Dataset Information

Autophagy is important to the acidogenic metabolism of Aspergillus niger.

ABSTRACT: Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvation in the A. niger ?atg1 strain. The acidogenic growth of the fungus consisted of a brief log phase followed by an extended autophagy-like state throughout the idiophase of fermentation. Mycelia in the idiophase were highly vacuolated and EGFP was localized to the vacuoles but no autolysis was observed. Both autophagy and acidogenesis are compromised in ?atg1 and ?atg8 strains of A. niger. The acidogenic growth of the fungus thus appears to mimic a condition of nutrient limitation and is associated with an extended autophagy-like state. This crucial role of autophagy in acidogenic A. niger physiology could be of value in improving citric acid fermentation.

SUBMITTER: Kaur B

PROVIDER: S-EPMC6788731 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Autophagy is important to the acidogenic metabolism of Aspergillus niger.

Kaur Baljinder B Punekar Narayan S NS

PloS one 20191011 10

Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvati ...[more]

PMID: 31603923

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Project description: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.

Dataset Information

Autophagy is important to the acidogenic metabolism of Aspergillus niger.

Publications

Autophagy is important to the acidogenic metabolism of Aspergillus niger.

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