Metabolic control analysis of the penicillin biosynthetic pathway in a high-yielding strain of Penicillium chrysogenum.

Metabolic control analysis is used to identify the rate-limiting step in the penicillin biosynthetic pathway in Penicillium chrysogenum. The analysis is carried out using a kinetic model for the first two steps in the pathway, i.e., the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS). The kinetic model is based on Michaelis-Menten type kinetics, with noncompetitive inhibition of the ACVS by ACV and competitive inhibition of the IPNS by glutathione. From measurements of the enzyme activities and intracellular metabolites during a fed-batch cultivation, the kinetic model is used to predict the flux through the pathway. The model prediction corresponds well with the measured rate of penicillin biosynthesis. From measurement of the activity of the acyl-CoA:isopenicillin acyltransferase, which catalyzes the third and last reaction in the pathway, it is concluded that the rate-limiting step is either at the ACVS or at the IPNS. From the kinetic model, the elasticity coefficients and the flux control coefficients are calculated throughout the fed-batch cultivations, and it is found that there is a shift in the flux control from the ACVS to the IPNS during the cultivation.