Enzymatic Cofactor Regeneration Research Articles

An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.

Co-Cross-Linked Aggregates of Baeyer–Villiger Monooxygenases and Formate Dehydrogenase for Repeated Use in Asymmetric Biooxidation

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts, but their applications are hindered by their poor stability and cofactor dependence. In this study, cross-linked enzyme aggregate (CLEA) technology was adopted to coimmobilize BVMO and its accessory cofactor-regeneration enzyme. Combi-CLEAs of a pyrmetazole monooxygenase from Acinetobacter calcoaceticus (AcPSMO) and a formate dehydrogenase from Burkholderia stabili (BstFDH) were prepared for the synthesis of (S)-omeprazole. After optimization, AcPSMO and BstFDH were coprecipitated with an activity ratio of 1:6 using ammonium sulfate and then cross-linked with glutaraldehyde (0.12% w/v). The activity recoveries of AcPSMO and BstFDH in the prepared combi-CLEAs were 43% and 38%, respectively. Compared with the free enzymes AcPSMO and BstFDH, the thermostabilities of AcPSMO and BstFDH in combi-CLEAs were improved by 2.5- and 1.6-fold, respectively. Both enzymes were more stable against alkaline buffer after being immobilized. The combi-CLEAs could be reused for seven cycles in the biooxidative synthesis of (S)-omeprazole without significant activity loss, indicating the excellent operational stability and reusability in repeated reactions for the enzymatic synthesis of (S)-omeprazole. Another two combi-CLEAs prepared under the same conditions, TmCHMO–BstFDH and RpBVMO–BstFDH, can be reused for at least 15 consecutive batches for the cyclohexanone mono-oxygenation reaction, which indicates the promising potential for coimmobilization of BVMOs and FDH with CLEA methodology.

Deep Eutectic Solvents as Smart Cosubstrate in Alcohol Dehydrogenase-Catalyzed Reductions

Alcohol dehydrogenase (ADH) catalyzed reductions in deep eutectic solvents (DESs) may become efficient and sustainable alternatives to afford alcohols. This paper successfully explores the ADH-catalyzed reduction of ketones and aldehydes in a DES composed of choline chloride and 1,4-butanediol, in combination with buffer (Tris-HCl, 20% v/v). 1,4-butanediol (a DES component), acts as a smart cosubstrate for the enzymatic cofactor regeneration, shifting the thermodynamic equilibrium to the product side. By means of the novel DES media, cyclohexanone reduction was optimized to yield maximum productivity with low enzyme amounts (in the range of 10 g L−1 d−1). Notably, with the herein developed reaction media, cinnamaldehyde was reduced to cinnamyl alcohol, an important compound for the fragrance industry, with promising high productivities of ~75 g L−1 d−1.

Round, round we go - strategies for enzymatic cofactor regeneration.

Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic organic chemistry. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogues have been synthesised that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the production of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogues, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.

Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase

AbstractThe monoterpenoid α‐isophorone is sourced from the available and renewable plant dry matter, as well as a waste recovery operation from acetone. This compound, can be hydroxylated to 4‐hydroxy‐isophorone which is the main precursor for the synthesis of ketoisophorone. On its turn, ketoisophorone is a key intermediate for the production of carotenoids and Vitamin E. Here, the enzymatic oxidation of 4‐hydroxy‐isophorone to ketoisophorone is demonstrated employing an alcohol dehydrogenase (ADHaa) from Artemisia annua and a NADPH oxidase (NOX), as a cofactor regeneration enzyme. After 24 h of reaction and an initial substrate concentration of 50 mM, 95.7 % yield and a space time yield of 6.52 g L−1 day−1 could be obtained. Furthermore, the immobilization of the alcohol dehydrogenase was studied on 17 different supports. An epoxy‐functionalized agarose resulted in the highest metrics, 100±0% immobilization yield and 58.2±3.5 % retained activity. Finally, the immobilized ADHaa was successfully implemented in 4 reaction cycles (96 h operation) presenting a biocatalyst yield of 23.4 g product g−1 of enzyme. It represents a 2.5‐fold increase compared with the reaction with soluble enzymes.

Trimethyl-ε-caprolactone synthesis with a novel immobilized glucose dehydrogenase and an immobilized thermostable cyclohexanone monooxygenase

An often associated drawback with Baeyer-Villiger monooxygenases, is its poor operational stability. Furthermore, these biocatalysts frequently suffer from substrate/product inhibition.In this work, a thermostable cyclohexanone monooxygenase (TmCHMO) was immobilized and used in the synthesis of trimethyl-ε-caprolactone (CHL). As a cofactor regeneration enzyme, a novel and highly active glucose dehydrogenase (GDH-01) was used immobilized for the first time. MANA-agarose was the carrier chosen since it presented an immobilization yield of 76.3 ± 0.7% and a retained activity of 62.6 ± 2.3%, the highest metrics among the supports tested.Both immobilized enzymes were studied either separately or together in six reaction cycles (30 mL; [substrate] =132.5 mM). A biocatalyst yield of 37.3 g g−1 of TmCHMO and 474.2 g g−1 of GDH-01 were obtained. These values represent a 3.6-fold and 1.9-fold increase respectively, compared with a model reaction where both enzymes were used in its soluble form.

An Enzymatic 2-Step Cofactor and Co-Product Recycling Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design.

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2‐diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co‐substrate is oxidized to the respective co‐product that accumulates in equal concentration to the desired target product. Co‐product removal during the course of the reaction shifts the reaction towards formation of the target product and minimizes undesired side effects. Here we describe an atom efficient enzymatic cofactor regeneration system where the co‐product of the ADH is recycled as a substrate in another reaction set. A 2‐step enzymatic cascade consisting of a thiamine diphosphate (ThDP)‐dependent carboligase and an alcohol dehydrogenase is presented here as a model reaction. In the first step benzaldehyde and acetaldehyde react to a chiral 2‐hydroxy ketone, which is subsequently reduced by to a 1,2‐diol. By choice of an appropriate co‐substrate (here: benzyl alcohol) for the cofactor regeneration in the alcohol dehydrogenases (ADH)‐catalyzed step, the co‐product (here: benzaldehyde) can be used as a substrate for the carboligation step. Even without any addition of benzaldehyde in the first reaction step, this cascade design yielded 1,2‐diol concentrations of >100 mM with optical purities (ee, de) of up to 99%. Moreover, this approach overcomes the low benzaldehyde solubility in aqueous systems and optimizes the atom economy of the reaction by reduced waste production. The example presented here for the 2‐step recycling cascade of (1R,2R)‐1‐phenylpropane‐1,2‐diol can be applied for any set of enzymes, where the co‐products of one process step serve as substrates for a coupled reaction.

Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation.

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogenation. However, the reduced cofactor flavin adenine dinucleotide (FADH2) has to be regenerated using a flavin reductase. Consequently, coupled biocatalytic steps are usually applied for cofactor regeneration. Nicotinamide adenine dinucleotide (NADH) mimics can be employed stoichiometrically to replace enzymatic cofactor regeneration in biocatalytic halogenation. Chlorination of l-tryptophan is successfully performed using such NADH mimics. The efficiency of this approach has been compared to the previously established enzymatic regeneration system using the two auxiliary enzymes flavin reductase (PrnF) and alcohol dehydrogenase (ADH). The reaction rates of some of the tested mimics were found to exceed that of the enzymatic system. Continuous enzymatic halogenation reaction for reaction scale-up is also possible.

Co‐immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self‐sufficient oxidative biocatalyst

AbstractBACKGROUNDThe oxy‐functionalization of non‐activated carbon bonds by the bacterial cytochrome P450 BM3 from Bacillus megaterium, presents a promising field in biosynthesis and it has gained much interest in recent decades. Nevertheless, the need for the expensive cofactor NADPH, together with low operational stability of the enzyme have made the implementation of this biocatalyst unfeasible in most cases for industry.RESULTSP450 BM3 and glucose dehydrogenase (GDH), as a cofactor regeneration enzyme, were successfully co‐immobilized obtaining a bi‐functional self‐sufficient oxidative biocatalyst. First, a broad screening of 13 different supports was carried out. Five selected agaroses with three different functionalities (epoxy, amine and aldehyde) were studied and their immobilization processes optimized. Finally, P450 BM3 and GDH were co‐immobilized on those supports showing the best performance for P450 BM3 immobilization: epoxy‐agarose (epoxy‐agarose‐UAB) presenting 83% and 20% retained activities respectively; AMINO‐agarose presenting 28% and 25%, and Lentikats® with which both enzymes retained 100% of the initial activity. Furthermore, the re‐utilization of the self‐sufficient immobilized derivatives was tested in five repeated cycles.CONCLUSIONSP450 BM3 and GDH have been successfully immobilized on three supports and their re‐usability has been tested in a model reaction. It represents a step forward for future P450 BM3 industrial implementations. © 2018 Society of Chemical Industry

Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration.

Enzymatic reduction of carbon dioxide (CO2 ) to methanol (CH3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3 OH, a TTN of 160 and BPR of 24 μmol CH3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency.

Kinetics of batch-wise enzymatic cycling system for mass production of chiral compound

Kinetics of batch-wise enzymatic cycling system (oxidoreductase-catalyzed reaction system involving enzyme-coupled cofactor regeneration) has been studied covering a broad range of the conserved total cofactor concentration, [C]0 (=NAD(P)+ + NAD(P)H), based on reasonable several assumptions. It is composed of two elementary reactions, i.e. product synthesis reaction and cofactor regeneration reaction, both of which have been expressed by Michaelis–Menten type rate equations. A novel dimensionless variable, r, has been introduced, which is defined as the concentration of one of the two cofactor components, [X] (NADH+ or NADPH+), divided by [C]0, i.e. r .e[X]/[C]0. The following results have been obtained. (1) The fundamental equation of the batch-wise enzymatic cycling system has been transformed to a differential equation whose formula is: dr/dT = N(r)/D(r) (N(r) and D(r) are quadratic equations of r having different coefficients). (2) It has been elucidated that the batch-wise enzymatic cycling system has two phases, an early short transient phase followed by a long phase in quasi-steady state (QSS). (3) In the enzymatic cycling system, r converges to a definite level regardless of any initial value of r. (4) In QSS, the definite level of r nearly equals the singular solution, rsingular, of the differential equation. (5) The actual rate of the targeted product (chiral compound) formation can be calculated by Michaelis–Menten equation in which the cofactor concentration is [C]0×rsingular instead of [C]0. rsingular has been proposed to name “redistribution factor”. (6) It is recommended that the “unit” of the cofactor regeneration enzyme be 2–3 times more used than the “unit” of the synthesis enzyme and that [C]0 be 15–25 times more than the Km value. Four special cases relating to the batch-wise enzymatic cycling system have been discussed.

Separation of xylose and glucose using an integrated membrane system for enzymatic cofactor regeneration and downstream purification

Mixtures of xylose, glucose and pyruvate were fed to a membrane bioreactor equipped with a charged NF membrane (NTR 7450). Value-added products were obtained in the reactor via enzymatic cofactor-dependent catalysis of glucose to gluconic acid and pyruvate to lactic acid, respectively. The initial cofactor (NADH) concentration could be decreased to 10% of the stoichiometric value (relative to glucose) without compromising process time and substrate conversion via i) efficient cofactor regeneration and ii) high retention of cofactor (R=0.98) in the membrane bioreactor. Furthermore, accumulation of xylose (R<0.1), lactic acid (R=0.38) and gluconic acid (R=0.63) was minimized. After separation of the cofactor in the membrane bioreactor, xylose, lactic acid and gluconic acid were separated based on charge repulsion and size exclusion in a sequence of two NF steps. Broad substrate specificity of glucose dehydrogenase (EC 1.1.1.47) (GDH) lead to partial conversion of xylose to xylonic acid, causing some loss of xylose, but the results obtained nevertheless showed that it is possible to build a robust system for conducting enzyme reactions by sequentially regenerating the cofactor and at the same time obtaining valuable products of high purity.

Enantioselective cascade biocatalysis for deracemization of 2-hydroxy acids using a three-enzyme system.

BackgroundEnantiopure 2-hydroxy acids are key intermediates for the synthesis of pharmaceuticals and fine chemicals. We present an enantioselective cascade biocatalysis using recombinant microbial cells for deracemization of racemic 2-hydroxy acids that allows for efficient production of enantiopure 2-hydroxy acids.ResultsThe method was realized by a single recombinant Escherichia coli strain coexpressing three enzymes: (S)-2-hydroxy acid dehydrogenase, (R)-2-keto acid reductase and glucose dehydrogenase. One enantiomer [(S)-2-hydroxy acid] is firstly oxidized to the keto acid with (S)-2-hydroxy acid dehydrogenase, while the other enantiomer [(R)-2-hydroxy acid] remains unchanged. Then, the keto acid obtained reduced to the opposite enantiomer with (R)-2-keto acid reductase plus cofactor regeneration enzyme glucose dehydrogenase subsequently. The recombinant E. coli strain coexpressing the three enzymes was proven to be a promising biocatalyst for the cascade bioconversion of a structurally diverse range of racemic 2-hydroxy acids, giving the corresponding (R)-2-hydroxy acids in up to 98.5 % conversion and >99 % enantiomeric excess.ConclusionsIn summary, a cascade biocatalysis was successfully developed to prepare valuable (R)-2-hydroxy acids with an efficient three-enzyme system. The developed elegant cascade biocatalysis possesses high atom efficiency and represents a promising strategy for production of highly valued (R)-2-hydroxy acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0560-1) contains supplementary material, which is available to authorized users.

Conversion of glycerol to 1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration.

To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA). In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3gl(-1) (GlyDH/LDH) and 2.2gl(-1) (GlyDH/LpNox1) with total turnover number (TTN) of NAD(+) recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH-LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10g glycerol l(-1) to DHA at 0.2-0.5gl(-1) in the presence of zero to 2mM exogenous NAD(+). The cell free extract of E. coli (GlyDH-LpNox) converted glycerol (2-50gl(-1)) to DHA from 0.5 to 4.0gl(-1) (8-25% conversion) without exogenous NAD(+). The disadvantage of the expensive consumption of NAD(+) for the production of DHA has been overcome.

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes.

New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc.

Process Development for Biocatalytic Oxidations Applying Alcohol Dehydrogenases

Alcohol dehydrogenases are able to catalyze the conversion of alcohols to aldehydes or ketones, simultaneously reducing the cofactor NAD+ or NADP+ to NAD(P)H. Because of the high costs of these pyridine cofactors, in situ cofactor regeneration is required for preparative applications in order to reach turnover numbers that are sufficient for economically viable processes. Here we present the development of a process for the enantioselective oxidation of rac-1-phenylethanol to acetophenone, applying an alcohol dehydrogenase coupled with an NAD(P)H oxidase for the enzymatic cofactor regeneration, which is active towards NADH as well as NADPH. The reaction system was investigated in view of various influential parameters with main focus on the external oxygen supply. We could show that a gassed stirred tank reactor is a promising reactor concept to run NAD(P)H oxidase-coupled alcohol dehydrogenase oxidations, including the possibility to scale-up the system.

A thermostable S-adenosylhomocysteine hydrolase from Thermotoga maritima: Properties and its application on S-adenosylhomocysteine production with enzymatic cofactor regeneration

S-adenosylhomocysteine (SAH) is an effective sedative, a good sleep modulator, and a new anticonvulsant. SAH can be synthesized from adenosine and homocysteine by using microbial S-adenosylhomocysteine hydrolase (SAHase). The extremely thermostable SAHase and lactate dehydrogenase (LDH) from Thermotoga maritima were successfully overexpressed in Escherichia coli, and purified by heat treatments. The SAHase exhibited the highest activity at 85°C and pH 8.0 with a specific activity of 6.2U/mg when NAD concentration was 1mM. However, optimal SAHase reaction conditions shifted to 100°C and pH 11.2, and its specific activity increased to 36.8U/mg after NAD concentration was raised to 8mM. Biosynthesis of SAH at 85°C largely increased the adenosine solubility which was a limiting factor for improving the titer of product. At 85°C and pH 8.0, 24μmol of SAH was obtained when 0.5mg of SAHase was applied to a 10ml reaction mixture. The SAH production was further increased to 153μmol by adding LDH and pyruvate into the reaction mixture for NAD regeneration. Therefore, extremely thermostable enzymes SAHase and LDH from T. maritima form an efficient NAD consumption and regeneration system for SAH biosynthesis. This method has great potential for industrial-scale enzymatic production of SAH.

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an L-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with D-glucitol oxidation to D-fructose but also converted L-glucitol to D-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K m value for L-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a D-sorbitol dehydrogenase (EC 1.1.1.14).

Purification of Xylitol Dehydrogenase and Improved Production of Xylitol by Increasing XDH Activity and NADH Supply in Gluconobacter oxydans

Gluconobacter oxydans is known to be a suitable candidate for producing xylitol from d-arabitol. In this study, the enzyme responsible for reducing d-xylulose to xylitol was purified from G. oxydans NH-10 and characterized as xylitol dehydrogenase. It has been reported that XDH depends exclusively on NAD(+)/NADH as cofactors with a relatively low activity, which was proposed to be the direct reason for its limiting the overall conversion process. To better produce xylitol, an engineered G. oxydans PXPG was constructed to coexpress the XDH gene and a cofactor regeneration enzyme (glucose dehydrogenase) gene from Bacillus subtilis. Activities for both enzymes were more than twofold higher in the G. oxydans PXPG than in the wild strain. Approximately 12.23 g/L xylitol was obtained from 30 g/L d-arabitol by resting cells of the engineered strain with a conversion yield of 40.8%, whereas only 7.56 g/L xylitol was produced by the wild strain with a yield of 25.2%. These results demonstrated that increasing the XDH activity and the cofactor NADH supply could improve the xylitol productivity notably.

Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from d-arabitol

The biotransformation of d-arabitol into xylitol was investigated with focus on the conversion of d-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted d-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91g/L xylitol was obtained from around 30g/L d-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD+, was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.