A bacterial checkpoint protein for ribosome assembly moonlights as an essential metabolite-proofreading enzyme

Sachla, Ankita J.; Helmann, John D.

doi:10.1038/s41467-019-09508-z

Cited by 33 publications

(36 citation statements)

References 45 publications

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“…We hypothesize that accumulation of 6-phosphogluconate leads to inhibition of the glycolytic enzyme PGI and redirection of the carbon flux into the oxidative PPP. Interestingly, partial inhibition of 6-PGD in Escherichia coli has also recently been shown to lead to a compensatory increased flux into the oxidative PPP through inhibition of upper glycolysis (24). These findings highlight how the parasite coordinates metabolic regulation via metabolite repair.…”

Section: Discussionmentioning

confidence: 99%

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

Dumont

Richardson

Peet

et al. 2019

mBio

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The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.

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Section: Discussionmentioning

confidence: 99%

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

Dumont

Richardson

Peet

et al. 2019

mBio

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“…Diverting the carbon flux towards the PPP at the branch node of G6P after overexpression of gsdA and gndA has in fact to lower the flux towards the EMP. One consequence would be that the 6PG and PEP pools increased (due to gsdA overexpression), which in turn inhibited the upper glycolytic pathway (feedback inhibition via 6PG [42] and PEP [43]). Another consequence would be the less carbon uptaken rate, which is indeed the case for the strain overexpressing gsdA ( Table 2).…”

Section: Metabolic Profiling Of Amino Acid Pools and Central Carbon Mmentioning

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.

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“…Diverting the carbon ux towards the PPP at the branch node of G6P after overexpression of gsdA and gndA has in fact to lower the ux towards the EMP. One consequence would be that the 6PG and PEP pools increased (due to gsdA overexpression), which in turn inhibited the upper glycolytic pathway (feedback inhibition via 6PG [42] and PEP [43]). Another consequence would be the less carbon uptaken rate, which is indeed the case for the strain overexpressing gsdA ( Table 2).…”

Section: Physiology and Gene Expression During Maltose-limited Chemosmentioning

confidence: 99%

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

Sui

Schütze

Ouyang

et al. 2020

Preprint

View full text Add to dashboard Cite

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.

show abstract

A bacterial checkpoint protein for ribosome assembly moonlights as an essential metabolite-proofreading enzyme

Cited by 33 publications

References 45 publications

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

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

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

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