Precursor and cofactor as a check valve for cephamycin biosynthesis in Streptomyces clavuligerus

The biosynthesis of secondary metabolites is closely linked to primary meta bolism via the supply of precursors, cofactors, and cellular energy. The av ailability of these precursors and cofactors can potentially be rate-limiti ng for secondary metabolism. A combined experimental and kinetic modeling a pproach was used to examine the regulation of flux in the cephamycin biosyn thetic pathway in Streptomyces clavuligerus. The kinetic parameters of lysi ne 6-aminotransferase (LAT), the first enzyme leading to cephamycin biosynt hesis and one which was previously identified as being a rate-limiting enzy me, were characterized. LAT converts lysine to alpha-aminoadipic acid using alpha-ketoglutarate as a cosubstrate. The K-m values for lysine and alpha- ketoglutarate were substantially higher than those for their intracellular concentrations, suggesting that lysine and alpha-ketoglutarate may play a k ey role in regulating the flux of cephamycin biosynthesis. The important ro le of this precursor/cosubstrate was supported by simulated results using a kinetic model. When the intracellular concentrations and high K-m values w ere taken into account, the predicted intermediate concentration was simila r to the experimental measurements. The results demonstrate the controlling roles that precursors and cofactors may play in the biosynthesis of second ary metabolites.