| Natural products have played a key role in the discovery of lead drugs for the treatment of human diseases. Soil-based microorganisms (bacterial and fungi) can biosynthesize secondary metabolites by enzymatically converting intracellular substrates to the final natural product. Polyketides are an extremely diverse and important class of bioactive secondary metabolites.; Experimental approaches have obtained a maximum molar yield of 1.4% for the production of the erythromycin precursor 6dEB in E. coli using propionate as the carbon source, which corresponds to the use of 6% of the input propionate. It also illustrates the main problem to heterologous natural product biosynthesis, i.e., the severely inefficient conversion of the precursor metabolites to the target natural product.; In this thesis, based on the annotated E. coli genome database and through the MATLAB linear programming package, we developed a stoichiometric model of metabolism for the engineered E. coli (BAP1) system, and then utilized FBA to calculate the flux distribution through several pathways. Two potential improvement target sites, the 2-oxobutanoate formate lyase reaction and the methylmalonyl-CoA mutase reaction, were identified.; Another transporter project is the first trial to study how E. coli transporter proteins affect heterologous polyketide production. Through chromosomal engineering, we constructed an acrAB mutation strain, and also used a plasmid system to over-expression macAB to test the effect on 6dEB production. MS-based 6-dEB comparison results showed that the acrAB mutation strain shows decreased 6dEB production, which discovered acrAB is the possible transporter for 6dEB.; Both of the two projects are examples of metabolic engineering to optimize natural product production. With advances in metabolic engineering, we can take a more targeted approach towards optimize natural production. |
