Mutant cells for protein secretion and lignocellulose degradation

Abstract

The present disclosure provides mutant cells for the secretion of proteins and for the degradation of lignocellulosic biomass. Methods for the use of these cells are also provided. Specifically, the utility of combined genetic deletions of β-glucosidases and the catabolite repressor gene creA/cre-1 for protein secretion in fungal and yeast cells is disclosed.

1 . A method for increasing secretion of a protein from a cell, the method comprising:
 (a) providing a mutant cell, wherein the mutant cell comprises inactivating mutations in two or more β-glucosidase genes; and   (b) contacting said mutant cell with cellulosic biomass, wherein said cellulosic biomass induces the mutant cell to secrete said protein.   
     
     
         2 . The method of  claim 1 , wherein the mutant cell further comprises an inactivating mutation in a cre-1 gene in said cell. 
     
     
         3 . A method for increasing secretion of a protein from a cell, the method comprising:
 (a) providing a mutant cell, wherein the mutant cell comprises an inactivating mutation in a cre-1 gene in said cell; and   (b) contacting said mutant cell with cellulosic biomass, wherein said cellulosic biomass induces the mutant cell to secrete said protein.   
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the cellulosic biomass comprises one or more of a polysaccharide, an oligosaccharide, cellulose, microcrystalline cellulose, cellodextrin, cellobiose, cellotriose, cellotetraose, cellopentose, and cellohexose. 
     
     
         6 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the secreted protein is selected from the group consisting of a cellulase, a GH61 enzyme, a cellobiose dehydrogenase, a lactonase, a carbohydrate esterase, a polysaccharide lyase, a cellulose binding domain-containing protein, and combinations thereof. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the secreted protein is encoded by a gene selected from the group consisting of NCU07340, NCU09680, NCU07898, NCU00762, NCU08760, NCU05057, NCU02240, NCU07190, NCU07898, NCU08760, NCU00206, NCU07143, NCU09491, NCU09664, NCU05598, NCU09764, and NCU05137. 
     
     
         17 .- 19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the cell is a recombinant cell. 
     
     
         21 . The method of  claim 1 , wherein the cell is a fungal or yeast cell. 
     
     
         22 .- 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the two or more β-glucosidase genes are three or more β-glucosidase genes comprising NCU00130, NCU04952, and NCU08755. 
     
     
         25 .- 67 . (canceled) 
     
     
         68 . A mutant cell comprising inactivating mutations in two or more β-glucosidase genes, wherein cellulosic biomass induces the cell to secrete higher levels of a protein than a corresponding cell lacking said mutation in the two or more β-glucosidase genes. 
     
     
         69 . The mutant cell of  claim 68 , further comprising an inactivating mutation in a cre-1 gene in said cell, wherein cellulosic biomass induces the cell to secrete higher levels of a protein than a corresponding cell lacking said mutation in the cre-1 gene. 
     
     
         70 . The mutant cell of  claim 68 , further comprising an inactivating mutation in at least one β-mannosidase gene in said cell, wherein cellulosic biomass induces the cell to secrete higher levels of a protein than a corresponding cell lacking said mutation in the at least one β-mannosidase gene. 
     
     
         71 . The mutant cell of  claim 68 , further comprising an inactivating mutation in at least one phospholipase gene or phospholipase-like gene in said cell, wherein cellulosic biomass induces the cell to secrete higher levels of a protein than a corresponding cell lacking said mutation in the at least one phospholipase gene or phospholipase-like gene. 
     
     
         72 . The mutant cell of  claim 68 , wherein the cellulosic biomass comprises one or more of a polysaccharide, an oligosaccharide, cellulose, microcrystalline cellulose, cellodextrin, cellobiose, cellotriose, cellotetraose, cellopentose, and cellohexose. 
     
     
         73 .- 80 . (canceled) 
     
     
         81 . The mutant cell of  claim 68 , wherein the secreted protein is selected from the group consisting of a cellulase, a GH61 enzyme, a cellobiose dehydrogenase, a lactonase, a carbohydrate esterase, a polysaccharide lyase, a cellulose binding domain-containing protein, and combinations thereof. 
     
     
         82 . (canceled) 
     
     
         83 . The mutant cell of  claim 68 , wherein the secreted protein is encoded by a gene selected from the group consisting of NCU07340, NCU09680, NCU07898, NCU00762, NCU08760, NCU05057, NCU02240, NCU07190, NCU07898, NCU08760, NCU00206, NCU07143, NCU09491, NCU09664, NCU05598, NCU09764, and NCU05137. 
     
     
         84 . (canceled) 
     
     
         85 . The mutant cell of  claim 68 , wherein the cell is a recombinant cell. 
     
     
         86 . The mutant cell of  claim 68 , wherein the cell is a fungal or yeast cell. 
     
     
         87 .- 88 . (canceled) 
     
     
         89 . The mutant cell of  claim 68 , wherein the two or more β-glucosidase genes are three or more β-glucosidase genes comprising NCU00130, NCU04952, and NCU08755. 
     
     
         90 .- 140 . (canceled) 
     
     
         141 . A mutant cell comprising an inactivating mutation in a cre-1 gene, wherein cellulosic biomass induces the cell to secrete higher levels of a protein than a corresponding cell lacking said mutation in the cre-1 gene. 
     
     
         142 . The mutant cell of  claim 141 , wherein the inactivating mutation is a deletion.