Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA

Canonaco, Fabrizio; Hess, Tracy A.; Heri, Sylvia; Wang, Taotao; Szyperski, Thomas; Sauer, Uwe

doi:10.1111/j.1574-6968.2001.tb10892.x

Cited by 161 publications

(151 citation statements)

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“…An additional factor in the redox-balancing capacity of E. coli is the presence of two kinds of transhydrogenases (Fuhrer & Sauer, 2009;Sauer et al, 2004). For example, it was observed that the overexpression of the transhydrogenase UdhA helps to dissipate the NADPH imbalance in the Dpgi mutant, increasing the growth rate of this mutant strain (Canonaco et al, 2001). Given the balancing capacity provided by the transhydrogenases, it is not clear why the dehydrogenases in the oxPPP from E. coli have evolved to be specific for NADP.…”

Section: Introductionmentioning

confidence: 99%

“…When these strains were grown aerobically with glucose as the sole carbon source, the mutant Dpgi-NAD-G6PDH showed a growth rate 59 % faster than that observed for the parental Dpgi (Table 2). Canonaco et al (2001) demonstrated that the impaired growth rate of the Dpgi strain is caused by the relative excess of NADPH produced when almost the entire assimilated glucose flows through the oxPPP. The results shown here are consistent with the results of Canonaco et al (2001) because the substitution of an NADPH-producing enzyme by an NADH-producing enzyme would partially relieve the relative excess of NADPH observed in Dpgi, improving its growth rate.…”

Section: Design and Kinetic Study Of An Nad-preferring G6pdhmentioning

confidence: 99%

“…The generation of NADPH must be balanced with its consumption to maintain optimal growth rates. For example, phosphoglucoisomerase (Dpgi) mutants grown on glucose as the sole carbon source exhibit reduced growth rates (Canonaco et al, 2001). In this case, all the glucose is channelled through oxPPP, leading to an unbalanced production of NADPH, and growth rates fall 75 % with respect to WT.…”

Section: Introductionmentioning

confidence: 99%

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Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

et al. 2014

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In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Dpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH R46E,Q47E . Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Dpgi E. coli strains were replaced by DNA encoding LmG6PDH R46E,Q47E . Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Dpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

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Section: Introductionmentioning

confidence: 99%

Section: Design and Kinetic Study Of An Nad-preferring G6pdhmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

et al. 2014

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“…Inhibition of respiratory growth due to excess NADPH accumulation has been documented in yeast (Boles et al 1993;Fiaux et al 2003;Nissen et al, 2001) and in a strain of E. coli lacking the cytoplasmic transhydrogenase UdhA (Sauer et al, 2004). UdhA catalyses the oxidation of NADPH, with NAD serving as the electron acceptor, and is essential for growth of E. coli under two conditions in which the rate of NADPH production exceeds its consumption: growth on glucose in the absence of the phosphoglucose isomerase and growth in minimal medium containing acetate as the sole electron donor and carbon source (Canonaco et al, 2001;Sauer et al, 2004). Thus, one possible explanation for the phenotype of the SfrAB-null strain is that SfrAB serves as a major route for NADP regeneration in G. sulfurreducens.…”

Section: Fe(iii) Reduction By Spheroplastsmentioning

confidence: 99%

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

et al. 2007

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A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism Geobacter sulfurreducens, suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an 'acetate-adapted' SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the 'acetate-adapted' SfrAB-null strain. Membrane and soluble fractions prepared from the 'acetate-adapted' strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.

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“…3 corresponds to the interconversion of NADPH and NADH catalysed by the soluble and membrane-bound transhydrogenases SthA and PntAB, respectively (Sauer et al, 2004;Canonaco et al, 2001). This flux is positive in MG1655 and in Pfl2, implying that the reduced cofactor NADH is converted to the biosynthetic reducing power NADPH.…”

Section: Resultsmentioning

confidence: 99%

Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

2010

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The fermentative metabolism of D-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDHdeficient strain (Pdh") exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH-and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh" were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD + ratio supported the proposed role of PDH. Moreover, the NADH : NAD + ratio in a strain deficient in both PDH and the ox-PPP (Pdh"Zwf") was even lower than that observed for Pdh". Strain Pdh"Zwf" also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh". Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh" mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD + ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

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Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA

Cited by 161 publications

References 18 publications

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction

Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

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