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 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.

Comparative genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein secretion capacity.

Aspergillus niger is widely used as a cell factory for homologous and heterologous protein production. As previous studies reported that reduced sporulation favors protein secretion in A. niger, in this study, we conducted a comparative genomic analysis of the non-sporulating industrially exploited A. niger strain LDM3 in China and the reference protein secretion strain CBS 513.88 to predict the key genes that might define the genetic basis of LDM3's high protein-producing potential in silico. After sequencing using a hybrid approach combining Illumina and PacBio sequencing platforms, a high-quality genome sequence of LDM3 was obtained which harbors 11,209 open reading frames (ORFs). LDM3 exhibits large chromosomal rearrangements in comparison to CBS 513.88. An alignment of the two genome sequences revealed that the majority of the 457 ORFs uniquely present in LDM3 possessed predicted functions in redox pathways, protein transport, and protein modification processes. In addition, bioinformatic analyses revealed the presence of 656 ORFs in LDM3 with non-synonymous mutations encoding for proteins related to protein translation, protein modification, protein secretion, metabolism, and energy production. We studied the impact of two of these on protein production in the established lab strain N402. Both tupA and prpA genes were selected because available literature suggested their involvement in asexual sporulation of A. niger. Our co-expression network analysis supportively predicted the role of tupA in protein secretion and the role of prpA in energy generation, respectively. By knockout experiments, we showed that the ΔtupA mutant displayed reduced sporulation (35%) accompanied by higher total protein secretion (65%) compared to its parental strain. Such an effect was, however, not observed in the ΔprpA mutant.