UC San Diego

Ansamycin polyketides from actinobacteria include the potent antibiotic and anticancer agents rifamycin SV, ansamitocin P-3, and geldanamycin. These natural product macrolactams are characterized by an mC₇N structural unit derived from the aromatic acid 3-amino-5-hydroxybenzoate, which is carboxy extended by multimodular polyketide synthases utilizing primarily acetate and propionate building blocks prior to macrolactam cyclization. Herein, I report a multidisciplinary investigation of the biosynthesis of two structurally fascinating natural products produced by ansamycin biosynthetic machinery. Saliniketals A and B are unusual bicyclic polyketides from the obligate marine actinomycete Salinispora arenicola that inhibit the cancer chemopreventative drug target ornithine decarboxylase. The saliniketals are noteworthy because of their structural similarity to the ansa portion of the rifamycin antibiotics, which co-occur in fermentation broth. Analysis of the S. arenicola genome identified a 106 kb rif gene cluster consisting of all the PKS and tailoring genes necessary for the biosynthesis of rifamycin B. No additional type I PKS with domain architecture consistent with saliniketal assembly could be identified, which suggested the saliniketals are products of the S. arenicola rif locus. Using stable isotope incorporation studies, PCR targeted mutagenesis, and chemical complementation with rifamycin pathway intermediates I have shown that the saliniketals and rifamycins share a single biosynthetic locus and a cytochrome P450 facilitates the divergence of the two compound classes. Additionally, in 2009 the novel ansamycin ansalactam A was isolated by the Fenical laboratory from an extract of a marine sediment-derived actinomycete of the genus Streptomyce. Unique from its predecessors, ansalactam A possesses a spiro-fused [gamma] -lactam moiety and an unusual isobutyrylmalonate polyketide extender unit. Using ¹³C-labeled precursors, I have shown that the unique branched chained polyketide extender unit of ansalactam A originates from the condensation of isobutyrate and acetate precursors. Further bioinformatic analysis of the partial ansalactam biosynthetic gene cluster identified a putative three gene cassette (KASII-HBDH-CCR) for the biosynthesis of isobutyrylmalonyl-CoA. Efforts towards the functional characterization of the novel polyketide biosynthetic genes are also discussed within