Amino acid producing microorganism and a method for producing an amino acid

Abstract

A microorganism is provided which has an ability to produce an L-amino acid such as L-lysine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine, and has been modified to increase the activity of pyruvate synthase or pyruvate:NADP + oxidoreductase. This microorganism is cultured in a medium containing ethanol or an aliphatic acid as the carbon source to produce and accumulate the L-amino acid in the medium or cells, and the L-amino acid is collected from the medium or the cells.

1 . A method for producing an L-amino acid selected from the group consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine, and L-serine comprising:
 A) culturing in a medium a microorganism which has an ability to produce the L-amino acid, and   B) collecting the L-amino acid from the medium or the microorganism, wherein said microorganism has been modified to increase an activity of NADH+ oxidoreductase by a method selected from the group consisting of:   i) increasing the copy number of a gene encoding NADH+ oxidoreductase,   ii) modifying an expression control sequence of the gene, and   iii) combinations thereof;   
       and wherein NADH+ oxidoreductase is selected from the group consisting of:
 (a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, and 
 (b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, but which includes between 1 and 20 substitutions, deletions, insertions, or additions, and has NADH+ oxidoreductase activity. 
 
     
     
         2 . The method according to  claim 1 , wherein the medium contains ethanol or an aliphatic acid as the carbon source. 
     
     
         3 . The method according to  claim 1 , wherein the gene encoding NADH+ oxidoreductase comprises a DNA selected from the group consisting of:
 (a) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 5, and   (b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 under stringent conditions comprising washing at 68° C., 0.1×SSC, 0.1% SDS and encoding a polypeptide having NADH+ oxidoreductase activity.   
     
     
         4 . The method according to  claim 1 , wherein the microorganism has been further modified to increase the activity of ferredoxin-NADP +  reductase by a method selected from the group consisting of:
 a) increasing the copy number of a gene encoding ferrodoxin-NADP +  reductase,   b) modifying an expression control sequence of the gene, and   c) combinations thereof.   
     
     
         5 . The method according to  claim 1 , wherein the microorganism has been further modified to increase production of ferredoxin or flavodoxin by a method selected from the group consisting of:
 a) increasing the copy number of a gene encoding ferredoxin or flavodoxin,   b) modifying an expression control sequence of the gene, and   c) combinations thereof.   
     
     
         6 . The method according to  claim 1 , wherein the microorganism has been further modified to decrease pyruvate dehydrogenase activity by a method selected from the group consisting of:
 a) introducing a deletion or mutation into a gene encoding pyruvate dehydrogenase,   b) introducing a deletion or mutation into an expression control sequence of the gene, and   c) combinations thereof.   
     
     
         7 . The method according to  claim 1 , wherein the microorganism has been further modified so that it can aerobically assimilate ethanol. 
     
     
         8 . The method according to  claim 1 , wherein the microorganism is a bacterium belonging to a genus selected from the group consisting of  Escherichia, Enterobacter, Pantoea, Klebsiella , and  Serratia.    
     
     
         9 . The method according to  claim 1 , wherein the microorganism is a coryneform bacterium. 
     
     
         10 . The method according to  claim 1 , wherein the microorganism is  Escherichia coli.    
     
     
         11 . A method for producing an L-amino acid selected from the group consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine comprising:
 A) culturing in a medium a microorganism which has an ability to produce the L-amino acid, and   B) collecting the L-amino acid from the medium or the microorganism, wherein said microorganism has been modified to increase an activity of NADH+ oxidoreductase by a method selected from the group consisting of:   i) increasing the copy number of a gene encoding NADH+ oxidoreductase,   ii) modifying an expression control sequence of the gene, and   iii) combinations thereof;   
       and wherein NADH+ oxidoreductase comprises the amino acid sequence shown in SEQ ID NO: 6. 
     
     
         12 . A method for producing an L-amino acid selected from the group consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine comprising:
 A) culturing in a medium a microorganism which has an ability to produce the L-amino acid, and   B) collecting the L-amino acid from the medium or the microorganism, wherein said microorganism has been modified to increase an activity of NADH+ oxidoreductase by method selected from the group consisting of:   i) increasing the copy number of a gene encoding NADH+ oxidoreductase,   ii) modifying an expression control sequence of the gene, and   iii) combinations thereof;   and wherein the gene encoding NADH+ oxidoreductase is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 5.