Novel nucleotide sequences coding the citE gene

Abstract

The invention relates to an isolated polynucleotide containing a polynucleotide sequence selected from the group a) polynucleotide that is at least 70% identical with a polynucleotide that codes for a polypeptide containing the amino acid sequence of SEQ ID No. 2, b) polynucleotide that codes for a polypeptide containing an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No. 2, c) polynucleotide that is complementary to the polynucleotides of a) or b), and d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c), and to a process for the production of L-amino acids by fermentation using coryneform bacteria in which at least the citE gene is present in attenuated form, and to the use of polynucleotides containing the sequences of the invention as hybridization probes.

1 . An isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence coding for the citE gene, selected from the group 
 a) polynucleotide that is at least 70% identical with a polynucleotide that codes for a polypeptide containing the amino acid sequence of SEQ ID No. 2,    b) polynucleotide that codes for a polypeptide containing an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No. 2,    c) polynucleotide that is complementary to the polynucleotides of a) or b), and    d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c), wherein    e) the polypeptide preferably exhibits the activity of citrate lyase E.    
     
     
         2 . The polynucleotide as claimed in  claim 1 , wherein the polynucleotide is a DNA, preferably recombinant DNA, that is replicatable in coryneform bacteria.  
     
     
         3 . The polynucleotide as claimed in  claim 1 , wherein the polynucleotide is an RNA.  
     
     
         4 . The polynucleotide as claimed in  claim 2 , containing the nucleic acid sequence as shown in SEQ ID No. 1.  
     
     
         5 . The replicatable DNA as claimed in  claim 2 , containing 
 (i) the nucleotide sequence shown in SEQ ID No. 1, or    (ii) at least one sequence that corresponds to sequence (i) within the region of the degeneration of the genetic code, or    (iii) at least one sequence that hybridizes with the sequence that is complementary to sequence (i) or (ii), and, optionally,    (iv) sense mutations in (i) that are neutral in terms of function.    
     
     
         6 . The replicatable DNA as claimed in  claim 5 , wherein the hybridization is carried out under a stringency corresponding to not more than 2× SSC.  
     
     
         7 . The polynucleotide sequence as claimed in  claim 1 , which codes for a polypeptide containing the amino acid sequence shown in SEQ ID No. 2.  
     
     
         8 . A coryneform bacterium in which the citE gene is attenuated, especially excluded.  
     
     
         9 . A process for the production of L-amino acids, especially L-lysine, by fermentation, which process comprises carrying out the following steps: 
 a) fermenting the coryneform bacteria producing the desired L-amino acid, in which bacteria at least the citE gene or nucleotide sequences coding therefor are attenuated, especially excluded;    b) concentrating the L-amino acid in the medium or in the cells of the bacteria, and    c) isolating the L-amino acid.    
     
     
         10 . The process as claimed in  claim 9 , wherein bacteria are used in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced.  
     
     
         11 . The process as claimed in  claim 9 , wherein bacteria are used in which at least some of the metabolic pathways that reduce formation of the desired L-amino acid are excluded.  
     
     
         12 . The process as claimed in  claim 9 , wherein expression of the polynucleotide(s) coding for the citE gene is attenuated, especially excluded.  
     
     
         13 . The process as claimed in  claim 9 , wherein the regulatory (or catalytic) properties of the polypeptide (enzyme protein) for which the polynucleotide citE codes are diminished.  
     
     
         14 . The process as claimed in  claim 9 , wherein, for the production of L-amino acids, coryneform microorganisms are fermented in which, at the same time, one or more genes selected from the group 
 14.1 The gene dapA coding for dihydropicolinate synthase,    14.2 the gene gap coding for glyceraldehyde 3-phosphate dehydrogenase,    14.3 the gene tpi coding for triose phosphate isomerase,    14.4 the gene pgk coding for 3-phosphoglycerate kinase,    14.5 the gene zwf coding for glucose-6-phosphate dehydrogenase,    14.6 the gene pyc coding for pyruvate carboxylase,    14.7 the gene mqo coding for malate quinone oxido-reductase,    14.8 the gene lysC coding for a feed-back resistant aspartate kinase,    14.9 the gene lysE coding for lysine export,    14.10 the gene zwa1 coding for the Zwa1 protein    are enhanced or overexpressed.    
     
     
         15 . The process as claimed in  claim 9 , wherein, for the production of L-amino acids, coryneform microorganisms are fermented in which, at the same time, one or more genes selected from the group 
 15.1 the gene pck coding for phosphoenol pyruvate carboxykinase,    15.2 the gene pgi coding for glucose-6-phosphate isomerase,    15.3 the gene poxB coding for pyruvate oxidase,    15.4 the gene zwa2 coding for the Zwa2 protein,    15.5 the gene hom coding for homoserine dehydrogenase,    15.6 the gene thrB coding for homoserine kinase, and    15.7 the gene panD coding for aspartate decarboxylase    are attenuated, in particular expression thereof is diminished.    
     
     
         16 . A coryneform bacterium containing a vector that carries portions of the polynucleotide as claimed in  claim 1 , but at least 15 consecutive nucleotides of the claimed sequence.  
     
     
         17 . An isolated microorganism of the  E. coli  strain Top10/pCR2.1citEint, deposited at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen in Braunschweig under number DSM 13981 in accordance with the Budapest Treaty.  
     
     
         18 . The process as claimed in one or more of the preceding claims, wherein microorganisms of the species  Corynebacterium glutamicum  are used.  
     
     
         19 . A method of finding RNA, cDNA and DNA in order to isolate nucleic acids, or polynucleotides or genes, that code for citrate lyase E or are very similar to the sequence of the citE gene, which method comprises using the polynucleotide containing the polynucleotide sequences as claimed in claims  1 ,  2 ,  3  or  4  as hybridization probe.  
     
     
         20 . The method as claimed in  claim 18 , wherein arrays, micro arrays or DNA chips are used.