Biochemical synthesis of 1,4-butanediamine

Abstract

The invention relates to a process for biochemical synthesis of 1,4-butanediamine in a microorganism having an increased level of an ornithine decarboxylase activity (increased ODC activity) as compared to the native level of the ornithine decarboxylase activity, wherein the increased ODC activity is obtained by means of overexpression of an ornithine decarboxylase encoding gene with increased translational and/or transcriptional efficiency, and wherein 1,4-butanediamine produced in the microorganism is excreted into a fermentation broth, and is recovered from the fermentation broth. In preferred embodiments also increased enzyme activity is obtained by of overexpression of either (i) an arginine decarboxylase encoding gene speA and an agmatinase encoding genespeB; or (ii) an arginine decarboxylase encoding gene speA and an agmatine iminohydrolase encoding gene aguA, and an N-carbamoylpotrescine amidohydrolase encoding geneaguB, and optionally also an agmatinase encoding gene speB. The invention also relates to vectors, plasmids and hosts carrying, at an increased level of activity, one or more of the enzyme activities as mentioned.

1 . Process for biochemical synthesis of 1,4-butanediamine in a microorganism having an increased level of an ornithine decarboxylase (ODC) activity as compared to the native level of the ornithine decarboxylase activity, wherein increased ornithine decarboxylase activity is obtained by overexpression of an ornithine decarboxylase encoding gene with increased translational and/or transcriptional efficiency, comprising:
 (a) excreting 1,4-butanediamine produced in the microorganism into a fermentation broth, and   (b) recovering said 1,4-butanediamine from the fermentation broth.   
     
     
         2 . Process according to  claim 1 , wherein the increased translational and/or transcriptional efficiency is obtained by the use of a strong, regulated promoter, preferably by use of a strong inducible promoter. 
     
     
         3 . Process according to  claim 2 , wherein the increased translational and/or transcriptional efficiency is obtained by the use of an isopropyl-β-D-thiogalactoside (IPTG) inducible strong promoter. 
     
     
         4 . Process according to  claim 1 , wherein the increased translational and/or transcriptional efficiency is obtained by the use of a promoter selected from the group consisting of T7, T5, ptac, and plac promoters. 
     
     
         5 . Process according to  claim 1 , wherein the ornithine decarboxylase encoding gene has a Ribosomal Binding Site (RBS) located up-stream of the coding region of the said gene which RBS is adapted to achieve better recognition of RNA-template by the ribosomes. 
     
     
         6 . Process according to  claim 1 , wherein the overexpressed ornithine decarboxylase encoding gene is an ornithine decarboxylase speF or speC gene (each belonging to E.C. 4.1.1.17). 
     
     
         7 . Process according to  claim 6 , wherein the overexpressed ornithine decarboxylase encoding gene is an ornithine decarboxylase speF gene. 
     
     
         8 . Process according to  claim 6 , wherein the overexpressed ornithine decarboxylase encoding gene is an ornithine decarboxylase gene speF or speC originating from one of the genera selected from the group consisting of  Escherichia, Shigella, Salmonella, Yersinia,  and  Shewanella.    
     
     
         9 . Process according to  claim 8 , wherein the overexpressed ornithine decarboxylase encoding gene is an ornithine decarboxylase gene originating from one of the species selected from the group consisting of  Escherichia coil, Shigella flexneri, Salmonella typhimutium Yersinia pestis,  and  Shewanella oneidensis.    
     
     
         10 . Process according to  claim 9 , wherein the overexpressed ornithine decarboxylase encoding gene is speF originating from one of the species selected from the group consisting of  Escherichia coli, Salmonella typhimutium,  and  Shewanella oneidensis.    
     
     
         11 . Process according to  claim 1 , wherein additionally to the increased ODC activity also increased enzyme activity is obtained for at least two other enzymes by means of overexpression of either
 (i) an arginine decarboxylase encoding gene speA (belonging to E.C. 4.1.1.19) and an agrnatinase encoding gene speB (belonging to E.C. 3.5.3.11; also referred to as agmatine ureahydrolase encoding gene); or   (ii) an arginine decarboxylase encoding gene speA (belonging to E.C. 4.1.1.19), and an agmatine iminohydrolase encoding gene aguA (belonging to E.C. 3.5.3.12; also referred to as agmatine deiminase encoding gene), and an N-carbamoylputrescine amidohydrolase encoding gene aguB (belonging to E.C. 3.5.1.53), and optionally also an agrnatinase encoding gene speB (belonging to E.C. 3.5.3.11).   
     
     
         12 . Process according to  claim 11 , wherein the overexpressed arginine decarboxylase encoding gene is an arginine decarboxylase gene speA originating from one of the genera selected from the group consisting of  Escherichia, Shigella, Salmonella, Yersinia, Pasteurella,  and  Neisseria.    
     
     
         13 . Process according to  claim 12 , wherein the overexpressed arginine decarboxylase encoding gene is an arginine decarboxylase gene speA originating from one of the species selected from the group consisting of  Escherichia coli, Shigella flexneri, Salmonella enterica, Yersinia pestis, Pasteurella multocida,  and  Neisseria meningitidis.    
     
     
         14 . Process according to  claim 13 , wherein the overexpressed agmatinase encoding gene is an agmatinase gene speB originating from one of the genera selected from the group consisting of  Escherichia, Salmonella, Proteus, Photorhabdus, Vibrio,  and  Neisseria.    
     
     
         15 . Process according to  claim 14 , wherein the overexpressed agmatinase encoding gene is an agmatinase gene speB originating from one of the species selected from the group consisting of  Escherichia coil, Salmonella enterica, Proteus mirabilis, Photorhabdus luminescens, Vibrio cholerae,  and  Neisseria meningitidis.    
     
     
         16 . Process according to  claim 15 , wherein the overexpressed agmatine iminohydrolase encoding gene and/or the overexpressed N-carbamoylputrescine amidohydrolase encoding gene is an agmatine iminohydrolase gene aguA and/or an N-carbamoylputrescine amidohydrolase gene aguB originating from one of the genera selected from the group consisting of  Pseudomonas, Streptococcus, Streptomyces, Azotobacter, Arabidopsis, Novosphingobium,  and  Bacillus.    
     
     
         17 . Process according to  claim 16 , wherein the overexpressed agmatine iminohydrolase encoding gene and/or the overexpressed N-carbamoylputrescine amidohydroiase encoding gene is an agmatine iminohydrolase gene aguA and/or an N-carbamoylputrescine amidohydroiase gene aguB originating from one of the species selected from the group consisting of  Pseudomonas aeruginosa, Streptococcus mutans, Streptomyces avermitilis, Azotobacter vinelandii, Arabidopsis thaliana, Novosphingobium aromaticivorans,  and  Bacillus cereus.    
     
     
         18 . Process according to  claim 1 , wherein
 the process is being carried out whilst ensuring an increased intracellular level of ornithine.   
     
     
         19 . Process according to  claim 1 , wherein the process is carried out in a host organism selected from the group consisting of  Saccharomyces  sp.,  Bacillus  sp.,  Corynebacterium  sp.,  Escherichia  sp., and  Pichia  sp. 
     
     
         20 . Process according to  claim 1 , wherein
 the process is carried out in a host organism selected from the group consisting of  Saccharomyces cerevisiae, Corynebacterium  sp., and  Escherichia  sp. and in that, apart from the increased level of activity of an ornithine decarboxylase, at least also the level of activity of an arginine decarboxylase in combination with an agmatinase and/or an agmatine iminohydrolase and an N-carbamoylputrescine amidohydroiase is increased.   
     
     
         21 . Vectors, plasmids and hosts carrying, at an increased level of activity, one or more of the enzyme activities as are mentioned in  claim 1 .