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Background: Since the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective.

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Production of ␤-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol ⅐ mol ؊1 , an increasing rate of formate oxidation via a cytosolic NAD ؉ -dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol ⅐ mol ؊1 , the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as ␤-lactams.The filamentous fungus Penicillium chrysogenum is applied on a large scale (Ͼ60,000 tons year Ϫ1 ) (8, 36) for the industrial production of ␤-lactam antibiotics, such as penicillin G and penicillin V, and for the production of the cephalosporin precursor adipoyl-7-ADCA. ␤-Lactam antibiotics are formed in a multistep process in which the first two steps are common for penicillins and cephalosporins. The three amino acids cysteine, valine, and ␣-aminoadipic acid, derived from central metabolism, are condensed to form the tripeptide ACV (␣-aminoadipyl-cysteinyl-valine). The next step is a ring closure that leads to the characteristic penam structure of isopenicillin N, the branch point intermediate at which penicillin biosynthesis diverges from cephalosporin biosynthesis. Penicillin G is formed from isopenicillin N by exchanging its ␣-aminoadipic acid side chain for phenylacetic acid, using phenylacetyl-coenzyme A as a side chain donor.Overproduction of secondary metabolites can have a large impact on central metabolism if it requires significant amounts of carbon precursors, reducing equivalents (NADH and NADPH), and free energy equivalents (ATP). Previous studies on penicillin G production in a high-producing industrial strain of P. chrysogenum have shown that constraints in central metabolism may reside in the supply and regeneration of the cofactor NADPH rather than in the supply of the carbon precursors, ␣-aminoadipic aci...