Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose

Abstract

The present invention is directed to cellulytic host cells. The host cells of the invention expressing heterologous cellulases and are able to produce ethanol from cellulose. According to the invention, host cells expressing a combination of heterologous cellulases can be used to produce ethanol from cellulose. In addition, multiple host cells expressing different heterologous cellulases can be co-cultured together and used to produce ethanol from cellulose. Furthermore, the invention demonstrates for the first time the ability of Kluyveromyces to produce ethanol from cellulose. The yeast strains and co-cultures of yeast strains of the invention can be used to produce ethanol on their own, or can also be used in combination with externally added cellulases to increase the efficiency of saccharification and fermentation processes.

1 - 211 . (canceled) 
     
     
         212 . A method of fermenting cellulose using a thermotolerant yeast host cell, said method comprising culturing said yeast host cell in a medium that contains insoluble cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose, and wherein said yeast host cell comprising:
 a. at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase;   b. at least one heterologous polynucleotide comprising a nucleic acid which encodes a β-glucosidase;   c. at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase I; and   d. at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase II;   wherein the yeast host cell can grow at a temperature of at least 35° C. or above, wherein the endoglucanase, β-glucosidase, cellobiohydrolase I, and cellobiohydrolase II are secreted; and   
       wherein the host cell is capable of producing ethanol from Avicel. 
     
     
         213 . The method of  claim 212 , wherein the β-glucosidase is a  Saccharomycopsis fibuligera  β-glucosidase comprising the amino acids of SEQ ID NO-40. 
     
     
         214 . The method of  claim 212 , wherein the cellobiohydrolase I is a  Talaromyces emersonii  cellobiohydrolase I comprising the amino acids of SEQ ID NO:23. 
     
     
         215 . The method of  claim 214 , wherein the  Talaromyces emersonii  cellobiohydrolase I is fused to a cellulose binding module (CBM). 
     
     
         216 . The method of  claim 215 , wherein the cellulose binding module (CBM) is the CBM of  Trichoderma reesei  Cbh1 comprising amino acids 503 to 535 of SEQ ID NO:27 or  Humicola grisea  Cbh1 comprising amino acids 492 to 525 of SEQ ID NO: 21. 
     
     
         217 . The method of  claim 212 , wherein the cellobiohydrolase II is a  Chrysosporium lucknowense  cellobiohydrolase II comprising the amino acids of SEQ ID NO:25. 
     
     
         218 . The method of  claim 212 , wherein the endoglucanase is an  Humicola grisea, Thermoascus aurantiacus, Talaromyces emersonii, Trichoderma reesei, Coptotermes lacteus, Coptotermes formosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis, Mastotermes darwinensis, Nasutitermes walkeri, Saccharomycopsis fibuligera, Chrysosporium lucknowense, Reticulitermes speratus, Thermobifida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Chaetomium thermophilum, Aspergillus terreus, Neurospora Crassa, Reticulitermes flavipes , or  Arabidopsis thaliana  endoglucanase. 
     
     
         219 . The method of  claim 212 , further comprising a heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase. 
     
     
         220 . The method of  claim 212 , wherein the β-glucosidase is a  Saccharomycopsis fibuligera  β-glucosidase comprising the amino acids of SEQ ID NO:40, the cellobiohydrolase I is a  Talaromyces emersonii  cellobiohydrolase I comprising the amino acids of SEQ ID NO:23, and the cellobiohydrolase II is a  Chrysosporium lucknowense  cellobiohydrolase II comprising the amino acids of SEQ ID NO 25. 
     
     
         221 . The method of  claim 220 , further comprising a second heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase, and wherein the endoglucanase and the second endoglucanase are selected from the group consisting of  Humicola grisea, Thermoascus aurantiacus, Talaromyces emersonii, Trichoderma reesei, Coptotermes lacteus, Coptotermes formosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis, Mastotermes darwinensis, Nasutitermes walkeri, Saccharomycopsis fibuligera, Chrysosporium lucknowense, Reticulitermes speratus, Thermobifida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Neosartorya fischeri, Chaetomium thermophilum, Aspergillus terreus, Neurospora Crassa, Reticulitermes flavipes , or  Arabidopsis thaliana  endoglucanases. 
     
     
         222 . The method of  claim 212 , wherein the host cell is a  Kluyveromyces  host cell. 
     
     
         223 . The method of  claim 212 , wherein the host cell is a  Saccharomyces cerevisiae  host cell. 
     
     
         224 . The method of  claim 212 , wherein the host cell is capable of fermenting pentose sugars. 
     
     
         225 . The method of  claim 224 , wherein the host cell is capable of fermenting xylose. 
     
     
         226 . The method of  claim 225 , wherein the host cell expresses xylose isomerase, xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase, transaldolase or a combination thereof. 
     
     
         227 . The method of  claim 226 , wherein the host cell expresses xylose isomerase, xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase. 
     
     
         228 . The method of  claim 224 , wherein the host cell does not express the GRE3 gene encoding aldose reductase. 
     
     
         229 . The method of  claim 212 , wherein the endoglucanase is an  Aspergillus kawachii, Heterodera schachtii, Hypocrea jecorina, Orpinomyces  sp.,  Irpex lacteus, Acremonium thermophilum, Chaetomium globosum , or  Aspergillus fumigatus  endoglucanase. 
     
     
         230 . The method of  claim 220 , further comprising a second heterologous polynucleotide comprising a nucleic acid which encodes a second endoglucanase, and wherein the endoglucanase and the second endoglucanase are selected from the group consisting of  Aspergillus kawachii, Heterodera schachtii, Hypocrea jecorina, Orpinomyces  sp.,  Irpex lacteus, Acremonium thermophilum, Chaetomium globosum , and  Aspergillus fumigatus  endoglucanase. 
     
     
         231 . The method of  claim 212 , wherein the endoglucanase is a  Neosartorya fischeri  endoglucanase comprising the amino acids of SEQ ID NO:37. 
     
     
         232 . The method of  claim 212 , wherein the thermotolerant yeast host cell can grow at temperatures of at least 38° C. or above. 
     
     
         233 . The method of  claim 212 , wherein the thermotolerant yeast host cell can grow at temperatures of at least 40° C. or above. 
     
     
         234 . The method of  claim 212 , wherein the thermotolerant yeast host cell can grow at temperature from 40° C. to 50° C. 
     
     
         235 . The method of  claim 220 , wherein the  Talaromyces emersonii  cellobiohydrolase I is fused to a cellulose binding module (CBM). 
     
     
         236 . A culture comprising the host cell of  claim 212  and cellulose.