Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phase E. coli

AbstractWe demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli. Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in α-ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126±7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a “central” metabolic pathway in stationary phase E. coli, which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels.HighlightsDynamic reduction in α-keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake.Dynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux.Pyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions.Production of citramalate to titers 126 ± 7g/L at > 90 % of theoretical yield.

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Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

10.1101/2020.07.27.222588 ◽

2020 ◽

Cited By ~ 2

Author(s):

Shuai Li ◽

Eirik A. Moreb ◽

Zhixia Ye ◽

Jennifer N. Hennigan ◽

Daniel Baez Castellanos ◽

...

Keyword(s):

Dynamic Control ◽

List Type ◽

E Coli ◽

Ferredoxin Oxidoreductase ◽

Ferredoxin Reductase ◽

Enoyl Acp Reductase ◽

Membrane Bound ◽

Pyruvate Ferredoxin Oxidoreductase ◽

Glucose 6 Phosphate Dehydrogenase

AbstractWe report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions.HighlightsDecreases in NADPH pools lead to increased NADPH fluxesPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH fluxXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock.

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Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

10.1101/2021.08.06.455447 ◽

2021 ◽

Author(s):

Joy Omini ◽

Izabela Wojciechowska ◽

Aleksandra Skirycz ◽

Hideaki Moriyama ◽

Toshihiro Obata

Keyword(s):

Malate Dehydrogenase ◽

Metabolic Flux ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Feedback Regulation ◽

Dynamic Structure ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Enzyme Complex ◽

Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

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Acetyl-CoA production by encapsulated pyruvate ferredoxin oxidoreductase in alginate hydrogels

Bioresource Technology ◽

10.1016/j.biortech.2016.12.051 ◽

2017 ◽

Vol 227 ◽

pp. 279-285 ◽

Cited By ~ 5

Author(s):

Makoto Takenaka ◽

Ki-Seok Yoon ◽

Takahiro Matsumoto ◽

Seiji Ogo

Keyword(s):

Acetyl Coa ◽

Ferredoxin Oxidoreductase ◽

Alginate Hydrogels ◽

Pyruvate Ferredoxin Oxidoreductase

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Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Biochemical Journal ◽

10.1042/bj1110279 ◽

1969 ◽

Vol 111 (3) ◽

pp. 279-286 ◽

Cited By ~ 8

Author(s):

M. B. Coukell ◽

W. J. Polglase

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Catabolite Repression ◽

Citrate Synthase ◽

Wild Type ◽

Experimental Conditions ◽

E Coli ◽

Equivalent Quantity ◽

Glucose 6 Phosphate Dehydrogenase ◽

Escherichia Coli B

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for β-galactosidase in the presence of glucose, although repression of β-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.

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Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in Escherichia coli via Regulation of thecsgD Gene

Journal of Bacteriology ◽

10.1128/jb.183.24.7213-7223.2001 ◽

2001 ◽

Vol 183 (24) ◽

pp. 7213-7223 ◽

Cited By ~ 267

Author(s):

Claire Prigent-Combaret ◽

Eva Brombacher ◽

Olivier Vidal ◽

Arnaud Ambert ◽

Philippe Lejeune ◽

...

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Biofilm Formation ◽

Regulatory System ◽

Feedback Regulation ◽

E Coli ◽

Transcription Regulator ◽

Medium Osmolarity ◽

Initial Adhesion ◽

Extracellular Structures

ABSTRACT The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompRgene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgDpromoter region and stimulating its transcription. ThecsgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBAoperon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR;cpxR-mediated repression of csgB andcsgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

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Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

Scientific Reports ◽

10.1038/s41598-021-98314-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Joy Omini ◽

Izabela Wojciechowska ◽

Aleksandra Skirycz ◽

Hideaki Moriyama ◽

Toshihiro Obata

Keyword(s):

Malate Dehydrogenase ◽

Metabolic Flux ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Feedback Regulation ◽

Dynamic Structure ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Enzyme Complex ◽

Acetyl Coa

AbstractMitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

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A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

Canadian Journal of Biochemistry ◽

10.1139/o80-098 ◽

1980 ◽

Vol 58 (9) ◽

pp. 696-706 ◽

Cited By ~ 9

Author(s):

David Morse ◽

Harry W. Duckworth

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Citrate Synthase ◽

Amino Acid Sequences ◽

Aerobic Bacterium ◽

Acetyl Coa ◽

Gram Negative ◽

E Coli ◽

Sulfhydryl Reagent ◽

Complete Inactivation

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, K1 = 2 μM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 μM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968)) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4′-dithiodipyridine (4,4′-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a rise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4′-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.

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