Compositions for enhanced enzyme production

Abstract

The present invention relates to compositions to induce production of proteins, e.g., enzymes, e.g., amylases or biomass degrading enzymes in a host cell, and methods for increasing the yield of the proteins, e.g., enzymes produced. Such compositions comprise a caramelized sugar product. The methods described herein can also be used to enhance processing of biomass materials, e.g., to produce sugar products.

What is claimed is: 
     
         1 . A method for inducing production of a biomass degrading enzyme comprising contacting a microorganism that produces the biomass degrading enzyme with a composition comprising a caramelized sugar product under conditions sufficient for production of a biomass degrading enzyme. 
     
     
         2 . The method of  claim 1 , wherein the microorganism is in a cell culture. 
     
     
         3 . The method of  claim 2 , wherein sugar is added to the cell culture prior to contacting the microorganism with the composition comprising a caramelized sugar product. 
     
     
         4 . The method of  claim 3 , wherein the microorganism is contacted with the composition comprising a caramelized sugar product when the cell culture is substantially free from sugar. 
     
     
         5 . The method of  claim 1  or  4 , wherein the caramelized sugar product is produced by caramelizing glucose, xylose, maltose, lactose, or a combination thereof. 
     
     
         6 . The method of  claim 5 , wherein the caramelized sugar product produced by caramelizing saccharified biomass comprises xylose and glucose. 
     
     
         7 . The method of  claim 1 ,  5  or  6 , wherein the caramelized sugar product comprises oligosaccharides, dehydration products of the oligosaccharides, hydration products of the oligosaccharides, disproportionation products of the oligosaccharides, colored aromatic products, or any combination thereof. 
     
     
         8 . The method of  claim 7 , wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasacchrides, hexasaccharides, or a combination thereof. 
     
     
         9 . The method of  claim 7 , wherein the caramelized sugar product is produced by caramelizing glucose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising glucose. 
     
     
         10 . The method of  claim 7 , wherein the caramelized sugar product is produced by caramelizing maltose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising maltose. 
     
     
         11 . The method of  claim 7 , wherein the caramelized sugar product is produced by caramelizing lactose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising lactose. 
     
     
         12 . The method of  claim 7 , wherein the caramelized sugar product is produced by caramelizing xylose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising xylose. 
     
     
         13 . The method of any of  claims 7 - 12 , wherein when the oligosaccharides comprise more than one species of oligosaccharides, trisaccharides are the most abundant species. 
     
     
         14 . The method of any of  claims 1 - 13 , wherein the biomass degrading enzyme comprises an amylase, e.g., an alpha, beta or gamma amylase, an endoglucanase, an exoglucanase, a cellobiase, a cellobiohydrolase, a xylanase, a ligninase, or a hemicellulase, or a combination thereof. 
     
     
         15 . The method of any of  claims 1 - 14 , wherein the composition further comprises an inducer biomass. 
     
     
         16 . The method of  claim 15 , wherein the inducer biomass comprises a starchy material or a starchy material that includes a cellulosic component. 
     
     
         17 . The method of  claim 15  or  16 , wherein the inducer biomass, e.g., starchy material or starchy material that includes a cellulosic component, comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof. 
     
     
         18 . The method of  claim 17 , wherein an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass,  miscanthus , cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof. 
     
     
         19 . The method of  claim 17 , wherein a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof. 
     
     
         20 . The method of  claim 17 , wherein a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof. 
     
     
         21 . The method of  claim 17 , wherein a general waste comprises manure, sewage, or offal, or a combination thereof. 
     
     
         22 . The method of any of  claims 15 - 21 , wherein the inducer biomass is pre-treated to reduce the recalcitrance of the inducer biomass, wherein the pre-treatment of the inducer biomass comprises exposure to an electron beam, bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. 
     
     
         23 . The method of  claim 1 , wherein the composition further comprises cellobiose, β-cellobiono-1,5-lactone, lactose, D-xylose, xylobiose, galactose, and sophorose. 
     
     
         24 . The method of any of  claims 1 - 23 , wherein the microorganism is a fungal cell. 
     
     
         25 . The method of any of  claims 1 - 23 , wherein the microorganism that produces a biomass degrading enzyme is from a species in the genera selected from  Bacillus, Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium  or  Trichoderma.    
     
     
         26 . The method of any of  claims 1 - 24 , wherein the microorganism that produces a biomass degrading enzyme is selected from  Aspergillus, Humicola insolens  ( Scytalidium thermophilum ),  Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Chrysosporium lucknowense, Trichoderma viride, Trichoderma reesei , or  Trichoderma koningii.    
     
     
         27 . The method of any of  claims 1 - 26 , wherein the microorganism is  T. reesei  or a variant thereof, e.g., RUT-NG14, PC3-7, QM9414, and RUT-C30. 
     
     
         28 . The method of any of  claims 1 - 27 , wherein the amount of biomass degrading enzyme produced is increased by at least 1-fold, e.g., at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, or more, compared the amount of biomass degrading enzyme produced by the microorganism without contacting with a caramelized sugar product. 
     
     
         29 . The method of any of  claims 1 - 27 , wherein the amount of biomass degrading enzyme produced is increased by at least 1-fold, e.g., at least 1.2-fold, 1.5 fold, 1.8-fold, 2-fold, compared the amount of biomass degrading enzyme produced by contacting the microorganism with a inducer biomass. 
     
     
         30 . The method of any of  claims 1 - 29 , wherein the biomass degrading enzyme comprises one or more, or all, of the enzymes listed in Table 1. 
     
     
         31 . The method of any of  claims 2 - 30 , further comprising separating the biomass degrading enzyme from a component of the cell culture, e.g., the microorganism or remaining inducer biomass, e.g., by chromatography or filtration. 
     
     
         32 . The method of  claim 31 , wherein the biomass degrading enzyme is purified from the cell culture. 
     
     
         33 . The method of any of  claims 1 - 32 , further comprising a step comprising:
 a) contacting the microorganism with a sugar in a first container under conditions such that the microorganism proliferates; and   b) transferring the microorganism to a second container, wherein the second container is larger, e.g., by volume, than the first container;   and wherein said step is performed prior to contacting the microorganism with the composition.   
     
     
         34 . The method of  claim 33 , wherein the step is repeated 1 or more times, e.g., 2, 3, 4, 5 times. 
     
     
         35 . A method for producing a product (e.g., hydrogen, a sugar, an alcohol) from a biomass, comprising:
 a) inducing the production of a biomass degrading enzyme using a method according to  claim 1 ;   b) providing a biomass; and   c) contacting the biomass with the microorganism of step (a) or the biomass degrading enzyme that has been separated or purified from the microorganism of step (a), under conditions suitable for production of the product.   
     
     
         36 . The method of  claim 35 , wherein the product is a sugar product. 
     
     
         37 . The method of  claim 36 , wherein the product is glucose and/or xylose. 
     
     
         38 . The method of any of  claims 35 - 37 , further comprising isolating the product. 
     
     
         39 . The method of  claim 38 , wherein the isolating of the product comprises precipitation, crystallization, chromatography, centrifugation, and/or extraction. 
     
     
         40 . The method of any of  claims 35 - 39 , wherein the biomass degrading enzyme is an endoglucanase, a cellobiase, a cellobiohydrolase, a xylanase, a ligninase, or a hemicellulase, or a combination thereof. 
     
     
         41 . The method of any of  claims 35 - 40 , wherein the biomass degrading enzyme comprises one or more, or all, of the enzymes listed in Table 1. 
     
     
         42 . The method of any of  claims 35 - 41 , further comprises a step of treating the biomass prior to step (c) to reduce the recalcitrance of the biomass. 
     
     
         43 . The method of  claim 42 , wherein the treating comprises exposure to an electron beam, bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze-grinding. 
     
     
         44 . The method of any of  claims 35 - 43 , wherein the biomass comprises a starchy material or a starchy material that includes a cellulosic component. 
     
     
         45 . The method any of  claims 35 - 44 , wherein the biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof; wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass,  miscanthus , cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         46 . The method of  claim 35 , wherein the caramelized sugar product is produced by caramelizing glucose, maltose, xylose, lactose, or a combination thereof. 
     
     
         47 . The method of  claim 46 , wherein the caramelized sugar product is produced by caramelizing saccharified biomass comprising xylose and glucose. 
     
     
         48 . The method of  claim 35  or  46 , wherein the caramelized sugar product comprises oligosaccharides, dehydration products of the oligosaccharides, hydration products of the oligosaccharides, disproportionation products of the oligosaccharides, colored aromatic products, or any combination thereof. 
     
     
         49 . The method of  claim 48 , wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasacchrides, hexasaccharides, or a combination thereof. 
     
     
         50 . The method of  claims 35 - 49  wherein the caramelized sugar product is produced by caramelizing glucose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising glucose. 
     
     
         51 . The method of  claims 35 - 49 , wherein the caramelized sugar product is produced by caramelizing maltose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising maltose. 
     
     
         52 . The method of  claims 35 - 49 , wherein the caramelized sugar product is produced by caramelizing lactose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising lactose. 
     
     
         53 . The method of  claims 35 - 49 , wherein the caramelized sugar product is produced by caramelizing xylose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising xylose. 
     
     
         54 . The method of any of  claims 48 - 53 , wherein when the oligosaccharides comprise more than one species of oligosaccharides, trisaccharides are the most abundant species. 
     
     
         55 . The method of any of  claims 47 - 54 , wherein the composition further comprises an inducer biomass. 
     
     
         56 . The method of  claim 55 , wherein the inducer biomass comprises a starchy material or a starchy material that includes a cellulosic component. 
     
     
         57 . The method of  claim 56 , wherein the inducer biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof; wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass,  miscanthus , cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         58 . The method of  claims 55 - 57 , wherein the inducer biomass is pre-treated to reduce the recalcitrance of the inducer biomass, wherein the pre-treatment of the biomass comprises exposure to an electron beam, bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. 
     
     
         59 . The method of any of  claims 35 - 58  wherein the inducer biomass is the same as the biomass provided in step (b). 
     
     
         60 . The method of  claim 35 , wherein the composition further comprises cellobiose, β-cellobiono-1,5-lactone, lactose, D-xylose, xylobiose, galactose, and sophorose. 
     
     
         61 . The method of any of  claims 35 - 60 , wherein the microorganism is a fungal cell. 
     
     
         62 . The method of any of  claims 35 - 60 , wherein the microorganism that produces a biomass degrading enzyme is from species in the genera selected from  Bacillus, Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium  or  Trichoderma.    
     
     
         63 . The method any of  claims 35 - 61 , wherein the microorganism that produces a biomass degrading enzyme is selected from  Aspergillus, Humicola insolens  ( Scytalidium thermophilum ),  Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Chrysosporium lucknowense, Trichoderma viride, Trichoderma reesei , or  Trichoderma koningii.    
     
     
         64 . The method of any of  claims 35 - 63  wherein the microorganism is  T. reesei  or a variant thereof. 
     
     
         65 . A composition comprising a caramelized sugar product for use in the method of any of  claims 1 - 64 . 
     
     
         66 . The composition of  claim 65 , wherein the caramelized sugar product is produced by caramelizing glucose, maltose, xylose, lactose, or a combination thereof. 
     
     
         67 . The composition of  claim 66 , wherein the caramelized sugar product is produced by caramelizing saccharified biomass comprising xylose and glucose. 
     
     
         68 . The composition of  claim 65  or  66 , wherein the caramelized sugar product comprises oligosaccharides, dehydration products of the oligosaccharides, hydration products of the oligosaccharides, disproportionation products of the oligosaccharides, colored aromatic products, or any combination thereof. 
     
     
         69 . The composition of  claim 68 , wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasacchrides, hexasaccharides, or a combination thereof. 
     
     
         70 . The composition of  claim 68 , wherein the caramelized sugar product is produced by caramelizing glucose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising glucose. 
     
     
         71 . The composition of  claim 68 , wherein the caramelized sugar product is produced by caramelizing maltose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising maltose. 
     
     
         72 . The composition of  claim 68 , wherein the caramelized sugar product is produced by caramelizing lactose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising lactose. 
     
     
         73 . The composition of  claim 68 , wherein the caramelized sugar product is produced by caramelizing xylose and wherein the oligosaccharides comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or a combination thereof, comprising xylose. 
     
     
         74 . The composition of any of  claims 68 - 73 , wherein when the oligosaccharides comprise more than one species of oligosaccharides, trisaccharides are the most abundant species. 
     
     
         75 . The composition of any of  claims 65 - 74  further comprising an inducer biomass. 
     
     
         76 . The composition of  claim 75 , wherein the inducer biomass comprises a starchy material comprising cellulose. 
     
     
         77 . The composition of  claim 76 , wherein the inducer biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof; wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass,  miscanthus , cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         78 . The composition of  claims 75 - 77  wherein the inducer biomass is pre-treated to reduce the recalcitrance of the inducer biomass, wherein the pre-treatment of the biomass comprises exposure to an electron beam, bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. 
     
     
         79 . The composition of any of  claims 75 - 77  wherein the composition further comprises cellobiose, β-cellobiono-1,5-lactone, lactose, D-xylose, xylobiose, galactose, and sophorose. 
     
     
         80 . A cell culture comprising a microorganism capable of producing a biomass degrading enzyme and a caramelized sugar product. 
     
     
         81 . The cell culture of  claim 80 , further comprising cell culture media. 
     
     
         82 . The cell culture of  claim 80  or  81 , wherein the biomass degrading enzyme is an endoglucanase, a cellobiase, a cellobiohydrolase, a xylanase, a ligninase, or a hemicellulase, or a combination thereof. 
     
     
         83 . The cell culture of any of  claims 80 - 82 , wherein the biomass degrading enzyme comprises one or more, or all, of the enzymes listed in Table 1. 
     
     
         84 . The cell culture of any of  claims 80 - 83 , wherein the caramelized sugar product is produced by caramelizing glucose, maltose, xylose, lactose, or a combination thereof. 
     
     
         85 . The cell culture of  claim 84 , wherein the caramelized sugar product is produced by caramelizing saccharified biomass comprising xylose and glucose. 
     
     
         86 . The cell culture of any of  claims 80 - 85 , wherein the caramelized sugar product comprises oligosaccharides, dehydration products of the oligosaccharides, hydration products of the oligosaccharides, disproportionation products of the oligosaccharides, colored aromatic products, or any combination thereof. 
     
     
         87 . The cell culture of any of  claims 80 - 86 , wherein the microorganism is  T. reesei  or a variant thereof, e.g., RUTC30. 
     
     
         88 . The cell culture of any of  claims 80 - 87 , further comprising an inducer biomass. 
     
     
         89 . The cell culture of  claim 88 , wherein the inducer biomass comprises a starchy material or a starchy material that includes a cellulosic component. 
     
     
         90 . The cell culture of  claim 89 , wherein the inducer biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof; wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass,  miscanthus , cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         91 . The cell culture of any of  claims 80 - 90 , further comprising a biomass degrading enzyme produced by the microorganism.