Thermophillic Organisms For Conversion Of Lignocellulosic Biomass To Ethanol

Abstract

Mutant thermophilic organisms that consume a variety of biomass derived substrates are disclosed herein. Strains of Thermoanaerobacterium saccharolyticum with acetate kinase and phosphotransacetylase expression eliminated are disclosed herein. Further, strain ALK1 has been engineered by site directed homologous recombination to knockout both acetic acid and lactic acid production. Continuous culture involving a substrate concentration challenge lead to evolution of ALK1, and formation of a more robust strain designated ALK2. Both organisms produce near theoretical ethanol yields without expressing pyruvate decarboxylase

1 . An isolated organism that ferments a cellulolytic substrate to produce ethanol in a concentration that is at least 90% of a theoretical yield, wherein the organism does not express pyruvate decarboxylase. 
     
     
         2 . A method for producing ethanol, said method comprising:
 transforming a native organism to produce the isolated organism of  claim 1  to provide a transformed host; and   culturing the transformed host in medium that contains a substrate including a material selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the substrate.   
     
     
         3 . A transformed organism comprising,
 a Gram-positive bacterium that in a native state contains at least one gene which confers upon the Gram-positive bacterium an ability to produce acetic acid as a fermentation product,   the Gram-positive bacterium being transformed to eliminate expression of the at least one gene.   
     
     
         4 . The Gram-positive bacterium according to  claim 3 , wherein the Gram-positive bacterium is a member of the Thermoanaerobacter genus. 
     
     
         5 . The Gram-positive bacterium according to  claim 3 , wherein the Gram-positive bacterium is a  Thermoanaerobacterium saccharolyticum.    
     
     
         6 . The Gram-positive bacterium according to  claim 3 , wherein the at least one gene codes for expression of acetate kinase. 
     
     
         7 . The Gram-positive bacterium according to  claim 3 , wherein the at least one gene codes for the expression of phosphotransacetylase. 
     
     
         8 . The Gram-positive bacterium according to  claim 3 , wherein the at least one gene includes a plurality of genes. 
     
     
         9 . The Gram-positive bacterium according to  claim 8 , wherein the plurality of genes code for expression of acetate kinase and phosphotransacetylase. 
     
     
         10 . The Gram-positive bacterium according to  claim 9 , further transformed to eliminate expression of one or more genes that confer upon the Gram-positive bacterium the ability to produce lactic acid as a fermentation product. 
     
     
         11 . The Gram-positive bacterium according to  claim 3 , further transformed to eliminate expression of one or more genes that confer upon the Gram-positive bacterium the ability to produce lactic acid as a fermentation product. 
     
     
         12 . The Gram-positive bacterium according to  claim 11 , wherein the at least one gene codes for the expression of lactate dehydrogenase. 
     
     
         13 . A method for producing ethanol, said method comprising:
 transforming a native organism to produce the Gram-positive bacterium of  claim 11  to produce a transformed bacterial host; and   culturing the transformed bacterial host in medium that contains a substrate including a material selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the substrate.   
     
     
         14 . The method according to  claim 13 , wherein said bacterial host is a  Thermoanaerobacterium saccharolyticum.    
     
     
         15 . The method according to  claim 13 , wherein the genes code for the expression of lactate dehydrogenase, acetate kinase, and phosphotransacetylase. 
     
     
         16 . A biologically pure culture of a microorganism designated ALK1 and deposited under Patent Deposit Designation No. PTA-7206. 
     
     
         17 . An isolated polynucleotide comprising:
 (a) a sequence of SEQ ID NO: 10;   (b) a sequence of SEQ ID NO: 9 and SEQ ID NO: 10; or   (c) a sequence having at least about 90% sequence identity with the sequence of (a) or (b).   
     
     
         18 . The polynucleotide of  claim 17 , having about 95% sequence identity with the sequence of (a) or (b). 
     
     
         19 . A vector comprising the isolated polynucleotide of  claim 18 . 
     
     
         20 . A host cell genetically engineered to express a compliment of the polynucleotide of  claim 18 . 
     
     
         21 . The host cell of  claim 20 , wherein the host cell is a bacterial cell. 
     
     
         22 . A method of producing ethanol comprising the step of:
 culturing a mutant bacterium according to  claim 21  in medium containing a substrate selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow fermentation of the substrate to ethanol.   
     
     
         23 . The method of  claim 22 , wherein the mutant bacterium is  Thermoanaerobacterium saccharolyticum.    
     
     
         24 . The method of  claim 23 , wherein the mutant bacterium is  Thermoanaerobacterium saccharolyticum  ALK1 (JW/SL-YS485 ALK1). 
     
     
         25 . A genetic construct comprising SEQ ID NO: 10 operably connected to a promoter expressible in a bacterium. 
     
     
         26 . A recombinant bacterium comprising the genetic construct of  claim 25 . 
     
     
         27 . The recombinant bacterium of  claim 26 , wherein said bacterium is  Thermoanaerobacterium saccharolyticum.