Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

Ketogluconate production by Gluconobacter strains: enzymes and biotechnological applications.

Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d-glucose to d-gluconate, which then diverges and splits into 2 pathways producing 5-keto-d-gluconate and 2-keto-d-gluconate and subsequently 2,5-diketo-d-gluconate. These transformations are facilitated by membrane-bound d-glucose dehydrogenase, glycerol dehydrogenase, d-gluconate dehydrogenase, and 2-keto-d-gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains.

The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis.

BACKGROUND: Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. RESULTS: The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. CONCLUSION: The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10-2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10-2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain.

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Semi-Continuous Flow Biocatalysis with Affinity Co-Immobilized Ketoreductase and Glucose Dehydrogenase.

The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.

Synthesis of Formate from CO2 Gas Catalyzed by an O2-Tolerant NAD-Dependent Formate Dehydrogenase and Glucose Dehydrogenase.

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

Azotobacters as biofertilizer.

Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L-1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L-1 h-1 g-1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.

An economically and environmentally acceptable synthesis of chiral drug intermediate L-pipecolic acid from biomass-derived lysine via artificially engineered microbes.

Deficiency in petroleum resources and increasing environmental concerns have pushed a bio-based economy to be built, employing a highly reproducible, metal contaminant free, sustainable and green biomanufacturing method. Here, a chiral drug intermediate L-pipecolic acid has been synthesized from biomass-derived lysine. This artificial bioconversion system involves the coexpression of four functional genes, which encode L-lysine α-oxidase from Scomber japonicus, glucose dehydrogenase from Bacillus subtilis, Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida, and lysine permease from Escherichia coli. Besides, a lysine degradation enzyme has been knocked out to strengthen the process in this microbe. The overexpression of LysP improved the L-pipecolic acid titer about 1.6-folds compared to the control. This engineered microbial factory showed the highest L-pipecolic acid production of 46.7 g/L reported to date and a higher productivity of 2.41 g/L h and a yield of 0.89 g/g. This biotechnological L-pipecolic acid production is a simple, economic, and green technology to replace the presently used chemical synthesis.

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.

Jinggangmycin-suppressed reproduction in the small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is mediated by glucose dehydrogenase (GDH).

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is a serious pest insect of rice, wheat, and maize in China. SBPH not only sucks plant sap but also transmits plant disease viruses, causing serious damage. These viruses include rice striped virus disease (RSV disease), black streaked dwarf, and maize rough disease virus. SBPH outbreaks are related to the overuse of pesticides in China. Some pesticides, such as triazophos, stimulate the reproduction of SBPH, but an antibiotic fungicide jinggangmycin (JGM) suppresses its reproduction. However, mechanisms of decreased reproduction of SBPH induced by JGM remain unclear. The present findings show that JGM suppressed reproduction of SBPH (↓approximately 35.7%) and resulted in the down-regulated expression of glucose dehydrogenase (GDH). GDH-silenced control females (control+dsGDH) show that the number of eggs laid was reduced by 48.6% compared to control females. Biochemical tests show that the total lipid and fatty acid contents in JGM-treated and control+dsGDH females decreased significantly. Thus, we propose that the suppression of reproduction in SBPH induced by JGM is mediated by GDH via metabolic pathways.

Biochemical and Computational Insights on a Novel Acid-Resistant and Thermal-Stable Glucose 1-Dehydrogenase.

Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in Escherichia coli BL21 (DE3) for primary screening. Of all the candidates, GDH from Bacillus sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically. It displayed maximum activity at 45 °C and pH 9.0, and was stable at temperatures below 50 °C. BzGDH also exhibited a broad pH stability, especially in the acidic region, which could maintain around 80% of its initial activity at the pH range of 4.0-8.5 after incubating for 1 hour. Molecular dynamics simulation was conducted for better understanding the stability feature of BzGDH against the structural context. The in-silico simulation shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 K, which is consistent with the biochemical studies. In brief, the robust stability of BzGDH made it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature.

Enantioselective Biosynthesis of l-Phenyllactic Acid by Whole Cells of Recombinant Escherichia coli.

BACKGROUND: l-Phenyllactic acid (l-PLA)-a valuable building block in the pharmaceutical and chemical industry-has recently emerged as an important monomer in the composition of the novel degradable biocompatible material of polyphenyllactic acid. However, both normally chemically synthesized and naturally occurring phenyllactic acid are racemic, and the product yields of reported l-PLA synthesis processes remain unsatisfactory. METHODS: We developed a novel recombinant Escherichia coli strain, co-expressing l-lactate dehydrogenase (l-LDH) from Lactobacillus plantarum subsp. plantarum and glucose dehydrogenase (GDH) from Bacillus megaterium, to construct a recombinant oxidation/reduction cycle for whole-cell biotransformation of phenylpyruvic acid (PPA) into chiral l-PLA in an enantioselective and continuous manner. RESULTS: During fed-batch bioconversion with intermittent PPA feeding, l-PLA yield reached 103.8 mM, with an excellent enantiomeric excess of 99.7%. The productivity of l-PLA was as high as 5.2 mM·h-1 per OD600 (optical density at 600 nm) of whole cells. These results demonstrate the efficient production of l-PLA by the one-pot biotransformation system. Therefore, this stereoselective biocatalytic process might be a promising alternative for l-PLA production.

Efficient synthesis of (S)-N-Boc-3-hydroxypiperidine using an (R)-specific carbonyl reductase from Candida parapsilosis.

(S)-N-Boc-3-hydroxypiperidine (S-NBHP) is a critical chiral intermediate in the synthesis of pharmaceuticals, including ibrutinib, the active pharmaceutical ingredient of the new drug Imbruvica approved for the treatment of lymphoma. An (R)-specific carbonyl reductase from Candida parapsilosis (CprCR, also known as R-specific alcohol dehydrogenase) that catalyzes asymmetric reduction to produce (S)-N-Boc-3-hydroxypiperidine (S-NBHP) was identified for the first time. When co-expressed with a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3), recombinant crude enzyme exhibited an activity of 9 U/mg with N-Boc-3-piperidone as the substrate and 12 U/mg with glucose as the substrate. The biocatalysis of N-Boc-3-piperidone to S-NBHP using recombinant whole-cell biocatalysts was processed in a water/butyl acetate system as well as an aqueous monophasic system without extra NAD+/NADH. This process showed great commercial potential, with a 100 g/l substrate concentration and a whole cells loading (w/v) of 10%, with the conversion of 97.8% and an e.e. of 99.8% in an aqueous monophasic system.