- Open Access
Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to corresponding (S)-enantiomer by fusion of carbonyl reductase and glucose dehydrogenase
© The Author(s) 2017
- Received: 1 April 2017
- Accepted: 23 April 2017
- Published: 2 May 2017
(S)-(−)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP) is a key intermediate for the preparation of (S)-duloxetine, an important antidepressant drug. However, so far, the catalytic efficiency of (S)-DHTP synthesis by asymmetric bioreduction is yet limited. The present study aims to develop an efficient system for synthesis of (S)-DHTP by bioreduction.
Various recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. The NADPH-dependent carbonyl reductase CR2 was identified as the suitable candidate, giving (S)-DHTP in absolute configuration. Then the fusion protein involving CR2 and glucose dehydrogenase (CR2-L-GDH) was constructed to further improve cofactor regeneration and resulted catalytic efficiency of the enzymatic reduction. By studying the effects of reaction conditions involving cofactor regeneration, suitable catalytic system was achieved for CR2-L-GDH catalyzing (S)-DHTP synthesis. Consequently, (S)-DHTP (>99.9% e.e.) with yield of 97.66% was obtained from 20 g L−1 DKTP within 8-h reaction, employing 40 g L−1 glucose and 0.1 mmol L−1 NADP+ to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L−1 h−1.
Optically pure (S)-DHTP with improved yield was obtained by fusion enzyme CR2-L-GDH. Fusion enzyme-mediated biocatalytic system would be promising to enhance reaction efficiency of enzyme-coupled system for preparation of optically active alcohols.
- Asymmetric reduction
- Carbonyl reductase
- Glucose dehydrogenase
- Fusion enzyme
- Cofactor regeneration
Optically active alcohols are valuable and promising chiral building blocks imposed in the production of pharmaceuticals, agrochemicals, functional materials, and fine chemicals (Munoz Solano et al. 2012; Ni and Xu 2012; Patel 2008). Optically active heterocyclic alcohols have been widely used as important precursors for the synthesis of chiral drugs (Huisman and Collier 2013; Pesti and DiCosimo 2003; Pollard and Woodley 2007). Using optically active heterocyclic alcohols as the key intermediate, (S)-duloxetine, the effective antidepressant and potent dual inhibitor of serotonin and norepinephrine reuptake (Bymaster et al. 2001), can be prepared in high optical purity.
The preparation of single enantiomers of chiral intermediates has become particularly prevalent in the pharmaceuticals industry (Patel 2003, 2008). Currently, enantiopure chiral alcohols can be acquired by ether chemical or biological strategies. Compared with chemical synthesis, biocatalytic route has become a subject of considerable interest due to its high chemo-, regio-, and enantioselectivities, and mild reaction conditions and environmental benignity (Lalonde 2016; Ni and Xu 2012; Wohlgemuth 2010). In recent years, enzymatic asymmetric reduction of prochiral ketones for preparation of optically active alcohols has gained increasing favor (Nakamura et al. 2003; Nealon et al. 2015; Noey et al. 2015; Sun et al. 2016; Wang et al. 2011).
Carbonyl reductases require stoichiometric amounts of nicotinamide cofactors (NADH or NADPH) as hydrogen donator for their activities. In situ cofactor regeneration would be feasible and prerequisite for practical application of carbonyl reductase-catalyzed ketone reductions. Of the cofactor regeneration approaches, enzyme-coupled system has been developed and is particular preferred for enzymatic reactions. Glucose dehydrogenase (GDH) as the coupled enzyme has been widely applied in NAD(P)H regeneration (Gao et al. 2013; Ye et al. 2010), due to its advantageous features of accessibility and practicability (Hall and Bommarius 2011). For enzyme coupling, construction of fusion protein has been developed as an effective method for cofactor regeneration, where two coding genes are combined by a short linker sequence to yield a single polypeptide exhibiting at least two functions (Calam et al. 2016; Farrow et al. 2015; Suehrer et al. 2014). The proximity effect of fusion protein generally reduces the intermediate diffusion distance and therefore increases the probability of intermediate undergoing a sequential reaction step before escaping by diffusion (Conrado et al. 2008; Dueber et al. 2009). In addition, the active sites of different enzymes for consecutive reactions can be brought in close proximity to accelerate the processing of intermediate through channeling (Castellana et al. 2014). Fusion protein of oxidoreductases would greatly facilitate the involved cofactor transfer between the two active centers of both the enzyme for catalysis and the coupled enzyme for cofactor regeneration, resulting in accumulation of necessary cofactor close to active center in some level and enhancement of desired biocatalytic reaction (Pazmino et al. 2008; Prachayasittikul et al. 2006).
In this study, various recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. Then the NADPH-dependent carbonyl reductase CR2 was obtained as the suitable candidate catalyzing asymmetric reduction. To efficiently improve the preparation of (S)-DHTP, GDH was introduced to construct the fusion enzyme CR2-L-GDH. Finally, the fusion enzyme-involved reaction conditions were optimized, and compared with free enzymes involving CR2 and GDH, the fusion enzyme system was employed for the production of the key intermediate of (S)-duloxetine (Fig. 1).
DKTP, (S)-DHTP, and (R)-DHTP were purchased from TCI (Shanghai) Development Co., Ltd. The cofactors including NAD(P)+ and NAD(P)H were obtained from Sigma-Aldrich (St. Louis, USA). Diethylamine, ethyl acetate, n-hexane, and isopropanol used for high-performance liquid chromatography were of chromatographic grade from Sigma-Aldrich (St. Louis, USA). All other chemicals used in this study were of analytical grade and commercially available.
Construction of fusion enzyme comprising CR2 and GDH
Fusion expression system containing CR2 (GenBank Accession Number: AB183149) and GDH (GenBank Accession Number: WP_013351020) was constructed using an aligned spacing sequence (GGGGSGGGGSGGGGS) as the peptide linker between them. The forward primer 5′-GGAATTCCATATGATGACATTTACAGTGGTGACAG GC-3′ and the reverse primer 5′-AGAGCCACCACCGCCAGAGCCACCACCGCCAG AGCCACCACCGCCCCCACGGTACGCGCC-3′ were used to amplify the fusion gene encoding CR2 and the linker. The forward primer 5′-GGCGGTGGTGGCTCTGGCGG TGGTGGCTCTGGCGGTGGTGGCTCTATGTATCCGGATTTAAAAGGAAAAG-3′ and the reverse primer 5′-ATAAGAATGCGGCCGCTTAACCGCGGCCTGC-3′ were used to amplify the fusion gene encoding the linker and GDH. The fusion gene cr2-l-gdh was cloned using the overlap-extension technique on the vector of pET-28a at the NdeI and NotI restriction sites. The recombinant plasmid pET-28a-cr2-l-gdh was transformed into the competent Escherichia coli BL21(DE3), and the positive E. coli BL21(DE3)/pET-28a-cr2-l-gdh was verified by DNA sequencing. The recombinant plasmid provides the fusion protein CR2-L-GDH with a six-His tag at the N-terminus.
Microorganisms and cultivation
Selection of the recombinant enzymes for catalyzing DKTP reduction to (S)-DHTP
Product configuration/yield (%)
Kizaki et al. (2001)
Kataoka et al. (2004)
Kataoka et al. (2004)
Weckbecker and Hummel (2006)
S/91.31 ± 0.45
Kataoka et al. (2006)
Yamamoto et al. (2004)
S/55.18 ± 0.25
Costello et al. (2000)
Kataoka et al. (2002)
Nie et al. (2008)
Nie et al. (2011)
Nie et al. (2011)
Nie et al. (2011)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Guo et al. (2014)
Preparation of crude enzyme
The recombinant cells were suspended in Triethanolamine-H3PO4 (TEA buffer) (0.1 mol L−1, pH 8.0) and disrupted by sonication with an ultrasonic oscillator (Sonic Materials Co., USA). The cell debris was removed by centrifugation at 4 °C and 18,000×g for 30 min, and the supernatant was used as the crude enzyme for catalyzing the asymmetric reduction of the substrate DKTP. The concentration of crude enzyme was expressed as 2 g L−1 total soluble protein for biocatalytic asymmetric reactions in this work.
Purification of recombinant enzymes
The harvested cells were suspended in TEA buffer (0.1 mol L−1, pH 8.0) and treated with sonication using an ultrasonic oscillator (Sonic Materials Co., USA). The cell debris were removed by centrifugation (18,000×g, 30 min) at 4 °C, and the supernatant was applied to a HisTrap HP affinity column (GE Healthcare, USA) equilibrated with the buffer (20 mmol L−1 Tris–HCl, 0.3 mol L−1 NaCl, 40 mmol L−1 imidazole, pH 8.0) on an ÄKTA purifier system (GE Healthcare, USA). Then the absorbed proteins were eluted with a 40-min linear imidazole gradient buffer (0–0.5 mol L−1 imidazole, 20 mmol L−1 Tris–HCl, 0.3 mol L−1 NaCl, pH 8.0) at a flow rate of 3 mL min−1 and the purified fractions were exchanged into the buffer (20 mmol L−1 Tris–HCl, 0.3 mol L−1 NaCl, pH 8.0) using disposable PD-10 desalting columns (GE Healthcare, USA) (Li et al. 2016). The preparations of purified enzymes were applied to activity assay and measurement of kinetic parameters.
Enzyme assay and kinetic parameters
The enzyme assay mixture in 100 μL for reducing DKTP or oxidating glucose activity of the purified CR2-L-GDH comprised 0.1 mol L−1 TEA buffer (pH 8.0), 5 mmol L−1 DKTP or 4 mmol L−1 glucose, 0.5 mmol L−1 NAD(P)H, or NAD(P)+, and appropriate amount of the purified CR2-L-GDH. The reactions for activity assay were carried out at 30 °C. The molar extinction coefficient of NAD(P)H was 6220 L mol−1 cm−1. One unit (U) of enzyme activity was defined as 1 μmol of NAD(P)H consumed or generated per minute under the assay conditions. The protein concentration was determined using Bradford reagents (Bio-Rad) as a standard.
Initial velocities at various concentrations of the substrate DKTP (0.05–1.0 mmol L−1) or the co-substrate glucose (0.15–1.2 mmol L−1) were measured at 30 °C to obtain the apparent K m values of the purified enzyme CR2-L-GDH. For activity assay to calculate kinetic parameters, the cofactor of NADPH or NADP+ at saturated concentration towards the enzyme was added in the reaction mixture. The kinetic parameters were further determined from Lineweaver–Burk plots.
Asymmetric reduction of DKTP and conditions optimization
The reaction involving free CR2 and GDH was carried out in 10 mL TEA buffer (0.1 mol L−1, pH 8.0) comprising 10 g L−1 DKTP, 100 g L−1 glucose, 0.02 mmol L−1 NADP+, appropriate amount of CR2 crude enzyme (total soluble protein 2 g L−1), and GDH crude enzyme with the activity equivalent to CR2. The fusion protein CR2-L-GDH-mediated reduction was carried out in 10 mL TEA buffer (0.1 mol L−1, pH 8.0) comprising 20 g L−1 DKTP, 40 g L−1 glucose, 0.2 mmol L−1 NADP+, and appropriate amount of CR2-L-GDH crude enzyme (total soluble protein 2 g L−1). The above reactions were conducted at 30 °C and 200 rpm for 12 h. After reaction, the mixture was centrifuged at 18,000×g for 30 min and the supernatant was extracted with ethyl acetate by vigorous mixing. The resulted organic layer was filtered through 0.22 μm PVDF syringe filter (Troody Technology, Shanghai, China) for further analysis.
The reaction conditions were optimized by analyzing the optical purity and yield of product under various parameters, including pH values ranging from 7.6 to 8.6 (0.1 mol L−1 TEA buffer), reaction temperature varying from 20 to 45 °C, and DKTP concentrations ranging from 10 to 50 g L−1.
To determine the optimal amounts of the added cofactor and co-substrate, the corresponding reaction parameters, involving NADP+ concentrations ranging from 0.005 to 0.2 mmol L−1 and glucose concentrations ranging from 5 to 200 g L−1, were analyzed for their effects on (S)-DHTP production from asymmetric reduction of DKTP by the catalytic system involving free CR2 and GDH or the fusion protein CR2-L-GDH.
The optical purity and yield of (S)-DHTP were determined by normal-phase high-performance liquid chromatography (HP 1100, Agilent, USA) equipped with a Chiralcel OZ-H column (4.6 mm × 250 mm; Daicel Chemical Ind., Ltd., Japan) and a ultraviolet absorption detector. The analysis was conducted with a mobile phase consisting of hexane and 2-propanol and diethyl amine (80:20:0.1, v/v/v) at a flow rate of 1.0 mL min−1 and detected at 241 nm. The column temperature was set at 30 °C. The retention times for (S)-DHTP, (R)-DHTP, and DKTP were 6.51, 8.13, and 5.87 min, respectively. The e.e. value of DHTP was calculated based on the peak areas of (S)- and (R)-DHTP (Soni and Banerjee 2005).
Selection of candidate enzyme for (S)-DHTP production from DKTP reduction
For the reaction system involving the crude enzymes of CR2 and GDH, the desired product (S)-DHTP with optical purity over 99.9% e.e. and yield of 90.43% was obtained from 10 g L−1 DKTP at 30 °C and pH 8.0, in the presence of 120 g L−1 glucose. When the substrate concentration was increased up to 20 g L−1, however, the yield of (S)-DHTP decreased significantly to 47.25%, which would be probably attributed to cofactor deficiency, besides the nature of substrate inhibition towards the functional enzyme.
Fusion expression of CR2 and GDH and its catalytic behavior
Kinetic parameters of the fusion enzyme CR2-L-GDH towards DKTP and glucose, respectively
V max (μmol min−1 mg−1)
K m (mmol L−1)
k cat (s−1)
k cat/K m (L mol−1 s−1)
6.75 ± 0.15
0.32 ± 0.05
4.35 ± 0.07
1.37 × 104
2.58 ± 0.12
0.47 ± 0.04
1.66 ± 0.12
3.54 × 103
Effects of pH and temperature on CR2-L-GDH catalyzing DKTP reduction
Reaction temperature would also have obvious effects on activity, selectivity, and stability of biocatalysts including whole cells and enzymes, and even reaction rate and equilibrium as well. As shown in Fig. 4b, within the tested temperature range from 20 to 45 °C, (S)-DHTP was prepared with high optical purity over 99.9% e.e. However, the yield of the product was improved with the increase of reaction temperature from 20 to 40 °C, and higher temperature above 40 °C led to a sharp drop in the yield, which would be attributed to partial inactivation of the enzyme at a relatively higher reaction temperature. Therefore, the reaction temperature at 40 °C was considered as the favorable factor for CR2-L-GDH catalyzing DKTP reduction.
Effect of substrate concentration on CR2-L-GDH catalyzing DKTP reduction
Process regulation of CR2-L-GDH catalyzing DKTP reduction involving cofactor regeneration
Effect of NADP+ concentration on CR2-L-GDH catalyzing asymmetric synthesis of (S)-DHTP
NADP+ (mmol L−1)
Optical purity (% e.e.)
92.75 ± 0.21
94.87 ± 0.15
83.35 ± 0.23
72.46 ± 0.18
64.64 ± 0.12
50.45 ± 0.28
Comparison of (S)-DHTP production efficiency between free CR2 and GDH and fusion enzyme CR2-L-GDH
Substrate concentration (g L−1)
Optical purity (% e.e.)
CR2 and GDH
90.43 ± 0.37
CR2 and GDH
47.25 ± 0.26
97.66 ± 0.16
The NADPH-dependent CR2 with excellent enantioselectivity was selected for catalyzing asymmetric reduction of DKTP to (S)-DHTP. To enhance the catalytic efficiency of CR2 catalyzing synthesis of (S)-DHTP via in situ cofactor regeneration, the chimeric gene encoding the fusion protein comprising CR2 and GDH was constructed and expressed as the fusion enzyme CR2-L-GDH. By regulation of the catalytic system and reaction process, the developed CR2-L-GDH catalytic system was achieved involving 20 g L−1 DKTP, 40 g L−1 glucose, and 0.1 mmol L−1 NADP+, under the optimized catalytic conditions at 40 °C and pH 8.4 for reaction 8 h. Consequently, optically pure (S)-DHTP (>99.9% e.e.) with the yield of 97.66% was obtained with increased substrate concentration of 20 g L−1 DKTP using the fusion enzyme CR2-L-GDH. Therefore, the fusion strategy for construction of multi-enzyme system would be promising in potential application for efficient biosynthesis of the precursor of (S)-duloxetine.
YN, DW, and YX designed the experiments; TS and BL performed the experiments; TS, YN, and DW wrote this manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Consent for publication
All authors have read and approved to submit it to Bioresources and Bioprocessing. There is no conflict of interest of any author in relation to the submission.
Financial supports from the National High Technology Research and Development Program of China (863 Program) (2015AA021004), the National Natural Science Foundation of China (NSFC) (21376107, 21336009, 21676120), the Natural Science Foundation of Jiangsu Province (BK20151124), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program (GDT20153200044), the Program for Advanced Talents within Six Industries of Jiangsu Province (2015-NY-007), the National Program for Support of Top-notch Young Professionals, the Fundamental Research Funds for the Central Universities (JUSRP51504), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program are greatly appreciated.
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- Bymaster FP, Dreshfield-Ahmad LJ, Threlkeld PG, Shaw JL, Thompson L, Nelson DL, Hemrick-Luecke SK, Wong DT (2001) Comparative affinity of duloxetine and venlafaxine for serotonin and norepinephrine transporters in vitro and in vivo, human serotonin receptor subtypes, and other neuronal receptors. Neuropsychopharmacology 25:871–880View ArticleGoogle Scholar
- Calam E, Gonzalez-Roca E, Fernandez MR, Dequin S, Pares X, Virgili A, Biosca JA (2016) Enantioselective synthesis of vicinal (R, R)-diols by Saccharomyces cerevisiae butanediol dehydrogenase. Appl Environ Microbiol 82:1706–1721View ArticleGoogle Scholar
- Castellana M, Wilson MZ, Xu Y, Joshi P, Cristea IM, Rabinowitz JD, Gitai Z, Wingreen NS (2014) Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat Biotechnol 32:1011–1018View ArticleGoogle Scholar
- Conrado RJ, Varner JD, DeLisa MP (2008) Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr Opin Biotechnol 19:492–499View ArticleGoogle Scholar
- Costello CA, Payson RA, Menke MA, Larson JL, Brown KA, Tanner JE, Kaiser RE, Hershberger CL, Zmijewski MJ (2000) Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii. Eur J Biochem 267:5493–5501View ArticleGoogle Scholar
- Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KLJ, Keasling JD (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27:753–759View ArticleGoogle Scholar
- Farrow SC, Hagel JM, Beaudoin GA, Burns DC, Facchini PJ (2015) Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat Chem Biol 11:728–732View ArticleGoogle Scholar
- Gao C, Zhang L, Xie Y, Hu C, Zhang Y, Li L, Wang Y, Ma C, Xu P (2013) Production of (3S)-acetoin from diacetyl by using stereoselective NADPH-dependent carbonyl reductase and glucose dehydrogenase. Bioresour Technol 137:111–115View ArticleGoogle Scholar
- Guo R, Nie Y, Mu XQ, Xu Y, Xiao R (2014) Genomic mining-based identification of novel stereospecific aldo-keto reductases toolbox from Candida parapsilosis for highly enantioselective reduction of carbonyl compounds. J Mol Catal B Enzym 105:66–73View ArticleGoogle Scholar
- Hall M, Bommarius AS (2011) Enantioenriched compounds via enzyme-catalyzed redox reactions. Chem Rev 111:4088–4110View ArticleGoogle Scholar
- Huisman GW, Collier SJ (2013) On the development of new biocatalytic processes for practical pharmaceutical synthesis. Curr Opin Chem Biol 17:284–292View ArticleGoogle Scholar
- Kataoka M, Kotaka A, Hasegawa A, Wada M, Yoshizumi A, Nakamori S, Shimizu S (2002) Old yellow enzyme from Candida macedoniensis catalyzes the stereospecific reduction of the C=C bond of ketoisophorone. Biosci Biotechnol Biochem 66:2651–2657View ArticleGoogle Scholar
- Kataoka M, Delacruz-Hidalgo A-R, Akond M, Sakuradani E, Kita K, Shimizu S (2004) Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis. Appl Microbiol Biotechnol 64:359–366View ArticleGoogle Scholar
- Kataoka M, Hoshino-Hasegawa A, Thiwthong R, Higuchi N, Ishige T, Shimizu S (2006) Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production. Enzyme Microb Technol 38:944–951View ArticleGoogle Scholar
- Kizaki N, Yasohara Y, Hasegawa J, Wada M, Kataoka M, Shimizu S (2001) Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes. Appl Microbiol Biotechnol 55:590–595View ArticleGoogle Scholar
- Lalonde J (2016) Highly engineered biocatalysts for efficient small molecule pharmaceutical synthesis. Curr Opin Biotechnol 42:152–158View ArticleGoogle Scholar
- Li B, Nie Y, Mu XQ, Xu Y (2016) De novo construction of multi-enzyme system for one-pot deracemization of (R, S)-1-phenyl-1,2-ethanediol by stereoinversion of (S)-enantiomer to the corresponding counterpart. J Mol Catal B Enzym 129:21–28View ArticleGoogle Scholar
- Liu W, Wang P (2007) Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol Adv 25:369–384View ArticleGoogle Scholar
- Munoz Solano D, Hoyos P, Hernaiz MJ, Alcantara AR, Sanchez-Montero JM (2012) Industrial biotransformations in the synthesis of building blocks leading to enantiopure drugs. Bioresour Technol 115:196–207View ArticleGoogle Scholar
- Nakamura K, Yamanaka R, Matsuda T, Harada T (2003) Recent developments in asymmetric reduction of ketones with biocatalysts. Tetrahedron Asymmetry 14:2659–2681View ArticleGoogle Scholar
- Nealon CM, Musa MM, Patel JM, Phillips RS (2015) Controlling substrate specificity and stereospecificity of alcohol dehydrogenases. ACS Catal 5:2100–2114View ArticleGoogle Scholar
- Ni Y, Xu JH (2012) Biocatalytic ketone reduction: a green and efficient access to enantiopure alcohols. Biotechnol Adv 30:1279–1288View ArticleGoogle Scholar
- Nie Y, Xu Y, Wang H, Xu N, Xiao R, Sun Z (2008) Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone. Biocatal Biotransform 26:210–219View ArticleGoogle Scholar
- Nie Y, Xiao R, Xu Y, Montelione GT (2011) Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones. Org Biomol Chem 9:4070–4078View ArticleGoogle Scholar
- Noey EL, Tibrewal N, Jiménez-Osés G, Osuna S, Park J, Bond CM, Cascio D, Liang J, Zhang X, Huisman GW, Tang Y, Houk KN (2015) Origins of stereoselectivity in evolved ketoreductases. Proc Natl Acad Sci USA 112:E7065–E7072Google Scholar
- Ou ZM, Zhao HB, Tang L, Zhang W, Yang GS (2014) Asymmetric synthesis of duloxetine intermediate (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol using immobilized Saccharomyces cerevisiae in liquid-core sodium alginate/chitosan/sodium alginate microcapsules. Bioprocess Biosyst Eng 37:2243–2250View ArticleGoogle Scholar
- Patel RN (2003) Microbial/enzymatic synthesis of chiral pharmaceutical intermediates. Curr Opin Drug Discov Devel 6:902–920Google Scholar
- Patel RN (2008) Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coord Chem Rev 252:659–701View ArticleGoogle Scholar
- Pazmino DET, Snajdrova R, Baas BJ, Ghobrial M, Mihovilovic MD, Fraaije MW (2008) Self-sufficient Baeyer–Villiger monooxygenases: effective coenzyme regeneration for biooxygenation by fusion engineering. Angew Chem Int Ed 47:2275–2278View ArticleGoogle Scholar
- Pesti J, DiCosimo R (2003) Recent progress in enzymatic resolution and desymmetrization of pharmaceuticals and their intermediates. Curr Opin Drug Discov Dev 6:884–901Google Scholar
- Pollard DJ, Woodley JM (2007) Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol 25:66–73View ArticleGoogle Scholar
- Prachayasittikul V, Ljung S, Isarankura-Na-Ayudhya C, Bulow L (2006) NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase. Int J Biol Sci 2:10–16View ArticleGoogle Scholar
- Ren Z-Q, Liu Y, Pei X-Q, Wang H-B, Wu Z-L (2015) Bioreductive production of enantiopure (S)-duloxetine intermediates catalyzed with ketoreductase ChKRED15. J Mol Catal B Enzym 113:76–81View ArticleGoogle Scholar
- Silva VD, Stambuk BU, da Graça Nascimento M (2012) Asymmetric reduction of (4R)-(−)-carvone catalyzed by baker’s yeast in aqueous mono-and biphasic systems. J Mol Catal B Enzym 77:98–104View ArticleGoogle Scholar
- Soni P, Banerjee U (2005) Biotransformations for the production of the chiral drug (S)-duloxetine catalyzed by a novel isolate of Candida tropicalis. Appl Microbiol Biotechnol 67:771–777View ArticleGoogle Scholar
- Suehrer I, Haslbeck M, Castiglione K (2014) Asymmetric synthesis of a fluoxetine precursor with an artificial fusion protein of a ketoreductase and a formate dehydrogenase. Process Biochem 49:1527–1532View ArticleGoogle Scholar
- Sun Z, Lonsdale R, Ilie A, Li G, Zhou J, Reetz MT (2016) Catalytic asymmetric reduction of difficult-to-reduce ketones: triple-code saturation mutagenesis of an alcohol dehydrogenase. ACS Catal 6:1598–1605View ArticleGoogle Scholar
- Tang C-G, Lin H, Zhang C, Liu Z-Q, Yang T, Wu Z-L (2011) Highly enantioselective bioreduction of N-methyl-3-oxo-3-(thiophen-2-yl) propanamide for the production of (S)-duloxetine. Biotechnol Lett 33:1435–1440View ArticleGoogle Scholar
- Wada M, Yoshizumi A, Furukawa Y, Kawabata H, Ueda M, Takagi H, Nakamori S (2004) Cloning and overexpression of the Exiguobacterium sp F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl) propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl) propanoate. Biosci Biotechnol Biochem 68:1481–1488View ArticleGoogle Scholar
- Wang LJ, Li CX, Ni Y, Zhang J, Liu X, Xu JH (2011) Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor. Bioresour Technol 102:7023–7028View ArticleGoogle Scholar
- Weckbecker A, Hummel W (2006) Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir. Biocatal Biotransform 24:380–389View ArticleGoogle Scholar
- Wohlgemuth R (2010) Asymmetric biocatalysis with microbial enzymes and cells. Curr Org Microbiol 13:283–292View ArticleGoogle Scholar
- Yamamoto H, Mitsuhashi K, Kimoto N, Matsuyama A, Esaki N, Kobayashi Y (2004) A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate. Biosci Biotechnol Biochem 68:638–649View ArticleGoogle Scholar
- Ye Q, Cao H, Yan M, Cao F, Zhang Y, Li X, Xu L, Chen Y, Xiong J, Ouyang P, Ying H (2010) Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate. Bioresour Technol 101:6761–6767View ArticleGoogle Scholar
- Zhang J, Tao S, Zhang B, Wu X, Chen Y (2014) Microparticle-based strategy for controlled release of substrate for the biocatalytic preparation of l-homophenylalanine. ACS Catal 4:1584–1587View ArticleGoogle Scholar
- Zhang D, Chen X, Chi J, Feng J, Wu Q, Zhu D (2015) Semi-rational engineering a carbonyl reductase for the enantioselective reduction of β-amino ketones. ACS Catal 5:2452–2457View ArticleGoogle Scholar
- Zhao H, van der Donk WA (2003) Regeneration of cofactors for use in biocatalysis. Curr Opin Biotechnol 14:583–589View ArticleGoogle Scholar