Six novel constitutive promoters for metabolic engineering of Aspergillus niger

ABSTRACT d-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of d-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi d-galacturonate is catabolized through a reductive pathway with a d-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on d-galacturonate. The genes of the pathway for d-galacturonate catabolism were upregulated in the presence of d-galacturonate in A. niger, even when the gene for d-galacturonate reductase was deleted, indicating that d-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a d-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of d-galacturonate to galactarate was introduced to both strains with disrupted d-galacturonate catabolism. Both strains converted d-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the d-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.

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Metabolic engineering and thermodynamic characterization of an extracellular -glucosidase produced by Aspergillus niger

AFRICAN JOURNAL OF BIOTECHNOLOGY ◽

10.5897/ajb11.186 ◽

2011 ◽

Vol 10 (41) ◽

pp. 8107-8116 ◽

Cited By ~ 8

Author(s):

Zahoor Sana ◽

Mohsin Javed Muhammad ◽

Aftab Saima ◽

Latif Farooq ◽

Ikram ul Haq

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Thermodynamic Characterization

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Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

FEBS Journal ◽

10.1111/j.1742-4658.2005.04554.x ◽

2005 ◽

Vol 272 (6) ◽

pp. 1313-1325 ◽

Cited By ~ 39

Author(s):

Bjarne R. Poulsen ◽

Jane Nøhr ◽

Stephen Douthwaite ◽

Line V. Hansen ◽

Jens J. L. Iversen ◽

...

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Pentose Phosphate Pathway ◽

Pentose Phosphate

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

10.21203/rs.3.rs-42423/v1 ◽

2020 ◽

Author(s):

Yu-fei Sui ◽

Tabea Schütze ◽

Li-Ming Ouyang ◽

Hong-zhong Lu ◽

Peng Liu ◽

...

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Gene Copy ◽

Cell Factory ◽

Flux Analysis ◽

Cofactor Engineering ◽

Nadph Regeneration ◽

Gene Copies ◽

Glaa Gene ◽

Glucoamylase Production

Abstract 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.

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Targeting enzymes to the right compartment: Metabolic engineering for itaconic acid production by Aspergillus niger

Metabolic Engineering ◽

10.1016/j.ymben.2013.05.003 ◽

2013 ◽

Vol 19 ◽

pp. 26-32 ◽

Cited By ~ 69

Author(s):

Marzena L. Blumhoff ◽

Matthias G. Steiger ◽

Diethard Mattanovich ◽

Michael Sauer

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Itaconic Acid ◽

Acid Production ◽

The Right

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Metabolic engineering of an industrial Aspergillus niger strain for itaconic acid production

3 Biotech ◽

10.1007/s13205-020-2080-2 ◽

2020 ◽

Vol 10 (3) ◽

Cited By ~ 5

Author(s):

Hui Xie ◽

Qinyuan Ma ◽

Dongzhi Wei ◽

Fengqing Wang

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Itaconic Acid ◽

Acid Production

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Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

Biotechnology for Biofuels ◽

10.1186/s13068-019-1577-6 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 4

Author(s):

Abeer H. Hossain ◽

Roy van Gerven ◽

Karin M. Overkamp ◽

Peter S. Lübeck ◽

Hatice Taşpınar ◽

...

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Acid Stress ◽

Weak Acid ◽

Glycolytic Flux ◽

Atp Citrate Lyase ◽

Nitrogen Supplementation ◽

Fermentation Conditions ◽

Citrate Lyase ◽

Weak Acid Stress

Abstract Background Bio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer Aspergillus terreus is currently used for industrial IA production; however, the filamentous fungus Aspergillus niger has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase citB in an A. niger strain carrying the full IA biosynthesis gene cluster from A. terreus, which resulted in the highest final titer reported for A. niger (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration. Results To increase the cytosolic acetyl-CoA pool, we have overexpressed genes acl1 and acl2 that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress acl12 show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors. Conclusion Ultimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in A. niger due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.

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Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era

Microbial Cell Factories ◽

10.1186/s12934-019-1064-6 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 20

Author(s):

Zhenyu Tong ◽

Xiaomei Zheng ◽

Yi Tong ◽

Yong-Cheng Shi ◽

Jibin Sun

Keyword(s):

Metabolic Engineering ◽

Citric Acid ◽

Aspergillus Niger ◽

Acid Production ◽

Citric Acid Production ◽

Systems Metabolic Engineering

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

10.21203/rs.3.rs-42423/v2 ◽

2020 ◽

Author(s):

Yu-fei Sui ◽

Tabea Schütze ◽

Li-Ming Ouyang ◽

Hong-zhong Lu ◽

Peng Liu ◽

...

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Pentose Phosphate Pathway ◽

Total Protein ◽

Tca Cycle ◽

Pentose Phosphate ◽

Gene Copy ◽

Cofactor Engineering ◽

Gene Copies ◽

Glucoamylase Production

Abstract 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 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)reverse TCA cycle (maeA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene) 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.

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