Mutant Acetolactate Synthase and a Method for Producing Branched-Chain L-Amino Acids

Abstract

A mutant bacterial acetolactate synthase (AHAS I) which is resistant to feedback inhibition by L-valine is described. Also described is a method for producing branched-chain L-amino acids using a bacterium from the Enterobacteriaceae family wherein the L-amino acid productivity of said bacterium is enhanced by the use of the acetolactate synthase (AHAS I) which is resistant to feedback inhibition by L-valine. This acetolactate synthase contains a mutant small subunit encoded by the mutant ilvN gene.

1 . A mutant small subunit of bacterial acetolactate synthase (AHAS I) comprising the wild-type acetolactate synthase small subunit of  Escherichia coli  comprising a mutation selected from the group consisting of:
 A) replacing the L-amino acid at position 17 and/or 30 in said wild-type acetolactate synthase small subunit with another L-amino acid,   B) replacing the N-terminus portion downstream from the L-amino acid at position 44 in said wild-type acetolactate synthase small subunit with several L-amino acids, and   C) combinations thereof,   and wherein said mutant small subunit of acetolactate synthase is desensitized to feedback inhibition by valine.   
     
     
         2 . The mutant small subunit of bacterial acetolactate synthase according to  claim 1 , wherein said L-amino acid at position 17, is replaced with a lysine residue. 
     
     
         3 . The mutant small subunit of bacterial acetolactate synthase according to  claim 1 , wherein said L-amino acid at position 30 is replaced with a proline residue. 
     
     
         4 . The mutant small subunit of bacterial acetolactate synthase according to  claim 1 , wherein said N-terminus portion downstream from the L-amino acid at position 44 is replaced with arginine and phenylalanine. 
     
     
         5 . The mutant small subunit of bacterial acetolactate synthase according to  claim 2 , wherein said L-amino acid at position 17 which is replaced with a lysine residue is asparagine. 
     
     
         6 . The mutant small subunit of bacterial acetolactate synthase according to  claim 3 , wherein said L-amino acid at position 30 which is replaced with a proline residue is alanine. 
     
     
         7 . The mutant small subunit of bacterial acetolactate synthase according to  claim 4 , wherein said L-amino acid at position 44 which is replaced with arginine and phenylalanine is isoleucine. 
     
     
         8 . A mutant acetolactate synthase comprising the small subunit according to  claim 1 . 
     
     
         9 . The mutant acetolactate synthase according to  claim 8 , wherein said mutant acetolactate synthase comprises the  Escherichia coli  large subunit. 
     
     
         10 . The mutant acetolactate synthase according to  claim 8 , wherein the small subunit includes deletions, substitutions, insertions, or additions of one or several amino acids at one or more positions other than position 17, 30 and/or 44, and wherein said mutant acetolactate synthase is desensitized to feedback inhibition by valine. 
     
     
         11 . A DNA coding for the mutant small subunit of the acetolactate synthase according to  claim 1 . 
     
     
         12 . A bacterium of the Enterobacteriaceae family, which contains the DNA according to  claim 11  and has the ability to produce branched-chain L-amino acids. 
     
     
         13 . The bacterium according to  claim 12 , wherein said branched-chain L-amino acids are selected from the group consisting of L-leucine, L-isoleucine, and L-valine. 
     
     
         14 . The bacterium according to  claim 13 , wherein the activity of the mutant acetolactate synthase is enhanced. 
     
     
         15 . The bacterium according to  claim 14 , wherein the bacterium belongs to the genus  Escherichia.    
     
     
         16 . The bacterium according to  claim 15 , wherein the activity of the mutant acetolactate synthase is enhanced by increasing the expression of the mutant acetolactate synthase gene. 
     
     
         17 . The bacterium according to  claim 16 , wherein the activity of the mutant acetolactate synthase is increased by a method selected from the group consisting of:
 a) increasing the copy number of the mutant acetolactate synthase gene,   b) modifying an expression control sequence of the gene so that the expression of the gene is enhanced, and   c) combinations thereof.   
     
     
         18 . The bacterium according to  claim 17 , wherein the copy number is increased by the integration of multiple copies of the mutant acetolactate synthase gene into the chromosome of the bacterium. 
     
     
         19 . A method for producing a branched-chain L-amino acid comprising cultivating the bacterium according to  claim 12  in a culture medium, and collecting the branched-chain L-amino acids from the culture medium. 
     
     
         20 . The method according to  claim 19 , wherein the bacterium has enhanced expression of genes involved in branched-chain L-amino acid biosynthesis. 
     
     
         21 . The method according to  claim 20 , wherein said branched-chain L-amino acid is selected from the group consisting of L-leucine, L-isoleucine, and L-valine.