Improvement of pyruvate production based on regulation of intracellular redox state in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The commercial demand for pyruvate has been expanding. However, some challenges need to be overcome in the microbial production of pyruvate, such as low glucose consumption caused by excessive accumulation of NADH. In this study, weakening or block of the TCA cycle, overexpression of foreign NADH oxidase, and carbon sources with different oxidation state was attempted to decrease NADH accumulation in engineered strain YP211. Results showed that blocking or weakening TCA cycle could not lower the intracellular redox state in strain YP211.Overexpressing NADH oxidase from Lactococcus lactis significantly decreased the intracellular NADH content and increased the consumption rate of glucose. However, the yield of pyruvate did not increase significantly. Compared with glucose as carbon source, sodium gluconate with a higher oxidation state resulted in a significant decrease of NADH/NAD⁺, and the concentration and yield of pyruvate increased by 62 and 6%, respectively. In the fed-batch fermentation, the yield of pyruvate increased to 0.78 g/g gluconate, and the concentration of pyruvate reached 78.8 g/L. It was suggested that sodium gluconate was a more ideal carbon source for strain YP211, which could effectively decrease NADH content and improve the pyruvate production.     

Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source

Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD⁺ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L.The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD⁺ ratio can be achieved by using carbon sources that have different oxidation states.Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid.     

Carbon nanotubes-polymer-redox mediator hybrid thin film for electrocatalytic sensing

Electrocatalytic sensing of NADH using a hybrid thin film derived from multi-wall carbon nanotubes (CNTs), Nafion (Nf) polymer and electrogenerated redox mediator is described. The redox mediator was electrochemically generated by the oxidation of serotonin on the hybrid thin film modified glassy carbon electrode (GC/Nf-CNT). Controlled potential electrolysis of serotonin at 0.1 V in neutral solution results in the generation of the redox mediator 5,5'-dihydroxy-4,4'-bitryptamine (DHB) on the hybrid thin film. The electrogenerated DHB has redox active quinone-imine structure and was electrochemically characterized by studying the pH dependent redox response. DHB on the hybrid thin film exhibits reversible redox peak at -0.05 V and the formal potential shifts by -55 mV while increasing the solution pH by 1 unit. The quinone-imine structure of DHB efficiently catalyzes the oxidation of NADH with a decrease in the overpotential of about 500 mV compared to the unmodified electrode. The CNTs of the hybrid thin film facilitates the mediated electrocatalytic oxidation of NADH. The hybrid thin film modified electrode exhibits stable amperometric response and it linearly responds to NADH (0.5-400 microM). This hybrid thin film modified electrode could detect NADH as low as 0.1 microM at -0.05 V with a sensitivity of 11.1 nA/microM in physiological pH.     

Oxidation of carbon monoxide and methane by Pseudomonas methanica.

The oxidation of carbon monoxide and methane by suspensions and ultrasonic extracts of Pseudomonas methanica was studied. A continuous assay for the oxidation of CO to CO2 was devised, using O2 and CO2 electrodes in combination. Stoicheiometries of CO-dependent CO2 formation, O2 consumption and NADH oxidation, and the partial stoicheiometries of methane-dependent NADH oxidation, suggest the involvement of a mono-oxygenase in these oxidations. Evidence is presented suggesting methane and CO oxidation are catalysed by a single enzyme system, distinct, at least in part, from the NADH oxidase present in extracts. Ethanol was able to provide the reductant necessary for CO oxidation by cell suspensions, though the metabolism of ethanol by P. methanica was found unlikely to result in substrate-level formation of NADH; the means whereby alcohol oxidation could supply reductant for the mono-oxygenase are discussed.     

Electrocatalytic properties of polypyrrole in amperometric electrodes

The electrocatalytic oxidation of NADH, ascorbate, urate, xanthine and H₂O₂ at different polypyrrole electrodes has been investigated. The conducting polymer was grown on platinum, glassy carbon, or graphite electrodes and modified by means of enclosed redox-active anions or other redox-active compounds covalently bound to either the N- or the β-position of the pyrrole. Copolymers of pyrrole and N-substituted pyrrole derivatives of chloranil or 2,3-dicholoro-1,4-naphthoquinone showed outstanding electrocatalytic properties for the oxidation of NADH. The application of these electrodes in amperometric steady-state measurements or flow-injection systems in combination with dehydrogenase reactions has been possible.     

Synergetic effect for NADH oxidation of ferrocene and zeolite in modified carbon paste electrodes. New approach for dehydrogenase based biosensors

The development of electrochemical biosensors using dehydrogenases associated with the corresponding cofactor is strongly related to the better understanding of NADH oxidation at the electrode surface. The aim is to lower the necessary overvoltage and consequently to escape interferences and electrode fouling. In this paper, we show that carbon paste electrode (CPE) modified with NaY zeolite fulfils this requirements thanks to its hydrophilic surface. Oxidation of NADH at ferrocene (FcH) modified carbon paste electrode exhibits a rather slow electrocatalytic effect. We demonstrated the existence of synergetic effect on the electrocatalytic oxidation of NADH when the CPE is doped with zeolite (NaY) and FcH mediator or with the zeolite exchanged beforehand with the mediator (Y-Ferricinium, YFcH). This cumulative effect permits to reach high sensitivity for NADH detection and offers new way for the development of enzymatic biosensors using dehydrogenases depending on NADH as cofactor.     

Effect of different levels of NADH availability on metabolite distribution in <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation in minimal and complex media

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.     

Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent

The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.     

A study on the mechanism of the vanadate-dependent NADH oxidation

The mechanism of the vanadate (V(_v))-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V(_v)) generated by the direct V(_v)-dependent oxidation of NADH formed a complex with V(_v). In phosphate buffers V(_v) autoxidized instead of forming a complex with V(_v). The generated superoxide radical (O₂⁻) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V(_v)-dependent NADH oxidation catalyzed by O₂⁻-generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate.     

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh ^– strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh ^– mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal. Electronic Publication     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Electrochemical investigations of the reaction mechanism and kinetics between NADH and redox-active (NC)2C6H3-NHOH/(NC)2C6H3-NO from 4-nitrophthalonitrile-(NC)2C6H3-NO2-modified electrode

A simple and sensitive method for the electrocatalytic detection of NADH on a carbon paste electrode modified with a redox-active (NC)(2)C(6)H(3)-NO/(NC)(2)C(6)H(3)-NHOH (NOPH/NHOHPH) electrogenerated in situ from 4-nitrophthalonitrile (4-NPHN) is presented. The electrode modified with 4-NPHN showed an efficient electrocatalytic activity towards the oxidation of NADH with activation overpotential of 0.12V vs. Ag/AgCl. The formation of an intermediate charge transfer complex is proposed for the charge transfer reaction between NADH and the 4-NPHN-resulting system. The second-order rate constant for electrocatalytic oxidation of NADH, kappa(obs), and the apparent Michaelis-Menten constant K(M), at pH 7.0 were evaluated with rotating disk electrode (RDE) experiments, giving 1.0x10(4)mol(-1)Ls(-1) and 2.7x10(-5)molL(-1), respectively. Employing the Koutecky-Levich approach indicated that the NADH oxidation reaction involves two electrons. The sensor provided a linear response range for NADH from 0.8 up to 8.5mumolL(-1) with sensitivity, detection, quantification limits and time response of 0.50muALmumol(-1), 0.25mumolL(-1), 0.82mumolL(-1) and 0.1s, respectively. The repeatability of the measurements with the same sensor and different sensors, evaluated in terms of relative standard deviation, were 4.1 and 5.0%, respectively, for n=10.     

Effects of carbon–dioxide-bicarbonate mixtures on oxidative phosphorylation by cauliflower mitochondria

1. Carbon dioxide-bicarbonate mixtures markedly inhibited oxidation and phosphorylation rates of mitochondria prepared from cauliflower. Inhibition occurred with succinate, malate, citrate, isocitrate and NADH as substrates. 2. Indophenol-reductase systems with malate, succinate, isocitrate and NADH as substrates were inhibited by 5% and 15% carbon dioxide. Cytochrome c oxidase was not inhibited by 15% carbon dioxide.     

Electrocatalytic oxidation of NADH with Meldola's blue functionalized carbon nanotubes electrodes

Meldola's blue (MB) functionalized carbon nanotubes (CNT) nanocomposite film (MB/CNT) electrode was prepared by non-covalent adsorbing MB on the surface of a carbon nanotubes modified glassy carbon electrode (CNT/GCE). Electrochemical behaviors of the resulting electrode were investigated thoroughly with cyclic voltammetry in the potential range of -0.6 to 0.2V, and two well-defined redox couples were clearly visualized. We also studied the electron transfer kinetics of MB loaded on CNT (MB/CNT) in comparison with that of MB on conventional graphite powder (MB/GP). The heterogeneous electron transfer rate constant (k(s)) of MB/CNT was calculated to be about three times larger than that of MB/GP. The accelerated electron transfer kinetics was attributed to the unique electrical and nanostructural properties of CNT supports as well as the interaction between MB and CNT. In connection with the oxidation of nicotinamide adenine dinucleotide (NADH), excellent electrocatalytic activities were observed at MB/CNT/GCE compared with MB/GP modified glassy carbon electrode (MB/GP/GCE). Based on the results, a new NADH sensor was successfully established using the MB/CNT/GCE. Under a lower operation potential of -0.1V, NADH could be detected linearly up to a concentration of 500 microM with an extremely lower detection limit of 0.048+/-0.02 microM estimated at a signal-to-noise ratio of 3. Sensitivity, selectivity, reproducibility and stability of the NADH sensor were also investigated and the main analytical data were also compared with those obtained with the MB/GP/GCE.     

Isolated durum wheat and potato cell mitochondria oxidize externally added NADH mostly via the malate/oxaloacetate shuttle with a rate that depends on the carrier-mediated transport

We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH. We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle. In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane. No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the alpha-glycerophosphate/dihydroxyacetonphosphate shuttles. We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 microm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively). The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.     

The effect of dibromothymoquinone on respiratory and photosynthetic electron transport in Rhodopseudomonas capsulata chromatophores

Dibromothymoquinone has been shown to inhibit light-induced cytochrome b reduction, and oxidation of succinate and NADH by chromatophores of Rhodopseudomonas capsulata. The half-inhibitory concentration of light-induced reactions and NADH oxidation is 2.5 M, but of succinate oxidation is 16 M. Hexane extraction inhibited oxidation of NADH and succinate equally. The results are interpreted to suggest that ubiquinone is concerned in all three processes described, but that the pools associated with NADH and succinate oxidation are not equally accessible to dibromothymoquinone.Abbreviations DBMIB Dibromothymoquinone - NADH Reduced nicotinamide adenine dinucleotide - Bchl Bacteriochlorophyll     

External Pathway of NADH Oxidation in the Liver of the River Lamprey Lampetra fluviatilis

A possibility of exogenous NADH oxidation via the external pathway has been shown on homogenates and isolated liver cells of the lamprey Lampetra fluviatilis in the presence of rotenone and antimycin A. The homogenates were incubated in isotonic and hypotonic sucrose media, while cells, in isotonic salt medium. At incubating the tissue preparations in isotonic media, digitonin was used to enhance membrane permeability to NADH and cytochrome c. In homogenates, the maximal rate of NADH oxidation via the external pathway in the presence of cytochrome c and digitonin was 5.3 nmol O₂/min/10 mg wet weight. This value in the cells amounted to 12.6, while without addition of exogenous NADH and cytochrome c, to 11.0 nmol O₂/min/10 million cells. Cyanide inhibited completely the NADH oxidation via the external pathway both in homogenates and in cells. The intact lamprey hepatocytes, unlike homogenates, are suggested to contain sufficient concentrations of cytochrome c and extramitochondrial NADH to provide maximal NADH oxidation rate in mitochondria through external pathway. This allows thinking that potential possibilities of NADH oxidation via the external pathway in Cyclostomata and mammals are qualitatively and quantitatively close.     

The membrane potential in everted vesicles of Escherichia coli.

Ion-selective electrodes were used to measure the equilibration of thiocyanate across the membrane of everted (“inside-out”) vesicles of Escherichia coli W1485. Membrane potentials, vesicle interior positive, generated by the oxidation of NADH, succinate, and d-lactate, or by the hydrolysis of ATP, fell in the range of 100–150 mV depending on the carbon source for cell growth and the substrate used to energize the membranes. There was no relationship between the rate of oxidation of different substrates and the membrane potential they generated. The membrane potential generated by oxidation of NADH was relatively constant between pH 7.0 and 8.5. Somewhat lower values obtained at pH 5.5 to 6.5 were attributed to the effect of pH on substrate oxidation.     

The enzymic reduction of glyoxylate and hydroxypyruvate in leaves of higher plants

Glyoxylate and hydroxypyruvate are metabolites involved in the pathway of carbon in photorespiration. The chief glyoxylate-reducing enzyme in leaves is now known to be a cytosolic glyoxylate reductase that uses NADPH as the preferred cofactor but can also use NADH. Glyoxylate reductase has been isolated from spinach leaves, purified to homogeneity, and characterized kinetically and structurally. Chloroplasts contain lower levels of glyoxylate reductase activity supported by both NADPH and NADH, but it is not yet known whether a single chloroplastic enzyme catalyzes glyoxylate reduction with both cofactors. The major hydroxypyruvate reductase activity of leaves has long been known to be a highly active enzyme located in peroxisomes; it uses NADH as the preferred cofactor. To a lesser extent, NADPH can also be used by the peroxisomal enzyme. A second hydroxypyruvate reductase enzyme is located in the cytosol; it preferentially uses NADPH but can also use NADH as cofactor. In a barley mutant deficient in peroxisomal hydroxypyruvate reductase, the NADPH-preferring cytosolic form of the enzyme permits sufficient rates of hydroxypyruvate reduction to support continued substrate flow through the terminal stages of the photosynthetic carbon oxidation (glycolate/glycerate) pathway. The properties and metabolic significance of the cytosolic and organelle-localized glyoxylate and hydroxypyruvate reductase enzymes are discussed.