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Sep 13, 2017 - Ketoreductase CgKR1. Gao-Wei Zheng,*. ,†,ǁ. Yuan-Yang Liu,. †,ǁ. Qi Chen,. †,ǁ. Lei Huang,. †. Hui-Lei Yu,. †. Wen-Yong Lou,. ‡. Chun-Xiu.
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Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1 Gao-Wei Zheng, Yuan-Yang Liu, Qi Chen, Lei Huang, Hui-Lei Yu, WenYong Lou, Chun-Xiu Li, Yun-Peng Bai, Ai-Tao Li, and Jian-He Xu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01933 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

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Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1 Gao-Wei Zheng,*,†,ǁ Yuan-Yang Liu,†,ǁ Qi Chen,†,ǁ Lei Huang,† Hui-Lei Yu,† Wen-Yong Lou,‡ Chun-Xiu Li,† Yun-Peng Bai,† Ai-Tao Li,§ and Jian-He Xu*,† †

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China ‡ Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China § Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr 45470, Germany ABSTRACT: Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. Based on computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1F92C/F94W variant was obtained that exhibited higher activity towards all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg−1 (54 to 775 U mg–1). Using CgKR1F92C/F94W as a catalyst, five substrates at high loading (>100 g–1 L–1) were reduced completely in gram-scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g–1 L–1 d–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1. KEYWORDS: asymmetric reduction, ketoreductase, chiral alcohol, protein engineering, substrate specificity, biocatalysis

INTRODUCTION Optically pure alcohols are highly valuable chiral intermediates used in the synthesis of fine chemicals and particularly pharmaceutical drugs. Asymmetric reduction of carbonyl compounds by biocatalysis to generate chiral alcohols represents a more environmentally friendly and sustainable process than traditional chemical synthesis methods. This approach operates under milder conditions and can generate products in 100% theoretical yield with exceptionally high stereoselectivity.1 Owing to its green credentials, considerable effort has been devoted to developing ketoreductases for the stereoselective synthesis of chiral alcohols of pharmaceutical interest.2 However, using naturally occurring enzymes as catalysts for the large-scale manufacture of chiral alcohols can suffer from intrinsic limitations, such as low catalytic activity (often 100 g–1 L–1), and high catalyst loading (often >20 g–1 L–1). Accordingly, only very few wild-type (WT) enzymes have been reported for the practical production of chiral alcohols.3,4 Although process engineering strategies5 can partially relieve these limitations, advances in protein engineering technologies have proven even much more powerful.6 Various enzymes have been successfully tailored by protein engineering to accept non-natural substrates,7 to catalyze novel reactions8 and to expand the range of substrates accepted.9 Regarding ketoreductases in particular, considerable effort has been

made in recent years to generate new enzymes by protein engineering in an attempt to more effectively reduce the desired carbonyl substrates under the practical process conditions,10 and facilitate their use in large-scale applications for the synthesis of optically active alcohols.11 Recently, we identified the versatile ketoreductase CgKR1 from Candida glabrata. The enzyme displays broad substrate specificity but very low catalytic activity towards the majority of substrates tested (for only one substrate was an activity over 50 U mg−1 observed).12 A CgKR1 variant (F92L/F94V/I99Y/G174A) was constructed by rational design, and this mutant enzyme exhibited enhanced activity towards aromatic α-keto esters such as methyl orthochlorobenzoylformate (7-fold increase) but significantly decreased activity towards aliphatic keto esters (24 U mg−1 for 10a compared with 114 U mg−1 for WT CgKR1).13 Very recently, You and co-workers also successfully engineered WT CgKR1 by modifying Phe92 and Tyr208 residues, affording mutants with higher activity and anti-Prelog stereoselectivity towards α-halo ketones.14,15 Although several positive CgKR1 mutants have been identified, these enzymes only display high specific activity towards a particular subset of substrates, and further engineering is required in order to obtain highly active mutants for more structurally diverse substrates. The main challenge now is to precisely identify the key residues that have a marked effect on the catalytic activity of CgKR1.

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Herein, we identified two residues probably affecting substrate binding in CgKR1 by molecular dynamics simulation and our previous work, and subsequently modified them to generate mutants with higher activity towards structurally diverse compounds. To highlight the industrial potential of the developed mutants, the large-scale synthesis of pharmaceutical intermediates was also carried out.

RESULTS AND DISCUSSION Exploring Key Residues of CgKR1 by Molecular Dynamics Simulations. To probe the key amino acid residues that may affect catalytic activity, molecular dynamics (MD) simulation was performed on WT CgKR1 in complex with the difficult-to-reduce ketone 17a (Figure 1A). Based on the binding mode shown in Figure 1A, although the substrate is stabilized in the binding pocket, no strong interactions between the catalytic triad (Ser134, Tyr175 and Lys179) and ketone 17a are observed throughout the entire simulation. Detailed analysis of the distance between the carbonyl group of the substrate and the catalytic Ser134 is shown in Figure S1, and the result demonstrates that the substrate binding position is distant from the catalytic residues. Furthermore, we found a strong π-π stacking interaction between the benzene ring of the Phe92 residue and the benzene ring of the catalytic Tyr175 residue, which forms a spatial barrier that blocks substrate binding with the catalytic residues, as has been observed previously for esterases.16 We speculated that F92 is a key residue affecting the activity of CgKR1, hence it was chosen for mutagenesis. Table 1. Kinetic Parameters of WT CgKR1 and its Variants for Substrate 17a Entry

Mutant

Km kcat kcat/Km (mM) (s–1) (s–1 mM–1) 6.50 ± 1 WT 10.4 ± 0.1 1.60 0.33 3.37 ± 2 F92T 8.72 ± 0.28 2.59 0.39 3.02 ± 3 F92I 21.2 ± 0.5 7.02 0.24 1.56 ± 4 F92M 41.2 ± 0.9 26.4 0.15 4.70 ± 5 F92L 156 ± 0.3 33.2 0.03 0.52 ± 6 F92C 128 ± 1 246 0.02 4.25 ± 7 F94W 9.25 ± 0.09 2.18 0.15 0.45 ± 8 F92C/F94W 250 ± 4 556 0.03 a Fold change improvement in kcat/Km over the WT enzyme.

Fold changea 1.0 1.6 4.4 17 21 154 1.4 348

Subsequently, a saturation mutagenesis library comprising all 20 natural amino acids at position 92 was initially constructed. We first examined the activities of cell-free extracts (CFEs) of all mutants toward 17a, and five mutants exhibited much higher activity than the WT enzyme. These five variants and the WT enzyme were purified and characterized by measuring kinetic parameters (Table 1). Compared with the WT enzyme, all five variants displayed increased catalytic efficiency (kcat/Km). Among them, variant F92L exhibited the highest kcat with a slight decrease in Km (Table 1, entry 5). Meanwhile, variant F92C displayed a decrease in Km by one order of magnitude and a 12-fold increase in kcat, leading to a 154-fold improvement in catalytic efficiency (Table 1, entry 6). The remarkable decrease in Km confirms that the F92C variant binds substrate 17a much more strongly than the WT enzyme.

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In addition, F94 surrounding the binding pocket is also likely to be an important residue affecting enzyme activity, based on our previous work.12,13 Therefore, a saturation mutagenesis library comprising all 20 amino acids at this position was also constructed. Interestingly, only F94W variant with an even larger side chain showed a slight increase in catalytic efficiency (Table 1, entry 7). Furthermore, the combination of F92C and F94W resulted in a positive synergistic effect on enzyme activity; the F92C/F94W double variant not only displayed a further decrease in Km, but also an additional 2-fold enhancement in kcat compared with the F92C variant, which resulted in a considerably higher kcat/Km of 556 s–1 mM–1, representing a 348-fold increase over the WT enzyme (Table 1, entry 8). Elucidating the Mechanism of the Enhanced Activity. To investigate the influence of these two residues on enzyme activity, an all-atoms MD simulation of the F92L, F92C and F92C/F94W mutants was performed in complex with substrate 17a. All four systems (CgKR1-WT, CgKR1-F92L, CgKR1F92C, and CgKR1-F92C/F94W) were simulated and a trajectory of about 50 ns for each system was analyzed in terms of root-mean-square deviations (RMSD) and root-mean-square fluctuation (RMSF) for all Cα atoms using the initial structure as template (Figure S2). With CgKR1-F92L, CgKR1-F92C and CgKR1-F92C/F94W mutants, the smaller side chains of L92 and C92 generate a larger binding pocket and cause a conformational rearrangement of the catalytic triad and the pyridine ring of the NADPH cofactor. In particular, the conformation of the side chain of Tyr175 shifts towards the inner surface of the binding pocket. Consequently, the substrate is bound more deeply in the pocket and forms stronger interactions than in the WT enzyme, as shown in Figure 1B, 1C and 1D. Compared with CgKR1-F92L variant, the Cys92 in the CgKR1-F92C and CgKR1-F92C/F94W variants affects the binding mode to a much greater extent than Leu92, not only because of the smaller side chain, but also by facilitating a more complex and stable hydrogen interaction network between the sulfur atom and other nearby atoms, as shown in Figure 1 and Table S2. In the CgKR1-F92C/F94W double mutant shown in Figure 1D, the indole ring of Trp 94 forms a stable π-π stacking interaction with the imidazole ring of His93 that is not observed in the CgKR1-F92C variant, as shown in Figure S3. This shift in the side chain of Trp 94 results in a significant spatial movement of the substrate. Furthermore, in addition to potentially stronger binding, the carbonyl group of the substrate faces towards the catalytic Ser134 and Tyr175 residues, and the substrate appears to be stabilized in the binding pocket in a more productive conformation. Therefore, the substrate assumes a more ‘activated’ conformation in the CgKR1-F92C/F94W mutant. The Molecular Mechanics Poisson Boltzmann (Generalized Born) surface area (MM-PB(GB)/SA) method was used to estimate the free binding energy of the substrate in four enzyme variants. As shown in Table S3, the binding free energy of substrate 17a was calculated to be −20.22 ± 3.04 kcal/mol in CgKR1-WT and −18.78 ± 2.32 kcal/mol in CgKR1-F92L. Interestingly, these values were increased to −12.9 ± 2.91 and −17.04 ± 3.63 kcal/mol with CgKR1-F92C and CgKR1F92C/F94W mutants, respectively. Although the binding free energy calculated for the WT enzyme was larger than for the enzyme variants, the substrate binds distant from the catalytic residues in the WT structure, as shown in Figure 1A, consistent with lower catalytic activity. Decomposition analysis of

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Figure 1. Analysis of the substrate binding mode in the four enzyme using MD simulations. (A) CgKR1-WT, (B) CgKR1-F92L, (C) CgKR1-F92C, (D) CgKR1-F92C/F94W. the total binding energy indicated that the van der Waals interactions (∆EVDW) contribute more to substrate binding than electrostatic interactions (∆EPB,elec). We noticed that although the calculated values differ from the experimental results, the overall trend in calculated the binding free energy is consistent with the experimentally determined Km values. The higher binding affinity of the substrate in the enzyme mutants was therefore partially responsible for the improved catalytic activity. Analysis of the Substrate Specificity of the Most Active Variant. A panel of carbonyl compounds (1a− −28a) with a broad range of structural features were used to characterize the substrate specificity of the WT reductase and the F92C/F94W variant. As shown in Table 2 and Table 3, the WT reductase exhibited an extremely low activity towards most substrates, and even no activity at all for some. Of the 28 substrates tested, only one was turned over with a specific activity of over 50 U mg−1 protein. By contrast, the F92C/F94W variant was considerably more active, and exhibited a specific activity above 50 U mg−1 for 13 substrates. For aliphatic substrates, the F92C/F94W variant also displayed improved activity of between 2.7- and 320-fold (up to 775 U mg−1) compared with WT CgKR1, which is far superior to the M3 variant developed in our previous work that showed enhanced activity towards aromatic α-keto esters but drastically lower activity towards aliphatic substrates (Table 2).13 For the best substrate 10a, the specific activity was further improved to 292 U mg−1 from 110 U mg−1. Meanwhile for azacyclic and aromatic substrates, the activity was 10- to 233-fold higher (up to 696 U mg−1) than that of the WT reductase (Table 3). Even for the challenging remote aliphatic ketoesters such as 13a, 14a and 15a,17 the F92C/F94W variant displayed improved activity. In addition, with the difficult-to-reduce ketones such as 16a-18a10c,18 and challenging α-halo ketones 22a and 23a,12,19 the F92C/F94W variant exhibited significantly improved activity (up to 301 U mg−1 with 17a).

Table 2. Specific Activity of WT CgKR1 and its F92C/F94W Variant Towards Aliphatic Ketones and Ketoesters O

O

O

1a

2a

O

O

O

O 9a

O

O

O

O

O 8a O

7a

6a

O

5a O

COOH

COOH

O

O

4a

3a

O

O

O

Cl

O

O O

10a

O

12a

11a O

O

O

O Cl

O

O

13a

Entry

O

Substrate

14a

O

15a

Specific activity (U/mg) WT

F92C/F94W

O

Fold change b

1

1a

0.09

7.7

86

2

2a

0.48

47

98

3

3a

0.79

34

43

4

4a

0.84

93

110

5

5a

4.1

58

14

6

6a

NAa

2.1

-

7

7a

NAa

0.6

-

8

8a

0.20

64

320

9

9a

22

775

35

10

10a

110

292

2.7

11

11a

0.88

20

23

12

12a

0.20

7.6

38

13

13a

0.98

24

25

14

14a

0.18

22

122

15 15a 0.08 15 188 a NA = no measurable activity. b Fold change improvement in the activity of the F92C/F94W variant over the WT enzyme.

Most pleasingly, the F92C/F94W variant displayed activity of more than 100 U mg−1 towards nine substrates with quite

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different structures, and therefore represents a promising biocatalyst for the industrial production of the corresponding chiral alcohols. These results confirmed our hypothesis that F92 and F94 are vital residues for improving the catalytic activity of CgKR1, and could also be modified to expand the substrate profile of other ketoreductases, particularly those possessing an active pocket similar to that in CgKR1, such as Gre2p from Sporobolomyces salmonicolor.20 Table 3. Specific Activity of WT CgKR1 and its F92C/F94W Variant Towards Azacyclic and Aromatic Ketones and Ketoesters

Entry

Substrate

Specific activity (U/mg) WT

F92C/F94W

Fold change a

16

16a

0.80

8.3

10

17

17a

1.4

301

215

18

18a

7.5

164

43

19

19a

0.15

2.88

19

20

20a

0.18

8.10

45

21

21a

0.30

31.8

106

22

22a

1.04

220

213

23

23a

0.11

25.0

233

24

24a

6.90

696

101

25

25a

16.1

453

28

26

26a

16.1

122

8

27

27a

24.0

470

20

28

28a

0.3

54

180

a

Fold change improvement in the activity of the F92C/F94W variant over the WT enzyme.

Preparative Bioreduction of Substrates Using the F92C/F94W Variant. Using the most active F92C/F94W variant, the feasibility for its use in the synthesis of pharmaceutically relevant chiral alcohols was further evaluated. Five substrates were chosen for asymmetric reduction on a gram scale. Lyophilized cells expressing the F92C/F94W variant were combined with glucose dehydrogenase (BmGDH) from Bacillus megaterium that was used to regenerate NADPH. (S)-3-hydrobutyrate [(S)-HBE] is an important chiral pharmaceutical building block for the synthesis of pheromones21 and antibiotics.22 Using the F92C/F94W variant, 130.1 g L−1 of substrate 9a was converted completely within 7 h, affording the corresponding product (S)-HBE with >99% ee and a space-

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time yield (STY) of 350 g L−1 d−1 (Table 4, entry 1). Table 4. Application of the F92C/F94W Variant for the Synthesis of Pharmaceutically Relevant Intermediates Entry Substrate /product

Substrate loading (g L−1)

Time (h)

Conversion /yield (%)

ee (%)

STY (g–1 L–1 d–1)

1

9a/9b

130.1

7

>99.0/77

>99.0 (S)

350

2

10a/10b

164.6

6

>99.0/78

97.4 (R)

520

3

17a/17b

100.0

4

>99.0/96

>99.0 (S)

583

4

25a/25b

100.0

7

>99.0/89

96.6 (R)

306

5

27a/27b

100.0

12

>99.0/86

>99.0 (R)

173

Ethyl (R)-4-chloro-3-hydroxybutyrate [(R)-CHBE] is a useful chiral precursor in the synthesis of biologically active compounds such as L-carnitine23 and (R)-4-amino-3hydroxybutyric acid.24 Recently, several ketoreductases have been developed for the asymmetric synthesis of (R)-CHBE.25 However, insufficient activity of these WT enzymes has hindered their practical applications. As ethyl 4-chloro-3oxobutanoate 10a is unstable in aqueous solution, a water/toluene biphasic system (1:1, v/v) was applied for the bioconversion of 10a. The F92C/F94W variant could efficiently catalyze the asymmetric reduction of 10a at 165 g L−1 loading in 50% toluene within 6 h, generating (R)-CHBE with 97.4% ee (Table 4, entry 2). Furthermore, the high STY of 520 g L−1 d−1 demonstrated that this bioreduction holds promise for the synthesis of (R)-CHBE. The piperidine moiety is prevalent in natural products, pharmaceutical drugs and biologically active molecules. For example, (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP] is a useful synthon in the synthesis of the drug Imbruvica that has been approved by the US in 2013 for the treatment of lymphoma. Recently, the biocatalytic synthesis of (S)-NBHP by asymmetric reduction of difficult-to-reduce ketone 17a has been developed.10c,26 In the present study, the F92C/F94W variant could completely reduce 100 g L−1 of 17a within 4 h (Table 4, entry 3), affording the corresponding product (S)NBHP with >99% ee in 96% isolated yield, and the STY of 583 g L−1 d−1 that is to our knowledge the highest value reported to date. (R)-Mandelic acid is the key chiral intermediate for the synthesis of semi-synthetic penicillins, cephalosporins, anti-tumor agents and anti-obesity drugs.27 In addition, it is also used as an important chiral resolving agent.28 Asymmetric reduction of 25a by the F92C/F94W variant proved to be an efficient process for the synthesis of (R)-mandelic acid; 100 g L−1 of 25a was converted to (R)-25a with 96.6% ee and 89% isolated yield (Table 4, entry 4). Ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is an important chiral intermediate for the synthesis of angiotensinconverting enzyme (ACE) inhibitors such as benazepril, cilazapril, enalapril and ramipril used to treat hypertension and congestive heart failure. Some efficient enzymatic asymmetric reduction processes with high substrate loading and STY have been reported.29 Herein, we also developed a highly efficient bioreduction process using the F92C/F94W variant as catalyst, in which (R)-HPBE was prepared with >99% ee and an STY

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of 173 g L−1 d−1 (Table 4, entry 5) without any further optimization. Finally, the biotransformation of 17a was scaled up to 100 mL to further confirm the feasibility of the process (Scheme 1). 17a (10 g) was converted completely to (S)-NBHP with >99% ee within 4 h. After extraction and normal work-up, 9.81 g of (S)-17b was isolated in 98% yield and an STY of 589 g L−1 d−1. This result indicates that the bioreduction is a practical process for the synthesis of the Imbruvica intermediate (S)NBHP.

Scheme 1. Biosynthesis of the Imbruvica intermediate 17b at 100 mL scale.

CONCLUSIONS MD simulation and previous literature analyses led to the identification of two residues, Phe92 and Phe94, that probably affect the activity of CgKR1. Mutagenesis of F92 and F94 resulted in the CgKR1-F92C/F94W variant that exhibited a marked improvement in activity towards structurally diverse substrates. Scale-up experiments confirmed the feasibility of the CgKR1-F92C/F94W variant for practical applications. In summary, we successfully engineered a useful and versatile ketoreductase for the practical synthesis of various chiral alcohols. Additionally, the findings provide guidance for the development of other similar ketoreductases and related enzymes.

EXPERIMENTAL SECTION Chemicals. 2-Pentanone (1a), 2-hexanone (2a), 2-octanone (3a), 2,5-hexanedione (5a) and N-Boc-3-pyrrolidinone (16a) were purchased from Aladdin Chemicals Co. Ltd. (Shanghai, China). Acetylacetone (4a), ethyl levulinate (11a), ethyl 4acetylbutyrate (13a), 4-chlorobutyrophenone (23a) and ethyl benzoylacetate (28a) were obtained from Alfa Aesar (Tianjing, China). Ethyl 4-chloroacetoacetate (10a) was obtained from Adamas Reagent, Ltd. 4-Oxodecanoic acid (6a), 5-oxodecanoic acid (7a), ethyl 4-chloro-3-oxobutanoate (12a) and ethyl 5-oxodecanoate (14a) were provided by Xiamen Bestally Biotechnology Co., Ltd. (Fujian, China). Ethyl acetoacetate (9a) and ethyl benzoylformate (22a) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Ethyl 8-chloro-6-oxooctanoate (15a) was obtained from Suzhou Fushilai Medicine & Chemical Co., Ltd. (Jiangsu, China). N-Boc-3-piperidinone (17a) was supplied by Shanghai Hobor Chemical Co., Ltd. (Shanghai, China). NBoc-4-piperidinone (18a), acetophenone (19a) and 2chloroacetophenone (22a) were purchased from J&K Chemi-

cal Co. Ltd. (Beijing, China), Methyl pyruvate (8a) was obtained from TCI Development Co. Ltd. (Shanghai, China). 4'Bromoacetophenone (20a) was obtained from Sigma-Aldrich (Shanghai, China). Butyrophenone (21a) was obtained from Energy Chemical Co. Ltd. (Shanghai, China). Methyl benzoylform (24a) was supplied by Jiangxi Keyuan Biopharm Co. Ltd. (Jiangxi, China). Ethyl 2-oxo-4-phenylbutyrate (27a) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methyl o-chloromandelate was synthesized by ourselves.12 (R)-9b, (S)-10b and (S)-17b were obtained from J&K Chemical Co. Ltd. (Beijing, China). All the other chemicals and reagents were purchased from authentic suppliers, at least of reagent grade and used without further purification. Mutagenesis. Site-directed mutagenesis was carried out by PCR using mutagenic primers (listed in Table S1) and plasmid pET28a-CgKR1 as template according to the manufacturer’s instructions of QuickChange (Stratagene). The PCR product (2 µL) digested by DpnI was transformed into 50 µL of Escherichia coli DH5α competent cells, and colonies after transformation were incubated for DNA sequencing until all the designed mutants were obtained. Subsequently, the plasmid of each mutant was extracted and transformed into E. coli BL21 (DE3) for target protein expression. Protein Expression and Purification. E. coli BL21 (DE3) cells carrying the recombinant plasmid were cultivated in 3 mL LB medium containing Kanamycin (50 µg mL−1) at 37°C and 180 rpm for 12 h. The overnight culture was inoculated into 100 mL LB medium and grown at 37 °C. When the culture’s optical density (OD600 nm) reached 0.6-0.8, 0.1 mM IPTG was added to induce the enzyme expression at 16 °C for additional 24 h. The cells were harvested by centrifugation (12,000 × g, 10 min) at 4 °C, washed with 0.9% NaCl solution and resuspended in sodium phosphate buffer (100 mM, pH 6.0). The cells were lysed by ultrasonication in an ice bath and the supernatant was collected by centrifugation at 28,000 × g for 30 min at 4 °C. The protein was purified using affinity chromatography with HisTrap FF crude column (GE, USA). The sample was loaded onto a standard Ni-NTA affinity column, and subsequently eluted with 20-500 mM imidazole solution in a flow rate of 2 ml/min. The target protein fractions were identified by SDS-PAGE before gathered. Protein concentration was measured with the Bradford protocol. Activity Assay. Enzyme activities of WT CgKR1 and its mutants were determined by measuring the decrease in the absorbance of NADPH at 340 nm (ε = 6.22 mM−1 cm−1) using an UV-Vis spectrophotometer (Shimadzu, Japan) at 30 °C. The assay mixture of 1 mL was composed of 962.5 µL potassium phosphate buffer (100 mM, pH 6.0), 10 µL substrates (200 mM), 17.5 µL NADPH (10 mM) and 10 µL purified protein with an appropriate concentration. One unit of enzyme activity is defined as the amount of enzyme catalyzing the oxidation of 1 µmol NADPH per minute. All experiments were conducted in triplicate. Determination of Kinetic Parameters. The kinetic parameters were determined by measuring the initial rate of enzymatic reaction at different substrate concentrations (0.2-10 mM) and 0.175 mM NADPH, and then calculated by nonlinear regression according to the Michaelis-Menten equation using OriginPro software. All experiments were conducted in triplicate. Molecular Dynamics Simulations. To elucidate the structure mechanism of the catalytic efficiency increase in different mutants, all-atoms molecular dynamics simulations were per-

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formed for WT CgKR1 and CgKR1-F92L, CgKR1-F92C CgKR1-F92C/F94W mutants. The 3D structure for each variant was constructed via homology modeling method using the structure PDB ID: 4PVD as a template with about 60% sequence similarity. AutoDock Vina was then used to obtain the starting structures of the wild type and CgKR1 mutants in complex with the substrate 17a. The LEaP module in Amber11 was used to add missing atoms and hydrogen. All the systems were solvated in an explicit TIP3p water box and extended with a thickness of at least 10 Å from the protein on each side of the box to generate the buffer between the protein and the periodic boundary in all direction. Sodium ions were added to neutralize each system. The topology and coordinate files for each system were generated via Amber FF99SB force filed and the force filed for the substrate was constructed using antechamber package and Gaussian 03. The force field parameters for the substrate were listed in Table S3. Finally, NAMD was used for running all the simulations. In the simulations, the Long-range electrostatic interactions were calculated by the particle-mesh Ewald (PME) method. The van der Waals (vdW) interactions were treated by smoothly turning off between 10.5 and 12 Å using a switching function. Each system was subjected to a four steps’ energy minimization with the steepest descents method. Each step was relaxed for 5000 steps. After minimization, each the simulation system was heated gradually from 0 K to 300 K in the NVT ensemble by Langevin dynamics. The NPT equilibration ensemble was then applied for the next 2 ns, followed by 50ns NPT production simulation. Trajectories generated from 0 ns-50 ns were gathered as conformational data for all the systems. Several analysis methods were applied for structural investigation such as Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and binding free energy estimation using the Molecular Mechanics Poisson Boltzmann (Generalized Born) surface area (MM-PB(GB)/SA) method. All the structural images were generated using VMD. Gram Scale Preparation of Chiral Alcohols Using the Most Active F92C/F94W Variant. Biotransformation was carried out on 10 ml scale by magnetic agitation at 30 °C. For substrates 9a, 17a, 25a and 27a, the reaction mixtures consist of a substrate with different loading, 50 mg lyophilized cells harboring CgKR1 F92C/F94W variant, 100 mg CFEs of BmGDH, 0.787 mg NADP+ (0.1 mM), 1.5 equivalents glucose, 9.5 mL phosphate buffer (100 mM, pH 6.0) and 0.5 mL EtOH. For substrate 10a, the reaction mixtures contain a substrate with different loading, 50 mg lyophilized cells harboring CgKR1 F92C/F94W variant, 100 mg CFEs of BmGDH, 0.787 mg NADP+ (0.1 mM), 1.5 equivalents glucose, 5 mL phosphate buffer (100 mM, pH 6.0) and 5 mL toluene. The reaction pH was maintained at 6.0 by adding 2 M Na2CO3 and the reaction was monitored by GC until the substrate was converted completely. The organic phase was extract with ethyl acetate (3 × 10 mL), dried using anhydrous Na2SO4 and evaporated under vacuum to obtained optically pure products. Synthesis of 9b. 1.30 g substrate 9a was converted completely within 7 h, generating 1.03 g (S)-9b as a colorless liquid in 77% isolated yield. 1H NMR (400 MHz, CDCl3) δ 4.25–4.11 (m, 3H), 2.69 (s, 1H), 2.44 (qd, J = 16.5, 6.1 Hz, 2H), 1.32– 1.17 (m, 6H). Synthesis of 10b. 1.65 g substrate 10a was converted completely within 6 h, generating 1.30 g (R)-10b as a colorless liquid in 78% isolated yield. 1H NMR (500 MHz, CDCl3). 1H

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NMR (400 MHz, CDCl3) δ 4.31–4.11 (m, 3H), 3.69–3.51 (m, 2H), 2.83 (s, 1H), 2.70–2.55 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H). Synthesis of 17b. 1.00 g substrate 17a was converted completely within 4 h, generating 0.97 g (S)-17b as a yellow oil in 96% isolated yield. 1H NMR (500 MHz, CDCl3). 1H NMR (400 MHz, CDCl3) δ 3.75 (dd, J = 12.9, 3.5 Hz, 2H), 3.58 (d, J = 26.5 Hz, 1H), 3.14–2.91 (m, 2H), 2.73 (s, 1H), 1.87 (s, 1H), 1.78–1.67 (m, 1H), 1.44 (d, J = 14.1 Hz, 11H). Synthesis of 25b. 1.00 g substrate 25a was converted completely within 7 h, generating 0.89 g (R)-25b as a light yellow liquid in 89% isolated yield. 1H NMR (500 MHz, CDCl3). 1H NMR (400 MHz, CDCl3) δ 7.46–7.28 (m, 5H), 5.16 (s, 1H), 4.33–4.11 (m, 2H), 3.44 (s, 1H), 1.23 (t, J = 7.1 Hz, 3H). Synthesis of 27b. 1.00 g substrate 27a was converted completely within 12 h, generating 0.87 g (R)-27b as a colorless liquid in 86% isolated yield. 1H NMR (500 MHz, CDCl3). 1H NMR (400 MHz, CDCl3) δ 7.32–7.27 (m, 2H), 7.20 (ddd, J = 7.2, 6.1, 1.8 Hz, 3H), 4.20 (ddd, J = 11.1, 9.2, 5.6 Hz, 3H), 2.86–2.64 (m, 3H), 2.12 (dddd, J = 13.7, 9.6, 7.2, 4.0 Hz, 1H), 1.95 (dddd, J = 13.8, 9.2, 7.8, 5.9 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H). Asymmetric Reduction of 17a at 100 mL Scale. 10.0 g ketone 17a (50 mmol) was added into 95 mL phosphate buffer ((100 mM, pH 6.0) containing 0.5 g lyophilized cells harboring CgKR1 F92C/F94W variant, 1.0 g dry CFEs of BmGDH, 7.87 mg NADP+ (0.1 mM), 13.5 g glucose (1.5 equiv.) and 5 mL EtOH. The bioconversion was performed by magnetic agitation at 30 °C for 6 h, and the pH was maintained at 6.0 using 2 M Na2CO3. After extraction and normal work-up, 9.81 g of (S)-17b was isolated in 97% yield.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Supplementary tables and figures for primers list, molecular dynamics simulation, HPLC and GC analytic conditions and SDS-PAGE of pure protein; HPLC and GC chromatograms of chiral alcohols; 1H NMR profiles with the chiral alcohols produced by the best mutant.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. * E-mail: [email protected]. Author Contributions ǁ

These authors contributed equally to this work (G.W.Z., Y.Y.L. and Q.C.).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 21472045, 21536004 & 21776085), the Fundamental Research Funds for the Central Universities (22A201514043) and Shanghai Pujiang Program (15PJ1401200).

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