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Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455

UNIGEN Center for Molecular Biology and Department of Biotechnology, NTNU,N-7489 Trondheim, Norway¹
Nord-Trondelag College, Dept of Engineering,N-7600 Levanger, Norway²
SINTEF Applied Chemistry, SINTEF, N-7034 Trondheim, Norway³

Author for correspondence: Sergey Zotchev. Tel: +47 73 59 86 79. Fax: +47 73 59 87 05. e-mail: sergey.zotchev{at}chembio.ntnu.no

Streptomyces noursei ATCC 11455 produces the antifungal polyene antibiotic nystatin containing the deoxysugar moiety mycosamine. Part of the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene (gdhA) known to be involved in deoxysugar biosynthesis was amplified by PCR from genomic DNA of S. noursei ATCC 11455. A gene library for S. noursei was made and screened with the gdhA probe. Several overlapping phage clones covering about 30 kb of the S. noursei genome were physically mapped. A partial DNA sequencing analysis of this region resulted in the identification of several putative genes typical of macrolide antibiotic biosynthetic gene clusters. A gene-transfer system for S. noursei has been established, and gene deletion or disruption experiments within the putative biosynthetic gene cluster were performed. All of the knock-out mutants retained the ability to produce nystatin, suggesting that the identified gene cluster is not involved in biosynthesis of this antibiotic. Culture extracts from the wild-type strain and three knock-out mutants were analysed by TLC followed by a bioassay against Micrococcus luteus. Two antibacterial compounds were found to be synthesized by the wild-type strain while only one was produced by the mutants. This provided evidence for the involvement of the identified gene cluster in the biosynthesis of a presumably novel antibacterial macrolide antibiotic in S. noursei.

Keywords: Streptomyces noursei, gene cluster, macrolide antibiotic

Abbreviations: ABC, ATP-binding cassette; PKS, polyketide synthase; TDP, deoxythymidyl diphosphate; WT, wild-type

The GenBank accession numbers for the sequences reported in this paper are AF071512 for ORF1, AF071513 for ORF2, AF071514 for ORF3, AF071515 for ORF4, AF071516 for ORF5, AF071517 for ORF6, AF071518 for ORF7, AF071519 for gdhA, AF071520 for ORF8, AF071521 for ORF9, AF071522 for ORF10 and AF071523 for ORF11.

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