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Cyclic heptapeptide microcystin biosynthesis requires the glutamate racemase gene

Laboratory of Molecular Genetics, School of Agriculture¹, and Gene Research Center², Ibaraki University, Ami, Ibaraki 300-0393, Japan
The United Graduate School, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan³

Author for correspondence: Makoto Shirai. Tel: +81 298 88 8652. Fax: +81 298 88 8653. e-mail: shirai{at}ipc.ibaraki.ac.jp

It was demonstrated previously that the operon consisting of the non-ribosomal peptide synthetase (NRPS) gene coupled with the polyketide synthase (PKS) gene involved in cyclic heptapeptide microcystin synthesis includes two different D-amino acid synthetase genes, an epimerization domain at the 3' end of module 2, and the racemase gene mcyF. To determine the role of mcyF in microcystin synthesis, gene-disruption and complementation analyses were carried out. Insertional mutagenesis in the mcyF gene, generated by homologous recombination, abolished only microcystin synthesis, but did not influence cell growth. Furthermore, McyF supported D-Glu-independent growth of a strain of Escherichia coli defective in D-Glu synthesis. It is concluded that mcyF is the glutamic acid racemase gene involved in the synthesis of D-Glu residues in the microcystin molecule. This is the first report of the racemase in prokaryotic NRPS.

Keywords: amino acid racemase, cyanobacteria, Microcystis, peptide synthetase gene, microcystin biosynthesis

Abbreviations: Adda, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4,6-dienoic acid; MCYST, microcystin; Mdha, N-methyldehydroalanine; D-MeAsp, D-erythro-ß-methylaspartic acid; NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; UDP-MurNAc-L-Ala-D-Glu, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate

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