The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.