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.     

NAD+ regeneration in a microreactor using permeabilized baker’s yeast cells

NAD(P)-dependent oxidoreductases represent a great interest in the field of biotechnology and biotransformation. Although they have many advantages, the biggest drawback and limitation of oxidoreductase usage is the price of the coenzymes. In order to solve this problem, many in situ methods for regeneration of coenzymes have been studied and developed. Unfortunately, although results indicate that those methods are suitable for regeneration procedure, most of the processes need additional optimization to make them more sustainable. As an alternative, microreactor technology could be used as a new technique for coenzyme regeneration processes due to many advantages.In this study regeneration of coenzyme NAD⁺ was carried out in a microreactor by acetaldehyde reduction to ethanol using enzyme alcohol dehydrogenase (ADH). Suspended and immobilized whole permeabilized baker’s yeast cells were used as the source of the ADH enzyme. A 65.3% conversion of NADH was achieved with suspended permeabilized baker’s yeast cells for a residence time of τ = 36 s and equimolar concentration of substrates (c_i,NADH = 5.5 mmol/dm³, c_{i,acetaldehyde} = 5.5 mmol/dm³). When working with immobilized cells, conversion achieved for the same residence time was 10 fold lower. When permeabilized baker’s yeast cells were used for coenzyme regeneration process was stabile for 6 days of continuous operation which makes this system a good alternative for coenzyme regeneration.     

Cloning,overexpression, purification,and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD⁺ as a cofactor. The recombinant caXDH has a K_M of 8.8 mM and 37.7 μM using the substrate xylitol and NAD⁺, respectively, and its catalytic efficiency is 53,200 min^?1 mM^?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD⁺ to NADP⁺ in which the K_M and k_cat/K_M towards NADP⁺ are 119 μM and 26,200 min^?1 mM^?1, respectively.     

Characterization of methylated azopyridine as a potential electron transfer mediator for electroenzymatic systems

N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD⁺- and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2 ± 0.1) × 10⁵ M⁻¹ s⁻¹, whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7 ± 0.1) × 10⁴ M⁻¹ s⁻¹. The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD⁺-dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3 × 10³. In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield l-lactic acid.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

Ethanol/O2 biofuel cell using a biocathode consisting of laccase/ HOOC-MWCNTs/polydiallyldimethylammonium chloride

In the present report we focused on the substitution of metallic catalysts by biocatalysts to develop a high efficient biofuel cell. A bioanode and a biocathode were designed using ADH and laccase, respectively. Carboxylated multiwall carbon nanotubes (HOOC-MWCNTs) and polydiallyldimethylammonium chloride (PDDA) were used for immobilizing the enzymes on either polymethylene green (PMG) modified glassy carbon or graphite electrodes. In this way, an ethanol–oxygen biofuel cell was designed in which PDDA/ADH/PDDA/HOOC-MWCNTs/PMG/GC and PDDA/Lac/PDDA/HOOC-MWCNTs/PMG/Gr operated as bioanode and biocathode, respectively. In the optimized condition of O₂ saturated PBS (0.1 M, pH 7.5) containing 1 mM ethanol and 1 mM NAD⁺ the open-circuit voltage reached to a plateau at 504 mV based of which the power density of 3.98 mW cm⁻² was obtained.     

Effects of redox potential control on succinic acid production by engineered Escherichia coli under anaerobic conditions

The effects of oxido-reduction potential (ORP) control on succinic acid production have been investigated in Escherichia coli LL016. In LL016, two CO₂ fixation pathways were achieved and NAD⁺ supply was enhanced by co-expression of heterologous pyruvate carboxylase (PYC) and nicotinic acid phosphoribosyltransferase (NAPRTase). During anaerobic fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of −200 to −400 mV. From the results, the ORP level of −400 mV was preferable, which resulted in the high succinic acid concentration (28.6 g/L) and high succinic acid productivity (0.33 g/L/h). Meanwhile, the yield of succinic acid at the ORP level of −400 mV was 39% higher than that at the ORP level of −200 mV. In addition, a higher NADH/NAD⁺ ratio and increased enzyme activities were also achieved by regulating the culture to a more reductive environment, which further enhanced the succinic acid production.     

Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD⁺-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L⁻¹ d⁻¹, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.     

Development of a multi-enzymatic desymmetrization and its application for the biosynthesis of l-norvaline from dl-norvaline

Perindopril is an effective antihypertensive drug in strong demand used to treat hypertension. l-norvaline is a vital intermediate of Perindopril production mainly produced by chemical synthesis with low purity. We developed an environmentally friendly method to produce l-norvaline with high purity based on a desymmetrization process. d-Norvaline was oxidized to the corresponding keto acid by d-amino acid oxidase from the substrate dl-norvaline. Asymmetric hydrogenation of the keto acid to l-norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD⁺. A NADH regeneration system was introduced by overexpressing a formate dehydrogenase. The unwanted H₂O₂ by-product generated during d-norvaline oxidation was removed by adding catalase. A total of 54.09 g/L of l-norvaline was achieved, with an enantiomeric excess over 99% under optimal conditions, with a 96.7% conversion rate. Our desymmetrization method provides an environmental friendly strategy for the production of enantiomerically pure l-norvaline in the pharmaceutical industry.     

Characterization of a highly thermostable ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824

Higher energy content and hydrophobicity make bio-based n-butanol a preferred building block for chemical and biofuels manufacturing. Butanol is obtained by Clostridium sp. based ABE fermentation process. While the ABE process is well understood, the enzyme systems involved have not been elucidated in detail. The important enzyme ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824 (Hbd) was purified and characterized. Surprisingly, Hbd shows extremely high temperature (T > 60 °C), pH (4–11) and solvent (1-butanol, isobutanol, ethanol) stability. Hbd catalyzes acetoacetyl CoA hydration to ß-hydroxybutyryl CoA up to pH 9.5, where the reaction is reversed. Substrate (acacCoA, ß-hbCoA) and cofactor (NADH, NAD⁺, NADPH and NADP⁺) specificities were determined. We identified NAD⁺ as an uncompetitive inhibitor. Identification of process relevant enzymes such as Hbd is key to optimize butanol production via cellular or cell-free enzymatic systems.     

Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD⁺, and a high titer of 371 mM (32 g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361 mM (32 g/L) R-BDO with a yield of 81% or 365 mM (33 g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.     

Effects of culture redox potential on succinic acid production by Corynebacterium crenatum under anaerobic conditions

During mixed-acid fermentation by Corynebacterium crenatum under anaerobic conditions, two moles of NADH are required to synthesize 1 mol of succinic acid. In this work, four controlled culture redox potentials and different carbon sources with different oxidation states were used to investigate the possibility of enhancing the succinic acid production by increasing the availability of NADH. When the culture redox potential was ?300 mV, the yield of succinic acid was 0.31 g/g, representing a 72% increase compared with the yield when the culture redox potential was ?40 mV. Meanwhile, the molar ratio of succinic acid/lactic acid increased from 0.27 to 0.48. When 0.1% neutral red was added to the acid production medium, the yield of succinic acid was 0.25 g/g, and the molar ratio of succinic acid/lactic acid was 0.38. Both values were higher than those obtained from glucose only (0.19 g/g, 0.26) or gluconate (0.05 g/g, 0.18). A higher NADH/NAD⁺ ratio and increased enzymatic activity could be achieved to enhance the succinic acid production by manipulating the culture to a more reductive environment.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

pH-rate profiles of l-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in l-xylulose production

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD⁺, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (k_cat) and the catalytic efficiency (k_cat/K_m) were 4200 min⁻¹ and 290 mM⁻¹ min⁻¹, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.     

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.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

The benzimidazole based drugs show good activity against T. gondii but poor activity against its proposed enoyl reductase enzyme target

The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD⁺ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD⁺ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC₅₀ for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.     

Improvement of acetoin reductase activity enhances bacitracin production by Bacillus licheniformis

Bacitracin fermentation by Bacillus licheniformis in this work showed three characteristics: (1) the extracellular propionate, butyrate, acetoin and 2,3-butanediol accumulates under conditions of low dissolved oxygen (zero after 4 h cultivation), reaching a total content of approximately 11.1 g/L; (2) cell growth occurs quickly subsequent to cell autolysis and the second growth; and (3) there is a low content of 2,3-butanediol, a reduced product of acetoin catalyzed by acetoin reductase, in the culture process. In this study, addition of MnCl₂ (0.3 mg/L) to the production medium increased the acetoin reductase activity, redirected the NADH oxidation partly from the propionate- and butyrate-production pathways to the 2,3-butanediol synthesis pathway, reduced the intracellular NADH/NAD⁺ ratio, and facilitated cell growth, ultimately achieving a 11.6% increase in bacitracin production (1076 U/mL) versus the control. The results provide useful information regarding large-scale bacitracin production by B. licheniformis.     

Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: A proof of concept

Dihydroxyacetone (DHA) has numerous industrial applications. In this work, we pursue the idea to produce DHA from sugars in the yeast Saccharomyces cerevisiae, via glycerol as an intermediate. Firstly, three glycerol dehydrogenase (GDH) genes from different microbial sources were expressed in yeast. Among them, the NAD⁺-dependent GDH of Hansenula polymorpha showed the highest glycerol-oxidizing activity. DHA concentration in shake-flask experiments was roughly 100 mg/l DHA from 20 g/l glucose, i.e. five times the wild-type level. This level was achieved only when cultures were subjected to osmotic stress, known to enhance glycerol production and accumulation in S. cerevisiae. Secondly, DHA kinase activity was abolished to prevent conversion of DHA to dihydroxyacetone phosphate (DHAP). The dak1Δdak2Δ double-deletion mutant overexpressing H. polymorpha gdh produced 700 mg/l DHA under the same conditions. Although current DHA yield and titer still need to be optimized, our approach provides the proof of concept for producing DHA from sugars in yeast.     

Heterologous production of extreme alkaline thermostable NAD+-dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila

NAD⁺-dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD⁺ or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile (MtFDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli, as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that MtFDH exhibits higher catalytic efficiency on NaHCO₃ compared to formate. Notable, recombinant MtFDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with T_m 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that MtFDH possess a huge potential for CO₂ reduction or NADH generation and under extreme alkaline conditions.