium is Clostridium sp. Q.D..
  • RNA codon table below (Table 4) shows the 64 codons and the amino acid for each.
  • the direction of the mRNA is 5' to 3'.
  • a microorganism is transformed with multiple genes encoding one or more enzymes.
  • a single transformed cell can contain exogenous nucleic acids encoding an entire biodegradation pathway.
  • a pathway can include genes encoding an ⁇ - ⁇ -glucanase, and endo-P-glucanase, and an endoxylanase.
  • Such cells transformed with entire pathways and/or enzymes extracted from them can saccharify certain components of biomass more rapidly than the naturally-occurring organism.
  • Constructs can contain multiple copies of the same gene, and/or multiple genes encoding the same enzyme from different organisms, and/or multiple genes with mutations in one or more parts of the coding sequences.
  • multiple copies of Cphy_3367 or Cphy_3368 can increase saccharification, thus increasing the rate and yield of fermentation products.
  • the nucleic acid sequences encoding the genes can be similar or identical to the endogenous gene. There can be a percent similarity of 70% or more in comparing the base pairs of the sequences.
  • a polynucleotides used in the processes described herein comprises all or a portion of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 44, or one or more polynucleotides that encodes one or more enzymes selected from the group consisting of Cphy_3202, Cphy_2058, Cphy_1163, Cphy_3367, CphyJ IOO, Cphy_1510, Cphy_3368, and Cphy_2128.
  • the recombinant microorganism useful for hydrolysis and fermentation during the processes described herein produces one or more hydrolytic enzyme encoded by a variant having a polynucleotide sequence with an identity of 70% or more compared to a sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28 or a combination thereof.
  • the products include one or more polynucleotides that are SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 44 or one or more polynucleotides encoding Cphy_3202, Cphy_2058, Cphy_1163, Cphy_3367, CphyJ IOO, Cphy_1510, Cphy_3368, or Cphy_2128.
  • vectors described herein can comprise a polynucleotide that encodes Cphy_3289 or Cphy 3290.
  • vectors described herein can also include one or more polynucleotides such as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 44 or one or more
  • vectors of this invention can encode biomass degrading enzymes such as Cphy_3202, Cphy_2058, Cphy_1163, Cphy_3367, CphyJ IOO, Cphy_1510, Cphy_3368, or Cphy_2128.
  • vectors described herein can comprise polynucleotides encoding a polypeptide of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • vectors described herein can be produced by utilizing primers comprising a polynucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 33.
  • a useful recombinant microorganisms with enhanced hydrolytic activity expresses one or more biomass degrading enzymes encoded by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or the chaperonin proteins encoded by SEQ ID NO: 31.
  • a recombinant microorganism with enhanced hydrolytic activity expresses one or more biomass degrading enzymes wherein the one or more biomass degrading enzymes are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or express the chaperonin proteins SEQ ID NO: 40 and SEQ ID NO: 41.
  • a recombinant microorganism expresses one or more biomass degrading enzymes wherein the one or more biomass degrading enzymes are Cphy_3202, Cphy_2058, Cphy_1163, Cphy_3367, CphyJ IOO, Cphy_1510, Cphy_3368, or Cphy_2128.
  • the recombinant microorganism can further comprise one or both of the chaperonin proteins encoded by all or a portion of SEQ ID NO: 31.
  • a expression of a expansin or a swollenin is regulated by a C.
  • phytofermentans transcriptional regulator In another embodiment expression of a heterologous polynucleotide is regulated by a C. phytofermentans transcriptional regulator. In another embodiment expression of an additional endogenous polynucleotide in a genetically modified microorganism is regulated by a C. phytofermentans transcriptional regulator.
  • Transcriptional regulators identified in C. phytofermentans include members of the AraC and PurR families. AraC regulators can include transcriptional activators of genes involved in carbon metabolism (Gallegos M. T. et al. "AraC/XylS Family of Transcriptional Regulators.” Microbiol. Mol. Biol. Rev. 61, 393-410 (1997)).
  • PurR regulators can include members of the lactose repressor family (Ramos, J. L. et al. "The TetR family of transcriptional repressors.” Microbiol. Mol. Biol. Rev. 69, 326-356 (2005)).
  • a C. phytofermentans transcriptional regulators is Cphy0029, Cphy0342, Cphy0385, Cphy0464, Cphy0572, Cphy0176, Cphy0461, Cphy0674, Cphy0709, Cphy0730, Cphy0768, Cphy0928, Cphy0971, Cphy0610, Cphyl l48, Cphyl l65, Cphyl l68, Cphy0667, Cphy0672, Cphyl364, Cphyl472, Cphyl367, Cphyl513, Cphyl528, Cphyl546, Cphyl633, Cphyl683, Cphyl706, Cphyl762, Cphyl837, Cphyl838, Cphyl856, Cphyl864, Cphyl910, Cphy2667, Cphy3395, Cphy3621, Cphyl915, Cphy2187, Cphy2230, Cphy2239, Cphy2338, Cphy2461, Cphy2556, Cphy0989, C
  • a fermentation end-product is a chemical additive, catalyst for fuel production, a food additive, an organic acid (e.g. acetic, lactic, formic, citric acid etc.), a derivative of an organic acid such as an ester (e.g. wax ester, glyceride, etc.), or a phytate such as phytic acid.
  • an organic acid e.g. acetic, lactic, formic, citric acid etc.
  • a derivative of an organic acid such as an ester (e.g. wax ester, glyceride, etc.)
  • a phytate such as phytic acid.
  • a microorganism produces a protein such as cellulase, polysaccharase, lipase, protease, ligninase, hemicellulase or other an enzyme involved in break down of carbohydrates.
  • a protein is an isolated, substantially
  • a protein is a mixture that is mixed with cellular components.
  • a protein is in a solution with detectable amount of impurities such as purification chemicals.
  • a protein is in a lyophilized form.
  • a fermentation end-product is a fuel or a biofuel.
  • a fermentation end-product is a raw material useful for producing a fuel or biofuel.
  • a fuel or biofuel is one or more compounds suitable for combustion or other means to produce energy such as electricity.
  • a fuel or biofuel is a liquid fuel, a gaseous fuel, or a solid fuel.
  • a fuel or biofuel is a hydrocarbon, hydrogen gas, methane gas, a hydroxy compound such as an alcohol (e.g. ethanol, butanol, propanol, methanol, etc.), or a carbonyl compound such as aldehyde or a ketone (e.g. acetone, formaldehyde, 1-propanal, etc.).
  • a fermentation end-product is a biological molecule such as a succinic acid, or a pyruvic acid.
  • a fermentation end-product can be produced through methods and compositions described herein such as saccharification and fermentation processes described herein.
  • An example of a fermentation end-product includes, but is not limited to, methane, methanol, ethane, ethene, ethanol, n-propane, 1-propene, 1 -propanol, propanal, acetone, propionate, n- butane, 1-butene, 1 -butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2- methylbutanol, 3-methylbutanal, 3-methylbutanol, 2-butene, 2-butanol, 2-butanone, 2,3- butanediol, 3-hydroxy-2-butanone, 2,3-butanedione, ethylbenzene, ethenylbenzene, 2- phenylethanol, phenylacetaldehyde,
  • phenylacetoaldehyde 1 ,4-diphenylbutane, 1,4-diphenyl-l-butene, 1 ,4-diphenyl-2-butene, 1,4- diphenyl-2-butanol, 1 ,4-diphenyl-2-butanone, l,4-diphenyl-2,3-butanediol, l,4-diphenyl-3- hydroxy-2-butanone, 1 -(4-hydeoxyphenyl)-4-phenylbutane, 1 -(4-hydeoxyphenyl)-4-phenyl- 1 - butene, l-(4-hydeoxyphenyl)-4-phenyl-2-butene, l-(4-hydeoxyphenyl)-4-phenyl-2-butanol, 1- (4-hydeoxyphenyl)-4-phenyl-2-butanone, 1 -(4-hydeoxy
  • one or more fermentation end-products can be produced by processes described herein.
  • a process includes a single step involved in the production of fermentation end-product to multiple combined steps.
  • multiple combined steps comprising a process can be a module that has a functional utility tied to the module.
  • a module can be a fermentation module, a saccharification module, or a separation module.
  • the process by which a fermentation end-product is produced can be described as an order of modules.
  • a module can be a functionally independent unit, which can be added, removed, inserted, multiplied or repeated in a production process.
  • a fermentation end-product is produced by a process comprising a fermentation module.
  • a fermentation module comprises multiple combined steps.
  • the module comprises a step of adding a microorganism described herein (e.g., natural or genetically modified).
  • the module comprises a step of culturing a microorganism described herein.
  • the culture is an aerobic culture.
  • the culture is an anaerobic culture.
  • the module comprises one or more steps of electronically or manually controlling a bioreactor in which the microorganism is cultured.
  • controlling comprises controlling the temperature, pH, and pressure of the bioreactor.
  • the culture is an anaerobic culture.
  • the culture has an aerobic phase and anaerobic phase.
  • the process comprises a first and second microorganism.
  • the process comprises the step of adding nutrients to the microorganism in the culture.
  • the process comprises a step of monitoring cell growth.
  • cell growth is monitored by measuring cell density in the culture.
  • cell density is measured by optical density of the culture, using a spectrophotometer capable of producing visible light (595 nm to 600 nm).
  • the process comprises a step of continuously adding microorganisms into the culture. In another embodiment, the process comprises a step of continuously adding nutrients, biomass, or both. In another embodiment, the process comprises a step of monitoring the production of ethanol. In another embodiment, the process comprises a step of extracting ethanol produced during fermentation. In another embodiment, the process comprises a loop that siphons off a portion of liquid culture, separates ethanol from the culture, and returns the liquid culture substantially free of ethanol to the bioreactor.
  • a fermentation end-product is produced by a process comprising a saccharification module.
  • a saccharification process comprises multiple combined steps.
  • the process comprises a step of converting biomass into polysaccharides separated from other components.
  • the process comprises a step of converting polysaccharide to lower molecular weight saccharides.
  • the process comprises a step of converting a lower molecular weight saccharide to a monosaccharide, disaccharide, or trisaccharide.
  • the process comprises a step of converting any saccharide to its derivative.
  • the process comprises a step of converting any saccharide to a combination of sugars and sugar derivatives.
  • the process comprises a step of incubating biomass with microorganisms described herein. In another embodiment, the incubation comprises the conversion of biomass by the microorganism as described herein. In another embodiment, the process comprises a step of digesting a saccharide by a microorganism. In another embodiment, the process comprises the steps of digesting saccharides by enzymes and proteins secreted by a microorganism. In another embodiment, the process comprises a step of adding purified protein or enzyme as described herein. In another embodiment, the purified protein is swollenin, expansin, or both. In another embodiment, the process comprises a step of adding a protein or enzyme composition as described herein.
  • the process comprises a step of adding a swollenin, expansin, or another enzyme as described herein. In another embodiment, the process comprises continuously adding a protein or enzyme as described herein. In another embodiment, the process comprises a step of adding two different types of microorganisms serving functionally different purposes.
  • a first microorganism performs saccharification while a second microorganism provides extracellular proteins or enzymes aiding the saccharification by the first microorganism.
  • a first microorganism performs saccharification while a second microorganism provides nutrients for the first microorganism.
  • a first microorganism converts biomass to
  • oligosaccharides and monosaccharides while a second microorganism converts these to fermentive-end products.
  • a fermentation end-product is produced by a process comprising a pretreatment of biomass.
  • pretreatment comprises multiple combined steps.
  • the pretreatment comprises a step of applying mechanical force to a biomass.
  • the mechanical force comprises shaking, shearing, crushing, mincing, chopping, breaking, filtering, squeezing, twisting, centrifuging, pounding, rupturing, pressurizing or cutting.
  • pretreatment comprises a step of treating biomass with one or more chemicals.
  • chemical pretreatment produces softening, smoothing, hydrolyzing, dehydrating, swelling, detoxifying, melting, liquefying, slurring, precipitating, separating, or dissolving of a biomass.
  • the chemical is an organic chemical. In another embodiment, the chemical is an inorganic chemical. In another embodiment, the chemical is a gas. In another embodiment, the chemical is an inert gas. In another embodiment, the pretreatment comprises a step of thermal conditioning. In another embodiment, the thermal conditioning comprises adding heat. In another embodiment, the thermal conditioning comprises removing heat. In another
  • the pretreatment comprises a step of enzymatic conditioning of a biomass.
  • Various enzymes that can be utilized include cellulase, amylase, ⁇ -glucosidase, xylanase, gluconase, and other polysaccharases; lysozyme; laccase, and other lignin-modifying enzymes; lipoxygenase, peroxidase, and other oxidative enzymes; proteases; and lipases.
  • a cellulase one or more cellulases
  • a C5/C6 hydrolyzing organism like C.phy or Clostridium sp.
  • the pretreatment comprises performing two or more steps at the same time.
  • pressurizing and adding heat is performed at the same time to sterilize a biomass.
  • sterilization is partial (pasteurization).
  • sterilization includes autoclaving a biomass.
  • pretreatment comprises two or more steps performed sequentially.
  • pretreatment comprises a step of increasing the access of microorganism, other proteins or cellulolytic enzymes to the polysaccharides in a biomass.
  • the pretreatment comprises a step of removing lignin.
  • the pretreatment comprises a step of disrupting cellulosic and/or hemicellulosic material. In another embodiment, the pretreatment comprises a step of performing steam explosion. In another embodiment, the pretreatment comprises a step of performing ammonia fiber expansion (or explosion) known as AFEX.
  • pretreatment can include removal or disruption of lignin so as to make the cellulose and hemicellulose polymers in the biomass more available to cellulolytic enzymes and/or microbes, for example, by treatment with acid or base.
  • the biomass is a plant biomass.
  • the biomass is an animal biomass.
  • pretreatment can include the use of a microorganism of one type to render plant polysaccharides more accessible to microorganisms of another type, for example, by treatment with acid or base.
  • pretreatment of a biomass comprises dilute acid hydrolysis. Example of dilute acid hydrolysis treatment are disclosed in T. A. Lloyd and C.
  • Acid hydrolysis of biomass can comprise steam and pressure but can also be conducted without any steam or pressure.
  • a pretreatment module comprises sulfite treatment, acid sulfite treatment, basic sulfite treatment, ammonia treatment, or hydrolysis. Other pretreatment methods are further described in WIPO applications PCT/US2010/026730 and PCT/US2010/40502, which are herein incorporated by reference in their entirety.
  • a fermentation end-product is produced by a process comprising a fed-batch module.
  • a fed-batch module comprises multiple combined steps.
  • feedstock such as biomass (pretreated or untreated) is added to the hydrolysis and/or the fermentation in steps.
  • the steps can be one step or many steps. For example, following and initial inoculation of a biomass slurry, another aliquot of biomass can be added to the fermentation mixture after a period of fermentation.
  • aliquots of biomass can be added to the fermentation mixture as the sugars from fermentation are converted to a fermentation end-product while keeping the fermenting microorganism in a continual growth stage.
  • the process comprises a step of installing self- seeding routine in a bioreactor where a portion of the microbial culture is set aside at the time of harvest and used as an inoculum.
  • the process comprises a step in a bioreactor wherein less than 100% of the culture is harvested.
  • partial harvest can be accomplished through a physical installation of pipes, connectors, reservoirs, gates, switches or regulators.
  • partial harvest can be done through software installation in which a computer-readable logic is executed to perform self-seeding or a partial-harvest routine. In another embodiment, partial harvest can be done manually wherein a bioreactor operator performs the self-seeding or partial-harvest routine.
  • the a fed batch system comprises a step of adding one or more biocatalysts. In another embodiment, a fed batch system comprises a step of adding extra broth. In another embodiment, a fed batch system comprises a step of continuously adding broth or biocatalyst while continuously harvesting the culture. In another embodiment, a fed batch system comprises a step of continuously harvesting the cultured medium.
  • a fed batch system comprises a mechanical return loop by which some of the material harvested from the bioreactor is returned to the bioreactor.
  • a return loop is connected to a reservoir in which the harvested material is mixed with other material, diluted with buffer or water, or processed by other systems described herein.
  • SSF simultaneous saccharification fermentation.
  • SHF sequential hydrolysis followed by subsequent fermentation.
  • a fermentation product can be produced by either of these methods.
  • the biomass is hydrolyzed and fermented at the same time and, normally, in the same
  • yield is measured and expressed as a percentage yield of a fermentation end- product from a starting mass of substrate.
  • the net reaction is generally accepted as: C 6 Hi 2 06 2 C 2 H 5 OH + 2 C0 2 .
  • the theoretical maximum yield or yield is 51% (wt.). Frequently, the yield will be referenced to the theoretical maximum such as, for example, "80% of the theoretical maximum.” In the case of conversion of glucose to ethanol, this statement would indicate a yield of 41% (wt.).
  • the context of the phrase will indicate the substrate and product intended to one of skill in the art.
  • the theoretical maximum yield of the biomass to ethanol is an average of the maximum conversion efficiencies of the individual carbon source constituents weighted by the relative concentration of each carbon source.
  • the theoretical maximum yield is calculated based on assumed saccharification efficiency.
  • the theoretical maximum yield may be calculated by assuming saccharification of the cellulose to the assimilable carbon source glucose of about 75% by weight.
  • lOg of cellulose may provide 7.5g of glucose which may provide a maximum theoretical yield of about 7.5g*51% or 3.8g of ethanol.
  • the efficiency of the saccharification step may be calculated or determined (i.e. measured). Saccharification efficiencies anticipated by the present invention include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or about 100% for any carbohydrate carbon sources larger than a single monosaccharide subunit.
  • a fermentation end-product is produced from a biomass by a process comprising the steps of: contacting a biomass with: (1) a microorganism that is capable of direct hydrolysis and fermentation of said biomass, and/or (2) an external source of one or more enzymes that are capable of enhancing said hydrolysis.
  • a fermentation end-product is produced from a biomass by a process comprising the steps of: contacting a biomass with: (1) a genetically modified microorganism that is capable of direct hydrolysis and fermentation of said biomass, and/or (2) an external source of one or more enzymes that are capable of enhancing said hydrolysis.
  • the genetically modified microorganism has been modified to express at least one swollenin or expansin.
  • the genetically modified microorganism has been modified to express at least one heterologous cellulase.
  • the genetically modified microorganism has been modified to express at least one additional copy of an endogenous cellulase.
  • an external source of one or more enzymes comprises a cellulase. In another embodiment the external source of one or more enzymes comprises a cellulase mixture. In one embodiment the cellulase mixture saccharifies cellulose to produce predominantly oligosaccharides. In another embodiment the cellulase mixture saccharifies cellulose to produce predominantly saccharides with higher than 5 degrees of polymerization (e.g. pentamers, hexamers, etc.). In another embodiment the cellulase mixture has predominantly endoglucanse activity. In another embodiment the cellulase mixture the cellulase mixture has predominantly endoglucanse activity and saccharifies cellulose to produce predominantly oligosaccharides. In another embodiment the cellulase mixture the cellulase mixture has predominantly
  • an external source of one or more enzymes comprises a expansin or a swollenin.
  • an expansin and/or a swollenin is added externally to a hydrolysis mixture that contacts a biomass.
  • an external source of one or more enzymes comprises a expansin or a swollenin and a cellulase.
  • an expansin and/or a swollenin is added externally to the hydrolysis mixture that contacts the biomass.
  • an external source of one or more enzymes comprises an expansin, a swollenin, a xylanase, a hemicellulase, a glucanase or glucosidase, or a cellulase.
  • an external source of one or more enzymes does not comprise a xylanase, a hemicellulase, a glucanase or glucosidase, but does comprise an expansin or swollenin protein or polypeptide.
  • an external source of one or more enzymes is used in a fermentation medium with a microorganism and a biomass; wherein the microorganism and the enzymes hydrolyze and ferment said biomass to produce a fermentation product such as a biofuel.
  • the biofuel is ethanol.
  • a microorganism is contacted with a pretreated or non-pretreated biomass containing cellulosic, hemicellulosic, and/or lignocellulosic material. Additional nutrients can be present or added to the biomass material to be processed by the microorganism including nitrogen-containing compounds such as amino acids, proteins, hydrolyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydrolyze yeast, autolyzed yeast, corn steep liquor, corn steep solids, monosodium glutamate, and/or other fermentation nitrogen sources, vitamins, and/or mineral supplements.
  • nitrogen-containing compounds such as amino acids, proteins, hydrolyzed proteins, ammonia, urea, nitrate, nitrite, soy, soy derivatives, casein, casein derivatives, milk powder, milk derivatives, whey, yeast extract, hydrolyze yeast, autoly
  • one or more additional lower molecular weight carbon sources can be added or be present such as glucose, sucrose, maltose, corn syrup, lactic acid, etc.
  • Such lower molecular weight carbon sources can serve multiple functions including providing an initial carbon source at the start of the fermentation period, help build cell count, control the carbon/nitrogen ratio, remove excess nitrogen, or some other function.
  • aerobic/anaerobic cycling is employed for the bioconversion of cellulosic/lignocellulosic material to fuels and chemicals.
  • the anaerobic microorganism can ferment biomass directly without the need of a pretreatment.
  • biomass is contacted with biocatalysts capable of breaking down plant-derived polymeric material into lower molecular weight products that can subsequently be transformed by biocatalysts to fuels and/or other desirable chemicals.
  • a biocatalyst is a cocktail of two or more catalytic enzymes added to a bioreactor.
  • a product for production of a biofuel comprises a carbonaceous biomass, a microorganism that is capable of direct hydrolysis and fermentation of said biomass, wherein the microorganism is genetically modified to express an expansin or swollenin or swollenin protein.
  • a modified organism of the invention is further modified to
  • product for production of fermentation end-products comprises: (a) a fermentation vessel comprising a carbonaceous biomass; (b) a genetically modified microorganism that is capable of direct hydrolysis and fermentation of said biomass; and (c) a source of one or more cellulase proteins that is external to said
  • reaction vessel is adapted to provide suitable conditions for fermentation of one or more carbohydrates into fermentation end- products.
  • a reaction vessel can be configured to separate one or more desired fermentation end- products.
  • biomass is hydro lyzed resulting in a greater concentration of cellobiose relative to monomeric carbohydrates.
  • a process comprises treating the biomass in a container with the microorganism and adding one or more proteins before, concurrent or after contacting the biomass with the microorganism, wherein the proteins added aid in the breakdown or detoxification of carbohydrates or lignocellulosic material.
  • protein activity is enhanced by modifying conditions in a reaction vessel, including but not limited to time, pH of a culture medium, temperature, concentration of nutrients and/or catalyst, or a combination thereof.
  • a fermentation end-product is produced by treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit (Figure 5).
  • the biomass can first be heated by addition of hot water or steam.
  • the biomass can be acidified by bubbling gaseous sulfur dioxide through the biomass that is suspended in water, or by adding a strong acid, e.g., sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition.
  • a strong acid e.g., sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition.
  • the pH is maintained at a low level, e.g., below about 5.
  • the temperature and pressure can be elevated after acid addition.
  • a metal salt such as ferrous sulfate, ferric sulfate, ferric chloride, aluminum sulfate, aluminum chloride, magnesium sulfate, or mixtures of these can be added to aid in the hydrolysis of the biomass.
  • the acid-impregnated biomass is fed into the hydrolysis section of the pretreatment unit.
  • Steam is injected into the hydrolysis portion of the pretreatment unit to directly contact and heat the biomass to the desired temperature.
  • the temperature of the biomass after steam addition is, e.g., between about 130° C and 220° C.
  • the hydrolysate is then discharged into the flash tank portion of the pretreatment unit, and is held in the tank for a period of time to further hydro lyze the biomass, e.g., into oligosaccharides and monomeric sugars. Steam explosion can also be used to further break down biomass.
  • the biomass can be subject to discharge through a pressure lock for any high- pressure pretreatment process. Hydrolysate is then discharged from the pretreatment reactor, with or without the addition of water, e.g., at solids concentrations between about 15% and 60%.
  • the biomass can be dewatered and/or washed with a quantity of water, e.g. by squeezing or by centrifugation, or by filtration using, e.g. a countercurrent extractor, wash press, filter press, pressure filter, a screw conveyor extractor, or a vacuum belt extractor to remove acidified fluid.
  • the acidified fluid with or without further treatment, e.g. addition of alkali (e.g. lime) and or ammonia (e.g.
  • ammonium phosphate can be re-used, e.g., in the acidification portion of the pretreatment unit, or added to the fermentation (acidification removes a considerable amount of hemicelluloses), or collected for other use/treatment.
  • Products can be derived from treatment of the acidified fluid, e.g., gypsum or ammonium phosphate.
  • Enzymes or a mixture of enzymes can be added during pretreatment to assist, e.g.
  • the fermentor is fed with hydrolyzed biomass (often neutralized to pH 6-8), any liquid fraction from biomass pretreatment, an active seed culture of a C5/C6 hydrolyzing microorganism, such as C. phy or Clostridium sp.
  • hydrolyzed biomass often neutralized to pH 6-8
  • any liquid fraction from biomass pretreatment an active seed culture of a C5/C6 hydrolyzing microorganism, such as C. phy or Clostridium sp.
  • Q.D cells either of which can be genetically modified to express one or more expansins, swollenins or cellulases
  • a co-fermenting microbe e.g., yeast or E. coli
  • nutrients to promote growth of C. phy or other microbes e.g., glucose, glucose, and, if required, nutrients to promote growth of C. phy or other microbes.
  • the pretreated biomass or liquid fraction can be split into multiple bioreactors, each containing a different strain of C. phy or Clostridium sp. Q.D and/or other microbes, and each operating under specific physical conditions. Fermentation is allowed to proceed for a period of time, e.g., between about 15 and 150 hours, while maintaining a temperature of, e.g., between about 25° C and 50° C.
  • Gas produced during the fermentation is swept from fermentor and is discharged, collected, or flared with or without additional processing, e.g. hydrogen gas can be collected and used as a power source or purified as a co-product.
  • the contents of the fermentor are transferred to product recovery. Products are extracted, e.g., ethanol is recovered through distillation and rectification.
  • fermentation end-products are produced by adding biomass to a fermentation vessel ( Figure 6).
  • the biomass can be allowed to soak for a period of time, with or without addition of heat, water, enzymes, or acid/alkali.
  • the pressure in the processing vessel can be maintained at or above atmospheric pressure.
  • Acid or alkali can be added at the end of the pretreatment period for neutralization.
  • pretreatment is carried out in another vessel and a neutralized and/or washed pretreated biomass is transferred to the fermentation vessel for consolidated bioprocessing (hydrolysis and fermentation).
  • an active seed culture of a C5/C6 hydrolyzing microorganism ⁇ e.g., C. phy or Clostridium sp.
  • a C5/C6 hydrolyzing microorganism ⁇ e.g., C. phy or Clostridium sp. Q.D
  • a C5/C6 hydrolyzing microorganism ⁇ e.g., C. phy or Clostridium sp. Q.D
  • a C5/C6 hydrolyzing microorganism can be used alone or together in combination with one or more other microbes ⁇ e.g. yeasts, fungi, or other bacteria).
  • productivity of fermentation end-product production by a microorganism is improved by culturing the microorganism in a medium comprising one or more compounds comprising hexose and/or pentose sugars.
  • a process comprises conversion of a starting material (such as a biomass) to a bio fuel, such as one or more alcohols.
  • methods comprise contacting the substrate biomass comprising both hexose (e.g. glucose, cellobiose) and pentose (e.g.
  • methods of the invention comprise contacting substrate comprising both hexose (e.g. glucose, cellobiose) and pentose (e.g. xylose, arabinose) saccharides with C. phytofermentans or Clostridium sp. Q.D to produce ethanol or other fermentation end-products.
  • the methods described herein provide uptake rates of about 0.1-8 g/L/h or more of hexose (e.g.
  • compositions and methods described herein batch fermentation with a microorganism (such as C. phy or Clostridium sp. Q.D) of a mixture of hexose and pentose saccharides using the methods of compositions and methods described herein provides uptake rates of about 0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or 6 g/L/h or more of hexose (e.g.
  • glucose, cellulose, cellobiose etc. glucose, cellulose, cellobiose etc.
  • pentose xylose, xylan, hemicellulose etc.
  • Example 1 Electroporation of C. phytofermentans
  • Plasmids comprising expansin or swollenin genes are constructed in accordance with Examples 2-5, with substitution of one or more of these genes for a cellulase.
  • a plasmid containing T. reesei swollenin gene is introduced to a strain of C. phytofermentans via electroporation.
  • Proper electroporation controls are prepared as follows: C. phy is electroporated without the plasmid to control for spontaneous antibiotic resistance; a dilution series of C. phy cells is prepared for viability control under the electroporation steps.
  • Electroporation Buffer EPB 270 mM Sucrose, 7 mM Sodium Phosphate, 1 mM MgS04
  • Electroporation Buffer EPB 270 mM Sucrose, 7 mM Sodium Phosphate, 1 mM MgS04
  • Electroporation Buffer EPB 270 mM Sucrose, 7 mM Sodium Phosphate, 1 mM MgS04
  • Disposable 0.4 cm electroporation cuvettes BioRadTM
  • 1 ug of plasmid, transfection-grade Sterile Plate
  • QM media Plates 4 Plates (standard QM Media) and 7 Plates (QM Media with antibiotics).
  • QM media is prepared as follows: Base media: ⁇ 2 ⁇ 0 4, 1.92 g/L, K 2 HP0 4 10.60 g/L, Ammonium sulfate 4.60 g/L, Sodium citrate tribasic 2H 2 0 3.00 g/L, Bacto yeast extract 6.00 g/L, Cysteine 2.00 g/L; 20x Substrate Stock: Maltose 400.00 g/L; 100X QM Salts solution: MgCl 2 6H 2 0 100 g/L, CaCl 2 2H 2 0 15 g/L, FeS0 4 7H 2 0 0.125 g/L.
  • the culture is washed in a buffered solution to remove media.
  • the culture is transferred to a 50 mL Falcon tube. The tube is spun at 8,500 RPM for 10 minutes. The supernatant is discarded in the anaerobic glove box and the pellet is
  • Plasmids suitable for use in Clostridium phytofermentans were constructed using portions of plasmids obtained from bacterial culture collections (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, InhoffenstraBe 7 B, 38124 Braunschweig, Germany, hereinafter "DSMZ"). Plasmid pIMPl is a non-conjugal shuttle vector that can replicate in E. coli, C. phy, and Clostridium sp. Q.D, Additionally, pIMPl (Fig. 1) encodes for resistance to erythromycin (Em R ).
  • the origin of transfer for the RK2 conjugal system was obtained from plasmid pRK290 (DSMZ) as DSM 3928, and the other conjugation functions of RK2 were obtained from pRK2013 (DSMZ) as DSM 5599.
  • Polymerase chain reaction (PCR) was used to amplify the 112 base pair origin of transfer region (oriT) from pRK290 using primers that added Clal restriction sites flanking the oriT region. This DNA fragment was inserted into the Clal site on pIMPl to yield plasmid pIMPT.
  • pIMPT was shown to be able to be transferred from one strain of E. coli to another when pRK2013 was also present to supply other conjugation functions.
  • PCR was used to amplify the promoter of the alcohol dehydrogenase (Adh) gene Cphy_1029 from the C. phy chromosome and it was used to replace the promoter of the erythromycin gene in pIMPT to create pIMPCphy (Fig. 2).
  • Adh alcohol dehydrogenase
  • Fig. 2 The successful transfer of pIMPCphy into C. phy or Clostridium sp. Q.D via electroporation was demonstrated by the ability to grow in the presence of 10 ⁇ / ⁇ . erythromycin.
  • Clostridium sp. Q.D confirmed that the same plasmid was isolated from C. phy or Clostridium sp. Q.D and transferred into E. coli and recovered.
  • Fresh cells E. coli culture and fresh cells of the C. phytofermentans or Clostridium sp. Q.D recipient culture were obtained by growth to mid-log phase using appropriate growth media (L broth and QMl media respectively). The two bacterial cultures were then centrifuged to yield cell pellets and the pellets resuspended in the same media to obtain cell suspensions that were concentrated about ten-fold having cell densities of about 10 10 cells per ml. These concentrated cell suspensions were then mixed to achieve a donor-to-recipient ratio of five-to-one, then the cell suspension was spotted onto QMl agar plates and incubated anaerobically at 30 °C for 24 hours.
  • the cell mixture was removed from the QMl plate and placed on solid or in liquid QMl media containing antibiotics that allow the survival of C. phytofermentans or Clostridium sp. Q.D recipient cells expressing erythromycin resistance. This was accomplished by using a combination of antibiotics consisting of trimethoprim (20 ⁇ g/ml), cycloserine (250 ⁇ g/ml), and erythromycin (10 ⁇ g/ml). The E. coli donor was unable to survive exposure to these
  • a promoter element can be selected by selecting key genes that would necessarily be involved in constitutive pathways ⁇ e.g., ribosomal genes, or for ethanol production, alcohol dehydrogenase genes).
  • promoters from such genes include but are not limited to: Cphy_1029: iron- containing alcohol dehydrogenase, Cphy_3510: Ig domain-containing protein and Cphy_3925: bifunctional acetaldehy de-Co A/alcohol dehydrogenase.
  • Cphy_1029 iron- containing alcohol dehydrogenase
  • Cphy_3510 Ig domain-containing protein
  • Cphy_3925 bifunctional acetaldehy de-Co A/alcohol dehydrogenase.
  • the primers were engineered to introduce restriction sites at the end of the promoter fragments that are present in the multiple cloning site of pIMPCphy but are otherwise not present in the promoter region itself, for example Sail, BamHI, Xmal, Smal, EcoRI.
  • the PCR reaction was performed with a commercially available PCR Kit, e.g. GoTaq® Green Master Mix (Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 USA), according to the manufacturer's conditions.
  • the reaction was run in a thermal cycler, e.g. Gene Amp System 2400 (PerkinElmer, 940 Winter St., Waltham MA 02451 USA).
  • the PCR products were purified with the GenEluteTM PCR Clean-Up Kit (Sigma-Aldrich Corp., St. Louis, MO, USA).
  • Both the purified PCR products as well as the plasmid pIMPCphy were then digested with the corresponding enzymes with the appropriate amounts according to the manufacturer's conditions (restriction enzymes from New England Biolabs, 240 County Road, Ipswich, MA 01938 USA and Promega).
  • the PCR products and the plasmid were then analyzed and gel-purified on a Recovery FlashGel (Lonza Biologies, Inc., 101 International Drive, Portsmouth, NH 03801 USA).
  • the PCR products were subsequently ligated to the plasmid with the Quick Ligation Kit (New England Biolabs) and competent cells of E.coli (DH5 ) are transformed with the ligation mixtures and plated on LB plates with 100 ⁇ g/ml ampicillin. The plates are incubated overnight at 37°C.
  • the bacteria mixture was either spread directly onto plates or first grown on liquid media for 6h to 18h and then plated.
  • the plates contain 10 ⁇ g/ml erythromycin as selective agent for C. phy and 10 ⁇ g/ml Trimethoprim, 150 ⁇ g/ml Cyclosporin and 100 ⁇ g/ml Nalidixic acid as counter selectable media for E .coli.
  • erythromycin-resistant colonies were picked from the plates and restreaked on fresh selective plates. Single colonies were picked and the presence of the plasmid confirmed by PCR reaction.
  • Two primers were chosen to amplify Cphy l 163 using C. phy genomic DNA as template.
  • the two primers were: cphy l 163F: 5' -CCG CGG AGG AGG GTT TTG TAT GAG TAA AAT CAG AAG AAT AGT TTC-3 (SEQ ID NO: 17), which contained a SacII restriction enzyme site and ribosomal site; and cphy l 163R: CCC GGG TTA GTG GTG GTG GTG GTG GTG TTT TCC ATA ATA TTG CCC TAA TGA (SEQ ID NO: 18), which containing a Xmal site and His-tag.
  • the amplified gene was cloned into Topo-TA first, then digested with SacII and Xmal, the cphy l 163 fragment was gel purified and ligated with pCPHY3510 (Fig. 3) digested with SacII and Xmal, respectively.
  • the plasmid was transformed into E.coli, purified and then transformed into C. phy by electroporation.
  • the plasmid map is shown in Figure 4.
  • the trans formants from the QM plates which contained 20 ⁇ g/ml of erythromycin, were transformed into QM liquid medium, which contained 2% cellobiose and 20 ⁇ g/ml of erythromycin.
  • the enzyme activities from the supernatant of overnight culture were assayed by CMC-congo red plate assay and Cellazyme T assay kit (Megazyme International Ireland, Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland).
  • genes encoding Cphy_3367, Cphy_3368, Cphy_3202 and Cphy_2058 were cloned into pCphy3510 to produce pCphy3510_3367, pCphy3510_3368, pCphy3510_3202, and pCphy3510_2058 respectively. These vectors were transformed into C. phy via electroporation as described supra.
  • genes encoding the heat shock chaperonin proteins, Cphy_3289 (GroES) and Cphy_3290 (GroEL) were incorporated into pCphy3510.
  • an enzymes described in table 5 were cloned into pCphy3510 to produce pCphy3510_3367, pCphy3510_3368, pCphy3510_3202, and pCphy3510_2058 respectively.
  • These vectors were transformed into C. phy via electroporation as described supra.
  • endogenous or exogenous gene from another bacteria or fungal cell i.e., an expansin or swollenin
  • an expansin or swollenin can be cloned into this vector and used to transform C. phy or Clostridium sp. Q.D..
  • Example 5 A process employing modified C. phy for producing ethanol
  • a modified C. phy genetically modified to express a swollenin protein is cultured overnight.
  • the modified C. phy contains an antibiotic resistance gene and the culture incubated with the respective antibiotic in order to insure swollenin expression.

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Abstract

La présente invention concerne des organismes modifiés améliorant la saccharification de la biomasse. Lesdits microorganismes sont modifiés pour renforcer l'activité d'une ou plusieurs protéines de type cellulase et/ou pour exprimer au moins un polypeptide de type expansine ou swollénine.
PCT/US2010/046866 2009-08-26 2010-08-26 Organismes modifiés utilisés pour une meilleure saccharification de la biomasse WO2011028623A2 (fr)

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WO2021181084A1 (fr) * 2020-03-10 2021-09-16 Marlow Foods Limited Procédés de production d'éthanol

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WO2007115723A2 (fr) * 2006-04-06 2007-10-18 Institut Français Du Petrole Proteines resultant de la fusion entre des enzymes degradant la paroi cellulaire de plantes et une swollenine et leurs utilisations
US20090068714A1 (en) * 2006-01-27 2009-03-12 University Of Massachusetts Systems and Methods for Producing Biofuels and Related Materials
US20090111154A1 (en) * 2007-04-04 2009-04-30 The Regents Of The University Of California Butanol production by recombinant microorganisms

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090068714A1 (en) * 2006-01-27 2009-03-12 University Of Massachusetts Systems and Methods for Producing Biofuels and Related Materials
WO2007115723A2 (fr) * 2006-04-06 2007-10-18 Institut Français Du Petrole Proteines resultant de la fusion entre des enzymes degradant la paroi cellulaire de plantes et une swollenine et leurs utilisations
US20090111154A1 (en) * 2007-04-04 2009-04-30 The Regents Of The University Of California Butanol production by recombinant microorganisms

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Title
STANLEY M. HARMON ET AL.: 'Beneficial effect of catalase treatment on growth of Clostridium perfringens' APPL. ENVIRON. MICROBIOL. vol. 32, no. 3, September 1976, pages 409 - 416 *

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