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Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

ABSTRACT: Noncanonical cofactors such as nicotinamide mononucleotide (NMN⁺) supplant the electron-transfer functionality of the natural cofactors, NAD(P)⁺, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN⁺-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN⁺. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN⁺-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ~3.4 × 10⁶-fold switch in cofactor specificity from its native cofactor NADP⁺ to NMN⁺. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN⁺, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN⁺-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.

SUBMITTER: King E

PROVIDER: S-EPMC10449333 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

King Edward E Cui Youtian Y Aspacio Derek D Nicklen Frances F Zhang Linyue L Maxel Sarah S Luo Ray R Siegel Justin B JB Aitchison Erick E Li Han H

ACS catalysis 20220705 14

Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron-transfer functionality of the natural cofactors, NAD(P)+, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN+-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydroge ...[more]

PMID: 37622090

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Project description:BackgroundThe halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens.ResultsFructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described.ConclusionsOverall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.

Project description:Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II(D213G) and thus by coupling to electron transport phosphorylation (ETP).

Project description:BackgroundIt is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner-Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden-Meyerhof-Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner-Doudoroff and Embden-Meyerhof-Parnas pathways to coexist.ResultsThree genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A 13C-labeling experiment using [1-13C] glucose indicated a distinctive 13C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD+ ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed.ConclusionsThe increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.

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Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

Publications

Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

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