Methods for improving the efficiency of simultaneous saccharification and fermentation reactions

Abstract

The present disclosure is directed, in a first aspect, to the use of inverting beta-xylosidase enzymes to reduce byproduct formation and increase the yield of fermentation products, as well as, in a second aspect, to the use of retaining beta-xylosidase enzymes to improve production of alkyl-beta-xylopyranoside compounds, in a simultaneous saccharification and fermentation reactions.

1 . A method for simultaneous saccharification and fermentation (SSF) comprising culturing a complete fermentation medium, said complete fermentation medium comprising at least one fermenting microorganism, at least one xylan-containing biomass, at least one cellulase, at least one hemicellulase, and at least one inverting β-xylosidase, for a period and under conditions suitable for producing a fermentation product and optionally recovering the fermentation product. 
     
     
         2 . The method of  claim 1 , wherein the complete fermentation medium comprises an effective amount of the inverting β-xylosidase such that the complete fermentation medium produces less short chain alkyl-β-xylopyranoside (“AXP”) than does a control fermentation medium lacking the inverting β-xylosidase. 
     
     
         3 . The method of  claim 1  or  2 , wherein the complete fermentation medium comprises an effective amount of the inverting β-xylosidase such that the complete fermentation medium produces at least 40% less AXP than does a control fermentation medium lacking the inverting β-xylosidase. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1  or  2 , wherein the AXP is a methyl-β-xylopyranoside (MXP), an ethyl-β-xylopyranoside (EXP), a propyl-β-xylopyranoside (PXP), or a butyl-β-xylopyranoside (BXP). 
     
     
         8 . The method of  claim 1  or  2 , wherein the complete fermentation medium comprises an effective amount of the inverting β-xylosidase to increase the yield of the fermentation product, as compared to the yield of the fermentation product from culturing a control fermentation medium lacking the inverting β-xylosidase. 
     
     
         9 . The method of  claim 8 , wherein the yield of the fermentation product is increased by at least 1%. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1  or  2 , wherein the fermentation product is an alcohol. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1  or  2 , wherein the inverting β-xylosidase is a GH43 family enzyme. 
     
     
         18 . The method of  claim 17 , wherein the inverting β-xylosidase is an Fv43D, a Pf43A, an Fv43E, an Fv43B, an Af43A, an Fo43A, a Gz43A, or a XynB3 polypeptide. 
     
     
         19 . The method of  claim 18 , wherein (a) the Fv43D polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:2, or to residues 21 to 350 of SEQ ID NO:2; (b) the Pf43A polypeptide has at least 90% sequence identity an amino acid sequence corresponding to SEQ ID NO:8, or to residues 21 to 445 of SEQ ID NO:8; (c) the Fv43E polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:10, or to residues 19 to 530 of SEQ ID NO:10; (d) the Fv43B polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:12, or to residues 17 to 574 of SEQ ID NO:12; (e) the Af43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:14, or to residues 15-558 of SEQ ID NO:14; (f) the Fo43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:24, or to residues 21-348 of SEQ ID NO:24; (g) the Gz43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:22, or to residues 19-340 of SEQ ID NO:22; or (h) the XynB3 polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:25. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . The method of any one of  claims 17 - 19 , wherein the inverting β-xylosidase is present in the complete fermentation medium at a concentration of 0.3 mg to 10 mg per gram of xylan in the xylan-containing biomass. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 1  or  2 , which is performed as a continuous, a batch, or a fed-batch SSF process. 
     
     
         31 . The method of  claim 1  or  2 , further comprising a step of forming the complete fermentation medium. 
     
     
         32 . The method of  claim 31 , wherein the step of forming the complete fermentation medium comprises combining (a) the fermenting microorganism, (b) the xylan-containing biomass, (c) the cellulase, (d) the hemicellulase, (e) the inverting β-xylosidase, and (f) a medium lacking one or more or all of (a)-(e). 
     
     
         33 . The method of  claim 32 , wherein the cellulase is present in the form of a whole cellulase preparation wherein the whole cellulase preparation optionally comprises a hemicellase. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 33 , wherein the whole cellulase preparation is a culture broth obtained from culturing a filamentous fungus wherein the filamentous fungus is optionally a  T. reesei.    
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein the  T. reesei  has been engineered such that the native β-xylosidase gene has been deleted or inactivated. 
     
     
         38 . The method of  claim 1  or  2 , wherein the fermenting microorganism is selected from the group of a fungus and a bacterium. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 1  or  2 , wherein the complete fermentation medium comprise a greater amount of inverting β-xylosidases than that of retaining β-xylosidases on a mole basis, molecular weight basis, or on both a mole basis and a molecular weight basis. 
     
     
         47 . The method of  claim 46 , wherein the ratio of inverting β-xylosidases to retaining β-xylosidases in the complete fermentation medium is at least 2:1, on a mole basis, molecular weight basis, or on both a mole basis and molecular weight basis. 
     
     
         48 . A  T. reesei  cell, which has been engineered such that the native β-xylosidase gene has been deleted or that it has no detectable β-xylosidase activity. 
     
     
         49 . The  T. reesei  cell of  claim 48 , which is engineered to recombinantly express a GH43 family enzyme. 
     
     
         50 . The  T. reesei  cell of  claim 48 , which is engineered to recombinantly express an Fv43D, a Pf43A, an Fv43E, an Fv43B, an Af43A, an Fo43A, a Gz43A, or a XynB3 polypeptide. 
     
     
         51 . The  T. reesei  cell of  claims 49 - 50 , wherein (a) the Fv43D polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:2, or to residues 21 to 350 of SEQ ID NO:2; (b) the Pf43A polypeptide has at least 90% sequence identity an amino acid sequence corresponding to SEQ ID NO:8, or to residues 21 to 445 of SEQ ID NO:8; (c) the Fv43E polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:10, or to residues 19 to 530 of SEQ ID NO:10; (d) the Fv43B polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:12, or to residues 17 to 574 of SEQ ID NO:12; (e) the Af43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:14, or to residues 15-558 of SEQ ID NO:14; (f) the Fo43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:24, or to residues 21-348 of SEQ ID NO:24; (g) the Gz43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:22, or to residues 19-340 of SEQ ID NO:22; or (h) the XynB3 polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:25. 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . A method for producing a cellulase preparation, comprising culturing the  T. reesei  cell of  claim 49  under conditions that result in the production of the cellulase preparation and optionally recovering the cellulase preparation. 
     
     
         60 . (canceled) 
     
     
         61 . (canceled) 
     
     
         62 . (canceled) 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . A composition comprising at least one fermenting microorganism, at least one xylan-containing biomass, at least one cellulase, at least one hemicellulase, and at least one inverting β-xylosidase. 
     
     
         66 . The composition of  claim 65 , wherein the inverting β-xylosidase is a GH43 family enzyme. 
     
     
         67 . The composition of  claim 65 , wherein the inverting β-xylosidase is an Fv43D, a Pf43A, an Fv43E, an Fv43B, an Af43A, an Fo43A, a Gz43A, or a XynB3 polypeptide. 
     
     
         68 . The composition of  claim 66  or  67 , wherein, if present, (a) the Fv43D polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:2, or to residues 21 to 350 of SEQ ID NO:2; (b) the Pf43A polypeptide has at least 90% sequence identity an amino acid sequence corresponding to SEQ ID NO:8, or to residues 21 to 445 of SEQ ID NO:8; (c) the Fv43E polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:10, or to residues 19 to 530 of SEQ ID NO:10; (d) the Fv43B polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:12, or to residues 17 to 574 of SEQ ID NO:12; (e) the Af43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:14, or to residues 15-558 of SEQ ID NO:14; (f) the Fo43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:24, or to residues 21-348 of SEQ ID NO:24; (g) the Gz43A polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:22, or to residues 19-340 of SEQ ID NO:22; or (h) the XynB3 polypeptide has at least 90% sequence identity to an amino acid sequence corresponding to SEQ ID NO:25. 
     
     
         69 . (canceled) 
     
     
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         77 . The composition of  claim 65  or  66 , wherein the fermenting microorganism is a fungus or a bacterium. 
     
     
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