RECOMBINANT MICROBIAL HOSTING CELL, PROCESSES TO PRODUCE A POLYPEPTIDE, TO PRODUCE A PROTEIN, TO DEG

专利摘要:
transgenic microbial host cell, processes to produce a polypeptide, to produce a mutant, to produce a protein, to degrade a cellulosic material, to synthesize a fermentation product, to ferment a cellulosic material, to produce a polypeptide with cellobiohydrolase activity, nucleic acid construct or expression vector, isolated polypeptide, and isolated polynucleotide. the present invention relates to isolated polypeptides with cellobiohydrolase activity and to the polynucleotides that encode the polypeptides. the invention also relates to the nucleic acid constructs, vectors, and host cells that comprise the polynucleotides, as well as the methods of producing and using the polypeptides.
公开号:BR112013018695B1
申请号:R112013018695-0
申请日:2012-01-26
公开日:2021-03-30
发明作者:Nikolaj Spodsberg
申请人:Novozymes A / S；Novozymes Inc；
IPC主号:

专利说明:

Declaration of Rights for Inventions Conducted in Research and Development Sponsored by the Federal Government
[001] This invention was made in part with the assistance of the Government in the cooperation agreement DE-FC36-08GO18080, granted by the Department of Energy. The Government has certain rights in this invention. Reference to a string listing
[002] This application contains a sequence listing in computer readable form, which is incorporated by reference here. Fundamentals of the Invention Field of the Invention
[003] The present invention relates to polypeptides with cellobiohydrolase activity and polynucleotides that encode the polypeptides. The invention also relates to the nucleic acid constructs, vectors and host cells that comprise the polynucleotides, as well as methods of producing and using the polypeptides. Description of the Related Art
[004] Cellulose is a glucose polymer bound by beta-1,4 binding. Many microorganisms produce enzymes that hydrolyze bound beta-glycans. These enzymes include endoglucanases, cellobiohydrolases and beta-glycosidases. Endoglucanases digest the cellulose polymer at random locations, opening it up to attack by cellobiohydrolases. Cellobiohydrolases sequentially release cellobiose molecules from the ends of the cellulose polymer. Cellobiosis is a dimer bound to water-soluble 1,4-beta glucose. Beta-glycosidases hydrolyze cellobiose into glucose.
[005] The conversion of lignocellulosic raw materials into ethanol has the advantages of the immediate availability of large quantities of raw material, the advantage of avoiding burning or grounding the materials, and the cleaning of fuel ethanol. Wood, agricultural residues, herbaceous crops and municipal solid residues are considered raw materials for the production of ethanol. These materials mainly consist of cellulose, hemicellulose, and lignin. Once cellulose is converted to glucose, glucose is easily fermented by yeast in ethanol. Whereas glucose is easily fermented in ethanol by a variety of yeasts, while cellobiosis is not, any cellobiosis that remains at the end of hydrolysis represents a loss of ethanol yield. More importantly, cellobiosis is a potent inhibitor of endoglucanases and cellobiohydrolases. The accumulation of cellobiosis during hydrolysis is undesirable for the production of ethanol.
[006] The present invention provides polypeptides with cellobiohydrolase activity and polynucleotides that encode the polypeptides.
[007] The polypeptide according to the invention that exhibits cellobiohydrolase activity shares 80.4% identity (excluding the intervals) with the deduced amino acid sequence of a predicted protein from the GH6 family of Aspergillus fumigatus (GENESEQP accession number: ABB80166 ). Summary of the Invention
The present invention relates to isolated polypeptides with cellobiohydrolase activity selected from the group consisting of: (a) a polypeptide that has at least 81% sequence identity with the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under conditions of low, medium, or medium-high, or high, or very high severity in (i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, ( ii) its cDNA sequence, or (iii) the full size complement of (i) or (ii); (c) a polypeptide encoded by a polynucleotide that has at least 60% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; or the cDNA sequence thereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2 which comprises a substitution, deletion and / or insertion at one or more (for example, several) positions; and (e) a fragment of the polypeptide of (a), (b), (c), or (d) that exhibits cellobiohydrolase activity.
[009] The present invention also relates to isolated polypeptides comprising a catalytic domain, selected from the group consisting of: (a) a catalytic domain that has at least 81% sequence identity with the catalytic domain of SEQ ID NO: 2 (for example, amino acids 105 to 464 of SEQ ID NO: 2); (b) a catalytic domain encoded by a polynucleotide that has at least 60% sequence identity with the sequence encoding the catalytic domain of SEQ ID NO: 1 (e.g., nucleotides 460-599, 678-931, 1001-1138 , 1210-1470, 1541-1782, and 1854-1895 of SEQ ID NO: 1); (c) a variant of a catalytic domain that comprises a substitution, deletion, and / or insertion of one or more (several) amino acids from the catalytic domain of SEQ ID NO: 2; and (d) a fragment of a catalytic domain of (a), (b), or (c), which exhibits cellobiohydrolase activity.
[0010] The present invention also relates to isolated polynucleotides that encode the polypeptides of the present invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells that comprise polynucleotides; and methods of producing the polypeptides.
[0011] The present invention also relates to processes for degrading a cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide that exhibits cellobiohydrolase activity of the present invention. In one aspect, the processes further comprise recovering the degraded or converted cellulosic material.
The present invention also relates to processes for producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide that exhibits the cellobiohydrolase activity of the present invention; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[0013] The present invention also relates to the processes of fermenting a cellulosic material comprising: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide that exhibits cellobiohydrolase activity of the present invention. In one aspect, the fermentation of the cellulosic material synthesizes a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.
The present invention also relates to a polynucleotide that encodes a signal peptide that comprises or consists of amino acids 1 to 18 of SEQ ID NO: 2, which is operably linked to a gene that encodes a protein; nucleic acid constructs, expression vectors, and recombinant host cells that comprise polynucleotides; and methods of producing a protein. Definitions
[0015] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glycan cellobiohydrolase (EC 3.2.1.91), which catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cell-oligosaccharides , or any polymer containing glucose linked to beta-1,4, which releases cellobiose from the reduced or unreduced ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160 -167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose , Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the method by Tomme et al. can be used to determine cellobiohydrolase activity.
[0016] In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%, or at least 100% of the cellobiohydrolase activity of the mature polypeptide of SEQ ID NO: 2.
[0017] Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-naphthyl acetate and p-acetate nitrophenyl. For purposes of the present invention, the activity of acetylxylan esterase is determined using 0.5 mM p-nitrophenylacetate as a substrate in 50 mM sodium acetate, pH 5.0, containing 0.01% TWEEN ™ 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 μmol of p-nitrophenolate anion per minute at pH 5, 25 ° C.
[0018] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene that occupies the same chromosomal locus. Allelic variation naturally increases through mutation, and can result in polymorphism in populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0019] Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of non-reduced alpha-L-arabinofuranoside residues in alpha- L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3) and / or (1,5) bonds, arabinoxylans and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, alpha-L-arabinofuranosidase polysaccharide, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L- arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate , pH 5, in a total volume of 200 μL for 30 minutes at 40 ° C, followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[0020] Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase is equal to the amount of enzyme capable of releasing 1 μmol of glucuronic acid or 4-O-methylglucuronic acid per minute at pH 5, 40 ° C.
[0021] Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (EC 3.2.1,21) that catalyzes the hydrolysis of beta-D-glucose terminal non-reducing residues with the release of beta -D- glucose. For purposes of the present invention, beta-glycosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as a substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glycosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 μmol of p-nitrophenolate anion produced per minute at 25 ° C, pH 4.8, from 1 mM p-nitrophenyl-beta-D-glucopyranoside as a substrate in citrate of 50 mM sodium containing 0.01% TWEEN® 20.
[0022] Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (EC 3.2.1.37) that catalyzes the exohydrolysis of short beta ^ (4) -xyl-oligosaccharides to remove successive residues of D-xylose from unreduced terminations. For the purposes of the present invention, a beta-xylosidase unit is defined as 1.0 μmol of p-nitrophenolate anion produced per minute at 40 ° C, pH 5, from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20.
[0023] DNAc: The term "DNAc" means a DNA molecule that can be prepared by reverse transcription of a mature, joined mRNA molecule obtained from a eukaryotic cell. CDNA needs intron sequences that can be present in the corresponding genomic DNA. The initial and primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
[0024] Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of the biomass is cellulose, the second most abundant is hemicellulose and the third is pectin. The secondary cell wall, produced after the cell stops growing, also contains polysaccharides and is reinforced by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is an anhydrocelobiose homopolymer and thus a linear beta- (1-4) -D-glycan, while hemicelluloses include a variety of compounds, such as xylans, xyloglycans, arabinoxylans and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphic, cellulose is found in plant tissue mainly as an insoluble crystalline matrix of parallel glycan chains. Hemicelluloses in general bind hydrogen to cellulose, as well as other hemicelluloses, which helps to stabilize the cell wall matrix.
[0025] Cellulose is found in general, for example, in the stems, leaves, sepals, barks and cobs of plants or leaves, branches and wood of trees. Cellulolytic material can be, but is not limited to, agricultural waste, herbaceous material (including energy-producing crops), municipal solid waste, pulp and ground paper waste, waste paper and wood (including forest waste) (see, for example) , Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington DC; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering / Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, New York). It is understood here that cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose and hemicellulose in a mixed matrix. In a preferred aspect, the cellulolytic material is any biomass material. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses and lignin.
[0026] In one aspect, cellulosic material is agricultural waste. In another aspect, the cellulosic material is herbaceous waste (including crops for energy production). In another aspect, cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and ground paper residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forest waste).
[0027] In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is ear of corn. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn residue. In another aspect, the cellulosic material is Miscanthus. In another aspect, the cellulosic material is orange house. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is yellow millet. In another aspect, the cellulosic material is wheat straw.
[0028] In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is pine cone. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
[0029] In another aspect, the cellulosic material is algae cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is cellulose treated with phosphoric acid.
[0030] In another aspect, the cellulosic material is an aquatic biomass. As used herein, the term "aquatic biomass" means biomass produced in an aquatic environment by a process of photosynthesis. Aquatic biomass can be seaweed, emerging plants, plants with floating leaves or submerged plants.
[0031] Cellulolytic material can be used as is, or can be subjected to pretreatment, using conventional methods known in the art, in the manner described here. In a preferred aspect, the cellulolytic material is pre-treated.
[0032] Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (for example, several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase (s), cellobiohydrolase (s), beta-glycosidase (s) or combinations thereof. The two basic approaches to measuring cellulolytic activity include: (1) measuring total cellulolytic activity, and (2) measuring individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glycosidases) in the manner reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is measured in general using insoluble substrates, including Whatman paper filter No. 1, microcrystalline cellulose, bacterial cellulose, algae cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the paper filter assay using Whatman No. 1 paper filter as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).
[0033] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulolytic material by the cellulolytic enzyme (s) under the following conditions: 1-50 mg of enzyme protein cellulolytic / g cellulose in PCS for 3-7 days at an appropriate temperature, for example at 50 ° C, 55 ° C or 60 ° C, compared to a control hydrolysis without the addition of cellulolytic enzyme protein. Typical conditions are 1 mL reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate at pH 5, 1 mM MnSO4, 50 ° C, 55 ° C or 60 ° C, 72 hours, analysis of sugar per AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[0034] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The limits of the coding sequence are generally determined by an open reading frame, which begins with an initial codon such as ATG, GTG, or TTG, and ends with a stop codon such as TAA, TAG or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination of these.
[0035] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide that encodes a mature polypeptide of the present invention. Each control sequence can be natural (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or natural or foreign to each other. Such control sequences include, but are not limited to, a major sequence, polyadenylation sequence, pro-peptide sequence, promoter, signal peptide sequence and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. The control sequences can be provided with ligands for the purpose of introducing specific restriction sites that facilitate the binding of the control sequences with the coding region of the polynucleotide that encodes a polypeptide.
[0036] Endoglucanase: The term "endoglucanase" means an endo-1,4- (1,3; 1,4) -beta-D-glycan 4-glycanhydrolase (EC 3.2.1.4), which catalyzes the endohydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenine, beta-1,4 bonds in mixed beta-1,3 glycans, such as beta-D-glycans or cereal xyloglycans and other plant material containing cellulolytic components. Endoglucanase activity can be determined by measuring the reduction in substrate viscosity or the increase in edge reduction determined by a sugar reduction assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as a substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40 ° C.
[0037] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.
[0038] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide that encodes a polypeptide, and is operably linked to control sequences that provide its expression.
[0039] Glycoside hydrolase family 61: The term "Glycoside hydrolase family 61" or "GH61 family" or "GH61" means a polypeptide that belongs to the glycoside hydrolase family 61 according to Henrissat, 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a family of glycoside hydrolase based on the measurement of the very weak activity of endo-1,4-beta-D-glucanase in a family member. The structure and mode of action of these enzymes are non-canonical and cannot be considered as authentic glycosidases. However, they are maintained in the CAZy classification based on their ability to improve the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
[0040] Feruloyl esterase: The term "feruloyl esterase" means a 4-hydroxy-3-methoxy-aminoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of the 4-hydroxy-3-methoxy-aminoyl (feruloyl) groups from esterified sugar, which is generally arabinose on “natural” substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).
[0041] Fragment: The term "fragment" means a polypeptide with one or more (for example, several) amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide; where the fragment has cellobiohydrolase activity. In one aspect, a fragment contains at least 360 amino acids. More particularly, in one embodiment, a fragment means a polypeptide comprising or consisting of amino acids 105 to 464 of SEQ ID NO: 2. In a further embodiment the linker, amino acids 56 to 104 of SEQ ID NO: 2, or a part thereof.
[0042] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases. Current Opinion in Microbiology 6 (3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetixylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannosidase , a xylanase and a xylosidase. The substrates of these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are linked by means of hydrogen bonds to the cellulose microfibrils in the plant cell wall, cross-linking them in a large network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses requires the combined action of many enzymes for their complete degradation. The catalytic modules of hemicellulases are both glycoside hydrolases (GHs), which hydrolyze glycosidic bonds, and carbohydrate esterases (CEs), which hydrolyze ester bonds of side groups of acetate or ferulic acid. These catalytic modules, based on the homology of their primary sequence, can be determined in the GH and CE families marked by numbers. Some families, with a similar complete fold, can be further grouped into clans, marked alphabetically (for example, GH-A). A more informative and up-to-date classification of these and other active carbohydrate enzymes is available in the CarbohydrateActive Enzymes (CAZy) database. The enzyme's hemicellulolytic activities can be measured according to Ghose and Bisaria, 1987, Pure & AppI. Chem. 59: 1739-1752, at a suitable temperature, for example, 50 ° C, 55 ° C or 60 ° C, and a suitable pH, for example, 5.0 or 5.5.
[0043] High severity conditions: The term "high severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared DNA from salmon sperm, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 ° C.
[0044] Host cell: The term "host cell" means any type of cell that is susceptible to transformation, transfection, transduction, or similar to a nucleic acid construct or expression vector that comprises a polynucleotide of the present invention. The term "host cell" includes any progeny of a mother cell that is not identical to the mother cell because of the mutations that occur during replication.
[0045] Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor that is at least partially removed from one or more, or all, the naturally occurring constituents with which it is associated in nature; (3) any substance modified by human manipulation related to that substance found in nature; or (4) any substance modified by increasing the amount of the substance related to other components with which it is naturally associated (for example, multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.
[0046] Low severity conditions: The term "low severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared DNA from salmon sperm, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each, for 15 minutes, using 2X SSC, 0.2% SDS at 50 ° C.
[0047] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form that allows for translation and any of the post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide has amino acids 19 to 464 of SEQ ID NO: 2, based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) which predicts that amino acids 1 to 18 of SEQ ID NO: 2 are a signal peptide. It is known in the art that a host cell can produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide.
[0048] Sequence encoding mature polypeptide: The term "sequence encoding mature polypeptide" means a polynucleotide encoding a mature polypeptide with cellobiohydrolase activity. In one aspect, the sequence encoding the mature polypeptide has nucleotides 55 to 1895 of SEQ ID NO: 1, or its cDNA sequence, based on the SignalP program (Nielsen et al., 1997, supra) which predicts that nucleotides 1 to 54 of SEQ ID NO: 1 encode a signal peptide. In a particular embodiment, the sequence encoding the mature polypeptide has nucleotides 55-76, 146-214, 290-599, 678-931, 1001-1138, 1210-1470, 1541-1782, 1854-1898 of SEQ ID NO : 1 (including the stop codon).
[0049] Catalytic domain: The term "catalytic domain" means the portion of an enzyme that contains the catalytic machinery of the enzyme. In a particular embodiment, the catalytic domain has amino acids 105-464 of SEQ ID NO: 2.
[0050] Cellulose binding domain: The term "cellulose binding domain" means the portion of an enzyme that mediates the binding of the enzyme in the amorphous regions of a cellulose substrate. The cellulose binding domain (CBD) is found at both the N-terminus and the C-terminus of an enzyme. A CBD is also referred to as a cellulose or CBM binding module. In one embodiment, CBM has amino acids 19 to 55 of SEQ ID NO: 2. CBM is separated from the catalytic domain by a ligand sequence. The linker, in one embodiment, has amino acids 56 to 104 of SEQ ID NO: 2.
[0051] Medium severity conditions: The term "medium severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55 ° C.
[0052] Medium-high severity conditions: The term “medium-high severity conditions” means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in SSPE 5X, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60 ° C.
[0053] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, both single-stranded and double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a way that may not otherwise exist in nature, or that is synthetic, that comprises one or more control sequences.
[0054] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed in an appropriate position, with respect to the coding sequence of a polynucleotide, in such a way that the control sequence directs the expression of the coding sequence.
[0055] Polypeptide with better cellulolytic activity: The term "polypeptide with better cellulolytic activity" means a GH61 polypeptide that catalyzes the improvement of the hydrolysis of a cellulosic material by an enzyme that has cellulolytic activity. For the purposes of the present invention, the best cellulolytic activity is determined by assessing the increase in reducing sugars, or the increase in total cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of protein total / g cellulose in PCS, where the total protein is comprised of 50-99.5% w / w of enzymatic cellulolytic protein and 0.5-50% w / w of protein of a GH61 polypeptide with better cellulolytic activity by 1-7 days at a suitable temperature, for example, 50 ° C, 55 ° C, or 60 ° C, and a suitable pH, for example, 5.0 or 5.5, compared to a control hydrolysis with total protein loading equal, without better cellulolytic activity (1-50 mg cellulolytic protein / g cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5 L (Novozymes A / S, Bagsv ^ rd, Denmark) in the presence of 2-3% by weight of total beta-glucosidase protein from Aspergillus oryzae (produced recombinantly) in Aspergillus oryzae according to WO 02/095014), or 2-3% by weight of total protein of Aspergillus fumigatus beta-glycosidase (produced recombinantly in Aspergillus oryzae in the manner described in WO 2002/095014) of the protein load cellulase is used as the source of cellulolytic activity.
[0056] GH61 polypeptides with better cellulolytic activity improve the hydrolysis of a cellulosic material, catalyzed by an enzyme that has cellulolytic activity, reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably at least 1.01 times, per example, at least 1.05 times, at least 1.10 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times.
[0057] Pre-treated corn residue: The term “PCS” or “pre-treated corn residue” means cellulolytic material derived from corn residue by heat treatment and diluted sulfuric acid, alkaline pretreatment or pretreatment neutral.
[0058] Sequence identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
[0059] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the replacement matrix EBLOSUM62 (EMBOSS version of BLOSUM62). The Needle yield marked as “best identity” (obtained using the non-summarized option) is used as the percentage identity and is calculated as follows: (Identical residuals x 100) / (Alignment size - Total number of intervals in the alignment )
[0060] For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), in the manner implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the substitution matrix EDNAFULL (EMBOSS version of NCBI NUC4.4). Needle yield marked as “best identity” (obtained using the non-summarized option) is used as the percentage identity and is calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment size - Total number of alignment intervals )
Subsequence: The term "subsequence" means a polynucleotide with one or more (for example, several) nucleotides missing at the 5 'and / or 3' end of a sequence encoding the mature polypeptide; wherein the subsequence encodes a fragment that exhibits cellobiohydrolase activity. In one aspect, a subsequence encodes a polypeptide that exhibits cellobiohydrolase activity, for example, a catalytic domain according to the invention. In one embodiment, a subsequence comprises or consists of nucleotides 460 to 1895 of SEQ ID NO: 1, and more particularly nucleotides 460-599, 678-931, 10011138, 1210-1470, 1541-1782, 1854-1895 of SEQ ID NO: 1.
[0062] Variant: The term "variant" means a polypeptide that exhibits cellobiohydrolase activity comprising an alteration, that is, a substitution, insertion, and / or elimination in one or more (for example, several) positions. A substitution means exchanging the amino acid that occupies a position for a different amino acid; an elimination means the removal of the amino acid that occupies a position; and an insertion means adding an amino acid adjacent and immediately after the amino acid that occupies a position.
[0063] Very high severity conditions: The term "very high severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in SSPE 5X, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70 ° C.
[0064] Very low severity conditions: The term "very low severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in SSPE 5X, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45 ° C.
[0065] Xylan-containing material: The term "xylan-containing material" means any material that comprises a plant cell wall polysaccharide, containing a major part of beta- (1-4) - residues bound to xylose. Terrestrial plant xylans are heteropolymers that have a major part of beta- (1-4) -D-xylopyranose, which is branched short chain carbohydrates. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose and / or various oligosaccharides composed of D-xylose, L-arabinose, D- or L-galactose and D-glucose. Xylan-like polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino) glucuronoxylans, (glucuron) arabinoxylans, arabinoxylans and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.
[0066] In the processes of the present invention, any material containing xylan can be used. In a preferred aspect, the material containing xylan is lignocellulose.
[0067] Activity that degrades xylan or xylanolitic activity: The term "activity that degrades xylan" or "xylanolitic activity" means a biological activity that hydrolyzes material containing xylan. The two basic approaches to measuring xylanolitic activity include: (1) measuring total xylanolitic activity, and (2) measuring individual xylanolitic activities (endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha- glucuronyl esterases). Recent progress in xylanolitic enzyme assays has been summarized in, for example, several publications, including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86 (11): 1636-1647; Spanikova and Biely, 2006, Glicuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580 (19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0068] The total activity that degrades xylan can be measured by determining the reduction of sugars formed from various types of xylan including, for example, oat spelled xylans, beech wood and larch wood, or by photometric determination of fragments stained xylans released from several covalently stained xylans. The most common total xylanolitic assay is based on the production of reduced sugars of polymeric 4-O-methyl glucuronoxylane, as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23 (3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as a substrate in TRITON® X-100 0.01% (4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol) and buffer 200 mM sodium sulfate pH 6 at 37 ° C. A unit of xylanase activity is defined as 1.0 μmol of azurine produced per minute at 37 ° C, pH 6, from AZCL-arabinoxylan 0.2% as a substrate in 200 mM sodium phosphate buffer, pH 6.
[0069] For purposes of the present invention, the activity that degrades xylan is determined by measuring the increase in birch xylan hydrolysis (Sigma Chemical Co., Inc., St. Louis, MO, USA) by enzyme (s) that degrades ( m) xylan in the following typical conditions: reactions of 1 ml, 5 mg / ml of substrate (total solids), 5 mg of xylanolitic protein / g of substrate, 50 mM sodium acetate at pH 5, 50 ° C, 24 hours , sugar analysis using the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.
[0070] Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endo-hydrolysis of 1,4-beta-D-xylosidic bonds in xylans. For purposes of the present invention, xylanase activity is determined with AZCL-arabinoxylan 0.2% as a substrate in TRITON X-100 0.01% and 200 mM sodium phosphate buffer, pH 6 at 37 ° C. One unit of xylanase activity is defined as 1.0 μmol of azurine produced per minute at 37 ° C, pH 6, from 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate buffer, pH 6. Detailed Description of the Invention Polypeptides with cellobiohydrolase activity
[0071] In one embodiment, the present invention relates to isolated polypeptides that have a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 81%, for example, at least 82%, at least 83%, at least 84%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which exhibit cellobiohydrolase activity. The polypeptides of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 100% of the cellobiohydrolase activity of the mature polypeptide of SEQ ID NO: 2. In one aspect, the polypeptides differ by no more than 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8 , or 9, of the mature polypeptide of SEQ ID NO: 2.
A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or it is a fragment of it with cellobiohydrolase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the polypeptide comprises or consists of amino acids 19 to 464 of SEQ ID NO: 2.
[0073] In another embodiment, the present invention relates to an isolated polypeptide that exhibits cellobiohydrolase activity encoded by a polynucleotide that hybridizes under conditions of medium severity, or conditions of medium-high severity, or conditions of high severity, or conditions of very high severity in (i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, (ii) its cDNA sequence, or (iii) the full size complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, UM Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).
[0074] The polynucleotide of SEQ ID NO: 1, or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, or a fragment thereof, can be used to determine the nucleic acid probes to identify and clone the polypeptides with cellobiohydrolase activity that encode DNA from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably smaller than the total sequence, but can be at least 15, for example, at least 25, at least 35, or at least 70 nucleotides in size. Preferably, the nucleic acid probe is at least 100 nucleotides in size, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in size. Both DNA and RNA probes can be used. The probes are typically labeled to detect the corresponding gene (for example, with 32P, 3H, 35S, biotin or avidin). Such probes are included by the present invention.
[0075] A genomic DNA or cDNA library prepared from such other strains can be selected for DNA that hybridizes to the probes described above and encodes a polypeptide with cellobiohydrolase activity. Genomic or other DNA, from such other strains, can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. The DNA from the libraries or the separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes to SEQ ID NO: 1, or a subsequence thereof, the carrier material is used in a Southern blot.
[0076] For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe that corresponds to (i) SEQ ID NO: 1; (ii) the sequence encoding the mature polypeptide of SEQ ID NO: 1; (iii) its cDNA sequence; (iv) the complement of its total size; or (v) a subsequence thereof; in conditions of medium to very high severity. The molecules on which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other means of detection known in the art.
[0077] In one aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2, its mature polypeptide or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1, or its cDNA sequence.
[0078] In another embodiment, the present invention relates to an isolated polypeptide, which exhibits cellobiohydrolase activity encoded by a polynucleotide, with a sequence identity to the sequence encoding the mature polypeptide of SEQ ID NO: 1, or the sequence of this cDNA, at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
[0079] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2, which comprise a substitution, deletion and / or insertion at one or more (for example, several) positions. In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 is no more than 10, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9. Amino acid changes may be of a lesser nature, that is, conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small binding peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.
[0080] Examples of conservative substitutions are in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.
[0081] Alternatively, the amino acid changes are of such a nature that the physicochemical properties of the polypeptides are altered. For example, amino acid changes can improve the thermal stability of the polypeptide, change the specificity of the substrate, change the ideal pH and the like.
[0082] The essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scan mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced and each residue in the molecule, and the resulting mutant molecules are tested for cellobiohydrolase activity to identify amino acid residues that are important for the molecule's activity. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, in the manner determined by such a technique as nuclear magnetic resonance, crystallography, electron diffraction, or photo affinity tagging, along with amino acid mutation at the supposed contact. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of the essential amino acids can also be inferred from the analysis of identities with polypeptides that are related to the parent polypeptide. The identity of the essential amino acids can also be inferred from an alignment with a related polypeptide.
[0083] Simple or single amino acid substitutions, deletions, and / or insertions can be performed and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant selection procedure, such as those revealed by Reidhaar- Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (for example, Lowman et al., 1991, Biochemistry 30: 10832-10837; US patent 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
[0084] Mutagenesis / scrambling methods can be combined with automated high-throughput selection methods to detect the activity of cloned and mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and quickly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
[0085] The polypeptide can be a hybrid polypeptide, in which a region of a polypeptide is fused to the N-terminus or to the C-terminus of a region of another polypeptide.
The polypeptide can be a fusion polypeptide or cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or at the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide that encodes another polypeptide with a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include linking the coding sequences that encode the polypeptides, so that they are in alignment and that the expression of the fusion polypeptide is in control of the same promoter (s) (s) and finisher (s). Fusion polypeptides can also be constructed using intein technology, in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266 : 776-779).
[0087] A fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48. Polypeptide sources with cellobiohydrolase activity
[0088] A polypeptide with cellobiohydrolase activity of the present invention can be obtained from microorganisms of any gender. For purposes of the present invention, the term "obtained from", as used herein in conjunction with a given source, can mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.
[0089] The polypeptide can be a Talaromyces polypeptide.
[0090] In another aspect, the polypeptide is a polypeptide from Talaromyces leycettanus, for example, a polypeptide obtained from the Talaromyces leycettanus strain CBS398.68.
[0091] It will be understood that, for the species mentioned above, the invention includes both the perfect and the imperfect stages, and other taxonomic equivalents, for example, anamorphs, without taking into account the name of the species for which they are known. Those skilled in the art will easily recognize the identity of suitable equivalents.
[0092] Strains of these species are easily accessible to the public in numerous culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
[0093] The polypeptide can be identified and obtained from other sources, including microorganisms isolated from nature (eg soil, compounds, water, etc.) or DNA samples obtained directly from natural materials (eg soil, compounds , water, etc.) using the aforementioned probes. Techniques for isolating microorganisms and DNA directly from their natural habitats are well known in the technology. A polynucleotide that encodes the polypeptide can then be obtained by similarly selecting a library of genomic DNA or cDNA from another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe (s), the polynucleotide can be isolated or cloned using techniques that are known to those skilled in the art (see, for example, Sambrook et al., 1989, supra). Catalytic domains
[0094] The present invention also relates to isolated polypeptides that comprise a catalytic domain selected from the group consisting of: (a) a catalytic domain that has at least 81% sequence identity with the catalytic domain of SEQ ID NO: 2 (for example, amino acids 105 to 464 of SEQ ID NO: 2); (b) a catalytic domain encoded by a polynucleotide that has at least 60% sequence identity with the sequence encoding the catalytic domain of SEQ ID NO: 1 (e.g., nucleotides 460-599, 678-931, 1001-1138 , 1210-1470, 1541-1782, and 1854-1895 of SEQ ID NO: 1); (c) a variant of a catalytic domain that comprises a substitution, deletion, and / or insertion of one or more (several) amino acids from the catalytic domain of SEQ ID NO: 2; and (d) a fragment of a catalytic domain of (a), (b), or (c), which has cellobiohydrolase activity.
[0095] The catalytic domain preferably has a degree of sequence identity with the catalytic domain of SEQ ID NO: 2 of at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In one aspect, the catalytic domain comprises an amino acid sequence that differs by ten amino acids, for example, by five amino acids, by four amino acids, by three amino acids, by two amino acids, and by one amino acid in the catalytic domain of SEQ ID NO: 2 .
[0096] The catalytic domain preferably comprises or consists of the catalytic domain of SEQ ID NO: 2 or an allelic variant thereof; or it is a fragment of it with cellobiohydrolase activity. In another preferred aspect, the catalytic domain comprises or consists of amino acids 105 to 464 of SEQ ID NO: 2.
[0097] In one embodiment, the catalytic domain can be encoded by a polynucleotide that hybridizes under conditions of medium severity, or conditions of medium-high severity, or conditions of high severity, or conditions of very high severity (as defined above) in (i) the sequence encoding the catalytic domain of SEQ ID NO: 1, (ii) the cDNA sequence contained in the sequence encoding the catalytic domain of SEQ ID NO: 1, or (iii) the complement of the size strip total of (i) or (ii) (J. Sambrook et al., 1989, supra).
[0098] The catalytic domain can be encoded by a polynucleotide with a degree of sequence identity with the sequence encoding the catalytic domain of SEQ ID NO: 1 of at least 60%, for example at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or 100%, which encodes a polypeptide with cellobiohydrolase activity.
[0099] In one aspect, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 460 to 1895 of SEQ ID NO: 1 or their cDNA sequence. In particular, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 460599, 678-931, 1001-1138, 1210-1470, 1541-1782, and 1854-1895 of SEQ ID NO: 1. Polynucleotides
The present invention also relates to isolated polynucleotides that encode a polypeptide of the present invention, in the manner described herein.
[00101] The techniques used to isolate or clone a polynucleotide are known in the art and include isolation of genomic DNA or cDNA, or a combination of these. The cloning of polynucleotides from genomic DNA can be performed, for example, using the well-known polymerase chain reaction (PCR) or antibody selection from expression libraries to detect cloned DNA fragments with shared structure characteristics. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures, such as ligase binding reaction (LCR), activated binding transcription (LAT) and nucleotide-based amplification (NASBA) can be used. Polynucleotides can be cloned from a Talaromyces strain or a related organism, and thus, for example, they can be an allelic or variant species of the polypeptide that encodes the polynucleotide region.
[00102] Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary to synthesize polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to the non-naturally occurring forms of the polypeptide. These polypeptides may differ, in some way modified by engineering, from the polypeptide isolated from its natural source, for example, variants that differ in specific activity, thermostability, ideal pH or the like. Variants can be constructed based on the polynucleotide shown as the sequence encoding the mature polypeptide of SEQ ID NO: 1, or on its cDNA sequence, for example, a subsequence of this, and / or by introducing nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the use of the intended host organ codon for the production of the enzyme, or by introducing nucleotide substitutions that may result in a different amino acid sequence. For a general description of nucleotide substitution, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107. Nucleic acid constructs
The present invention also relates to nucleic acid constructs that comprise a polynucleotide of the present invention operably linked to one or more control sequences, which direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
[00104] A polynucleotide can be manipulated in a variety of ways to provide expression of the polypeptide. The manipulation of the polynucleotide before insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.
The control sequence can be a promoter, a polynucleotide that is recognized by a host cell for the expression of a polynucleotide that encodes a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate polypeptide expression. The promoter can be any polynucleotide that shows transcriptional activity in the host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are both homologous and heterologous to the host cell.
[00106] Examples of promoters suitable for directing the transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levanosaccharase gene (sacB), Bacillus subtilis xylA and xylB gene, Bacillus thylase gene and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene ( dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2125). Additional promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American 242: 7494; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
[00107] Examples of promoters suitable for directing the transcription of the nucleic acid constructs of the present invention, in a host cell of filamentous fungus, are the promoters obtained from the genes for Aspergillus nidulans acetamidase, neutral alpha-amylase from Aspergillus niger, alfa - stable acidic amylase from Aspergillus niger, glucoamylase from Aspergillus niger or Aspergillus awamori (glaA), TAKA amylase from Aspergillus oryzae, alkaline protease from Aspergillus oryzae, triose phosphate isomerase from Aspergillus oryasee protease type 77, protease type 7 ), Fusarium venenatum amyloglycosidase (WO 00/56900), Fusarium venenatum daria (WO 00/56900), Fusarium venenatum quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichode rma reesei, endoglucanase III of Trichoderma reesei, endoglucanase IV of Trichoderma reesei, endoglucanase V of Trichoderma reesei, xylanase I of Trichoderma reesei, xylanase II of Trichoderma reesei, beta-xylidasidase of Trichoderma reesei, as well as the promoter NA-promoter2 modified from a neutral Aspergillus alpha-amylase gene, in which the main untranslated part has been replaced by an untranslated main part, from an Aspergillus phosphate isomerase triose gene; non-limiting examples include promoters modified from an Aspergillus niger neutral alpha-amylase gene, in which the untranslated main part has been replaced by an untranslated main part of an Aspergillus nidulans or Aspergillus oryzae triosis phosphate isomerase gene) ; and mutant, truncated and hybrid promoters thereof.
[00108] In a host yeast, the promoters used are obtained from the genes for Saccharomyces cerevisiae (ENO-1) enolase, Saccharomyces cerevisiae galactokinase (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase, Saccharomyces cerevisiae (AD2H2, AD2H2) / GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1) and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters used for yeast as host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
[00109] The control sequence can also be a transcription terminator, which is recognized by a host cell to terminate transcription. The finisher is operably linked at the 3 'end of the polynucleotide encoding the polypeptide. Any finisher that is functional in the host cell can be used in the present invention.
The preferred finalizers for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL) and Escherichia coli ribosomal RNA (rrnB).
The preferred finalizers for filamentous fungus host cells are obtained from Aspergillus nidulans anthranilate synthase genes, Aspergillus niger glucoamylase, Aspergillus niger alpha-glycosidase, Aspergillus oryzae TAKA amylase and Fusarium oxysum trypsin protease.
The preferred finalizers for yeast as host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other finishers used for yeast as host cells are described by Romanos et al., 1992, supra.
[00113] The control sequence can also be a stabilizing region of mRNA downstream of a promoter and upstream of the coding sequence of a gene that increases the expression of the gene.
[00114] Examples of suitable mRNA stabilizing regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal de Bacteriology 177: 3465- 3471).
[00115] The control sequence can also be a major part, a region of an untranslated mRNA, which is important for translation by the host cell. The main part is operably linked to the 5 'end of the polynucleotide that encodes the polypeptide. Any major part that is functional in the host cell can be used.
[00116] The main preferred parts for the host cells of filamentous fungi are obtained from the genes for TAKA amylase from Aspergillus oryzae and triose phosphate isomerase from Aspergillus nidulans.
[00117] The main parts suitable for yeast as host cells are obtained from the genes for Saccharomyces cerevisiae (ENO-1) enolase, Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor and alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase Saccharomyces cerevisiae (ADH2 / GAP).
The control sequence can also be a polyadenylation sequence, a sequence operably linked to the 3 'end of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.
[00119] The preferred polyadenylation sequences for the host cells of filamentous fungi are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glycoamylase, Aspergillus niger alpha-glycosidase, TAKA amylase of the aspergillus oryzae protease and trypsin protease of Fusarium oxysporum.
[00120] The polyadenylation sequences used for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
[00121] The control sequence can also be a region that encodes a signal peptide, that encodes a signal peptide attached to the N-terminus of a polypeptide, and directs the polypeptide in the cell's secretion pathway. The 5 'end of the polynucleotide coding sequence can intrinsically contain a coding signal peptide sequence naturally linked in the open reading frame, with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5 'end of the coding sequence can contain a coding signal peptide sequence that is foreign to the coding sequence. A foreign coding signal peptide sequence may be required where the coding sequence does not naturally contain a coding signal peptide sequence. Alternatively, a foreign signal peptide coding sequence can simply replace the natural signal peptide coding sequence in order to improve secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell can be used.
[00122] The efficient peptide signal coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for NCIB 11837 Bacillus maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus licheniformis alpha-amylase and Bacillus stilus amylase , neutral proteases from Bacillus stearothermophilus (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[00123] Efficient coding signal peptide sequences for filamentous fungus host cells are the signal encoding peptide sequences obtained from the genes for neutral amylase from Aspergillus niger, Aspergillus niger glucoamylase, TAKA amylase from Aspergillus oryzae, Humicucola insolens cellulase, endicoglucan insolens, endogenous V for Humicola insolens, Humicola lanuginosa lipase and Rhizomucor miehei aspartic proteinase.
[00124] The signal peptides used for yeast as host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other sequences of signal coding peptides used are described by Romanos et al., 1992, supra.
The control sequence can also be a coding propeptide sequence that encodes a pro-peptide positioned at the N-terminus of a polypeptide. The resulting polypeptide is known as a pro-enzyme or pro-polypeptide (or a zymogen in some cases). A pro- polypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the pro-peptide from the pro- polypeptide. The coding pro-peptide sequence can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase and alpha factor Saccharomyces cerevisiae.
[00126] Where both the signal peptide and the sequence of propeptides are present, the pro-peptide sequence is positioned close to the N-terminus of a polypeptide, and the signal peptide sequence is positioned close to the N-terminus of the pro-sequence -peptide.
[00127] It may also be desirable to add regulatory sequences that regulate polypeptide expression with respect to host cell growth. Examples of regulatory systems are those that cause gene expression to turn on and off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include lac, tac and trp operator systems. In yeast, the ADH2 system or GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter and Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide can be operably linked with the regulatory sequence. Expression Vectors
[00128] The present invention also relates to recombinant expression vectors that comprise a polynucleotide of the present invention linked to one or more control sequences, for example, a promoter and transcriptional and translational stop signals, which direct the production of the polypeptide in a expression host. The various nucleotides and control sequences can be combined to produce a recombinant expression vector that can include one or more convenient restriction sites to allow insertion or replacement of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide, or a nucleic acid construct that comprises the polynucleotide, into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector in such a way that the coding sequence is operably linked with the appropriate control sequences for expression.
[00129] The recombinant expression vector can be any vector (for example, a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the polynucleotide. The choice of the vector will typically depend on the vector's compatibility with the host cell into which the vector will be introduced. The vector can be a closed linear or circular plasmid.
[00130] The vector can be a vector that replicates autonomously, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or a artificial chromosome. The vector can contain any means to guarantee self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicated along with the chromosome (s) into which it has been integrated. In addition, a single vector, or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell's genome, or a transposon, can be used.
[00131] The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a product of the gene that provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophic and the like.
[00132] Examples of selectable bacterial markers are the genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Suitable markers for use in a filamentous fungus host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenyltransferase sulfate), and trpC (anthranilate synthase), as well as their equivalents. The preferred genes for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and a bar gene of Streptomyces hygroscopicus.
[00133] The vector preferably contains an element (s) that allows integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome.
[00134] For integration into the host cell genome, the vector may depend on the polynucleotide sequence encoding the polypeptide, or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration by homologous recombination into the host cell genome at an exact location (s) on the chromosome (s). To increase the likelihood of integration in a precise location, the integrational elements can contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to improve the likelihood of homologous recombination. The integrational elements can be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the host cell genome by non-homologous recombination.
[00135] For autonomous replication, the vector may additionally comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any replicator plasmid that mediates autonomous replication that functions in a cell. The term "origin of replication" or "replicator plasmid" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
[00136] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUB110, pE194, pTA1060, and pAMβl that allow replication in Bacillus.
[00137] Examples of origins of replication for use in a yeast as a host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
[00138] Examples of origins of replication used in a filamentary cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and the construction of plasmids or vectors comprising the gene can be carried out according to the methods disclosed in WO 00/24883.
[00139] More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase production of a polypeptide. An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including a selectable marker gene amplifiable with the polynucleotide, where cells containing amplified copies of the selectable marker gene and, through In addition, additional copies of the polynucleotide can be selected by culturing the cells in the presence of the appropriate selectable agent.
[00140] The procedures used to link the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra). Host cells
The present invention also relates to recombinant host cells, which comprise a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell in such a way that the construct or vector is maintained as a chromosomal integral or as an extra-chromosomal self-replicating vector in the manner described above. The term "host cell" includes any progeny of a mother cell that is not identical to the mother cell because of the mutations that occur during replication. The choice of a host cell will depend heavily on the gene that encodes the polypeptide and its source.
The host cell can be any cell used in the recombinant production of a polypeptide of the present invention, for example, a prokaryote or a eukaryote.
[00143] The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
[00144] The bacterial host cell can be any Bacillus cell including, but not limited to, Bacillus alkalophilus cells, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lentus Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.
The bacterial host cell can also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis cells, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus.
The bacterial host cell can also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes cells, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans.
[00147] The introduction of DNA into a Bacillus cell can be carried out by protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see , for example, Young and Spizizen, 1961, J. Bacteriol. 81: 823829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, for example, Shigekawa and Dower , 1988, Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell can be carried out by transformation of protoplasts (see, for example, Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell can be carried out by protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, for example , Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell can be performed by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell can be performed by natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), transformation of protoplasts (see, for example, Catt and Jollick , 1991, Microbios 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
[00148] The host cell can also be a eukaryote, such as a mammalian cell, insect, plant or fungus.
[00149] The host cell can be a fungus cell. “Fungi” as used herein includes the phylum Ascomycota, Basidiomycota, Chitridiomycota and Zygomycota, as well as the Oomicota and all mitosporic fungi (as defined by Hawksworth et al., In Ainsworth and Bisby's Dictionary of the Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
[00150] The fungal host cell can be a yeast cell. “Yeast”, as used here, includes ascosporogenic yeasts (Endomycetales), basidiosporogenic yeasts and yeasts that belong to imperfect fungi (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeasts can be defined in the manner described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, publishers, Soc. App. Bacteriol. Symposium number of series 9, 1980).
[00151] Yeast as a host cell can be a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as a cell of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyyces, Saccharomyy , Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
[00152] The fungal host cell can be a filamentous fungus cell. “Filamentous fungi” include all filamentous forms in the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glycan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by stretching hyphae and carbon catabolism is mandatory aerobic. On the contrary, the vegetative growth of yeasts, such as Saccharomyces cerevisiae, is by budding of a single-celled stem and the carbon catabolism can be fermentative.
[00153] The host cells of filamentous fungus can be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Neocor, Mycelio, Mycelio, Mycelio Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.
[00154] For example, the host cells of the filamentous fungus may be a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Ceriororisisis, Cerjorisisiporis, Ceriporiopsis pannocinta, rivulose Ceriporiopsis, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, lucknowense Chrysosporium, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum cinereus Coprinus, Coriolus hirsutus, Fusarium bactridioides, cerealis Fusarium, Fusarium crookwellense , Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusa rium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotielusury, terrestrial, terrarium, terrarium , Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
[00155] Fungal cells can be transformed by a process that involves the formation of protoplasts, transformation of protoplasts and regeneration of the cell wall in a manner known by itself. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio / Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, JN and Simon, MI, editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc. , New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920. Production Methods
The present invention also relates to methods of producing a polypeptide of the present invention comprising: (a) cultivating a cell, which in its wild type produces the polypeptide, under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is a Talaromyces cell. In a more preferred aspect, the cell is a Talaromyces leycettanus cell. In a most preferred aspect, the cell is Talaromyces leycettanus strain CBS398.68.
The present invention also relates to methods of producing a polypeptide of the present invention, which comprises (a) culturing a recombinant host cell of the present invention under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[00158] Host cells are cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, the cell can be grown by shaking flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed batch, or solid fermentation) in laboratory or industrial fermenters, in a medium and suitable conditions that allow the polypeptide to be expressed and / or isolated. Cultivation takes place in a suitable nutrient medium that comprises sources of carbon and nitrogen and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers, or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
[00159] The polypeptide can be detected using methods known in the art that are specific to the polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the polypeptide.
[00160] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.
[00161] The polypeptide can be purified by a variety of procedures known in the art including, but not limited to, chromatography (for example, ion exchange, affinity, hydrophobic, isoelectric focusing and size exclusion), electrophoretic procedures ( for example, preparative isoelectric focusing), differential solubility (for example, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
[00162] In an alternative aspect, the polypeptide is not recovered, but certainly a host cell of the present invention that expresses the polypeptide is used as a source of the polypeptide. Plants
[00163] The present invention also relates to isolated plants, for example, a transgenic plant, part of the plant or plant cell, which comprises a polynucleotide of the present invention in order to express and produce a polypeptide or domain in recoverable amounts. The polypeptide or domain can be recovered from the plant or part of the plant. Alternatively, the plant or part of the plant containing the polypeptide or domain can be used as such to improve the quality of a food or feed, for example, to improve nutritional value, palatability and rheological properties, or to destroy an anti-nutritive factor.
[00164] The transgenic plant can be dicotyledonous (a dicotyledonous) or monocotyledonous (a monocotyledonous). Examples of monocotyledonous plants are grasses, such as meadow grass (blue grass, Poa), forage grasses such as Fescue, Lolium, temperate grasses such as Agrostis, and cereals, for example, wheat, oats, rye, barley, rice, sorghum and corn (corn grain).
[00165] Examples of dicotyledonous plants are tobacco, vegetables such as lupins, potatoes, beets, peas, beans and soybeans, and cruciferous plants (Brassicaceae family), such as cauliflower, canola and the closely related model organism Arabidopsis thaliana.
[00166] Examples of plant parts are stem, callus, leaves, root, fruit, seeds and tubers, as well as the individual tissues that comprise these parts, for example, epidermis, mesophile, parenchyma, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a part of the plant. Furthermore, any plant cell, regardless of the origin of the tissue, is considered to be a part of the plant. Likewise, parts of the plant such as specific tissues and isolated cells to facilitate the use of the invention are also considered parts of the plant, for example, embryos, endosperm, aleurone and seed coatings.
[00167] Also included in the scope of the present invention are the progeny of such plants, parts of the plant and plant cells.
[00168] The transgenic plant or plant cell that expresses the polypeptide or domain can be constructed according to methods known in the art. In summary, the plant or plant cell is constructed by incorporating one or more expression constructs that encode the polypeptide or domain in the genome of the host plant or chloroplast genome, and which propagate the resulting modified plant or plant cell in a transgenic plant or plant cell.
The expression construct is conveniently a nucleic acid construct, which comprises a polynucleotide that encodes the polypeptide or domain operably linked to appropriate regulatory sequences required for the expression of the polynucleotide in the plant or part of the plant of choice. Furthermore, the expression construct can comprise a selectable marker used to identify plant cells, into which the expression construct has been integrated and the DNA sequences necessary for the introduction of the construct into the plant in question (the latter depends on the method of introduction of DNA to be used).
[00170] The choice of regulatory sequences, such as promoter and terminator sequences and, optionally, signal or transit sequences, is determined, for example, based on where, when and how to express the polypeptide or domain. For example, the expression of the gene encoding a polypeptide or domain can be constitutive or inducible, or it can be developmental, stage or tissue specific, and the gene product can be targeted to a specific tissue or part of the plant such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
[00171] For constitutive expression, 35S-CaMV, corn ubiquitin 1 or rice actin 1 promoter can be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992 , Plant Mol. Biol. 18: 675689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters can be, for example, a promoter of storage tissues such as seeds, potato tubers and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or of tissue from metabolic storage such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a specific seed promoter such as the promoter of glutelin, prolamine, globulin, or rice albumin (Wu et al. , 1998, Plant Cell Physiol. 39: 885889), a Vicia faba promoter from legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711) , a promoter of a seed oil body protein Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the Brassica napus napA storage protein promoter, or any other seed specific promoter known in the art, for example, in the manner described in WO 91/14772. Furthermore, the promoter may be a leaf-specific promoter such as the rbcs rice or tomato promoter (Kyozuka et al., 1993, Plant Physiol. 102: 991-1,000), the promoter of the chlorella virus adenine methyltransferase gene ( Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the promoter of the rice aldP gene (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or an inducible promoter of wound such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter can be induced by abiotic treatments, such as temperature, dryness, or changes in salinity, or induced by exogenously applied substances that activate the promoter, for example, ethanol, estrogen, plant hormones, such as ethylene, acid abscisic and gibberellic acid and heavy metals.
[00172] A promoter enhancing element can also be used to achieve greater expression of a polypeptide or domain in the plant. For example, the enhancer element of the promoter may be an intron that is placed between the promoter and the polynucleotide that encodes a polypeptide or domain. For example, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to improve expression.
[00173] The selectable marker gene and any other parts of the expression construct can be chosen from those available in the art.
[00174] The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biological transformation and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio / Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
[00175] Agrobacterium tumefaciens-mediated gene transfer is a method for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transforming monocots, although other methods of transformation can be used by these plants. One method for generating transgenic monocots is to bombard particles (microscopic gold or tungsten particles coated with transforming DNA) from embryonic calluses or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr Opin Biotechnol 5: 158-162; Vasil et al., 1992, Bio / Technology 10: 667-674). An alternative method for the transformation of monocots is based on the transformation of protoplasts, in the manner described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformation methods include those described in U.S. patents 6,395,966 and 7,151,204 (both of which are incorporated herein by reference in their entirety).
[00176] After transformation, transformants with the incorporated expression construct are selected and regenerated in complete plants according to methods well known in the art. In general, the transformation procedure is designed for the selective elimination of selection genes both during regeneration and after generations using, for example, cotransformation with two separate DNA-T constructs or excision of the specific selection gene by a recombinase specific.
[00177] In addition to directing the transformation of a particular plant genotype with a construct of the present invention, transgenic plants can be prepared by crossing a plant with the construct on a second plant that needs the construct. For example, a construct that encodes a polypeptide or domain can be introduced into a particular plant variety by crossing, without the need to directly transform a plant of that given variety. Therefore, the present invention includes not only a plant regenerated directly from the cells that have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny can refer to the offspring of any generation of a mother plant prepared in accordance with the present invention. Such a progeny can include a DNA construct prepared in accordance with the present invention. Intersections result in the introduction of a transgene into a plant strain cross-pollinating an initial strain with a donor plant strain. Non-limiting examples of such steps are described in U.S. Patent 7,151,204.
[00178] Plants can be generated through a backcross conversion process. For example, plants include plants referred to as a genotype, lineage, innate, hybrid converted by backcross.
[00179] Genetic markers can be used to assist in the introgression of one or more transgenes of the invention from one genetic background in another. Marker-assisted selection offers advantages over conventional reproduction, in which it can be used to avoid errors caused by phenotypic variations. In addition, genetic markers can provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait, which otherwise has an agronomically undesirable genetic background, is crossed with an elite mother, genetic markers can be used to select the progeny that have not only the trait of interest, but they also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits in a particular genetic background is minimized.
The present invention also relates to methods of producing a polypeptide or domain of the present invention comprising: (a) cultivating a transgenic plant or a plant cell, comprising a polynucleotide encoding the polypeptide or domain, under conditions that lead to the production of the polypeptide or domain; and (b) recovering the polypeptide or domain.
[00181] Examples of preferred uses of the polypeptide compositions of the invention are provided below. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used can be determined based on methods known in the art.
[00182] The present invention is also concerned with the following processes for using polypeptides with cellobiohydrolase activity, or compositions thereof.
[00183] The present invention also relates to processes for degrading a cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide that exhibits cellobiohydrolase activity of the present invention. In one aspect, the processes further comprise recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of cellulosic material can be separated from insoluble cellulosic material using a method known in the art as such, for example, centrifugation, filtration or gravity environment.
[00184] The present invention also relates to processes for producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide that exhibits the cellobiohydrolase activity of the present invention; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[00185] The present invention also relates to the processes of fermenting a cellulosic material comprising: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide that exhibits cellobiohydrolase activity of the present invention. In one aspect, the fermentation of the cellulosic material synthesizes a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.
[00186] The processes of the present invention can be used to saccharize cellulosic material into fermentable sugars, and to convert fermentable sugars into many usable fermentation products, for example fuel, potable ethanol and / or platform chemicals (for example , acids, alcohols, ketones, gases and the like). The production of a desired fermentation product from cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification) and fermentation.
[00187] The processing of the cellulosic material according to the present invention can be carried out using conventional methods in the art. Furthermore, the processes of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
[00188] Hydrolysis (saccharification) and fermentation, separate or simultaneous include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and cofermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and cofermentation (SHCF); hybrid hydrolysis and cofermentation (HHCF) and direct microbial conversion (DMC), sometimes also called consolidated bioprocessing (CBP). SHF uses the separate process steps to first enzymatically hydrolyze the cellulosic material into fermentable sugars, for example, the glucose, cellobiose and pentose monomers, and then ferment the fermentable sugars in ethanol. In SSF, the enzymatic hydrolysis of the cellulosic material and the fermentation of sugars in ethanol are combined in one step (Philippidis, GP, 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, CE, ed., Taylor & Francis, Washington, DC, 179-212). SSCF involves cofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and teh environment: A strategic perspective on the US Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817827). HHF involves a separate hydrolysis step and, in addition, a simultaneous saccharification and hydrolysis step that can be carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, that is, enzymatic saccharification at an elevated temperature, followed by SSF at a lower temperature than the fermentation strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis and fermentation) in one or more (for example, several) steps, where the same organism is used to produce the enzymes for converting cellulosic material into fermentable sugars, and to convert the fermentable sugars in a final product (Lynd, LR, Weimer, PJ, van Zyl, WH, and Pretorius, IS, 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood here that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination of these, can be used to carry out the processes of the present invention.
[00189] A conventional apparatus may include a batch fed agitated reactor, a batch agitated reactor, a continuous flow agitated reactor with ultrafiltration, and / or a continuous piston flow column reactor (Fernanda de Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, AV, and Sinitsyn, AP, 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), a friction reactor (Ryu, SK, and Lee, J. M, 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive agitation induced by an electromagnetic field (Gusakov, AV, Sinitsyn, AP, Davydkin, IY, Davydkin, VY, Protas, OV, 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor wit h intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional types of reactors include: fluidized bed, upstream flow, immobilized and extruder reactors for hydrolysis and / or fermentation.
[00190] Pre-treatment. In the practice of the processes of the present invention, any pretreatment process known in the art can be used to break up cell wall components of plants of cellulosic material and / or containing xylan (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis enzymatic of lignocellulosics Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of cellulosic lignomaterial for efficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41 -65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment od lignocellulosic biomass, Bioresource Technol. 96: 673 -686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. de Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: The key to un locking low-cost cellulosic ethanol, Biofuels Bioproducts nd Biorefining-Biofpr. 2: 26-40).
[00191] Cellulosic material can also be subjected to particle size reduction, sieving, pre-soaking, humidification, washing and / or conditioning before pretreatment using methods known in the art.
[00192] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), diluted acid pretreatment, hot water pretreatment, alkaline pretreatment, pretreatment with lime, wet oxidation, wet explosion, explosion with ammonia fiber, pretreatment with organosolve and biological pretreatment. Additional pretreatments include pretreatments with percolation with ammonia, ultrasound, electroporation, microwaves, supercritical CO2, supercritical H2O, ozone, ionic liquid and gamma irradiation.
[00193] The cellulosic material can be pre-treated before hydrolysis and / or fermentation. Pre-treatment is preferably carried out before hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzymatic hydrolysis to release fermentable sugars, such as glucose, xylose and / or cellobiosis. In many cases, the pre-treatment step itself results in some conversion of biomass into fermentable sugars (even in the absence of enzymes).
[00194] Pre-treatment with steam. In the steam pretreatment, the cellulosic material is heated to break up the components of the cell walls of plants, including lignin, hemicellulose and cellulose, to make cellulose and other fractions, for example, hemicellulose, accessible to enzymes. The cellulosic material passes through a reaction vessel where the steam is injected to increase the temperature to the required temperature and pressure and is maintained there for the desired reaction time. Pre-treatment with steam is preferably carried out at 140-250 ° C, for example, 160-200 ° C or 170-190 ° C, where the ideal temperature range depends on the addition of a chemical catalyst. The residence time for pre-treatment with steam is preferably 1-60 minutes, for example, 1-30 minutes, 1-20 minutes, 3-12 minutes or 4-10 minutes, where the ideal residence time depends on the range temperature and the addition of a chemical catalyst. Pre-treatment with steam allows relatively high solid loads, in such a way that the cellulosic material is generally moist only during the pre-treatment. Steam pretreatment is often combined with an explosive discharge of the material after pretreatment, which is known as a steam explosion, that is, rapid burning at atmospheric pressure and turbulent flow of the material to increase the surface area accessible by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; US patent application 2002/0164730). During steam pretreatment, the acetyl hemicellulose groups are cleaved and the resulting acid self-catalyzes the partial hydrolysis of hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to a limited extent only.
[00195] Chemical pre-treatment: The term "chemical treatment" refers to any chemical pre-treatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin. Such a treatment can convert crystalline cellulose into amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, pretreatment with dilute acid, pretreatment with lime, wet oxidation, fiber explosion with ammonia / freezing (AFEX), percolation with ionic liquid ammonia (APR) and pretreatments with organosolve.
[00196] A catalyst such as H2SO4 or SO2 (typically 0.3 to 5% w / w) is often added prior to steam pretreatment which decreases time and temperature, increases recovery and improves enzymatic hydrolysis ( Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762). In the pre-treatment with diluted acid, the cellulosic material is mixed with the diluted acid, typically H2SO4, and water to form a slurry, heated by steam at the desired temperature, and after a residence time it is burned at atmospheric pressure. Pretreatment with diluted acid can be carried out with numerous reactor designs, for example, piston flow reactors, counter current reactors, or agitated bed reactors against direct current (Duff and Murray, 1996, supra; Schell et al. ., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[00197] Various pre-treatment methods in alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, percolation with ammonia (APR), and fiber burst with ammonia / freezing (AFEX).
[00198] Pre-treatment with lime is carried out with calcium oxide or calcium hydroxide at temperatures of 85-150 ° C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose the pretreatment methods using ammonia.
[00199] Wet oxidation is a thermal pretreatment typically carried out at 180-200 ° C for 5-15 minutes with the addition of an oxidizing agent, such as hydrogen peroxide or oxygen super pressure (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). Pre-treatment is preferably carried out on 1-40% dry material, for example, 2-30% dry material or 5-20% dry material, and often the initial pH is increased by the addition of alkali, such as carbonate sodium.
[00200] A modification of the pre-treatment method with wet oxidation, known as wet explosion (combination of wet oxidation and steam explosion), can handle dry material up to 30%. In the wet explosion, the oxidizing agent is introduced during the pre-treatment after a certain period of residence. The pre-treatment is then finished by burning at atmospheric pressure (WO 2006/032282).
[00201] The fiber explosion with ammonia (AFEX) involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures, such as 90-150 ° C, and high pressure such as 17-20 bar for 5-10 minutes, where the dry material content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). During pretreatment with AFEX, the cellulose and hemicellulose remain relatively intact. Lignin-carbohydrate complexes are cleaved.
[00202] Pretreatment with organosolve delignifies cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200 ° C for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulfuric acid is generally added as a catalyst. In pretreatment with organosolve, most of the hemicellulose and lignin is removed.
[00203] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. 105-108: 6985, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. published application 2002/0164730.
[00204] In one aspect, the chemical pretreatment is preferably carried out as a diluted acid treatment, and more preferably as a continuous diluted acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride or mixtures thereof. Weak acid treatment is conducted in the pH range of preferably 1-5, for example, 1-4 or 1-2.5. In one aspect, the acid concentration is in the range of preferably 0.01 to 10% by weight of acid, for example, 0.05 to 5% by weight of acid or 0.1 to 2% by weight of acid. The acid is brought into contact with the cellulosic material and maintained at a temperature in the range of preferably 140-200 ° C, for example, 165-190 ° C, for periods ranging from 1 to 60 minutes.
[00205] In another aspect, the pre-treatment takes place in an aqueous sludge. In preferred aspects, the cellulosic material is present during pre-treatment in amounts preferably between 10-80% by weight, for example, 20-70% by weight or 30-60% by weight, such as around 40% by weight. Weight. The pre-treated cellulosic material can be unwashed or washed using any method known in the art, for example, washed with water.
[00206] Mechanical pretreatment or physical pretreatment: The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes particle size reduction. For example, such pre-treatment can involve various types of crushing or grinding (for example, dry grinding, wet grinding, or vibrating ball grinding).
[00207] Cellulosic material can be pretreated both physically (mechanically) and chemically. The mechanical or physical pretreatment can be coupled with vapor / steam explosion, hydrothermolysis, treatment with dilute or weak acid, treatment with high temperature and high pressure, irradiation (for example, microwave irradiation) or combinations of these. In one aspect, high pressure means pressure in the range of preferably about 100 to about 2757.9 KPa, for example, about 1034.2 to about 1723.7 KPa. In another aspect, the elevated temperature means temperatures in the range of about 100 to about 300 ° C, for example, about 140 to about 200 ° C. In a preferred aspect, mechanical or physical pretreatment is carried out in a batch process using a steam gun hydrolyzer system that uses high pressure and high temperature in the manner defined above, for example, a Sunds hydrolyzer available from Sunds Defibrator AB , Sweden. Physical and chemical pretreatments can be carried out sequentially or simultaneously, if desired.
[00208] Thus, in a preferred aspect, the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination of these, to promote the separation and / or release of cellulose, hemicellulose and / or lignin.
[00209] Biological pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin from cellulosic material or containing xylan. Biological pretreatment techniques may involve applying microorganisms and / or enzymes that solubilize lignin (see, for example, Hsu, T.-A., 1996, Pretreatments of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, CE , ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic / microbial conversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, JD , 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, ME, Baker, JO, and Overend, RP, eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15 ; Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany , 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulo sic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from cellulosic lignomaterial: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
[00210] Saccharification. In the hydrolysis stage, also known as saccharification, the cellulosic or xylan-containing material, for example, pre-treated, is hydrolyzed to break down cellulose and / or hemicellulose into fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, soluble mannose, galactose and / or oligosaccharides. Hydrolysis is carried out enzymatically by an enzyme composition in the presence of a polypeptide with cellobiohydrolase activity of the present invention. The enzymes in the compositions can be added simultaneously or sequentially.
[00211] Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be easily determined by those skilled in the art. In one aspect, hydrolysis is carried out under conditions suitable for the activity of the enzyme (s), that is, ideal for the enzyme (s). Hydrolysis can be carried out as a batch or continuous batch process, where the cellulosic material is fed gradually, for example, by an enzyme containing the hydrolysis solution.
[00212] Saccharification is carried out in general in reactors or fermenters in agitated tanks under controlled conditions of pH, temperature and mixture. The suitable conditions of process time, temperature and pH can be easily determined by those skilled in the art. For example, saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 120 hours, for example, about 16 to about 72 hours or about 24 to about 48 hours. The temperature is preferably in the range of about 25 ° C to about 70 ° C, for example, about 30 ° C to about 65 ° C, about 40 ° C to about 60 ° C, or about 50 ° C to about 55 ° C. The pH is preferably in the range of about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is preferably in the range of about 5 to about 50% by weight, for example, about 10 to about 40% by weight or about 20 to about 30% by weight.
[00213] The present invention also relates to enzyme compositions that comprise a polypeptide of the present invention. Preferably, the compositions are enriched in a polypeptide like this. The term "enriched" indicates that the cellobiohydrolase activity of the composition has been increased, for example, with an enrichment factor of at least 1.1.
[00214] The composition may comprise a polypeptide of the present invention as the main enzyme component, for example, a monocomponent composition. Alternatively, the composition may comprise multiple enzyme activities, such as one or more (for example, several) enzymes selected from the group consisting of a cellulase, a hemicellulase, a GH61 polypeptide, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, peroxidase, protease and swolenin.
[00215] In a preferred embodiment, the enzyme composition comprises at least the cellobiohydrolase of the invention, at least one endoglucanase, at least one beta-glycosidase and at least one GH61 polypeptide with better cellulolytic activity.
[00216] Polypeptide compositions can be prepared according to methods known in the art, and can be in the form of a liquid or dry composition. For example, the polypeptide composition can be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition can be stabilized according to methods known in the art.
[00217] Enzyme compositions can comprise any protein used in the degradation of cellulosic material.
[00218] In one aspect, the enzyme composition comprises or additionally comprises one or more (for example, several) proteins selected from the group consisting of a cellulase, a GH61 polypeptide with better cellulolytic activity, a hemicellulase, an esterase, an expandin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. In another aspect, cellulase is preferably one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, an additional cellobiohydrolase and a beta-glucosidase. In another aspect, hemicellulase is preferably one or more (for example, several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase and a xylosidase.
[00219] In another aspect, the enzyme composition comprises one or more (for example, several) cellulolytic enzyme. In another aspect, the enzyme composition further comprises or comprises one or more (for example, several) hemicellulolytic enzyme. In another aspect, the enzyme composition comprises one or more (for example, several) cellulolytic enzyme and one or more (for example, several) hemicellulolytic enzyme. In another aspect, the enzyme composition comprises one or more (for example, several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises a beta-glycosidase. In another aspect, the enzyme composition comprises a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises a cellobiohydrolase and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises a beta-glycosidase and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glycosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a beta-glycosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises a cellobiohydrolase, a beta-glycosidase, and a polypeptide with better cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a beta-glycosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glycosidase, and a polypeptide with better cellulolytic activity.
[00220] In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylane esterase. In another aspect, the enzyme composition comprises an arabinanase (for example, alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (for example, alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises coumaric acid esterase. In another aspect, the enzyme composition comprises a feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (for example, alpha-galactosidase and / or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (for example, alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises mannanase. In another aspect, the enzyme composition comprises a mannosidase (for example, beta-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, xylanase is a family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., beta-xylosidase).
[00221] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expansin. In another aspect, the enzyme composition comprises a laccase. In another aspect, the enzyme composition comprises a lignolytic enzyme. In a preferred aspect, the lignolytic enzyme is a manganese peroxidase. In another preferred aspect, the lignolytic enzyme is a lignin peroxidase. In another preferred aspect, the lignolytic enzyme is an H2O2-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swolenin.
[00222] In the processes of the present invention, the enzyme (s) can be added before or during fermentation, for example, during saccharification, or during or after the propagation of the microorganism (s) fermenter (s).
[00223] One or more (for example, several) components of the enzyme composition can be wild type proteins, recombinant proteins, or a combination of wild type proteins and recombinant proteins. For example, one or more (for example, several) components can be natural proteins of a cell, which are used as a host cell to recombinantly express one or more (for example, several) other components of the enzyme composition. One or more (for example, several) components of the enzyme composition can be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition can be a combination of multi-component and single-component protein preparations.
[00224] The enzymes used in the processes of the present invention can be in any form suitable for use such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cell debris, a semipurified enzyme preparation or purified, or a host cell as a source of the enzymes. The enzyme composition can be a dry or granulated powder, a dust-free granulate, a liquid, a stabilized liquid, or a stabilized protected protein. Liquid enzyme preparations, for example, can be stabilized by adding stabilizers such as a sugar, sugar alcohol or other polyol, and / or lactic acid or other organic acid according to the established processes.
[00225] The ideal amounts of enzymes and polypeptides with cellobiohydrolase activity depend on several factors including, but not limited to, the cellulolytic enzyme component mix, the cellulosic material, the concentration of cellulosic material, the pre- treatment (s) of cellulosic material, temperature, time, pH, and inclusion of fermenting organism (for example, yeast for saccharification and simultaneous fermentation).
[00226] In one aspect, an efficient amount of cellulolytic or hemicellulolytic enzyme for cellulosic material is about 0.5 to about 50 mg, for example, about 0.5 to about 40 mg, about 0.5 about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg per gram of cellulosic material.
[00227] In another aspect, an efficient amount of a polypeptide with cellobiohydrolase activity for the cellulosic material is about 0.01 to about 50.0 mg, for example, about 0.01 to about 40 mg, about from 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1 , 5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1 , 25 mg, or about 0.25 to about 1.0 mg per gram of cellulosic material.
[00228] In another aspect, an efficient amount of a polypeptide with cellobiohydrolase activity for the cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g, for example, about 0.01 to about 1.0 g , about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0, 25 g, or about 0.05 to about 0.2 g per gram of cellulolytic or hemicellulolytic enzyme.
[00229] Polypeptides with cellulolytic enzyme activity or hemicellulolytic enzyme activity, as well as other proteins / polypeptides used in the degradation of cellulosic material, for example, GH61 polypeptides with better cellulolytic activity (collectively "polypeptides with enzymatic activity") , can be derived from or obtained from any suitable source, including bacterial, fungal, yeast, plant or mammal origin. The term "obtained" also means here that the enzyme may have been produced recombinantly in a host organism, employing methods described herein, in which the enzyme produced recombinantly is either natural or foreign to the host organism, or has a modified sequence of amino acids, for example. example, with one or more (for example, several) amino acids that are deleted, inserted and / or substituted, i.e., a recombinantly produced enzyme that is a mutant and / or a fragment of a natural amino acid sequence, or an enzyme produced by nucleic acid scrambling processes known in the art. Natural variants are included in the meaning of a natural enzyme, and in the meaning of a foreign enzyme are the variants obtained recombinantly, such as by site-directed mutagenesis or shuffling.
[00230] Chemically modified or protein engineered mutants can also be used.
[00231] One or more (several) components of the enzyme composition can be a recombinant component, that is, produced by cloning a DNA sequence that encodes the single component and the subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host is preferably a heterologous host (the enzyme is foreign to the host), but the host can also, under certain conditions, be a homologous host (the enzyme is natural to the host). Mono-component cellulolytic proteins can also be prepared by purifying a protein like this from a fermentation broth.
[00232] In one aspect, the one or more (for example, several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC ™ CTec (Novozymes A / S), CELLIC ™ CTec2 (Novozymes A / S), CELLUCLAST ™ (Novozymes A / S), NOVOZYM ™ 188 (Novozymes A / S), CELLUZYME ™ (Novozymes A / S), CEREFLO ™ (Novozymes A / S), and ULTRAFLO ™ (Novozymes A / S), ACCELERASE ™ (Genencor Int.), LAMINEX ™ (Genencor Int. ), SPEZYME ™ CP (Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S / L 100 (DSM), ROHAMENT ™ 7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International, Inc .). Cellulase enzymes are added in efficient amounts of about 0.001 to about 5.0% by weight of solids, for example, about 0.025 to about 4.0% by weight of solids or about 0.005 to about 2, 0% by weight of solids.
[00233] Examples of bacterial endoglucanases that can be used in the processes of the present invention include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; US patent 5,275,944; WO 96 / 02551; US patent 5,536,655, WO 00/70031, WO 05/093050); endoglucanase III from Thermobifida fusca (WO 05/093050) and endoglucanase V from Thermobifida fusca (WO 05/093050).
[00234] Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase ( access to GENBANK ™ number M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63: 11-22), Trichoderma reesei Cel5A endoglucanase II (access to GENBANK ™ number M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563, access to GENBANK ™ number AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, access GENBANK ™ number Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanases (Sakamoto et al., 1995, Current Genetics 27: 435-439), endoglucanase de Erwinia carotovara (Saarilahti et al., 1990, Gene 90: 9-14), Fusar endoglucanase ium oxysporum (access to GENBANK ™ number L29381), endoglucanase from Humicola grisea var. thermoidea (access to GENBANK ™ number AB003107), endoglucanases of Melanocarpus albomyces (access to GENBANK ™ number MAL515703), endoglucanases of Neurospora crassa (access to GENBANK ™ number XM_324477), endoglucanase V of Humicolahys.6 basidiomycete CBS 495.95, endoglucanases of CBS 494.95, endoglucanases of Thielavia terrestris NRRL 8126 CEL6B, endoglucanases of Thielavia terrestris NRRL 8126 CEL6C, endoglucanases of Thielavia terrestris NRRL 8126 CEL7C endoglucanases from Cladorrhinum foecundissimum ATCC 62373 CEL7A and endoglucanase from Trichoderma reesei strain number VTT-D-80133 (access to GENBANK ™ number M15665).
Examples of other cellobiohydrolases used in the present invention include, but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO 2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II, Humobiohydrolase I, cellobiohydrolase II from Myceliophthora thermophila (WO 2009/042871), cellobiohydrolase II from Thielavia hyrcanie (WO 2010/141325), cellobiohydrolase II from Thielavia terrestris (CEL6A, WO 2006/074435), cellobiohydrolase I from Trichoderma reesei and celobioma reesei and celobioma reesei cellobiohydrolase II of Trichophaea saccata (WO 2010/057086).
[00236] Examples of beta-glycosidases used in the present invention include, but are not limited to, beta-glycosidases from Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO 2005/047499 ), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO 2011/035029) and Trichophaea saccata (WO 2007/019442).
[00237] Beta-glucosidase can be a fusion protein. In one aspect, beta-glucosidase is a BG variant fusion protein of Aspergillus oryzae beta-glucosidase (WO 2008/057637) or a Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).
[00238] Other endoglucanases, cellobiohydrolases and beta-glycosides used are disclosed in several families of glycosyl hydrolase, using the classification according to Henrissat, 1991, The classification of glycosil hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosil hydrolases, Biochem. J. 316: 695-696.
[00239] Other cellulolytic enzymes that can be used in the present invention are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255 , WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592 , WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793 , US patent 5,457,046, US patent 5,648,263 and US patent 5,686,593.
[00240] In the processes of the present invention, any GH61 polypeptide with better cellulolytic activity can be used.
[00241] Examples of GH61 polypeptides with better cellulolytic activity used in the processes of the present invention include, but are not limited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647, WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085868, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754), GH61 polypeptides from Penicillium pinophilum (WO 2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397) and Thermoascus crustaceous (WO 2011/041504).
[00242] In one aspect, the GH61 polypeptide with the best cellulolytic activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, for example, manganese sulfate.
[00243] In one aspect, the GH61 polypeptide with the best cellulolytic activity is used in the presence of a dioxy compound, a bicyclic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pre-treated cellulosic material, such as pre-treated corn residue (PCS).
[00244] The dioxy compound can include any suitable compound containing two or more oxygen atoms. In some respects, the dioxy compounds contain a substituted aryl fraction, as described herein. Dioxy compounds can comprise one or more (for example, several) hydroxyls and / or hydroxyl derivatives, but also include substituted aryl fractions that do not contain hydroxyl and hydroxyl derivatives. Non-limiting examples of dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; synapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butino-1,4-diol; (crochonic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethioxy-1,2-propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid and methyl-3,5-dimethoxy-4-hydroxybenzoate, or a salt or solvate of themselves.
[00245] The bicyclic compound can include any suitable substituted fused ring system, as described herein. The compounds may comprise one or more (for example, several) additional rings, and are not limited to a specific number of rings, unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally substituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavilium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivatives thereof. Non-limiting examples of bicyclic compounds include epicatechin; quercetin; myricetin; taxifoline; caempferol; morina; acacetin; naringenin; isoramnetine; apigenin; cyanidin; cyanine; curomanine; kerakyanine or a salt or solvate thereof.
The heterocyclic compound can be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, in the manner described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl fraction, or an optionally substituted heteroaryl fraction. In another aspect, the optionally substituted heterocycloalkyl fraction, or optionally substituted heteroaryl fraction, is an optionally substituted 5-element heterocycloalkyl or an optionally substituted 5-element heteroaryl fraction. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, pyridine, thiazolyl, pyridine, thiazolyl, pyridine, thiazolyl, pyridine, thiazolyl, pyridine, triazole, triazole, tianaftenil, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazylyl, dimethyl, pyridine, pyridine, pyridine piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyl fraction, or optionally substituted heteroaryl fraction, is an optionally substituted furanyl. Non-limiting examples of heterocyclic compounds include (1,2-dihydroxyethyl) -3,4-dihydroxyfuran-2 (5H) - one; 4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2 (5H) -furanone; [1,2-dihydroxyethyl] furan-2,3,4 (5H) -trione; a-hydroxy — Y — butyrolactone; Y-lactone ribonic; aldoexuronic acid Y — lactone; gluconic acid δ — lactone; 4 - hydroxycoumarin; dihydrobenzofuran; 5— (hydroxymethyl) furfural; furoin; 2 (5H) —furanone; 5.6 — dihydro — 2H — piran — 2 — one; and 5.6 — dihydro — 4 — hydroxy — 6 — methyl— 2H — pyran — 2 — one; or a salt or solvate thereof.
[00247] The nitrogen-containing compound can be any suitable compound with one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine or nitroxide fraction. Non-limiting examples of nitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin and maleamic acid; or a salt or solvate thereof.
[00248] The quinone compound can be any suitable compound that comprises a fraction of quinone in the manner described herein. Non-limiting examples of quinone compounds include 1,4-benzoquinone; 1,4- naphthoquinone; 2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q0; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; 1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; quinone pyrroloquinoline; or a salt or solvate thereof.
The sulfur-containing compound can be any suitable compound comprising one or more sulfur atoms. In one aspect, the sulfur-containing compound comprises a selected fraction of thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.
[00250] In one aspect, an efficient amount of a compound like this described above for cellulosic material as a molar ratio in cellulose glucosyl units is about 10-6 to about 10, for example, about 10-6 about 7.5, about 10-6 to about 5, about 10-6 to about 2.5, about 10-6 to about 1, about 10-5 to about 1, about 10 -5 to about 10-1, about 10-4 to about 10-1, about 10-3 to about 10-1, or about 10-3 to about 10-2. In another aspect, an efficient amount of a compound like the one described above is about 0.1 μM to about 1 M, for example, about 0.5 μM to about 0.75 M, about 0.75 μM at about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM to about 1 mM.
[00251] The term "liquor" means the phase of the solution, both aqueous, organic, and a combination thereof, which arises from the treatment of a material with lignocellulose and / or hemicellulose in a sludge, or monosaccharides of these, for example, xylose, arabinose, mannose, etc., in conditions as described herein, and their soluble contents. A liquor for cellulolytic improvement of a GH61 polypeptide can be produced by treating a material with lignocellulose or hemicellulose (or raw material), applying heat and / or pressure, optionally in the presence of a catalyst, for example, acid, optionally in the presence of a solvent organic, and optionally in combination with physical interruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic improvement, obtained by combining liquor and a GH61 polypeptide, during the hydrolysis of a cellulosic substrate by a cellulase preparation. The liquor can be separated from the treated material using a standard method in the art, such as filtration, sedimentation or centrifugation.
[00252] In one aspect, an efficient amount of the cellulose liquor is about 10-6 to about 10 g per g of cellulose, for example, about 10-6 to about 7.5 g, about 10- 6 to about 5, about 10-6 to about 2.5 g, about 10-6 to about 1 g, about 10-5 to about 1 g, about 10-5 to about 10-1 g, about 10-4 to about 10-1 g, about 10-3 to about 10-1 g, or about 10-3 to about 10-2 g per g of cellulose.
[00253] In one aspect, the one or more (for example, several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME ™ (Novozymes A / S), CELLIC ™ HTec (Novozymes A / S), CELLIC ™ HTec2 (Novozymes A / S), VISCOZYME® (Novozymes A / S), ULTRAFLO® (Novozymes A / S), PULPZYME® HC (Novozymes A / S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX -200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL ™ 333P (Biocatalysts Limit, Wales, United Kingdom), DEPOL ™ 740L. (Biocatalysts Limit, Wales, United Kingdom), and DEPOL ™ 762P (Biocatalysts Limit, Wales, United Kingdom).
[00254] Examples of xylanases used in the processes of the present invention include, but are not limited to, Aspergillus aculeatus xylanases (GeneSeqP: AAR63790; WO 94/21785), Aspergillus fumigatus (WO 2006/078256), Penicillium pinophilum (WO 2011 / 041405), Penicillium sp. (WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210) and Trichophaea saccata GH10 (WO 2011/057083).
[00255] Examples of beta-xylosidases used in the processes of the present invention include, but are not limited to, beta-xylosidases from Neurospora crassa (SwissProt access number Q7SOW4), Trichoderma reesei (access number to UniProtKB / TrEMBL Q92458) , and Talaromyces emersonii (SwissProt Q8X212 accession number).
[00256] Examples of acetylxylan esterases used in the processes of the present invention include, but are not limited to, acetylxylan esterases of Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile (accession number GeneSeqP AAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (UniProt access number q7s259), Phaeosphaeria node (one access number) Q0UHJ1) and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[00257] Examples of feruloyl esterases (ferulic acid esterases) used in the processes of the present invention include, but are not limited to, feruloyl esterases of Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt access number A1D9T4) , Neurospora crassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729) and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).
[00258] Examples of arabinofuranosidases used in the processes of the present invention include, but are not limited to, Aspergillus niger arabinofuranosidases (GeneSeqP accession number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383) and M giganteus (WO 2006/114094).
[00259] Examples of alpha-glucuronidases used in the processes of the present invention include, but are not limited to, alpha-glucuronidases of Aspergillus clavatus (UniProt access number alcc12), Aspergillus fumigatus (SwissProt access number Q4WW45), Aspergillus niger ( Uniprot access number Q96WX9), Aspergillus terreus (SwissProt access number Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii (UniProt access number Q8X211) and Trichoderma number access Uniprot Q99024).
[00260] Polypeptides with enzymatic activity, used in the processes of the present invention, can be produced by fermenting the microbial strains mentioned above in a nutrient medium containing suitable sources of carbon and nitrogen and inorganic salts, using procedures known in the art (see, for example, example, Bennett, JW and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers, or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). Temperature ranges and other conditions suitable for enzyme growth and production are known in the art (see, for example, Bailey, J.E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).
[00261] Fermentation can be any method of culturing a cell that results in the expression or isolation of an enzyme or protein. Therefore, fermentation can be understood as one that comprises shaking bottle cultivation, or small or large scale fermentation (including continuous, batch, fed batch or solid fermentation) in laboratory or industrial fermenters, carried out in a appropriate medium and under conditions that allow the enzyme to be expressed or isolated. The resulting enzymes produced by the methods described above can be recovered from the fermentation medium and purified by conventional procedures.
[00262] Fermentation. The fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (for example, several) fermenting microorganisms capable of fermenting the sugars directly or indirectly in a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process that comprises a fermentation step. Fermentation processes also include fermentation processes used in the alcohol consumption industry (for example, beer and wine), the bakery industry (for example, fermented bakery products), the leather industry and the tobacco industry. The fermentation conditions depend on the desired fermentation product and the fermenting organism, and can be easily determined by those skilled in the art.
[00263] In the fermentation step, the sugars, released from the cellulosic material as a result of the pre-treatment and enzymatic hydrolysis steps, are fermented in a product, for example, ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separated or simultaneous, as described here.
[00264] Any suitable hydrolyzed cellulosic material can be used in the fermentation step in the practice of the present invention. The material is in general selected on the basis of the desired fermentation product, that is, the substance to be obtained from the fermentation, and in the process employed, in a manner well known in the art.
[00265] It is understood here that the term "fermentation medium" refers to a medium before the fermenting microorganism (s) is (are) added, such as a medium that results from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
[00266] "Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and / or pentose fermenting organisms, or a combination of these. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of fermenting, that is, converting sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose and / or oligosaccharides, directly or indirectly into the desired fermentation product.
[00267] Examples of bacterial and fungal fermenting organisms that produce ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[00268] Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeasts include strains of Candida, Kluyveromyces and Saccharomyces, for example, Candida sonorensis, Kluyveromyces marxianus and Saccharomyces cerevisiae.
[00269] Examples of fermenting organisms that can ferment pentose sugars in their natural state include bacterial and fungal organisms, such as some yeasts. Preferred xylose fermenting yeasts include strains of Candida, preferably C. sheatae or C. sonorensis; and Pichia strains, preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeasts include Pachysolen strains, preferably P. tannophilus. Organisms that are not capable of fermenting pentose sugars, such as xylose and arabinose, can be genetically modified to accomplish this by methods known in the art.
[00270] Examples of bacteria that can efficiently ferment hexose and pentose in ethanol include, for example, Bacillus coagulans, Clostridium acetobutilicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum and Zymomonas mobilis (Philippidis, supra).
[00271] Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis and C. scehatae; Clostridium, such as C. acetobutilicum, C. thermocellum and C. phytofermentans; E. coli, especially strains of E. coli that have been genetically modified to improve ethanol yield; Geobacillus sp .; Hansenula, such as Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum and Zymomonas, such as Zymomonas mobilis.
[00272] In a preferred aspect, yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In a preferred aspect, the yeast is Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.
[00273] In a preferred aspect, the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutilicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacilus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.
[00274] Yeasts commercially available and suitable for ethanol production include, for example, BIOFERM ™ AFT and XR (NABC - North American Bioproducts Corporation, GA, United States), yeast ETANOL RED ™ (Fermentis / Lesaffre, United States) , FALI ™ (Fleischmann's Yeast, United States), FERMIOL ™ (DSM Specialties), GERT STRAND ™ (Gert Strand AB, Sweden), and SUPERSTART ™ and THERMOSACC ™ fresh yeast (Ethanol Technology, WI, United States).
[00275] In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as microorganisms that use xylose, which use arabinose, and which coutilize xylose and arabinose.
[00276] The cloning of heterologous genes in various fermenting microorganisms led to the construction of organisms capable of converting hexoses and pentoses into ethanol (cofermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993 , Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase and transaldolase and applaldases. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle , FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).
[00277] In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.
[00278] It is well known in the art that the organisms described above can also be used to produce other substances, in the manner described herein.
[00279] The fermenting microorganism is typically added to the degraded or hydrolyzed cellulosic material, and fermentation is carried out for about 8 to about 96 hours, for example, about 24 to about 60 hours. The temperature is typically between about 26 ° C to about 60 ° C, for example, about 32 ° C or 50 ° C, and about pH 3 to about pH 8, for example, pH 4-5, 6 , or 7.
[00280] In one aspect, yeast and / or another microorganism are applied to the degraded cellulosic material, and fermentation is carried out for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably between about 20 ° C to about 60 ° C, for example, about 25 ° C to about 50 ° C, about 32 ° C to about 50 ° C, or about from 32 ° C to about 50 ° C, and the pH is generally about pH 3 to about pH 7, for example, about pH 4 to about pH 7. However, some fermenting organisms, for example, bacteria , have a higher ideal fermentation temperature. Yeast or other microorganism is preferably applied in amounts of approximately 105 to 1012, preferably in approximately 107 to 1010, essentially approximately 2 x 108 viable cell counts per ml of fermentation broth. Additional guidance regarding the use of yeast for fermentation can be found in, for example, “The Alcohol Textbook” (Editors K. Jacques, TP Lyons and DR Kelsall, Nottingham University Press, UK 1999), which is incorporated by reference.
[00281] Regarding the production of ethanol, after fermentation, the fermented sludge is distilled to extract the ethanol. The ethanol obtained according to the processes of the invention can be used, for example, as fuel ethanol, ethanol for beverages, i.e., potable neutral spirits, or industrial ethanol.
[00282] A fermentation stimulator can be used in combination with any of the processes described here to further improve the fermentation process and, in particular, the performance of the fermenting microorganism, such as improving the ethanol rate and yield. A "fermentation stimulator" refers to stimulators for the growth of fermenting microorganisms, in particular, yeasts. Preferable fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin and vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients that comprise P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.
[00283] Fermentation products: A fermentation product can be any substance derived from fermentation. The fermentation product can be, without limitation, an alcohol (for example, arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [ropylene glycol], butanediol, glycerin, sorbitol and xylitol ); an alkane (for example, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (for example, cyclopentane, cyclohexane, cycloheptane and cyclooctane), an alkene (for example pentene, hexene, heptene and octene); an amino acid (for example, aspartic acid, glutamic acid, glycine, lysine, serine and threonine); a gas (for example, methane, hydrogen (H2), carbon dioxide (CO2) and carbon monoxide (CO)); isoprene; a ketone (for example, acetone); an organic acid (eg, acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diceto-D-gluconic acid, formic acid, fumaric acid, gluconic acid, gluconic acid, glucuronic acid, glutaric acid , 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid and xylonic acid) and polyketide. The fermentation product can also be protein as a high-value product.
[00284] In a preferred aspect, the fermentation product is an alcohol. It will be well understood that the term "alcohol" includes a substance that contains one or more hydroxyl fractions. In a more preferred aspect, the alcohol is n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is 1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira and Jonas, 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processs for fermentative production of xylitol - a sugar substitute, Process Biochemistry 30 (2): 117124; Ezeji et al., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.
[00285] In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or branched alkane. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is dean. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.
[00286] In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, cycloalkane is cyclopentane. In another more preferred aspect, cycloalkane is cyclohexane. In another more preferred aspect, cycloalkane is cycloheptane. In another more preferred aspect, cycloalkane is cycloctane.
[00287] In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.
[00288] In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, for example, Richard and Margaritis, 2004, Empirical modeling of batch fermentation kinetics for poly (glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.
[00289] In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H2. In another more preferred aspect, the gas is CO2. In another more preferred aspect, the gas is CO. See, for example, Kataoka et al., 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V.N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.
[00290] In another preferred aspect, the fermentation product is isoprene.
[00291] In another preferred aspect, the fermentation product is a ketone. It will be well understood that the term "ketone" includes a substance that contains one or more fractions of ketone. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
[00292] In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diceto-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen and Lee, 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435- 448.
[00293] In another preferred aspect, the fermentation product is polyketide.
[00294] Recovery. The fermentation product (s) can optionally be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation or extraction. For example, the alcohol is separated from the fermented cellulosic material and purified by conventional distillation methods. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used, for example, as ethanol fuel, ethanol for beverages, that is, neutral potable spirits or industrial ethanol. Sign peptide
[00295] The present invention also relates to an isolated polynucleotide that encodes a signal peptide, comprising or consisting of amino acids 1 to 18 of SEQ ID NO: 2. The polynucleotide may additionally comprise a gene that encodes a protein, which is operably linked to the signal peptide. The protein is preferably foreign to the signal peptide. In one aspect, the polynucleotide encoding the signal peptide has nucleotides 1 to 54 of SEQ ID NO: 1.
The present invention also relates to the nucleic acid constructs, expression vectors and recombinant host cells that comprise such polynucleotides.
[00297] The present invention also relates to methods of producing a protein that comprise: (a) culturing a recombinant host cell that comprises such a polynucleotide; and (b) recovering the protein.
[00298] The protein can be natural or heterologous to a host cell. The term "protein" does not mean here that it refers to a specific size of the encoded product and therefore includes peptides, oligopeptides and polypeptides. The term "protein" also includes two or more polypeptides combined to form the encoded product. Proteins also include hybrid polypeptides and fused polypeptides.
[00299] Preferably, the protein is a hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, for example, an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin, glycosyltransferase, glycosyltransferase, deglucose esterase, alpha-galactosidase, beta-galactosidase, glycoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, ribonuclease beta-xylosidase.
[00300] The gene can be obtained from any prokaryote, eukaryote or other source. List of preferred modalities
[00301] Modality 1. An isolated polypeptide that has cellobiohydrolase activity, selected from the group consisting of: (a) a polypeptide that has at least 81%, for example, at least 82%, at least 83%, at least 84 %, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under conditions of medium severity, or conditions of medium-high severity, or conditions of high severity, or conditions of very high severity in (i) the sequence encoding the mature SEQ polypeptide ID NO: 1, (ii) its cDNA sequence, or (iii) the full size complement of (i) or (ii); (c) a polypeptide encoded by a polynucleotide that has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; or the cDNA sequence thereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2, which comprises a substitution, deletion, and / or insertion at one or more positions; and (e) a fragment of the polypeptide of (a), (b), (c), or (d) that exhibits cellobiohydrolase activity.
[00302] Modality 2. The polypeptide of modality 1, which presents at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 87%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the mature polypeptide of SEQ ID NO: 2.
[00303] Modality 3. The polypeptide of modality 1 or 2, which is encoded by a polynucleotide that hybridizes under conditions of medium severity, or conditions of medium-high severity, or conditions of high severity, or conditions of very high severity in ( i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, (ii) its cDNA sequence, or (iii) the full length complement of (i) or (ii).
[00304] Mode 4. The polypeptide of any of the modalities 13, which is encoded by a polynucleotide that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or 100% sequence identity to the sequence encoding the mature polypeptide of SEQ ID NO: 1 or its cDNA sequence.
[00305] Mode 5. The polypeptide of any of the modalities 14, which comprises or consists of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2.
[00306] Modality 6. The polypeptide of modality 5, in which the mature polypeptide has amino acids 19 to 464 of SEQ ID NO: 2.
[00307] Mode 7. The polypeptide of any of the modalities 14, which is a variant of the mature polypeptide of SEQ ID NO: 2 which comprises a substitution, elimination, and / or insertion in one or more positions.
[00308] Modality 8. The polypeptide of modality 1, which is a fragment of SEQ ID NO: 2, in which the fragment exhibits cellobiohydrolase activity.
[00309] Modality 9. An isolated polypeptide comprising a catalytic domain selected from the group consisting of: (a) a catalytic domain that has at least 81% sequence identity with the catalytic domain of SEQ ID NO: 2; (b) a catalytic domain encoded by a polynucleotide that has at least 60% sequence identity with the sequence encoding the catalytic domain of SEQ ID NO: 1; (c) a variant of a catalytic domain that comprises a substitution, deletion, and / or insertion of one or more (several) amino acids from the catalytic domain of SEQ ID NO: 2; and (d) a fragment of a catalytic domain of (a), (b), or (c), which exhibits cellobiohydrolase activity.
[00310] Mode 10. The polypeptide of mode 9, which comprises or consists of the catalytic domain of SEQ ID NO: 2.
[00311] Mode 11. The polypeptide of mode 10, wherein the catalytic domain has amino acids 105 to 464 of SEQ ID NO: 2.
[00312] Modality 12. The polypeptide of any of the 9-11 modalities, which further comprises a cellulose binding domain.
[00313] Mode 13. The polypeptide of any of the modalities 1-12, which is encoded by the polynucleotide contained in the Talaromyces leycettanus strain CBS398.68.
[00314] Modality 14. An enzyme composition comprising the polypeptide of any of the modalities 1-13.
[00315] Mode 15. An isolated polynucleotide that encodes the polypeptide of any of the modalities 1-13.
[00316] Mode 16. A nucleic acid construct or expression vector comprising the polynucleotide of modality 15 operably linked to one or more control sequences, which direct the production of the polypeptide in an expression host.
[00317] Modality 17. A recombinant host cell comprising the polynucleotide of modality 15 operably linked to one or more control sequences, which direct the production of the polypeptide.
[00318] Mode 18. A method for producing the polypeptide of any of the modalities 1-13 comprising: (a) cultivating a cell, which in its wild type produces the polypeptide, under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[00319] Modality 19. A method for producing a polypeptide that exhibits cellobiohydrolase activity comprising: (a) cultivating the modality 17 host cell under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[00320] Mode 20. A transgenic plant, part of a plant or plant cell transformed with a polynucleotide that encodes the polypeptide of any of the modalities 1-13.
[00321] Modality 21. A method for producing a polypeptide that exhibits cellobiohydrolase activity comprising: (a) cultivating the transgenic plant or modality 20 plant cell under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[00322] Mode 22. An isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 18 of SEQ ID NO: 2.
[00323] Modality 23. A nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of modality 22, wherein the gene is foreign to the polynucleotide encoding the signal peptide.
[00324] Modality 24. A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of modality 22, wherein the gene is foreign to the polynucleotide encoding the signal peptide.
[00325] Modality 25. A method for producing a protein comprising: (a) cultivating a recombinant host cell comprising a gene encoding a protein operably linked to the mode 22 polynucleotide, wherein the gene is foreign to the polynucleotide encoding the peptide signal, in conditions that lead to the production of the protein; and (b) recovering the protein.
[00326] Mode 26. A process for degrading a cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of the polypeptide that has cellobiohydrolase activity of any of the modalities 1-13.
[00327] Mode 27. The process of mode 26, in which cellulosic material is pre-treated.
[00328] Mode 28. The process of mode 26 or 27, in which the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with better cellulolytic activity, a hemicellulase, an esterase, an expandin , a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin.
[00329] Mode 29. The process of mode 28, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[00330] Mode 30. The process of mode 28, in which hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase.
[00331] Mode 31. The process of any of the 2630 modes, which additionally comprises recovering the degraded cellulosic material.
[00332] Mode 32. The process of mode 31, in which the degraded cellulosic material is sugar.
[00333] Modality 33. The 32 modality process, in which sugar is selected from the group consisting of glucose, xylose, mannose, galactose and arabinose.
[00334] Modality 34. A process for synthesizing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of the polypeptide that exhibits cellobiohydrolase activity of any of the modalities 1-13; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[00335] Mode 35. The process of mode 34, in which the cellulosic material is pre-treated.
[00336] Mode 36. The process of mode 34 or 35, in which the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with better cellulolytic activity, a hemicellulase, an esterase, an expandin , a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin.
[00337] Mode 37. The process of mode 36, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[00338] Mode 38. The process of mode 36, in which hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase.
[00339] Mode 39. The process of any of the 3438 modes, in which steps (a) and (b) are carried out simultaneously in a simultaneous saccharification and fermentation.
[00340] Mode 40. The process of any of the 3439 modes, in which the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid or polyketide.
[00341] Modality 41. A process of fermenting a cellulosic material comprising: fermenting the cellulosic material with one or more fermenting microorganisms, in which the cellulosic material is saccharified with an enzyme composition in the presence of the polypeptide that has cellobiohydrolase activity of any of the modalities 1-13.
[00342] Mode 42. The process of mode 41, in which the fermentation of the cellulosic material synthesizes a fermentation product.
[00343] Mode 43. The process of mode 42, which additionally comprises recovering the fermentation product from fermentation.
[00344] Modality 44. The process of any of the 4143 modalities, in which the cellulosic material is pre-treated before saccharification.
[00345] Mode 45. The process of any of the 4144 modalities, in which the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with better cellulolytic activity, a hemicellulase, an esterase, an expansin , a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin.
[00346] Mode 46. The process of mode 45, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[00347] Mode 47. The process of mode 45, in which hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
[00348] Mode 48. The process of any of the 4147 modes, in which the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide .
[00349] The invention described and claimed herein should not be limited in scope by the specific aspects disclosed herein, since these aspects are intended as illustrations of various aspects of the invention. Any of the equivalent aspects are intended to be within the scope of this invention. In fact, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the preceding description. Such modifications are also intended to be within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail. Material Examples
[00350] The chemicals used as buffers and substrates were commercial products of at least reagent grade. Strains
[00351] The Talaromyces leycettanus strain CBS398.68 was used as the source of a polypeptide that exhibits cellobiohydrolase activity. The strain Aspergillus oryzae MT3568 was used for the expression of the Talaromyces leycettanus gene that encodes the polypeptide that has cellobiohydrolase activity. A. oryzae MT3568 is a derivative with the interrupted amdS (acetamidase) gene from Aspergillus oryzae JaL355 (WO 2002/40694), in which the pyrG auxotrophy was recovered by interrupting the A. oryzae acetamidase (amdS) gene. Means and solutions
[00352] The YP + 2% glucose medium was composed of 1% yeast extract, 2% peptone and 2% glucose.
[00353] The PDA agar plates were composed of potato infusion (the potato infusion was carried out by boiling 300 g of chopped potatoes, washed, but not peeled) in water for 30 minutes, and then decanting or squeezing the broth through of calico. The distilled water was then added until the total volume of the suspension reached one liter, followed by 20 g of dextrose and 20 g of powdered agar. The medium was sterilized by autoclaving at 103.4 KPa for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).
[00354] The LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and deionized water for 1 liter. The medium was sterilized by autoclaving at 103.4 KPa for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).
[00355] The CVE sucrose plates were composed of 342 g of sucrose (Sigma S-9378), 20 g of powdered agar, 20 ml of Cove salt solution (26 g of MgSO4.7H2O, 26 g of KCL, 26 g of KH2PO4, 50 mL of trace metal solution Cove) and deionized water to 1 liter. The medium was sterilized by autoclaving at 103.4 KPa for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The medium was cooled to 60 ° C and 10 mM acetamide, 15 mM CsCl, Triton X-100 (50 μL / 500 ml) was added.
[00356] The Cove metal trace solution was composed of 0.04 g of Na2B4O7.10H2O, 0.4 g of CuSO4.5H2O, 1.2 g of FeSO4.7H2O, 0.7 g of MnSO4.H2O, 0, 8 g of Na2MoO4.2H2O, 10 g of ZnSO4.7H2O, and deionized water to 1 liter.
[00357] Dap-4C medium consisted of 20 g dextrose, 10 g maltose, 11 g MgSO4.7H2O, 1 g KH2PO4, 2 g citric acid, 5.2 g K3PO4.H2O, 0.5 g of yeast extract (Difco), 1 ml of Dowfax 63N10 (Dow Chemical Company), 0.5 ml of trace metal KU6 solution, 2.5 g of CaCO3, and deionized water for 1 liter. The medium was sterilized by autoclaving at 103.4 KPa for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Before use, Dap-4C 3.5 ml of 50% sterile (NH4) 2HPO4 and 5 ml of 20% sterile lactic acid per 150 ml medium were added to the medium.
[00358] The KU6 trace metal solution was composed of 0.13 g of NiCl2, 2.5 g of CuSO4.5H2O, 13.9 g of FeSO4.7H2O, 8.45 g of MnSO4.H2O, 6.8 g of ZnCl2, 3g of citric acid, and deionized water to 1 liter. Example 1: Source of DNA sequence information for the Talaromyces leycettanus strain CBS398.68
[00359] Genomic sequence information was generated by DNA sequencing in Illumina at the Beijing Genome Institute (BGI) in Beijing, China, from genomic DNA isolated from the Talaromyces leycettanus strain CBS398.68. A preliminary genome assembly was analyzed using the Pedant-ProTM Sequence Analysis Suite (Biomax Informatics AG, Martinsried, Germany). The gene models built by the software were used as a starting point to detect GH6 counterparts in the genome. The most accurate gene models were constructed manually using the multiple GH6 protein sequences and known as a guideline. Example 2: Extraction of genomic DNA from the Talaromyces leycettanus strain CBS398.68
[00360] To obtain the genomic DNA for PCR amplification, the Talaromyces leycettanus CBS398.68 strain was propagated on PDA agar plates growing at 26 ° C for 7 days. The spores collected from the PDA plates were used to inoculate 25 mL of YP medium + 2% glucose in a shaking flask with protrusions at the bottom, and incubated at 30 ° C for 72 hours with shaking at 85 rpm.
[00361] Genomic DNA was isolated according to a modified protocol from the DNeasy Plant Maxi kit (Qiagen Danmark, Copenhagen, Denmark). The fungal material from the previous culture was collected by centrifugation at 14,000 x g for 2 minutes. The supernatant was removed and 0.5 g of the precipitate was frozen in liquid nitrogen with quartz sand and ground into a fine powder in a pre-cooled mortar. The powder was transferred to a 15 mL centrifuge tube and 5 mL of AP1 buffer (preheated to 65 ° C) and 10 μL of RNase A stock solution (100 mg / mL) were added, followed by vigorous vortexing. . After incubating for 10 minutes at 65 ° C with regular inversion of the tube, 1.8 mL of AP2 buffer was added to the lysate by mixing gently, followed by incubation on ice for 10 minutes. The lysate was then centrifuged at 3,000 x g for 5 minutes at room temperature, and the supernatant was decanted into a QIAshredder maxi spin column placed in a 50 mL collection tube. This was followed by centrifugation at 3,000 x g for 5 minutes at room temperature. The flow obtained was transferred to a new 50 ml tube and 1.5 volumes of AP3 / E buffer were added, followed by vortexing. 15 mL of the sample was transferred to a DNeasy Maxi spin column placed in a 50 mL collection tube, and centrifuged at 3,000 x g for 5 minutes at room temperature. The flow obtained was discarded and 12 mL of AW buffer was added to the DNeasy Maxi spin column placed in a 50 mL collection tube, and centrifuged at 3,000 x g for 10 minutes at room temperature. After discarding the flow obtained, the centrifugation was repeated to eliminate the remainder of the alcohol. The DNeasy Maxi spin column was transferred to a new 50 ml tube and 0.5 ml of AE buffer (preheated to 70 ° C) was added. After incubation for 5 minutes at room temperature, the sample was eluted by centrifugation at 3,000 x g for 5 minutes at room temperature. The elution was repeated with an additional 0.5 ml of AE buffer and the eluates were combined. The concentration of the collected DNA was evaluated by a UV spectrophotometer at 260 nm. Example 3: Construction of an Aspergillus oryzae expression vector containing the genomic sequence of the strain Talaromyces leycettanus CBS398.68 that encodes a polypeptide of the GH6 family, which has cellobiohydrolase activity
[00362] Two synthetic primer oligonucleotides shown below were determined to PCR amplify the Talaromyces leycettanus strain CBS398.68 P23YSW gene, from the genomic DNA prepared in example 2. An IN-FUSION ™ cloning kit (BD Biosciences, Palo Alto, CA, USA) was added to clone the fragment directly into the expression vector pDau109 (WO 2005/042735). F-P23YSW 5'-ACACAACTGGGGATCCACCATGCGGTCTCTCCTGG CT -3 '(SEQ ID NO: 3) R-P23YSW 5' -CCCTCTAGATCTCGAGCAGAATGAGGCAGAGTTAC GAGA -3 ’(SEQ ID NO: 4)
[00363] The bold letters represent the sequence of the gene. The underlined sequence is homologous to the pDau109 insertion sites.
[00364] An MJ Research PTC-200 DNA apparatus was used to perform the PCR reaction. A Phusion® High-Fidelity PCR kit (Finnzymes Oy, Espoo, Finland) was used for PCR amplification. The PCR reaction was composed of 5 μL of HF 5X buffer (Finnzymes Oy, Espoo, Finland), 0.5 μL of dNTPs (10 mM), 0.5 μL of Phusion® DNA polymerase (0.2 units / μL) (Finnzymes Oy, Espoo, Finland), 1 μL of primer oligonucleotide F-P23YSW (5 μM), 1 μL of primer oligonucleotide R-P23YSW (5 μM), 0.5 μL of genomic DNA from Talaromyces leycettanus (100 ng / μL ), and 16.5 μL of deionized water in a total volume of 25 μL. The PCR conditions were 1 cycle at 95 ° C for 2 minutes. 35 cycles each at 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 72 ° C for 2 minutes; and 1 cycle at 72 ° C for 10 minutes. The sample was then kept at 12 ° C until removed from the PCR machine.
[00365] The reaction products were isolated by electrophoresis on 1.0% agarose gel using 40 mM Tris base buffer, 20 mM sodium acetate, 1 mM disodium EDTA (TAE), where a 1,977 bp product band was excised from the gel and purified using an Illustra GFX® PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences, Brondby, Denmark) according to the manufacturer's instructions. The fragment was then cloned into Bam HI and Xho I digested with pDau109, using an IN-FUSION ™ cloning kit that results in plasmid pP23YSW. Cloning of the P23YSW gene in Bam HI-Xho I digested with pDau109 resulted in the transcription of the Talaromyces leycettanus P23YSW gene in the control of a NA2-tpi double promoter. NA2-tpi is a promoter modified from the gene that encodes the neutral alpha-amylase of Aspergillus niger, in which the untranslated main part has been replaced by an untranslated main part of the gene encoding the Aspergillus nidulans phosphate isomerase triosis.
[00366] The cloning protocol was performed according to the instructions of the IN-FUSION ™ cloning kit, which generated a P23YSW GH6 construct. The treated plasmid and the insert were transformed into chemically competent One Shot® TOP10F 'E. coli cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and placed in LB plates supplemented with 0.1 mg ampicillin per ml. After incubation at 37 ° C overnight, colonies were observed to grow in selection on the LB plates with ampicillin. Four colonies transformed with the P23YSW GH6 construct were cultured in LB medium supplemented with 0.1 mg of ampicillin per mL, and the plasmid was isolated with a QIAprep Spin Miniprep kit (QIAGEN Inc., Valencia, CA, USA) according to manufacturer's protocol.
The isolated plasmids were sequenced with the vector primer oligonucleotides and the specific primer oligonucleotides of the P23YSW gene, in order to determine a representative expression plasmid clone that was free of PCR errors. Example 4: Characterization of the genomic sequence of Talaromyces leycettanus CBS398.68 which encodes a P23YSW GH6 polypeptide, which has cellobiohydrolase activity
[00368] DNA sequencing of the genomic clone of Talaromyces leycettanus CBS398.68 P23YSW GH6 was performed with an automated DNA sequencer Applied Biosystems Model 3700, using version 3.1 BIG-DYE ™ terminator chemistry (Applied Biosystems, Inc., Foster City , CA, USA) and the walking primer oligonucleotide strategy. The nucleotide sequence data were analyzed for quality and all sequences were compared with each other with the aid of the PHRED / PHRAP software (University of Washington, Seattle, WA, United States). The obtained sequence was identical to the BGI sequence.
The nucleotide sequence and the deduced amino acid sequence of the Talaromyces leycettanus P23YSY gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coding sequence has 1,898 bp, including eight introns and the stop codon. The predicted encoded protein has 464 amino acids. Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), an 18-residue signal peptide was predicted. The predicted mature protein contains 446 amino acids with a predicted molecular mass of 47 kDa and an isoelectric pH of 4.41.
[00370] A global alignment to the comparative pairs of amino acid sequences was determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), with a gap gap penalty of 10 , 0.5 range extension penalty, and the EBLOSUM62 matrix. The alignment showed that the deduced amino acid sequence of the Talaromyces leycettanus gene encoding the P23YSW GH6 polypeptide, which exhibits cellobiohydrolase activity, shares 8.4% identity (excluding intervals) with the deduced amino acid sequence of a predicted protein in the GH6 family Aspergillus fumigatus (GENESEQP accession number: ABB80166), with cellobiohydrolase activity. Example 5: Expression of the GH6 P23YSW cellobiohydrolase from Talaromyces leycettanus
[00371] The expression plasmid pP23YSW was transformed into Aspergillus oryzae MT3568. Aspergillus oryzae MT3568 is a derivative of AMDS (acetamidase) interrupted from JaL355 (WO 2002/40694), in which the pyrG auxotrophy was recovered in the process of neutralizing the A. oryzae acetamidase gene (AMDS). MT3568 protoplasts are prepared according to the method of the European patent, EP0238023, pages 14-15, which is incorporated herein by reference.
[00372] The transformants were purified in sucrose selection plates of COVE, by means of simple conidia, before they sporulated in PDA plates. The production of the GH6 polypeptide from Talaromyces leycettanus by the transformants was analyzed from the culture supernatants of the cultures in stationary phase, in 96-well deep plates of 1 mL, at 30 ° C in YP medium + 2% glucose. Expression was verified on a 48-well 8% E-Page SDS-PAGE gel (Invitrogen, Carlsbad, CA, USA) by Coomassie staining. A transformant was selected to function more and Aspergillus oryzae 78.2 was determined.
[00373] For large-scale production, the spores of Aspergillus oryzae 78.2 were dispersed on a PDA plate and incubated for five days at 37 ° C. The confluent spore plate was washed twice with 5 mL of TWEEN® 20 0.01% to maximize the number of spores collected. The spore suspension was then used to inoculate twenty-five 500 ml vials containing 100 ml of Dap-4C medium. The culture was incubated at 30 ° C with constant shaking at 100 rpm. On the fourth day after inoculation, the culture broth was collected by filtration through a 0.2 μm PES filter on the top of the MF75 Supor MachV flask (Thermo Fisher Scientific, Roskilde, Denmark). The fresh culture broth from this transformant produced a GH6 protein band of approximately 70 kDa. The identity of this band as the GH5 polypeptide of Talaromyces leycettanus was verified by peptide sequencing. Example 6: Alternative method to produce the cellobiohydrolase GH6 P23YSW from Talaromyces leycettanus
[00374] Based on the nucleotide sequence identified as SEQ ID NO: 1, a synthetic gene can be obtained from numerous commercial sources, such as Gene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053, Regensburg , Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). The synthetic gene can be determined to incorporate more DNA sequences, such as restriction sites or regions of homologous recombination, to facilitate cloning into an expression vector.
[00375] Using the two synthetic primers oligonucleotides F-P23YSW and F-P23YSW described previously, a simple PCR reaction can be used to amplify the open frame of full size reading from the synthetic gene. The gene can then be cloned into an expression vector, for example, in the manner described above, and expressed in a host cell, for example, in Aspergillus oryzae in the manner previously described. Example 7: Purification of GH6 P23YSW cellobiohydrolase from Talaromyces leycettanus
[00376] 1,000 mL of broth from the Aspergillus oryzae 78.2 expression strain was adjusted to pH 7.0 and filtered through a 0.22 μm PES filter (Thermo Fisher Scientific, Roskilde, Denmark). Then, the filtrate was added to 1.8 M ammonium sulfate. The filtrate was loaded onto a Phenyl Sepharose ™ 6 (sub-high) fast flow column (GE Healthcare, Piscataway, NJ, USA) (with a column volume 60 mL) equilibrated with 1.8 M ammonium sulfate pH 7.0, 25 mM HEPES pH 7.0. After application, the column was washed with 3 column volumes of equilibration buffer, followed by 7 column volumes of 1 M ammonium sulphate column (the protein maintaining link with the column). Then, the protein was eluted with 5 column volumes of 25 mM HEPES pH 7.0, at a flow rate of 15 mL / min. The 10 mL fractions were collected and analyzed by SDS-page. The fractions were grouped and applied to a Sephadex ™ G-25 (medium) column (GE Healthcare, Piscataway, NJ, USA) balanced in HEPES 25 mM pH 7.0. The fractions were applied to a SOURCE ™ 15Q column (GE Healthcare, Piscataway, NJ, USA) equilibrated in 25 mM HEPES pH 7.0 (60 mL column volumes). After application, the column was washed with 3 column volumes with equilibration buffer, and the bound proteins were eluted with a linear gradient in 20 column volumes of 0-500 mM sodium chloride. The 10 mL fractions were collected and analyzed by SDS-page, and the fractions with the protein were grouped. The protein concentration was determined by absorbance A280 / A260. Example 8: Pre-treated corn residue hydrolysis test
[00377] The corn residue was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1.4% by weight of sulfuric acid at 165 ° C and 737.7 KPa for 8 minutes. The water-insoluble solids in the pre-treated corn residue (PCS) contained 56.5% cellulose, 4.6% hemicellulose and 28.4% lignin. Cellulose and hemicellulose were determined by a hydrolysis of sulfuric acid in two stages, with subsequent analysis of sugars by high performance liquid chromatography, using the standard analytical procedure NREL # 002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using the standard analytical procedure NREL # 003.
[00378] The unwashed and unground PCS (the complete PCS sludge) was prepared by adjusting the PCS pH to 5.0 by adding 10 M NaOH with extensive mixing, and then autoclaving for 20 minutes at 120 ° C. The dry weight of the complete PCS sludge was 29%. The PCS was used either washed or not washed with water. The ground, unwashed PCS (dry weight 32.35%) was prepared by grinding the complete PCS sludge in a wet Cosmos ICMG 40 multi-purpose grinder (EssEmm Corporation, Tamil Nadu, India). The washed and ground PCS (dry weight 32.35%) was prepared in the same way, with subsequent washing with deionized water and decanting the supernatant fraction repeatedly.
[00379] PCS hydrolysis was conducted using 2.2 mL deep well plates (Axygen, Union City, CA, United States), in a total reaction volume of 1.0 mL. Hydrolysis was carried out with 50 mg of insoluble PCS solids per ml of 50 mM sodium acetate buffer pH 5.0, containing 1 mM manganese sulphate and several protein loads of various enzyme compositions (expressed as mg of protein per gram cellulose). Enzyme compositions were prepared and then added simultaneously to all wells, in a volume ranging from 50 μL to 200 μL, to a final volume of 1 mL in each reaction. The plate was then sealed using an ALPS-300 ™ hot plate sealer (Abgene, Epsom, UK), thoroughly mixed and incubated at a specific temperature for 72 hours. All reported experiments were carried out in triplicate.
[00380] After hydrolysis, the samples were filtered using a 96-well plate with 0.45 μm MULTISCREEN® filter (Millipore, Bedford, MA, United States), and the filtrates were analyzed for sugar content as described below. When not used immediately, the filtered aliquots were frozen at -20 ° C. Sugar concentrations of samples diluted in 0.005 M H2SO4 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, United States) by elution with 0.05 % w / w of benzoic acid H2SO4 0.005 M at 65 ° C, at a flow rate of 0.6 mL per minute, and quantification by integrating the signals of glucose, cellobiosis and xylose from the detection of the refractive index (CHEMSTATION®, AGILENT ® 1100 HPLC, Agilent Technologies, Santa Clara, CA, United States) was calibrated by pure sugar samples. The resulting glucose and cellobiose equivalents were used to calculate the percentage of cellulose conversion for each reaction.
[00381] Glucose, cellobiosis and xylose were measured individually. The measured sugar concentrations were adjusted with the appropriate dilution factor. The net concentrations of sugars, produced enzymatically from unwashed PCS, were determined by adjusting the measured sugar concentrations to the corresponding previous sugar concentrations in unwashed PCS at time point zero. All HPLC data processing was performed using the MICROSOFT EXCEL ™ software (Microsoft, Richland, WA, United States).
[00382] The degree of conversion from cellulose to glucose was calculated using the following equation:% conversion = (glucose concentration / glucose concentration in a limit digest) x 100. In order to calculate the conversion%, a 100% conversion was adjusted based on a cellulase control (100 mg of Trichoderma reesei cellulase per gram of cellulose), and all values were divided by this number and then multiplied by 100. The triplicate data points had the calculated average and the standard deviation were calculated. Example 9: Preparation of an enzyme composition
[00383] Aspergillus fumigatus cellobiohydrolase I (SEQ ID NO: 5 and SEQ ID NO: 6) was prepared recombinantly in Aspergillus oryzae in the manner described in WO 2011/057140. The filtered cellobiohydrolase I GH7A broth from Aspergillus fumigatus was concentrated and the buffer was exchanged using a tangential flow concentrator (Pall Filtron, Northborough, MA, USA), equipped with a 10 kDa polyethersulfone membrane (Pall Filtron, Northborough, MA , USA) with 20 mM Tris-HCl pH 8.0. The desalinated cellobiohydrolase I GH7A broth from Aspergillus fumigatus was purified on a Q SEPHAROSE ™ ion exchange chromatography column (GE Healthcare, Piscataway, NJ, USA)) in 20 mM Tris-HCl pH 8, with a linear NaCl gradient of 0 to 1 M. Fractions were collected and fractions containing cellobiohydrolase I cellulase were grouped based on SDS-PAGE without 8-16% CRITERION® dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[00384] Preparation of GH61A polypeptide with better cellulolytic activity of Penicillium sp. (emersonii). The GH61A polypeptide from Penicillium sp. (emersonii) (SEQ ID NO: 7 and SEQ ID NO: 8) was prepared recombinantly according to WO 2011/041397. The GH61A polypeptide from Penicillium sp. (emersonii) was purified according to WO 2011/041397.
[00385] Preparation of endoglucanase II GH5 from Trichoderma reesei. Trichoderma reesei endoglucanase II GH5 (SEQ ID NO: 9 and SEQ ID NO: 10) was prepared recombinantly according to WO 2011/057140, using Aspergillus oryzae as a host. The filtered trichoderma reesei endoglucanase II GH5 broth was desalted and the buffer was exchanged for 20 mM Tris pH 8.0, using tangential flow (10K membrane, Pall Filtron, Northborough, MA, USA) according to the manufacturer's instructions.
[00386] Preparation of GH10 xylanase from Aspergillus fumigatus NN055679. GH10 xylanase from Aspergillus fumigatus NN055679 (xyn3) (SEQ ID NO: 11 and SEQ ID NO: 12) was prepared recombinantly according to WO 2006/078256, using Aspergillus oryzae BECh2 (WO 2000/39322) as a host. The filtered xylanase broth GH10 from Aspergillus fumigatus NN055679 (xyn3) was desalted and the buffer was exchanged with 50 mM sodium acetate pH 5.0 using a HIPREP® 26/10 desalination column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.
[00387] Preparation of beta-glucosidase Cel3A from Aspergillus fumigatus NN055679. (SEQ ID NO: 13 and SEQ ID NO: 14) was prepared recombinantly according to WO 2005/047499, using Aspergillus oryzae as a host. The filtered broth was adjusted to pH 8.0 with 20% sodium acetate, which left the solution cloudy. To remove turbidity, the solution was centrifuged (20,000 x g, 20 minutes), and the supernatant was filtered through a 0.2 μm filtration unit (Nalgene, Rochester, NY, USA). The filtrate was diluted with deionized water to achieve the same conductivity as in 50 mM Tris / HCl, pH 8.0. The adjusted enzyme solution was applied to a Q SEPHAROSE ™ rapid flow column (GE Healthcare, Piscataway, NJ, USA), equilibrated in 50 mM Tris-HCl, pH 8.0, and eluted with a linear sodium chloride gradient 0 to 500 mM. The fractions were pooled and treated with 1% (w / v) activated charcoal to remove the color from the beta-glucosidase pool. The charcoal was removed by filtration of the suspension through a 0.2 μm filtration unit (Nalgene, Rochester, NY, USA). The filtrate was adjusted to pH 5.0 with 20% acetic acid and diluted 10 times with deionized water. The adjusted filtrate was applied to a SP SEPHAROSE ™ rapid flow column (GE Healthcare, Piscataway, NJ, USA), equilibrated in 10 mM succinic acid, pH 5.0, and eluted with a 0 to 500 linear sodium chloride gradient mM.
[00388] Preparation of GH3 beta-xylosidase from Aspergillus fumigatus NN051616. Beta-xylosidase GH3 from Aspergillus fumigatus NN051616 (SEQ ID NO: 15 and SEQ ID NO: 16) was prepared recombinantly in Aspergillus oryzae, as described in WO 2011/057140. The filtered beta-xylosidase broth GH3 from Aspergillus fumigatus NN051616 was desalted and the buffer was exchanged with 50 mM sodium acetate pH 5.0 using a HIPREP® 26/10 desalination column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.
[00389] The protein concentration for each of the monocomponents described above was determined using a BCA ™ microplate protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA), in which serum bovine albumin was used as a standard protein. An enzyme composition was composed of each monocomponent, prepared in the manner described above, as follows: 37% cellobiohydrolase I Cel7A from Aspergillus fumigatus, 15% GH61A polypeptide with the best cellulolytic activity of Penicillium emersonii, 10% endoglucanase II GH5 Trichoderma reesei, 5% GH10 xylanase from Aspergillus fumigatus, 5% beta-glucosidase from Aspergillus fumigatus and 3% beta-xylidasidase from Aspergillus fumigatus. The enzyme composition is determined here as the "enzyme composition without cellobiohydrolase II". Example 10: Preparation of cellobiohydrolase II from Aspergillus fumigatus
[00390] Preparation of cellobiohydrolase II from Aspergillus fumigatus NN055679. The cellobiohydrolase II GH6A from Aspergillus fumigatus (SEQ ID NO: 17 and SEQ ID NO: 18) was prepared recombinantly in Aspergillus oryzae, as described in WO 2011/057140. The filtered cellobiohydrolase II GH6A broth from Aspergillus fumigatus had its buffer exchanged in 20 mM Tris pH 8.0, using a 400 mL SEPHADEX ™ G-25 column (GE Healthcare, United Kingdom) according to the manufacturer's instructions. The fractions were grouped and adjusted in 1.2 M-ammonium sulfate 20 mM pH 8.0. The balanced protein was placed on a PHENYL SEPHAROSE ™ 6 (sub high) fast flow column (GE Healthcare, Piscataway, NJ, USA), equilibrated in 20 mM Tris pH 8.0 with 1.2 M ammonium sulfate, and the Bound proteins were eluted with 20 mM Tris pH 8.0 without any ammonium sulfate. The fractions were grouped. The protein concentration was determined using a BCA ™ microplate protein assay kit with bovine serum albumin as a protein standard. Example 11: Effect of the cellobiohydrolase of the GH6 family of Talaromyces leycettanus (P23YSW) on the hydrolysis of ground and unwashed PCS at 50-65 ° C, by an enzyme composition.
[00391] The cellobiohydrolase of the GH6 family of Talaromyces leycettanus (P23YSW) was evaluated in an enzyme composition without cellobiohydrolase II at 50 ° C, 55 ° C, 60 ° C, and 65 ° C, using crushed and unwashed PCS as a substrate. The enzyme composition without cellobiohydrolase II (Example 9) was added to the PCS hydrolysis reactions in 2.25 mg of total protein per g of cellulose, and the hydrolysis results were compared with the results of an enzyme composition at elevated temperature similar with and without cellobiohydrolase II GH6 added (3.0 mg protein per g cellulose).
[00392] The test was carried out in the manner described in example 8. The reactions of 1 mL with ground and unwashed PCS (5% insoluble solids) were carried out for 72 hours in 50 mM sodium acetate buffer pH 5.0, containing 1 mM manganese sulfate. All reactions were performed in triplicate and involved simple mixing at the beginning of the hydrolysis.
[00393] As shown in Table 1 below, the enzyme composition that included cellobiohydrolase II of the GH6 family of Talaromyces leycettanus (P23YSW) significantly outperformed the enzyme composition without cellobiohydrolase II (2.25 mg protein / g of cellulose and 3.0 mg protein / g cellulose) at 50 ° C, 55 ° C, 60 ° C and 65 ° C (since the degree of conversion of cellulose into glucose for cellobiohydrolase II of the GH6 family of Talaromyces leycettanus (P23YSW) was greater than the enzyme composition without cellobiohydrolase II at 50 ° C, 55 ° C 60 ° C and 65 ° C). The results in Table 1 below show that the enzyme composition that included the cellobiohydrolase II of the GH6 family of Talaromyces leycettanus (P23YSW) outperformed the enzyme composition that included the cellobiohydrolase II of the GH6 family of Aspergillus fumigatus at 50 ° C, 55 ° C, 60 ° C and 65 ° C (since the degree of conversion of cellulose into glucose for cellobiohydrolase II of the GH6 family of Talaromyces leycettanus (P23YSW) was greater than the enzyme composition containing cellobiohydrolase II of the GH6 family of Aspergillus fumigatus at 50 ° C, 55 ° C, 60 ° C and 65 ° C). Table 1.
Example 12: Evaluation of two cellobiohydrolases II in PCS ground and washed at 50-65 ° C
[00394] Two cellobiohydrolases II were evaluated at 1 mg of protein per g of cellulose at 50 ° C, 55 ° C, 60 ° C and 65 ° C, using ground and washed PCS as a substrate, with 1 mg of protein per g beta-glucosidase cellulose of the GH3 family of Aspergillus fumigatus. The following cellobiohydrolases II were tested: cellobiohydrolase GH6 from Talaromyces leycettanus (P23YSW) and cellobiohydrolase II GH6A from Aspergillus fumigatus.
[00395] The test was carried out in the manner described in example 8. The reactions of 1 ml with ground and washed PCS (5% insoluble solids) were carried out for 72 hours in 50 mM sodium acetate buffer pH 5.0, containing 1 mM manganese sulfate. All reactions were performed in triplicate and involved simple mixing at the beginning of the hydrolysis.
[00396] The results shown in Table 2 below demonstrated that, at 50 ° C, 55 ° C, 60 ° C and 65 ° C, the GH6 cellobiohydrolase from Talaromyces leycettanus (P23YSW) showed a cellulose to glucose conversion significantly greater than cellobiohydrolase II GH6 from Aspergillus fumigatus. Table 2.

[00397] The invention described and claimed herein should not be limited in scope by the specific aspects disclosed herein, since these aspects are intended as illustrations of various aspects of the invention. Any of the equivalent aspects are intended to be within the scope of this invention. In fact, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the preceding description. Such modifications are also intended to be within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail.

权利要求:
Claims (8)
[0001]
1. Recombinant microbial host cell, characterized by the fact that it comprises a polynucleotide that encodes a polypeptide with cellobiohydrolase activity, in which the polynucleotide consists of SEQ ID NO: 1 or the sequence of nucleotides 55 to 1895 of SEQ ID NO: 1, and wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
[0002]
2. Recombinant microbial host cell according to claim 1, characterized in that the polypeptide with cellobiohydrolase activity consists of SEQ ID NO: 2 or amino acids 19 to 464 of SEQ ID NO: 2.
[0003]
3. Process for producing a polypeptide, characterized by the fact that it comprises: (a) culturing the recombinant microbial host cell as defined in claim 1 or 2 under conditions suitable for producing the polypeptide; and (b) recovering the polypeptide.
[0004]
4. Recombinant microbial host cell, characterized by the fact that it comprises a nucleic acid construct or an expression vector, which comprises a gene that encodes a protein operably linked to the polynucleotide sequence of nucleotides 1 to 54 of SEQ ID NO: 1; wherein the gene is heterologous to the polynucleotide that encodes the signal peptide.
[0005]
5. Process for producing a protein, characterized by the fact that it comprises: (a) cultivating the recombinant microbial host cell as defined in claim 4 under conditions that lead to the production of the protein; and (b) protein recovery.
[0006]
6. Process to degrade a cellulosic material, characterized by the fact that it comprises treating the cellulosic material with an enzyme composition in the presence of a polypeptide with cellobiohydrolase activity consisting of SEQ ID NO: 2 or amino acids 19 to 464 of SEQ ID NO : two.
[0007]
7. Process according to claim 6, characterized by the fact that it still comprises recovering the degraded cellulosic material.
[0008]
8. Nucleic acid construct or an expression vector, characterized by the fact that it comprises a polynucleotide that encodes a polypeptide with cellobiohydrolase activity, in which the polynucleotide consists of SEQ ID NO: 1, or the sequence of nucleotides 55 to 1895 of SEQ ID NO: 1, operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-02-04| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-09-15| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-02-02| B09A| Decision: intention to grant|

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2021-03-30| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 26/01/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11152252.0|2011-01-26|

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EP11152252|2011-01-26|

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EP11250698|2011-08-04|

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EP11250698.5|2011-08-04|

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EP11191773|2011-12-02|

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EP11191773.8|2011-12-02|

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PCT/US2012/022653|WO2012103288A1|2011-01-26|2012-01-26|Polypeptides having cellobiohydrolase activity and polynucleotides encoding same|

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