Development of high-functional production processes using microbial cyclic-amide metabolism

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Novel processes for the production of useful compounds using microbial cyclic-amide metabolism were developed. A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using the cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 286 mM pyruvate was produced from 500 mM fumarate in 27 h. Bromopyruvate-resistant mutants were derived from P. putida s52, and their pyruvate production was examined. One of the mutants, strain No. 15160 showed much higher pyruvate production than the parent strain. Using the mutant cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 770 mM pyruvate was produced from 1000 mM fumrate in 72 h. Brevibacterium linens C-l was found to produce a novel cyclic amidohydrolase which stereoselectively hydrolyzes 5-substituted thiazolidinedione and wasuseful for the production of optically active α-mercapto acids. The gene encoding the enzyme was cloned from the chromosomal DNA of B. lines C-1 and sequenced. It contains an open reading frame consisting of 936 nucleotides corresponding to 312 amino acid residues. The deduced amino acid sequence exhibited no apparent homology with other protein sequences in the protein databases. The enzyme was expressed in recombinant E. coli cells. The E. coli transformant, E. coli BL21/pETBTH, was a good catalyst for stereo selective production of .S-3-phenyl-2-mercaptopropionic (S-PMPA) from RS-5-Benzyl-thiazolidinedione. The optical purity of S-PMPA produced was approximately 80% e.e.