Characterization of pathway engineered strains of filamentous fungi in submerged cultures

Filamentous fungi are used by the industry for making a variety of products, e.g. penicillin, high-protein food, washing powder and soft drinks. Synthesis of many of these products requires reducing equivalents in the form of NADPH, which is a potential limiting factor. The pentose phosphate pathway (PPP) is believed to be the major source of NADPH. Our main goal has been to make strains ofAspergillus nigerwith increased NADPH availability and/or increased flux through the PPP by overexpression of enzymes in this pathway. Cloning of genes encoding for these enzymes enabled transformation to obtain multicopy transformants. To measure the effect(s) of overexpression, comparison with wild type in well-defined cultures is necessary. A bioreactor was designed especially for cultures of filamentous fungi and techniques were developed for automatic sampling of culture filtrate, rate analysis of culture data, morphology profiling and continuous culturing. NADPH availability was increased in multicopy transformants of the gene encoding for 6-phosphogluconate dehydrogenase.We succeeded to reach our main goal: to increase the availability of NADPH in A. niger. This was obtained by overexpression of 6-phosphogluconate dehydrogenase, but also overexpression of glucose 6-phosphate dehydrogenase and transketolase showed interesting effects and a combination of these overexpressions might result in even higher availability of NADPH and/or flux through the PPP. It has been confirmed by NMR studies that the flux through the PPP is increased in the 6-phosphogluconate dehydrogenase overexpressing strain [Hesse SJA, Dijkema C, de Graaf AA, Ruijter GJG, Visser J, unpublished results].