Precursor and Cofactor as a Check Valve for Cephamycin Biosynthesis in Streptomyces clavuligerus

The biosynthesis of secondary metabolites is closely linked to primary metabolism via the supply of precursors, cofactors, and cellular energy. The availability of these precursors and cofactors can potentially be rate-limiting for secondary metabolism. A combined experimental and kinetic modeling approach was used to examine the regulation of flux in the cephamycin biosynthetic pathway in Streptomyces clavuligerus. The kinetic parameters oflysine 6-aminotransferase (LAT), the first enzyme leading to cephamycin biosynthesis and one which was previously identified as being a ratelimiting enzyme, were characterized. LAT converts lysine to (alpha)-aminoadipic acid using (alpha)-ketoglutarate as a cosubstrate. The Km values for lysine and (alpha)-ketoglutarate were substantially higher than those for their intracellular concentrations, suggesting that lysine and a-ketoglutarate may play a key role in regulating the flux of cephamycin biosynthesis. The important role of this precursor/cosubstrate was supported by simulated results using a kinetic model. When the intracellular concentrations and high Km values were taken into account, the predicted intermediate concentration was similar to the experimental measurements. The results demonstrate the controlling roles that precursors and cofactors may play in the biosynthesis of secondary metabolites.