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Appl. Environ. Microbiol. doi:10.1128/AEM.01751-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK288 in Chromobacterium violaceum No. 968

Department of Biological Sciences, Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, PO Box 413, Wisconsin 53201, USA

^* To whom correspondence should be addressed. Email: ycheng{at}uwm.edu.

	Abstract

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned and partially characterized in Chromobacterium violaceum No. 968. First, a genome scanning approach was applied to identify three distinctive C. violaceum No. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate those genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative FAD-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze a disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system will enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum No. 968 for the generation of structural analogs as anticancer drug candidates.