Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

On the significance of electron transport systems for growth of Rhodospirillum rubrum

The inhibitory effects of 2-hydroxybiphenyl on various electron transport reactions of isolated membranes and growth in the presence of malate of either phototrophic or chemotrophic cells of Rhodospirillum rubrum were studied. 50% inhibition of both oxygen uptake of whole cells and growth under chemotrophic conditions (i.e. aerobiosis in the dark) was achieved in the presence of 0.09 mM 2-hydroxybiphenyl. With isolated membranes the same effect on NADH oxidase was obtained with 0.08 mM of inhibitor. Succinate dependent respiratory reactions were inhibited by 50% at a concentration of 0.36 mM. Growth under phototrophic conditions (i.e. anaerobiosis in the light) was inhibited by 50% in the presence of 0.17 mM (wild type strain) or 0.21 mM (blue-green mutant, strain VI) of 2-hydroxybiphenyl. Photophosphorylation and light dependent NAD⁺ reduction by succinate were inhibited by 50% at concentrations of 0.21 mM and 0.03 mM of inhibitor, respectively. After phototrophic growth of the organisms for about five doublings of cell mass in the presence of 0.18 mM of 2-hydroxybiphenyl coloured carotenoids could no longer be detected. Membrane fractions of such cultures exhibited normal activities of succinate cytochrome c reductase but activities of NADH cytochrome c reductase were decreased by 80%. In comparison with a blue green mutant, strain VI, of R. rubrum light induced absorbance changes at 865 nm as well as activities of photophosphorylation were unaffected. However, no activity of light dependent NAD⁺ reduction with succinate could be detected. The data indicate that cellular respiration as well as chemotrophic growth depend largely on NADH dependent respiration. Phototrophic growth, on the other hand, is limited by photophosphorylation while energy dependent reversed electron flow to NAD⁺, if at all, is of rathe minor importance.Abbreviation BChl bacteriochlorophyll     

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.     

Production of nadh by microbial reduction of added nad

Summary Of thirteen bacterial strains and four strains of yeast-like organisms, permeabilized cells of two bacterial and one yeast strain effectively converted added NAD⁺ into NADH in the presence of glucose as substrate.Arthrobacter ureafaciens reduced more than 90% of 10 mM NAD⁺ into NADH during 1h.     

Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction

The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH APAD⁺ transhydrogenase reactions and energy-linked NAD⁺ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K _i = 1 M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K _i = 5 M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD⁺. The type of the Tinopals' effect on the NADH APAD⁺ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD⁺ and NADH, correspondingly). The competition between 5BM-GX and APAD⁺ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD⁺ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH APAD⁺ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD⁺ in the reverse electron transfer reaction.     

Effect of monovalent cations on the inhibition by NAD+ of NADH oxidation in submitochondrial particles.

Oxidation of NADH in submitochondrial particles, with O₂ or ferricyanide as electron acceptor, was inhibited by micromolar concentrations of NAD⁺ when measured in 240 mM sucrose or, in a lesser extent, in 120 mM NaCl or LiCl. In 120 mM solutions of either KCl, RbCl, CsCl or NH₄Cl the inhibition by up to 100 μM concentrations of NAD⁺ did not occur. The inhibition observed in the sucrose medium disappeared after solubilization of the particles with detergents and re-appeared when the membranes were reconstituted. The inhibitory effect was potentiated by palmitoyl-CoA. The possibility is discussed that the inhibition of NADH oxidation by low concentrations of NAD⁺ and its release by K⁺, Rb⁺, Cs⁺ and NH₄⁺ depend on the interaction between NAD⁺ and the negatively charged mitochondrial membrane.     

Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D‐proline reductase (PR), a Stickland reaction that regenerates NAD⁺ from NADH. Many Clostridium spp. use Stickland metabolism (co‐fermentation of pairs of amino acids) to generate ATP and NAD⁺. Synthesis of PR is activated by PrdR, a proline‐responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD⁺‐generating pathways, such as the glycine reductase and succinate‐acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox‐sensing regulator that responds to the NAD⁺/NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD⁺ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR‐regulated Stickland metabolism in the virulence of C. difficile.     

A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

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.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Optimization of the mediated electrocatalytic reduction of NAD+ by cyclic voltammetry and construction of electrochemically driven enzyme bioreactor

The optimal concentrations of diaphorase, methyl viologen (MV²⁺) and NAD⁺ in the mediated electrocatalytic reduction of NAD⁺ were decided by applying cyclic voltammetry. The steady-state catalytic current was achieved under the conditions of 1.5 U diaphorase ml^–1, 0.2 mM MV²⁺, and 4.8 mM NAD⁺ at the scan rate of 2 mV s^–1, suggesting that the rate of the electrocatalytic reaction is the highest under the former conditions. However, NAD⁺ was effective at 0.3 mM as it was at 4.8 mM when the electrocatalysis is coupled with an enzymatic synthesis requiring NADH. In effect, the electrochemical procedure under the conditions of 1.5 U diaphorase ml^–1, 0.2 mM MV²⁺, and 0.3 mM NAD⁺ worked satisfactorily to drive an enzymatic reduction of pyruvate to d-lactate in the presence of d-lactate dehydrogenase.     

Electrochemical characterization of immobilized NAD+

Nicotinamide adenine dinucleotide (NAD⁺) has been covalently attached to alginic acid using carbodiimide coupling, thereby producing a macromolecular adduct of NAD, which can be rendered either soluble or insoluble by adjustment of pH. It was found that this NAD⁺ · alginic acid complex was enzymatically active, and also that the oxidized form could be electrochemically reduced without loss in enzymatic activity. This NAD⁺ adduct has now also been polarographically characterized as to its two-step reduction waves, which are slightly shifted toward more cathodic potential as compared to free NAD⁺. When controlled electrolysis was conducted to reduce the bound NAD⁺ at the cathode, the NADH so formed by electrochemical action was found to be again oxidizable either enzymatically or electrochemically without loss in co-enzymic function. The NADH adduct produced by electrochemical reduction of the NAD⁺ adduct has also been characterized by voltammetry.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.     

Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD⁺-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B₁₂, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD⁺ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD⁺ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD⁺ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.