Yeast Expressing Saccharolytic Enzymes for Consolidated Bioprocessing Using Starch and Cellulose

Abstract

The present invention is directed to a yeast strain, or strains, secreting a full suite, or any subset of that full suite, of enzymes to hydrolyze corn starch, corn fiber, lignocellulose, (including enzymes that hydrolyze linkages in cellulose, hemicellulose, and between lignin and carbohydrates) and to utilize pentose sugars (xylose and arabinose). The invention is also directed to the set of proteins that are well expressed in yeast for each category of enzymatic activity. The resulting strain, or strains can be used to hydrolyze starch and cellulose simultaneously. The resulting strain, or strains can be also metabolically engineered to produce less glycerol and uptake acetate. The resulting strain, or strains can also be used to produce ethanol from granular starch without liquefaction. The resulting strain, or strains, can be further used to reduce the amount of external enzyme needed to hydrolyze a biomass feedstock during an Simultaneous Saccharification and Fermentation (SSF) process, or to increase the yield of ethanol during SSF at current saccharolytic enzyme loadings. In addition, multiple enzymes of the present invention can be co-expressed in cells of the invention to provide synergistic digestive action on biomass feedstock. In some aspects, host cells expressing different heterologous saccharolytic enzymes can also be co-cultured together and used to produce ethanol from biomass feedstock.

1 - 149 . (canceled) 
     
     
         150 . A recombinant yeast host cell comprising two or more heterologous polynucleotides encoding two or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 288, SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 289, and SEQ ID NO: 219. 
     
     
         151 . The recombinant yeast host cell of  claim 150 , comprising three or more heterologous polynucleotides encoding three or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 288, SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 289, and SEQ ID NO: 219. 
     
     
         152 . The recombinant yeast host cell of  claim 150 , comprising four or more heterologous polynucleotides encoding four or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 288, SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 289, and SEQ ID NO: 219. 
     
     
         153 . The recombinant yeast host cell of  claim 150 , comprising five or more heterologous polynucleotides encoding five or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 288, SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 289, and SEQ ID NO: 219. 
     
     
         154 . The recombinant yeast host cell of  claim 150 , comprising each of the heterologous polynucleotides encoding each of the polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 288, SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 289, and SEQ ID NO: 219. 
     
     
         155 . The recombinant yeast host cell of  claim 150 , further comprising one or more heterologous polynucleotides encoding one or more polypeptides selected from the group consisting of: an α-glucuronidase isolated from  Aspergillus , a β-mannosidase isolated from  Neosartorya fischeri  and a beta-glucosidase isolated from  Aspergillus.    
     
     
         156 . A recombinant yeast host cell comprising each of the heterologous polynucleotides encoding the polypeptides comprising an amino acid sequence at least 90% identical to the amino acid sequences encoded by: SEQ ID NO: 466 and SEQ ID NO: 467, and further comprising one or more heterologous polynucleotides encoding one or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 219, and SEQ ID NO: 248. 
     
     
         157 . The recombinant host cell of  claim 156 , comprising two or more heterologous polynucleotides encoding two or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 219, and SEQ ID NO: 248. 
     
     
         158 . The recombinant host cell of  claim 156 , comprising three or more heterologous polynucleotides encoding three or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 219, and SEQ ID NO: 248. 
     
     
         159 . The recombinant host cell of  claim 156 , comprising four or more heterologous polynucleotides encoding four or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 219, and SEQ ID NO: 248. 
     
     
         160 . The recombinant host cell of  claim 156 , comprising each of the heterologous polynucleotides encoding each of the polypeptides comprising amino acid sequences at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 278, SEQ ID NO: 450, SEQ ID NO: 468, SEQ ID NO: 219, and SEQ ID NO: 248. 
     
     
         161 . The recombinant host cell of  claim 156 , further comprising a heterologous polynucleotide encoding a polypeptide encoding a beta-glucosidase isolated from  Aspergillus.    
     
     
         162 . The recombinant host cell of any one of  claims 150  or  156 , further comprising one or more heterologous polynucleotides encoding one or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 461, SEQ ID NO: 457, SEQ ID NO: 460, SEQ ID NO: 459, and SEQ ID NO: 454. 
     
     
         163 . The recombinant host cell of any one of  claims 150  or  156 , further comprising two or more heterologous polynucleotides encoding two or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 461, SEQ ID NO: 457, SEQ ID NO: 460, SEQ ID NO: 459, and SEQ ID NO: 454. 
     
     
         164 . The recombinant host cell of any one of  claims 150  or  156 , further comprising three or more heterologous polynucleotides encoding three or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 461, SEQ ID NO: 457, SEQ ID NO: 460, SEQ ID NO: 459, and SEQ ID NO: 454. 
     
     
         165 . The recombinant host cell of any one of  claims 150  or  156 , further comprising four or more heterologous polynucleotides encoding four or more polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 461, SEQ ID NO: 457, SEQ ID NO: 460, SEQ ID NO: 459, and SEQ ID NO: 454. 
     
     
         166 . The recombinant host cell of any one of  claims 150  or  156 , further comprising each of the heterologous polynucleotides encoding each of the polypeptides comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 461, SEQ ID NO: 457, SEQ ID NO: 460, SEQ ID NO: 459, and SEQ ID NO: 454. 
     
     
         167 . The recombinant yeast host cell of any one of  claims 150  or  156 , further comprising a down-regulation of at least one gene encoding an endogenous glycerol-producing enzyme selected from the group consisting of: glycerol 3-phosphate dehydrogenase 1 and glycerol-3 phosphate dehydrogenase 2. 
     
     
         168 . The recombinant yeast host cell of any one of  claims 150  or  156 , further comprising one or more heterologous polynucleotides encoding one or more polypeptides encoding an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide encoding SEQ ID NO: 449. 
     
     
         169 . The recombinant yeast host cell of any one of  claims 150  or  156 , wherein the yeast strain is a  Saccharomyces cerevisiae  strain. 
     
     
         170 . The recombinant yeast host cell of any one of  claims 150  or  156 , wherein the recombinant yeast host cell produces ethanol at a rate of at least about 0.5 g per hour per liter. 
     
     
         171 . A method of producing ethanol comprising: (a) providing the microorganism of any one of  claims 150  or  156 ; and (b) culturing said microorganism in the presence of a carbon containing feedstock for sufficient time to produce ethanol. 
     
     
         172 . A co-culture comprising at least two microorganisms wherein
 i. one of the microorganisms comprises a microorganism from any one of  claims 150  or  156 ; and,   ii. one of the microorganisms is genetically distinct from (i).   
     
     
         173 . The co-culture of  claim 172 , wherein the genetically distinct microorganism is a yeast or bacterium. 
     
     
         174 . The co-culture of  claim 172 , wherein the genetically distinct microorganism is any organism selected from the group consisting of:  Issatchenkia, Pichia, Clavispora, Candida, Hansenula, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Kloeckera, Schwanniomyces, Blakeslea, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon  and  Yarrowia.