Ethanol production by recombinant hosts
First Claim
1. A method for producing ethanol from cellulose-containing biomass, comprising the steps of:
- A. contacting, in a first reaction vessel, said biomass with a polysaccharase enzyme such that the cellulose in said biomass is broken down into simpler oligosaccharides and/or monosaccharides;
wherein said contacting is carried out at a temperature of from about 40°
C. to about 60°
C. and a pH of from about 4.5 to about 5.0;
B. producing from said first reaction vessel a sugar solution comprising at least glucose, other cellulose-derived sugars and hemicellulose-derived sugars;
C. reacting said sugar solution of Step B to biologically consume a portion of the sugar solution thereby producing (i) a reaction product stream and (ii) a depleted sugar solution;
D. introducing said depleted sugar solution into a fermentor which comprises gram-negative enteric recombinant bacteria capable of fermenting the sugars present in said depleted sugar solution; and
E. fermenting said sugars in the depleted sugar solution into ethanol at a temperature of from about 30°
C. to about 35°
C. and a pH of aboutwherein said bacteria are capable of fermenting said sugars in the depleted sugar solution into ethanol, and comprises a recombinant host, other than Escherichia coli, comprising a first heterologous DNA coding for alcohol dehydrogenase and pyruvate decarboxylase, wherein said heterologous DNA is from Zymomonas mobilis and wherein said host expresses said heterologous DNA at a sufficient functional level so as to facilitate the production of ethanol as the primary fermentation product by said host,wherein said host also produces a polysaccharase, and said host further comprises a second heterologous DNA segment, the expression product of which is said polysaccharase.
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Abstract
Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.
125 Citations
45 Claims
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1. A method for producing ethanol from cellulose-containing biomass, comprising the steps of:
-
A. contacting, in a first reaction vessel, said biomass with a polysaccharase enzyme such that the cellulose in said biomass is broken down into simpler oligosaccharides and/or monosaccharides; wherein said contacting is carried out at a temperature of from about 40°
C. to about 60°
C. and a pH of from about 4.5 to about 5.0;B. producing from said first reaction vessel a sugar solution comprising at least glucose, other cellulose-derived sugars and hemicellulose-derived sugars; C. reacting said sugar solution of Step B to biologically consume a portion of the sugar solution thereby producing (i) a reaction product stream and (ii) a depleted sugar solution; D. introducing said depleted sugar solution into a fermentor which comprises gram-negative enteric recombinant bacteria capable of fermenting the sugars present in said depleted sugar solution; and E. fermenting said sugars in the depleted sugar solution into ethanol at a temperature of from about 30°
C. to about 35°
C. and a pH of aboutwherein said bacteria are capable of fermenting said sugars in the depleted sugar solution into ethanol, and comprises a recombinant host, other than Escherichia coli, comprising a first heterologous DNA coding for alcohol dehydrogenase and pyruvate decarboxylase, wherein said heterologous DNA is from Zymomonas mobilis and wherein said host expresses said heterologous DNA at a sufficient functional level so as to facilitate the production of ethanol as the primary fermentation product by said host, wherein said host also produces a polysaccharase, and said host further comprises a second heterologous DNA segment, the expression product of which is said polysaccharase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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Specification