Hosts and fermentation processes for cellulase production
First Claim
1. A fed-batch or continuous fermentation process for the production of a cellulase mixture, said process comprising:
- a) providing a modified host filamentous fungus that overexpresses a Xyr1 transcription factor, said Xyr1 transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;
27 (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX2CX6CX5-12CX2CX6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)3-like motif within cellulase and/or hemicellulase promoter sequences; and
b) culturing the modified host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 60 wt % to about 100 wt % of a hemicellulose-derived carbohydrate selected from the group consisting of xylo-oligosaccharides, arabinoxylo-oligosaccharides, D-xylose, xylobiose, L-arabinose, and combinations thereof, and about 0 wt % of a cellulase-inducing carbohydrate selected from the group consisting of cellulose, lactose, cellobiose, sophorose, gentiobiose and a combination thereof, to produce a cellulase mixture;
wherein the fermentation process produces at least 10 g/L of the cellulase mixture, cellulase components represent at least 40 wt % of the total protein in the cellulase mixture, and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium.
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Abstract
A fermentation process for the production of cellulase mixtures is provided. The process comprises providing a genetically modified host filamentous fungus that overexpresses a Xyr1 transcription factor and/or that is partially or completely deficient in expressing one or more hemicellulase enzyme. The host filamentous fungus is cultured in a medium comprising a carbon source. The carbon source contains from about 60 wt % to about 100 wt % hemicellulose-derived carbohydrate and less than 5% of a cellulase-inducing carbohydrate or contains from about 25 wt % to about 100% wt % hemicellulose-derived sugar alcohol in combination with from about 0 wt % to about 75 wt % glucose, glycerol or a combination thereof.
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Citations
46 Claims
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1. A fed-batch or continuous fermentation process for the production of a cellulase mixture, said process comprising:
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a) providing a modified host filamentous fungus that overexpresses a Xyr1 transcription factor, said Xyr1 transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;
27 (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX2CX6CX5-12CX2CX6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)3-like motif within cellulase and/or hemicellulase promoter sequences; andb) culturing the modified host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 60 wt % to about 100 wt % of a hemicellulose-derived carbohydrate selected from the group consisting of xylo-oligosaccharides, arabinoxylo-oligosaccharides, D-xylose, xylobiose, L-arabinose, and combinations thereof, and about 0 wt % of a cellulase-inducing carbohydrate selected from the group consisting of cellulose, lactose, cellobiose, sophorose, gentiobiose and a combination thereof, to produce a cellulase mixture; wherein the fermentation process produces at least 10 g/L of the cellulase mixture, cellulase components represent at least 40 wt % of the total protein in the cellulase mixture, and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium. - View Dependent Claims (2, 3, 4, 5, 15, 17, 18, 21, 22, 23, 27, 29, 31, 33, 35, 37, 38, 44)
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6. A fermentation process for the production of a cellulase mixture, said process comprising:
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a) genetically modifying a host filamentous fungus to overexpress a Xyr1 transcription factor, said Xyl transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;
27 (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX2CX6CX5-12CX2CX6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)3-like motif within cellulase and/or hemicellulase promoter sequences; andb) culturing the host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 60 wt % to about 100 wt % of a hemicellulose-derived carbohydrate selected from the group consisting of xylo-oligosaccharides, arabinoxylo-oligosaccharides, D-xylose, xylobiose, L-arabinose, and combinations thereof, and about 0 wt % of a cellulase-inducing carbohydrate selected from the group consisting of cellulose, lactose, cellobiose, sophorose, gentiobiose and a combination thereof, to produce a cellulase mixture; wherein the fermentation process produces at least 10 g/L of the cellulase mixture, and wherein cellulase components represent at least 40 wt % of the total protein in the cellulase mixture and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 16, 19, 20, 24, 25, 26, 28, 30, 32, 34, 36, 39, 40, 45)
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41. A fermentation process for the production of a cellulase mixture, said process comprising:
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a. providing a modified host filamentous fungus that overexpresses a Xyr1 transcription factor, said Xyr1 transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;
27, (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX2CX6CX5-12CX2CX6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)3-like motif within cellulase and/or hemicellulase promoter sequences; andb. culturing the modified host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 25 wt % to about 100 wt % of a hemicellulose-derived sugar alcohol, about 0 wt % of a cellulase-inducing carbohydrate and from about 0 wt % to about 75 wt % glucose, glycerol or a combination thereof, to produce a cellulase mixture; wherein the fermentation process produces at least 10 g/L of the cellulase mixture, cellulase components represent at least 40 wt % of the total protein in the cellulase mixture, and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium. - View Dependent Claims (42, 43, 46)
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Specification