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.     

Whole-cell bioreduction of aromatic α-keto esters using <Emphasis Type="Italic">Candida tenuis</Emphasis> xylose reductase and <Emphasis Type="Italic">Candida boidinii</Emphasis> formate dehydrogenase co-expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP⁺ or NAD⁺ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR.     

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.     

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.     

Physiological and quantitative proteomic analyses unraveling potassium deficiency stress response in alligator weed (<Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> L.) root

Key message

KEG is involved in mediating the proteasome-dependent degradation of FDH, a stress-responsive enzyme. The UPS may function to suppress FDH mediated stress responses under favorable growth conditions.

Abstract

Formate dehydrogenase (FDH) has been studied in bacteria and yeasts for the purpose of industrial application of NADH co-factor regeneration. In plants, FDH is regarded as a universal stress protein involved in responses to various abiotic and biotic stresses. Here we show that FDH abundance is regulated by the ubiquitin proteasome system (UPS). FDH is ubiquitinated in planta and degraded by the 26S proteasome. Interaction assays identified FDH as a potential substrate for the RING-type ubiquitin ligase Keep on Going (KEG). KEG is capable of attaching ubiquitin to FDH in in vitro assays and the turnover of FDH was increased when co-expressed with a functional KEG in planta, suggesting that KEG contributes to FDH degradation. Consistent with a role in regulating FDH abundance, transgenic plants overexpressing KEG were more sensitive to the inhibitory effects of formate. In addition, FDH is a phosphoprotein and dephosphorylation was found to increase the stability of FDH in degradation assays. Based on results from this and previous studies, we propose a model where KEG mediates the ubiquitination and subsequent degradation of phosphorylated FDH and, in response to unfavourable growth conditions, reduction in FDH phosphorylation levels may prohibit turnover allowing the stabilized FDH to facilitate stress responses.

     

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost‐effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co‐immobilized them on modified poly‐p‐hydroxybutyrate synthase (PhaC)‐inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled‐coil interaction. Enzyme‐loaded particles could be easily purified by centrifugation. Total conversion of 4'‐chloroacetophenone to (S)‐4‐chloro‐α‐methylbenzyl alcohol could be accomplished using enzyme‐loaded particles, catalytic amounts of NAD⁺ and formate as substrates for FDH. Chiral GC‐MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost‐effective alternative to coupled reactions using purified enzymes.     

Mechanistic model for prediction of formate dehydrogenase kinetics under industrially relevant conditions

Formate dehydrogenase (FDH) from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme‐catalyzed reductions. The mathematical model that is currently applied to predict progress curves during (semi‐)batch reactions has been derived from initial rate studies. Here, it is demonstrated that such extrapolation from initial reaction rates to performance during a complete batch leads to considerable prediction errors. This observation can be attributed to an invalid simplification during the development of the literature model. A novel mechanistic model that describes the course and performance of FDH‐catalyzed NADH regeneration under industrially relevant process conditions is introduced and evaluated. Based on progress curve instead of initial reaction rate measurements, it was discriminated from a comprehensive set of mechanistic model candidates. For the prediction of reaction courses on long time horizons (>1 h), decomposition of NADH has to be considered. The model accurately describes the regeneration reaction under all conditions, even at high concentrations of the substrate formate and thus is clearly superior to the existing model. As a result, for the first time, course and performance of NADH regeneration in industrial enzyme‐catalyzed reductions can be accurately predicted and used to optimize the cost efficiency of the respective processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co²⁺ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD⁺ with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.     

Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-dependent formate dehydrogenase

The NAD⁺-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.     

Growth and recombinant protein expression with <Emphasis Type="Italic">Escherichia coli</Emphasis> in different batch cultivation media

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L⁻¹. EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L⁻¹. The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.     

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.     

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO₂) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO₂ for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO₂ and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO₂ into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO₂ reduction. Structural analysis of the enzyme through homology modeling is also presented.     

Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst

Abstract

The oxygen-tolerant NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 has been described as a promising catalyst for cofactor regeneration in biocatalysed reductions. In this study, the actual potential of this enzyme for application in technical synthesis was evaluated. An overproduced, purified version of the enzyme was coupled to the carbonyl reductase from Candida parapsilosis (CPCR), where it allowed an almost quantitative conversion of the model substrate; total turnover numbers (TTN: n_product/n_enzyme) of up to 143,666 were achieved. This was distinctly superior to the commonly used NADH regenerating enzyme formate dehydrogenase (FDH) from Candida boidinii. In a systematic quantitative approach, maximum activity for NAD⁺ reduction was observed at 35 °C and pH 8, which corresponds to that of native SH. The half-life of the enzyme under these conditions was 5.3 hours. In the presence of sodium salts, distinct inhibitory effects were observed while ammonium and potassium ions increased the enzyme stability. Overall, a high but not unusual sensitivity of SH for changes in temperature, pH and mechanical stress in a reactor was found. Technical application in chemical synthesis can therefore be considered a feasible goal.     

Construction and evaluation of a novel bifunctional phenylalanine–formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration

Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of l-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of l-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH–FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5–9.0). The bifunctional enzyme can produce 153.9 mM l-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to l-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH–FDH with coenzyme regeneration for phenylpyruvic acid analysis and l-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field.     

Multi‐enzymatic one‐pot reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid with whole‐cell biocatalysts

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non‐surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven‐step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo‐enzymatic preparation of UDCA—the two‐step reduction of dehydrocholic acid (DHCA) to 12‐keto‐ursodeoxycholic acid using a mutant of 7β‐hydroxysteroid dehydrogenase (7β‐HSDH) from Collinsella aerofaciens and 3α‐hydroxysteroid dehydrogenase (3α‐HSDH) from Comamonas testosteroni. Three different one‐pot reaction approaches were investigated using whole‐cell biocatalysts in simple batch processes. We applied one‐biocatalyst systems, where 3α‐HSDH, 7β‐HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two‐biocatalyst systems, where 3α‐HSDH and 7β‐HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one‐pot reaction. The best result was achieved by the one‐biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12‐keto‐UDCA within 4.5 h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 68–77. © 2012 Wiley Periodicals, Inc.     

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF₆]: Butyl-methyl-imidazoliumhexafluorophosphate     

Biological nitrogen and organic matter removal from tannery wastewater in pilot plant operations in Ethiopia

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of d-fructose to d-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce d-mannitol from d-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form d-mannitol from d-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up d-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM d-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD⁺-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y _{D-mannitol/D-fructose}was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.     

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.     

Improved synthesis of chiral alcohols with <Emphasis Type="Italic">Escherichia coli</Emphasis> cells co-expressing pyridine nucleotide transhydrogenase,NADP<Superscript>+</Superscript>-dependent alcohol dehydrogenase and NAD<Superscript>+</Superscript>-dependent formate dehydrogenase

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD⁺ and NADPH. The pnta and pntb genes encoding for the - and -subunits were cloned and co-expressed with NADP⁺-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.     

A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate

To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.