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Background  Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory  Aspergillus niger  indicated that low availability of NADPH might be a limiting factor for GlaA overproduction.    Results  We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and  13 C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two  A. niger  recipient strains, one carrying a single and one carrying seven  glaA  gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards  glaA  biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the  gsdA  gene (glucose 6-phosphate dehydrogenase),  gndA  gene (6-phosphogluconate dehydrogenase) and  maeA  gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven  glaA  gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the  gndA  or  maeA  gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the  gsdA  gene had a negative effect on both total protein and glucoamylase production.    Conclusions  This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway ( gndA  gene) followed by engineering the flux through the reverse TCA cycle ( maeA  gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of  A. niger  to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger