| A general approach to metabolic engineering of biosynthetic pathways is developed in an effort to understand the basic biological processes of secondary metabolism. This has been pursued using the integrated approach of theoretical analysis and genetic manipulation. To demonstrate such an approach, the biosynthesis of {dollar}beta{dollar}-lactams was chosen as the model system.; The biosynthesis of {dollar}beta{dollar}-lactams is one of the most thoroughly studied antibiotic pathways. However, there remains a fundamental lack of knowledge about control of carbon flow into the synthesis of these metabolites. To understand the control of {dollar}alpha{dollar}-lactam biosynthesis, a theoretical kinetic model was constructed to predict the rate-limiting enzymatic steps. Sensitivity analysis revealed that the availability of precursor {dollar}alpha{dollar}-aminoadipic acid and enzymatic activity of ACV synthetase were the potential rate-limiting steps. This precursor limitation was alleviated by integration of an additional copy of the gene encoding lysine {dollar}varepsilon{dollar}-aminotransferase (lat) into the chromosome. As predicted, the recombinant strain produced higher levels of cephamycin C than its parental strain. The intracellular levels of several metabolites were measured and compared between the engineered and the wild-type strains. Differences between these strains revealed and demonstrated that ACV synthetase was an additional rate-limiting enzyme.; An extended kinetic model was constructed based on the additional kinetic data measured. Discrepancy in the predicted and measured values of the intracellular metabolites suggests that additional biosynthetic reactions are critical in controlling the biosynthetic rates. It is proposed that the level of the cofactor required for enzymatic activity of lysine {dollar}varepsilon{dollar}-aminotransferase can influence the biosynthesis of {dollar}alpha{dollar}-aminoadipic acid. Furthermore, dimerization of ACV tripeptide can potentially impede the biosynthesis of cephamycin C. |
