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Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments^b

Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8,D-72076 Tübingen, Germany¹
Institut für Pflanzenbiochemie, Weinberg 3, 06120 Halle (Saale), Germany²

Author for correspondence: Shu-Ming Li. Tel: +49 7071 2976995. Fax: +49 7071 295250. e-mail: shuming.li{at}uni-tuebingen.de

The coumarin antibiotic coumermycin A₁ contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A₁ biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A₁ production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A₁, respectively. C-methylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the non-methylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A₁ in the wild-type.

Keywords: aminocoumarin antibiotics, biosynthesis, methyltransferase

Abbreviations: CID, collision-induced dissociation; ESI, electrospray ionization

^b The GenBank accession number for the sequence reported in this paper is AF235050.

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