TRANSGENIC MICROBIAL HOSTING CELL, METHODS TO PRODUCE A POLYPEPTIDE, TO PRODUCE A PROTEIN, TO DEGRAD

专利摘要:
transgenic microbial host cell, methods for producing a polypeptide, for producing a mutant of an originating cell, for producing a protein, for degrading a cellulosic material, for producing a fermentation product, and for fermenting a cellulosic material, building nucleic acid or expression vector, isolated polypeptide, and isolated polynucleotide. the present invention relates to isolated polypeptides with cellobiohydrolase activity, catalytic domains and cellulose binding domains, and to polynucleotides encoding polypeptides, catalytic domains and cellulose binding domains. the invention also relates to nucleic acid constructs, vectors and host cells that comprise polynucleotides, as well as methods of producing and using polypeptides, catalytic domains, or cellulose-binding domains.
公开号:BR112013019038B1
申请号:R112013019038-8
申请日:2012-01-26
公开日:2021-03-30
发明作者:Mary Ann Stringer；Brett McBrayer
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
[0001] This invention was carried out 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
[0002] This application contains a sequence listing in computer readable form, which is incorporated by reference here. Reference to a Biological Material Deposit
[0003] This application contains a reference to a deposit of biological material, the deposit of which is incorporated by reference here. Fundamentals of the Invention Field of the Invention
[0004] The present invention relates to polypeptides with cellobiohydrolase activity, catalytic domains and cellulose binding domains, and to polynucleotides encoding polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells that comprise polynucleotides, as well as methods of producing and using polypeptides, catalytic domains, and cellulose-binding domains. Description of the Related Art
[0005] Cellulose is a simple sugar glucose polymer, covalently linked by beta-1,4 bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glycans. These enzymes include endoglucanases, cellobriohydrolases and beta-glycosidases. Endoglucanases digest the cellulose polymer at random locations, opening it up to attack by cellobriohydrolases. 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.
[0006] 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. Since ligonocellulose is converted into fermentable sugars, for example, glucose, fermentable sugars are easily fermented by yeast in ethanol.
[0007] The present invention provides polypeptides with cellobiohydrolase activity and polynucleotides that encode the polypeptides. Summary of the Invention
[0008] The present invention relates to isolated polypeptides with cellobiohydrolase activity selected from the group consisting of:
[0009] (a) a polypeptide with at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 2;
[00010] (b) a polypeptide encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, (ii) its DNAc sequence, or (iii ) the total size complement of (i) or (ii);
[00011] (c) a polypeptide encoded by a polynucleotide having at least 85% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1 or the cDNA sequence thereof;
[00012] (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
[00013] (e) a fragment of the polypeptide of (a), (b), (c) or (d) which exhibits cellobiohydrolase activity.
[00014] The present invention also relates to isolated polypeptides that comprise a catalytic domain selected from the group consisting of:
[00015] (a) a catalytic domain with at least 90% sequence identity with amino acids 98 to 456 of SEQ ID NO: 2;
[00016] (b) a catalytic domain encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) nucleotides 397 to 1786 of SEQ ID NO: 1, (ii) its DNAc sequence, or (iii) the full size complement of (i) or (ii);
[00017] (c) a catalytic domain encoded by a polynucleotide having at least 90% sequence identity with nucleotides 397 to 1786 of SEQ ID NO: 1 or the cDNA sequence thereof;
[00018] (d) an amino acid variant 98 to 456 of SEQ ID NO: 2 comprising substitution, deletion and / or insertion at one or more (for example, several) positions; and
[00019] (e) a fragment of the catalytic domain of (a), (b), (c), or (d) that exhibits cellobiohydrolase activity.
[00020] The present invention also relates to isolated polypeptides that comprise a cellulose-binding domain selected from the group consisting of:
[00021] (a) a cellulose binding domain with at least 90% sequence identity with amino acids 20 to 56 of SEQ ID NO: 2;
[00022] (b) a cellulose binding domain encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) nucleotides 58 to 273 of SEQ ID NO: 1, (ii) its DNAc sequence, or ( iii) the total size complement of (i) or (ii);
[00023] (c) a cellulose binding domain encoded by a polynucleotide with at least 90% sequence identity with nucleotides 58 to 273 of SEQ ID NO: 1 or the DNAc sequence thereof;
[00024] (d) an amino acid variant 20 to 56 of SEQ ID NO: 2 comprising substitution, deletion and / or insertion at one or more (for example, several) positions; and
[00025] (e) a fragment of the cellulose-binding domain of (a), (b), (c), or (d) which exhibits cellulose-binding activity.
[00026] The present invention also relates to isolated polynucleotides that encode the polypeptides of the present invention; nucleic acid constructs, recombinant expression vectors, and recombinant host cells that comprise polynucleotides; and methods of producing the polypeptides.
[00027] The present invention also relates to methods for degrading or converting a cellulosic material which comprises: treating the cellulosic material with an enzymatic composition in the presence of a polypeptide with cellobiohydrolase activity of the present invention.
[00028] The present invention also relates to methods of producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzymatic composition in the presence of a polypeptide with 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.
[00029] The present invention also relates to methods 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 enzymatic composition in the presence of a polypeptide with cellobiohydrolase activity of the present invention.
[00030] The present invention also relates to a polynucleotide that encodes a signal peptide that comprises or consists of amino acids 1 to 19 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. Brief Description of the Figures
[00031] Figure 1 shows the effect of a cellobiohydrolase of the GH6 family of Talaromyces byssochlamydoides on the hydrolysis of unwashed PCS ground at 50-65 ° C, by an enzyme composition at elevated temperature (HTM).
[00032] Figure 2 shows a comparison of a GH6 cellobiohydrolase from Talaromyces byssochlamydoides (Tb6), cellobiohydrolase from Aspergillus fumigatus (Af6A) and cellobiohydrolase GH6A (Mt6A) from Myceliophthora thermophila to the hydrolysis of a 50 ° hydrolyzed hydrolysis of a hydrolyzed 50 ° hydrolysis of a hydrolyzed 50 ° hydrolysis. and pH 4.0-5.0, in the presence of Aspergillus fumigatus beta-glucosidase. Definitions
[00033] 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-naptyl acetate and p-acetate nitrodenyl. 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.
[00034] 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.
[00035] Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase” means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of alpha-L-arabinofuranoside non-reducing 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 the 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 analysis of arabinose by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, United States).
[00036] 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 per minute at pH 5, 40 ° C.
[00037] Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glycoside glucohydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of non-terminal reducing beta-D-glucose residues, with the release of beta- D-glucose. For purposes of the present invention, beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glycopyranoside as a substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glycosidase 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-glycopyranoside as a substrate in citrate of 50 mM sodium containing 0.01% TWEEN® 20.
[00038] Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (EC 3.2.1.37), which catalyzes the exohydrolysis of short beta (i ^ 4) -xyl-oligosaccharides to remove successive D-xylose residues from non-reducing 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.
[00039] Catalytic domain: The term "catalytic domain" means the region of an enzyme containing the catalytic machinery of the enzyme.
[00040] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature and joined mRNA molecule, obtained from a eukaryotic or prokaryotic cell. CDNA does not have intron sequences that may 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 and spliced mRNA.
[00041] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glycan cellobiohydrolase (EC 3.2.1.91 and DC 3.2.1.176) that catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose , cell-oligosaccharides, or any beta-1,4-linked glucose containing polymer, which releases cellobiose from the reducing or non-reducing 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 cellullose , Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described by 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.
[00042] 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.
[00043] 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 assess cellulolytic activity include: (1) measuring total cellulolytic activity, and (2) measuring individual cellulolytic activities (endoglucanases, cellobiohydrolases and beta-glucosidases) in the manner reviewed in Zhang et al., Outlook for cellullase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452481. Total cellulolytic activity is assessed in general using insoluble substrates, including Whatman N «1 filter paper, microcrystalline cellulose, bacterial cellulose, algae cellulose, cotton, pre lignocellulose -treated, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N «1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellullase activities, Pure Appl. Chem. 59: 25768).
[00044] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme (s) under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at an appropriate temperature, for example, 50 ° C, 55 ° C, or 60 ° C, compared to a control hydrolysis without the addition of cellulolytic enzymatic protein . Typical conditions are 1 mL reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO4, 50 ° C, 55 ° C, or 60 ° C, 72 hours, analysis of sugar per column AMINEX® HPX-87H (Bio-Rad Laboratories, Inc., Hercules, CA, United States).
[00045] Cellulose binding domain: The term "cellulose binding domain" means the region of an enzyme that can mediate the binding of the enzyme in amorphous regions of a cellulose substrate. The cellulose binding domain (CBD) is typically seen at both the N-terminal and the C-terminal end of an enzyme.
[00046] 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 is 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 it is generally polymorphic, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glycan chains. Hemicelluloses in general bind hydrogen to cellulose, as well as other hemicelluloses, which help to stabilize the cell wall matrix.
[00047] Cellulose is generally found, for example, in the stems, leaves, sepals, barks and cobs of plants or leaves, branches and wood of trees. Cellulosic material can be, but is not limited to, agricultural waste, herbaceous material (including crops intended for energy production), municipal solid waste, pulp and ground paper waste, paper and wood waste (including forest waste) (see, for example) 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: 695719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering / Biotechnology, T. Scheper, executive 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 cellulosic material is any material from the biomass. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses and lignin.
[00048] In one aspect, cellulosic material is agricultural waste. In another aspect, cellulosic material is herbaceous material (including crops intended 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).
[00049] 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 peel. 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.
[00050] 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. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
[00051] 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 lint. 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.
[00052] In another aspect, the cellulosic material is an aquatic biomass. As used here, the term "aquatic biomass" means biomass produced in an aquatic environment by a process of photosynthesis. Aquatic biomass can be algae, emerging plants, floating leaf plants, or submerged plants.
[00053] The cellulosic material can be used as is, or can be subjected to pretreatment using conventional methods known in the art, in the manner described herein. In a preferred aspect, the cellulosic material is pre-treated.
[00054] Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The ends of the coding sequence are generally determined by an open reading frame, which begins with a start 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.
[00055] 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, propeptide 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 linkers, with 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.
[00056] Endoglucanase: The term "endoglucanase" means an endo-1,4- (1,3; 1,4) -beta-D-glycan 4-glycanhydrolase (EC 3.2.1.4) that catalyzes the bonding endohydrolysis 1,4-beta-D-glycosides in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenine, beta-1,4 bonds in mixed beta-1,3 glycan such as beta-D-glycans or xyloglycans cereal, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring the reduction in substrate viscosity, or the increase in reducing ends determined by a reducing sugar 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.
[00057] Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.
[00058] 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 the control sequences that provide its expression.
[00059] 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 B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 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 very weak endo-1,4-beta-D-glycanase activity 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.
[00060] Feruloyl esterase: The term "feruloyl esterase" means a hydrolysis of the 4-hydroxy-3-methoxycinnamyl sugar (EC 3.1.1.73) which catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamyl (feruloyl) groups from the sugar esterified, which is generally arabinose on natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as a substrate in 50 mM sodium acetate, pH 5.0. One unit of feruloyl esterase is equivalent to the amount of enzyme capable of releasing 1 μmol of p-nitrophenolate anion per minute at pH 5, 25 ° C.
[00061] Fragment: The term "fragment" means a polypeptide or a cellulose-binding or catalytic domain, with one or more (for example, several) amino acids missing at the amino and / or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has cellobiohydrolase or cellulose binding activity. In one aspect, a fragment contains at least 375 amino acid residues, for example, at least 395 amino acid residues or at least 415 amino acid residues.
[00062] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (for example, several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicelullases. 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 acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronyl esterase, a mannanase, a mannosidase, a mannosidase, a mannosidase, an 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 strong network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a very 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 acetate ester bonds or lateral groups of ferulic acid. These catalytic modes, based on the homology of their primary sequence, can be determined in the GH and CE families. Some families, with a similar complete folding, can be further grouped into clans, marked alphabetically (for example, GH-A). A more informative classification and the updated classification of these and other active enzymes in the carbohydrate are available in the Carbohydrate-Active Enzymes (CAZy) database. The activities of the hemicellulolytic enzyme can be evaluated 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 at pH, for example, 5.0 or 5.5.
[00063] 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.
[00064] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" includes any progeny of a parental cell that is not identical to the parental cell because of the mutations that occur during replication.
[00065] 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). The GH61 polypeptide of the present invention can be used in industrial applications, in the form of a fermentation broth product, that is, the polypeptide of the present invention is a component of a fermentation broth used as a product in industrial applications (for example, ethanol production). The fermentation broth product, in addition to the polypeptide of the present invention, will comprise additional ingredients used in the fermentation process such as, for example, cells (including, host cells containing the gene encoding the polypeptide of the present invention, which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and / or fermentation products. The fermentation broth can optionally be subjected to one or more purification steps (including filtration), to remove or reduce more components of a fermentation process. In this way, an isolated substance can be present in a fermentation broth product like this.
[00066] 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.
[00067] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after 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 20 to 456 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 19 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 or more different mature polypeptides (i.e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide.
[00068] Sequence encoding the mature polypeptide: The term "sequence encoding the mature polypeptide" means a polynucleotide that encodes a mature polypeptide with cellobiohydrolase activity. In one aspect, the sequence encoding the mature polypeptide has nucleotides 58 to 1786 of SEQ ID NO: 1, or the DNAc sequence thereof, based on the SignalP program (Nielsen et al., 1997, supra) which predicts that nucleotides 1 to 57 of SEQ ID NO: 1 encode a signal peptide.
[00069] 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.
[00070] 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 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 60 ° C.
[00071] 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 nucleic acids in a way that could not exist in nature, or that is synthetic, that comprises one or more control sequences.
[00072] 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.
[00073] 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 with cellulolytic activity. For the purposes of the present invention, the best cellulolytic activity is determined by measuring the increase in reducing sugars, or the increase in the 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 cellulolytic enzyme protein and 0.550% w / w protein of a GH61 polypeptide with better cellulolytic activity for 1- 7 days at an appropriate temperature, for example, 50 ° C, 55 ° C, or 60 ° C and pH, for example, 5.0 or 5.5, compared to a control hydrolysis with total protein load without better cellulolytic activity equal (1-50 mg cellulolytic protein / g cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5 L (Novozymes A / S, Bagsv; ml. Denmark), in the presence of 2-3% total weight of beta-glucosidase protein from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014), or 2-3% by weight of Aspergillus fumigatus beta-glucosidase protein (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014), of the cellulase protein load is used as the source of cellulolytic activity.
[00074] GH61 polypeptides with better cellulolytic activity improve the hydrolysis of a cellulosic material, catalyzed by enzyme with cellulolytic activity, reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably at least 1.01 times, for 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 at least 10 times, or at least 20 times.
[00075] Pre-treated corn residue: The term “PCS” or “Pre-treated corn residue” means cellulosic material derived from corn residue and heat treatment and dilute sulfuric acid, alkaline pretreatment or pretreatment neutral.
[00076] Sequence identity: the relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
[00077] 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:
[00078] (Identical residues x 100) / (Alignment size - Total number of intervals in the alignment)
[00079] 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). The Needle yield marked as “best identity” (obtained using the non-summarized option) is used as the percentage identity, and is calculated as follows:
[00080] (Identical deoxyribonucleotides x 100) / (Alignment size - Total number of alignment intervals)
[00081] Substring: 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 with cellobiohydrolase activity. In one aspect, a subsequence contains at least 1,125 nucleotides, for example, at least 1,185 nucleotides or at least 1,245 nucleotides; or their cDNA sequence.
[00082] 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.
[00083] 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.
[00084] Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a major part of xylose residues bound to beta- (1-4). Terrestrial plant xylans are heteropolymers that have a major part of beta- (1-4) -D-xylopyranose, which is branched by short carbohydrate chains. 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-type 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.
[00085] In the methods of the present invention, any material containing xylan can be used. In a preferred aspect, the material containing xylan is lignocellulose.
[00086] 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 assess xylanolitic activity include: (1) measuring total xylanolitic activity, and (2) measuring individual xylanolitic activities (for example, endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases , and alpha-glucuronyl esterases). Recent progress in xylanolitic enzyme assays has been summarized in several publications, including Biely and Puchard, 2006, Recent progress in the assays of xilanolytics enzymes, Journal of the Science of Food and Agriculture 86 (11): 1636-1647; Spanikova and Biely, 2006, Glycuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580 (19): 4597-4601; Herrmann et al., 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[00087] The activity that degrades total xylan can be assessed by determining the reducing 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 activity assay is based on the production of reducing sugars from polymeric 4-O-methyl glucuronoxylane, described in Bailey et al., 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23 (3): 257-270. Xylanase activity can also be determined with AZCL-0.2% arabinoxylan as a substrate in TRITON® X-100 (4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol) at 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 AZCL-arabinoxylan 0.2% as substrate, in 200 mM sodium phosphate buffer pH 6.
[00088] 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, United States) 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 hour 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.
[00089] Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (EC 3.2.1,8) that catalyzes the endo-hydrolysis of the 1,4-beta-D-xylosidic bonds in xylans . For the purposes of the present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan as a substrate in 0.01% TRITON® X-100, 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 AZCL-0.2% arabinoxylan as a substrate in 200 mM sodium phosphate buffer pH 6 . Detailed Description of the Invention Polypeptides with cellobiohydrolase activity
[00090] In one embodiment, the present invention relates to isolated polypeptides with a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 85%, for example, at least 86%, at least 87%, at least at least 88%, at least 89%, 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 minus 98%, at least 99% or 100%, which has cellobiohydrolase activity. In one aspect, polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, from the mature polypeptide of SEQ ID NO: 2.
[00091] 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 20 to 456 of SEQ ID NO: 2.
[00092] In another embodiment, the present invention relates to isolated polypeptides with cellobiohydrolase activity that are encoded by polynucleotides that hybridize under conditions of very high severity, conditions of low severity, conditions of medium severity, conditions of medium-high severity , 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 DNAc sequence, or (iii) the full size complement of ( i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).
[00093] 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 nucleic acid probes to identify and clone DNA encoding polypeptides with activity cellobiohydrolase from strains of different genera or species, according to methods well known in the art. In particular, such probes can be used for hybridization to 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 in the present invention.
[00094] 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.
[00095] 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 very low 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.
[00096] In one aspect, the nucleic acid probe has nucleotides 58 to 1786 of SEQ ID NO: 1 or their cDNA. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or the mature polypeptide thereof; or a fragment of it. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or its cDNA sequence. In another aspect, the nucleic acid probe is the polynucleotide contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474, in which the polynucleotide encodes a polypeptide with cellobiohydrolase activity. In another aspect, the nucleic acid probe is the region that encodes the mature polypeptide contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474.
[00097] In another embodiment, the present invention relates to isolated polypeptides with cellobiohydrolase activity, encoded by polynucleotides with a sequence identity, with the sequence encoding the mature polypeptide of SEQ ID NO: 1 or the DNAc sequence thereof. at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
[00098] 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 up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 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 another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.
[00099] 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), amino acids aromatics (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.
[000100] 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.
[000101] 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 techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photo affinity tagging, along with amino acid mutation at the site. 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 identity of the essential amino acids can also be inferred from an alignment with the related polypeptide.
[000102] Single 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. United States 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).
[000103] 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.
[000104] The polypeptide can be a hybrid polypeptide in which a region of a polypeptide is fused at the N-terminus or at the C-terminus of a region of another polypeptide.
[000105] The polypeptide can be a fusion polypeptide or cleavable fusion polypeptide, in which 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, such that they are in alignment, and that the expression of the fusion polypeptide is in control of the same (s) ) promoter (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).
[000106] 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
[000107] A polypeptide with cellobiohydrolase activity of the present invention can be obtained from microorganisms of any genus. 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.
[000108] The polypeptide can be a bacterial polypeptide. For example, the polypeptide can be a polypeptide of Gram-positive bacteria, such as a polypeptide with cellobiohydrolase activity from Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Streptococcus, or Gram-negative, such as a polypeptide from Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.
[000109] In one aspect, the polypeptide is a polypeptide from Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lichenifis stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.
[000110] In another aspect, the polypeptide is a polypeptide from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus.
[000111] In another aspect, the polypeptide is a polypeptide from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.
[000112] The polypeptide can also be a fungal polypeptide. For example, the polypeptide can be a yeast polypeptide, such as a polypeptide from Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia; or a filamentous fungus polypeptide, such as an Acremonium polypeptide, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptoteria, Cryptography, Cryptography, Cryptography, Cryptocephaly Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poomy, Ponyerichy, Poomyras Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.
[000113] In another aspect, the polypeptide is a polypeptide from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.
[000114] In another aspect, the polypeptide is a polypeptide from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusporn, keratosisporus, keratosispor Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heteramporumum, Fusarium, fusarium, Fusarium, fusarium, Fusarium, fusarium, Fusarium , Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crimea, Neurospora funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Talaromyces byssochlamydoides, Talaromyces emersonii, Talaromyces stipitatus, Thielavia achromatica, Thielavia albopces, Thielavia albopilosa, Thielavia australeinsis, Thielaviaaviaporiel, Thielaviaaviaporiel, Thielaviaaviaporiel, Thielaviaaviapori Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride or Trichophaea saccata.
[000115] In another aspect, the polypeptide is a polypeptide from Talaromyces byssochlamydoides, for example, a polypeptide obtained from Talaromyces byssochlamydoides CBS 413.71.
[000116] It will be understood that, for the aforementioned species, the invention includes both the perfect and the imperfect stages, and other taxonomic equivalents, for example, anamorphs, without regard to the name of the species for which they are known. Those skilled in the art will easily recognize the identity of suitable equivalents.
[000117] 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).
[000118] 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 natural habitats are well known in the art. A polynucleotide encoding 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
[000119] In one embodiment, the present invention also relates to catalytic domains, with a sequence identity with amino acids 98 to 456 of SEQ ID NO: 2 of at least 90%, for example, 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%. In another embodiment, the catalytic domains comprise sequences of amino acids that differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, from amino acids 98 to 456 of SEQ ID NO: 2.
[000120] In a preferred embodiment, the catalytic domain preferably comprises or consists of amino acids 98 to 456 of SEQ ID NO: 2 or an allelic variant thereof; or it is a fragment of it with cellobiohydrolase activity.
[000121] In another embodiment, the present invention also concerns the catalytic domains encoded by polynucleotides that hybridize under conditions of very high severity, conditions of low severity, conditions of medium severity, conditions of medium-high severity, conditions of high severity , or conditions of very high severity (as defined above) in (i) nucleotides 397 to 1786 of SEQ ID NO: 1, (ii) their cDNA sequence, or (iii) the full size complement of (i) or (ii) (Sambrook et al., 1989, supra).
[000122] In another embodiment, the present invention also relates to the catalytic domains encoded by polynucleotides having a sequence identity with nucleotides 397 to 1786 of SEQ ID NO: 1, or the DNAc thereof, of at least 90%, for example , 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%.
[000123] In a preferred embodiment, the polynucleotide encoding the catalytic domain comprises or preferably consists of nucleotides 397 to 1786 of SEQ ID NO: 1 or is the sequence contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474 .
[000124] In another embodiment, the present invention also relates to variants of the catalytic domain of amino acids 98 to 456 of SEQ ID NO: 2, which comprise a substitution, deletion and / or insertion in one or more (for example, several ) positions. In another embodiment, the number of amino acid substitutions, deletions and / or insertions introduced in the amino acid sequence 98 to 456 of SEQ ID NO: 2 is 10, for example, 1, 2, 3, 4, 5, 6, 8 , 9 or 10. Linking domains
[000125] In one embodiment, the present invention also relates to cellulose-binding domains, with a sequence identity with amino acids 20 to 56 of SEQ ID NO: 2 of at least 90%, for example, 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%. In another embodiment, cellulose-binding domains comprise sequences of amino acids that differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, from amino acids 20 to 56 of SEQ ID NO: 2.
[000126] In a preferred embodiment, the cellulose-binding domain comprises or preferably consists of amino acids 20 to 56 of SEQ ID NO: 2, or an allelic variant thereof; or it is a fragment of it with cellulose binding activity.
[000127] In another embodiment, the present invention also relates to cellulose-binding domains encoded by polynucleotides that hybridize under conditions of very high severity, conditions of low severity, conditions of medium severity, conditions of medium-high severity, conditions high severity, or very high severity conditions (as defined above) in (i) nucleotides 58 to 273 of SEQ ID NO: 1, (ii) the DNA sequence of nucleotides 58 to 273 of SEQ ID NO: 1 , or (iii) the full size complement of (i) or (ii) (Sambrook et al., 1989, supra).
[000128] In another embodiment, the present invention also relates to cellulose binding domains, encoded by polynucleotides with a sequence identity with nucleotides 58 to 273 of SEQ ID NO: 1 or the DNAc thereof of at least 90%, for example, 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% .
[000129] In a preferred embodiment, the polynucleotide encoding the cellulose binding domain comprises or consists of nucleotides 58 to 273 of SEQ ID NO: 1, or is the sequence contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474.
[000130] In another embodiment, the present invention also relates to variants of the cellulose-binding domain of amino acids 20 to 56 of SEQ ID NO: 2, which comprise a substitution, elimination and / or insertion in one or more ( for example, several) positions. In another embodiment, the number of amino acid substitutions, deletions and / or insertions introduced in the amino acid sequence 20 to 56 of SEQ ID NO: 2 is 10, for example, 1, 2, 3, 4, 5, 6, 8 , 9 or 10.
[000131] A catalytic domain operably linked to the binding domain can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, for example, an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase , cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, enzyme, pectinolysis, enzyme pectinol proteolytic, ribonuclease, transglutaminase, xylanase or beta-xylosidase. The polynucleotide encoding the catalytic domain can be obtained from any prokaryote, eukaryote or other source. Polynucleotides
[000132] The present invention also relates to isolated polynucleotides that encode a polypeptide, a catalytic domain, or a cellulose-binding domain of the present invention, in the manner described herein.
[000133] The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic or cDNA, or a combination thereof. 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 similar structural 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 chain reaction (LCR), ligation-activated transcription (LAT) and polynucleotide-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 species or variant of the polynucleotide region that encodes the polypeptide.
[000134] 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 genetically modified 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 the cDNA sequence thereof, and / or by introducing nucleotide substitutions that do not result in a change in the sequence of amino acids of the polypeptide, but which correspond to the use of the codon of the intended host organism for the production of the enzyme, or by the introduction of nucleotide substitutions that can give rise to a different sequence of amino acids. For a general description of nucleotide substitution see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107. Nucleic acid constructs
[000135] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell, under conditions compatible with the control sequences.
[000136] The polynucleotide can be manipulated in a variety of ways to provide expression of a 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.
[000137] 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, both homologous and heterologous to the host cell.
[000138] 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 alpha-amylase gene of Bacillus amyloliquefaciens (amyQ), alpha-amylase gene from Bacillus licheniformis (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus (amyM) maltogenic amylase gene, Bacillus subtilis (sacB) levansucrase gene, Bacillus thilisis subtilis (xylA and xylB genes, gene subtilis, gene and xylB) 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 (dagA) agarase gene and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. United States 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. United States 80: 21-25). 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.
[000139] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the present invention, in a host cell of filamentous fungus, are promoters obtained from the genes for Aspergillus nidulans acetamidase, neutral alpha-amylase from Aspergillus niger, alpha- Aspergillus niger acid-stable amylase, Aspergillus niger glycoamylase (glaA) or Aspergillus awamori, TAKA Aspergillus oryzae amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae oxide protease tyase7 of 77 protease type of protease, 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, Trichoderma reesei beta-glucosidase , cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichoder ma reesei, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylidasidase and lengthening factor as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene, where the untranslated main sequence has been replaced by a major region from an Aspergillus triose phosphate isomerase gene; non-limiting examples include promoters modified from an Aspergillus niger neutral alpha-amylase gene, in which the untranslated main region has been replaced by an untranslated main sequence from an Aspergillus nidulans or Aspergillus oryzae isomerase triose gene) and mutant, truncated and hybrid promoters thereof. Other promoters are described in U.S. patent 6,011,147.
[000140] In a host yeast, the promoters used are obtained from Saccharomyces cerevisiae enolase (ENO-1) genes, Saccharomyces cerevisiae galactokinase (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP) Saccharomyces cerevisiae, triose phosphate isomerase (TPI) from Saccharomyces cerevisiae, metallothionein (CUP1) from Saccharomyces cerevisiae and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters used for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
[000141] 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.
[000142] Preferred finalizers for bacterial host cells are obtained from Bacillus clausii alkaline protease (aprH) genes, Bacillus licheniformis alpha-amylase (amyL) and Escherichia coli ribosomal RNA (rrnB).
[000143] The preferred finishers for filamentous fungi host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glycoamylase, Aspergillus niger alpha-glycosidase, TAKA amylase, aspergine type of Aspergine from Fusarium oxysporum, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichoderma reesei, endoglucanase III from Trichoderma reesei, endoglucanase III from Trichoderma reese Trichoderma reesei, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase and Trichoderma reesei translation elongation factor.
[000144] Preferred finalizers for yeast host cells are obtained from Saccharomyces cerevisiae enolase genes, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other finalizers used for yeast host cells are described by Romanos et al., 1992, supra.
[000145] 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 gene expression.
[000146] 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 of Bacteriology 177: 3465-3471) .
[000147] The control sequence can also be a main sequence, an untranslated region of an mRNA that is important for translation by the host cell. The main sequence is operably linked at the 5 'end of the polynucleotide that encodes the polypeptide. Any major sequence that is functional in the host cell can be used.
[000148] The preferred main sequences for host cells of filamentous fungi are obtained from the genes for TAKA amylase from Aspergillus oryzae and triose phosphate isomerase from Aspergillus nidulans.
[000149] The appropriate leader sequences for yeast host cells are obtained from Saccharomyces cerevisiae enolase (ENO-1) genes, Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor and dehydrogenase / glyceraldehyde-3-phosphate alcohol dehydrogenase (ADH2 / GAP) of Saccharomyces cerevisiae.
The control sequence can also be a polyadenylation sequence, a sequence operably linked at the 3 'end of the polypeptide 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.
[000151] The preferred polyadenylation sequences for filamentous fungus host cells are obtained from the genes for anthranilate synthase of Aspergillus nidulans, glycoamylase of Aspergillus niger, alpha glycosidase of Aspergillus niger, TAKA amylase of Aspergillus oryzae and protease type of tyrosine oxide and tyrosine protease .
[000152] The polyadenylation sequences used for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
[000153] The control sequence can also be a signal peptide coding region that encodes a signal peptide attached at the N-terminus of a polypeptide, and directs the polypeptide in the cell's secretory pathway. The 5 'end of the polynucleotide coding sequence may inherently contain a coding signal peptide sequence, naturally linked to the 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 the secretion of the polypeptide. However, any signal encoding peptide sequence, which directs the expressed polypeptide into the secretory pathway of a host cell, can be used.
[000154] The efficient signal peptide sequences encoding bacterial host cells are the coding signal peptide sequences obtained from the genes for Bacillus maltogenic amylase NCIB 11837, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus alpha-amylase stearothermophilus, neutral proteases from Bacillus stearothermophilus (nprT, nprS, nprM) and prsA from Bacillus subtilis. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[000155] The efficient signal peptide sequences that encode filamentous fungus host cells are the signal peptide sequences encoding obtained from the genes for Aspergillus niger amylase, Aspergillus niger glycoamylase, TAKA Aspergillus oryzae amylase, Humicola insolens cellulase, endoglucanase, endoglucan Humicola insolens, Humicola lanuginosa lipase and Rhizomucor miehei aspartic proteinase.
[000156] The signal peptides used for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other coding signal peptide sequences used are described by Romanos et al., 1992, supra.
[000157] 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 auto-catalytic cleavage of the pro-peptide from the pro-polypeptide. The coding propeptide sequence can be obtained from 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.
[000158] Where both the signal peptide and the propeptide sequence are present, the pro-peptide sequence is positioned close to the N-terminus of the polypeptide, and the signal peptide sequence is positioned close to the N-terminus of the pro-peptide sequence peptide.
[000159] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide, with respect to the growth of the host cell. Examples of regulatory sequences are those that cause gene expression to be triggered and deactivated in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glycoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glycoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter and Troboderma reesei cellobiohydrolase promoter. Other examples of regulatory sequences are those that allow the amplification of the gene. In eukaryotic systems, these regulatory sequences include the di-idrofolate 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 to the regulatory sequence. Expression Vectors
[000160] The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences can be joined to produce a recombinant expression vector, which 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 comprising the polynucleotide, into a vector for the appropriate expression. In the creation of the expression vector, the coding sequence is located in the vector, such that the coding sequence is operably linked in the appropriate control sequences for the expression.
[000161] 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.
[000162] 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 chromosome artificial. 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.
[000163] 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.
[000164] Examples of selectable bacterial markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer resistance to antibiotics, such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in a filamentous fungus host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar , hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenyl transferase sulfate), and trpC (anthranylate synthase), as well as equivalents thereof. Preferred 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. The genes adeA, adeB, amdS, hph and pyrG are preferred for use in a Trichoderma cell.
[000165] The selectable marker can be a double selectable marker system, as described in WO 2010/039889. In one aspect, the double selectable marker is a hph-tk double selectable marker system.
[000166] 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.
[000167] 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.
[000168] 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.
[000169] 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.
[000170] 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.
[000171] 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.
[000172] 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.
[000173] 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
[000174] 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.
[000175] 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.
[000176] 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.
[000177] The bacterial host cell may 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 lentus, Bacillus lenten, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lenten , Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.
[000178] 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.
[000179] 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.
[000180] 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. United States 98: 62896294). 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, Microbes 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.
[000181] The host cell can also be from a eukaryote, such as a mammalian cell, insect, plant or fungus.
[000182] The host cell can be a fungal cell. “Fungi”, as used herein, includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all mythosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of the Fungi, 8th edition , 1995, CAB International, University Press, Cambridge, United Kingdom).
[000183] 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, RR, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[000184] Yeast as a host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis cell, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyy , Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
[000185] 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 from a single-celled stalk and the carbon catabolism can be fermentative.
[000186] The host cells of the 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.
[000187] 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, Cerjorisilipis, 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, Fusarium arium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotielusyon, terrestrial, terrarium, terrarium , Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
[000188] 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 per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. United States 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. United States 75: 1920. Production methods
[000189] 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 one aspect, the cell is a Talaromyces cell. In another aspect, the cell is a cell of Talaromyces byssochlamydoides. In another aspect, the cell is Talaromyces byssochlamydoides CBS 413.71.
[000190] The present invention also relates to methods of producing a polypeptide of the present invention comprising: (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.
[000191] The cell is grown 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.
[000192] 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.
[000193] 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.
[000194] 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 focus), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Janson and Ryden, publishers, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
[000195] 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
[000196] 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 the nutritional value, palatability and rheological properties, or to destroy an anti-nutritive factor.
[000197] The transgenic plant can be dicotyledonous (a dicotyledon) or monocotyledonous (a monocotyledon). 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).
[000198] Examples of dicotyledonous plants are tobacco, vegetables such as lupines, potatoes, beets, peas, beans and soybeans, and cruciferous plants (Brassicaceae family), such as cauliflower, canola and the closely related model organism Arabidopsis thaliana.
[000199] Examples of part of the plant 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 use of the invention are also considered parts of the plant, for example, embryos, endosperm, aleurone and seed coatings.
[000200] The progeny of such plants, plant parts and plant cells are also included in the scope of the present invention.
[000201] 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 host plant genome, or chloroplast genome, and which propagate the resulting modified plant or plant cell in a transgenic plant or plant cell.
[000202] The expression construct is conveniently a nucleic acid construct, which comprises a polynucleotide that encodes a 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).
[000203] 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 the polypeptide or domain is to be expressed. 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.
[000204] 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.
[000205] 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.
[000206] The selectable marker gene and any other parts of the expression construct can be chosen from those available in the art.
[000207] 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, biolistic transformation and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio / Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
[000208] Agrobacterium tumefaciens-mediated gene transfer is the 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 processing plants can be used for these plants. One method of choice for generating transgenic monocots is particle bombardment (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 methods of transformation include those described in U.S. patents 6,395,966 and 7,151,204 (both of which are incorporated herein in full by reference).
[000209] After the transformation, the transformers that incorporated the expression construct are selected and regenerated in the complete plants, according to methods well known in the technology. Often, the transformation procedure is determined for the selective elimination of selection genes both during regeneration and in subsequent generations using, for example, co-transformation with two separate DNA-T constructs or site-specific excision of the selection gene by a specific recombinase.
[000210] In addition to the direct transformation of a particular plant genotype with a construct of the present invention, transgenic plants can be prepared by crossing one plant with the construct and a second plant that does not have 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 always directly transform a plant of that variety provided. 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 from the progeny of such plants. As used herein, progeny can refer to the descendants of any generation of a parent plant prepared in accordance with the present invention. Such a progeny can include a DNA construct prepared in accordance with the present invention. Crossbreeding results in the introduction of a transgene into a plant strain by cross-pollinating an initial strain with a donor plant strain. Non-limiting examples of such steps are further described in U.S. Patent 7,151,204.
[000211] 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.
[000212] Genetic markers can be used to assist in the introgression of one or more transgenes of the invention from a genetic background into 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.
[000213] 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, which encodes the polypeptide or domain under conditions that lead to for the production of the polypeptide or domain; and (b) recovering the polypeptide or domain. Removal or reduction of cellobiohydrolase activity
[000214] The present invention also relates to methods of producing a mutant from a mother cell, which comprise interrupting or deleting a polynucleotide, or a portion thereof, encoding a polypeptide of the present invention, which results in the mutant cell that produces less of the polypeptide. than the mother cell when grown under the same conditions.
[000215] The mutant cell can be constructed by reducing or eliminating polynucleotide expression using methods well known in the art, for example, insertions, interruptions, substitutions or deletions. In a preferred aspect, the polynucleotide is inactivated. The polynucleotide to be modified or inactivated can be, for example, the coding region, or a part of it, essential for the activity, or a regulatory element required for the expression of the coding region. An example of a regulatory or control sequence like this can be a promoter sequence or a functional part of it, that is, a part that is sufficient to affect the expression of the polynucleotide. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, signal peptide sequence, transcription terminator and transcriptional activator.
[000216] The modification or inactivation of the polynucleotide can be carried out by subjecting the mother cell to mutagenesis and selecting mutant cells in which the expression of the polynucleotide has been reduced or eliminated. Mutagenesis, which can be specific or random, can be performed, for example, by using a suitable physical or chemical mutagen, by using a suitable oligonucleotide, or by submitting the DNA sequence to mutagenesis generated by PCR. Furthermore, mutagenesis can be performed using any combination of these mutagens.
[000217] Examples of a physical or chemical mutagen suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulfonate (EMS), sodium bisulfite, formic acid and nucleotide analogs.
[000218] When such agents are used, mutagenesis is typically performed by incubating the mother cell to be mutagenized in the presence of the mutagen of choice under suitable conditions, and by screening and / or selecting mutant cells that exhibit reduced or no expression of the gene.
[000219] The modification or inactivation of the polynucleotide can be carried out by inserting, replacing or eliminating one or more nucleotides in the gene, or a regulatory element required for its transcription or translation. For example, nucleotides can be inserted or removed in a way that results in the introduction of a stop codon, removal of the start codon, or a change in the open reading frame. Such modification or inactivation can be carried out by site-directed mutagenesis or mutagenesis generated by PCR according to methods known in the art. Although the modification can initially be carried out in vivo, that is, directly in the cell expressing the polynucleotide to be modified, it is preferable that the modification is carried out in vitro in the manner exemplified below.
[000220] An example of a convenient way to eliminate or reduce the expression of a polynucleotide is based on techniques of gene replacement, gene deletion or gene disruption. For example, in the gene disruption method, a nucleic acid sequence that corresponds to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the mother cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that can be used for the selection of transformants in which the polynucleotide has been modified or destroyed. In one aspect, the polynucleotide is disrupted with a selectable marker such as those described herein.
[000221] The present invention also relates to methods of inhibiting the expression of a polypeptide with cellobiohydrolase activity in a cell, which comprises administering to the cell or expressing in the cell a double-stranded RNA molecule (RNAds), in which the RNAds comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, RNAds are about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in size.
[000222] RNAds are preferably a small interference RNA (RNAsi) or a micro RNA (RNAmi). In a preferred aspect, RNAds are small interfering RNAs to inhibit transcription. In another preferred aspect, RNAds are micro RNA to inhibit translation.
[000223] The present invention also relates to such double-stranded RNA molecules (RNAds) that comprise a portion of the sequence encoding the mature polypeptide of SEQ ID NO: 1 to inhibit expression of the polypeptide in a cell. Although the present invention is not limited by any particular mechanism of action, RNAds can enter a cell and cause degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous RNAms. When a cell is exposed to RNAds, the homologous gene's mRNA is selectively degraded by a process called interference RNA (RNAi).
[000224] The RNAsds of the present invention can be used in gene silencing. In one aspect, the invention provides methods for selectively degrading RNA using an RNAids of the present invention. The process can be practiced in vitro, ex vivo or in vivo. In one respect, RNAds molecules can be used to generate a loss-of-function mutation in a cell, an organ or an animal. Methods of preparing and using RNAd molecules to selectively degrade RNA are well known in the art; see, for example, U.S. patents 6,489,127, 6,506,559, 6,511,824 and 6,515,109.
[000225] The present invention additionally relates to a mutant cell of a mother cell comprising an interruption or deletion of a polynucleotide encoding the polypeptide, or a control sequence thereof, or a muted gene encoding the polypeptide, which results in the cell mutant processing less of the polypeptide or no polypeptide, compared to the parent cell.
[000226] Polypeptide-deficient mutant cells are particularly used in host cells for the expression of natural and heterologous polypeptides. Therefore, the present invention further relates to methods of producing a heterologous or natural polypeptide which comprises: (a) cultivating the mutant cell under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptides" means polypeptides that are not natural to the host cell, for example, a variant of a natural protein. The host cell can comprise more than one copy of a polynucleotide that encodes the heterologous or natural polypeptide.
[000227] The methods used for the cultivation and purification of the product of interest can be carried out by methods known in the art.
[000228] The methods of the present invention for producing a product essentially free of cellobiohydrolase are of particular interest in the production of eukaryotic polypeptides, in particular, fungal proteins such as enzymes. Cellobiohydrolase-deficient cells can also be used to express heterologous proteins of pharmaceutical interest, such as hormones, growth factors, receptors and the like. The term “eukaryotic polypeptides” includes not only natural polypeptides, but also those polypeptides, for example, enzymes, which have been modified by amino acid substitutions, deletions or additions, or other such modifications to improve activity, thermostability, pH tolerance and similar.
[000229] In a further aspect, the present invention relates to a protein product essentially free of cellobiohydrolase activity which is synthesized by a method of the present invention. Compositions
[000230] The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched with a polypeptide like this. The term "enriched" indicates that the cellobiohydrolase of the composition has been improved, for example, with an enrichment factor of at least 1.1.
[000231] The compositions may comprise a polypeptide of the present invention as the main enzyme component, for example, a mono-component composition. Alternatively, the compositions may comprise multiple enzyme activities, such as one or more (for example, several) additional enzymes selected from the group consisting of a cellulase, a 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.
[000232] The compositions can be prepared according to methods known in the art, and can be in the form of a liquid or dry composition. The compositions can be stabilized according to methods known in the art.
[000233] In one embodiment, the composition further comprises one or more enzymes selected from the group consisting of one or more (for example, several) xylanases, mannases, glucanases, cellulases, lipases, esterases, proteases, endoglycosidases, endo-beta- 1,4- glucanases, beta-glucanases, endo-beta-1,3 (4) -glucanases, cutinases, peroxidases, catalases, laccases, amylases, glucoamylases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pululanases, arabinases, hemicellulases , mannanases, xyloglucanases, xylanases, mannanases, glucanases, pectin acetyl esterases, ramnogalacturonan acetyl esterases, polygalacturonases, ramnogalacturonases, galactanases, pectin liases, pectin methylesterases and transglutaminases.
[000234] The compositions can be prepared according to methods known in the art, and can have any physical appearance such as liquid, paste or solid. For example, the polypeptide composition can be formulated using methods known in the art to formulate enzymes and / or pharmaceutical products, for example, in coated or uncoated granules or microgranules. The polypeptide to be included in the composition can be stabilized according to methods known in the art, for example, stabilizing the polypeptide in the composition by adding an antioxidant or reducing agent to limit oxidation, or the polypeptide can be stabilized by adding polymers such as PVP, PVA, PEG or other suitable polymers known to be beneficial to the stability of polypeptides in solid or liquid compositions.
[000235] The compositions can be a fermentation broth formulation or a cell composition in the manner described herein. Consequently, the present invention also relates to the fermentation broth formulations and cell compositions comprising a polypeptide with cellobiohydrolase, endoglucanase, best cellulolytic or xylanase activity of the present invention. In some embodiments, the composition is a complete broth of dead cells containing organic acid (s), dead cells and / or cell debris, and culture medium.
[000236] The term "fermentation broth", as used here, refers to a preparation produced by cell fermentation that undergoes minimal or no purification and / or recovery. For example, fermentation broths are produced when microbial cultures are grown in saturation, incubated under conditions of carbon limitation, to allow protein synthesis (for example, expression of enzymes by host cells) and secretion in a culture medium of cells. The fermentation broth may contain unfractionated or fractionated contents, from the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the culture medium used and the cell debris present after the microbial cells (for example, filamentous fungus cells) are removed, for example, by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes and viable and / or non-viable microbial cells.
[000237] In one embodiment, the fermentation broth formulation and cellular compositions comprise a first organic acid component, comprising at least one 1-5 carbon organic acid and / or a salt thereof, and a second organic acid component comprising at least an organic acid of 6 or more carbons and / or a salt thereof. In a specific embodiment, the first component of organic acid is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the above, and the second component of organic acid is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
[000238] In one aspect, the composition contains organic acid (s) and, optionally, contains dead cells and / or additional cell debris. In one embodiment, dead cells and / or cell debris are removed from a broth complete with dead cells to provide a composition that is free of these components.
[000239] Fermentation broth formulations or cell compositions may additionally comprise a preservative and / or antimicrobial agent (e.g., bacteriostatic), including, but not limited to, sorbitol, sodium chloride, potassium sorbate and others known in the art.
[000240] The broth or complete composition with dead cells may further comprise one or more enzyme activities, such as acetylxylan esterase, alpha-arabinofuranosidase, alpha-galactosidase, alpha-glucuronidase, amylase, arabinanase, arabinofuranosidase, beta-galactosidase, beta-galactosidase, beta- glycosity, cellobiohydrolase, endoglucanase, endo-beta-1,3 (4) -glucanase, ferrulic acid esterase, galactanase, glucoamylase, glycohydrolase, hybrid peroxidases, with combined properties of lignin peroxidases and peroxidases dependent on manganese, laccase, lignin peroxidase manganese-dependent, mannanase, mannan acetyl esterase, mannosidase, pectate lyase, pectin acetyl esterase, pectinase lyase, pectin methyl esterase, polygalacturonase, protease, ramnogalacturonana liase, ramnogalacturonan acetyl esterase, ramnogalacturase, ramnogalacturase, ramnogalacturase, ramnogalacturase.
[000241] In some embodiments, the broth or complete composition of the dead cell or composition includes cellulolytic enzymes including, but not limited to, (i) endoglucanases (EG) or 1,4-D-glucan-4-glucanohydrolases (EC 3.2.1.4 ), (ii) exoglucanases, including 1,4-D-glucan glucanohydrolases (also known as cellodextanases) (EC 3.2.1.74) and 1,4-D-glucan cellobriohydrolases (exo-cellobriohydrolases, CBH) (EC 3.2.1.91) , and (iii) beta-glucosidase (BG) or beta-glycoside glucohydrolases (EC 3.2.1.21).
[000242] In some embodiments, the broth or complete composition of dead cells includes cellulolytic enzyme including, but not limited to, (i) endoglucanases (EG) or 1,4-D-glucan-4-glycanhydrolases (EC 3.2.1.4), (ii) exoglucanases, including 1,4-D-glucan glycanohydrolases (also known as cellodextanases) (EC 3.2.1.74) and 1,4-D-glucan cellobiohydrolases (exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) beta-glycosidase (BG) or beta-glycoside glycohydrolases (EC 3.2.1.21).
[000243] The broth or complete composition of dead cells may contain the unfractionated contents of the fermentation materials derived from the end of the fermentation. Typically, the broth or complete composition of dead cells contains the culture medium used and the cell debris present after the microbial cells (for example, filamentous fungus cells) grow by saturation, are incubated under conditions of carbon limitation to allow synthesis protein (for example, cellulase and / or glycosidase enzyme expression (s)). In some embodiments, the broth or complete composition of dead cells contains the culture medium used, extracellular enzymes and dead cells of filamentous fungus. In some embodiments, the microbial cells present in the broth or complete composition of dead cells can be permeabilized and / or lysed using methods known in the art.
[000244] A complete cell composition or broth, as described herein, is typically a liquid, but may contain insoluble components, such as dead cells, cell debris, components of the culture medium, and / or insoluble enzyme (s) ( s). In some embodiments, insoluble components can be removed to provide a clear liquid composition.
[000245] The full-broth formulations and cellular compositions of the present invention can be produced by a method described in WO 90/15861 or WO 2010/096673.
[000246] The examples provided below are for preferred uses of the compositions of the present invention. The dosage of the composition, and other conditions under which the composition is used, can be determined based on methods known in the art. Uses
[000247] The present invention is also concerned with the following methods for using polypeptides with cellobiohydrolase activity, or compositions thereof.
[000248] The present invention also relates to methods for degrading a cellulosic material comprising: treating the cellulosic material with an enzymatic composition in the presence of a polypeptide with cellobiohydrolase activity of the present invention. In one aspect, the methods 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, such as, for example, centrifugation, filtration or gravity setting.
[000249] The present invention also relates to methods of producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzyme composition, in the presence of a polypeptide with cellobiohydrolase activity of the present invention; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from fermentation.
[000250] The present invention also relates to methods of fermenting a cellulosic material which comprise: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzymatic composition in the presence of a polypeptide with cellobiohydrolase activity of the present invention. In one aspect, the fermentation of the cellulosic material synthesizes a fermentation product. In another aspect, the methods further comprise recovering the fermentation product from the fermentation.
[000251] The methods 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.
[000252] The processing of cellulosic material, according to the present invention, can be carried out using conventional methods in the art. Furthermore, the methods of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
[000253] 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 co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF) and direct microbial conversion (DMC), sometimes also called consolidated bioprocessing (CBP). SHF uses separate process steps to enzymatically hydrolyze cellulosic material into fermentable sugars, for example, glucose, cellobiose and pentose monomers, and then ferment fermentable sugars in ethanol. In SSF, the enzymatic hydrolysis of 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 the co-fermentation of multiple sugars (Sheehan and Himmel, 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. 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 high temperature followed by SSF at a temperature lower than the fermentation strain can tolerate. DMC combines all three processes (production, hydrolysis and enzymatic 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 sugars fermentable in a final product (Lynd et al., 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 which comprises pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination of these, can be used in the practice of the methods of the present invention.
[000254] 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 (Corazza et al., 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov and Sinitsyn, 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 and Lee, 1983, Bioconversion of waste cellulose using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a field-induced intensive agitation reactor electromagnetic (Gusakov et al., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with 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.
[000255] Pre-treatment. In the practice of the methods of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components from cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis enzyme of lignocellulosics Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin. / Biotechnol. 108: 41-65; Hendriks e 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 e 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 unlocking low-cost cell ulosic ethanol, Biofuels Bioproducts nd Biorefining-Biofpr. 2: 26-40).
[000256] 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.
[000257] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), diluted acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment , wet oxidation, wet explosion, explosion with ammonia fiber, pretreatment with organosolv and biological pretreatment. Additional pre-treatments include percolation treatments with ammonia, ultrasound, electroporation, microwaves, supercritical CO2, supercritical H2O, ozone, ionic liquid and gamma irradiation.
[000258] 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).
[000259] 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 170190 ° 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 20020164730). During the steam pretreatment, the acetyl hemicellulose groups are cleaved and the resulting acid autocatalyzes the partial hydrolysis of hemicellulose into monosaccharides and oligosaccharides. Lignin is removed to a limited extent only.
[000260] Chemical pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin. Such a pretreatment 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 burst with ammonia / freezing (AFEX), percolation with ionic liquid ammonia (APR) and pre-treatments with organosolv.
[000261] A catalyst, such as H2SO4 or SO2 (typically 0.3 to 5% w / w), is generally added to the 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).
[000262] 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).
[000263] Pre-treatment with lime is carried out with calcium oxide or calcium hydroxide at temperatures of 85-150 ° C and residence time from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 19591966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatment methods that use ammonia.
[000264] Wet oxidation is a heat 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.
[000265] 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).
[000266] 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.
[000267] Pretreatment with organosolv 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 pre-treatment with organosolv, most of the hemicellulose and lignin is removed.
[000268] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. published application 2002/0164730.
[000269] 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.
[000270] 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.
[000271] 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).
[000272] Cellulosic material can be pretreated both physically (mechanically) and chemically. The mechanical or physical pretreatment can be coupled with vapor / steam explosion, hydrothermolysis, weak or diluted acid treatment, high temperature, high pressure treatment, 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 400 psi, for example, about 150 to about 250 psi. In another aspect, 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. Chemical or physical pretreatments can be carried out sequentially or simultaneously, if desired.
[000273] 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.
[000274] 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. Biological pretreatment techniques may involve applying microorganisms and / or enzymes that solubilize lignin (see, for example, Hsu, T.-A., 1996, Pretreatment 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 lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from material lignocelullosics: State of art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
[000275] Saccharification. In the hydrolysis stage, also known as saccharification, the cellulosic material, for example, pre-treated, is hydrolyzed to break down cellulose and / or hemicellulose into fermentable sugars, such as glucose, cellobiosis, xylose, xylulose, arabinose, mannose, galactose , and / or soluble oligosaccharides. Hydrolysis is carried out enzymatically by an enzymatic 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.
[000276] 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 continuous or batch fed process, where the cellulosic material is fed gradually, for example, to an enzyme containing hydrolysis solution.
[000277] Saccharification is performed 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.
[000278] Enzyme compositions can comprise any protein used in the degradation of cellulosic material.
[000279] 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, a 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.
[000280] 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-glycosidase, 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.
[000281] 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 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 a 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).
[000282] 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.
[000283] In the methods of the present invention, the enzyme (s) can be added before or during saccharification, saccharification and fermentation, or fermentation.
[000284] 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.
[000285] The enzymes used in the methods of the present invention can be in any form suitable for use, such as, for example, a fermentation broth formulation or cell composition, a cell lysate with or without cell debris, a preparation of semi-purified or purified enzyme, or a host cell as a source of the enzymes. The enzyme composition can be a dry powder or granulate, a non-powdered granulate, a liquid, a stabilized liquid, or a protected stabilized enzyme. Liquid enzyme preparations, for example, can be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and / or lactic acid or another organic acid, according to the established procedures.
[000286] The ideal amounts of enzymes and polypeptides with cellobiohydrolase activity depend on several factors including, but not limited to, the mixture of cellulolytic component enzymes and / or hemicellulolytic enzymes, cellulosic material, cellulosic material concentration, (s) ) pre-treatment (s) of the cellulosic material, temperature, time, pH and inclusion of fermenting organism (for example, yeast for simultaneous saccharification and fermentation).
[000287] 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.
[000288] 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 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.
[000289] 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.
[000290] 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.
[000291] A polypeptide with enzymatic activity can be a bacterial polypeptide. For example, the polypeptide can be a polypeptide from Gram positive bacteria, such as a polypeptide with enzymatic activity from Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, Caldicellulosiria, Therdicellothermidid, Therdacellusidid, Thermo, a Gram negative bacterial polypeptide such as a polypeptide with enzymatic activity from E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter Neisseria or Ureaplasma.
[000292] In one aspect, the polypeptide is a polypeptide with enzymatic activity from Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus. pumilus, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis.
[000293] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberi, or Streptococcus equi subsp. Zooepidemicus.
[000294] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus or Streptomyces lividans.
[000295] The polypeptide with enzymatic activity can also be a fungal polypeptide, and more preferably a yeast polypeptide such as a polypeptide with enzymatic activity from Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia; or more preferably a filamentous fungus polypeptide such as a polypeptide with enzymatic activity of Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Cryptography, Cryptography, Cryptography, Cryptography, Cryptography, Cryptography Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillis, Picherich, Picherich, Picherich, Picherich, Picher Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella or Xylaria.
[000296] In one aspect, the polypeptide is a polypeptide with enzymatic activity from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis.
[000297] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulus, Aspergillus nerisillis, kermisillispor, Gillispor Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminearum, Fusarium graminumum, Fusarium graminum, Fusarium graminum, Fusarium graminum, Fusarium graminum, Fusarium graminum, Fusarium graminum, Fusarium graminum , Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthoram, Myceliophthoram spora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia ovispora, Thielavia ovispora, Thielavia terris, peria, , Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride or Trichophaea saccata.
[000298] Chemically modified or protein engineered mutants can also be used.
[000299] 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.
[000300] 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.
[000301] Examples of bacterial endoglucanases that can be used in the methods 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 d e Thermobifida fusca (WO 05/093050) and endoglucanase V from Thermobifida fusca (WO 05/093050).
[000302] 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 to 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), Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14), Fusariu endoglucanase m oxysporum (access 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).
[000303] Examples of 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, Humicola cellobiohydrolase II, Humicola cellulose, Humicola Myceliophthora thermophila (WO 2009/042871), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei cellobiohydrolase II, Trichoderma cellobiohydrolase II and Trichoderma cellulose II Trichophaea saccata (WO 2010/057086).
[000304] 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: 49734980), 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).
[000305] 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).
[000306] 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.
[000307] 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.
[000308] In the methods of the present invention, any GH61 polypeptide with better cellulolytic activity can be used as a component of the enzyme composition.
[000309] Examples of GH61 polypeptides with better cellulolytic activity used in the methods 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 2009/085864, 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).
[000310] 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 or copper sulfate.
[000311] In another 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).
[000312] 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; di-idroxifumaric 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 thereof.
[000313] 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 of these.
[000314] 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, thiazylyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thyrolyl. -pyrazolyl, tianaftenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinolinyl, isoindolyl, acridinyl, pyridine, pyrethyl, pyridyl, pyridine , tiepinila, piperidinila, and oxepinila. 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-di-idroxyethyl) -3,4-di-idroxifuran-2 (5H) -one; 4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2 (5H) -furanone; [1,2-di-idroxyethyl] furan-2,3,4 (5H) -trione; α-hydroxy-Y-butyrolactone; ribonic Y — lactone; aldoexuronic acid Y — lactone; δ-lactone gluconic acid; 4-hydroxycoumarin; di-idrobenzofuran; 5- (hydroxymethyl) furfural; furoin; 2 (5H) -furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate of these.
[000315] 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 of these.
[000316] 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 of these.
[000317] 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 of these.
[000318] 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 to 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.
[000319] 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.
[000320] 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.
[000321] 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).
[000322] Examples of xylanases used in the methods 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).
[000323] Examples of beta-xylosidases used in the methods of the present invention include, but are not limited to, beta-xylosidases from Neurospora crassa (SwissProt access number Q7SOW4), Trichoderma reesei (UniProtKB / TrEMBL accession number Q92458), and Talaromyces emersonii (SwissProt Q8X212 accession number).
[000324] Examples of acetylxylan esterases used in the methods of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile (GeneSeqP accession number AAB82124 ), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (accession number UniProt q7s259), Phaeosphaeria nodorum (accession number UniprotQU00U and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[000325] Examples of feruloyl esterases (ferulic acid esterases) used in the methods of the present invention include, but are not limited to, feruloyl esterases of Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (accession number UniProt A1D9T4), Neurospora crassa (accession number UniProt Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729) and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).
[000326] Examples of arabinofuranosidases used in the methods 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).
[000327] Examples of alpha-glucuronidases used in the methods of the present invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus (UniProt access number alcc12), Aspergillus fumigatus (SwissProt access number Q4WW45), Aspergillus niger (number of access Uniprot 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 access number Q99024).
[000328] Polypeptides with enzymatic activity, used in the methods of the present invention, can be produced by fermenting the aforementioned microbial strains 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).
[000329] 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.
[000330] 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.
[000331] 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.
[000332] 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.
[000333] 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).
[000334] "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.
[000335] Examples of bacterial and fungal fermenting organisms that produce ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[000336] 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.
[000337] 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.
[000338] 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 (Philippid.,).
[000339] 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.
[000340] 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 another 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.
[000341] 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.
[000342] 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).
[000343] 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 co-use xylose and arabinose.
[000344] The cloning of heterologous genes into various fermenting microorganisms led to the construction of organisms capable of converting hexoses and pentoses into ethanol (co-fermentation) (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 transketolase and Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of princip le, 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).
[000345] 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.
[000346] 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.
[000347] 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.
[000348] In one aspect, yeast and / or another microorganism is 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 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 another microorganism is preferably applied in amounts of approximately 105 to 1012, preferably in approximately 107 to 1010, essentially approximately counts of 2 x 108 viable cells 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.
[000349] 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.
[000350] 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.
[000351] 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, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400408; Nigam and Singh, 1995, Processs for fermentative production of xylitol - a sugar substitute, Process Biochemistry 30 (2): 117-124; 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.
[000352] 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.
[000353] 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.
[000354] 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.
[000355] 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.
[000356] 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, 1997 in Biomass and Bioenergy, 13 (1-2): 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.
[000357] In another preferred aspect, the fermentation product is isoprene.
[000358] 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.
[000359] 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, 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.
[000360] In another preferred aspect, the fermentation product is polyketide.
[000361] 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
[000362] The present invention also relates to an isolated polynucleotide that encodes a signal peptide comprising or consisting of amino acids 1 to 19 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 for the signal peptide has nucleotides 1 to 57 of SEQ ID NO: 1.
[000363] The present invention also relates to the nucleic acid constructs, expression vectors and recombinant host cells that comprise such polynucleotides.
[000364] 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.
[000365] 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.
[000366] 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 alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylidasidase, carboidrase , carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, proteinase, proteinase, enzyme pectin transglutaminase or xylanase.
[000367] The gene can be obtained from any prokaryote, eukaryote or other source.
[000368] The present invention is further described by the following examples, which are not to be construed as limiting the scope of the invention. Examples Means
[000369] COVE N medium was composed of 218 g of sorbitol, 50 ml of COVE salt solution, 10 g of dextrose, 2.02 g of KNO3, 25 g of agar, and deionized water for 1 liter.
[000370] The COVE salt solution was composed of 26 g of MgSO4 ^ 7H2O, 26 g of KCl, 76 g of KH2PO4, 50 ml of trace metal COVE solution, and deionized water to 1 liter.
[000371] The trace metal solution of COVE was composed of 0.04 g of Na2B4O 40H2O, 0.4 g of ('uSO / 5112 (O 1.2 g FeSO / 7 | I2 (O, 0.7 g of MnSO4 ^ H2O, 0.8 g of Na2MoO. ^ 2H2O, 10 g of ZnS (K7112 (), and deionized water to 1 liter.
[000372] DAP4C-1 medium was composed of 11 g of MgSO4 ^ 7H2O, 1 g of KH2P) 4, 2 g of citric acid monohydrate, 20 g of dextrose, 10 g of maltose, 6 g of K3PO4 ^ 3H2O, 0 , 5 g of yeast extract, 0.5 ml of trace metal solution, 1 ml of pluronic, and deionized water for 1 liter. The medium was divided into flasks, adding 250 mg of CaCO3 to each 150 mL portion. The medium was sterilized in an autoclave. After cooling, 150 ml of medium was added next: 3.5 ml of (NH4) 2HPO4 sterilized by 50% w / v filtration, and 5.0 ml of 20% lactic acid sterilized by filtration.
[000373] The trace metal solution of the DAP4C-1 medium was composed of 6.8 g of ZnCl2, 2.5 g of CiiSíbol F (O, 0.24 g of iCk6lFO, 13.9 g of F'cSOrVIFO, 8.45 g of MnSOrlFO, 3 g of citric acid monohydrate, and deionized water for 1 liter.
[000374] The MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO4 ^ 7H2O, 1 g of NaCl, 2 g of K2SO4, 12 g of KH2PO4, 7 g of yeast extract, 2 g of urea, 0.5 mL of AMG trace metal solution, and deionized water to 1 liter; pI 5.0.
[000375] The AMG trace metal solution was composed of 14.3 g of ZnSO4 ^ 7H2O, 2.5 g of CuSO4 ^ 5H2O, 0.5 g of NiClróHO, 13.8 g of FeSO4 ^ 7H2O, 8.5 g of MnSO4 ^ 7H2O, 3 g of citric acid, and deionized water to 1 liter.
[000376] MY25 medium consisted of 25 g of maltodextrin, 2 g of MgSO4 ^ 7H2O, 10 g of KH2PO4, 2 g of citric acid, 2 g of K2SO4, 2 g of urea, 10 g of yeast extract , 1.5 mL of AMG trace metal solution, and deionized water for 1 liter; adjusted to pI 6.
[000377] SY50 medium was composed of 50 g of sucrose, 2 g of MgSO4 ^ 7H2O, 10 g of anhydrous KH2PO4, 2 g of K2SO4, 2 g of citric acid, 10 g of yeast extract, 2 g of urea, 0.5 g of CaC ^ 2H2O, and 0.5 g of solution of trace metals AMG 200X, and deionized water for 1 liter; pI 6.0.
[000378] The AMG 200X trace metal solution was composed of 3 g of citric acid, 14.3 g of ZnSO4 ^ 7H2O, 2.5 g of CuSO4 ^ 5H2O, 13.8 g of FeSO4 ^ 7H2O, 8.5 g of MnSO4 ^ H2O, and deionized water to 1 liter. Example 1: PCR amplification of a cellobiohydrolase gene from the genomic DNA of Talaromyces byssochlamydoides CBS 413.71
[000379] A gene encoding cellobiohydrolase was amplified by PCR from the genomic DNA of Talaromyces byssochlamydoides CBS 413.71, in a two-step process. First, a central fragment of the gene was amplified by PCR using degenerate primer oligonucleotides and determined to combine two conserved regions of sequence into genes encoding cellobiohydrolase enzymes of the GH6 family. After amplification of the internal fragment, the sequence of the fragment was determined and used to determine the gene-specific oligonucleotide primers for a gene that walks in either the 5 'or 3' direction, to obtain the complete coding sequence.
[000380] The internal gene fragment was amplified by PCR using the degenerate primers 859 and 860, shown below in a touch-down PCR protocol in which the initial annealing temperature of 67 ° C was decreased by 1 ° C in each successive cycle, for a total of 10 cycles, until an annealing temperature of 57 ° C was reached. The PCR amplification was then completed with an additional 29 cycles using an annealing temperature of 57 ° C. Oligonucleotide primer 859: TKCCYGAYCGYGAYTGYGC (SEQ ID NO: 3) Oligonucleotide primer 860: TCRCCACCKGGCTTKAYCCA (SEQ ID NO: 4)
[000381] Amplification was performed using a REDDYMIX ™ PCR master mix (ABgene Ltd, Epsom, UK). The amplification reaction consisted of 1 μL of T. byssochlamydoides CBS 413.71 genomic DNA as a template, 50 pmol each of the 859 and 860 primer oligonucleotides, and 12.5 μL of REDDYMIX ™ PCR master mix in a final volume of 25 μL. Genomic DNA from T. byssochlamydoides was extracted from fresh mycelium using a FastDNA® SPIN protocol (Qbiogene, Inc., Carlsbad, CA, United States). The amplification was performed in a thermocycler programmed for an initial mold denaturation step at 94 ° C for 2 minutes; 11 cycles with denaturation at 94 ° C for 45 seconds, annealing at 67 ° C for 45 seconds with a decrease of 1 ° C for each subsequent cycle, and stretching at 72 ° C for 1 minute; and 29 cycles with denaturation at 94 ° C for 45 seconds, annealing at 57 ° C for 45 seconds, and extension at 72 ° C for 1 minute. A final stretching was carried out at 72 ° C for 7 minutes.
[000382] The reaction products were determined by electrophoresis on 1% agarose gel, where a PCR product band of approximately 700-800 bp was observed. The band was cut from the gel and the DNA was purified using an ILLUSTRATM GFXTM PCR DNA and gel band purification kit (GE Healthcare, Little Chalfont, UK). The purified PCR fragment was cloned into the pCR®2.1-TOPO® vector (Invitrogen, Life Technologies, Carlsbad, CA, United States) using a TOPO® TA CLONING® kit (Invitrogen, Life Technologies, Carlsbad, CA, United States), according to the manufacturer's instructions, and then transformed into chemically competent E. coli cells (Invitrogen, Life Technologies, Carlsbad, CA, United States) according to the manufacturer's instructions.
[000383] The sequence of the PCR product was determined directly with the primers 859 and 860, and sequencing 4 individual clones of the PCR product with the primers oligonucleotides of the sense M13 and reverse M13 shown below. M13 sense: TGTAAAACGACGGCCAGT (SEQ ID NO: 5) M13 reverse: AGCGGATAACAATTTCACACAGG (SEQ ID NO: 6)
[000384] The sequence was compared with the known sequences using the BLAST search tool (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) and confirmed as similar to the known gene encoding cellobiohydrolases.
[000385] The partial sequence of the Talaromyces byssochlamydoides gene encoding cellobiohydrolase was used to determine the gene specific oligonucleotides 934, 935, 1044 and 1045, shown below, to enable the gene to walk from both ends of the sequence. Oligonucleotide 934: AGAGTCTCGTCTCAGTACATG (SEQ ID NO: 7) Oligonucleotide 935: CGAATACGTCACCAGCCAC (SEQ ID NO: 8) Oligonucleotide 1044: AATTGCTGAGCTGTTTCAGC (SEQ ID NO: 9)
[000386] The walk along the gene was performed using a DNA Walking SPEEDUP ™ Premix kit (Seegene, Seoul, Korea), based on the manufacturer's protocol with some modifications. Only the first two sets of PCR reactions described in the protocol were used, which included an initial set of amplifications with a specific gene primer and four different return primers, and a set of reactions nested with a second specific gene primer . Half of the recommended reaction volumes were used for the first set of reactions.
[000387] To walk in the 5 'direction, the first set of PCR reactions was performed with the specific gene 934 primer oligonucleotide. After amplification, the reactions were diluted with 150 μL of water, and 5 μL of the dilution were used as a template in the second set of nested PCR reactions, with the specific gene 935 primer oligonucleotide. The second amplifications were performed in the manner described by the Walking SPEEDUP ™ Premix DNA kit protocol, with an annealing temperature of 58 ° C. The reaction products were determined using 1% agarose gel electrophoresis, where a single weak band of approximately 1,000 bp was observed in one of the four nested reactions. The 1,000 bp fragment was re-amplified twice, first by repeating the nested PCR reaction using 1 μL of the reaction, including the 1,000 bp product as a template. The reaction products were determined by electrophoresis on 1% agarose gel, and a second re-amplification was carried out from there by removing a small piece of the 1000 bp band from the gel with a pipette tip, which was used as a template in a PCR reaction under the same conditions. The reaction products were determined by electrophoresis on 1% agarose gel using 40 mM Tris base buffer - 20 mM sodium acetate - 1 mM disodium EDTA (TAE), and the 1000 bp band was cut from the gel and the DNA was purified using an ILLUSTRATM GFXTM PCR DNA and gel band purification kit. The sequence of the PCR product was determined using the 935 primer oligonucleotide.
[000388] To walk in the 3 'direction, the first set of PCR reactions was performed with the 1044 specific gene oligonucleotide. After amplification, the reactions were diluted with 150 μL of water, and 5 μL of the dilution were used as a template in the second set of PCR reactions nested with the specific 1045 gene primer oligonucleotide. The second amplifications were performed in the manner described by the Walking SPEEDUP ™ Premix DNA kit protocol, with a ring temperature of 56 ° C. The reaction products were purified from the PCR reaction components using an ILLUSTRATM GFXTM DNA for PCR and gel band purification kit, and were concentrated by eluting in 10 μL of elution buffer provided with the kit. The products were analyzed by first cloning 4 μL of each purified PCR reaction directly into pCR®2.1-TOPO®, using a TOPO TA CLONING® kit reaction and chemically transforming the reactions of the TOPO TA CLONING® kit into E. coli TOP10 cells competent authorities (Invitrogen, Life Technologies, Carlsbad, CA, United States) according to the manufacturer's instructions. The obtained clones were selected for insertions by means of restriction digestion, and those containing inserts were sequenced with the primers oligonucleotides of the sense vector M13 (SEQ ID NO: 5) and reverse M13 (SEQ ID NO: 6). Four individual clones, each approximately 800 bp, provided the 3 'sequence for the gene encoding T. byssochlamydoides cellobiohydrolase. All sequences were assembled in a single contiguous.
[000389] The genomic DNA sequence and the amino acid sequence deduced from the sequence encoding Talaromyces byssochlamydoides cellobiohydrolase are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The 1789 bp genomic DNA sequence (including the stop codon) contains 7 introns located at nucleotides 80 to 131, 201 to 253, 540 to 592, 847 to 897, 1036 to 1095, 1354 to 1443, and 1686 to 1744 from SEQ ID NO: 1. The genomic DNA fragment encodes a polypeptide of 456 amino acids. The% G + C content of the sequence encoding the mature polypeptide is 56%. Using the software program SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6), a 19-residue signal peptide was predicted. The predicted mature protein contains 437 amino acids with a predicted molecular mass of 46 kDa and an isoelectric point of 4.0. The protein contains a cellulose binding module of the CBM1 type at the N-terminus (amino acids 20 to 56 of SEQ ID NO: 2). The catalytic domain has amino acids 98 to 456.
[000390] A comparative alignment of the mature cellobiohydrolase amino acid sequences, without the signal peptides, was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the EMBOSS Needle program, with a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that the deduced amino acid sequence of Talaromyces byssochlamydoides cellobiohydrolase (mature polypeptide) shares 84% identity (excluding the intervals) with the deduced amino acid sequence of a Talaromyces emersonii cellobiohydrolase (UNIPROT: Q8NIB5). Example 2: Cloning of the Talaromyces byssochlamydoides gene that encodes cellobiohydrolase in an Aspergillus expression vector
[000391] The gene encoding T. byssochlamydoides cellobiohydrolase was cloned into the Aspergillus pMStr57 expression vector (WO 2004/032648), by PCR amplification of the protein coding sequence from the genomic DNA with two synthetic primers shown to follow. The vector pMStr57 contains the sequences for selection and propagation in E. coli, and selection and expression in Aspergillus. Selection in Aspergillus is facilitated by the amdS gene of Aspergillus nidulans which allows the use of acetamide as a single source of nitrogen. Expression in Aspergillus is mediated by a modified neutral amylase II (NA2) promoter from Aspergillus niger, which is fused to the 5 'main sequence of the gene encoding Aspergillus nidulans triose phosphate isomerase (tpi), and to the terminator of the gene encoding amyloglycosidase from Aspergillus niger. 1167 Primer Oligonucleotide: ACACAACTGGGGATCCTCACCATGCGAAATATTCTTG (SEQ ID NO: 11) 1168 Primer Oligonucleotide: CCCTCTAGATCTCGAGCTAGAATGACGGATTGGCGTT (SEQ ID NO: 12)
[000392] Amplification was performed using the IPXOF ™ 2X high-fidelity master mix (Bio-Rad Laboratories, Inc., Hercules, CA, United States), following the manufacturer's instructions. The amplification reaction was composed of T. byssochlamydoides CBS 413.71 genomic DNA as a template, 25 pmol of each of the 1167 and 1168 primer oligonucleotides, and 25 μL of IPROOF ™ high-fidelity 2X master mix in a final volume of 50 μL. The amplification was performed in a thermocycler programmed for an initial mold denaturation step at 98 ° C for 2 minutes; 5 cycles each with denaturation at 98 ° C for 10 seconds, annealing at 65 ° C for 10 seconds, and stretching at 72 ° C for 1 minute; and 30 cycles each with denaturation at 98 ° C for 10 seconds, and combined annealing and extension at 72 ° C for 1 minute. A final stretch was performed at 72 ° C for 10 minutes.
[000393] A PCR product of approximately 2,000 bp was separated from the residual reaction components using a GFX® PCR DNA and gel band purification kit, according to the manufacturer's instructions. The purified PCR fragment was sequenced, and the sequence was completely similar to the SEQ sequence. ID NO. 1.
[000394] The PCR fragment was cloned into pMStr57 digested with Bam HI and Xho I, using an IN-FUSION ™ DryDown PCR cloning kit (Clontech Laboratories, Inc., Mountain View, CA, United States), according to the manufacturer's instructions. The DNA encoding Talaromyces byssochlamydoides cellobiohydrolase from the resulting Aspergillus expression construct, pMStr215, was sequenced and the sequence was completely in accordance with the sequence of SEQ ID NO: 1. Example 3: Expression of the gene encoding Talaromyces byssochlamydoides cellobiohydrolase in Aspergillus oryzae MT3568
[000395] The fungal host strain of expression Aspergillus oryzae MT3568 was transformed with pMStr215, according to Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO 2004/032648. Aspergillus oryzae MT3568 has a disrupted amdS (acetamidase) gene derived from Aspergillus oryzae JaL355 (WO 2002/40694), in which the pyrG auxotrophy has been restored in the process of neutralizing the A. oryzae amdS gene. Eight transformants were cultured for 4 days at 30 ° C in 750 μL of DAP2C-1 medium (WO 2004/032648). The samples were monitored by SDS-PAGE using gels at 8% E-PAGE 48 with SeeBlue Plus2 molecular weight standards (Invitrogen, Life Technologies, Carlsbad, CA, United States), according to the manufacturer's instructions. The gel was stained with INSTANTBLUE ™ (Expedeon Protein Solutions, Cambridge, UK). Six transformants produced an unprecedented protein band of approximately 55 kDa.
[000396] Two of these transformants, determined Aspergillus oryzae MStr390 and MStr391, were isolated twice by inoculating conidia by dilution in selective medium (amdS) containing TRITON® X-100 0.01% to limit the size of the colony. Example 4: Shake flask cultures of recombinant Aspergillus oryzae MStr391 for the production of Talaromyces byssochlamydoides cellobiohydrolase
[000397] The spores of two confluent Aspergillus oryzae MStr391 grown in inclined tubes with COVE N, determined EXP03666, were collected with a 0.01% solution of TWEEN® 20 and used to inoculate 10 shake flasks each containing 150 mL of DAP4C-1 medium. The flasks were incubated at 30 ° C for 4 days, with constant shaking at 200 rpm. Mycelia and fungal spores were removed during collection by first filtering the fermentation broth using a sandwich of 3 glass microfiber filters, with increasing particle retention sizes of 1.6 μm, 1.2 μm and 0, 7 μm, and then filtering through a 0.45 μm groundwater filter.
[000398] The filtered broth was added to the 1.8 M ammonium sulfate and adjusted to pH 5.6. After filtration on a 0.22 μm PES filter (Nalge Nunc International, Rochester, NY, United States) the filtrate was placed on a Fenil Sepharose ™ 6 (sub-high) fast flow column (GE Healthcare, Piscataway, NJ, United States), equilibrated with 1.8 M ammonium sulfate pH 5.6, and the bound proteins were eluted with 25 mM HEPES pH 7.0. The fractions were grouped and applied to a SEPHADEX ™ G-25 (medium) column (GE Healthcare, Piscataway, NJ, United States), equilibrated in 50 mM HEPES pH 7.0. The fractions were applied to a SOURCE ™ 15Q column (GE Healthcare, Piscataway, NJ, United States), equilibrated in 50 mM HEPES pH 7.0, and the bound proteins were eluted with a linear 0-1,000 mM sodium chloride gradient . Protein concentration was determined using a Microplate BCA ™ protein assay kit (Thermo Fischer Scientific, Waltham, MA, United States), in which bovine serum albumin was used as a protein standard. Example 5: Pre-treated corn residue hydrolysis test
[000399] The corn residue was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1.4% by weight sulfuric acid at 165 ° C and 107 psi 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.
[000400] 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 ground, unwashed PCS (dry weight 32.35%) was prepared by grinding the complete PCS sludge in a wet multi-purpose grinder Cosmos ICMG 40 (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.
[000401] 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.
[000402] After hydrolysis, 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.
[000403] 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).
[000404] 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. To calculate the total conversion, the glucose and cellobiose values have been combined. The cellobiose concentration was multiplied by 1.053, in order to convert it into glucose equivalents and add it to the glucose concentration. The degree of total cellulose conversion was calculated using the following equation:% conversion = ([glucose concentration + 1.053 x (cellobiose concentration)] / [(glucose concentration + 1.053 x (cellobiose concentration) in a limit digestion ]) x 100. The 1.053 factor for cellobiosis takes into account the increase in mass when cellobiose is converted to glucose. In order to calculate the% conversion, a 100% conversion point was adjusted based on a cellulase control (50-100 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 were averaged and the standard deviation was calculated. Example 6: Preparation of cellobiohydrolase I from Aspergillus fumigatus NN055679 Cel7A
[000405] A tfasty search (Pearson et al., 1997, Genomics 46: 24-36) of the partial sequence of the Aspergillus fumigatus genome (The Institute for Genomic Research, Rockville, MD) was performed using a cellobiohydrolase protein sequence as input Cel7 from Trichoderma reesei (accession number P00725). Several genes have been identified as possible homologues of the GH7 family, based on a high degree of similarity with the input sequence at the amino acid level. A genomic region with significant identity for the input sequence was chosen for further study, and the corresponding gene was called cel7A.
[000406] The two synthetic primer oligonucleotides shown below were determined to PCR amplify a cellobiohydrolase I cel7A gene from Aspergillus fumigatus NN055679 (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence] ), from the genomic DNA of Aspergillus fumigatus prepared in the manner described in WO 2005/047499. Sense primer oligonucleotide: 5'-gggcATGCTGGCCTCCACCTTCTCC-3 '(SEQ ID NO: 15) reverse primer oligonucleotide: 5'-gggttaattaaCTACAGGCACTGAGAGTAA-3 ’(SEQ ID NO: 16)
[000407] Capital letters represent the coding sequence. The remainder of the sequence provides restriction endonuclease sites for Sph I and Pac I in the sense and reverse sequences, respectively. Using these primer oligonucleotides, the Aspergillus fumigatus cel7A gene was amplified using standard PCR methods and the reaction product was isolated by 1% agarose gel electrophoresis using TAE buffer and was purified using a QIAQUICK gel extraction kit ® (QIAGEN Inc., Valencia, CA, United States), according to the manufacturer's instructions.
[000408] The fragment was digested with Sph I and Pac I and ligated into the expression vector pAlLo2, also digested with Sph I and Pac I, according to standard procedures. The ligation products were transformed into E. coli XL10 SOLOPACK® cells (Stratagene, La Jolla, CA, United States) according to the manufacturer's instructions. A transforming E. coli containing a plasmid of the correct size was detected by restricted digestion, and DNA from the plasmid was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, CA, United States). DNA sequencing of the inserted gene from this plasmid was performed with an automated DNA sequencer Applied Biosystems Model 377 XL (Perkin-Elmer / Applied Biosystems, Inc., Foster City, CA, United States) using finisher dye chemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) 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 nucleotide sequence has been shown to combine with the genomic sequence determined by TIGR (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence]). The resulting plasmid was called pEJG93.
[000409] Aspergillus oryzae JaL250 protoplasts (WO 99/61651) prepared according to the method of Christensen et al., 1988, supra and transformed with 5 μg of pEJG93 (as well as pAlLo2 as a control vector) were used to transform Aspergillus oryzae JaL250. The transformation yielded about 100 transformants. Ten transformants were isolated on individual PDA plates.
[000410] The confluent PFA plates from five of the ten transformants were washed with 5 ml of 0.01% TWEEN® 20, and separately inoculated in 25 ml of MDU2BP medium in 125 ml glass shake flasks and incubated at 34 ° C. ° C, 250 rpm. Five days after incubation, 0.5 μL of the supernatant from each culture was analyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad, CA, United States), according to the manufacturer's instructions. The SDS-PAGE profiles of the cultures showed that one of the transformants had a main band of approximately 70 kDa. This transformant was called Aspergillus oryzae JaL250EJG93.
[000411] Five hundred ml of medium in the shake flask was added to a 2800 ml shake flask. The medium in the shake flask was composed of 45 g of maltose, 2 g of K2HPO4, 12 g of KH2PO4, 1 g of NaCl, 1 g of MgSOrVIFO, 7 g of yeast extract, 2 g of urea, 0.5 ml of trace element solution, and deionized water to 1 liter. The trace element solution was composed of 13.8 g of FcSOrVI I2O, 14.3 g of ZnSO ^ IFO, 8.5 g of MnSO ^ O, 2.5 g of ('iiSO-51 FO, 0.5 g of NiCk-óHO, 3 g of citric acid, and deionized water to 1 liter Two shaking flasks were inoculated with a suspension of a PDA plate of Aspergillus oryzae JaL250EJG93 with 0.01% TWEEN® 80, and incubated at 34 ° C on an orbital shaker at 200 rpm for 120 hours The broth was filtered using a 0.7 μm Whatman GF / F glass filter (Whatman, Piscataway, NJ, United States), followed by a 0, 22 μm EXPRESS ™ Plus (Millipore, Bedford, MA, United States).
[000412] The filtered broth was concentrated and the buffer was exchanged for 20 mM Tris-HCl pH 8.5 using a tangential flow concentrator (Pall Filtron, Northborough, MA, United States), equipped with a 10 kDa polyethersulfone membrane (Pall Filtron, Northborough, MA, United States). Protein concentration was determined using a BCA ™ microplate protein assay kit, in which bovine serum albumin was used as a protein standard. Example 7: Preparation of endoglucanase II from Thermoascus aurantiacus CGMCC 0670 Cel5A
[000413] Thermoascus aurantiacus CGMCC 0670 DNA, which encodes a Cel5A endoglucanase II (SEQ ID NO: 17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid sequence]), was cloned according to the procedure a follow. The T. aurantiacus strain was grown in 80 mL of CBH1 medium (2.5% AVICEL®, 0.5% glucose, 0.14% (NH4) 2SO4) in Erlenmeyer flasks with grooves in the bottom of 500 mL, at 45 ° C for 3 days, with stirring at 165 rpm. Mycelia were collected by centrifugation at 7,000 rpm for 30 minutes and stored at -80 ° C before use for RNA extraction. RNA was isolated from 100 mg of mycelia using an RNEASY® Plant kit (QIAGEN Inc., Valencia, CA, United States).
[000414] The cDNA for Thermoascus aurantiacus endoglucanase was isolated by RT PCR using a 3 'RACE system and a 5' RACE system (Invitrogen, Life Technologies, Carlsbad, CA, United States), and the BG025-1 primer oligonucleotides, BG025-2, BG025-3, and BG025-4 shown below with the N-terminal amino acids. Primer oligonucleotide BG025-1: 5'- AA (T / C) GA (A / G) TC (T / C / A / G) GG (T / C / A / G) GC (T / C / A / G ) GAATT-3 '(SEQ ID NO: 19) Oligonucleotide primer BG025-2: 5'- AA (T / C) GA (A / G) TC (T / C / A / G) GG (T / C / A / G) GC (T / C / A / G) GAGTT-3 '(SEQ ID NO: 20) BG025-3 primer oligonucleotide: 5'- AA (T / C) GA (A / G) AG (T / C ) GG (T / C / A / G) GC (T / C / A / G) GAATT-3 '(SEQ ID NO: 21) Primer BG025-4 oligonucleotide: 5'-AA (T / C) GA (A / G) AG (T / C) GG (T / C / A / G) GC (T / C / A / G) GAGTT-3 '(SEQ ID NO: 22)
[000415] RT PCR products were ligated into plasmid pGEM®-T using a pGEM®-T vector system (Promega, Madison, WI, United States) and transformed into the E. coli strain JM109. A single clone carrying a plasmid called pBGC1009, containing the endoglucanase cDNA, was isolated.
[000416] PCR primer oligonucleotides were determined to amplify the cDNA encoding T aurantiacus endoglucanase from plasmid pBGC1009. The restriction enzyme Bsp HI and Pac I sites were incorporated for cloning in alignment on the Aspergillus oryzae expression plasmid, pBM120a (WO 2006/039541). Oligonucleotide primer 996261: 5'-GATCTCATGAAGCTCGGCTCTCTCGT-3 '(SEQ ID NO: 23) BspHI Oligonucleotide primer 996167: 5'-TTAATTAATCAAAGATACGGAGTCAAAATAGG- 3 ’(SEQ ID NO: 24)
[000417] The fragment of interest was amplified by PCR using an EXPAND ™ high-fidelity PCR system. The PCR amplification reaction mixture contained 1 μL of pBGC1009 0.09 μg / μL, 1 μL of primer oligonucleotide 996261 (50 pmol / μL), 1 μL of primer oligonucleotide 996167 (50 pmol / μL), 5 μL of buffer of 10X PCR with 15 mM MgCl2, 1 μL of dNTP mixture (10 mM each), 37.25 μL of water, and 0.75 μL (3.5 U / μL) of DNA polymerase mixture. An EPPENDORF® MASTERCYCLER® thermocycler (Eppendorf Scientific, Inc., Westbury, NY, United States) was used to amplify the fragment and programmed for 1 cycle at 94 ° C for 2 minutes; 10 cycles each at 94 ° C for 15 seconds, 55 ° C for 30 seconds, 72 ° C for 1.5 minutes; 15 cycles each at 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 72 ° C for 1.5 minutes, and 5 seconds of stretching in each successive cycle; 1 cycle at 72 ° C for 7 minutes; and a retention at 4 ° C.
[000418] The 1008 bp PCR product was purified by 1% agarose gel electrophoresis using TAE buffer, excised from the gel and purified using a QIAQUICK® gel purification kit (QIAGEN Inc., Valencia, CA, States United). The purified product was connected directly to pCR®2.1-TOPO®, according to the manufacturer's instructions. The resulting plasmid was called pBM124a.
[000419] Plasmid pBM124a was digested with Bsp HI and Pac I, purified by 1% agarose gel electrophoresis using TAE buffer, excised from the gel, and purified using a QIAQUICK® gel purification kit. The plasmid fragment was ligated to the vector pBM120a, which was digested with Nco I and Pac I. The resulting expression plasmid was determined to be pBM123a. Plasmid pBM123a contains a duplicate NA2-TPI promoter that directs the expression of the Thermoascus aurantiacus endoglucanase cDNA clone, the AMG finisher, and amdS as a selectable marker.
[000420] Aspergillus oryzae BECh2 protoplasts (WO 2000/139322) were prepared according to the method of Christensen et al., 1988, supra, and transformed with 6 μg of pBM123a. The main transformants were selected on COVE plates for 5 days. The transformants had their spores purified twice before analyzing the shake flask.
[000421] The spores of the transformants were inoculated in 25 ml of MY25 medium in 125 ml shake flasks. The cultures were incubated at 34 ° C, 200 rpm on a platform shaker for five days. On day 3 and day 5, culture supernatants were collected and clarified by centrifugation to remove mycelia. Twenty microliters of supernatant from the three transformants were analyzed using a 10-20% gradient SDS-PAGE gel without CRITERION® dye (Bio-Rad Laboratories, Inc., Hercules, CA, United States), according to the manufacturer's instructions . The SDS-PAGE profiles showed that all transformants had a new main band of approximately 32 kDa. A transformant was chosen and named A. oryzae EXP00858.
[000422] 500 mL plastic flasks, without grooves in the bottom, containing 100 mL of SY50 medium were inoculated with 0.1 mL of a spore stock of A. oryzae EXP00858, and incubated at 34 ° C, 200 rpm for 24 hours to produce a germ culture. Fifty mL of the germ culture was inoculated in a 2-liter fermentation tank, containing 2 liters of medium composed of 0.5 g of pluronic acid, 30 g of sucrose, 2 g of MgSOr7II2O. 2 g of anhydrous KH2PO4, 1 g of citric acid ^ 2 g of (NH4) 2SO4, 1 g of K2SO4, 20 g of yeast extract, and 0.5 g of trace metal solution AMG 200X, pH 5.0. The fermentation was fed with a supply of maltose. The pH was controlled using 5 N H3PO4 and 15% NH4OH and was kept at 5.0, and then increased to 5.25. The temperature was maintained at 34.0 ° C +/- 1.0 ° C. The agitation was 1,000 rpm. The air flow was 1.0 vvm.
[000423] A 200 ml volume of the cellless supernatant was diluted to 1 liter with deionized water. The pH was adjusted to 8 and the sample was sterilized by filtration using a 0.22 μm polyethersulfone filter (PES). The sterilized filtered sample was loaded onto a 250 mL Q SEPHAROSE ™ rapid flow column (GE Healthcare, Piscataway, NJ, United States) pre-equilibrated with 25 mM Tris pH 8. The enzyme was eluted from the column with a 0 to 1 M NaOH gradient in the same buffer. Fractions containing beta-glucosidase activity were grouped (400 mL) and the enzyme concentration was calculated from the theoretical extinction coefficient and the sample absorbance at 280 nm. Example 8: Preparation of Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide with better cellulolytic activity
[000424] The GH61A polypeptide from Thermoascus aurantiacus CGMCC 0583 with better cellulolytic activity (SEQ ID NO: 25 [DNA sequence] and SEQ ID NO: 26 [deduced amino acid sequence]) was prepared recombinantly, according to WO 2005 / 074656, using Aspergillus oryzae JaL250 as a host. The recombinantly produced Thermoascus aurantiacus GH61A polypeptide was first concentrated by ultrafiltration using a 10 kDa membrane, the buffer was exchanged for 20 mM Tris-HCl pH 8.0, and then purified using a 100 mL Q SEPHAROSE® Big column Beads (GE Healthcare, Piscataway, NJ, United States) with 600 mL of a linear 0-600 mM NaCl gradient in the same buffer. The 10 mL fractions were collected and grouped based on SDS-PAGE.
[000425] The pooled fractions (90 mL) were then further purified using a 20 mL MONO Q® column (GE Healthcare, Piscataway, NJ, United States), with 500 mL of a linear 0-500 mM NaCl gradient in the same plug. The 6 mL fractions were collected and grouped based on SDS-PAGE. The pooled fractions (24 mL) were concentrated by ultrafiltration using a 10 kDa membrane, and chromatography was performed using a 320 mL SUPERDEX® 75 SEC column (GE Healthcare, Piscataway, NJ, United States) with isocratic elution of approximately 1 , 3 liters of 150 mM NaCl - 20 mM Tris-HCl, pH 8.0. The 20 mL fractions were collected and grouped based on SDS-PAGE. Protein concentration was determined using a BCA ™ microplate protein assay kit, in which bovine serum albumin was used as a protein standard. Example 9: Preparation of beta-glucosidase Cel3A from Aspergillus fumigatus NN055679
[000426] Aspergillus fumigatus Cel3A beta-glycosidase NN055679 (SEQ ID NO: 27 [DNA sequence] and SEQ ID NO: 28 [deduced amino acid sequence]) was prepared recombinantly, according to WO 2005/047499, using Trichoderma reesei RutC30 as a host.
[000427] The filtered broth was concentrated and the buffer was exchanged using a tangential flow concentrator, equipped with a 10 kDa polyethersulfone membrane with 20 mM Tris-HCl pH 8.5. The sample was loaded onto a high-performance Q SEPHAROSE® column (GE Healthcare, Piscataway, NJ, United States), equilibrated in 20 mM Tris, pH 8.5, and bound proteins were eluted with a linear sodium chloride gradient 0-600 mM. The fractions were concentrated and loaded onto a SUPERDEX® 75 HR 26/60 column from GE Healthcare, Piscataway, NJ, United States, equilibrated with 20 mM Tris - 150 mM sodium chloride, pH 8.5. Protein concentration was determined using a BCA ™ microplate protein assay kit, in which bovine serum albumin was used as a protein standard. Example 10: Preparation of GH10 xylanase from Aspergillus fumigatus NN055679
[000428] Aspergillus fumigatus GH10 xylanase NN055679 (xyn3) (SEQ ID NO: 29 [DNA sequence] and SEQ ID NO: 30 [deduced amino acid sequence]) was prepared recombinantly, according to WO 2006/078256, using Aspergillus oryzae BECh2 as a host.
[000429] The filtered broth was desalted and the buffer was exchanged for 20 mM Tris - 150 mM NaCl pH 8.5, using a HIPREP® 26/10 desalination column (GE Healthcare, Piscataway, NJ, United States) according to the manufacturer's instructions. Protein concentration was determined using a BCA ™ microplate protein assay kit, with bovine serum albumin as a protein standard. Example 11: Effect of cellobiohydrolase of the GH6 family of Talaromyces byssochlamydoides on the hydrolysis of unwashed PCS ground at 50-65 ° C, by an enzymatic composition at elevated temperature
[000430] The cellobiohydrolase of the GH6 family of Talaromyces byssochlamydoides was evaluated in an enzymatic composition at elevated temperature at 50 ° C, 55 ° C, 60 ° C and 65 ° C, using ground non-washed PCS as a substrate. The enzyme composition at elevated temperature included 40% cellobiohydrolase I Cel7A from Aspergillus fumigatus, 25% cellobiohydrolase from the GH6 family of Talaromyces byssochlamydoides polypeptide, 10% endoglucanase II Cel5A from Thermoascus aurantiacus, 15% cellulose GH Thermoascus aurantiacus, 5% beta-glucosidase Cel3A from Aspergillus fumigatus, and 5% GH10 xylanase from Aspergillus fumigatus (xyn3). The enzyme composition at elevated temperature was added to the PCS hydrolysis reactions in 3.0 mg of total protein per g of cellulose, and the hydrolysis results were compared with the results for a similar enzyme composition at elevated temperature, without the GH6 cellobiohydrolase. of T. byssochlamydoides (2.25 mg of protein per g of cellulose).
[000431] The assay was carried out in the manner described in example 5. The reactions of 1 ml with ground non-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 a single mixture at the beginning of hydrolysis.
[000432] The results shown in figure 1 demonstrated that at 50 ° C, 55 ° C, 60 ° C and 65 ° C the enzyme composition at elevated temperature, which included 25% cellobiohydrolase from the GH6 family of T. byssochlamydoides, significantly exceeded the enzymatic composition without T. byssochlamydoides cellobiohydrolase. Example 12: Preparation of Aspergillus fumigatus cellobiohydrolase
[000433] Aspergillus fumigatus cellobiohydrolase NN055679 (SEQ ID NO: 31 [DNA sequence] and SEQ ID NO: 32 [deduced amino acid sequence]) was prepared recombinantly in Aspergillus oryzae, as described in WO 2011/057140 . The filtered cellobiohydrolase broth GH6A from Aspergillus fumigatus had its buffer exchanged for 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 for 1.2 M ammonium sulfate - 20 mM Tris pH 8.0. The balanced protein was loaded onto a PHENYL SEPHAROSE ™ 6 (sub high) fast flow column (GE Healthcare, Piscataway, NJ, United States), 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. Protein concentration was determined using a BCA ™ microplate protein assay kit, with bovine serum albumin as a protein standard. Example 13: Preparation of Myceliophthora thermophila CBS 202.75 cellobiohydrolase GH6A
[000434] Myceliophthora thermophila CBS 117.65 cellobiohydrolase GH6A (SEQ ID NO: 33 [DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) was prepared recombinantly in Aspergillus oryzae, as described in WO 2011 / 057140. The filtered cellobiohydrolase broth GH6A from Myceliophthora thermophila had its buffer exchanged for 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 broth with exchanged buffer was adjusted to 20 mM Tris-HCl pH 8.0 with 1.2 M ammonium sulfate, and applied to a PHENYL SEPHAROSE ™ 6 (sub-high) fast flow column (GE Healthcare, Piscataway, NJ, United States), balanced with 20 mM Tris pH 8.0 and 1.2 M ammonium sulfate. The bound proteins were eluted with 20 mM Tris pH 8.0 without any ammonium sulfate and the fractions were grouped. The pooled fractions were concentrated and the buffer was exchanged for 20 mM Tris pH 8.0, using a 10 kDa MWCO Amicon Ultra centrifugal concentrator (Millipore, Bedford, MA, United States). Protein concentration was determined using a BCA ™ microplate protein assay kit, with bovine serum albumin as a protein standard. Example 14: Evaluation of three cellobriohydrolases in washed and ground PCS at 50-65 ° C
[000435] Three cellobriohydrolases (Talaromyces byssochlamydoides cellobiohydrolase, Aspergillus fumigatus cellobiohydrolase GH6A and Myceliophthora thermophila cellobiohydrolase GH6A) were evaluated at 1 mg of protein per g of cellulose at 65 ° C, 55 ° C, 55 ° C, 55 ° C, 50 ° C, 55 ° C, C, using washed and ground PCS as a substrate with 1 mg of protein per g of cellulose of beta-glucosidase from Aspergillus fumigatus.
[000436] The test was carried out in the manner described in example 5. The reactions of 1 ml with washed and ground PCS (5% insoluble solids) were carried out for 72 hours in 50 mM sodium acetate buffer, pH 4.0, 4.5 and 5.0, containing 1 mM manganese sulfate. All reactions were carried out in triplicate and involved a single mixture at the beginning of the hydrolysis.
[000437] The results shown in figure 2 demonstrated that at 50 ° C, 55 ° C, 60 ° C and 65 ° C, and pH 4.0, 4.5 and 5.0, the GH6 cellobiohydrolase from Talaromyces byssochlamydoides (Tb6 ) yielded a greater conversion of washed and milled PCS than the cellobiohydrolase GH6A of Aspergillus fumigatus (Af6A) or cellobiohydrolase GH6A of Myceliophthora thermophila (Mt6A), when compared under the same temperature and pH conditions. Example 15: Determination of Td by differential scanning calorimetry
[000438] The thermostability of Talaromyces byssochlamydoides cellobiohydrolase GH6 was determined by differential scanning calorimetry (DSC), using a VP-Capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, United States). The thermal denaturation temperature, Td (° C), was obtained as the highest denaturation peak (main endothermic peak) in thermograms (Cp vs. T), obtained after heating the enzyme solution in 50 mM sodium acetate, pH 5.0, at a constant programmed heating rate of 200 K / hr. The sample and reference solutions (approximately 0.2 mL) were placed in the calorimeter (reference: buffer without enzyme) from the storage conditions at 10 ° C, and were thermally pre-equilibrated for 20 minutes at 20 ° C, before of the DSC scan from 20 ° C to 110 ° C. The Td for Talaromyces byssochlamydoides cellobiohydrolase GH6 was determined to be 74 ° C +/- 1 ° C at pH 5.0. Biological Material Deposit
[000439] The following biological material was deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, IL, United States, and provided the following accession number : Deposit Access number Deposit date E. coli (pAJ227) NRRL B-50474 March 1, 2011
[000440] The strain was deposited under conditions that guarantee that access to the crop will be available pending this patent application determined by foreign patent laws to be entitled to it. The deposit represents the substantially pure culture of the deposited strain. The deposit is available in the manner required by foreign patent laws in countries where duplicates of the submitted application, or their progeny, are deposited. However, it can be understood that the availability of a deposit does not constitute a license to practice the invention submitted in derogation from the patent rights granted by government action.
[000441] The present invention is further described by the following numbered paragraphs:
[000442] [1] A polypeptide isolate with cellobiohydrolase activity, selected from the group consisting of: (a) a polypeptide with at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, 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 mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, (ii) its DNAc sequence, or (iii) the complement total size of (i) or (ii); (c) a polypeptide encoded by a polynucleotide of at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, 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 its cDNA sequence; (d) a variant of the mature polypeptide of SEQ ID NO: 2 which comprises a substitution, elimination 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.
[000443] [2] The polypeptide of paragraph 1, with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 SEQ polypeptide ID NO: 2.
[000444] [3] The polypeptide of paragraph 1 or 2, which is encoded by a polynucleotide that hybridizes under conditions of very high severity in (i) the sequence encoding the mature polypeptide of SEQ ID NO: 1, (ii) a its cDNA sequence, or (iii) the full size complement of (i) or (ii).
[000445] [4] The polypeptide of any of paragraphs 1-3, which is encoded by a polynucleotide with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% of sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1 or the cDNA sequence thereof.
[000446] [5] The polypeptide of any of paragraphs 1-4, which comprises or consists of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2.
[000447] [6] The polypeptide of paragraph 5, wherein the mature polypeptide has amino acids 20 to 456 of SEQ ID NO: 2.
[000448] [7] The polypeptide of any of paragraphs 1-4, which is a variant of the mature polypeptide of SEQ ID NO: 2 which comprises a substitution, deletion and / or insertion at one or more positions.
[000449] [8] The polypeptide of paragraph 1, which is a fragment of SEQ ID NO: 2, in which the fragment exhibits cellobiohydrolase activity.
[000450] [9] The polypeptide of paragraph 5, which is encoded by a polynucleotide that is identical to the polynucleotide contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474.
[000451] [10] The polypeptide of paragraph 5, which is identical to the polypeptide encoded by the polynucleotide contained in plasmid pAJ227, which is contained in E. coli NRRL B-50474.
[000452] [11] A polypeptide isolate comprising a catalytic domain selected from the group consisting of: (a) a catalytic domain with at least 90%, for example, 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 amino acids 98 to 456 of SEQ ID NO: 2; (b) a catalytic domain encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) nucleotides 397 to 1786 of SEQ ID NO: 1, (ii) its DNAc sequence, or (iii) the size complement total of (i) or (ii); (c) a catalytic domain encoded by a polynucleotide of at least 90%, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99% or 100% sequence identity with nucleotides 397 to 1786 of SEQ ID NO: 1 or the cDNA sequence thereof; (d) an amino acid variant 98 to 456 of SEQ ID NO: 2 which comprises a substitution, deletion and / or insertion at one or more positions; and (e) a fragment of a catalytic domain of (a), (b), (c), or (d) that exhibits cellobiohydrolase activity.
[000453] [12] The polypeptide of paragraph 11, which further comprises a cellulose binding domain.
[000454] [13] A polypeptide isolate comprising a cellulose binding domain operably linked to a catalytic domain, wherein the cellulose binding domain is selected from the group consisting of: (a) a cellulose binding domain with at least 90%, for example, 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 amino acids 20 to 56 of SEQ ID NO: 2; (b) a cellulose binding domain encoded by a polynucleotide that hybridizes under conditions of very high severity to (i) nucleotides 58 to 273 of SEQ ID NO: 1, (ii) its DNAc sequence, or (iii) the full size complement of (i) or (ii); (c) a cellulose binding domain encoded by a polynucleotide of at least 90%, for example, 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 nucleotides 58 to 273 of SEQ ID NO: 1 or the cDNA sequence thereof; (d) an amino acid variant 20 to 56 of SEQ ID NO: 2 which comprises a substitution, deletion and / or insertion at one or more positions; and (e) a fragment of (a), (b), (c), or (d) that exhibits cellulose binding activity.
[000455] [14] The polypeptide of paragraph 13, wherein the catalytic domain is obtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, for example, an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, oxidase, laccase, laccase, lipase peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase or beta-xylosidase.
[000456] [15] A composition that comprises the polypeptide of any of paragraphs 1-14.
[000457] [16] An isolated polynucleotide that encodes the polypeptide of any of paragraphs 1-14.
[000458] [17] A nucleic acid construct or expression vector that comprises the polynucleotide of paragraph 16 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
[000459] [18] A recombinant host cell comprising the polynucleotide of paragraph 16 operably linked to one or more control sequences that direct the production of the polypeptide.
[000460] [19] A method of producing the polypeptide of any of paragraphs 1-14 which comprises: (a) cultivating a cell, which in its wild type produces the polypeptide, under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.
[000461] [20] A method of producing a polypeptide with cellobiohydrolase activity that comprises: (a) culturing the host cell of paragraph 18 under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[000462] [21] A transgenic plant, part of the plant or plant cell transformed with a polynucleotide that encodes the polypeptide of any of paragraphs 1-14.
[000463] [22] A method of producing a polypeptide with cellobiohydrolase activity that comprises: (a) cultivating the transgenic plant or plant cell of paragraph 21 under conditions that lead to the production of the polypeptide; and (b) recovering the polypeptide.
[000464] [23] A method of producing a mutant of a parental cell that comprises inactivating a polynucleotide that encodes the polypeptide of any of paragraphs 1-14, which results in the mutant that produces less of the polypeptide than the parental cell.
[000465] [24] A mutant cell produced by the method of paragraph 23.
[000466] [25] The mutant cell of paragraph 24, which further comprises a gene encoding a natural or heterologous protein.
[000467] [26] A method of producing a protein comprising: (a) cultivating the mutant cell of paragraph 24 or 25 under conditions that lead to the production of the protein; and (b) recovering the protein.
[000468] [27] A double-stranded inhibitory RNA molecule (RNAds) comprising a subsequence of the polynucleotide of paragraph 16, where optionally the RNAds is an RNAsi or an RNAmi molecule.
[000469] [28] The double-stranded inhibitory RNA molecule (RNAds) of paragraph 27, which has about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in size.
[000470] [29] A method of inhibiting the expression of a polypeptide with cellobiohydrolase activity in a cell, comprising administering to the cell or expressing in the cell the double-stranded inhibitory RNA molecule (RNAds) of paragraph 27 or 28.
[000471] [30] A cell produced by the method of paragraph 29.
[000472] [31] The cell in paragraph 30, which further comprises a gene encoding a natural or heterologous protein.
[000473] [32] A method of producing a protein comprising: (a) cultivating the cell of paragraph 30 or 31 under conditions that lead to the production of the protein; and (b) recovering the protein.
[000474] [33] An isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2.
[000475] [34] A nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 33, wherein the gene is foreign to the polynucleotide encoding the signal peptide.
[000476] [35] A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 33, wherein the gene is foreign to the polynucleotide encoding the signal peptide.
[000477] [36] A method of producing a protein comprising: (a) culturing a recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 33, wherein the gene is foreign to the polynucleotide encoding the signal peptide, under conditions that lead to the production of the protein; and (b) recovering the protein.
[000478] [37] A method for degrading or converting a cellulosic material comprising: treating the cellulosic material with an enzymatic composition in the presence of the polypeptide with cellobiohydrolase activity of any of paragraphs 1-14.
[000479] [38] The method of paragraph 37, in which the cellulosic material is pre-treated.
[000480] [39] The method of paragraph 37 or 38, wherein 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.
[000481] [40] The method of paragraph 39, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[000482] [41] The method of paragraph 39, wherein hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
[000483] [42] The method of any of paragraphs 37-41, which further comprises recovering degraded cellulosic material.
[000484] [43] The method of paragraph 42, wherein the degraded cellulosic material is sugar.
[000485] [44] The method of paragraph 43, in which sugar is selected from the group consisting of glucose, xylose, mannose, galactose and arabinose.
[000486] [45] A method of producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzymatic composition in the presence of the polypeptide with cellobiohydrolase activity of any of paragraphs 1-14; (b) fermenting the saccharified cellulosic material with one or more of the fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[000487] [46] The method of paragraph 45, in which the cellulosic material is pre-treated.
[000488] [47] The method of paragraph 45 or 46, wherein 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.
[000489] [48] The method of paragraph 47, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[000490] [49] The method of paragraph 47, in which hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
[000491] [50] The method of any of paragraphs 45-49, in which steps (a) and (b) are carried out simultaneously in simultaneous saccharification and fermentation.
[000492] [51] The method of any of paragraphs 45-50, wherein 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.
[000493] [52] A method of fermenting a cellulosic material comprising: fermenting the cellulosic material with one or more of the fermenting microorganisms, wherein the cellulosic material is saccharified with an enzymatic composition in the presence of the polypeptide with cellobiohydrolase activity of any of the paragraphs 1-14.
[000494] [53] The method of paragraph 52, in which the fermentation of the cellulosic material synthesizes a fermentation product.
[000495] [54] The method of paragraph 53, which further comprises recovering the fermentation product from fermentation.
[000496] [55] The method of any of paragraphs 52-54, in which the cellulosic material is pre-treated before saccharification.
[000497] [56] The method of any of paragraphs 52-55, wherein 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, a expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin.
[000498] [57] The method of paragraph 56, in which cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[000499] [58] The method of paragraph 56, in which hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
[000500] [59] The method of any of paragraphs 53-58, wherein 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.
[000501] [60] A complete broth formulation or cell culture composition comprising a polypeptide from any of paragraphs 1-14.
[000502] 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 aspect is intended to be within the scope of this invention. In fact, various modifications of the invention, in addition to those shown and described here, 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 (12)
[0001]
1. Transgenic microbial host cell, characterized by the fact that it comprises the polynucleotide that encodes a polypeptide having cellobiohydrolase activity, in which the polynucleotide consists of SEQ ID NO: 1 or nucleotides 58 to 1786 of SEQ ID NO: 1.
[0002]
2. Transgenic microbial host cell according to claim 1, characterized by the fact that the polypeptide having cellobiohydrolase activity consists of SEQ ID NO: 2 or amino acids 20 to 456 of SEQ ID NO: 2.
[0003]
3. Method for producing a polypeptide, characterized by the fact that it comprises: (a) cultivating the transgenic microbial host cell as defined in claim 1 or 2, under conditions suitable for the production of the polypeptide; and (b) recovering the polypeptide.
[0004]
4. Transgenic microbial host cell, characterized by the fact that it comprises a nucleic acid construct or an expression vector comprising a gene encoding a protein, operationally linked to a polynucleotide that encodes a signal peptide consisting of amino acids 1 to 19 of SEQ ID NO : 2, where the gene is foreign to the polynucleotide that encodes the signal peptide.
[0005]
5. Method for producing a protein, characterized by the fact that it comprises: (a) cultivating the transgenic microbial host cell as defined in claim 4, under conditions that lead to the production of the protein; and (b) recovering the protein.
[0006]
6. Method for degrading a cellulosic material, characterized by the fact that it comprises: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having cellobiohydrolase activity, consisting of SEQ ID NO: 2 or amino acids 20 to 456 of SEQ ID NO: 2.
[0007]
Method according to claim 6, characterized by the fact that it additionally comprises recovering the degraded cellulosic material.
[0008]
8. Method for producing a fermentation product, characterized by the fact that it comprises: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellobiohydrolase activity consisting of SEQ ID NO: 2 or amino acids 20 to 456 SEQ ID NO: 2; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from fermentation.
[0009]
9. Method for fermenting a cellulosic material, characterized by the fact that it comprises: 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 a polypeptide having cellobiohydrolase activity, consisting of SEQ ID NO: 2 or the mature polypeptide thereof.
[0010]
10. Method according to claim 9, characterized by the fact that the fermentation of the cellulosic material produces a fermentation product.
[0011]
11. Method according to claim 10, characterized by the fact that it additionally comprises recovering the fermentation product from the fermentation.
[0012]
12. Nucleic acid construction or an expression vector, characterized by the fact that it comprises a polynucleotide that encodes a polypeptide having cellobiohydrolase activity, in which the polynucleotide consists of SEQ ID NO: 1 or nucleotides 58 to 1786 of SEQ ID NO: 1 ; operationally 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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2019-06-18| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-01-14| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-07-21| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-02-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-30| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|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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US201161450494P| true| 2011-03-08|2011-03-08|

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US61/450,494|2011-03-08|

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PCT/US2012/022749|WO2012103350A1|2011-01-26|2012-01-26|Polypeptides having cellobiohydrolase activity and polynucleotides encoding same|

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