Process for Preparing L-amino Acids

Abstract

The invention relates to isolated polynucleotides comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b), and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the citA gene is present in attenuated form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

1 - 19 . (canceled)  
     
     
         20 . A process for producing L-amino acids, which comprises 
 fermentation of coryneform bacterium which produce the desired L-amino acid in which the intracellular activity of a polypeptide encoded by the citA gene comprising the amino acid sequence of SEQ ID NO: 2 is eliminated,    wherein elimination is achieved by one or more methods of mutagenesis selected from the group consisting of deletion, insertional mutagenesis due to homologous recombination, and transition or transversion mutagenesis with incorporation of a non-sense mutation in the citA gene.    
     
     
         21 . The process according to  claim 20 , wherein the citA gene of said coryneform bacterium comprises a polynucleotide having the nucleotide sequence of nucleotides 201 to 1853 of SEQ ID NO:1.  
     
     
         22 . The process according to  claim 20 , wherein the citA gene of said coryneform bacterium comprises a polynucleotide having the nucleotide sequence shown in SEQ ID NO:1.  
     
     
         23 . The process according to  claim 20 , further comprising: 
 a) concentrating the desired L-amino acid produced, in the medium or in the cells of the bacterium; and    b) isolating the L-amino acid produced.    
     
     
         24 . The process according to  claim 20 , wherein the L-amino acid produced is selected from the group consisting of L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, and L-arginine.  
     
     
         25 . The process according to  claim 24 , wherein the L-amino acid produced is L-lysine.  
     
     
         26 . The process according to  claim 20 , wherein bacteria are employed in which one or more further gene or genes of the biosynthesis pathway of the desired L-amino acid is additionally enhanced.  
     
     
         27 . The process according to  claim 20 , wherein one or more coryneform bacterium genes selected from the group consisting of: 
 (a) the dapA gene which codes for dihydrodipicolinate synthase,    (b) the gap gene which codes for glycerolaldehyde 3-phosphate dehydrogenase,    (c) the zwf gene which codes for glucose 6-phosphate dehydrogenase,    (d) the pyc gene which codes for pyruvate carboxylase,    (e) the lysE gene which codes for a protein of lysine export,    (f) the lysC gene which codes for a feed back resistant aspartate kinase,    (g) the zwal gene which codes for the zwal protein    is overexpressed by increasing the copy number or placing said gene under a strong promoter during fermentation for the production of the desired L-amino acid.    
     
     
         28 . The process according to  claim 27 , wherein the L-amino acid produced is L-lysine.  
     
     
         29 . The process according to  claim 20 , wherein bacteria are employed in which metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated.  
     
     
         30 . The process according to  claim 20 , wherein the activity (activities) of the polypeptide(s) encoded by one or more coryneform bacterium genes selected from the group consisting of: 
 (a) the pck gene which codes for pyhosphoenol pyruvate carbosykinase,    (b) the pgi gene which codes for glucose 6-phosphate isomerase,    (c) the poxB gene which codes for pyruvate oxidase    (d) the zwa2 gene which codes for the zwa2 protein    is attenuated.    
     
     
         31 . The process according to  claim 30 , wherein the expression of one or more coryneform bacterium genes selected from the group consisting of: 
 (a) the pck gene which codes for pyhosphoenol pyruvate carbosykinase,    (b) the pgi gene which codes for glucose 6-phosphate isomerase,    (c) the poxB gene which codes for pyruvate oxidase    (d) the zwa2 gene which codes for the zwa2 protein    is reduced or eliminated.    
     
     
         32 . The process according to  claim 30 , wherein the catalytic properties of the polypeptide(s) encoded by one or more coryneform bacterium genes selected from the group consisting of: 
 (a) the pck gene which codes for pyhosphoenol pyruvate carbosykinase,    (b) the pgi gene which codes for glucose 6-phosphate isomerase,    (c) the poxB gene which codes for pyruvate oxidase    (d) the zwa2 gene which codes for the zwa2 protein    are reduced or eliminated.    
     
     
         33 . The process according to  claim 20 , wherein microorganisms of the genus  Corynebacterium  are employed.  
     
     
         34 . The process according to  claim 33 , wherein microorganisms of the species  Corynebacterium glutamicum  are employed.