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

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 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH regeneration enzymes under the control of 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 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 reverse TCA cycle ( maeA gene) followed by engineering the flux through the pentose phosphate pathway ( gndA 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.

Cloning of cDNA glucoamylase Aspergillus awamori into yeast integrative expression vector

We constructed pKLAC2-based integrative expression plasmid pKGLA-1 with glaA gene from Aspergillus awamori 466. The PCR amplification of the target gene glaA and restriction analysis proved pKGLA-1 construction. Linearised plasmid was used for the integrative transformation of chemically competent Kluyveromyces lactis GG799 cells. Colonies of cells transformed with pKGLA-1 plasmid were selected by growth on agar plates containing 5 mmol/L acetamide. Expression of the heterologous gene in K. lactis cells was visually assessed using medium containing 2 % starch. K. lactis cells containing integrated pKGLA-1 DNA secreted recombinant protein glucoamylase with a native N-terminus.