Method for microbial production of amino acids of the spartate and/or glutamate family and agents which can be used in said method

Abstract

A method is disclosed for microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: culturing a strain of Escherichia coli that produces said L-amino acids in a medium suitable for the production of an L-amino acid, whereby said Escherichia coli strain is transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.

1 . A method of microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: 
 culturing a strain of  Escherichia coli  that produces said L-amino acids in a medium suitable for the production of an L-amino acid whereby said  Escherichia coli  strain is transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.    
     
     
         2 . A method of microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: 
 culturing a strain of  Escherichia coli  that produces said L-amino acids in a medium suitable for the production of an L-amino acid, whereby said  Escherichia coli  strain comprises an isolated polynucleotide, whereby said polynucledtide encoding an enzyme with pyruvate carboxylase activity comprises the amino acid sequence having SEQ ID NO: 2.    
     
     
         3 . The method according to  claim 2  wherein said polynucleotide encoding an enzyme with pyruvate carboxylase activity comprises SEQ ID NO:1 or a nucleotide sequence with positions:165 to 3587 of SEQ ID NO:1.  
     
     
         4 . The method according to  claim 2  wherein said polynucleotide is isolated from a microorganism of the genus  Corynebacterium.    
     
     
         5 . The method according to  claim 2  wherein a strain of  Escherichia coli  is transformed with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         6 . The method according to  claim 3  wherein a strain of  Escherichia coli  is transformed with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         7 . The method according to  claim 2  whereby the amino acid of the aspartate and/or glutamate family produced is selected from the group consisting of L-lysine, L-threonine, L-arginine, and glutamate.  
     
     
         8 . A method of microbially producing L-amino acids of aspartate and/or glutamate family, which comprises the step of: 
 transforming a strain of  Escherichia coli  with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising a polynucleotide having the amino acid sequence of SEQ ID NO:2; and    culturing said transformed strain in a medium suitable for the production of an L-amino acid and-producing said L-amino acids.    
     
     
         9 . The method according to  claim 8  whereby the L-amino acid of the aspartate and/or glutamate family produced is selected from the group consisting of L-lysine, L-threonine, L-arginine, and glutamate.  
     
     
         10 . A method for microbially overproducing L-threonine in a culture medium, which comprises the step of: 
 (a) providing a strain of  Escherichia coli  transformed with an isolated polynucleotide encoding a pyruvate carboxylase polypeptide comprising the amino acid sequence of SEQ ID NO:2 operatively linked to a promoter and capable of producing threonine;    (b) culturing the transformed strain of  Escherichia coli  under conditions which permit expression of the pyruvate carboxylase polypeptide to cause the overproduction of threonine compared to an untransformed form of the strain of  Escherichia coli;  and    (c) isolating the threonine produced by the transformed strain of  Escherichia coli.      
     
     
         11 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (b) the expression by the isolated polynucleotide of the pyruvate carboxylase polypeptide is increased by increasing a copy number of the isolated polynucleotide.  
     
     
         12 . The method for microbially overproducing L-threonine defined in  claim 11 , wherein increasing the copy number of the isolated polynucleotide is achieved by transforming said  Escherichia coli  with a vector comprising the isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 in association with a regulatory sequence.  
     
     
         13 . The method for microbially overproducing L-threonine defined in  claim 12 , wherein said isolated polynucleotide comprises the nucleotide sequence of nucleotides 165 to 3587 of SEQ ID NO:1.  
     
     
         14 . The method for microbially overproducing L-threonine defined in  claim 13 , wherein said isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO:1.  
     
     
         15 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (b) the pyruvate carboxylase having SEQ ID NO:2 participating in the synthesis of L-threonine is deregulated and/or wherein an enhanced expert carrier activity is shown for the threonine.  
     
     
         16 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (a) the transformed  Escherichia coli  has a high proportion of central metabolism metabolites of the L-threonine participating in the synthesis.  
     
     
         17 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (a) the transformed  Escherichia coli  has biosynthesis paths which run with reduced activity when competing with biosynthesis paths of L-threonine.  
     
     
         18 . The method for microbially overproducing L-threonine defined in  claim 17  wherein according to step (a) the isolated polynucleotide encoding a pyruvate carboxylase polypeptide comprising the amino acid sequence of SEQ-ID NO:2 is isolated from a microorganism strain of the variety  Corynebacterium.    
     
     
         19 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (a) the expression of the polynucleotide encoding the polypeptide having SEQ ID NO:2 is increased by reinforcement of the transcription signal.  
     
     
         20 . The method for microbially overproducing L-threonine defined in  claim 10  wherein according to step (a) the promoter linked to the polynucleotide encoding the polypeptide of SEQ ID NO:2 is the tac promoter located ahead of the polynucleotide.  
     
     
         21 . The method for microbially overproducing L-threonine defined in  claim 20  wherein the tac promoter is associated with regulatory-sequences.  
     
     
         22 . The method for microbially overproducing L-threonine defined in  claim 10  wherein the transformed  Escherichia coli  contains a vector pVWEX1pyc.  
     
     
         23 . The method for microbially overproducing L-threonine defined in  claim 12  wherein the vector comprising the isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is pVWEX1pyc.  
     
     
         24 . An  Escherichia coli  cell that produces L-amino acids of the aspartate and/or glutamate families, transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.  
     
     
         25 . The  Escherichia coli  cell defined in  claim 24  transformed with an isolated polynucleotide encoding an enzyme with pyruvate carboxylase activity comprising the amino acid sequence having SEQ ID NO:2.  
     
     
         26 . The  Escherichia coli  cell defined in  claim 24  transformed with the vector pVWEX1pyc.  
     
     
         27 . A method of making a transformed  Escherichia coli  cell that produces L-amino acids of the aspartate and/or glutamate families, which comprises the step of transforming the  Escherichia coli  cell with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.  
     
     
         28 . The method of making a transformed  Escherichia coli  cell defined in  claim 26  wherein the polynucleotide is an isolated polynucleotide encoding an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO: 2  
     
     
         29 . The method of making a transformed  Escherichia coli  cell defined in  claim 26  wherein the  Escherichia coli  cell is transformed with the vector pVWEX1pyc.