Application of metabolic flux analysis for the identification of metabolicbottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G p roduction in Penicillium chrysogenum. From an a priori metabolic flux analy sis using this model it appeared that penicillin production requires signif icant changes in fluxes through the primary metabolic pathways. This is bro ught about by the biosynthesis of carbon precursors for the beta-lactan nuc leus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principa l nodes in primary metabolism. These are located at: (1) glucose-6-phosphat e; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondria l isocitrate. These nodes should be regarded as potential bottlenecks for i ncreased productivity. The flexibility of these principal nodes was investi gated by experimental manipulation of the fluxes through the central metabo lic pathways using a high-producing strain of P. chrysogenum. Metabolic flu xes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measur ed input and output fluxes, was used to calculate the fluxes around the pri ncipal nodes. It was found that, for growth on glucose, ethanol, and acetat e, the flux partitioning around these nodes differed significantly. However , this had hardly any effect on penicillin productivity, showing that prima ry carbon metabolism is not likely to contain potential bottlenecks. Furthe r experiments were performed to manipulate the total metabolic demand for t he cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH dema nd was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen s ource, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in pr imary metabolism reside in the supply/regeneration of cofactors (NADPH) rat her than in the supply of carbon precursors. (C) 2000 John Wiley & Sons, In c.