| Methymycin, neomethymycin, narbomycin, and pikromycin are structurally-related macrolide antibiotics. In this thesis the biosynthetic gene cluster ( pik) for these macrolides has been cloned, sequenced, and characterized in Streptomyces venezuelae ATCC 15439. The cluster consists of five genetic loci: pikA, pikB ( des), pikC, pikD, and pikR. At center of the cluster is the polyketide synthase (PKS) locus, pikA, which encodes a six-module PKS complex in four multifunctional proteins, and a type II thioesterase (TEII). Immediately downstream is pikB (des), which contains a set of genes for desosamine biosynthesis and transfer. PikC encodes a P450 mono-oxygenase responsible for hydoxylations in methymcyin, neomethymycin, and pikromycin. Besides these biosynthetic genes, pikD encodes a transcriptional activator, and pikR encodes for rRNA methyltransferases that confer the bacteria self resistance to the antibiotics produced.; A unique feature of the pik gene cluster is that the single Pik PKS system generates two macrolactones of different chain length. It is demonstrated in this thesis that PikAIV, the multifunctional PKS responsible for the final condensation cycle in the polyketide assembly, is translated as two different forms under two different culture conditions. The expression of full-length PikAIV corresponds to the production of 14-membered ring macrolactone narbonolide, whereas an N-terminal truncated form is responsible for the creation of 12-membered ring macrolactone 10-deoxymethynolide. Overall, the pik cluster represents the least complex yet most versatile modular PKS system identified. Differential expression of the cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate multiple metabolites. |
