Targeted gene insertion methodology was used to study the effect of perturbing α-aminoadipic acid precursor flux on the overall production rate of β-lactam biosynthesis in Streptomyces clavuligerus. A high-copy-number plasmid containing the lysine ε-aminotransferase gene (lat) was constructed and used to transform S. clavuligerus. The resulting recombinant strain (LHM100) contained an additional complete copy of lat located adjacent to the corresponding wild-type gene in the chromosome. Biological activity and production levels of β-lactam antibiotics were two to five times greater than in wild-type S. clavuligerus. Although levels of lysine ε- aminotransferase were elevated fourfold in LHM100, the level of ACV synthetase, whose gene is located just downstream of lat, remained unchanged. These data strongly support the notion that direct perturbation of α- aminoadipic acid precursor flux resulted in increased antibiotic production. This strategy represents a successful application of metabolic engineering based on theoretical predictions of precursor flux in a secondary metabolic pathway.