title: Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase.
abstract: Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in beta-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP(+)-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP(+)-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP(+)-linked acetaldehyde dehydrogenase and NADP(+)-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and beta-lactam production and may provide an interesting target for metabolic engineering.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Harris
authors.forename: Jasper A
authors.initials: JA
authors.lastname: Diderich
authors.forename: Zita A
authors.initials: ZA
authors.lastname: van der Krogt
authors.forename: Marijke A H
authors.initials: MA
authors.lastname: Luttik
authors.forename: Léonie M
authors.initials: LM
authors.lastname: Raamsdonk
authors.forename: Roel A L
authors.initials: RA
authors.lastname: Bovenberg
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
authors.forename: Johannes P
authors.initials: JP
authors.lastname: van Dijken
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
mesh_terms: D049109:Cell Proliferation; D003198:Computer Simulation; D004734:Energy Metabolism / Q000502:physiology; D004789:Enzyme Activation; D008928:Mitochondria / Q000201:enzymology*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000166:cytology* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D047090:beta-Lactams; D009249:NADP; D009252:NADPH Dehydrogenase