Global transcriptional response of Aspergillus niger in the process of glucoamylase fermentation

Sui, Yufei; Ouyang, Liming; Chu, Ju; Cao, Wenjun; Liang, Lifeng; Zhuang, Yingping; Cheng, Shu; Norrman, Henk; Zhang, Siliang; Zhang, Gengyun

doi:10.1186/s40643-017-0160-x

Cited by 7 publications

(7 citation statements)

References 32 publications

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“…Figure 5 and Additional file 1 : Table S5 summarize the amino acid pools in all three overexpression strains compared to B36. In general, Ala, Glu, Gly, Leu, and Lys are the top five amino acids in the biomass of A. niger , whereas Ser, Thr, Ala, Leu, and Gly account for about 50% of the total amino acids in GlaA [ 40 ]. Our metabolomics analyses uncovered that in all four strains, amino acids from the glutamate family are most and aromatic amino acids are least abundant, which is in general agreement to previously reported amino acid pools in A. niger [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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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 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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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“…Based on our previous transcriptome analysis of oxygen-limited fermentation for glucoamylase production in A. niger [ 13 ], 5 genes, related to fatty acids degradation and continuously up-regulated during oxygen-limitation period, were selected as candidate genes (Table 2 ). Five genes named fadE (encoding Acyl-CoA dehydrogenase), fadA (encoding Acetyl-CoA C-acyltransferase), hyp (encoding hypothetical protein with hydrolase activity), cyp (encoding cytochrome P450 monooxygenase), acy (encoding acyltransferases) were overexpressed in the pyrG locus of A. niger YS20.2 genome under the control of Tet-on system (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, multi-omics integrative analysis of glucoamylase fermentation process in A. niger in our lab [ 13 ] showed that fatty acids degradation and precursor amino acids synthesis were elevated under oxygen limitation, whereas fatty acid synthesis was diminished. While it is well known that lipids, as essential components of cell membranes, play an important role in many processes such as cell signaling, energy supply and cell growth [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

Xiao

Ouyang

et al. 2022

Microb Cell Fact

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Background Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. Results Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. Conclusion This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

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“…Table S5 summarize the amino acid pools in all three overexpression strains compared to B36. In general, Ala, Glu, Gly, Leu, and Lys are the top five amino acids in the biomass of A. niger, whereas Ser, Thr, Ala, Leu, and Gly account for about 50% of the total amino acids in GlaA [40]. Our metabolomics analyses uncovered that in all four strains, amino acids from the glutamate family are most and aromatic amino acids are least abundant, which is in general agreement to previously reported amino acid pools in A. niger [41].…”

Section: Metabolic Differences Revealed By Multivariate Statistical Amentioning

confidence: 99%

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

Sui

Schütze

Ouyang

et al. 2020

Preprint

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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 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.

show abstract

Global transcriptional response of Aspergillus niger in the process of glucoamylase fermentation

Cited by 7 publications

References 32 publications

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

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

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

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

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