Process for the production of L-amino acids using strains of the Enterobacteriaceae family

Abstract

The present invention relates to a process for the production of L-amino acids by fermentation of recombinant microorganisms of the Enterobacteriaceae family, wherein a) the yfiD ORF and/or the pflB gene or nucleotide sequences coding for the gene products are overexpressed in the microorganisms producing the desired L-amino acid, and the microorganisms are cultured in a medium under conditions in which the desired L-amino acid is enriched in the medium or in the cells; and b) the desired L-amino acid is isolated, in a manner such that constituents of the fermentation broth and/or the biomass in its entirety or in portions (>0 to 100%) either remain in the isolated product or are completely removed.

1 . A process for the production of an L-amino acid product by fermentation comprising: 
 a) culturing a recombinant microorganism from the Enterobacteriaceae family in a fermentation medium, wherein said recombinant microorganism produces said L-amino acid and wherein the pflB gene is overexpressed in said recombinant microorganism or another nucleotide sequence that codes for the pflB gene product is expressed in said recombinant microorganism;    b) enriching said L-amino acid in said fermentation medium or in said recombinant microorganism; and    c) isolating said L-amino acid to produce said L-amino acid product.    
     
     
         2 . The process of  claim 1  wherein some or all of the constituents of said fermentation medium and/or the biomass of said recombinant microorganism remain in said L-amino acid product.  
     
     
         3 . The process of  claim 1 , wherein said recombinant microorganism is made by the transformation of a microorganism of the Enterobacteriaceae family with a vector containing the plfB gene.  
     
     
         4 . The process of  claim 1 , wherein the number of copies of said pflB gene in said recombinant microorganism is increased by at least 1.  
     
     
         5 . The process of  claim 4 , wherein the increase in the number of copies of the pflB gene by at least 1 is achieved by integration of said gene or ORF into the chromosome of said recombinant microorganism.  
     
     
         6 . The process of  claim 4 , wherein the increase in the number of copies of the pflB gene by at least 1 is achieved by means of an extra-chromosomally replicating vector.  
     
     
         7 . The process of  claim 1 , wherein said overexpression is achieved by: 
 a) mutating the promoter or the ribosome binding site upstream of said pflB gene; or    b) incorporating an expression cassette or promoter upstream of said pflB gene.    
     
     
         8 . The process of  claim 1 , wherein said recombinant microorganism is made by the transformation of a microorganism with a polynucleotide coding for the pflB gene product and wherein the expression of said polynucleotide is under the control of a promoter.  
     
     
         9 . The process of  claim 1 , wherein, through the recombinant engineering of the pflB gene, the concentration or activity of the pflB gene product (protein) is increased by at least 10%, relative to the activity or concentration of the gene product in the initial strain.  
     
     
         10 . The process of  claim 1 , wherein the genus of said recombinant microorganism is selected from the group consisting of:  Escherichia; Erwinia; Providencia;  and  Serratia.    
     
     
         11 . The process of  claim 1 , wherein, said microorganism overexpresses said pflB gene, and, in addition, at least one gene in the biosynthesis pathway of said L-amino acid is also overexpressed.  
     
     
         12 . The process of  claim 1 , wherein said microorganism overexpresses said pflB gene, and, in addition, the activity of one or more additional genes is enhanced, said one or more additional genes being selected from the group consisting of: 
 a) the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase;    b) the pyc gene coding for pyruvate carboxylase;    c) the pps gene for phosphoenolpyruvate synthase;    d) the ppc gene coding for phosphoenolpyruvate carboxylase;    e) the genes pntA and pntB coding for transhydrogenase;    f) the rhtB gene imparting homoserine resistance;    g) the mqo gene coding for malate:quinone oxidoreductase;    h) the rhtC gene imparting threonine resistance;    i) the thrE gene coding for the threonine-export protein;    j) the gdhA gene coding for glutamate dehydrogenase;    k) the hns gene coding for the DNA binding protein HLP-II;    l) the pgm gene coding for phosphoglucomutase;    m) the fba gene coding for fructose biphosphate aldolase;    n) the ptsH gene coding for phosphohistidine protein hexose phosphotransferase;    o) the ptsI gene coding for enzyme I of the phosphotransferase system;    p) the crr gene coding for the glucose-specific IIA component;    q) the ptsG gene coding for the glucose-specific IIBC component;    r) the lrp gene coding for the regulator of the leucine regulon;    s) the csrA gene coding for the global regulator Csr;    t) the fadR gene coding for the regulator of the fad regulon;    u) the iclR gene coding for the regulator of central intermediary metabolism;    v) the mopB gene coding for the 10 kDa chaperon;    w) the ahpC gene coding for the small subunit of alkyl hydroperoxide reductase;    x) the ahpF gene coding for the large subunit of alkyl hydroperoxide reductase;    y) the cysK gene coding for cysteine synthase A;    z) the cysB gene coding for the regulator of the cys regulon;    aa) the cysJ gene coding for the flavoprotein of NADPH sulfite reductase;    bb) the cysI gene coding for the haemoprotein of NADPH sulfite reductase;    cc) the cysH gene coding for adenylyl sulfate reductase;    dd) the phoB gene coding for the positive regulator PhoB of the pho regulon;    ee) the phoR gene coding for the sensor protein of the pho regulon;    ff) the phoE gene coding for protein E of the outer cell membrane;    gg) the pykF gene coding for pyruvate kinase I, which is stimulated by fructose;    hh) the pfkB gene coding for 6-phosphofructokinase II;    ii) the malE gene coding for the periplasmic binding protein of maltose transport;    jj) the sodA gene coding for superoxide dismutase;    kk) the rseA gene coding for a membrane protein with anti-sigmaE activity;    ll) the rseC gene coding for a global regulator of the sigmaE factor;    mm) the sucA gene coding for the decarboxylase subunit of 2-ketoglutarate dehydrogenase;    nn) the sucB gene coding for the dihydrolipoyl transsuccinase E2 subunit of 2-ketoglutarate dehydrogenase;    oo) the sucC gene coding for the β-subunit of succinyl-CoA synthetase;    pp) the sucD gene coding for the a-subunit of succinyl-CoA synthetase;    qq) the adk gene coding for adenylate kinase;    rr) the hdeA gene coding for a periplasmic protein with chaperonin-type function;    ss) the hdeB gene coding for a periplasmic protein with chaperonin-type function;    tt) the icd gene coding for isocitrate dehydrogenase;    uu) the mglB gene coding for the periplasmic, galactose-binding transport protein;    vv) the lpd gene coding for dihydrolipoamide dehydrogenase;    ww) the aceE gene coding for the E1 component of the pyruvate-dehydrogenase complex;    xx) the aceF gene coding for the E2 component of the pyruvate-dehydrogenase complex;    yy) the pepB gene coding for aminopeptidase B;    zz) the aldH gene coding for aldehyde dehydrogenase,    aaa) the bfr gene coding for the iron-storage homoprotein;    bbb) the udp gene coding for uridine phosphorylase; and    ccc) the rseB gene coding for the regulator of sigmaE-factor activity.    
     
     
         13 - 14 . (canceled)  
     
     
         15 . The process of  claim 1 , wherein said L-amino acid is selected from the group consisting of: L-asparagine, L-serine, L-glutamate, 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.  
     
     
         16 . The process of  claim 1 , wherein said L-amino acid is selected from the group consisting of: L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine are produced.  
     
     
         17 . The process of of  claim 1 , wherein said L-amino acid is L-threonine.  
     
     
         18 - 20 . (canceled)  
     
     
         21 . The process of  claim 17 , wherein some or all of the constituents of said fermentation medium and/or the biomass of said recombinant microorganism remain in said L-amino acid product.  
     
     
         22 . The process of  claim 21 , wherein said recombinant microorganism is made by the transformation of a microorganism of the Enterobacteriaceae family with a vector containing the plfB gene.  
     
     
         23 . The process of  claim 21 , wherein the number of copies of said pflB gene in said recombinant microorganism is increased by at least 1.  
     
     
         24 . The process of  claim 17 , wherein overexpression is achieved by: 
 a) mutating the promoter or the ribosome binding site upstream of said pflB gene; or    b) incorporating an expression cassette or promoter upstream of said pflB gene.    
     
     
         25 . The process of  claim 17 , wherein said recombinant microorganism is made by the transformation of a microorganism with a polynucleotide coding for the pflB gene product and wherein the expression of said polynucleotide is under the control of a promoter.