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Cloning and functional analysis of a phosphopantetheinyl transferase superfamily gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230

Department of Biology, Dalhousie University, Halifax, Nova Scotia, CanadaB3H 4J1¹

Author for correspondence: Leo C. Vining. Tel: +1 902 494 2040. Fax: +1 902 494 3736. e-mail: leo.vining{at}dal.ca

Sequence analysis of a XhoI/SacI fragment of chromosomal DNA downstream of jadL in the Streptomyces venezuelae ISP5230 gene cluster for jadomycin biosynthesis detected a partial ORF similar in its deduced amino acid sequence to the hetI product involved in synthesizing a regulator of heterocyst spacing in Anabaena. By probing a phage library of S. venezuelae DNA with the XhoI/SacI fragment, the authors identified and isolated a hybridizing clone. The nucleotide sequence of its DNA contained three complete ORFs (jadM, N and X) and one incomplete ORF (jadO). The jadM ORF lay immediately downstream of, and partially overlapped, jadL. It contained 786 nucleotides encoding an amino acid sequence like those of enzymes in the phosphopantetheinyl transferase family. The jadN ORF contained 1794 nucleotides and encoded an amino acid sequence resembling acyl-CoA decarboxylases, thus suggesting a role in polyketide condensation reactions.The jadX ORF was not identified, but the partial jadO showed marked similarities in its deduced amino acid sequence to NDP-hexose-2,3-dehydratases, indicating a role in forming the sugar component of jadomycin B. Expression of jadM in Escherichia coli and examination of the product by SDS-PAGE established that the ORF encoded a 29·1 kDa protein, corresponding in size to the 262 amino acid polypeptide deduced from the jadM sequence. Evidence from a Northern hybridization indicated that jadM expression is correlated with jadomycin B synthesis. Cultures of S. venezuelae ISP5230 disrupted in jadM produced only 2–5% of the wild-type titre of jadomycin B, but grew well and produced chloramphenicol normally. The authors conclude that jadM encodes a holo-ACP synthase needed primarily for jadomycin B biosynthesis.

Keywords: phosphopantetheinyl transferase gene, jad genes, polyketide antibiotic, acyl carrier protein synthase

Abbreviations: ACP, acyl carrier protein; ACPS, holo-acyl carrier protein synthase; act, actinorhodin biosynthesis gene; ArCP, aryl carrier protein; CLF, chain-length factor; cml, chloramphenicol biosynthesis gene; fren, frenolicin biosynthesis gene; gra, granaticin biosynthesis gene; jad, jadomycin biosynthesis gene; KS, ketosynthase; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein; PKS, polyketide synthase; Ppan, phosphopantetheine; PPTase, phosphopantetheinyl transferase

The GenBank accession numbers for the jadM and jadNOX sequences reported in this paper are AF222693 and AY026363, respectively.

^a Present address: Department of Medicine, McMaster University, Hamilton, Ontario, Canada.

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