Engineering of Acyl-CoA Metabolism for Production of Branched Chain Fatty Acids and Pikromycin in Streptomyces

Abstract

In this thesis, branched chain fatty acids (BCFA) and polyketide antibiotics, pikromycin (PKM), were overproduced in Streptomyces species. Primarily, branched chain amino acids (BCAA) catabolism were examined and engineered for synthesis of BCFA in Streptomyces coelicolor, and PKM in Streptomyces venezuelae. Increases in productions of BCFAs were achieved by overexpressing branched chain α-keto acid dehydrogenase (BCDH) and 3-ketoacyl acyl carrier protein synthase III (KASIII) in polyketide synthase gene clusters deleted S. coelicolor strain. Deletion of fadD of β-oxidation was also performed to block degradation of BCFAs.For enhancing productions of PKM from S. venezuelae, three key enzymes in BCAA catabolism, KASIII, acyl-CoA dehydrogenase (ACAD), and BCDH were manipulated. First, using promoter and 5UTR sets developed in S. coelicolor, KASIII was repressed to lower fatty acid (FA) synthesis, but to increase polyketide synthesis on the other hand. Unfortunately, growth defects from KASIII repression was too severe that the PKM production decreased. As a result, BCDH and ACAD were overexpressed to increase the fluxes towards methylmalonyl-CoA productions. It was compared to PKM productions from central carbon metabolism with Methylmalonyl-CoA mutase (MCM) overexpression. It appeared that methylmalonyl-CoA productions from central carbon metabolism was more effective than that from BCAA catabolism. When BCDH and MCM were overexpressed together, precursor supplementation was no longer a bottleneck, but activities of pikC P450 monooxygenase limited synthesis of pikromycin. In short, this study explored less-assessed area of BCAA metabolism by engineering, to generate precursors of polyketide antibiotics and fatty acids. It would provide general insights to design methodologies for productions of various metabolites composed of acyl-CoAs in various Streptomyces sp..