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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

Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to... Background: (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 synthe - sis of (S)-DHTP by bioreduction. Results: 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 identi- fied 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% −1 −1 e.e.) with yield of 97.66% was obtained from 20 g L DKTP within 8-h reaction, employing 40 g L glucose and −1 + −1 −1 0.1 mmol L NADP to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L h . Conclusion: 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. Keywords: Asymmetric reduction, Carbonyl reductase, Glucose dehydrogenase, Fusion enzyme, Cofactor regeneration pharmaceuticals, agrochemicals, functional materials, Background and fine chemicals (Munoz Solano et al. 2012; Ni and Xu Optically active alcohols are valuable and promising 2012; Patel 2008). Optically active heterocyclic alcohols chiral building blocks imposed in the production of have been widely used as important precursors for the synthesis of chiral drugs (Huisman and Collier 2013; Pesti *Correspondence: ynie@jiangnan.edu.cn; dwang@jiangnan.edu.cn and DiCosimo 2003; Pollard and Woodley 2007). Using Taiqiang Sun and Bin Li contributed equally to this work optically active heterocyclic alcohols as the key inter- School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu mediate, (S)-duloxetine, the effective antidepressant and Road, Wuxi 214122, China potent dual inhibitor of serotonin and norepinephrine Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 2 of 10 reuptake (Bymaster et al. 2001), can be prepared in high Carbonyl reductases require stoichiometric amounts of optical purity. nicotinamide cofactors (NADH or NADPH) as hydrogen The preparation of single enantiomers of chiral inter - donator for their activities. In  situ cofactor regeneration mediates has become particularly prevalent in the phar- would be feasible and prerequisite for practical applica- maceuticals industry (Patel 2003, 2008). Currently, tion of carbonyl reductase-catalyzed ketone reductions. enantiopure chiral alcohols can be acquired by ether Of the cofactor regeneration approaches, enzyme-cou- chemical or biological strategies. Compared with chemi- pled system has been developed and is particular pre- cal synthesis, biocatalytic route has become a subject of ferred for enzymatic reactions. Glucose dehydrogenase considerable interest due to its high chemo-, regio-, and (GDH) as the coupled enzyme has been widely applied in enantioselectivities, and mild reaction conditions and NAD(P)H regeneration (Gao et  al. 2013; Ye et  al. 2010), environmental benignity (Lalonde 2016; Ni and Xu 2012; due to its advantageous features of accessibility and Wohlgemuth 2010). In recent years, enzymatic asym- practicability (Hall and Bommarius 2011). For enzyme metric reduction of prochiral ketones for preparation coupling, construction of fusion protein has been devel- of optically active alcohols has gained increasing favor oped as an effective method for cofactor regeneration, (Nakamura et  al. 2003; Nealon et  al. 2015; Noey et  al. where two coding genes are combined by a short linker 2015; Sun et al. 2016; Wang et al. 2011). sequence to yield a single polypeptide exhibiting at least Based on the retrosynthetic strategy, biosynthesis two functions (Calam et  al. 2016; Farrow et  al. 2015; of (S)-(−)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1- Suehrer et  al. 2014). The proximity effect of fusion pro - propanamine (DHTP) or its substituted derivatives has tein generally reduces the intermediate diffusion distance been identified as the straightforward and efficient way and therefore increases the probability of intermediate for the preparation of (S)-duloxetine (Fig.  1) (Ren et  al. undergoing a sequential reaction step before escaping 2015; Tang et  al. 2011; Wada et  al. 2004; Zhang et  al. by diffusion (Conrado et  al. 2008; Dueber et  al. 2009). 2015). Immobilized cells of Saccharomyces cerevisiae In addition, the active sites of different enzymes for have been applied to produce (S)-(−)-3-N-methylamino- consecutive reactions can be brought in close proxim- 1-(2-thienyl)-1-propanol with the optical purity of ity to accelerate the processing of intermediate through 99% enantiomeric excess (e.e.) and conversion of 100% channeling (Castellana et  al. 2014). Fusion protein of −1 under the substrate concentration of 5  g  L after reac- oxidoreductases would greatly facilitate the involved tion for 48  h (Ou et  al. 2014). Otherwise, the whole-cell cofactor transfer between the two active centers of both catalytic system using Candida tropicalis has been devel- the enzyme for catalysis and the coupled enzyme for oped to produce (S)-DHTP with 99% e.e. at the conver- cofactor regeneration, resulting in accumulation of nec- sion of 84%, while the substrate tolerance of this system essary cofactor close to active center in some level and −1 was merely 1  g  L (Soni and Banerjee 2005). Although enhancement of desired biocatalytic reaction (Pazmino optically pure (S)-DHTP can be prepared by biocatalytic et al. 2008; Prachayasittikul et al. 2006). reductions, the substrate concentration and the corre- In this study, various recombinant carbonyl reductases sponding space–time yield of the whole-cell bioprocesses were evaluated for asymmetric reduction of N,N-dime- are yet limited. Therefore, it would be necessary to thyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to pro- develop suitable carbonyl reductase and the correspond- duce (S)-DHTP. Then the NADPH-dependent carbonyl ing enzymatic system for efficient synthesis of (S)-DHTP. reductase CR2 was obtained as the suitable candidate Fig. 1 Scheme for (S)-duloxetine synthesis from bioreduction of DKTP to (S)-DHTP involving fusion-based enzyme-coupled system Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 3 of 10 catalyzing asymmetric reduction. To efficiently improve strains cultivation was Luria–Bertani (LB) broth contain- −1 the preparation of (S)-DHTP, GDH was introduced to ing 50 μg mL kanamycin, and for CR2, the recombinant −1 construct the fusion enzyme CR2-L-GDH. Finally, the cells were cultured in LB broth containing 100  μg  mL fusion enzyme-involved reaction conditions were opti- ampicillin. Regarding all the recombinants used in this mized, and compared with free enzymes involving CR2 study, the cells were grown in 5  mL LB broth compris- and GDH, the fusion enzyme system was employed for ing the corresponding antibiotic at 37 °C and 200 rpm for the production of the key intermediate of (S)-duloxetine 8–10 h. Then the culture was transferred into a 2000 mL (Fig. 1). Erlenmeyer flask containing 500  mL fresh LB medium with the corresponding antibiotic. When the OD value −1 Methods of the culture reached 0.6–0.8, 1.0  mmol  L isopropyl Materials β-d-thiogalactopyranoside (IPTG) was added to induce DKTP, (S)-DHTP, and (R)-DHTP were purchased from protein expression. The cultures were cultivated at 17 °C TCI (Shanghai) Development Co., Ltd. The cofactors for 14 h, and then the cells were harvested by centrifuga- including NAD(P) and NAD(P)H were obtained from tion and washed three times with physiological saline for Sigma-Aldrich (St. Louis, USA). Diethylamine, ethyl further use. acetate, n-hexane, and isopropanol used for high-perfor- mance liquid chromatography were of chromatographic Preparation of crude enzyme grade from Sigma-Aldrich (St. Louis, USA). All other The recombinant cells were suspended in Triethanola - −1 chemicals used in this study were of analytical grade and mine-H PO (TEA buffer) (0.1 mol L , pH 8.0) and dis- 3 4 commercially available. rupted by sonication with an ultrasonic oscillator (Sonic Materials Co., USA). The cell debris was removed by Construction of fusion enzyme comprising CR2 and GDH centrifugation at 4  °C and 18,000×g for 30  min, and the Fusion expression system containing CR2 (GenBank supernatant was used as the crude enzyme for catalyz- Accession Number: AB183149) and GDH (GenBank ing the asymmetric reduction of the substrate DKTP. The −1 Accession Number: WP_013351020) was constructed concentration of crude enzyme was expressed as 2 g L using an aligned spacing sequence (GGGGSGGGGSG total soluble protein for biocatalytic asymmetric reac- GGGS) as the peptide linker between them. The forward tions in this work. primer 5′-GGAATTCCATATGATGACATTTACAGTGG TGACAG GC-3′ and the reverse primer 5′-AGAGCCAC Purification of recombinant enzymes CACCGCCAGAGCCACCACCGCCAG AGCCACCAC The harvested cells were suspended in TEA buffer −1 CGCCCCCACGGTACGCGCC-3′ were used to amplify (0.1  mol  L , pH 8.0) and treated with sonication using the fusion gene encoding CR2 and the linker. The for - an ultrasonic oscillator (Sonic Materials Co., USA). The ward primer 5′-GGCGGTGGTGGCTCTGGCGG TGGT cell debris were removed by centrifugation (18,000×g, GGCTCTGGCGGTGGTGGCTCTATGTATCCGGATT 30  min) at 4  °C, and the supernatant was applied to TAAAAGGAAAAG-3′ and the reverse primer 5′-ATAAGA a HisTrap HP affinity column (GE Healthcare, USA) −1 ATGCGGCCGCTTAACCGCGGCCTGC-3′ were used equilibrated with the buffer (20  mmol  L Tris–HCl, −1 −1 to amplify the fusion gene encoding the linker and GDH. 0.3  mol  L NaCl, 40  mmol  L imidazole, pH 8.0) on The fusion gene cr2- l-gdh was cloned using the overlap- an ÄKTA purifier system (GE Healthcare, USA). Then extension technique on the vector of pET-28a at the NdeI the absorbed proteins were eluted with a 40-min lin- −1 and NotI restriction sites. The recombinant plasmid pET- ear imidazole gradient buffer (0–0.5  mol  L imida- −1 −1 28a-cr2-l-gdh was transformed into the competent Escher- zole, 20  mmol  L Tris–HCl, 0.3  mol  L NaCl, pH 8.0) −1 ichia coli BL21(DE3), and the positive E. coli BL21(DE3)/ at a flow rate of 3  mL  min and the purified fractions −1 pET-28a-cr2-l-gdh was verified by DNA sequencing. The were exchanged into the buffer (20 mmol L Tris–HCl, −1 recombinant plasmid provides the fusion protein CR2-L- 0.3 mol L NaCl, pH 8.0) using disposable PD-10 desalt- GDH with a six-His tag at the N-terminus. ing columns (GE Healthcare, USA) (Li et  al. 2016). The preparations of purified enzymes were applied to activity Microorganisms and cultivation assay and measurement of kinetic parameters. The recombinant strains expressing candidate enzymes for DKTP reduction and the recombinant E. coli Enzyme assay and kinetic parameters BL21(DE3)/pET-28a-gdh expressing GDH used in this The enzyme assay mixture in 100 μL for reducing DKTP study were constructed and expressed previously in our or oxidating glucose activity of the purified CR2-L-GDH −1 −1 laboratory (Table  1) (Li et  al. 2016). For GDH and the comprised 0.1 mol L TEA buffer (pH 8.0), 5 mmol L −1 −1 fusion protein CR2-L-GDH, the medium for recombinant DKTP or 4  mmol  L glucose, 0.5  mmol  L NAD(P) Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 4 of 10 Table 1 Selection of the recombinant enzymes for catalyzing DKTP reduction to (S)-DHTP Enzyme GenBank ID Source Product configuration/yield (%) References S1 AB036927 Candida magnoliae – Kizaki et al. (2001) C1 AB084515 C. parapsilosis – Kataoka et al. (2004) C2 AB084516 C. parapsilosis – Kataoka et al. (2004) ADHR AY267012 Lactobacillus kefiri – Weckbecker and Hummel (2006) CR2 AB183149 Kluyveromyces marxianus S/91.31 ± 0.45 Kataoka et al. (2006) CR4 E59061 K. aestuarii – Yamamoto et al. (2004) KRD AF178079 Zygosaccharomyces rouxii S/55.18 ± 0.25 Costello et al. (2000) OYE AB126227 K. marxianus – Kataoka et al. (2002) RCR DQ295067 C. parapsilosis – Nie et al. (2008) SCR DQ675534 C. parapsilosis – Nie et al. (2011) SCR1 FJ939565 C. parapsilosis – Nie et al. (2011) SCR3 FJ939564 C. parapsilosis – Nie et al. (2011) CPAR1 JX512911 C. parapsilosis – Guo et al. (2014) CPAR2 JX512912 C. parapsilosis – Guo et al. (2014) CPAR3 JX512913 C. parapsilosis – Guo et al. (2014) CPAR4 JX512915 C. parapsilosis – Guo et al. (2014) CPAR5 JX512916 C. parapsilosis – Guo et al. (2014) CPAR6 JX512917 C. parapsilosis – Guo et al. (2014) CPAR7 JX512918 C. parapsilosis – Guo et al. (2014) CPAR8 JX512919 C. parapsilosis – Guo et al. (2014) −1 −1 −1 −1 + All reactions were carried out in 2 mL Tris–HCl buffer (0.05 mol L , pH 8.0) comprising 1 g L DKTP with addition of 10 g L glucose and 0.02 mmol L NADP at 30 °C and 200 rpm for 12 h. All the results were the average of three parallel replicates H, or NAD(P) , and appropriate amount of the puri- protein CR2-L-GDH-mediated reduction was carried −1 fied CR2-L-GDH. The reactions for activity assay were out in 10  mL TEA buffer (0.1  mol  L , pH 8.0) com- −1 −1 −1 carried out at 30  °C. The molar extinction coefficient prising 20  g  L DKTP, 40  g  L glucose, 0.2  mmol  L −1 −1 + of NAD(P)H was 6220  L  mol   cm . One unit (U) of NADP , and appropriate amount of CR2-L-GDH crude −1 enzyme activity was defined as 1 μmol of NAD(P)H con - enzyme (total soluble protein 2  g  L ). The above reac - sumed or generated per minute under the assay condi- tions were conducted at 30  °C and 200  rpm for 12  h. tions. The protein concentration was determined using After reaction, the mixture was centrifuged at 18,000×g Bradford reagents (Bio-Rad) as a standard. for 30  min and the supernatant was extracted with Initial velocities at various concentrations of the sub- ethyl acetate by vigorous mixing. The resulted organic −1 strate DKTP (0.05–1.0  mmol  L ) or the co-substrate layer was filtered through 0.22  μm PVDF syringe fil - −1 glucose (0.15–1.2  mmol  L ) were measured at 30  °C to ter (Troody Technology, Shanghai, China) for further obtain the apparent K values of the purified enzyme analysis. CR2-L-GDH. For activity assay to calculate kinetic The reaction conditions were optimized by analyz - parameters, the cofactor of NADPH or NA DP at satu- ing the optical purity and yield of product under various rated concentration towards the enzyme was added in parameters, including pH values ranging from 7.6 to 8.6 −1 the reaction mixture. The kinetic parameters were fur - (0.1  mol  L TEA buffer), reaction temperature varying ther determined from Lineweaver–Burk plots. from 20 to 45 °C, and DKTP concentrations ranging from −1 10 to 50 g L . Asymmetric reduction of DKTP and conditions To determine the optimal amounts of the added cofac- optimization tor and co-substrate, the corresponding reaction param- The reaction involving free CR2 and GDH was carried eters, involving NADP concentrations ranging from −1 −1 out in 10 mL TEA buffer (0.1 mol L , pH 8.0) compris- 0.005 to 0.2 mmol L and glucose concentrations rang- −1 −1 −1 −1 ing 10  g  L DKTP, 100  g  L glucose, 0.02  mmol  L ing from 5 to 200  g  L , were analyzed for their effects NADP , appropriate amount of CR2 crude enzyme on (S)-DHTP production from asymmetric reduction −1 (total soluble protein 2  g  L ), and GDH crude of DKTP by the catalytic system involving free CR2 and enzyme with the activity equivalent to CR2. The fusion GDH or the fusion protein CR2-L-GDH. Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 5 of 10 Analytical methods The optical purity and yield of (S)-DHTP were deter - mined by normal-phase high-performance liquid chro- matography (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 −1 (80:20:0.1, v/v/v) at a flow rate of 1.0  mL  min 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). Results and discussion Selection of candidate enzyme for (S)‑DHTP production from DKTP reduction The recombinant enzymes used in this study were expressed according to the protocol involving optimized conditions as described previously (Li et  al. 2016). Then the cell-free extracts of recombinant enzymes were employed for catalyzing asymmetric reduction of DKTP to produce (S)-DHTP. As shown in Table  1, only CR2 and KRD were active towards the substrate DKTP to produce the optically active DHTP in (S)-configuration (Fig.  2), while other enzymes showed no catalytic capa- bility towards the substrate, indicating that most of the involved enzymes do not have obvious capabilities of act- Fig. 2 Asymmetric reduction of DKTP by recombinant carbonyl ing to the substrate possessing heterocyclic group. Com- reductase. a Standard samples of DKTP, (S)-DHTP and (R)-PED; b reac- pared to KRD, CR2 was more efficient in the perspective tion products of productivity (Table  1), and hence the carbonyl reduc- tase CR2 was selected as the suitable enzyme for further biocatalysis. −1 pET-28a-cr2-l-gdh cells were induced with 1.0 mmol L For the reaction system involving the crude enzymes IPTG and supplied for SDS-PAGE analysis. Comparison of CR2 and GDH, the desired product (S)-DHTP with analysis between CR2 and the fusion protein on SDS- optical purity over 99.9% e.e. and yield of 90.43% was PAGE suggested that the fusion enzyme CR2-L-GDH −1 obtained from 10  g  L DKTP at 30  °C and pH 8.0, in was successfully constructed and expressed with the −1 the presence of 120  g  L glucose. When the substrate expected molecular weight, corresponding to the sum of −1 concentration was increased up to 20  g  L , however, both the theoretical molecular weights of CR2 (39.8 kDa) the yield of (S)-DHTP decreased significantly to 47.25%, and GDH (28.5 kDa) (Fig. 3). which would be probably attributed to cofactor defi - As the fusion enzyme CR2-L-GDH involving both ciency, besides the nature of substrate inhibition towards CR2 and GDH can be active towards both the substrate the functional enzyme. DKTP and the coupled co-substrate glucose, it would be necessary to evaluate the fusion enzyme by measuring its Fusion expression of CR2 and GDH and its catalytic activities and kinetic parameters towards the two sub- behavior strates, respectively. As shown in Table  2, the K values To further enhance the production of (S)-DHTP from of CR2-L-GDH towards DKTP and glucose were 0.32 and DKTP reduction, GDH, as the coupled enzyme for −1 0.47  mmol  L , respectively, suggesting its comparable recycling of the necessary cofactor, was supposed to affinities for DKTP and glucose. On the other hand, CR2- be integratedly expressed with CR2 in a fusion form L-GDH exhibited higher k /K value to DKTP than to cat m by introducing the peptide linker of aligned spac- glucose, indicating the fusion enzyme was more favora- ing sequence. The recombinant E. coli BL21(DE3)/ ble for catalyzing the bioreduction of DKTP and more Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 6 of 10 or cell-free systems, which in turn affects the synthesis of desired product in terms of optical purity and yield con- sequently (Silva et  al. 2012). For CR2-L-GDH catalyzing DKTP reduction, the optical purity of final product kept constant with the change of initial pH values, while the yield of (S)-DHTP was increased with the alteration of pH from 7.6 to 8.4 (Fig.  4a), which might be due to the generation of gluconic acid from GDH-catalyzed glu- cose oxidation. Thus, basic condition at initial pH 8.4 was favorable for CR2-L-GDH catalyzing DKTP reduction. Reaction temperature would also have obvious effects on activity, selectivity, and stability of biocatalysts includ- ing 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 Fig. 3 SDS-PAGE analysis of the recombinant CR2 and the fusion the enzyme at a relatively higher reaction temperature. protein CR2-L-GDH. Lane M Protein molecular weight marker; lane 1 Therefore, the reaction temperature at 40 °C was consid - recombinant CR2; lane 2 fusion protein CR2-L-GDH ered as the favorable factor for CR2-L-GDH catalyzing DKTP reduction. glucose should be loaded in the reaction system. Using the cell-free extract of recombinant E. coli BL21(DE3)/ Eec ff t of substrate concentration on CR2‑L‑GDH catalyzing −1 pET-28a-cr2-l-gdh as the catalyst, with DKTP of 20 g L , DKTP reduction optically pure (S)-DHTP (>99.9% e.e.) was obtained at The amount of substrate loading is a key issue for biocat - the yield of 83.26% at 30  °C and pH 8.0 (TEA buffer). alytic application potential. However, substrate inhibition These results indicated that it would be much potential has generally become a common issue for almost all of to improve the reaction efficiency under increased sub - the biocatalytic processes (Zhang et al. 2014). u Th s, effect −1 strate concentration of 20  g  L by the fusion enzyme of concentration of the substrate DKTP on the fusion CR2-L-GDH. enzyme CR2-L-GDH catalyzing asymmetric reduction was studied here to establish an efficient biocatalytic sys - Eec ff ts of pH and temperature on CR2‑L‑GDH catalyzing tem for the production of (S)-DHTP. As shown in Fig. 5, DKTP reduction substrate concentration did not have much impact on It is well known that pH plays a crucial role in biocatalytic optical purity of the product, while the yield of (S)-DHTP reactions. The ionic state of substrate and enzyme, espe - decreased obviously with the increase of substrate con- cially the local polarity of active site in enzyme, would be centration, especially when the amount of DKTP was −1 generally determined by the pH condition of correspond- increased from 20 to 25 g L in the reaction system. The ing reaction. Change of pH value of reaction buffer could effect of substrate inhibition might be resulted from the not only influence the selectivity and activity of the func - toxicity of the non-natural substrate towards the enzyme. tional enzymes involved in the reaction, but also the effi - To further enhance the reaction efficiency of DKTP −1 ciency of the required cofactor regeneration in whole-cell bioreduction, the substrate concentration of 20 g L was Table 2 Kinetic parameters of the fusion enzyme CR2-L-GDH towards DKTP and glucose, respectively −1 −1 −1 −1 −1 −1 Substrate V (μmol min  mg ) K (mmol L ) k (s ) k /K (L mol  s ) max m cat cat m DKTP 6.75 ± 0.15 0.32 ± 0.05 4.35 ± 0.07 1.37 × 10 Glucose 2.58 ± 0.12 0.47 ± 0.04 1.66 ± 0.12 3.54 × 10 −1 All reactions for the calculation of kinetic parameters were carried out in 0.1 mol L TEA buffer (pH 8.0) at 30 °C. For activity assay to calculate kinetic parameters, the cofactor of NADPH or NADP was added in the reaction mixture at saturated concentration towards the enzyme. All the results were the average of three parallel replicates Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 7 of 10 Fig. 5 Eec ff t of DKTP concentration on CR2-L-GDH catalyzing asym- metric synthesis of (S)-DHTP. All reactions were carried out in 10 mL −1 −1 TEA buffer (0.1 mol L , pH 8.4) comprising 10–50 g L DKTP with −1 + addition of 0.2 mmol L NADP at 40 °C and 200 rpm for 12 h. The concentration of glucose added in the reaction was 10 folds of that of DKTP accordingly. All the results were the average of three parallel replicates reduction. As shown in Fig.  6, by studying the effect of glucose concentration on DKTP reduction using the −1 crude enzyme of CR2-L-GDH, addition of 40  g  L glu- cose in the reaction resulted in synthesis of (S)-DHTP −1 (>99.9% e.e.) with the yield of 93.26% under 20  g  L DKTP after 10-h reaction. For cofactor regeneration, total turnover number (TTN) of the cofactor is defined as the number of moles Fig. 4 Eec ff t of pH (a) and temperature (b) on CR2-L-GDH catalyz- of product formed from per mole of cofactor during the ing asymmetric synthesis of (S)-DHTP. All reactions were carried out −1 −1 in 10 mL TEA buffer (0.1 mol L ) comprising 20 g L DKTP with −1 −1 + addition of 40 g L glucose and 0.2 mmol L NADP at 30 °C and 200 rpm for 12 h. All the results were the average of three parallel replicates adopted for the production of (S)-DHTP in term of the −1 −1 relatively higher space–time yield of 1.85 g L  h . Process regulation of CR2‑L‑GDH catalyzing DKTP reduction involving cofactor regeneration Coenzyme recycling is one of the most important issues encountered in biocatalytic reductions. Glucose as the co-substrate with equal molar ratio to the substrate DKTP would be theoretically enough to drive the biosyn- thesis by the fusion enzyme CR2-L-GDH. For the cell- free system involving the fusion enzyme, however, there Fig. 6 Eec ff t of glucose concentration on CR2-L-GDH catalyzing would be a series of biochemical pathways of metaboliz- asymmetric synthesis of (S)-DHTP. All reactions were carried out in −1 −1 ing glucose. Therefore, excess amount of glucose should 10 mL TEA buffer (0.1 mol L , pH 8.4) comprising 20 g L DKTP with −1 + be added into the reaction system to completely promote addition of 0.2 mmol L NADP at 40 °C and 200 rpm for 10 h. All the results were the average of three parallel replicates cofactor regeneration for CR2-L-GDH catalyzing DKTP Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 8 of 10 course of a complete reaction (Liu and Wang 2007). TTN generally indicates loss of cofactor due to degradation or incorrect regiochemistry of regeneration, and reac- tion rate and turnover number of biocatalyst (Zhao and van der Donk 2003). As shown in Table  3, the addition of less cofactor led to the increase of TTN. For the fixed −1 substrate concentration of 20 g L , however, addition of −1 + 0.1 mmol L NADP facilitated CR2-L-GDH catalyzing DKTP reduction, giving (S)-DHTP in the yield of 94.87%. After adjusting the fusion enzyme-catalyzed reac- tion by adding suitable amount of cofactor and glucose, during the reaction progress, optically pure (S)-DHTP −1 (>99.9% e.e.) was produced from 20  g  L DKTP and accumulated at the yield reaching 97.66% under the opti- mal catalytic conditions after reaction for 8  h (Fig.  7), Fig. 7 Reaction process of CR2-L-GDH catalyzing asymmetric syn- −1 −1 resulting in the space–time yield of 2.44  g  L  h . In thesis of (S)-DHTP. The reaction was carried out in 10 mL TEA buffer −1 −1 (0.1 mol L , pH 8.4) comprising 20 g L DKTP with addition of comparison with the previously reported literature (Ou −1 −1 + 40 g L glucose and 0.1 mmol L NADP at 40 °C and 200 rpm. All et  al. 2014), the substrate concentration was increased the results were the average of three parallel replicates 5 times with 24-fold increase of space–time yield. Con- sequently, compared with the catalytic system involving free CR2 and GDH, the fusion enzyme CR2-L-GDH was more efficient for catalyzing asymmetric reduction of Table 4 Comparison of  (S)-DHTP production efficiency DKTP to produce optically pure (S)-DHTP, giving 2.1- between free CR2 and GDH and fusion enzyme CR2-L-GDH −1 fold increase of the production yield towards 20  g  L Enzyme Substrate concentra‑ Yield (%) Optical purity −1 DKTP. On the other hand, (S)-DHTP was synthesized tion (g L ) (% e.e.) at the yield over 90% with the substrate concentration −1 CR2 and GDH 10 90.43 ± 0.37 >99.9 increased from 10 to 20 g L (Table  4). Since the crude −1 CR2 and GDH 20 47.25 ± 0.26 >99.9 enzyme (total soluble protein 2  g  L ) was used at the CR2-L-GDH 20 97.66 ± 0.16 >99.9 fixed enzyme loading level, it would be promising to fur - ther improve the synthesis of optically pure (S)-DHTP under higher substrate concentration by employing more efficiency of CR2 catalyzing synthesis of (S)-DHTP via fusion enzyme of CR2-L-GDH and regulating the corre- in  situ cofactor regeneration, the chimeric gene encod- sponding reaction process. ing the fusion protein comprising CR2 and GDH was constructed and expressed as the fusion enzyme CR2- Conclusion L-GDH. By regulation of the catalytic system and reac- The NADPH-dependent CR2 with excellent enantiose - tion process, the developed CR2-L-GDH catalytic system lectivity was selected for catalyzing asymmetric reduc- −1 −1 was achieved involving 20 g L DKTP, 40 g L glucose, tion of DKTP to (S)-DHTP. To enhance the catalytic −1 + and 0.1 mmol L NADP , under the optimized catalytic conditions at 40  °C and pH 8.4 for reaction  8  h. Conse- quently, optically pure (S)-DHTP (>99.9% e.e.) with the + yield of 97.66% was obtained with increased substrate Table 3 Eecfft of  NADP concentration on  CR2-L-GDH cat- −1 concentration of 20 g L DKTP using the fusion enzyme alyzing asymmetric synthesis of (S)-DHTP CR2-L-GDH. Therefore, the fusion strategy for construc - + −1 NADP (mmol L ) Optical purity (% e.e.) Yield (%) TTN tion of multi-enzyme system would be promising in 0.2 >99.9 92.75 ± 0.21 501 potential application for efficient biosynthesis of the pre - 0.1 >99.9 94.87 ± 0.15 1024 cursor of (S)-duloxetine. 0.05 >99.9 83.35 ± 0.23 1799 Authors’ contributions 0.02 >99.9 72.46 ± 0.18 3911 YN, DW, and YX designed the experiments; TS and BL performed the experi- ments; TS, YN, and DW wrote this manuscript. All authors read and approved 0.01 >99.9 64.64 ± 0.12 6977 the final manuscript. 0.005 >99.9 50.45 ± 0.28 10,891 −1 Author details All reactions were carried out in 10 mL TEA buffer (0.1 mol L , pH 8.4) −1 −1 1 comprising 20 g L DKTP with addition of 100 g L glucose at 40 °C and School of Biotechnology and Key Laboratory of Industrial Biotechnology, 200 rpm for 10 h. All the results were the average of three parallel replicates Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 9 of 10 China. State Key Laboratory of Food Science and Technology, Jiangnan carbonyl reductase and glucose dehydrogenase. Bioresour Technol University, 1800 Lihu Road, Wuxi 214122, China. 137:111–115 Guo R, Nie Y, Mu XQ, Xu Y, Xiao R (2014) Genomic mining-based identification Acknowledgements of novel stereospecific aldo-keto reductases toolbox from Candida par - Not applicable. apsilosis for highly enantioselective reduction of carbonyl compounds. J Mol Catal B Enzym 105:66–73 Competing interests Hall M, Bommarius AS (2011) Enantioenriched compounds via enzyme-cata- The authors declare that they have no competing interests. lyzed redox reactions. Chem Rev 111:4088–4110 Huisman GW, Collier SJ (2013) On the development of new biocatalytic Availability of data and materials processes for practical pharmaceutical synthesis. Curr Opin Chem Biol All data generated or analyzed during this study are included in this article. 17:284–292 Kataoka M, Kotaka A, Hasegawa A, Wada M, Yoshizumi A, Nakamori S, Shimizu Consent for publication S (2002) Old yellow enzyme from Candida macedoniensis catalyzes the All authors have read and approved to submit it to Bioresources and stereospecific reduction of the C=C bond of ketoisophorone. Biosci Bioprocessing. There is no conflict of interest of any author in relation to the Biotechnol Biochem 66:2651–2657 submission. Kataoka M, Delacruz-Hidalgo A-R, Akond M, Sakuradani E, Kita K, Shimizu S (2004) Gene cloning and overexpression of two conjugated polyketone Funding reductases, novel aldo-keto reductase family enzymes, of Candida parap- Financial supports from the National High Technology Research and Develop- silosis. 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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

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Springer Journals
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2017 The Author(s)
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2197-4365
DOI
10.1186/s40643-017-0151-y
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Abstract

Background: (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 synthe - sis of (S)-DHTP by bioreduction. Results: 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 identi- fied 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% −1 −1 e.e.) with yield of 97.66% was obtained from 20 g L DKTP within 8-h reaction, employing 40 g L glucose and −1 + −1 −1 0.1 mmol L NADP to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L h . Conclusion: 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. Keywords: Asymmetric reduction, Carbonyl reductase, Glucose dehydrogenase, Fusion enzyme, Cofactor regeneration pharmaceuticals, agrochemicals, functional materials, Background and fine chemicals (Munoz Solano et al. 2012; Ni and Xu Optically active alcohols are valuable and promising 2012; Patel 2008). Optically active heterocyclic alcohols chiral building blocks imposed in the production of have been widely used as important precursors for the synthesis of chiral drugs (Huisman and Collier 2013; Pesti *Correspondence: ynie@jiangnan.edu.cn; dwang@jiangnan.edu.cn and DiCosimo 2003; Pollard and Woodley 2007). Using Taiqiang Sun and Bin Li contributed equally to this work optically active heterocyclic alcohols as the key inter- School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu mediate, (S)-duloxetine, the effective antidepressant and Road, Wuxi 214122, China potent dual inhibitor of serotonin and norepinephrine Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 2 of 10 reuptake (Bymaster et al. 2001), can be prepared in high Carbonyl reductases require stoichiometric amounts of optical purity. nicotinamide cofactors (NADH or NADPH) as hydrogen The preparation of single enantiomers of chiral inter - donator for their activities. In  situ cofactor regeneration mediates has become particularly prevalent in the phar- would be feasible and prerequisite for practical applica- maceuticals industry (Patel 2003, 2008). Currently, tion of carbonyl reductase-catalyzed ketone reductions. enantiopure chiral alcohols can be acquired by ether Of the cofactor regeneration approaches, enzyme-cou- chemical or biological strategies. Compared with chemi- pled system has been developed and is particular pre- cal synthesis, biocatalytic route has become a subject of ferred for enzymatic reactions. Glucose dehydrogenase considerable interest due to its high chemo-, regio-, and (GDH) as the coupled enzyme has been widely applied in enantioselectivities, and mild reaction conditions and NAD(P)H regeneration (Gao et  al. 2013; Ye et  al. 2010), environmental benignity (Lalonde 2016; Ni and Xu 2012; due to its advantageous features of accessibility and Wohlgemuth 2010). In recent years, enzymatic asym- practicability (Hall and Bommarius 2011). For enzyme metric reduction of prochiral ketones for preparation coupling, construction of fusion protein has been devel- of optically active alcohols has gained increasing favor oped as an effective method for cofactor regeneration, (Nakamura et  al. 2003; Nealon et  al. 2015; Noey et  al. where two coding genes are combined by a short linker 2015; Sun et al. 2016; Wang et al. 2011). sequence to yield a single polypeptide exhibiting at least Based on the retrosynthetic strategy, biosynthesis two functions (Calam et  al. 2016; Farrow et  al. 2015; of (S)-(−)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1- Suehrer et  al. 2014). The proximity effect of fusion pro - propanamine (DHTP) or its substituted derivatives has tein generally reduces the intermediate diffusion distance been identified as the straightforward and efficient way and therefore increases the probability of intermediate for the preparation of (S)-duloxetine (Fig.  1) (Ren et  al. undergoing a sequential reaction step before escaping 2015; Tang et  al. 2011; Wada et  al. 2004; Zhang et  al. by diffusion (Conrado et  al. 2008; Dueber et  al. 2009). 2015). Immobilized cells of Saccharomyces cerevisiae In addition, the active sites of different enzymes for have been applied to produce (S)-(−)-3-N-methylamino- consecutive reactions can be brought in close proxim- 1-(2-thienyl)-1-propanol with the optical purity of ity to accelerate the processing of intermediate through 99% enantiomeric excess (e.e.) and conversion of 100% channeling (Castellana et  al. 2014). Fusion protein of −1 under the substrate concentration of 5  g  L after reac- oxidoreductases would greatly facilitate the involved tion for 48  h (Ou et  al. 2014). Otherwise, the whole-cell cofactor transfer between the two active centers of both catalytic system using Candida tropicalis has been devel- the enzyme for catalysis and the coupled enzyme for oped to produce (S)-DHTP with 99% e.e. at the conver- cofactor regeneration, resulting in accumulation of nec- sion of 84%, while the substrate tolerance of this system essary cofactor close to active center in some level and −1 was merely 1  g  L (Soni and Banerjee 2005). Although enhancement of desired biocatalytic reaction (Pazmino optically pure (S)-DHTP can be prepared by biocatalytic et al. 2008; Prachayasittikul et al. 2006). reductions, the substrate concentration and the corre- In this study, various recombinant carbonyl reductases sponding space–time yield of the whole-cell bioprocesses were evaluated for asymmetric reduction of N,N-dime- are yet limited. Therefore, it would be necessary to thyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to pro- develop suitable carbonyl reductase and the correspond- duce (S)-DHTP. Then the NADPH-dependent carbonyl ing enzymatic system for efficient synthesis of (S)-DHTP. reductase CR2 was obtained as the suitable candidate Fig. 1 Scheme for (S)-duloxetine synthesis from bioreduction of DKTP to (S)-DHTP involving fusion-based enzyme-coupled system Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 3 of 10 catalyzing asymmetric reduction. To efficiently improve strains cultivation was Luria–Bertani (LB) broth contain- −1 the preparation of (S)-DHTP, GDH was introduced to ing 50 μg mL kanamycin, and for CR2, the recombinant −1 construct the fusion enzyme CR2-L-GDH. Finally, the cells were cultured in LB broth containing 100  μg  mL fusion enzyme-involved reaction conditions were opti- ampicillin. Regarding all the recombinants used in this mized, and compared with free enzymes involving CR2 study, the cells were grown in 5  mL LB broth compris- and GDH, the fusion enzyme system was employed for ing the corresponding antibiotic at 37 °C and 200 rpm for the production of the key intermediate of (S)-duloxetine 8–10 h. Then the culture was transferred into a 2000 mL (Fig. 1). Erlenmeyer flask containing 500  mL fresh LB medium with the corresponding antibiotic. When the OD value −1 Methods of the culture reached 0.6–0.8, 1.0  mmol  L isopropyl Materials β-d-thiogalactopyranoside (IPTG) was added to induce DKTP, (S)-DHTP, and (R)-DHTP were purchased from protein expression. The cultures were cultivated at 17 °C TCI (Shanghai) Development Co., Ltd. The cofactors for 14 h, and then the cells were harvested by centrifuga- including NAD(P) and NAD(P)H were obtained from tion and washed three times with physiological saline for Sigma-Aldrich (St. Louis, USA). Diethylamine, ethyl further use. acetate, n-hexane, and isopropanol used for high-perfor- mance liquid chromatography were of chromatographic Preparation of crude enzyme grade from Sigma-Aldrich (St. Louis, USA). All other The recombinant cells were suspended in Triethanola - −1 chemicals used in this study were of analytical grade and mine-H PO (TEA buffer) (0.1 mol L , pH 8.0) and dis- 3 4 commercially available. rupted by sonication with an ultrasonic oscillator (Sonic Materials Co., USA). The cell debris was removed by Construction of fusion enzyme comprising CR2 and GDH centrifugation at 4  °C and 18,000×g for 30  min, and the Fusion expression system containing CR2 (GenBank supernatant was used as the crude enzyme for catalyz- Accession Number: AB183149) and GDH (GenBank ing the asymmetric reduction of the substrate DKTP. The −1 Accession Number: WP_013351020) was constructed concentration of crude enzyme was expressed as 2 g L using an aligned spacing sequence (GGGGSGGGGSG total soluble protein for biocatalytic asymmetric reac- GGGS) as the peptide linker between them. The forward tions in this work. primer 5′-GGAATTCCATATGATGACATTTACAGTGG TGACAG GC-3′ and the reverse primer 5′-AGAGCCAC Purification of recombinant enzymes CACCGCCAGAGCCACCACCGCCAG AGCCACCAC The harvested cells were suspended in TEA buffer −1 CGCCCCCACGGTACGCGCC-3′ were used to amplify (0.1  mol  L , pH 8.0) and treated with sonication using the fusion gene encoding CR2 and the linker. The for - an ultrasonic oscillator (Sonic Materials Co., USA). The ward primer 5′-GGCGGTGGTGGCTCTGGCGG TGGT cell debris were removed by centrifugation (18,000×g, GGCTCTGGCGGTGGTGGCTCTATGTATCCGGATT 30  min) at 4  °C, and the supernatant was applied to TAAAAGGAAAAG-3′ and the reverse primer 5′-ATAAGA a HisTrap HP affinity column (GE Healthcare, USA) −1 ATGCGGCCGCTTAACCGCGGCCTGC-3′ were used equilibrated with the buffer (20  mmol  L Tris–HCl, −1 −1 to amplify the fusion gene encoding the linker and GDH. 0.3  mol  L NaCl, 40  mmol  L imidazole, pH 8.0) on The fusion gene cr2- l-gdh was cloned using the overlap- an ÄKTA purifier system (GE Healthcare, USA). Then extension technique on the vector of pET-28a at the NdeI the absorbed proteins were eluted with a 40-min lin- −1 and NotI restriction sites. The recombinant plasmid pET- ear imidazole gradient buffer (0–0.5  mol  L imida- −1 −1 28a-cr2-l-gdh was transformed into the competent Escher- zole, 20  mmol  L Tris–HCl, 0.3  mol  L NaCl, pH 8.0) −1 ichia coli BL21(DE3), and the positive E. coli BL21(DE3)/ at a flow rate of 3  mL  min and the purified fractions −1 pET-28a-cr2-l-gdh was verified by DNA sequencing. The were exchanged into the buffer (20 mmol L Tris–HCl, −1 recombinant plasmid provides the fusion protein CR2-L- 0.3 mol L NaCl, pH 8.0) using disposable PD-10 desalt- GDH with a six-His tag at the N-terminus. ing columns (GE Healthcare, USA) (Li et  al. 2016). The preparations of purified enzymes were applied to activity Microorganisms and cultivation assay and measurement of kinetic parameters. The recombinant strains expressing candidate enzymes for DKTP reduction and the recombinant E. coli Enzyme assay and kinetic parameters BL21(DE3)/pET-28a-gdh expressing GDH used in this The enzyme assay mixture in 100 μL for reducing DKTP study were constructed and expressed previously in our or oxidating glucose activity of the purified CR2-L-GDH −1 −1 laboratory (Table  1) (Li et  al. 2016). For GDH and the comprised 0.1 mol L TEA buffer (pH 8.0), 5 mmol L −1 −1 fusion protein CR2-L-GDH, the medium for recombinant DKTP or 4  mmol  L glucose, 0.5  mmol  L NAD(P) Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 4 of 10 Table 1 Selection of the recombinant enzymes for catalyzing DKTP reduction to (S)-DHTP Enzyme GenBank ID Source Product configuration/yield (%) References S1 AB036927 Candida magnoliae – Kizaki et al. (2001) C1 AB084515 C. parapsilosis – Kataoka et al. (2004) C2 AB084516 C. parapsilosis – Kataoka et al. (2004) ADHR AY267012 Lactobacillus kefiri – Weckbecker and Hummel (2006) CR2 AB183149 Kluyveromyces marxianus S/91.31 ± 0.45 Kataoka et al. (2006) CR4 E59061 K. aestuarii – Yamamoto et al. (2004) KRD AF178079 Zygosaccharomyces rouxii S/55.18 ± 0.25 Costello et al. (2000) OYE AB126227 K. marxianus – Kataoka et al. (2002) RCR DQ295067 C. parapsilosis – Nie et al. (2008) SCR DQ675534 C. parapsilosis – Nie et al. (2011) SCR1 FJ939565 C. parapsilosis – Nie et al. (2011) SCR3 FJ939564 C. parapsilosis – Nie et al. (2011) CPAR1 JX512911 C. parapsilosis – Guo et al. (2014) CPAR2 JX512912 C. parapsilosis – Guo et al. (2014) CPAR3 JX512913 C. parapsilosis – Guo et al. (2014) CPAR4 JX512915 C. parapsilosis – Guo et al. (2014) CPAR5 JX512916 C. parapsilosis – Guo et al. (2014) CPAR6 JX512917 C. parapsilosis – Guo et al. (2014) CPAR7 JX512918 C. parapsilosis – Guo et al. (2014) CPAR8 JX512919 C. parapsilosis – Guo et al. (2014) −1 −1 −1 −1 + All reactions were carried out in 2 mL Tris–HCl buffer (0.05 mol L , pH 8.0) comprising 1 g L DKTP with addition of 10 g L glucose and 0.02 mmol L NADP at 30 °C and 200 rpm for 12 h. All the results were the average of three parallel replicates H, or NAD(P) , and appropriate amount of the puri- protein CR2-L-GDH-mediated reduction was carried −1 fied CR2-L-GDH. The reactions for activity assay were out in 10  mL TEA buffer (0.1  mol  L , pH 8.0) com- −1 −1 −1 carried out at 30  °C. The molar extinction coefficient prising 20  g  L DKTP, 40  g  L glucose, 0.2  mmol  L −1 −1 + of NAD(P)H was 6220  L  mol   cm . One unit (U) of NADP , and appropriate amount of CR2-L-GDH crude −1 enzyme activity was defined as 1 μmol of NAD(P)H con - enzyme (total soluble protein 2  g  L ). The above reac - sumed or generated per minute under the assay condi- tions were conducted at 30  °C and 200  rpm for 12  h. tions. The protein concentration was determined using After reaction, the mixture was centrifuged at 18,000×g Bradford reagents (Bio-Rad) as a standard. for 30  min and the supernatant was extracted with Initial velocities at various concentrations of the sub- ethyl acetate by vigorous mixing. The resulted organic −1 strate DKTP (0.05–1.0  mmol  L ) or the co-substrate layer was filtered through 0.22  μm PVDF syringe fil - −1 glucose (0.15–1.2  mmol  L ) were measured at 30  °C to ter (Troody Technology, Shanghai, China) for further obtain the apparent K values of the purified enzyme analysis. CR2-L-GDH. For activity assay to calculate kinetic The reaction conditions were optimized by analyz - parameters, the cofactor of NADPH or NA DP at satu- ing the optical purity and yield of product under various rated concentration towards the enzyme was added in parameters, including pH values ranging from 7.6 to 8.6 −1 the reaction mixture. The kinetic parameters were fur - (0.1  mol  L TEA buffer), reaction temperature varying ther determined from Lineweaver–Burk plots. from 20 to 45 °C, and DKTP concentrations ranging from −1 10 to 50 g L . Asymmetric reduction of DKTP and conditions To determine the optimal amounts of the added cofac- optimization tor and co-substrate, the corresponding reaction param- The reaction involving free CR2 and GDH was carried eters, involving NADP concentrations ranging from −1 −1 out in 10 mL TEA buffer (0.1 mol L , pH 8.0) compris- 0.005 to 0.2 mmol L and glucose concentrations rang- −1 −1 −1 −1 ing 10  g  L DKTP, 100  g  L glucose, 0.02  mmol  L ing from 5 to 200  g  L , were analyzed for their effects NADP , appropriate amount of CR2 crude enzyme on (S)-DHTP production from asymmetric reduction −1 (total soluble protein 2  g  L ), and GDH crude of DKTP by the catalytic system involving free CR2 and enzyme with the activity equivalent to CR2. The fusion GDH or the fusion protein CR2-L-GDH. Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 5 of 10 Analytical methods The optical purity and yield of (S)-DHTP were deter - mined by normal-phase high-performance liquid chro- matography (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 −1 (80:20:0.1, v/v/v) at a flow rate of 1.0  mL  min 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). Results and discussion Selection of candidate enzyme for (S)‑DHTP production from DKTP reduction The recombinant enzymes used in this study were expressed according to the protocol involving optimized conditions as described previously (Li et  al. 2016). Then the cell-free extracts of recombinant enzymes were employed for catalyzing asymmetric reduction of DKTP to produce (S)-DHTP. As shown in Table  1, only CR2 and KRD were active towards the substrate DKTP to produce the optically active DHTP in (S)-configuration (Fig.  2), while other enzymes showed no catalytic capa- bility towards the substrate, indicating that most of the involved enzymes do not have obvious capabilities of act- Fig. 2 Asymmetric reduction of DKTP by recombinant carbonyl ing to the substrate possessing heterocyclic group. Com- reductase. a Standard samples of DKTP, (S)-DHTP and (R)-PED; b reac- pared to KRD, CR2 was more efficient in the perspective tion products of productivity (Table  1), and hence the carbonyl reduc- tase CR2 was selected as the suitable enzyme for further biocatalysis. −1 pET-28a-cr2-l-gdh cells were induced with 1.0 mmol L For the reaction system involving the crude enzymes IPTG and supplied for SDS-PAGE analysis. Comparison of CR2 and GDH, the desired product (S)-DHTP with analysis between CR2 and the fusion protein on SDS- optical purity over 99.9% e.e. and yield of 90.43% was PAGE suggested that the fusion enzyme CR2-L-GDH −1 obtained from 10  g  L DKTP at 30  °C and pH 8.0, in was successfully constructed and expressed with the −1 the presence of 120  g  L glucose. When the substrate expected molecular weight, corresponding to the sum of −1 concentration was increased up to 20  g  L , however, both the theoretical molecular weights of CR2 (39.8 kDa) the yield of (S)-DHTP decreased significantly to 47.25%, and GDH (28.5 kDa) (Fig. 3). which would be probably attributed to cofactor defi - As the fusion enzyme CR2-L-GDH involving both ciency, besides the nature of substrate inhibition towards CR2 and GDH can be active towards both the substrate the functional enzyme. DKTP and the coupled co-substrate glucose, it would be necessary to evaluate the fusion enzyme by measuring its Fusion expression of CR2 and GDH and its catalytic activities and kinetic parameters towards the two sub- behavior strates, respectively. As shown in Table  2, the K values To further enhance the production of (S)-DHTP from of CR2-L-GDH towards DKTP and glucose were 0.32 and DKTP reduction, GDH, as the coupled enzyme for −1 0.47  mmol  L , respectively, suggesting its comparable recycling of the necessary cofactor, was supposed to affinities for DKTP and glucose. On the other hand, CR2- be integratedly expressed with CR2 in a fusion form L-GDH exhibited higher k /K value to DKTP than to cat m by introducing the peptide linker of aligned spac- glucose, indicating the fusion enzyme was more favora- ing sequence. The recombinant E. coli BL21(DE3)/ ble for catalyzing the bioreduction of DKTP and more Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 6 of 10 or cell-free systems, which in turn affects the synthesis of desired product in terms of optical purity and yield con- sequently (Silva et  al. 2012). For CR2-L-GDH catalyzing DKTP reduction, the optical purity of final product kept constant with the change of initial pH values, while the yield of (S)-DHTP was increased with the alteration of pH from 7.6 to 8.4 (Fig.  4a), which might be due to the generation of gluconic acid from GDH-catalyzed glu- cose oxidation. Thus, basic condition at initial pH 8.4 was favorable for CR2-L-GDH catalyzing DKTP reduction. Reaction temperature would also have obvious effects on activity, selectivity, and stability of biocatalysts includ- ing 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 Fig. 3 SDS-PAGE analysis of the recombinant CR2 and the fusion the enzyme at a relatively higher reaction temperature. protein CR2-L-GDH. Lane M Protein molecular weight marker; lane 1 Therefore, the reaction temperature at 40 °C was consid - recombinant CR2; lane 2 fusion protein CR2-L-GDH ered as the favorable factor for CR2-L-GDH catalyzing DKTP reduction. glucose should be loaded in the reaction system. Using the cell-free extract of recombinant E. coli BL21(DE3)/ Eec ff t of substrate concentration on CR2‑L‑GDH catalyzing −1 pET-28a-cr2-l-gdh as the catalyst, with DKTP of 20 g L , DKTP reduction optically pure (S)-DHTP (>99.9% e.e.) was obtained at The amount of substrate loading is a key issue for biocat - the yield of 83.26% at 30  °C and pH 8.0 (TEA buffer). alytic application potential. However, substrate inhibition These results indicated that it would be much potential has generally become a common issue for almost all of to improve the reaction efficiency under increased sub - the biocatalytic processes (Zhang et al. 2014). u Th s, effect −1 strate concentration of 20  g  L by the fusion enzyme of concentration of the substrate DKTP on the fusion CR2-L-GDH. enzyme CR2-L-GDH catalyzing asymmetric reduction was studied here to establish an efficient biocatalytic sys - Eec ff ts of pH and temperature on CR2‑L‑GDH catalyzing tem for the production of (S)-DHTP. As shown in Fig. 5, DKTP reduction substrate concentration did not have much impact on It is well known that pH plays a crucial role in biocatalytic optical purity of the product, while the yield of (S)-DHTP reactions. The ionic state of substrate and enzyme, espe - decreased obviously with the increase of substrate con- cially the local polarity of active site in enzyme, would be centration, especially when the amount of DKTP was −1 generally determined by the pH condition of correspond- increased from 20 to 25 g L in the reaction system. The ing reaction. Change of pH value of reaction buffer could effect of substrate inhibition might be resulted from the not only influence the selectivity and activity of the func - toxicity of the non-natural substrate towards the enzyme. tional enzymes involved in the reaction, but also the effi - To further enhance the reaction efficiency of DKTP −1 ciency of the required cofactor regeneration in whole-cell bioreduction, the substrate concentration of 20 g L was Table 2 Kinetic parameters of the fusion enzyme CR2-L-GDH towards DKTP and glucose, respectively −1 −1 −1 −1 −1 −1 Substrate V (μmol min  mg ) K (mmol L ) k (s ) k /K (L mol  s ) max m cat cat m DKTP 6.75 ± 0.15 0.32 ± 0.05 4.35 ± 0.07 1.37 × 10 Glucose 2.58 ± 0.12 0.47 ± 0.04 1.66 ± 0.12 3.54 × 10 −1 All reactions for the calculation of kinetic parameters were carried out in 0.1 mol L TEA buffer (pH 8.0) at 30 °C. For activity assay to calculate kinetic parameters, the cofactor of NADPH or NADP was added in the reaction mixture at saturated concentration towards the enzyme. All the results were the average of three parallel replicates Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 7 of 10 Fig. 5 Eec ff t of DKTP concentration on CR2-L-GDH catalyzing asym- metric synthesis of (S)-DHTP. All reactions were carried out in 10 mL −1 −1 TEA buffer (0.1 mol L , pH 8.4) comprising 10–50 g L DKTP with −1 + addition of 0.2 mmol L NADP at 40 °C and 200 rpm for 12 h. The concentration of glucose added in the reaction was 10 folds of that of DKTP accordingly. All the results were the average of three parallel replicates reduction. As shown in Fig.  6, by studying the effect of glucose concentration on DKTP reduction using the −1 crude enzyme of CR2-L-GDH, addition of 40  g  L glu- cose in the reaction resulted in synthesis of (S)-DHTP −1 (>99.9% e.e.) with the yield of 93.26% under 20  g  L DKTP after 10-h reaction. For cofactor regeneration, total turnover number (TTN) of the cofactor is defined as the number of moles Fig. 4 Eec ff t of pH (a) and temperature (b) on CR2-L-GDH catalyz- of product formed from per mole of cofactor during the ing asymmetric synthesis of (S)-DHTP. All reactions were carried out −1 −1 in 10 mL TEA buffer (0.1 mol L ) comprising 20 g L DKTP with −1 −1 + addition of 40 g L glucose and 0.2 mmol L NADP at 30 °C and 200 rpm for 12 h. All the results were the average of three parallel replicates adopted for the production of (S)-DHTP in term of the −1 −1 relatively higher space–time yield of 1.85 g L  h . Process regulation of CR2‑L‑GDH catalyzing DKTP reduction involving cofactor regeneration Coenzyme recycling is one of the most important issues encountered in biocatalytic reductions. Glucose as the co-substrate with equal molar ratio to the substrate DKTP would be theoretically enough to drive the biosyn- thesis by the fusion enzyme CR2-L-GDH. For the cell- free system involving the fusion enzyme, however, there Fig. 6 Eec ff t of glucose concentration on CR2-L-GDH catalyzing would be a series of biochemical pathways of metaboliz- asymmetric synthesis of (S)-DHTP. All reactions were carried out in −1 −1 ing glucose. Therefore, excess amount of glucose should 10 mL TEA buffer (0.1 mol L , pH 8.4) comprising 20 g L DKTP with −1 + be added into the reaction system to completely promote addition of 0.2 mmol L NADP at 40 °C and 200 rpm for 10 h. All the results were the average of three parallel replicates cofactor regeneration for CR2-L-GDH catalyzing DKTP Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 8 of 10 course of a complete reaction (Liu and Wang 2007). TTN generally indicates loss of cofactor due to degradation or incorrect regiochemistry of regeneration, and reac- tion rate and turnover number of biocatalyst (Zhao and van der Donk 2003). As shown in Table  3, the addition of less cofactor led to the increase of TTN. For the fixed −1 substrate concentration of 20 g L , however, addition of −1 + 0.1 mmol L NADP facilitated CR2-L-GDH catalyzing DKTP reduction, giving (S)-DHTP in the yield of 94.87%. After adjusting the fusion enzyme-catalyzed reac- tion by adding suitable amount of cofactor and glucose, during the reaction progress, optically pure (S)-DHTP −1 (>99.9% e.e.) was produced from 20  g  L DKTP and accumulated at the yield reaching 97.66% under the opti- mal catalytic conditions after reaction for 8  h (Fig.  7), Fig. 7 Reaction process of CR2-L-GDH catalyzing asymmetric syn- −1 −1 resulting in the space–time yield of 2.44  g  L  h . In thesis of (S)-DHTP. The reaction was carried out in 10 mL TEA buffer −1 −1 (0.1 mol L , pH 8.4) comprising 20 g L DKTP with addition of comparison with the previously reported literature (Ou −1 −1 + 40 g L glucose and 0.1 mmol L NADP at 40 °C and 200 rpm. All et  al. 2014), the substrate concentration was increased the results were the average of three parallel replicates 5 times with 24-fold increase of space–time yield. Con- sequently, compared with the catalytic system involving free CR2 and GDH, the fusion enzyme CR2-L-GDH was more efficient for catalyzing asymmetric reduction of Table 4 Comparison of  (S)-DHTP production efficiency DKTP to produce optically pure (S)-DHTP, giving 2.1- between free CR2 and GDH and fusion enzyme CR2-L-GDH −1 fold increase of the production yield towards 20  g  L Enzyme Substrate concentra‑ Yield (%) Optical purity −1 DKTP. On the other hand, (S)-DHTP was synthesized tion (g L ) (% e.e.) at the yield over 90% with the substrate concentration −1 CR2 and GDH 10 90.43 ± 0.37 >99.9 increased from 10 to 20 g L (Table  4). Since the crude −1 CR2 and GDH 20 47.25 ± 0.26 >99.9 enzyme (total soluble protein 2  g  L ) was used at the CR2-L-GDH 20 97.66 ± 0.16 >99.9 fixed enzyme loading level, it would be promising to fur - ther improve the synthesis of optically pure (S)-DHTP under higher substrate concentration by employing more efficiency of CR2 catalyzing synthesis of (S)-DHTP via fusion enzyme of CR2-L-GDH and regulating the corre- in  situ cofactor regeneration, the chimeric gene encod- sponding reaction process. ing the fusion protein comprising CR2 and GDH was constructed and expressed as the fusion enzyme CR2- Conclusion L-GDH. By regulation of the catalytic system and reac- The NADPH-dependent CR2 with excellent enantiose - tion process, the developed CR2-L-GDH catalytic system lectivity was selected for catalyzing asymmetric reduc- −1 −1 was achieved involving 20 g L DKTP, 40 g L glucose, tion of DKTP to (S)-DHTP. To enhance the catalytic −1 + and 0.1 mmol L NADP , under the optimized catalytic conditions at 40  °C and pH 8.4 for reaction  8  h. Conse- quently, optically pure (S)-DHTP (>99.9% e.e.) with the + yield of 97.66% was obtained with increased substrate Table 3 Eecfft of  NADP concentration on  CR2-L-GDH cat- −1 concentration of 20 g L DKTP using the fusion enzyme alyzing asymmetric synthesis of (S)-DHTP CR2-L-GDH. Therefore, the fusion strategy for construc - + −1 NADP (mmol L ) Optical purity (% e.e.) Yield (%) TTN tion of multi-enzyme system would be promising in 0.2 >99.9 92.75 ± 0.21 501 potential application for efficient biosynthesis of the pre - 0.1 >99.9 94.87 ± 0.15 1024 cursor of (S)-duloxetine. 0.05 >99.9 83.35 ± 0.23 1799 Authors’ contributions 0.02 >99.9 72.46 ± 0.18 3911 YN, DW, and YX designed the experiments; TS and BL performed the experi- ments; TS, YN, and DW wrote this manuscript. All authors read and approved 0.01 >99.9 64.64 ± 0.12 6977 the final manuscript. 0.005 >99.9 50.45 ± 0.28 10,891 −1 Author details All reactions were carried out in 10 mL TEA buffer (0.1 mol L , pH 8.4) −1 −1 1 comprising 20 g L DKTP with addition of 100 g L glucose at 40 °C and School of Biotechnology and Key Laboratory of Industrial Biotechnology, 200 rpm for 10 h. All the results were the average of three parallel replicates Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Sun et al. Bioresour. Bioprocess. (2017) 4:21 Page 9 of 10 China. State Key Laboratory of Food Science and Technology, Jiangnan carbonyl reductase and glucose dehydrogenase. Bioresour Technol University, 1800 Lihu Road, Wuxi 214122, China. 137:111–115 Guo R, Nie Y, Mu XQ, Xu Y, Xiao R (2014) Genomic mining-based identification Acknowledgements of novel stereospecific aldo-keto reductases toolbox from Candida par - Not applicable. apsilosis for highly enantioselective reduction of carbonyl compounds. J Mol Catal B Enzym 105:66–73 Competing interests Hall M, Bommarius AS (2011) Enantioenriched compounds via enzyme-cata- The authors declare that they have no competing interests. lyzed redox reactions. Chem Rev 111:4088–4110 Huisman GW, Collier SJ (2013) On the development of new biocatalytic Availability of data and materials processes for practical pharmaceutical synthesis. Curr Opin Chem Biol All data generated or analyzed during this study are included in this article. 17:284–292 Kataoka M, Kotaka A, Hasegawa A, Wada M, Yoshizumi A, Nakamori S, Shimizu Consent for publication S (2002) Old yellow enzyme from Candida macedoniensis catalyzes the All authors have read and approved to submit it to Bioresources and stereospecific reduction of the C=C bond of ketoisophorone. Biosci Bioprocessing. There is no conflict of interest of any author in relation to the Biotechnol Biochem 66:2651–2657 submission. Kataoka M, Delacruz-Hidalgo A-R, Akond M, Sakuradani E, Kita K, Shimizu S (2004) Gene cloning and overexpression of two conjugated polyketone Funding reductases, novel aldo-keto reductase family enzymes, of Candida parap- Financial supports from the National High Technology Research and Develop- silosis. Appl Microbiol Biotechnol 64:359–366 ment Program of China (863 Program) (2015AA021004), the National Natural Kataoka M, Hoshino-Hasegawa A, Thiwthong R, Higuchi N, Ishige T, Shimizu Science Foundation of China (NSFC) (21376107, 21336009, 21676120), the S (2006) Gene cloning of an NADPH-dependent menadione reductase Natural Science Foundation of Jiangsu Province (BK20151124), the 111 from Candida macedoniensis, and its application to chiral alcohol produc- Project (111-2-06), the High-end Foreign Experts Recruitment Program tion. 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Journal

"Bioresources and Bioprocessing"Springer Journals

Published: Dec 1, 2017

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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