Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.     

The effect of NAPRTase overexpression on the total levels of NAD,the NADH/NAD+ ratio,and the distribution of metabolites in Escherichia coli

Escherichia coli (E. coli) maintains its total NADH/NAD+ intracellular pool by synthesizing NAD through the de novo pathway and the pyridine nucleotide salvage pathway. The salvage pathway recycles intracellular NAD breakdown products and preformed pyridine compounds from the environment, such as nicotinic acid (NA). The enzyme nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11), encoded by the pncB gene, catalyzes the formation of nicotinate mononucleotide (NAMN), a direct precursor of NAD, from NA. This reaction is believed to be the rate-limiting step in the NAD salvage pathway. The current study investigates the effect of overexpressing the pncB gene from Salmonella typhimurium on the total levels of NAD, the NADH/NAD+ ratio, and the production of different metabolites in E. coli under anaerobic chemostat conditions and anaerobic tube experiments. In addition, this paper studies the effect of combining the overexpression of the pncB gene with an NADH regeneration strategy that increases intracellular NADH availability, as we have previously shown. (The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, 230-237; Metabolic engineering of Escherichia coli: Increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229.) Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, overexpression of the pncB gene led to a significant shift in the metabolic patterns as evidenced by a decrease in lactate production and an increase as high as two-fold in the ethanol-to-acetate (Et/Ac) ratio. These results suggest that under chemostat conditions the total level of NAD is not limiting and the metabolic rates are fixed by the system at steady state. On the other hand, under transient conditions (such as those in batch cultivation) the increase in the total level of NAD can increase the rate of NADH-dependent pathways (ethanol) and therefore change the final distribution of metabolites. The effect of combining overexpression of the pncB gene with the substitution of the native cofactor-independent formate dehydrogenase (FDH) with an NAD(+)-dependent FDH was also investigated under anaerobic tube conditions. This manipulation produced a metabolic pattern that combines a high Et/Ac ratio similar to that obtained with the new FDH with an intermediate lactate level similar to that obtained with the overexpression of the pncB gene. It was found that addition of the pncB gene to the FDH system does not increase further the production of reduced metabolites because the system for NADH regeneration already reached the maximum theoretical yield of approximately 4 mol NADH/mol of glucose.     

Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning.

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.     

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2)

We describe the identification of Rex, a novel redox-sensing repressor that appears to be widespread among Gram-positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. The DNA-binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA-binding activity of Rex. However, NAD+ competes with NADH for Rex binding, allowing Rex to sense redox poise over a range of NAD(H) concentrations. Rex is predicted to include a pyridine nucleotide-binding domain (Rossmann fold), and residues that might play key structural and nucleotide binding roles are highly conserved. In support of this, the central glycine in the signature motif (GlyXGlyXXGly) is shown to be essential for redox sensing. Rex homologues exist in most Gram-positive bacteria, including human pathogens such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts.

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli.

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.     

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.     

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.     

Quantum dots as nano plug-in's for efficient NADH resonance energy routing

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD(+)/NADH with QDs was used to monitor the reaction of NAD(+)-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000-0.01 ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD(+) and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028-0.4 U). Hence, it was possible to detect formaldehyde in the range 1000-0.01 ng/mL with a limit of detection (LOD) at 0.01 ng/mL and regression coefficient R(2)=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD(+)-dependent enzymes and detection of dehydrogenases in general.     

Complexes of NADH with betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus L

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD(+) adding first. NADH is a noncompetitive inhibitor against NAD(+), which was interpreted before as evidence of an iso mechanism, in which NAD(+) and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD(+). Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD(+).NADH would produce an uncompetitive effect in the inhibition of NADH against NAD(+), the iso mechanism cannot be conclusively discarded.