engleski

Polyketide chain length is influenced by post-polyketide gene disruption

The gene cluster for biosynthesis of the polyketide antibiotic oxytetracycline (OTC) has been cloned from Streptomyces rimosus. A systematic strategy to characterize each gene is to identify the metabolite(s) made by a recombinant which has that gene disrupted. Disruption of otcC, who hydroxylates the tetracyclic nucleus many steps after the nascent polyketide chain has been synthesized and folded, was used as a model to validate such a strategy. Disruption was achieved by introduction of a gentamicin (gmr) resistance cassette into a cloned copy of the gene, then homogenotization of the S.rimosus chromosome. This strain no longer produced OTC but three novel compounds were visualized by tlc. Structural elucidation by 2D homonuclear and heteronuclear NMR showed that they had backbones of 17 carbon atoms whereas OTC has 19. The OTC skeleton is synthesized from a 3-carbon starter unit followed by the iterative condensation of eight acetates, whereas these novel products are the result of condensation of seven. Thus, the otcC::gmr strain has lost its intrinsic functionality to make a polyketide of the usual 19-carbon chain length. Others (reviewed, Hopwood, Chem. Rev. 1997, 97, 2465) have shown that metabolites like OTC are made by ‘Type II’ polyketide synthase (PKS) complexes comprising three to six different mono- or bifunctional monomers. Only three subunit types (termed the ‘minimal PKS’) are required to biosynthesize a polyketide of correct chain length and coexpression of other components primarily influences the folding of the nascent polyketide. Disruption of otcC, which lies outwith the transcription units of all genes involved in biosynthesis and folding of the OTC backbone, should not affect expression of these genes. Our results show that disruption in the producer strain results in ‘derailment’ of the ability of the PKS to form a chain of the usual length - which implies that normally OtcC must contact and influence the quaternary structure of the PKS. Thus, a gene product involved in a late biosynthetic step, post-polyketide formation, has a substantial influence on the capability of the PKS to determine the length of chain made. Disruption of this hydroxylase gene results in a metabolite whose structure would not have been predicted from knowledge of the function of the gene.

Streptomyces rimosus; oxytetracycline; novel polyketides

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