Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD+-dependent formate dehydrogenase

Balzer, Grant J.; Thakker, Chandresh; Bennett, George N.; San, Ka‐Yiu

doi:10.1016/j.ymben.2013.07.005

Cited by 90 publications

(57 citation statements)

References 26 publications

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“…Since the creC mutant produced larger amounts of succinate than the parental strain, additional strategies were tested to increase the titer of the metabolite in E. coli DC1060. Two plasmids were introduced into the strains: plasmid pEcPpc, carrying the carboxylating enzyme phosphoenolpyruvate carboxylase (Ppc Ec ) from E. coli (42), and plasmid pSBF2, carrying a NAD ϩ -dependent formate dehydrogenase from the methylotrophic yeast Candida boidinii (FDH1 Cb ) (43)(44)(45). In both plasmids, the corresponding genes are under the control of the lac promoter, and their expression can be induced by IPTG.…”

Section: Resultsmentioning

confidence: 99%

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Godoy

Nikel

Gomez

et al. 2016

Appl Environ Microbiol

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The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ⌬creC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ⌬creC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P) ؉ ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ⌬creC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ⌬creC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.T he survival of an organism depends, at least in part, on its ability to sense and respond to changes in the environment. In bacteria, global regulators control the transcription of genes in response to specific external stimuli and metabolic signals, finely tuning different aspects of their physiology to overcome environmental challenges. In Escherichia coli, seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate the expression of about one-half of all genes (1). This facultative aerobe is able to adapt its metabolism to different oxygen availability conditions through the concerted actions of a network of regulators, including the global regulators ArcAB and Fnr (2-4). These regulators affect many metabolic pathways, allowing the cells to reach an adequate redox balance under any given conditions. There is a very close association between carbon and electron flows, and even small differences in oxygen availability have been observed to elicit profound effects on the distribution of carbon fluxes (5).CreBC (for carbon source responsive) is a global sensing and regulation system affecting genes involved in a variety of functions, including enzymes of intermediary catabolism (6). Previous studies have shown that the creABCD operon is activated (i) during growth in minimal mediu...

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Section: Resultsmentioning

confidence: 99%

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Godoy

Nikel

Gomez

et al. 2016

Appl Environ Microbiol

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“…In addition, the levels of TCA cycle intermediates citrate and 2-oxoglutarate were among the highest. E. coli C has indeed previously been successfully used as the base strain for succinate production from glucose under aerobic and anaerobic conditions (Balzer et al, 2013;Jantama et al, 2008;Lin et al, 2005aLin et al, , 2005bSánchez et al, 2005). Interestingly, E. coli DH5a had the highest levels of citrate, aconitate, isocitrate, and 2-oxoglutarate.…”

Section: Rapidrip Vs Fullripmentioning

confidence: 99%

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

McCloskey

Schrübbers

et al. 2018

Metabolic Engineering

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A B S T R A C TFast metabolite quantification methods are required for high throughput screening of microbial strains obtained by combinatorial or evolutionary engineering approaches. In this study, a rapid RIP-LC-MS/MS (RapidRIP) method for high-throughput quantitative metabolomics was developed and validated that was capable of quantifying 102 metabolites from central, amino acid, energy, nucleotide, and cofactor metabolism in less than 5 minutes. The method was shown to have comparable sensitivity and resolving capability as compared to a full length RIP-LC-MS/MS method (FullRIP). The RapidRIP method was used to quantify the metabolome of seven industrial strains of E. coli revealing significant differences in glycolytic, pentose phosphate, TCA cycle, amino acid, and energy and cofactor metabolites were found. These differences translated to statistically and biologically significant differences in thermodynamics of biochemical reactions between strains that could have implications when choosing a host for bioprocessing.

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“…Due to the CO 2 fixation step in the production of SA it is theoretically possible to obtain 1.12 grams of SA per gram of glucose (van Heerden and Nicol 2013a). Modified E. coli batch fermentations have exceeded the 1 g g -1 threshold (Balzer et al 2013;Jantama et al 2008) although no report exists where this threshold has been exceeded with a continuous culture. A.…”

Section: Introductionmentioning

confidence: 99%

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility, stability and productivity

Maharaj

Bradfield

Nicol

2014

Appl Microbiol Biotechnol

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Continuous anaerobic fermentations were performed in a biofilm reactor packed with Poraver® beads. Dilution rates (D) varied between 0.054 h -1 and 0.72 h -1 and D-glucose and CO 2 gas were used as carbon substrates. Steady-state conditions were shown to be repeatable and independent of the operational history. Production stability was achieved over periods exceeding 80 h at values of D below 0.32 h -1 . In these situations, steady-state variation (expressed as fluctuations in NaOH neutralisation flow rates) exhibited a standard deviation of less than 5% while no indication of biofilm deactivation was detected. The total biomass amount was found to be independent of the dilution rate with an average dry concentration of

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Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD+-dependent formate dehydrogenase

Cited by 90 publications

References 26 publications

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility, stability and productivity

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