Process for the production of gamma-aminobutyric acid

Abstract

The present invention relates to a novel method for the fermentative production of gamma-aminobutyric acid (GABA) by cultivating a recombinant microorganism expressing an enzyme having a glutamate decarboxylase activity. The present invention also relates to corresponding recombinant hosts, recombinant vectors, expression cassettes and nucleic acids suitable for preparing such hosts as well as to a method for preparing polyamides making use of GABA as obtained fermentative production.

We claim: 
     
         1 . A method for the fermentative production of gamma-aminobutyric acid (GABA), comprising cultivating a recombinant microorganism derived from a parent microorganism having the ability to produce glutamate and additionally having the ability to express a heterologous glutamate decarboxylase (E.C. 4.1.1.15), wherein said microorganism is a  Corynebacterium , so that glutamate is converted to GABA. 
     
     
         2 . The method of  claim 1 , wherein the microorganism is  Corynebacterium glutamicum.    
     
     
         3 . The method of  claim 1 , wherein said heterologous glutamate decarboxylase is of eukaryotic origin. 
     
     
         4 . The method of  claim 3 , wherein said heterologous glutamate decarboxylase is a plant glutamate decarboxylase or a chimeric glutamate decarboxylase comprising amino acid sequence portions derived from plant glutamate decarboxylase. 
     
     
         5 . The method of  claim 3 , wherein said heterologous glutamate decarboxylase is a decarboxylase of a plant of the genus  Solanum , in particular from  Solanum tuberosum.    
     
     
         6 . The method of  claim 3 , wherein the heterologous glutamate decarboxylase is from  Solanum tuberosum  and comprises an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or a sequence having at least 92% identity thereto. 
     
     
         7 . The method of  claim 6 , wherein the heterologous glutamate decarboxylase is N-terminally and/or C-terminally supplemented by the corresponding terminal amino acid sequences of a glutamate decarboxylase from  Solanum tuberosum.    
     
     
         8 . The method of  claim 6 , wherein the heterologous glutamate decarboxylase is N-terminally and/or C-terminally supplemented by the corresponding terminal amino acid sequences of a glutamate decarboxylase of a second plant different from  Solanum tuberosum.    
     
     
         9 . The method of  claim 8 , wherein said second plant is  Solanum lycopersicum.    
     
     
         10 . The method of  claim 9 , wherein said glutamate decarboxylase comprises an amino acid sequence according to SEQ ID NO: 2 or a sequence having at least 80% identity thereto. 
     
     
         11 . The method of  claim 1 , wherein the enzyme is encoded by a nucleic acid sequence, which is adapted to the codon usage of said parent microorganism having the ability to produce glutamate having glutamate decarboxylase activity. 
     
     
         12 . The method of  claim 1 , wherein the enzyme having glutamate decarboxylase activity is encoded by a nucleic acid sequence comprising a coding sequence selected from the group consisting of
 a) position 472 to 1200 according to SEQ ID NO:1 or from position 193 to 1605 according to SEQ ID NO:1;   b) a coding sequence encoding a glutamate decarboxylase of a plant of the genus  Solanum , in particular from  Solanum tuberosum;      c) a coding sequence encoding a glutamate decarboxylase from  Solanum tuberosum  and comprising an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or a sequence having at least 92% identity thereto;   d) a coding sequence encoding a glutamate decarboxylase that is N-terminally and/or C-terminally supplemented by the corresponding terminal amino acid sequences of a glutamate decarboxylase from  Solanum tuberosum;      e) a coding sequence encoding a glutamate decarboxylase that is N-terminally and/or C-terminally supplemented by the corresponding terminal amino acid sequences of a glutamate decarboxylase of a second plant different from  Solanum tuberosum;      f) a coding sequence encoding a glutamate decarboxylase that is N-terminally and/or C-terminally supplemented by the corresponding terminal amino acid sequences of a glutamate decarboxylase of  Solanum lycopersicum;      g) a coding sequence encoding a glutamate decarboxylase comprising an amino acid sequence according to SEQ ID NO: 2 or a sequence having at least 80% identity thereto, and   h) a coding sequence encoding a glutamate decarboxylase, wherein the coding sequence is adapted to the codon usage of said parent microorganism having the ability to produce glutamate having glutamate decarboxylase activity.   
     
     
         13 . A glutamate decarboxylase as defined in  claim 5 . 
     
     
         14 . A nucleic acid sequence comprising the coding sequence for a glutamate decarboxylase as claimed in  claim 13 . 
     
     
         15 . An expression cassette, comprising at least one nucleic acid sequence as claimed in  claim 14 , which sequence is operatively linked to at least one regulatory nucleic acid sequence. 
     
     
         16 . A recombinant vector, comprising at least one expression cassette as claimed in  claim 15 . 
     
     
         17 . A prokaryotic or eukaryotic host, transformed with at least one vector as claimed in  claim 16 . 
     
     
         18 . The host of  claim 17 , selected from a recombinant  Corynebacterium.    
     
     
         19 . The host of  claim 18 , which is recombinant  Corynebacterium glutamicum.    
     
     
         20 . The method of  claim 1 , wherein the GABA thus produced is isolated from the fermentation broth. 
     
     
         21 . A method of preparing a polyamide, which method comprises
 a) preparing GABA by the method of  claim 1 ;   b) isolating GABA; and   c) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent co-monomer, selected from aminocarboxylic acids, and hydroxycarboxylic acids.   
     
     
         22 . The method of  claim 1 , wherein the recombinant microorganism further comprises at least one deregulated gene selected from the group consisting of:
 i) amplification of isocitrate dehydrogenase;   ii) amplification of glutamate dehydrogenase;   iii) amplification of phosphoenolpyruvate carboxylase;   iv) releasing feedback inhibition by point mutation and amplification of pyruvate carboxylase;   v) attenuation of 2-oxoglutarate dehydrogenase;   vi) attenuation of isocitrate lyase;   vii) attenuation of phosphoenolpyruvate carboxykinase;   viii) attenuation of glutamine synthetase; and   ix) attenuation of glutamate exporter.