Feedback resistant acetohydroxy acid synthethase mutants

Abstract

The present invention provides nucleotide sequences coding for acetohydroxy acid synthetase (AHAS) mutants, the mutated enzymes themselves and a process for the fermentative production of branched-chain amino acids using these enzymes in specific hosts in which genes which code for the modified acetohydroxy acid synthetase (AHAS) are expressed.

1 - 11 . (canceled) 
     
     
         12 . A process for preparing an enantiomer-enriched branched chain amino acid comprising:
 fermenting a suitable medium with a cell encoding a polypeptide having acetohydroxy acid synthetase (AHAS) activity and encoding a polypeptide that does not contain the sequence Gly Ile Ile at the positions corresponding to residues 20-22 of SEQ ID NO: 2 comprising:   (a) SEQ ID NO: 1,   (b) SEQ ID NO: 3, or   (c) a polynucleotide sequence that is at least 95% homologous to SEQ ID NO: 1 or 3;   wherein said polynucleotide sequence does not encode a protein comprising the sequence Gly Ile Ile at the positions corresponding to residues 20-22 of SEQ ID NO: 2.   
     
     
         13 . The process of  claim 12 , wherein said polynucleotide encodes the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         14 . The process of  claim 12 , wherein said polynucleotide comprises SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
         15 . The process of  claim 12 , wherein said polynucleotide encodes a polypeptide having Asp and Phe at the positions corresponding to residues 21 and 22 of SEQ ID NO: 2. 
     
     
         16 . The process of  claim 12 , wherein said cornyneform bacteria have been transformed with a vector expressing said polypeptide. 
     
     
         17 . The process of  claim 12 , wherein said cornyneform bacteria have been transformed with a vector expressing said polypeptide that is pECKA or pECKA/ilvBNC. 
     
     
         18 . The process of  claim 12 , wherein the cell is  corynebacterium.    
     
     
         19 . The process of  claim 12 , wherein said cell is  Corynebacterium.    
     
     
         20 . The process of  claim 12 , wherein said cell is  Corynebacterium glutamicum.    
     
     
         21 . The process of  claim 12 , wherein said cell is  Brevibacterium.    
     
     
         22 . The process of  claim 12 , wherein said cell is selected from the group consisting of DSM15652, DSM 15561 and DSM15650. 
     
     
         23 . The process of  claim 12 , wherein said branched chain amino acid is valine. 
     
     
         24 . The process of  claim 12 , wherein said branched chain amino acid is leucine. 
     
     
         25 . The process of  claim 12 , wherein said branched chain amino acid is isoleucine. 
     
     
         26 . A process for preparing an enantiomer-enriched branched chain amino acid comprising:
 fermenting a suitable medium with a cell transformed with polynucleotide encoding a polypeptide having acetohydroxy acid synthetase (AHAS) activity, said polynucleotide comprising:   (a) SEQ ID NO: 1,   (b) SEQ ID NO: 3, or   (c) a polynucleotide sequence that is at least 95% homologous to SEQ ID NO: 1 or SEQ ID NO: 3;   wherein said polynucleotide is over-expressed by increasing its copy number, modifying its promoter, regulatory region or ribosome binding site, or by upstream incorporation of an expression cassette.   
     
     
         27 . The process of  claim 26 , wherein said polynucleotide sequence encodes a protein comprising the sequence Gly Ile Ile at the positions corresponding to residues 20-22 of SEQ ID NO: 2