Use of serine proteases for improving ethanol yield

Abstract

The present invention relates to S53 proteases and to the use of S53 protease in processes for converting starch to ethanol.

1 . A process for producing a fermentation product from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzymes and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material in the presence of a serine protease belonging to the S53 family. 
     
     
         2 . A process for producing a fermentation product from starch-containing material comprising the steps of:
 (a) liquefying starch-containing material in the presence of an alpha-amylase;   (b) saccharifying the liquefied material obtained in step (a) using a carbohydrate-source generating enzyme;   (c) fermenting using a fermenting organism;   
       wherein a serine protease belonging to family 53 is present during step b) and/or c). 
     
     
         3 . The process of  claim 1  or  2 , wherein the S53 protease is derived from a strain of the genus  Meripilus, Trametes , Dichomitus, Polyporus, Lenzites,  Ganoderma , Neolentinus, Lecanicillium,  Talaromyces  or  Bacillus , more particularly  Meripilus giganteus, Trametes versicolor, Dichomitus squalens, Polyporus arcularius, Lenzites betulinus, Ganoderma lucidum, Neolentinus lepideus , Lecanicillium sp WMM742,  Talaromyces proteolyticus  or  Bacillus  sp. 19138. 
     
     
         4 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 3; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1. 
 
     
     
         5 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 6; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 4. 
 
     
     
         6 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 9; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7. 
 
     
     
         7 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 12; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 10. 
 
     
     
         8 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 15; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13. 
 
     
     
         9 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 26; 
 (c) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25. 
 
     
     
         10 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 30; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29. 
 
     
     
         11 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 34; 
 (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 33. 
 
     
     
         12 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 38, such as amino acids 195 to 560 or 190 to 560 of SEQ ID NO: 38. 
 
     
     
         13 . The process of  claim 1  or  2 , wherein the S53 protease is a polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 39, such as amino acids 226 to 647 or 221 to 647 of SEQ ID NO: 39. 
 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The process of  claim 1 , wherein the fermentation product is an alcohol. 
     
     
         18 . A composition comprising a S53 protease and a carbohydrate-source generating enzyme and optionally an alpha-amylase. 
     
     
         19 . The composition of  claim 18 , wherein the S53 protease is derived from a strain of the genus  Meripilus, Trametes, Dichomitus, Polyporus, Lenzites, Ganoderma, Neolentinus Lecanicillium, Talaromyces  or  Bacillus , more particularly  Meripilus giganteus, Trametes versicolor, Dichomitus squalens, Polyporus arcularius, Lenzites betulinus, Ganoderma lucidum, Neolentinus lepideus, Lecanicillium  sp WMM742,  Talaromyces proteolyticus  or  Bacillus  sp. 19138. 
     
     
         20 . (canceled) 
     
     
         21 . A polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;   (b) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 10;   (c) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 15;   (d) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13;   (e) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 27;   (f) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25;   (g) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 31; and   (h) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 29   
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . A polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 38, such as amino acids 195 to 560 or 190 to 560 of SEQ ID NO: 38.   
     
     
         26 . A polypeptide having serine protease activity, selected from the group consisting of:
 (a) a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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: 39, such as amino acids 226 to 647 or 221 to 647 of SEQ ID NO: 39.   
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide of  claim 21 , wherein the polypeptide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host. 
     
     
         30 . A recombinant host cell comprising the polynucleotide of claim  21  operably linked to one or more control sequences that direct the production of the polypeptide. 
     
     
         31 . The process according to  claim 1  wherein the S53 protease is an endo-protease.