Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase.
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.
Harris
Diana M
DM
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Diderich
Jasper A
JA
van der Krogt
Zita A
ZA
Luttik
Marijke A H
MA
Raamsdonk
Léonie M
LM
Bovenberg
Roel A L
RA
van Gulik
Walter M
WM
van Dijken
Johannes P
JP
Pronk
Jack T
JT
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*
D009097:Multienzyme Complexes; D047090:beta-Lactams; D009249:NADP; D009252:NADPH Dehydrogenase