Process for preparing L-amino acids using improved strains of the enterobacteriaceae family

Abstract

The invention relates to a process for preparing L-amino acids by fermenting recombinant microorganisms of the Enterobacteriaceae family, characterized in that a) the desired L-amino acid-producing microorganisms, in which the yjcG-ORF, or nucleotide sequences or alleles encoding the gene product, is/are enhanced, in particular overexpressed, is cultured in a medium under conditions under which the desired L-amino acid is accumulated in the medium or in the cells, and b) the desired L-amino acid is isolated, with, where appropriate, constituents of the fermentation broth, and/or the biomass remaining in its/their entirety or in portions (from ≧0 to 100%) in the isolated product or being removed completely.

1 . A recombinant microorganism which contains an enhanced or overexpressed yjcG-ORF, the gene product of which has acetate permease activity.  
     
     
         2 . A microorganism as claimed in  claim 1 , in which a polynucleotide which corresponds to yjcG-ORF and encodes a polypeptide whose amino acid sequence is at least 90% identical to an amino acid sequence selected from the group SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6 is enhanced, the polypeptide having acetate permease activity.  
     
     
         3 . A microorganism as claimed in  claim 2 , characterized in that it contains an overexpressed or enhanced polynucleotide which corresponds to ycjG-ORF and which is selected from the group: 
 a) polynucleotide having a nucleotide sequence, selected from SEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 3 and the nucleotide sequences complementary thereto;    b) polynucleotide having a nucleotide sequence which corresponds to SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 within the limits of the degeneracy of the genetic code;    c) polynucleotide sequence having a sequence which hybridizes, under stringent conditions, with the sequence which is complementary to the sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5, with the stringent conditions being achieved by means of a washing step in which the temperature extends over a range of from 64° C. to 68° C. and the salt concentration of the buffer extends over a range of from 2×SSC to 0.1×SSC;    d) polynucleotide having a sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 which contains functionally neutral sense mutants.    
     
     
         4 . A microorganism as claimed in  claim 2 , characterized in that the polypeptide possesses an amino acid sequence which is at least 95% identical to one of the sequences selected from the group SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6.  
     
     
         5 . A microorganism as claimed in  claim 2 , characterized in that the polypeptide possesses the amino acid sequence which is 100% identical to that of one of the sequences selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No.6.  
     
     
         6 . A microorganism as claimed in  claim 1 , characterized in that it is produced by transformation, transduction or conjugation, or a combination of these methods, with a vector which contains the yjcG-ORF, an allele of this ORF, or parts thereof, and/or a promotor.  
     
     
         7 . A microorganism as claimed in  claim 1 , in which the copy number of the yjcG-ORF or the alleles has been increased by at least 1.  
     
     
         8 . The microorganism as claimed in  claim 7 , characterized in that the increase in the copy number of the yjcG-ORF by at least 1 is achieved by integrating the ORF or the alleles into the chromosome of the microorganism.  
     
     
         9 . The microorganism as claimed in  claim 7 , characterized in that the increase in the copy number of the yjcG-ORF by at least 1 is achieved by means of a vector which replicates extrachromosomally.  
     
     
         10 . The microorganism as claimed in  claim 1 , characterized in that, in order to achieve the enhancement, 
 a) the promoter and regulatory region or the ribosomal binding site upstream of the yjcG-ORF is mutated, or    b) expression cassettes or promoters are incorporated upstream of the yjcG-ORF.    
     
     
         11 . The microorganism as claimed in  claim 1 , characterized in that the expression of the yjcG-ORF is under the control of a promoter enhancing the expression of the ORF.  
     
     
         12 . The microorganism as claimed in  claim 1 , characterized in that enhancing the yjcG-ORF increases the concentration or activity of the yjcG gene product (protein) by at least 10%, based on the activity or concentration of the gene product in the parent strain or microorganism not recombinant for the yjcG-ORF.  
     
     
         13 . The microorganism as claimed in  claim 1 , characterized in that the microorganism is selected from the genera  Escherichia, Erwinia, Providencia  and  Serratia.    
     
     
         14 . The microorganism as claimed in  claim 13 , characterized in that other genes of the pathway for the biosynthesis of the desired L-amino acid are also present in enhanced, in particular overexpressed, form.  
     
     
         15 . The microorganism as claimed in  claim 1 , characterized in that it produces L-threonine.  
     
     
         16 . A process for preparing L-amino acids by fermenting recombinant microorganisms of the Enterobacteriaceae family, characterized in that 
 a) the desired L-amino acid-producing microorganisms as claimed in  claim 1  are cultured in a medium under conditions under which the desired L-amino acid is accumulated in the medium or in the cells, and    b) the desired L-amino acid is isolated, with constituents of the fermentation broth, and/or the biomass remaining in its/their entirety or in portions (from ≧0 to 100%) in the isolated product or being removed completely.    
     
     
         17 . The process as claimed in  claim 16 , characterized in that, for the purpose of preparing L-threonine, microorganisms are fermented in which one or more of the genes selected from the group: 
 a) at least one gene of the thrABC operon encoding aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase,    b) the pyruvate carboxylase-encoding  Corynebacterium glutamicum  pyc gene,    c) the phosphoenolpyruvate synthase-encoding pps gene,    d) the phosphoenolpyruvate carboxylase-encoding ppc gene,    e) the pntA and pntB genes encoding the subunits of pyridine transhydrogenase,    f) the rhtC gene encoding the threonine resistance-mediating protein,    g) the threonine export carrier protein-encoding  Corynebacterium glutamicum  thrE gene,    h) the glutamate dehydrogenase-encoding gdhA gene,    i) the ptsH gene encoding the phosphohistidine protein hexose phosphotransferase,    j) the ptsI gene encoding enzyme I of the phosphotransferase system,    k) the crr gene encoding the glucose-specific IIA component,    l) the ptsG gene encoding the glucose-specific IIBC component,    m) the cysteine synthase A-encoding cysK gene,    n) the cysB gene encoding the regulator of the cys regulon,    o) the cysJ gene encoding the NADPH sulfite reductase flavoprotein,    p) the cysI gene encoding the NADPH sulfite reductase hemoprotein,    q) the adenylyl sulfate reductase-encoding cysH gene,    r) the sucA gene encoding the decarboxylase subunit of 2-ketoglutarate dehydrogenase,    s) the sucB gene encoding the dihydrolipoyl-transsuccinase E2 subunit of 2-ketoglutarate dehydrogenase,    t) the sucC gene encoding the β-subunit of succinyl-CoA synthetase,    u) the sucD gene encoding the α-subunit of succinyl-CoA synthetase,    v) the gene product of the  Escherichia coli  yibD open reading frame (ORF), and    w) the acs encoding the acetyl coenzyme A synthetase    is/are additionally, at the same time, enhanced, in particular overexpressed.    
     
     
         18 . The process as claimed in  claim 16 , characterized in that use is made of microorganisms in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partially attenuated.  
     
     
         19 . The process as claimed in  claim 18 , characterized in that, for the purpose of preparing L-threonine, microorganisms are fermented in which one or more of the genes selected from the group: 
 a) the threonine dehydrogenase-encoding tdh gene,    b) the malate dehydrogenase-encoding mdh gene, c) the gene product of the  Escherichia coli  yjfA open reading frame (ORF),    d) the gene product of the  Escherichia coli  ytfP open reading frame (ORF),    e) the pckA gene encoding the phosphoenolpyruvate carboxykinase,    f) the pyruvate oxidase-encoding poxB gene,    g) the dgsA gene encoding the DgsA regulator of the phosphotransferase system,    h) the fruR gene encoding the fructose repressor,    i) the rpoS gene encoding the sigma 38  factor, and,    j) the aspartate ammonium lyase-encoding aspA gene,    is/are additionally, at the same time, attenuated, in particular eliminated, or their expression is reduced.    
     
     
         20 . The process as claimed in  claim 16 , characterized in that L-amino acids selected from the group L-asparagine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-proline, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and L-homoserine are prepared.  
     
     
         21 . The process as claimed in  claim 16 , characterized in that L-amino acids selected from the group L-isoleucine, L-valine, L-methionine, L-homoserine, L-tryptophan and L-lysine are prepared.