Methods and compositions for simultaneous saccharification and fermentation

Abstract

The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates

1 - 65 . (canceled)  
     
     
         66 . A method of making a recombinant host cell suitable for degrading an oligosaccharide comprising: 
 introducing into said host cell a first heterologous polynucleotide segment encoding a first endoglucanase having a first degrading activity, wherein said segment is under the transcriptional control of a surrogate promoter; and    a second heterologous polynucleotide segment comprising a sequence encoding a second endoglucanase having a second degrading activity, wherein said segment is under the transcriptional control of a surrogate promoter,    wherein said first and second endoglucanases are expressed such that said first and said second degrading activities are present in a ratio such that the degrading of said oligosaccharide by said first and second endoglucanases is synergized.    
     
     
         67 . The method of claim  65 , wherein said first endoglucanase or said second endoglucanase or both said first and second endoglucanases are secreted.  
     
     
         68 . The method of  claim 66 , wherein said host cell is ethanologenic.  
     
     
         69 . The method of  claim 66 , wherein said first endoglucanase is encoded by celZ and said second endoglucanase is encoded by celY, wherein celZ and celY are derived from  Erwinia.    
     
     
         70 . The method of  claim 66 , wherein said surrogate promoter of said first heterologous polynucleotide segment or said second heterologous polynucleotide segment or both said first and second polynucleotide segments, comprises a polynucleotide fragment derived from  Zymomonas mobilis.    
     
     
         71 . The method of  claim 68 , wherein said recombinant host cell is suitable for simultaneous saccharification and fermentation.  
     
     
         72 . The method of  claim 70  or  71 , wherein said host cell is ethanologenic.  
     
     
         73 . A method of making a recombinant host cell integrant comprising, 
 introducing into said host cell a vector comprising the polynucleotide sequence of pLOI2352 (SEQ ID NO: 17); and    identifying a host cell having said vector stably integrated.    
     
     
         74 . A method for expressing a endoglucanase in a host cell comprising: 
 introducing into said host cell a vector comprising the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12); and    identifying a host cell expressing said endoglucanase.    
     
     
         75 . A method for producing ethanol from an oligosaccharide source comprising, contacting said oligosaccharide source with a ethanologenic host cell comprising: 
 a first heterologous polynucleotide segment encoding a first endoglucanase having a first degrading activity, wherein said segment is under the transcriptional control of a surrogate promoter; and    a second heterologous polynucleotide segment encoding a second endoglucanase having a second degrading activity, wherein said segment is under the transcriptional control of a surrogate promoter,    wherein said first and second endoglucanases are expressed so that said first and said second degrading activities are present in a ratio such that the degrading of said oligosaccharide by said first and second endoglucanases is synergized resulting in a degraded oligosaccharide that is fermented into ethanol.    
     
     
         76 . The method of  claim 75 , wherein said first endoglucanase is encoded by celZ and said second endoglucanase is encoded by celY gene, wherein celZ and celY are derived from  Erwinia.    
     
     
         77 . The method of  claim 75 , further said host cell further comprising a heterologous polynucleotide segment encoding at least one pul gene or out gene.  
     
     
         78 . The method of  claim 75 , wherein said host cell is selected from the family Enterobacteriaceae.  
     
     
         79 . The method of  claim 75 , wherein said host cell is  Escherichia  or  Klebsiella.    
     
     
         80 . The method of  claim 79 , wherein said host cell is selected from the group consisting of  E. coli  KO4 (ATCC 55123),  E. coli  KO11 (ATCC 55124),  E. coli  KO12 (ATCC 55125), and  K. oxytoca  P2 (ATCC 55307).  
     
     
         81 . The method of  claim 75 , wherein said method is conducted in an aqueous solution.  
     
     
         82 . The method of  claim 75 , wherein said oligosaccharide is selected from the group consisting of cellooligosaccharide, lignocellulose, hemicellulose, cellulose, pectin, and any combination thereof.  
     
     
         83 . The method of  claim 75 , wherein said heterologous polynucleotide segment is, or derived from, of pLOI2352 (SEQ ID NO: 17).  
     
     
         84 . The method of  claim 75 , wherein said first endoglucanase is EGZ and said second endoglucanase is EGY.  
     
     
         85 . The method of  claim 75  wherein said surrogate promoter of said first polynucleotide segment or said second polynucleotide segment, or both said first second polynucleotide segments comprises a polynucleotide fragment derived from  Zymomonas mobilis.    
     
     
         86 . A vector comprising the polynucleotide sequence of a plasmid, or fragment thereof, selected from group consisting of pLOI2311, pLOI1620, pLOI2316, pLOI2317, pLOI2318, pLOI2319, pLOI2320, pLOI2323, pLOI2342, pLOI2348, pLOI2349, pLOI2350, pLOI2352, pLOI2353, pLOI2354, pLOI2355, pLOI2356, pLOI2357, pLO12358, and pLO2359.  
     
     
         87 . A host cell comprising a vector having the polynucleotide sequence of a plasmid, of fragment thereof, selected from the group consisting of pLOI2311, pLOI1620, pLOI2316, pLOI2317, pLOI2318, pLOI2319, pLOI2320, pLOI2323, pLOI2342, pLOI2348, pLOI2349, pLOI2350, pLOI2352, pLOI2353, pLOI2354, pLOI2355, pLOI2356, pLOI2357, pLOI2358, and pLO2359.  
     
     
         88 . The host cell of  claim 87 , wherein said host is selected from the group comprising  Klebsiella oxytoca  strain P2 (pCPP2006),  Klebsiella oxytoca  strain SZ6 (pCPP2006),  Klebsiella oxytoca  strain SZ21 (pCPP2006), and  Klebsiella oxytoca  strain SZ22 (pCPP2006).  
     
     
         89 - 110 . (canceled)