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Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in Streptomyces ambofaciens

Fatma Karray

Hélène Dominguez

Karine Tuphile

CNRS UMR8621, Université Paris-Sud, Institut de Génétique et Microbiologie, Bâtiment 400, F-91405 Orsay Cedex, France

Correspondence
Jean-Luc Pernodet
jean-luc.pernodet{at}igmors.u-psud.fr

Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.

Present address: Centre de Biotechnologie de Sfax, B.P ‘K’, 3038 Sfax, Tunisia.

Present address: Université d'Avignon, IUT, Site Agroparc, BP 1207, F-84911 Avignon Cedex 9, France.

Present address: Unité Bactéries Lactiques et Pathogènes Opportunistes, Bâtiment 222, INRA, F-78352 Jouy en Josas Cedex, France.

The GenBank/EMBL/DDBJ accession numbers for the sequences of the regions downstream and upstream of the PKS genes are AM709783 and AM709784.

Details of the construction of pWED2 and pOSV238 are available as supplementary material with the online version of this paper.