Method for zymotic production of fine chemicals containing sulphur (metA)

Abstract

The invention relates to methods for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine, by using bacteria which express a nucleotide sequence coding for a methionine synthase (meta) gene.

1 . A method for the fermentative production of L-methionine, which comprises the following steps: 
 a) fermenting in a medium cells of a coryneform bacterium for producing L-methionine, the coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes for a protein with homoserine O-acetyltransferase (metA) activity, wherein said heterologous nucleotide sequence comprises a nucleotide sequence encoding a metA protein having an amino acid sequence with 95% homology or more to the sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46;    b) concentrating L-methionine in the medium or in the bacterial cells; and    c) isolating L-methionine.    
     
     
         2 . The method of  claim 1 , wherein the metA-encoding sequence is derived from any of the following organisms:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                 
                     
                   
                     Mycobacterium leprae 
                   
                   ATCC 43910 
                 
                     
                     Mycobacterium tuberculosis  CDC1551 
                   ATCC 25584 
                 
                     
                   
                     Chlorobium tepidum 
                   
                   ATCC 49652 
                 
                     
                   
                     Pseudomonas aeruginosa 
                   
                   ATCC 17933 
                 
                     
                   
                     Caulobacter crescentus 
                   
                   ATCC 19089 
                 
                     
                   
                     Neisseria gonorrhoeae 
                   
                   ATCC 53420 
                 
                     
                   
                     Neisseria meningitidis 
                   
                   ATCC 53414 
                 
                     
                   
                     Pseudomonas fluorescens 
                   
                   ATCC 13525 
                 
                     
                   
                     Burkholderia cepacia 
                   
                   ATCC 25416 
                 
                     
                   
                     Nitrosomonas europaea 
                   
                   ATCC 19718 
                 
                     
                   
                     Haemophilus influenzae 
                   
                   ATCC 51907 
                 
                     
                     Halobacterium  sp NRC1 
                   ATCC 33170 
                 
                     
                   
                     Thermus thermophilus 
                   
                   ATCC 27634 
                 
                     
                   
                     Deinococcus radiodurans 
                   
                   ATCC 13939 
                 
                     
                   
                     Saccharomyces cerevisiae 
                   
                   ATCC 10751 
                 
                     
                   
                     Schizosaccharomyces pombe 
                   
                   ATCC 24969 
                 
                     
                   
                     Xylella fastidiosa 
                   
                   ATCC 35881 
                 
                     
                   
                     Emericella nidulans 
                   
                   ATCC 36104 
                 
                     
                   
                     Mesorhizobium loti 
                   
                   ATCC 35173 
                 
                     
                   
                     Acremonium crysogenum 
                   
                   ATCC 11550 
                 
                     
                   
                     Pseudomonas putida 
                   
                   ATCC 47054 
                 
                     
                   
                     Staphylococcus aureus 
                   
                   ATCC 35556 
                 
                     
                     
                 
                     
                     
                 
             
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . The method of  claim 1 , wherein the meta-encoding sequence comprises a coding sequence as set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45.  
     
     
         4 . The method of  claim 1 , wherein the meta-encoding sequence encodes for a protein with meta activity, said protein comprising an amino acid sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46.  
     
     
         5 . The method of  claim 1 , wherein the meta encoding sequence is a DNA or RNA which can be replicated in coryneform bacteria or is stably integrated into the chromosome.  
     
     
         6 . The method of  claim 5 , wherein 
 a) a bacteria strain transformed with a plasmid vector carrying at least one copy of the meta encoding sequence under the control of regulatory sequences is used, or    b) a strain in which the meta encoding sequence has been integrated into the bacteria chromosome is used.    
     
     
         7 . The method of  claim 1 , wherein the meta encoding sequence is overexpressed.  
     
     
         8 . The method of  claim 1 , wherein bacteria are fermented in which additionally at least one further gene of the biosynthetic pathway of L-methionine has been overexpressed or mutated such that its activity is not influenced by metabolic metabolites.  
     
     
         9 . The method of  claim 1 , wherein bacteria are fermented in which at least one metabolic pathway, which reduces the production of L-methionine, is at least partially switched off.  
     
     
         10 . The method of  claim 1 , wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among 
 a) a lysC gene derived from a coryneform bacterium, which encodes an aspartate kinase,    b) a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gap,    c) a 3-phosphoglycerate kinase-encoding gene pgk,    d) a pyruvate carboxylase-encoding gene pyc,    e) a triose phosphate isomerase-encoding gene tpi,    f) a methylene tetrahydrofolate reductase-encoding gene metF,    g) a cystathionine gamma-synthase-encoding gene metB,    h) a cystathionine gamma-lyase-encoding gene metC,    i) a serine hydroxymethyltransferase-encoding gene glyA,    j) a O-acetylhomoserine sulfhydrylase-encoding gene metY,    k) a vitamin B12-dependent methionine synthase-encoding gene metH,    l) a phosphoserine aminotransferase-encoding gene serC,    m) a phosphoserine phosphatase-encoding gene serB,    n) a serine acetyltransferase-encoding gene cysE, and    o) a gene hom, which encodes a homoserine dehydrogenase,    is overexpressed or mutated in such a way that the activity of the corresponding proteins is influenced by metabolic metabolites to a smaller extent, if at all, compared to nonmutated proteins.    
     
     
         11 . The method of  claim 1 , wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among 
 a) a homoserine kinase-encoding gene thrB,    b) a threonine dehydratase-encoding gene ilvA,    c) a threonine synthase-encoding gene thrC,    d) a meso-diaminopimelate D-dehydrogenase-encoding gene ddh,    e) a phosphoenolpyruvate carboxykinase-encoding gene pck,    f) a glucose-6-phosphate 6-isomerase-encoding gene pgi,    g) a pyruvate oxidase-encoding gene poxB,    h) a dihydrodipicolinate synthase-encoding gene dapA,    i) a dihydrodipicolinate reductase-encoding gene dapB; and    j) a diaminopicolinate decarboxylase-encoding gene,    is attenuated by changing the rate of expression or by introducing a specific mutation.    
     
     
         12 . The method of  claim 1 , wherein the coryneform bacterium is of the species  Corynebacterium glutamicum.    
     
     
         13 . A method for producing an L-methionine-containing animal feed additive from a fermentation medium, comprising 
 a) fermenting in a fermentation medium cells of a coryneform bacterium for producing L-methionine, the coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes for a protein with homoserine O-acetyltransferase (meta) activity, wherein said heterologous nucleotide sequence comprises a nucleotide sequence encoding a meta protein having an amino acid sequence with 95% homology or more to the sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46;    b) removing water from the L-methionine-containing fermentation medium and/or removing from 0 to 100% by weight of biomass formed during fermentation; and    c) drying the medium to obtain an L-methionine-containing animal feed additive in a powder or a granule form.    
     
     
         14 . A method for the production of L-methionine, which comprises the following steps: 
 a) fermenting in a medium cells of a coryneform bacterium for producing L-methionine, said coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes a protein with homoserine O-acetyltransferase (meta) activity, wherein the heterologous nucleotide sequence comprises a nucleotide sequence having 95% homology or more to the sequence as set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45;    b) concentrating L-methionine in the medium or in the bacterial cells; and    c) isolating L-methionine.    
     
     
         15 . The method of  claim 14 , wherein the meta-encoding nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45.  
     
     
         16 . The method of  claim 14 , wherein the meta-encoding nucleotide sequence encodes a protein with meta activity, said protein comprising an amino acid sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46.  
     
     
         17 . The method of  claim 14 , wherein the metA encoding sequence is a DNA or RNA which can be replicated in coryneform bacteria or is stably integrated into the chromosome.  
     
     
         18 . The method of  claim 14 , wherein 
 a) a bacteria strain transformed with a plasmid vector carrying at least one copy of the metA encoding sequence under the control of regulatory sequences is used, or    b) a strain in which the meta encoding sequence has been integrated into the bacteria chromosome is used.    
     
     
         19 . The method of  claim 14 , wherein the meta encoding sequence is overexpressed.  
     
     
         20 . The method of  claim 14 , wherein bacteria are fermented in which additionally at least one further gene of the biosynthetic pathway of L-methionine has been overexpressed or mutated such that its activity is not influenced by metabolic metabolites.  
     
     
         21 . The method of  claim 14 , wherein the coryneform bacterium is of the species  Corynebacterium glutamicum.    
     
     
         22 . A method for producing an L-methionine-containing animal feed additive from a fermentation medium, comprising 
 a) fermenting in a medium cells of a coryneform bacterium for producing L-methionine, said coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes a protein with homoserine O-acetyltransferase (meta) activity, wherein the heterologous nucleotide sequence comprises a nucleotide sequence having 95% homology or more to the sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45;    b) removing water from the L-methionine-containing fermentation medium and/or removing from 0 to 100% by weight of biomass formed during fermentation; and    c) drying the medium to obtain an L-methionine-containing animal feed additive in a powder or a granule form.    
     
     
         23 . A method for the fermentative production of L-methionine, which comprises the following steps: 
 a) fermenting in a medium cells of a coryneform bacterium for producing L-methionine, the coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes for a protein with homoserine O-acetyltransferase (meta) activity, wherein said heterologous nucleotide sequence comprises a nucleotide sequence encoding a metA protein having an amino acid sequence with 95% homology or more to the sequence as set forth in SEQ ID NO: 2;    b) concentrating L-methionine in the medium or in the bacterial cells; and    c) isolating L-methionine; wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among    a) a gene lysC derived from a coryneform bacterium, which encodes an aspartate kinase,    a) a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gap,    b) a 3-phosphoglycerate kinase-encoding gene pgk,    c) a pyruvate carboxylase-encoding gene pyc,    d) a triose phosphate isomerase-encoding gene tpi,    e) a methylene tetrahydrofolate reductase-encoding gene metF,    f) a cystathionine gamma-synthase-encoding gene metB,    g) a cystathionine gamma-lyase-encoding gene metC,    h) a serine hydroxymethyltransferase-encoding gene glyA,    i) a O-acetylhomoserine sulfhydrylase-encoding gene metY,    j) a vitamin B12-dependent methionine synthase-encoding gene metH,    k) a phosphoserine aminotransferase-encoding gene serC,    l) a phosphoserine phosphatase-encoding gene serB,    m) a serine acetyltransferase-encoding gene cysE, and    n) a gene hom, which encodes a homoserine dehydrogenase,    is overexpressed or mutated in such a way that the activity of the corresponding proteins is influenced by metabolic metabolites to a smaller extent, if at all, compared to nonmutated proteins.    
     
     
         24 . A method for the fermentative production of L-methionine, which comprises the following steps: 
 a) fermenting in a medium cells of a coryneform bacterium for producing L-methionine, the coryneform bacteria expressing at least one heterologous nucleotide sequence which encodes for a protein with homoserine O-acetyltransferase (metA) activity, wherein said heterologous nucleotide sequence comprises a nucleotide sequence encoding a metA protein having an amino acid sequence with 95% homology or more to the sequence as set forth in SEQ ID NO: 2;    b) concentrating L-methionine in the medium or in the bacterial cells; and    c) isolating L-methionine;    wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among    a) a homoserine kinase-encoding gene thrB,    b) a threonine dehydratase-encoding gene ilvA,    c) a threonine synthase-encoding gene thrC,    d) a meso-diaminopimelate D-dehydrogenase-encoding gene ddh,    e) a phosphoenolpyruvate carboxykinase-encoding gene pck,    f) a glucose-6-phosphate 6-isomerase-encoding gene pgi,    g) a pyruvate oxidase-encoding gene poxB,    h) a dihydrodipicolinate synthase-encoding gene dapA,    i) a dihydrodipicolinate reductase-encoding gene dapB; and    j) a diaminopicolinate decarboxylase-encoding gene,    is attenuated by changing the rate of expression or by introducing a specific mutation.