Biosynthesis studies and mutasynthesis of myxobacterial secondary metabolites

Graduation date: 2007Myxobacteria represent an interesting class of Gram-negative soil bacteria. These bacteria utilize organic materials from the environment as a food source by the action of their extracellular hydrolytic enzymes. They grow vegetatively in the presence of adequate nutrients. During starvation conditions, however, they aggregate and form multicellular structures called fruiting bodies. In addition, myxobacteria have been shown to be prolific producers of biologically active secondary metabolites. A fundamental goal of our research is to understand and utilize the biosynthetic machineries used by these organisms to build biologically active molecules. \ud The first part of this research focuses on the investigation of a novel biosynthetic pathway to isovaleryl-CoA, which is involved in myxothiazol biosynthesis in S. aurantiaca. In this study, we identified a novel biosynthetic route to isovaleryl-CoA. Isovaleryl-CoA is a starter unit of branched-chain fatty acid (BCFA) and many other microbially derived polyketide natural products, including myxothiazol. BCFA compose the majority of the cellular fatty acids of myxobacteria. They are formed from branched-chain starter units (isovaleryl-CoA, isobutyryl-CoA, and 2-methylbutyryl-CoA), which in turn are derived from the degradation of leucine, valine, and isoleucine, respectively. Inactivation of the branched-chain α-keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, gave a mutant that lost the ability to utilize leucine as a precursor of isovaleryl-CoA but still produced myxothiazol and BCFA. Feeding experiments with isotopically labeled precursors as well as cell-free extract experiments provided significant evidence for a novel branch of the mevalonate pathway responsible for the formation of isovaleryl-CoA.\ud The focus of the second part of this research centered on the biosynthesis of the myxalamids in S. aurantiaca strain Sg a15. Myxalamid is synthesized by a combination of multimodular/multifunctional enzymes, the polyketide synthases (PKS) and the nonribosomal peptide synthetases (NRPS). Preliminary studies have provided evidence on the feasibility of manipulating the biosynthetic processes in the myxalamid producer using unnatural starter units that can be accepted by both PKSs and NRPSs of myxalamid biosynthesis to generate novel natural products. Using this knowledge, we have generated novel analogs of the myxalamids via mutagenetic approaches. The studies showed that a combination of genetic engineering and biochemical methods used to dissect the substrate acquisition and incorporation processes can lead to the development of new bioactive natural products with potential pharmaceutical uses