Increased productivity of L-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of L-threonine deaminase.

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.

Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis.

(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.

Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy.

OBJECTIVES: To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL]. RESULTS: The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%. CONCLUSIONS: Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

OBJECTIVES: To find an efficient and cheap system for NAD(+) regeneration RESULTS: A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 µM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains.

An NADPH-dependent sorbose reductase from Candida albicans was identified to catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE). The activity of the recombinant enzyme toward COBE was 6.2 U/mg. The asymmetric reduction of COBE was performed with two coexisting recombinant Escherichia coli strains, in which the recombinant E. coli expressing glucose dehydrogenase was used as an NADPH regenerator. An optical purity of 99% (e.e.) and a maximum yield of 1240 mM (S)-4-chloro-3-hydroxybutanoate were obtained under an optimal biomass ratio of 1:2. A highest turnover number of 53,900 was achieved without adding extra NADP(+)/NADPH compared with those known COBE-catalytic systems.

Catalytic Synthesis of (S)-CHBE by Directional Coupling and Immobilization of Carbonyl Reductase and Glucose Dehydrogenase.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.

A Novel Strategy for Whole-Cell Biotransformation Enabling Simultaneous l-Phenyllactic Acid Production and Coenzyme Regeneration.

l-Phenyllactic acid (l-PLA) is a small molecular organic acid that exhibits a powerful capacity for inhibition against foodborne pathogens. In this work, we developed a new cost-effective and environmentally friendly process for the biosynthesis of l-PLA. This strategy designed a novel whole-cell biotransformation system employing two heterologous enzymes, namely, phenylalanine dehydrogenase (PheDH) and l-hydroxyisocaproate dehydrogenase (l-HicDH). The novelty of this strategy lies in the first-time utilization of these two enzymes, which not only enables cascade catalysis for the production of l-PLA but also facilitates the regeneration of the coenzymes (NAD+/NADH) using only two enzymes rather than introducing more heterologous enzymes to the system. Consequently, this strategy can effectively simplify the biosynthesis process of l-PLA and minimize production costs. The initial l-PLA yield using this process achieved 2.53 ± 0.07 g/L. Furthermore, through meticulous optimization of the parameters for inducible enzyme expression and l-PLA biosynthesis, the l-PLA yield was successfully increased to 4.68 ± 0.04 g/L with a yield rate of 64.54 ± 0.29%. Moreover, this novel strategy is versatile in the biosynthesis of other organic acids, which can be achieved by easily modulating the combinations of substrates and enzymes.

[Construction of multi-enzyme cascade reactions and its application in the synthesis of bifunctional chemicals].

The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.

Asymmetric Hydrogenation of C = C Bonds in a SpinChem Reactor by Immobilized Old Yellow Enzyme and Glucose Dehydrogenase.

The application of immobilized enzymes in pharmaceutical and bulk chemical production has been shown to be economically viable. We demonstrate the exceptional performance of a method that immobilizes the old yellow enzyme YqjM and glucose dehydrogenase (GDH) on resin for the asymmetric hydrogenation (AH) of C = C bonds in a SpinChem reactor. When immobilized YqjM and GDH are reused 10 times, the conversion of 2-methylcyclopentenone could reach 78%. Which is because the rotor of the SpinChem reactor effectively reduces catalyst damage caused by shear force in the reaction system. When the substrate concentration is 175 mM, an 87% conversion of 2-methylcyclopentenone is obtained. The method is also observed to perform well for the AH of C = C bonds in other unsaturated carbonyl compounds with the SpinChem reactor. Thus, this method has great potential for application in the enzymatic production of chiral compounds.

Enantioselective Biosynthesis of L-Phenyllactic Acid From Phenylpyruvic Acid In Vitro by L-Lactate Dehydrogenase Coupling With Glucose Dehydrogenase.

As a valuable versatile building block, L-phenyllactic acid (L-PLA) has numerous applications in the fields of agriculture, pharmaceuticals, and biodegradable plastics. However, both normally chemically synthesized and naturally occurring PLA are racemic, and the production titer of L-PLA is not satisfactory. To improve L-PLA production and reduce the high cost of NADH, an in vitro coenzyme regeneration system of NADH was achieved using the glucose dehydrogenase variant LsGDHD255C and introduced into the L-PLA production process. Here an NADH-dependent L-lactate dehydrogenase-encoding variant gene (L-Lcldh1Q88A/I229A) was expressed in Pichia pastoris GS115. The specific activity of L-LcLDH1Q88A/I229A (Pp) was as high as 447.6 U/mg at the optimum temperature and pH of 40°C and 5.0, which was 38.26-fold higher than that of wild-type L-LcLDH1 (Pp). The catalytic efficiency (k cat/K m) of L-LcLDH1Q88A/I229A (Pp) was 94.3 mM-1 s-1, which was 67.4- and 25.5-fold higher than that of L-LcLDH1(Pp) and L-LcLDH1Q88A/I229A (Ec) expressed in Escherichia coli, respectively. Optimum reactions of L-PLA production by dual-enzyme catalysis were at 40°C and pH 5.0 with 10.0 U/ml L-LcLDH1Q88A/I229A (Pp) and 4.0 U/ml LsGDHD255C. Using 0.1 mM NAD+, 400 mM (65.66 g/L) phenylpyruvic acid was completely hydrolyzed by fed-batch process within 6 h, affording L-PLA with 90.0% yield and over 99.9% ee p. This work would be a promising technical strategy for the preparation of L-PLA at an industrial scale.

Microbial redox coenzyme engineering and applications in biosynthesis.

Many biosynthetic processes, either in vivo or in vitro, involve redox reactions catalyzed by oxidoreductases - which depend on coenzymes as electron carriers. Redox balance is regulated mainly by coenzymes NAD(P)+ and NAD(P)H and is essential for biosynthesis. New techniques for the regulation and regeneration of coenzymes have recently advanced our understanding of, and demonstrated promising applications in, synthetic biology.

[Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate].

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.

Coenzyme Binding Site Analysis of an Isopropanol Dehydrogenase with Wide Substrate Spectrum and Excellent Organic Solvent Tolerance.

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.

Biochemical characterization of a novel azoreductase from Streptomyces sp.: Application in eco-friendly decolorization of azo dye wastewater.

Azo dyes are the most widely applied chemical dyes that have also raised great concerns for environmental contamination and human health issues. There has been a growing interest in discovering bioremediation methods to degrade azo dyes for environmental and economic purposes. Azoreductases are key enzymes evolved in nature capable of degrading azo dyes. The current work reports the identification, expression, and properties of a novel azoreductase (AzoRed2) from Streptomyces sp. S27 which shows an excellent stability against pH change and organic solvents. To overcome the requirements of coenzyme while degrading azo dyes, we introduced a coenzyme regeneration enzyme, Bacillus subtilis glucose 1-dehydrogenase (BsGDH), to construct a recycling system in living cells. The whole-cell biocatalyst containing AzoRed2 and BsGDH was used to degrade a representative azo dye methyl red. The degradation rate of methyl red was up to 99% in 120 min with high substrate concentration (250 µM) and no external coenzyme added. The degradation rate was still 98% in the third batch trial. To sum up, a novel azoreductase with good properties was found, which was applied to construct whole-cell biocatalyst. Both the enzymes and whole-cell biocatalysts are good candidates for the industrial wastewater treatment and environmental restoration.

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues.

Engineering flavin-free NAD(P)+-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD+s) is of importance for eliminating the need for costly NAD(P)+ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD+ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD+-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD+-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD+-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN+). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (k cat/K M) and a specific activity of 17.7 U/mg on NMN+, which is comparable to the wild-type enzyme on its natural coenzyme, NADP+. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD+-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD+s similar to the levels of natural enzyme/coenzyme pairs.

Full-time dynamics of batch-wise enzymatic cycling system composed of two kinds of dehydrogenase mediated by NAD(P)H for mass production of chiral hydroxyl compounds.

Enzymatic cycling system (coupled dehydrogenase-catalyzed biosystem being composed of two elementary enzymatic reactions mediated by NAD(P)H + NAD(P)+) is industrially attractive for reducing prochiral carbonyl compounds to the corresponding chiral hydroxyl compounds. The reaction rate equation of the batch-wise biosystem was generally derived by ordered Bi Bi mechanism of two-substrate enzyme reaction on several reasonable assumptions. The rate equations of the batch-wise biosystem was generalized by transforming them into the dimensionless forms. The dimensionless forms were solved numerically. It was revealed that the batch-wise biosystem was generally made up of unique 3 phases, i.e., phases I, II and III. Phase I was very short transient so that the biosystem entered rapidly phase II. In phase II the consumption rate dynamically balanced with its formation rate so that the concentration of NAD(P)H was invariable with time (and hence NAD(P)+ concentration was, too). Phase III was substrate-exhausting phase, and the coenzyme concentration became finally only [NAD(P)+] or only [NAD(P)H] depending on the initial molar ratio of the prochiral carbonyl compound to the substrate of the coenzyme regeneration reaction ( [Formula: see text] ) > or <1.0. In phases I and II the numerically calculated values of state variables were very close to the analytical but approximate ones. Preferable initial conditions of the batch-wise enzymatic cycling system, i.e., the initial coenzyme species = NAD(P)+ and [Formula: see text] , were proposed. As the main assumption irreversibility of the two elemental enzymatic reactions was discussed. Validity of the proposed rate equations was mentioned.

An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design.

Although most in vitro (cell-free) synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products, in vitro synthetic biology platform, which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, has been proposed for low-cost biomanufacturing of bioenergy, food, biochemicals, and nutraceuticals. In addition to the most important advantage-high product yield, in vitro synthetic biology platform features several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this article, we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks (thermoenzymes and/or immobilized enzymes) to building modules (e.g., enzyme complexes or multiple enzymes as a module) with specific functions. With development in thermostable building blocks-BioBricks and modules, the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.

Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.).

NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO2 reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli DH5α-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 ± 0.07 mM) and NAD+ (0.06 ± 0.01 mM). At the same time, the affinity (1.98 ± 0.4 mM) and catalytic efficiency (0.0041) values of the enzyme for NADP+ show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The Tm value of GhFDH1 is 53.3 °C and the highest enzyme activity is measured at 30 °C with a half-life of 61 h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P)H regeneration for its prominent thermostability and NADP+ specificity.

Review on optofluidic microreactors for artificial photosynthesis.

Artificial photosynthesis (APS) mimics natural photosynthesis (NPS) to store solar energy in chemical compounds for applications such as water splitting, CO2 fixation and coenzyme regeneration. NPS is naturally an optofluidic system since the cells (typical size 10 to 100 µm) of green plants, algae, and cyanobacteria enable light capture, biochemical and enzymatic reactions and the related material transport in a microscale, aqueous environment. The long history of evolution has equipped NPS with the remarkable merits of a large surface-area-to-volume ratio, fast small molecule diffusion and precise control of mass transfer. APS is expected to share many of the same advantages of NPS and could even provide more functionality if optofluidic technology is introduced. Recently, many studies have reported on optofluidic APS systems, but there is still a lack of an in-depth review. This article will start with a brief introduction of the physical mechanisms and will then review recent progresses in water splitting, CO2 fixation and coenzyme regeneration in optofluidic APS systems, followed by discussions on pending problems for real applications.

Improved Xylitol Production from D-Arabitol by Enhancing the Coenzyme Regeneration Efficiency of the Pentose Phosphate Pathway in Gluconobacter oxydans.

Gluconobacter oxydans is used to produce xylitol from D-arabitol. This study aims to improve xylitol production by increasing the coenzyme regeneration efficiency of the pentose phosphate pathway in G. oxydans. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were overexpressed in G. oxydans. Real-time PCR and enzyme activity assays revealed that G6PDH/6PGDH activity and coenzyme regeneration efficiency increased in the recombinant G. oxydans strains. Approximately 29.3 g/L xylitol was obtained, with a yield of 73.2%, from 40 g/L d-arabitol in the batch biotransformation with the G. oxydans PZ strain. Moreover, the xylitol productivity (0.62 g/L/h) was 3.26-fold of the wild type strain (0.19 g/L/h). In repetitive batch biotransformation, the G. oxydans PZ cells were used for five cycles without incurring a significant loss in productivity. These results indicate that the recombinant G. oxydans PZ strain is economically feasible for xylitol production in industrial bioconversion.