CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Fabrication and characterization of a novel zein/pectin/pumpkin seed oil Pickering emulsion and the effects of myricetin on oxidation stability.

In this study, zein/pectin/pumpkin seed oil (PSO) Pickering emulsions (ZPPEs) were fabricated loading with myricetin (MYT), and the quality control methods of oxidation stability were innovatively investigated. The microstructure and particle properties of zein-pectin particles were determined. The zein to pectin ratio of 5:3 and oil phase fraction (φ = 50 %) turned out as the most optimal conditions for the stabilization of myricetin-loaded ZPPEs. The expected oil-in-water emulsion-type structure was confirmed by confocal laser scanning microscopy (CLSM). The internal 3D structure of Pickering emulsions (Lugol's solution improved the water-phase contrast) was imaged by micro-computed tomography (Micro-CT) for the first time. Results showed a sponge like structure of water phase in emulsion with 42 µm as mean droplet size. Light-induced oxidation was evaluated with the PetroOxy method and malondialdehyde (MDA) assays. Encapsuling ZPPEs with MYT could prevent the light induced oxidation, especially, loading of MYT at the core of the emulsion. The analysis of Electronic nose (E-nose) was used to analyze the odor before and after UV-induced oxidation, and showed a good discrimination. This study provided a new approach to prepare ZPPEs with high oxidation stability. Micro-CT, PetroOxy and E-nose could be new methods for characterization and quality assessment of Pickering emulsions.

Lab-Scale Cultivation of Cupriavidus necator on Explosive Gas Mixtures: Carbon Dioxide Fixation into Polyhydroxybutyrate.

Aerobic, hydrogen oxidizing bacteria are capable of efficient, non-phototrophic CO2 assimilation, using H2 as a reducing agent. The presence of explosive gas mixtures requires strict safety measures for bioreactor and process design. Here, we report a simplified, reproducible, and safe cultivation method to produce Cupriavidus necator H16 on a gram scale. Conditions for long-term strain maintenance and mineral media composition were optimized. Cultivations on the gaseous substrates H2, O2, and CO2 were accomplished in an explosion-proof bioreactor situated in a strong, grounded fume hood. Cells grew under O2 control and H2 and CO2 excess. The starting gas mixture was H2:CO2:O2 in a ratio of 85:10:2 (partial pressure of O2 0.02 atm). Dissolved oxygen was measured online and was kept below 1.6 mg/L by a stepwise increase of the O2 supply. Use of gas compositions within the explosion limits of oxyhydrogen facilitated production of 13.1 ± 0.4 g/L total biomass (gram cell dry mass) with a content of 79 ± 2% poly-(R)-3-hydroxybutyrate in a simple cultivation set-up with dissolved oxygen as the single controlled parameter. Approximately 98% of the obtained PHB was formed from CO2.

Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol.

BACKGROUND: Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase. RESULTS: A single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·103 s-1 M-1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee. CONCLUSION: The current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.

Food Ingredients and Nutraceuticals from Microalgae: Main Product Classes and Biotechnological Production.

Microalgal products are an emerging class of food, feed, and nutraceuticals. They include dewatered or dried biomass, isolated pigments, and extracted fat. The oil, protein, and antioxidant-rich microalgal biomass is used as a feed and food supplement formulated as pastes, powders, tablets, capsules, or flakes designed for daily use. Pigments such as astaxanthin (red), lutein (yellow), chlorophyll (green), or phycocyanin (bright blue) are natural food dyes used as isolated pigments or pigment-rich biomass. Algal fat extracted from certain marine microalgae represents a vegetarian source of n-3-fatty acids (eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), γ-linolenic acid (GLA)). Gaining an overview of the production of microalgal products is a time-consuming task. Here, requirements and options of microalgae cultivation are summarized in a concise manner, including light and nutrient requirements, growth conditions, and cultivation systems. The rentability of microalgal products remains the major obstacle in industrial application. Key challenges are the high costs of commercial-scale cultivation, harvesting (and dewatering), and product quality assurance (toxin analysis). High-value food ingredients are commonly regarded as profitable despite significant capital expenditures and energy inputs. Improvements in capital and operational costs shall enable economic production of low-value food products going down to fishmeal replacement in the future economy.

Pushing the limits: Cyclodextrin-based intensification of bioreductions.

The asymmetric reduction of ketones is a frequently used synthesis route towards chiral alcohols. Amongst available chemo- and biocatalysts the latter stand out in terms of product enantiopurity. Their application is, however, restricted by low reaction output, often rooted in limited enzyme stability under operational conditions. Here, addition of 2-hydroxypropyl-ß-cyclodextrin to bioreductions of o-chloroacetophenone enabled product concentrations of up to 29 % w/v at full conversion and 99.97 % e.e. The catalyst was an E. coli strain co-expressing NADH-dependent Candida tenuis xylose reductase and a yeast formate dehydrogenase for coenzyme recycling. Analysis of the lyophilized biocatalyst showed that E. coli cells were leaky with catalytic activity found as free-floating enzymes and associated with the biomass. The biocatalyst was stabilized and activated in the reaction mixture by 2-hydroxypropyl-ß-cyclodextrin. Substitution of the wild-type xylose reductase by a D51A mutant further improved bioreductions. In previous optimization strategies, hexane was added as second phase to protect the labile catalyst from adverse effects of hydrophobic substrate and product. The addition of 2-hydroxypropyl-ß-cyclodextrin (11 % w/v) instead of hexane (20 % v/v) increased the yield on biocatalyst 6.3-fold. A literature survey suggests that bioreduction enhancement by addition of cyclodextrins is not restricted to specific enzyme classes, catalyst forms or substrates.

Enzymes revolutionize the bioproduction of value-added compounds: From enzyme discovery to special applications.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Integration of whole-cell reaction and product isolation: Highly hydrophobic solvents promote in situ substrate supply and simplify extractive product isolation.

Product isolation from aqueous-organic reaction mixtures that contain high concentrations of whole cells constitutes a challenging task in bioprocessing. Stirring of the biphasic reaction media leads to the formation of solvent droplets coated by cells and other surface active components and an emulsion forms. We used an early focus on phase separation to simplify a whole-cell bioreduction. Octanol, heptanol, hexanol, hexane and dipropylether were tested as co-solvents in the E. coli catalyzed reduction of o-chloroacetophenone. All solvents showed very similar performance in bioreductions and highest yields were obtained with low organic-to-aqueous phase ratios. Reaction mixtures were directly investigated for organic-phase recovery. Phase separation was optimized in small-scale settling experiments and confirmed by the isolation of 20.4g (S)-1-(2-chlorophenyl)ethanol from a 0.5L batch reduction containing 40gCDW/L whole-cell catalyst. Solvent consumption during product isolation could be halved by the simple addition of sodium hydroxide prior to product extraction. Basification to pH 13.5 and three extraction steps with a total of 1.2v/v hexane led to an isolated yield of 87% (97% reduction yield). A general emulsion destabilizing effect under harsh conditions, as extreme pH values and presence of toxic reactants, was observed.

Rules for biocatalyst and reaction engineering to implement effective, NAD(P)H-dependent, whole cell bioreductions.

Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to "test" for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.

Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: stereoselectivity, cell activity, in situ substrate supply and product removal.

Generally, recombinant and native microorganisms can be employed as whole-cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H-dependent reduction of o-chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co-expressing xylose reductase and a dehydrogenase for co-enzyme recycling. Xylose-grown cells of C. tenuis and P. stipitis displayed specific o-chloroacetophenone reductase activities of 366 and 90 U gCDW (-1) , respectively, in the cell-free extracts. Fresh biomass was employed in batch reductions of 100 mM o-chloroacetophenone using glucose as co-substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)-1-(2-chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 gCDW L(-1) of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7-fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo-like kinase 1 inhibitors).

Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering.

Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase.

Scale-up and intensification of (S)-1-(2-chlorophenyl)ethanol bioproduction: economic evaluation of whole cell-catalyzed reduction of o-chloroacetophenone.

Escherichia coli cells co-expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o-chloroacetophenone with in situ coenzyme recycling. The product, (S)-1-(2-chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo-like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi-gram scale requires intensification and scale-up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9-L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)-1-(2-chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)-1-(2-chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)-1-(2-chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated.

Comparison of Scheffersomyces stipitis strains CBS 5773 and CBS 6054 with regard to their xylose metabolism: implications for xylose fermentation.

The various strains of Scheffersomyces stipitis (Pichia stipitis) differ substantially with respect to their ability to ferment xylose into ethanol. Two P. stipitis strains CBS 5773 and CBS 6054 have been most often used in literature but comparison of their performance in xylose fermentation under identical conditions has not been reported so far. Conversion of xylose (22 g/L) by each of these P. stipitis strain was analyzed under anaerobic and microaerobic conditions. Ethanol yields of ∼0.41 g/g were independent of strain and conditions used. Glycerol and acetate were formed in constant yields of 0.006 g/g and 0.02 g/g, respectively. Xylitol formation decreased from ∼0.08 g/g to ∼0.05 g/g upon switch from anaerobic to microaerobic conditions. Specific activities of enzymes of the two-step oxidoreductive xylose conversion pathway (xylose reductase and xylitol dehydrogenase) matched for both strains within limits of error. When xylose was offered at 76 g/L under microaerobic reaction conditions, ethanol yields were still high (0.37-0.39 g/g) for both strains even though the xylitol yields (0.12-0.13 g/g) were increased as compared to the conditions of low xylose concentration. P. stipitis strains CBS 5773 and CBS 6054 are therefore identical by the criteria selected and show useful performance during conversion of xylose into ethanol, irrespective of the supply of oxygen.

Candida tenuis xylose reductase catalyzed reduction of aryl ketones for enantioselective synthesis of active oxetine derivatives.

Candida tenuis xylose reductase shows high catalytic efficiencies in carbonyl reduction of acetophenone and 1-phenyl-1-propanone derivatives. The quite low substrate solubility in aqueous buffer systems is circumvented by addition of methanol or by two-phase solvent systems. In the latter, methanol improves the substrate phase transfer as solvent mediator and leads to reasonable space/time yields. Resulting enantiomerically pure chiral alcohols are key intermediates for synthesis of active pharmaceutical ingredients. (R)-Atomoxetine is exemplarily synthesized in four steps, and the further use for generation of other oxetine derivatives and a polo-like kinase 1 inhibitor are discussed.

Host cell and expression engineering for development of an E. coli ketoreductase catalyst: enhancement of formate dehydrogenase activity for regeneration of NADH.

BACKGROUND: Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone. RESULTS: As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/g(CDW), co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/g(CDW) was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/g(CDW) while reducing the XR activity by just 19% (1140 U/g(CDW)). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/g(CDW)) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane. CONCLUSIONS: Expression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.

Bioprocess design guided by in situ substrate supply and product removal: process intensification for synthesis of (S)-1-(2-chlorophenyl)ethanol.

We report herein on bioprocess development guided by the hydrophobicities of substrate and product. Bioreductions of o-chloroacetophenone are severely limited by instability of the catalyst in the presence of aromatic substrate and (S)-1-(2-chlorophenyl)ethanol. In situ substrate supply and product removal was used to protect the utilized Escherichia coli whole cell catalyst based on Candida tenuis xylose reductase during the reaction. Further engineering at the levels of the catalyst and the reaction media was matched to low substrate concentrations in the aqueous phase. Productivities obtained in aqueous batch reductions were 21-fold improved by addition of 20% (v/v) hexane, NAD(+), expression engineering, cell permeabilization and pH optimization. Reduction of 300 mM substrate was accomplished in 97% yield and use of the co-solvent hexane in subsequent extraction steps led to 88% recovery. Product loss due to high catalyst loading was minimized by using the same extractant in bioreduction and product isolation.

Candida tenuis xylose reductase catalysed reduction of acetophenones: the effect of ring-substituents on catalytic efficiency.

The catalytic efficiencies of Candida tenuis xylose reductase catalysed reductions of mono-substituted acetophenones are in reasonable correlation with the σ-Hammett coefficients of the substituted phenyl groups. Variations of the substrate transformation rates are hence mainly caused by mesomeric and inductive effects of the substituents, while differences in substrate binding have a secondary relevance. Some substrate (1)H NMR chemical shifts and carbonyl IR absorption bands are in reasonable accordance with the catalytic activities and allow the estimation of the transformation rates with good accuracy. The resulting substituted (S)-1-phenyl ethanols are generated in very high enantiomeric excess.

Enzyme identification and development of a whole-cell biotransformation for asymmetric reduction of o-chloroacetophenone.

Chiral 1-(o-chlorophenyl)-ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o-chloroacetophenone is a preferred method of production but well investigated chemo- and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o-chloroacetophenone with useful specificity (kcat/Km=340 M(-1) s(-1)) and perfect S-stereoselectivity, we developed whole-cell catalysts from Escherichia coli and Saccharomyces cerevisiae co-expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o-Chloroacetophenone was unexpectedly "toxic," and catalyst half-life times of only 20 min (E. coli) and 30 min (S. cerevisiae) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o-chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD+ (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S-1-(o-chlorophenyl)-ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity.

The role of Cys108 in Trigonopsis variabilis d-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D.

Oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in D-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 degrees C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of D-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

Whole-cell bioreduction of aromatic alpha-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli.

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 alpha-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. RESULTS: Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (rS) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the alpha-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L), we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to congruent with100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e.e. of >/= 99.9%. Modifications to the recombinant E. coli (cell permeabilisation; addition of exogenous NAD+) and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised E. coli cells that expressed solely CbFDH (410 U/gCDW). The positive effect on yield (18% --> 62%; 100 mM substrate) caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%. CONCLUSION: A whole-cell system based on E. coli co-expressing CtXR and CbFDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement (>/= 10-fold) of the intracellular level of FDH activity.