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 host cell comprising at least one heterologous polynucleotide comprising a nucleic acid encoding a cellulose, wherein the cellulase has an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 54. 
     
     
         213 . The host cell of  claim 212 , wherein the cellulase has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 54. 
     
     
         214 . The host cell of  claim 212 , wherein the cellulase has an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 54. 
     
     
         215 . The host cell of  claim 212 , wherein the nucleic acid is at least 90% identical to the nucleotide sequence of SEQ ID NO: 49. 
     
     
         216 . The host cell of  claim 212 , wherein the nucleic acid is codon-optimized. 
     
     
         217 . The host cell of  claim 212 , wherein the host cell is a  Saccharomyces cerevisiae  host cell. 
     
     
         218 . The host cell of  claim 212 , wherein the host cell is a  Kluyveromyces  host cell. 
     
     
         219 . The host cell of  claim 212 , wherein the host cell is thermotolerant. 
     
     
         220 . The host cell of  claim 212 , wherein the host cell is a xylose-utilizing host cell. 
     
     
         221 . The host cell of  claim 212 , wherein the host cell can hydrolyze Avicel. 
     
     
         222 . The host cell of  claim 212 , wherein the host cell further comprises a heterologous polynucleotide comprising a nucleic acid encoding a .beta.-glucosidase I, a heterologous polynucleotide comprising a nucleic acid encoding a cellobiohydrolase I; and a heterologous polynucleotide comprising a nucleic acid encoding a cellobiohydrase II. 
     
     
         223 . The host cell of  claim 222 , wherein the host cell further comprises a heterologous polynucleotide comprising a nucleic acid encoding an  S. fibuligera  .beta.-glucosidase I, a heterologous polynucleotide comprising a nucleic acid encoding a  T. emersonii  cellobiohydrolase I; and a heterologous polynucleotide comprising a nucleic acid encoding a  C. lucknowense  cellobiohydrase II. 
     
     
         224 . The host cell of  claim 222 , wherein the host cell can produce ethanol when grown using cellulose as a carbon source. 
     
     
         225 . A method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell according to  claim 212 . 
     
     
         226 . The method of  claim 225 , wherein said cellulosic substrate is a lignocellulosic biomass selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, Agave, and combinations thereof. 
     
     
         227 . The method of  claim 225 , further comprising contacting said cellulosic substrate with externally produced cellulase enzymes. 
     
     
         228 . The method of  claim 225 , wherein said host cell produces ethanol. 
     
     
         229 . The method of  claim 228 , wherein ethanol is produced at a rate of at least about 10 mg per hour per liter. 
     
     
         230 . A method of fermenting cellulose using the host cell of  claim 212 , said method comprising culturing said transformed host cell in medium that contains insoluble cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose. 
     
     
         231 . The method of  claim 230 , wherein said culturing is at a temperature of at least about 37° C.