Directed Evolution of Carbonyl Reductase from Rhodosporidium

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Directed evolution of carbonyl reductase from Rhodosporidium toruloides and its application in stereoselective synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate Zhi-Qiang Liu, Lin Wu, Xiao-Jian Zhang, Ya-Ping Xue, and Yu-Guo Zheng J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

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Journal of Agricultural and Food Chemistry

Title: Directed evolution of carbonyl reductase from Rhodosporidium toruloides and its application

in

stereoselective

synthesis

of

tert-butyl

(3R,5S)-6-chloro-

3,5-dihydroxyhexanoate

Authors and Affiliation: Zhi-Qiang Liua,b, Lin Wua,b, Xiao-Jian Zhang a,b, Ya-Ping Xue a,b and Yu-Guo Zheng a,b * a

Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and

Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China b

Engineering Research Center of Bioconversion and Biopurification of Ministry of Education,

Zhejiang University of Technology, Hangzhou 310014, China

*Corresponding author: Fax: +86-571-88320630; Tel: +86-571-88320630; E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH)

2

is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase

3

RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward

4

tert-butyl (S)-6-chloro-3-hydoxy-5-oxohexanoate ((S)-CHOH). To improve the

5

activity of RtSCR9, random mutagenesis and site-saturation mutagenesis were

6

performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys and

7

mut-Phe156Gln). These mutants exhibited 1.94-, 3.03- and 1.61-fold, and 1.93-, 3.15-

8

and 1.97-fold improvement on the specific activity and kcat/Km, respectively.

9

Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose

10

dehydrogenase (GDH) was conducted. The yield and enantiomeric excess of

11

(3R,5S)-CDHH respectively reached 98% and 99% after 8-h bioconversion in a single

12

batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol

13

L-1 h-1 g-1 wet cell weight. This study presents a new carbonyl reductase for efficient

14

synthesis of (3R,5S)-CDHH.

15

KEYWORDS:

16

reductase, random mutagenesis, site-saturation mutagenesis, single batch reaction

tert-butyl

(3R,5S)-6-chloro-3,5-dihydroxyhexanoate,

2


carbonyl

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17

INTRODUCTION

18

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a

19

promising intermediate applied in the side chain synthesis of atorvastatin and

20

rosuvastatin, cholesterol regulation drugs used in preventing cardiovascular diseases,

21

such as hypercholesterolemia, atherosclerosis and coronary heart disease.1-3 Chemical

22

synthesis of (3R,5S)-CDHH typically starts with (S)-epichlorohydrin followed by a

23

series of catalytic reactions involving expensive metal catalysts and environmentally

24

unfriendly organic solvents. To introduce the second chiral center in the chemical

25

synthesis of (3R,5S)-CDHH, sodium borohydride (NaBH4) is used to reduce the

26

carbonyl at C-3 under Parasad’s conditions (Et2BOMe, -80°C to -75°C).4,5 Although

27

(3R,5S)-CDHH can be totally synthesized within a short processing period by

28

chemical synthesis, difficulties remain in producing a final product with sufficient

29

enantiomeric purity. In addition, complex protection-deprotection processes, low

30

overall yield and high environmental pressure have made chemical synthesis less

31

competitive.6 Compared to chemical synthesis, biosynthesis has been an alternative in

32

the preparation of (3R,5S)-CDHH, along with the merits of low catalyst cost, high

33

enantioselectivities and a broad functional group tolerability, which is dominant in

34

large-scale pharmaceutical manufacture of important chiral intermediates.7-9

35

Carbonyl reductases (EC 1.1.1.148) have been demonstrated to be valuable

36

biocatalysts in the reduction of ketones to enantiopure alcohols with advantages

37

including mild catalytic conditions, chemo-, regio- and stereo-selectivity, and no

38

heavy metal contamination. So far, considerable studies have been performed on 3



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39

biotransformation of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) and tert-butyl

40

(S)-6-chloro-3-hydoxy-5-oxohexanoate ((S)-CHOH) by carbonyl reductases to

41

produce

42

enantioselectivity in the reduction of CDOH at C-5 to produce (S)-CHOH with over

43

99.5% enantiomeric excess (e.e.).11-13 Previously, a two-step biotransformation

44

process was established using LkADH1 and LkADH2 from Lactobacillus kefir in a

45

simple batch process to produce (3R,5S)-CDHH with >99% e.e. and 47.5% yield.14-18

46

Subsequently, fed batch processes and two-phase biotransformation systems were

47

established.19-21 A recombinant carbonyl reductase from Candida magnoliae

48

coexpressed with a glucose dehydrogenase highly efficiently catalyzed the reduction

49

of 200 g/L (S)-CHOH to prepare (3R,5S)-CDHH with 97.2% yield and 98.6%

50

diastereoisomeric excess (d.e.).22,23 Recently, liquid-core immobilized Saccharomyces

51

cerevisiae CGMCC No.2233 was used as catalyst to accomplish 100% conversion of

52

(S)-CHOH to produce (3R,5S)-CDHH with >99% d.e. when initial substrate

53

concentration was less than 50 g/L.24 Enzymatic asymmetric reductions for

54

commercial application have in general been, however, limited to poor substrate

55

tolerance, insufficient stability and low volumetric productivity. Thus, biocatalysts

56

with evolved characteristics are still urgently demanded to meet the large-scale

57

production of (3R,5S)-CDHH.

(3R,5S)-CDHH.10

Lactobacillus

brevis

(LbADH)

showed

a

high

58

Modification of carbonyl reductases is mainly focused on the improvement of

59

catalytic activity, stereoselectivity, stability and cofactor specificity.25-28 Codexis

60

Corporation (Redwood, CA) engineered a ketoreductase gene from Saccharomyces 4


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61

cerevisiae by rounds of directed evolution, resulting in a 99.8% conversion of 13 g/L

62

substrate (S)-CHOH with 99.2% d.e. in a 4-h reaction compared to wild type

63

ketoreductase with 37% yield, 97.3% d.e. in a 20-h reaction.29 A tetrad mutant of

64

LkADH through rational design was screened, exhibiting a 3.7- and 42-fold

65

improvement in specific activity toward CDOH over LbADH and wild-type LkADH,

66

respectively.30 Recently, the catalytic efficiency of aldo-keto reductase toward

67

tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate was improved 11.25-fold through

68

rational protein modification. The mutant (KIAKR-Tyr295Trp/Trp296Leu) catalyzed

69

the asymmetric reduction with tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate

70

accumulated up to 162.7 mM with >99.5% d.e..31

71

In our previous work, a carbonyl reductase RtSCR9 (Genbank accession No.

72

EMS 18622.1) from Rhodosporidium toruloides was screened and showed

73

considerable activity toward (S)-CHOH.32 To improve the catalytic efficiency for

74

industrial application, random mutagenesis and site-saturation mutagenesis were

75

performed. Three positive variants showing 1.94-, 3.03- and 1.61-fold improvement in

76

specific activity were obtained. Apparent kinetic parameters, molecular modeling and

77

docking experiments were used to illustrate the mechanism of the changes in enzyme

78

characteristics. Bioconversion of 1 M (S)-CHOH was conducted in a single batch

79

reaction. The variant mut-Ile144Lys exhibited an enhanced catalytic ability

80

with >98% yield and >99% e.e. within 8 h of reaction, indicating its potential

81

application in upscale production of (3R,5S)-CDHH.

82

MATERIALS AND METHODS 5



83

Microorganism, media and chemicals. Escherichia coli BL21(DE3)

84

(Invitrogen, Carlsbad, CA) was used as the host cell and plasmid pET-28a(+)

85

(Novagen, Darmstadt, Germany) was used for cloning and expression. E. coli

86

BL21(DE3)/pET28a-RtSCR9 was used for expression of carbonyl reductase gene

87

RtSCR9. E. coli BL21(DE3)/pET28a-GDH carrying a glucose dehydrogenase (GDH)

88

gene was used for the “enzyme-coupled” cofactor regeneration system.33 E. coli cells

89

were cultured in Luria-Bertani (LB) medium containing 5 g of yeast extract, 10 g of

90

tryptone, and 10 g of NaCl per liter. Kanamycin (Kan) and the inducer

91

isopropylthio-β-galactoside (IPTG) were purchased from Sango Biotech (Shanghai,

92

China). Nicotinamide adenine dimucleotide phosphate (NADPH) was obtained from

93

Roche (Karlsruhe, Germany). Standards of (S)-CHOH and (3R,5S)-CDHH were

94

purchased from J&K Scientific Ltd. (Shanghai, China). All other chemicals were of

95

analytical grade purity and commercially available.

96

Analytical methods. The concentrations of (S)-CHOH and (3R,5S)-CDHH

97

were determined by high-performance liquid chromatography (HPLC) (Shimadzu Co.,

98

Kyoto, Japan) using an Agilent Zorbax SB-C8 column (150×4.6 mm, particle size 5

99

µm, Agilent Technologies Co., Santa Clara, CA) with UV detection at 210 nm. The

100

mobile phase was a mixture of acetonitrile and ultrapure water (30:70 v/v). The

101

retention times of (S)-CHOH and (3R,5S)-CDHH were 6.11 min and 9.66 min,

102

respectively, at 40°C, with a flow rate of 1 mL/min (Figure S1).

103

The e.e. values of (3R,5S)-CDHH were determined on a Chiracel OD-H column

104

(250×4.6 mm, particle size 5 µm, Daicel Chemical Industries, Tokyo, Japan) at 215 6


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105

nm using hexane and isopropanol (85:15 v/v) as the mobile phase.30 The column

106

conditions were 40°C at a flow rate of 1 mL/min. Samples from the aqueous reaction

107

mixture containing hydroxyl keto ester were extracted into the mobile phase by

108

vigorous mixing. After centrifuging at 12,000×g for 3 min, the organic phase was

109

collected and filtered before HPLC analysis. The retention times of (S)-CHOH and

110

(3R,5S)-CDHH were 5.09 min and 5.90 min, respectively (Figure S2).

111

Construction of mutant library. A random mutagenesis library was

112

established through error-prone PCR and megaprimer PCR. The recombinant plasmid

113

pET28a-RtSCR9 was used as template DNA and the primers for error-prone PCR

114

were

115

5’-TCTACCATGGCAAGAACGTCC-3’. Appropriate amounts of Mg2+ and Mn2+

116

were added to the PCR reaction mixture in order to control the mismatch rate of base

117

pairing. The final error-prone PCR reaction mixture contained MgCl2 1.5 mM, MnCl2

118

0.3 mM, dATP 0.1 mM, dGTP 0.1 mM, dTTP 0.5 mM, dCTP 0.5 mM, 30 pmol each

119

primer, 10 ng template DNA, and 5 U Taq polymerase, diluted to a final volume of 50

120

µL by adding ddH2O. The error-prone PCR reaction condition started at 94°C for 3

121

min followed by amplification for 30 cycles: 95°C 30 s, 55°C 30 s, and 72°C 1 min,

122

followed by elongation at 72°C for 10 min. Subsequently, the target gene with

123

mutations from error-prone PCR was used as megaprimer to create random

124

mutagenesis libraries through megaprimer PCR of the whole plasmid (MEGAWHOP).

125

The 50 µL megaprimer PCR reaction mixture contained 2×PCR buffer 25 µL, 10 mM

126

dNTP 1 µL, 10 ng template DNA, 2 µL megaprimer, 2.5 U Phanta Max super-Fidelity

5’-TATGTCTTCGCCTACTCCCAAC-3’

7


and


127

DNA polymerase and 20.5 µL ddH2O following the procedure of denaturation at 98°C

128

for 3 min, 26 cycles for amplification 10 s at 98°C, 5 s at 55°C and 6 min at 72°C,

129

then elongation at 72°C for 10 min. The PCR products were treated with 40 U DpnI at

130

37°C for 3 h to remove the original template plasmid. The treated PCR products were

131

transformed to E. coli BL21(DE3) by heat-shock treatment. Transformants were

132

plated on LB-agar medium supplemented with 50 µg/mL of Kan and incubated for 12

133

h at 37°C. The mutations were determined by DNA sequencing.

134

High throughput screening and HPLC assay. A two-step screening

135

method based on the high-throughput screening combined with HPLC analysis was

136

designed to select positive mutants. Specifically, after transformants were obtained,

137

single colonies were picked into 800 µL LB medium (50 µg/mL Kan) in a well of a

138

96-well plate and incubated for 12 h at 37°C, 150 rpm. Fifty µL from each well of the

139

preculture was transferred to another 96-well plate with each well containing 800 µL

140

LB (50 µg/mL Kan), to which was added 300 µL 30% glycerol. After addition of

141

IPTG (0.1 mM final concentration), the main culture was incubated at 37°C for 3 h

142

(150 rpm) and then cultivated at 28°C for 16 h (150 rpm). Cells were harvested by

143

centrifugation at 1,500×g for 20 min, washed with phosphate buffer, pH 7.0, and

144

stored in a -80°C freezer. The cells were taken through three freeze-thaw cycles for

145

disruption and then treated with 2 g/L lysozyme at 28°C for 2 h. The lysate was

146

collected after centrifugation at 1,500×g for 20 min. The enzyme activity was

147

determined in a 200 µL reaction mixture containing 0.5 mM NADPH and 2 mM

148

(S)-CHOH within a quartz 96-well plate. Fifty µL of the resulting supernatant was 8


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149

added and the decrease absorbance of optical density at 340 nm (OD340) in 3 min after

150

shaking 3 seconds was monitored on SpectraMax 5 (Molecular Devices, Sunnyvale,

151

CA). Positive mutants with improved activity consumed NADPH faster than negative

152

ones. This high-throughput screening method was used as preliminary step for

153

screening positive mutants, which shortened the screening time greatly.

154

Continually, the positive mutants obtained by high-throughput screening

155

methods were further tested according to the following procedures. Mutants with

156

higher activities during the first round of screening were inoculated into 8 mL LB

157

culture medium (50 µg/mL Kan, 37°C, 150 rpm, 8 h) from the primary seed plate and

158

transferred to 500 mL Erlenmeyer flasks containing 100 mL of LB medium (50

159

µg/mL Kan). IPTG (final concentration 0.1 mM) was added into the culture to induce

160

protein expression at 28°C on a rotary shaker at 150 rpm when OD600 reached 0.6-0.8.

161

After 12-h of induction, the cells were centrifuged at 8,000×g for 10 min and washed

162

with 30 mL phosphate buffer (pH 7.0). The bioconversion reaction system in 10 mL

163

contained 5 g/L variant, 5 g/L GDH, 100 mM (S)-CHOH and 140 mM glucose. The

164

bioconversion reactions were conducted at 30°C, 150 rpm on a water bath shaker for

165

30 min and terminated by adding 1 mL acetonitrile. The reaction mixture was

166

vigorously mixed before parallel samples were extracted and diluted to a proper

167

concentration before HPLC analysis of (3R,5S)-CDHH. Positive mutants were

168

preserved and sequenced.

169

Site-saturation mutagenesis. Amino acid sites 95, 144 and 156 were

170

subjected to site-saturation mutagenesis using the plasmid pET28a-RtSCR9 as the 9



171

template in order to determine the best variant at each position. The primers are listed

172

in Table S1. PCR reaction was carried out with Phanta Max super-Fidelity DNA

173

Polymerase in a 50 µL reaction volume containing 0.2 mM of both primers, 10 ng

174

template DNA, 1.0 mM MgCl2, 0.2 mM dNTP, and 2.5 U of Phanta DNA polymerase.

175

Twenty-six cycles of PCR were carried out at 98°C for 10 s, 55°C for 5 s and 72°C

176

for 6 min. The PCR product was digested with DpnI restriction enzyme to remove

177

template plasmid and then transformed into competent cells of E. coli BL21(DE3).

178

The transformants were sequenced to identify the variations at each site. The relative

179

activities of mutants with varied amino acid substitutions at each site were determined

180

in 10 mL reaction mixtures under the same condition as described above.

181

Expression and purification of recombinant carbonyl reductase. E.

182

coli cells were cultured in 50 mL LB medium containing 50 µg/mL Kan for 8 h at

183

37°C on a rotary shaker at 150 rpm, and then IPTG (0.1 mM final concentration) was

184

added into the culture when the OD600 reached 0.6-0.8 to induce the expression of

185

RtSCR9 at 28°C, 150 rpm. After 12-h of induction, the cells were centrifuged at 8,000

186

×g for 10 min, washed with buffer A (50 mM Na2HPO4-NaH2PO4, 300 mM NaCl, pH

187

8.0). The harvested cells were resuspended in buffer A and disrupted by sonication in

188

an ice water bath. The cell debris was removed by centrifugation at 12,000×g for 30

189

min. The supernatant was loaded onto a Nickel-NTA column (Bio-Rad, Hercules, CA)

190

at 1 mL/min, equilibrated with buffer A at 1 mL/min. Nonspecifically bound protein

191

was washed from the column with buffer B (buffer A containing 15 mM imidazole,

192

pH 8.0), and target protein was eluted with buffer C (buffer A containing 400 mM 10


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193

imidazole, pH 8.0). The collected protein was washed with ddH2O and concentrated

194

with a 10 kDa Millipore filter. The purity and molecular weight were analyzed by

195

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).34

196

Enzyme activity assay and kinetic parameters measurement. The

197

enzyme activities of purified recombinant RtSCR9 and variants were measured at

198

30°C in 100 mM phosphate buffer pH 7.0 containing 1 mM NADPH and 5 mM

199

(S)-CHOH. The reaction mixtures were preheated for 3 min at 30°C. The reactions

200

were performed at 30°C for 3 min and terminated by adding 1 mL of 30% aqueous

201

acetonitrile. One mL of the reacting solution was extracted and centrifuged at

202

12,000×g for 3 min. The supernatant was filtered and analyzed by HPLC. One unit (U)

203

of enzyme activity was defined as the amount of enzyme required to catalyze the

204

production of 1 µmol (3R,5S)-CDHH per minute under the standard enzyme activity

205

assay conditions. To determine kinetic parameters, (S)-CHOH was used as substrate,

206

with concentrations ranging from 0 to 10 mM, and the concentration of the cofactor

207

NADPH was varied from 0.5-2.5 mM. The maximal reaction rate (Vmax) and apparent

208

Michaelis-Menten constant (Km) were calculated based on the Lineweaver-Burk plot

209

according to the following equation:

210

v=

Vmax ⋅ [A][B] [A][B] + [B] K mA + [A]K Bm + K sA K Bm

211

where [A] and [B] are concentrations of NADPH and (S)-CHOH; Vmax is the

212

maximal reaction rate; K Am and K Bm correspond to apparent kinetic parameters of

11



213

RtSCR9 towards NADPH and (S)-CHOH; K sA represents the dissociation constant

214

between RtSCR9 and NADPH.

215

Effects of pH and temperature on recombinant carbonyl reductase.

216

To determine the optimal catalytic conditions of wild and mutant enzymes, the effect

217

of temperature and pH were investigated under standard enzyme activity assay

218

conditions. The optimal temperatures for recombinant RtSCR9 and its variants were

219

determined at the range of 25°C to 65°C. The thermostability of recombinant RtSCR9

220

was examined by preincubating the purified enzymes in 100 mM phosphate buffer,

221

pH 7.0, at 30°C, 40°C and 50°C for required time intervals. The residual activity was

222

detected with non-preheated enzyme as control (100% of activity). The optimum pH

223

was evaluated at 30°C with the assay mixture in different buffers including

224

citrate-Na2HPO4 (pH 4.0-6.5), KH2PO4-K2HPO4 (pH 6.0-8.0) and Tris-HCl (pH

225

7.5-9.0). The pH stabilities were examined by preincubating the purified enzyme in

226

different KH2PO4-K2HPO4 (100 mM, pH 5.0-9.0) for 1 h and then the residual

227

activities of the enzymes were detected. The non-preinbubated enzyme was taken as

228

control (100% of activity).

229

Molecular modeling. The crystal structure of Thermotoga maritima SCR

230

(PDB-code: 1 VL8) in complex with NADPH was used as template for

231

three-dimensional (3D) analysis of RtSCR9 structure. The MODELLER 9.12 program

232

(http://www.salilab.rog) was used to construct the relaxed models of RtSCR9. Models

233

with the highest scores were selected and further evaluated using the PROCHECK

234

program.35 The final models were used for docking analysis with the AutoDock 4.2.1 12


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235

program (Scripps Res. Inst., La Jolla, CA). The 3D structure of (S)-CHOH and

236

NADPH were created using ChemDraw 8.0 (CambridgeSoft, Cambridge, UK). The

237

structures were visualized with the PyMOL program (http://www.pymol.org).

238

Asymmetric bioreduction of (S)-CHOH in a single batch reaction. The

239

bioconversion of (S)-CHOH to (3R,5S)-CDHH using RtSCR9 and mut-Ile144Lys was

240

investigated using whole E. coli cells in single batch reactions. Recombinant GDH

241

was used to complete the “enzyme-coupled” cofactor regenerating system (Figure S1).

242

The reaction mixture (30 mL) containing 0.9 g RtSCR9 (or mut-Ile144Lys) wet cell,

243

0.9 g GDH wet cell, 8.25 g (S)-CHOH and 8.77 g glucose was preheated for 5 min at

244

30°C on a magnetic stirring apparatus. During the process of biotransformation, 50 µL

245

samples were extracted per hour and diluted 200 times with 30% acetonitrile

246

(acetonitrile: water, 30:70 v/v) to a proper concentration and filtered before HPLC

247

analysis. The pH of the reaction mixture was controlled by an automatic regulation

248

system 902 Titrando Titrator (Metrohm Inc., Herisau, Switzerland) through titrating

249

an 8.0 M NaOH solution at a flow rate of 0.2 mL/min. It is noteworthy that the

250

volume of (S)-CHOH and titrated NaOH solution were calculated into the total

251

volume of the whole biotransformation reaction. The amount of products were

252

calculated according to the standard curves of (S)-CHOH and (3R,5S)-CDHH. The

253

yield represents the ratio between the formed (3R,5S)-CDHH and the original

254

(S)-CHOH.

255

After filtering, the reaction mixture was saturated by 5 g NaCl and then extracted

256

three times with 30 mL ethyl acetate. The product in organic phase was thoroughly 13



257

dried with 5 g anhydrous Na2SO4. The solvent was removed by rotary evaporation

258

under 40°C until the weight of product maintains to a constant, and then the yield of

259

pure product was calculated according to the following equation:

260

Yield (%) =

purified product (g) ×100 8.32 (g)

261

Statistical analysis. If not specifically noted, all experiments in this study

262

were performed in triplicate. Analysis of variance (ANOVA) was carried out using

263

the SAS program version 8.1 (SAS Institute Inc., Cary, NC). Least significant

264

difference (LSD) was computed at p < 0.05. All the Figures in this study were drawn

265

using the origin software version 8.0 (OriginLab Corp., Northampton, MA).

266

RESULTS AND DISCUSSION

267

Construction and screening of mutant libraries. Directed evolution does

268

not require detailed information of secondary structure or catalytic mechanism of

269

proteins.36 Site saturation mutagenesis at hotspot is a unique method for creating

270

focused libraries whereby each amino acid is substituted by the other 19 naturally

271

occurring amino acids.37

272

To improve the enzyme activity of RtSCR9, random mutagenesis strategy was

273

initially adopted through error-prone PCR and megaprimer PCR to mimic in vitro

274

Darwinian evolution. Random mutations were introduced into the gene of RtSCR9 by

275

error-prone PCR. The genes carrying the mutations were then cloned into the

276

expression vector by megaprimer PCR (Figure S3). Over 10,000 colonies were picked

277

in an initial round of mutagenesis. The majority of transformants carrying negative 14


Page 14 of 42

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278

mutations scarcely showed any detectable activity, while only a few numbers of

279

mutants exhibited similar or improved activity compared with wild type strain (Figure

280

S4). Mutants with higher activities were selected for a secondary screening by

281

determining the bioconversion of (S)-CHOH to (3R,5S)-CDHH in a 10-mL reaction

282

mixture and further determined using HPLC. Three positive mutations (Gln95Asp,

283

Ile144Leu and Phe156Asn) at three different residual sites were obtained, displaying

284

1.96-, 1.20- and 1.65-fold increased specific activity, respectively (Table S2).

285

Though three hotspots were identified through random mutagenesis, molecular

286

understanding of the improved properties of mutants was still vague and the best

287

substitutions at sites 95, 144 and 156 could not be identified. Site-saturation

288

mutagenesis was performed therefore to further explore the most beneficial

289

substitutions in this study (Table S3). After site-saturation mutagenesis, enzyme

290

activities of different mutants were also determined by HPLC. Through site-saturation

291

mutagenesis, there was more than one mutant that showed improved activity at each

292

site (Figure 1). Among the mutants, Gln95Cys, Gln95Asp, Gln95Glu and Gln95Asn

293

showed a more significant improvement of activity (Figure 1a); Ile144Lys was

294

obviously superior to other substitutions in the conversion of the substrate (S)-CHOH.

295

In addition, Ile144Ala, Ile144Leu and Ile144Gln also showed a little improvement of

296

the enzyme activity (Figure 1b). For substitutions of Phe156, Phe156Cys, Phe156Lys,

297

Phe156Asn, Phe156Gln, Phe156Ser, Phe156Thr and Phe156Arg all showed improved

298

enzyme activity (Figure 1c). Based on the above, Gln95Asp, Ile144Lys and

299

Phe156Gln were finally selected for further studies. 15



Page 16 of 42

300

Kinetic studies. The kinetic parameters were determined using (S)-CHOH as

301

substrate at different concentrations (0-10 mM) and cofactor NADPH with a varied

302

concentration (0.5-2.5 mM). Compared with the wild-type RtSCR9, mut-Gln95Asp

303

and mut-Ile144Lys showed similar Km values, mut-Phe156Gln gave a 1.23-fold

304

decrease of Km. The Vmax of Gln95Asp, Ile144Lys and Phe156Gln were improved

305

1.94-, 3.03- and 1.61-fold, respectively, compared to wild type (Table 1). All three

306

mutants exhibited increased kcat values, giving 1.94-, 3.03- and 1.61-fold improvement.

307

The kcat/Km values increased to 1.93-, 3.15- and 1.97-fold. Improved kcat and kcat/Km

308

values mainly contributed to the enhancement of the catalytic efficiency.

309

Purification and biochemical properties of purified wild-type and

310

evolved RtSCR9. Recombinant RtSCR9 and its variants were purified through

311

immobilized metal affinity chromatography. Both crude protein and the purified

312

enzyme were examined by SDS-PAGE analysis (Figure 2). There are no big

313

differences

314

mut-Ile144Lys and mut-Phe156Gln. Single site amino acid substitution might not

315

affect the correct protein folding and soluble expression. In addition, purified proteins

316

gave only one band on the SDS-PAGE gel without any undesired protein

317

contamination. The protein molecular weights of mut-Gln95Asp, mut-Ile144Lys and

318

mut-Phe156Gln were found nearly 27 kDa, which was identical to the wild-type

319

RtSCR9.

in

the

protein

expression

levels

of

RtSCR9,

mut-Gln95Asp,

320

The effects of different metal ions and the metal chelator EDTA on the activity

321

of RtSCR9 were reported in our previous work32, indicating the non-metal 16


Page 17 of 42


322

dependence of RtSCR9, which differs from the most representative carbonyl reductase

323

LbADH.38 The effects of temperature and pH on the enzymes catalyzing the

324

conversion of (S)-CHOH to (3R,5S)-CDHH were investigated. Temperature

325

influences molecule movement and the protonation process during interactions

326

between (S)-CHOH molecules and amino acid residures.39 The optimum catalytic

327

temperature of both RtSCR9 and its variants appeared to be 30°C. The activities of

328

wild type RtSCR9 and mutants were maintained at over 90% between 25°C and 40°C,

329

decreased rapidly between 45°C and 55°C and were completely lost when the

330

temperature went over 60°C (Figure 3a). The studies showed that the amino acid

331

substitutions at position 95 and 144 did not change the thermostability of the enzyme.

332

Phe156Gln did, however, retain >80% activity after incubation in phosphate buffer,

333

pH 7.0, for 24 h at 30°C, which was better than other mutants. Thermostability tests

334

under 40°C also demonstrated that mut-Phe156Gln had a slightly better stability

335

(Figure 3b). Besides protolysis, pH conditions also influence the hydrolysis of

336

ketoester substrates.40 To investigate the effect of pH on enzyme activity, reactions

337

were performed in buffers at pH 4.0 to 9.0. RtSCR9, mut-Gln95Asp, mut-Ile144Lys

338

and mut-Phe156Gln showed almost the same trends of activities under different pH

339

conditions. The activities of RtSCR9 and mutants increased slowly from pH 4.0 to 6.0

340

and the maximum activities were observed at 6.5 and 7.0, then began to decrease at

341

7.5 to 9.0 (Figure 3c). The investigation of pH stability of wild type RtSCR9 and its

342

variants showed that there was no apparent difference before and after protein

343

engineering. RtSCR9, Gln95Asp and Ile144Lys showed good stability between pH 17



344

6.5 and pH 8.5, retaining >90% of original activity, while Phe156Gln had a wider

345

range of pH tolerance, from pH 5.5 to pH 8.5 (Figure 3d). The effects of temperature

346

and pH indicated that mutant Phe156Gln was more thermostable and had a wider pH

347

catalytic condition than the others.

348

Homology modeling and analysis of mutants based on molecular

349

simulation. To elucidate the mechanism for the enhancement of activity regarding

350

the amino acid substitutions, homology modeling of the enzyme structures and

351

docking between enzymes and substrates were performed. Based on structure (PDB: 1

352

VL8), the three-dimensional structures of RtSCR9 and mutants were built.41

353

Validation with the PROCHECK program revealed that 92.8% of the residues were

354

presented in the most favored regions of the structure (Figure S5), and no residues

355

were in disallowed regions, suggesting that the built model was of high quality and

356

suitable for further analysis. The catalytic tetrads of RtSCR9 were Asn115, Thr143,

357

Tyr161 and Lys165. The cofactor NADPH was embedded in the coenzyme binding

358

area (Figure S6). The traits of substrate binding pockets play a key role in binding

359

process between ligands and receptors and thus impact the catalytic efficiency of

360

enzymes.42 Generally, an appropriately sized pocket is beneficial for substrate binding

361

and product release. Large ligands encountering a narrow pocket might not enter the

362

catalytic center, while a loose pocket environment can cause wrong positioning of

363

ligands, leading to the opposite stereo-configuration of the products.43

364

It was observed that the chloromethyl group of (S)-CHOH entered the substrate

365

pocket first and embedded into a hydrophobic cavity composed of Ile144, Ala145, 18


Page 18 of 42

Page 19 of 42


366

Ala149 and Ser150 (Figure 4a, e). The terminal ester group with its large steric

367

hindrance pointed away from the substrate pocket, which consisted of Ser94, Gln95,

368

Ser97, Met196, Ile197 and Gln200. Thr143 and Tyr161 stabilized (S)-CHOH by

369

forming H-bonds with C-3 carbonyl oxygen atom, which was within attacking

370

distance from a hydrogen atom NADPH C-4 at the si-face.

371

Gln95, located on the inlet of the substrate pocket, influenced the entrance of

372

substrate molecules. When replaced with Asp, the carboxyl of Asp oriented down

373

toward the entryway of ligands and reduced the inlet size of the pocket (Figure 4b, f).

374

This change could lead to a well-orientated tert-butyl ester in the binding pocket, as

375

the motility of the terminal ester group impacts the correct positioning of the

376

C3-carbonyl. Positive substitutions (Cys, Asp, Glu and Asn) from the site-saturation

377

mutagenesis experiments had similar spatial structures with the original Gln. It is

378

concluded that the dramatic change in steric hindrance of the amino acid in this site

379

could cause irregular features of the outer pocket, blocking the entrance of substrate

380

molecules.

381

When (S)-CHOH molecules entered the pocket in the simulations, the

382

chloromethyl group positioned next to the Ile144, and the methyl group substitution

383

on the β’ carbon atom of Ile increased the steric hindrance. When the chlorine atom

384

stretched upward towards the deeper hydrophobic cavity, the carbonyl moiety of the

385

substrate was put in a point where interactions between C-3 carbonyl oxygen and

386

active residues (Thr143 and Tyr156) were weakened. Substitution of Ile with the long

387

chain amino acid Lys showed that a small arc could form between the β’ and γ’ 19



388

carbon atoms, making room for the chloromethyl group (Figure 4c, g). The distance

389

from the hydrogen atom of NADPH to the substrate carbonyl oxygen atom shortened

390

from 3.3 Å to 3.2 Å after substitution of Ile to Lys. Ala, Leu, and Gln substitutions at

391

site 144 also improved the activity of the enzyme. Ala reduced the steric hindrance of

392

hydrophobic cavity, which facilitated the entrance of chloromethyl group. Leu has

393

similar steric hindrance but with a methyl substitution at γ’ carbon atom. It is

394

presumed that the β’-substitution has bigger impact on the positioning of

395

chloromethyl group than γ’-substitution. Most substitutions at this site were, however,

396

negative. The reason might be that site 144 is next to the catalytic tetrad and amino

397

acid variations at this site influence the enzyme function.

398

The third mutation, Phe156Gln, was on the outer surface of the enzyme (Figure

399

4d), which is associated with the stability of RtSCR9. When Phe156 bound by

400

substrate was mutated with the uncharged polar amino acids Gly, Ser, Thr, Cys, Asn,

401

Tyr, and Gln, substitutions of Ser, Thr, Cys, Asn, and Gln exhibited improved enzyme

402

activity. Gly156 had no activity, which might be due to the tremendous difference in

403

both the structure and properties of the two amino acids. Substitutions of Phe156 with

404

nonpolar amino acids Ala, Vla, Leu, Ile, Pro, Trp, and Met lost activity completely

405

except for Phe156Trp. Since Phe, Tyr and Trp are all aromatic amino acids sharing

406

close conformations and physicochemical characteristics, Phe156Tyr and Phe156Trp

407

could retain the original activity of the wild enzyme. In addition, substitutions with

408

negatively charged amino acids, Phe156Asp and Phe156Glu, also weakened the

409

enzyme activity. Oppositely, positively charged amino acids Lys and Arg enhanced 20


Page 20 of 42

Page 21 of 42


410

the catalytic activity of the enzyme. Thermostability tests (Figure 3b) and B-factor

411

analysis (Table S4) of Phe156Gln also confirmed that it displayed more stability than

412

the other enzymes.44-46 Pro157 and Val158 linked to the Phe156, as well as several

413

other amino acids, constituted the inner surface of the substrate pocket (Figure 4 h).

414

Substitution at site 156 also influenced the conformations of sites 157 and 158, thus

415

further changing the shape of the inner surface.

416

Whole cell asymmetric reduction of (S)-CHOH in a single batch

417

reaction. To verify the potential application of mut-Ile144Lys, a time course

418

bioconversion process was conducted to verify its catalytic efficiency in single batch

419

reactions. GDH was used as a coupled enzyme to complete the cofactor regeneration

420

(Figure S7).47-49 Glucose was added as co-substrate. The reaction pH was controlled at

421

approximately 7.0 by continuous titration with 8.0 M NaOH. Both RtSCR9 and

422

mut-Ile144Lys displayed outstanding catalytic ability towards (S)-CHOH (Figure 5).

423

During the first 2 h, the yield of product grew slowly because of mass transfer

424

resistance, and then the catalytic efficiency increased rapidly. When the yield of

425

(3R,5S)-CDHH was over 90%, the reaction rate slowed down gradually and finally

426

leveled at >95% after 12-h biotransformation. In comparison, the asymmetric

427

reduction process for mut-Ile144Lys presented a higher product yield, reaching 98.9%

428

after an 8-h reaction. The highest space-time yield of (3R,5S)-CDHH increased from

429

245.17 mmol L-1 h-1 g-1 wet cell weight (WCW) by RtSCR9 to 542.83 mmol L-1 h-1 g-1

430

WCW by mut-Ile144Lys. After isolation, 7.84 g (3R,5S)-CDHH was finally harvested,

21



431

total yield of product reached 95.1%. These results demonstrated the high catalytic

432

efficiency and the potential for industrial application of mut-Ile144Lys.

433

In conclusion, compared with wild type enzyme, the mut-Ile144Lys displayed

434

3.03- and 3.15-fold improvement of specific activity and kcat/Km respectively.

435

Potential mechanisms for the improvements in catalytic efficiency were studied

436

through homology modeling and molecular docking, indicating that there is still

437

opportunity for further evolution on catalytic activity and stabilities. Mut-Ile144Lys

438

catalyzed 542.83 mmol L-1 h-1 g-1 WCW during a single batch reaction, demonstrating

439

its great potential for industrial application.

440

ASSOCIATED CONTENT

441 442 443

The Supporting Information is available free of charge on the ACS Publications website at DOI:

444

Figure S1: HPLC detection of (3R,5S)-CDHH and (S)-CHOH. Figure S2:

445

enantiomeric excess detection of (3R,5S)-CDHH and (S)-CHOH. Figure S3: agarose

446

gel electrophoresis of error-prone PCR and megaprimer PCR. Figure S4: screening of

447

clones in a 96-well plate by measuring the absorbance of NADPH at OD340. Figure S5:

448

ramachandran plot of RtSCR9 chain A. Figure S6: homology modeled structure of

449

RtSCR9 monomer and the location of mutant sites. Figure S7: asymmetric reduction

450

of (S)-CHOH by recombinant E. coli BL21(DE3)/pET28a-RtSCR9 coupled with E.

451

coli BL21(DE3)/pET28a-GDH. Table S1: primers for PCR amplification. Table S2:

452

specific activities of positive mutants obtained from random mutagenesis. Table S3: 22


Page 22 of 42

Page 23 of 42


453

relative activity and enantioselectivity towards (3R,5S)-CDHH of mutants generated

454

by saturation mutagenesis. Table S4: the 20 amino acids of RtSCR9 chain A with the

455

highest B-values.

456

AUTHOR INFORMATION

457 458

*Phone: +86-571-88320630, E-mail: [email protected]

459 460

This study was financially supported by the National Natural Science Foundation of

461

China (no. 21672190) and the Program of Science and Technology of Zhejiang

462

Province (no. 2015C33137).

463 464

The authors declare no competing financial interest.

465

23



Page 24 of 42

466

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δ-dihydroxy hexanoate. Chem. Eur. J. 2001, 7, 4562-4571.

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4476-4478.

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4306-4308.

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thereof and method of using the same. US Patent 6645746 B1, 2003.

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2-[6-(hydroxymethyl)-1,3-dioxan-4yl] acetic acid derivatives. US Patent 6472544 B1,

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Saccharomyces cerevisiae CGMCC No 2233. Korean J. Chem. Eng. 2013, 30,

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Semi–rational engineering a carbonyl reductase for the enantioselective eeduction of

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β-amino ketones. ACS Catal. 2015, 5, 2452-2457.

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7β-hydroxysteroid dehydrogenase for enhanced ursodeoxycholic acid production by

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multiobjective directed evolution. J. Agric. Food Chem. 2017, 65, 1178-1185.

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feruloyl esterase EstF27 by directed evolution and the underlying structural basis. J.

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Agric. Food Chem. 2015, 63, 8225-8233.

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J.; Behrouzian, B.; Grate, J. H.; Lalonde, J. Polynucleotides encoding ketoreductases

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for producing stereoisomerically pure statins and synthetic intermediates therefor. US

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synthesis of tert-butyl (S)-6-cholro-5-hydroxy-3-oxohexanoate with a mutant alcohol

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dehydrogenase of Lactobacillus kefir. Appl. Microbiol. Biotechnol. 2015, 99,

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8963-8975.

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Kluyveromyces lactic aldo-keto reductase KIAKR via rational design. J. Biotecnol.

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30


Page 30 of 42

Page 31 of 42


FIGURE CAPTIONS Figure 1 Relative activities of mut-Gln95 (a), mut-Ile144 (b) and mut-Phe156 (c) mutants generated by site-saturation mutagenesis, comparing the wild-type, are presented in the bar graph.

Figure 2 SDS-PAGE analysis of wild type and mutants. Lane M: Protein Marker; Lane 1: Supernatant extract of BL21-pET28a; Lane 2: Supernatant extract of wild type RtSCR9; Lane 3: Supernatant extract of mut-Gln95Asp; Lane 4: Supernatant extract of mut-Ile144Lys; Lane 5: Supernatant extract of mut-Phe156Gln; Lane 6: Purified wild type RtSCR9. Lane 7: Purified mut-Gln95Asp; Lane 8: Purified mut-Ile144Lys; Lane 9: Purified mut-Phe156Gln.

Figure 3 Effects of temperature and pH on recombinant enzyme activities. a. Effects of temperature on enzymatic activities; b. Thermostabilities of RtSCR9 and mutants; c. Effects of pH on enzyme activities; d. pH stability of RtSCR9 and mutants.

Figure 4 Molecular docking analysis. Conformations of (S)-CHOH (cyan sticks) within RtSCR9 (a, e), mut-Gln95Asp (b, f), mut-Ile144Lys (c, g), mut-Phe156Gln (e, h). a, b, c, and d show the interaction of (S)-CHOH with active sites and NADPH. H-bonds are shown as red dashes, and the distance of (S)-CHOH and the C4 atom of the nicotinamide ring are shown as black dashes. Three mutants (green), active sites (white) and NADPH (gray) are displayed as sticks. Key amino acids in the substrate binding pocket are drawn in purple lines. e, f, g and h show the general conformation of the substrate pocket when (S)-CHOH is embedded.

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Figure 5 Time course of asymmetric reduction of (S)-CHOH in a batch reaction catalyzed by wild-type RtSCR9 and mut-Ile144Lys.

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Table 1. Apparent kinetic parameters of RtSCR9 and mutants. a

kcat

kcat/Km

e.e.

(mM)

(s-1)

(mM-1 s-1)

(%)

62.84 ± 4.54

0.83 ± 0.05

28.28 ± 4.83

33.99 ± 2.06 >99

mut-Gln95Asp

122.15 ± 4.73

0.84 ± 0.06

54.97 ± 4.67

65.44 ± 3.27 >99

mut-Ile144Lys

190.55 ± 7.71

0.80 ± 0.03

85.75 ± 5.12

107.19 ± 3.58 >99

mut-Phe156Gln

101.14 ± 5.02

0.68 ± 0.05

45.51 ± 3.29

67.12 ± 4.14 >99

Vmax

Km

(µmol min-1) RtSCR9

Enzyme

a

kcat, where [E] is the molar concentration of the enzymes.

33


AL CYA AS S G P L PHU G E LY H I IL S LYE LE S M U E AS T PR N G O L A N RG SE THR VAR TRL TY P R

Relative activity (%) AL CYA AS S G P L PHU G E LY H I IL S LYE LE S M U E AS T PR N G O L A N RG SE THR VAR TRL TY P R

Relative activity (%)


FIGURE GRAPHICS

400 Wild Variants

300

200

100

0

Figure 1a

400 Wild Variants

300

200

100

0

Figure 1b

34


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400 Wild Variants


300

200

100

AL CA Y AS S G P L PHU G E LY H I IL S LYE LE S M U E AS T PR N G O L AR N SEG THR VAR TRL TY P R

0

Figure 1c

35



Figure 2

36


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120 Relative activity

e.e.

100

80

60

60

40 30 20

0

0 20

30

40

50

60

o

Temperature ( C)

Figure 3a

120


90

60

30

o

o

RtSCR9-30 C o Gln95Asp-30 C o Ile144Lys-30 C o Phe156Gln-30 C

RtSCR9-40 C o Gln95Asp-40 C o Ile144Lys-40 C o Phe156Gln-40 C

0 0

4

8

12

16

20

24

Time (h)

Figure 3b

37


e.e. (%)


90


120

Citrate buffer

Phosphate buffer

Tris-HCl buffer

Page 38 of 42

e.e.

90

75

60

50

30

25

0

0 4

5

6

7

8

9

pH of aqueous phase

Figure 3c

120


90

60

RtSCR9 Gln95Asp Ile144Lys Phe156Gln

30

0 5

6

7

8

9

pH of aqueous phase

Figure 3d

38


10

e.e. (%)


100

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a

b

c

d

Figure 4a-d

39



e

f

g

h

Figure 4e-h

40


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Yield of (3R,5S)-CDHH (%)

120

90 Wild-type RtSCR9 RtSCR9-Ile144Lys

60

30

0

0

4

8

12

16

20

24

Time (h)

Figure 5

41



TOC Graphic

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Directed Evolution of Carbonyl Reductase from Rhodosporidium

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