Directed Evolution of Carbonyl Reductase from Rhodosporidium toruloides and Its Application in Stereoselective Synthesis of tert-Butyl (3R,5S)-6-Chloro-3,5-dihydroxyhexanoate

Article abstract of DOI:10.1021/acs.jafc.7b00866

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and k_cat/K_m, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L^-1 h^-1 g^-1 wet cell weight. This study presents a new carbonyl reductase for efficient synthesis of (3R,5S)-CDHH.

Full text of DOI:10.1021/acs.jafc.7b00866

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Article
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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Page 1 of 42
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
*
aKey Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and
Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
bEngineering 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: zhengyg@zjut.edu.cn
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ABSTRACT: tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH)
is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase
RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward
tert-butyl (S)-6-chloro-3-hydoxy-5-oxohexanoate ((S)-CHOH). To improve the
activity of RtSCR9, random mutagenesis and site-saturation mutagenesis were
performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys and
mut-Phe156Gln). These mutants exhibited 1.94-, 3.03- and 1.61-fold, and 1.93-, 3.15-
and 1.97-fold improvement on the specific activity and kcat/Km, respectively.
Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose
dehydrogenase (GDH) was conducted. The yield and enantiomeric excess of
(3R,5S)-CDHH respectively reached 98% and 99% after 8-h bioconversion in a single
batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol
L-1 h-1 g-1 wet cell weight. This study presents a new carbonyl reductase for efficient
synthesis of (3R,5S)-CDHH.
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KEYWORDS: tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, carbonyl
reductase, random mutagenesis, site-saturation mutagenesis, single batch reaction
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INTRODUCTION
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tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a
promising intermediate applied in the side chain synthesis of atorvastatin and
rosuvastatin, cholesterol regulation drugs used in preventing cardiovascular diseases,
such as hypercholesterolemia, atherosclerosis and coronary heart disease.1-3 Chemical
synthesis of (3R,5S)-CDHH typically starts with (S)-epichlorohydrin followed by a
series of catalytic reactions involving expensive metal catalysts and environmentally
unfriendly organic solvents. To introduce the second chiral center in the chemical
synthesis of (3R,5S)-CDHH, sodium borohydride (NaBH4) is used to reduce the
carbonyl at C-3 under Parasad’s conditions (Et2BOMe, -80°C to -75°C).4,5 Although
(3R,5S)-CDHH can be totally synthesized within a short processing period by
chemical synthesis, difficulties remain in producing a final product with sufficient
enantiomeric purity. In addition, complex protection-deprotection processes, low
overall yield and high environmental pressure have made chemical synthesis less
competitive.6 Compared to chemical synthesis, biosynthesis has been an alternative in
the preparation of (3R,5S)-CDHH, along with the merits of low catalyst cost, high
enantioselectivities and a broad functional group tolerability, which is dominant in
large-scale pharmaceutical manufacture of important chiral intermediates.7-9
Carbonyl reductases (EC 1.1.1.148) have been demonstrated to be valuable
biocatalysts in the reduction of ketones to enantiopure alcohols with advantages
including mild catalytic conditions, chemo-, regio- and stereo-selectivity, and no
heavy metal contamination. So far, considerable studies have been performed on
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biotransformation of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) and tert-butyl
(S)-6-chloro-3-hydoxy-5-oxohexanoate ((S)-CHOH) by carbonyl reductases to
produce (3R,5S)-CDHH.10 Lactobacillus brevis (LbADH) showed
a
high
enantioselectivity in the reduction of CDOH at C-5 to produce (S)-CHOH with over
99.5% enantiomeric excess (e.e.).11-13 Previously, a two-step biotransformation
process was established using LkADH1 and LkADH2 from Lactobacillus kefir in a
simple batch process to produce (3R,5S)-CDHH with >99% e.e. and 47.5% yield.14-18
Subsequently, fed batch processes and two-phase biotransformation systems were
established.19-21 A recombinant carbonyl reductase from Candida magnoliae
coexpressed with a glucose dehydrogenase highly efficiently catalyzed the reduction
of 200 g/L (S)-CHOH to prepare (3R,5S)-CDHH with 97.2% yield and 98.6%
diastereoisomeric excess (d.e.).22,23 Recently, liquid-core immobilized Saccharomyces
cerevisiae CGMCC No.2233 was used as catalyst to accomplish 100% conversion of
(S)-CHOH to produce (3R,5S)-CDHH with >99% d.e. when initial substrate
concentration was less than 50 g/L.24 Enzymatic asymmetric reductions for
commercial application have in general been, however, limited to poor substrate
tolerance, insufficient stability and low volumetric productivity. Thus, biocatalysts
with evolved characteristics are still urgently demanded to meet the large-scale
production of (3R,5S)-CDHH.
Modification of carbonyl reductases is mainly focused on the improvement of
catalytic activity, stereoselectivity, stability and cofactor specificity.25-28 Codexis
Corporation (Redwood, CA) engineered a ketoreductase gene from Saccharomyces
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cerevisiae by rounds of directed evolution, resulting in a 99.8% conversion of 13 g/L
substrate (S)-CHOH with 99.2% d.e. in a 4-h reaction compared to wild type
ketoreductase with 37% yield, 97.3% d.e. in a 20-h reaction.29 A tetrad mutant of
LkADH through rational design was screened, exhibiting a 3.7- and 42-fold
improvement in specific activity toward CDOH over LbADH and wild-type LkADH,
respectively.30 Recently, the catalytic efficiency of aldo-keto reductase toward
tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate was improved 11.25-fold through
rational protein modification. The mutant (KIAKR-Tyr295Trp/Trp296Leu) catalyzed
the asymmetric reduction with tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate
accumulated up to 162.7 mM with >99.5% d.e..31
In our previous work, a carbonyl reductase RtSCR9 (Genbank accession No.
EMS 18622.1) from Rhodosporidium toruloides was screened and showed
considerable activity toward (S)-CHOH.32 To improve the catalytic efficiency for
industrial application, random mutagenesis and site-saturation mutagenesis were
performed. Three positive variants showing 1.94-, 3.03- and 1.61-fold improvement in
specific activity were obtained. Apparent kinetic parameters, molecular modeling and
docking experiments were used to illustrate the mechanism of the changes in enzyme
characteristics. Bioconversion of 1 M (S)-CHOH was conducted in a single batch
reaction. The variant mut-Ile144Lys exhibited an enhanced catalytic ability
with >98% yield and >99% e.e. within 8 h of reaction, indicating its potential
application in upscale production of (3R,5S)-CDHH.
MATERIALS AND METHODS
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Microorganism, media and chemicals. Escherichia coli BL21(DE3)
(Invitrogen, Carlsbad, CA) was used as the host cell and plasmid pET-28a(+)
(Novagen, Darmstadt, Germany) was used for cloning and expression. E. coli
BL21(DE3)/pET28a-RtSCR9 was used for expression of carbonyl reductase gene
RtSCR9. E. coli BL21(DE3)/pET28a-GDH carrying a glucose dehydrogenase (GDH)
gene was used for the “enzyme-coupled” cofactor regeneration system.33 E. coli cells
were cultured in Luria-Bertani (LB) medium containing 5 g of yeast extract, 10 g of
tryptone, and 10 g of NaCl per liter. Kanamycin (Kan) and the inducer
isopropylthio-β-galactoside (IPTG) were purchased from Sango Biotech (Shanghai,
China). Nicotinamide adenine dimucleotide phosphate (NADPH) was obtained from
Roche (Karlsruhe, Germany). Standards of (S)-CHOH and (3R,5S)-CDHH were
purchased from J&K Scientific Ltd. (Shanghai, China). All other chemicals were of
analytical grade purity and commercially available.
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Analytical methods. The concentrations of (S)-CHOH and (3R,5S)-CDHH
were determined by high-performance liquid chromatography (HPLC) (Shimadzu Co.,
Kyoto, Japan) using an Agilent Zorbax SB-C8 column (150×4.6 mm, particle size 5
µm, Agilent Technologies Co., Santa Clara, CA) with UV detection at 210 nm. The
mobile phase was a mixture of acetonitrile and ultrapure water (30:70 v/v). The
retention times of (S)-CHOH and (3R,5S)-CDHH were 6.11 min and 9.66 min,
respectively, at 40°C, with a flow rate of 1 mL/min (Figure S1).
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The e.e. values of (3R,5S)-CDHH were determined on a Chiracel OD-H column
(250×4.6 mm, particle size 5 µm, Daicel Chemical Industries, Tokyo, Japan) at 215
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nm using hexane and isopropanol (85:15 v/v) as the mobile phase.30 The column
conditions were 40°C at a flow rate of 1 mL/min. Samples from the aqueous reaction
mixture containing hydroxyl keto ester were extracted into the mobile phase by
vigorous mixing. After centrifuging at 12,000×g for 3 min, the organic phase was
collected and filtered before HPLC analysis. The retention times of (S)-CHOH and
(3R,5S)-CDHH were 5.09 min and 5.90 min, respectively (Figure S2).
Construction of mutant library. A random mutagenesis library was
established through error-prone PCR and megaprimer PCR. The recombinant plasmid
pET28a-RtSCR9 was used as template DNA and the primers for error-prone PCR
were
5’-TATGTCTTCGCCTACTCCCAAC-3’
and
5’-TCTACCATGGCAAGAACGTCC-3’. Appropriate amounts of Mg2+ and Mn2+
were added to the PCR reaction mixture in order to control the mismatch rate of base
pairing. The final error-prone PCR reaction mixture contained MgCl2 1.5 mM, MnCl2
0.3 mM, dATP 0.1 mM, dGTP 0.1 mM, dTTP 0.5 mM, dCTP 0.5 mM, 30 pmol each
primer, 10 ng template DNA, and 5 U Taq polymerase, diluted to a final volume of 50
µL by adding ddH2O. The error-prone PCR reaction condition started at 94°C for 3
min followed by amplification for 30 cycles: 95°C 30 s, 55°C 30 s, and 72°C 1 min,
followed by elongation at 72°C for 10 min. Subsequently, the target gene with
mutations from error-prone PCR was used as megaprimer to create random
mutagenesis libraries through megaprimer PCR of the whole plasmid (MEGAWHOP).
The 50 µL megaprimer PCR reaction mixture contained 2×PCR buffer 25 µL, 10 mM
dNTP 1 µL, 10 ng template DNA, 2 µL megaprimer, 2.5 U Phanta Max super-Fidelity
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DNA polymerase and 20.5 µL ddH2O following the procedure of denaturation at 98°C
for 3 min, 26 cycles for amplification 10 s at 98°C, 5 s at 55°C and 6 min at 72°C,
then elongation at 72°C for 10 min. The PCR products were treated with 40 U DpnI at
37°C for 3 h to remove the original template plasmid. The treated PCR products were
transformed to E. coli BL21(DE3) by heat-shock treatment. Transformants were
plated on LB-agar medium supplemented with 50 µg/mL of Kan and incubated for 12
h at 37°C. The mutations were determined by DNA sequencing.
High throughput screening and HPLC assay. A two-step screening
method based on the high-throughput screening combined with HPLC analysis was
designed to select positive mutants. Specifically, after transformants were obtained,
single colonies were picked into 800 µL LB medium (50 µg/mL Kan) in a well of a
96-well plate and incubated for 12 h at 37°C, 150 rpm. Fifty µL from each well of the
preculture was transferred to another 96-well plate with each well containing 800 µL
LB (50 µg/mL Kan), to which was added 300 µL 30% glycerol. After addition of
IPTG (0.1 mM final concentration), the main culture was incubated at 37°C for 3 h
(150 rpm) and then cultivated at 28°C for 16 h (150 rpm). Cells were harvested by
centrifugation at 1,500×g for 20 min, washed with phosphate buffer, pH 7.0, and
stored in a -80°C freezer. The cells were taken through three freeze-thaw cycles for
disruption and then treated with 2 g/L lysozyme at 28°C for 2 h. The lysate was
collected after centrifugation at 1,500×g for 20 min. The enzyme activity was
determined in a 200 µL reaction mixture containing 0.5 mM NADPH and 2 mM
(S)-CHOH within a quartz 96-well plate. Fifty µL of the resulting supernatant was
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added and the decrease absorbance of optical density at 340 nm (OD340) in 3 min after
shaking 3 seconds was monitored on SpectraMax 5 (Molecular Devices, Sunnyvale,
CA). Positive mutants with improved activity consumed NADPH faster than negative
ones. This high-throughput screening method was used as preliminary step for
screening positive mutants, which shortened the screening time greatly.
Continually, the positive mutants obtained by high-throughput screening
methods were further tested according to the following procedures. Mutants with
higher activities during the first round of screening were inoculated into 8 mL LB
culture medium (50 µg/mL Kan, 37°C, 150 rpm, 8 h) from the primary seed plate and
transferred to 500 mL Erlenmeyer flasks containing 100 mL of LB medium (50
µg/mL Kan). IPTG (final concentration 0.1 mM) was added into the culture to induce
protein expression at 28°C on a rotary shaker at 150 rpm when OD600 reached 0.6-0.8.
After 12-h of induction, the cells were centrifuged at 8,000×g for 10 min and washed
with 30 mL phosphate buffer (pH 7.0). The bioconversion reaction system in 10 mL
contained 5 g/L variant, 5 g/L GDH, 100 mM (S)-CHOH and 140 mM glucose. The
bioconversion reactions were conducted at 30°C, 150 rpm on a water bath shaker for
30 min and terminated by adding 1 mL acetonitrile. The reaction mixture was
vigorously mixed before parallel samples were extracted and diluted to a proper
concentration before HPLC analysis of (3R,5S)-CDHH. Positive mutants were
preserved and sequenced.
Site-saturation mutagenesis. Amino acid sites 95, 144 and 156 were
subjected to site-saturation mutagenesis using the plasmid pET28a-RtSCR9 as the
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template in order to determine the best variant at each position. The primers are listed
in Table S1. PCR reaction was carried out with Phanta Max super-Fidelity DNA
Polymerase in a 50 µL reaction volume containing 0.2 mM of both primers, 10 ng
template DNA, 1.0 mM MgCl2, 0.2 mM dNTP, and 2.5 U of Phanta DNA polymerase.
Twenty-six cycles of PCR were carried out at 98°C for 10 s, 55°C for 5 s and 72°C
for 6 min. The PCR product was digested with DpnI restriction enzyme to remove
template plasmid and then transformed into competent cells of E. coli BL21(DE3).
The transformants were sequenced to identify the variations at each site. The relative
activities of mutants with varied amino acid substitutions at each site were determined
in 10 mL reaction mixtures under the same condition as described above.
Expression and purification of recombinant carbonyl reductase. E.
coli cells were cultured in 50 mL LB medium containing 50 µg/mL Kan for 8 h at
37°C on a rotary shaker at 150 rpm, and then IPTG (0.1 mM final concentration) was
added into the culture when the OD600 reached 0.6-0.8 to induce the expression of
RtSCR9 at 28°C, 150 rpm. After 12-h of induction, the cells were centrifuged at 8,000
×g for 10 min, washed with buffer A (50 mM Na2HPO4-NaH2PO4, 300 mM NaCl, pH
8.0). The harvested cells were resuspended in buffer A and disrupted by sonication in
an ice water bath. The cell debris was removed by centrifugation at 12,000×g for 30
min. The supernatant was loaded onto a Nickel-NTA column (Bio-Rad, Hercules, CA)
at 1 mL/min, equilibrated with buffer A at 1 mL/min. Nonspecifically bound protein
was washed from the column with buffer B (buffer A containing 15 mM imidazole,
pH 8.0), and target protein was eluted with buffer C (buffer A containing 400 mM
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imidazole, pH 8.0). The collected protein was washed with ddH2O and concentrated
with a 10 kDa Millipore filter. The purity and molecular weight were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).34
Enzyme activity assay and kinetic parameters measurement. The
enzyme activities of purified recombinant RtSCR9 and variants were measured at
30°C in 100 mM phosphate buffer(pH 7.0) containing 1 mM NADPH and 5 mM
(S)-CHOH. The reaction mixtures were preheated for 3 min at 30°C. The reactions
were performed at 30°C for 3 min and terminated by adding 1 mL of 30% aqueous
acetonitrile. One mL of the reacting solution was extracted and centrifuged at
12,000×g for 3 min. The supernatant was filtered and analyzed by HPLC. One unit (U)
of enzyme activity was defined as the amount of enzyme required to catalyze the
production of 1 µmol (3R,5S)-CDHH per minute under the standard enzyme activity
assay conditions. To determine kinetic parameters, (S)-CHOH was used as substrate,
with concentrations ranging from 0 to 10 mM, and the concentration of the cofactor
NADPH was varied from 0.5-2.5 mM. The maximal reaction rate (Vmax) and apparent
Michaelis-Menten constant (Km) were calculated based on the Lineweaver-Burk plot
according to the following equation:
V
max ⋅[A][B]
210
v =
A +[A]KB
A
B
[A][B]+[B]
+
Ks Km
Km
m
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212
where [A] and [B] are concentrations of NADPH and (S)-CHOH; Vmax is the
KmA
KmB
correspond to apparent kinetic parameters of
maximal reaction rate;
and
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KsA
represents the dissociation constant
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RtSCR9 towards NADPH and (S)-CHOH;
between RtSCR9 and NADPH.
Effects of pH and temperature on recombinant carbonyl reductase.
To determine the optimal catalytic conditions of wild and mutant enzymes, the effect
of temperature and pH were investigated under standard enzyme activity assay
conditions. The optimal temperatures for recombinant RtSCR9 and its variants were
determined at the range of 25°C to 65°C. The thermostability of recombinant RtSCR9
was examined by preincubating the purified enzymes in 100 mM phosphate buffer,
pH 7.0, at 30°C, 40°C and 50°C for required time intervals. The residual activity was
detected with non-preheated enzyme as control (100% of activity). The optimum pH
was evaluated at 30°C with the assay mixture in different buffers including
citrate-Na2HPO4 (pH 4.0-6.5), KH2PO4-K2HPO4 (pH 6.0-8.0) and Tris-HCl (pH
7.5-9.0). The pH stabilities were examined by preincubating the purified enzyme in
different KH2PO4-K2HPO4 (100 mM, pH 5.0-9.0) for 1 h and then the residual
activities of the enzymes were detected. The non-preinbubated enzyme was taken as
control (100% of activity).
Molecular modeling. The crystal structure of Thermotoga maritima SCR
(PDB-code: 1 VL8) in complex with NADPH was used as template for
three-dimensional (3D) analysis of RtSCR9 structure. The MODELLER 9.12 program
(http://www.salilab.rog) was used to construct the relaxed models of RtSCR9. Models
with the highest scores were selected and further evaluated using the PROCHECK
program.35 The final models were used for docking analysis with the AutoDock 4.2.1
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program (Scripps Res. Inst., La Jolla, CA). The 3D structure of (S)-CHOH and
NADPH were created using ChemDraw 8.0 (CambridgeSoft, Cambridge, UK). The
structures were visualized with the PyMOL program (http://www.pymol.org).
Asymmetric bioreduction of (S)-CHOH in a single batch reaction. The
bioconversion of (S)-CHOH to (3R,5S)-CDHH using RtSCR9 and mut-Ile144Lys was
investigated using whole E. coli cells in single batch reactions. Recombinant GDH
was used to complete the “enzyme-coupled” cofactor regenerating system (Figure S1).
The reaction mixture (30 mL) containing 0.9 g RtSCR9 (or mut-Ile144Lys) wet cell,
0.9 g GDH wet cell, 8.25 g (S)-CHOH and 8.77 g glucose was preheated for 5 min at
30°C on a magnetic stirring apparatus. During the process of biotransformation, 50 µL
samples were extracted per hour and diluted 200 times with 30% acetonitrile
(acetonitrile: water, 30:70 v/v) to a proper concentration and filtered before HPLC
analysis. The pH of the reaction mixture was controlled by an automatic regulation
system 902 Titrando Titrator (Metrohm Inc., Herisau, Switzerland) through titrating
an 8.0 M NaOH solution at a flow rate of 0.2 mL/min. It is noteworthy that the
volume of (
volume of the whole biotransformation reaction. The amount of products were
calculated according to the standard curves of ( )-CHOH and (3 ,5 )-CDHH. The
yield represents the ratio between the formed (3 ,5 )-CDHH and the original
)-CHOH.
After filtering, the reaction mixture was saturated by 5 g NaCl and then extracted
S)-CHOH and titrated NaOH solution were calculated into the total
S
R S
R
S
(S
three times with 30 mL ethyl acetate. The product in organic phase was thoroughly
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dried with 5 g anhydrous Na2SO4. The solvent was removed by rotary evaporation
under 40°C until the weight of product maintains to a constant, and then the yield of
pure product was calculated according to the following equation:
purified product (g)
260
Yield (%) =
×100
8.32 (g)
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Statistical analysis. If not specifically noted, all experiments in this study
were performed in triplicate. Analysis of variance (ANOVA) was carried out using
the SAS program version 8.1 (SAS Institute Inc., Cary, NC). Least significant
difference (LSD) was computed at
p < 0.05. All the Figures in this study were drawn
using the origin software version 8.0 (OriginLab Corp., Northampton, MA).
RESULTS AND DISCUSSION
Construction and screening of mutant libraries. Directed evolution does
not require detailed information of secondary structure or catalytic mechanism of
proteins.36 Site saturation mutagenesis at hotspot is a unique method for creating
focused libraries whereby each amino acid is substituted by the other 19 naturally
occurring amino acids.37
To improve the enzyme activity of RtSCR9, random mutagenesis strategy was
initially adopted through error-prone PCR and megaprimer PCR to mimic in vitro
Darwinian evolution. Random mutations were introduced into the gene of RtSCR9 by
error-prone PCR. The genes carrying the mutations were then cloned into the
expression vector by megaprimer PCR (Figure S3). Over 10,000 colonies were picked
in an initial round of mutagenesis. The majority of transformants carrying negative
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mutations scarcely showed any detectable activity, while only a few numbers of
mutants exhibited similar or improved activity compared with wild type strain (Figure
S4). Mutants with higher activities were selected for a secondary screening by
determining the bioconversion of (S)-CHOH to (3R,5S)-CDHH in a 10-mL reaction
mixture and further determined using HPLC. Three positive mutations (Gln95Asp,
Ile144Leu and Phe156Asn) at three different residual sites were obtained, displaying
1.96-, 1.20- and 1.65-fold increased specific activity, respectively (Table S2).
Though three hotspots were identified through random mutagenesis, molecular
understanding of the improved properties of mutants was still vague and the best
substitutions at sites 95, 144 and 156 could not be identified. Site-saturation
mutagenesis was performed therefore to further explore the most beneficial
substitutions in this study (Table S3). After site-saturation mutagenesis, enzyme
activities of different mutants were also determined by HPLC. Through site-saturation
mutagenesis, there was more than one mutant that showed improved activity at each
site (Figure 1). Among the mutants, Gln95Cys, Gln95Asp, Gln95Glu and Gln95Asn
showed a more significant improvement of activity (Figure 1a); Ile144Lys was
obviously superior to other substitutions in the conversion of the substrate (S)-CHOH.
In addition, Ile144Ala, Ile144Leu and Ile144Gln also showed a little improvement of
the enzyme activity (Figure 1b). For substitutions of Phe156, Phe156Cys, Phe156Lys,
Phe156Asn, Phe156Gln, Phe156Ser, Phe156Thr and Phe156Arg all showed improved
enzyme activity (Figure 1c). Based on the above, Gln95Asp, Ile144Lys and
Phe156Gln were finally selected for further studies.
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Kinetic studies. The kinetic parameters were determined using (S)-CHOH as
substrate at different concentrations (0-10 mM) and cofactor NADPH with a varied
concentration (0.5-2.5 mM). Compared with the wild-type RtSCR9, mut-Gln95Asp
and mut-Ile144Lys showed similar Km values, mut-Phe156Gln gave a 1.23-fold
decrease of Km. The Vmax of Gln95Asp, Ile144Lys and Phe156Gln were improved
1.94-, 3.03- and 1.61-fold, respectively, compared to wild type (Table 1). All three
mutants exhibited increased kcat values, giving 1.94-, 3.03- and 1.61-fold improvement.
The kcat
/Km values increased to 1.93-, 3.15- and 1.97-fold. Improved kcat and kcat/Km
values mainly contributed to the enhancement of the catalytic efficiency.
Purification and biochemical properties of purified wild-type and
evolved RtSCR9. Recombinant RtSCR9 and its variants were purified through
immobilized metal affinity chromatography. Both crude protein and the purified
enzyme were examined by SDS-PAGE analysis (Figure 2). There are no big
differences in the protein expression levels of RtSCR9, mut-Gln95Asp,
mut-Ile144Lys and mut-Phe156Gln. Single site amino acid substitution might not
affect the correct protein folding and soluble expression. In addition, purified proteins
gave only one band on the SDS-PAGE gel without any undesired protein
contamination. The protein molecular weights of mut-Gln95Asp, mut-Ile144Lys and
mut-Phe156Gln were found nearly 27 kDa, which was identical to the wild-type
RtSCR9.
The effects of different metal ions and the metal chelator EDTA on the activity
of RtSCR9 were reported in our previous work32, indicating the non-metal
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dependence of RtSCR9, which differs from the most representative carbonyl reductase
LbADH.38 The effects of temperature and pH on the enzymes catalyzing the
conversion of (
influences molecule movement and the protonation process during interactions
between (
)-CHOH molecules and amino acid residures.39 The optimum catalytic
S)-CHOH to (3R,5S)-CDHH were investigated. Temperature
S
temperature of both RtSCR9 and its variants appeared to be 30°C. The activities of
wild type RtSCR9 and mutants were maintained at over 90% between 25°C and 40°C,
decreased rapidly between 45°C and 55°C and were completely lost when the
temperature went over 60°C (Figure 3a). The studies showed that the amino acid
substitutions at position 95 and 144 did not change the thermostability of the enzyme.
Phe156Gln did, however, retain >80% activity after incubation in phosphate buffer,
pH 7.0, for 24 h at 30°C, which was better than other mutants. Thermostability tests
under 40°C also demonstrated that mut-Phe156Gln had a slightly better stability
(Figure 3b). Besides protolysis, pH conditions also influence the hydrolysis of
ketoester substrates.40 To investigate the effect of pH on enzyme activity, reactions
were performed in buffers at pH 4.0 to 9.0. RtSCR9, mut-Gln95Asp, mut-Ile144Lys
and mut-Phe156Gln showed almost the same trends of activities under different pH
conditions. The activities of RtSCR9 and mutants increased slowly from pH 4.0 to 6.0
and the maximum activities were observed at 6.5 and 7.0, then began to decrease at
7.5 to 9.0 (Figure 3c). The investigation of pH stability of wild type RtSCR9 and its
variants showed that there was no apparent difference before and after protein
engineering. RtSCR9, Gln95Asp and Ile144Lys showed good stability between pH
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365
6.5 and pH 8.5, retaining >90% of original activity, while Phe156Gln had a wider
range of pH tolerance, from pH 5.5 to pH 8.5 (Figure 3d). The effects of temperature
and pH indicated that mutant Phe156Gln was more thermostable and had a wider pH
catalytic condition than the others.
Homology modeling and analysis of mutants based on molecular
simulation. To elucidate the mechanism for the enhancement of activity regarding
the amino acid substitutions, homology modeling of the enzyme structures and
docking between enzymes and substrates were performed. Based on structure (PDB: 1
VL8), the three-dimensional structures of RtSCR9 and mutants were built.41
Validation with the PROCHECK program revealed that 92.8% of the residues were
presented in the most favored regions of the structure (Figure S5), and no residues
were in disallowed regions, suggesting that the built model was of high quality and
suitable for further analysis. The catalytic tetrads of RtSCR9 were Asn115, Thr143,
Tyr161 and Lys165. The cofactor NADPH was embedded in the coenzyme binding
area (Figure S6). The traits of substrate binding pockets play a key role in binding
process between ligands and receptors and thus impact the catalytic efficiency of
enzymes.42 Generally, an appropriately sized pocket is beneficial for substrate binding
and product release. Large ligands encountering a narrow pocket might not enter the
catalytic center, while a loose pocket environment can cause wrong positioning of
ligands, leading to the opposite stereo-configuration of the products.43
It was observed that the chloromethyl group of (S)-CHOH entered the substrate
pocket first and embedded into a hydrophobic cavity composed of Ile144, Ala145,
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387
Ala149 and Ser150 (Figure 4a, e). The terminal ester group with its large steric
hindrance pointed away from the substrate pocket, which consisted of Ser94, Gln95,
Ser97, Met196, Ile197 and Gln200. Thr143 and Tyr161 stabilized (S)-CHOH by
forming H-bonds with C-3 carbonyl oxygen atom, which was within attacking
distance from a hydrogen atom NADPH C-4 at the si-face.
Gln95, located on the inlet of the substrate pocket, influenced the entrance of
substrate molecules. When replaced with Asp, the carboxyl of Asp oriented down
toward the entryway of ligands and reduced the inlet size of the pocket (Figure 4b, f).
This change could lead to a well-orientated tert-butyl ester in the binding pocket, as
the motility of the terminal ester group impacts the correct positioning of the
C3-carbonyl. Positive substitutions (Cys, Asp, Glu and Asn) from the site-saturation
mutagenesis experiments had similar spatial structures with the original Gln. It is
concluded that the dramatic change in steric hindrance of the amino acid in this site
could cause irregular features of the outer pocket, blocking the entrance of substrate
molecules.
When (S)-CHOH molecules entered the pocket in the simulations, the
chloromethyl group positioned next to the Ile144, and the methyl group substitution
on the β’ carbon atom of Ile increased the steric hindrance. When the chlorine atom
stretched upward towards the deeper hydrophobic cavity, the carbonyl moiety of the
substrate was put in a point where interactions between C-3 carbonyl oxygen and
active residues (Thr143 and Tyr156) were weakened. Substitution of Ile with the long
chain amino acid Lys showed that a small arc could form between the β’ and γ’
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409
carbon atoms, making room for the chloromethyl group (Figure 4c, g). The distance
from the hydrogen atom of NADPH to the substrate carbonyl oxygen atom shortened
from 3.3 Å to 3.2 Å after substitution of Ile to Lys. Ala, Leu, and Gln substitutions at
site 144 also improved the activity of the enzyme. Ala reduced the steric hindrance of
hydrophobic cavity, which facilitated the entrance of chloromethyl group. Leu has
similar steric hindrance but with a methyl substitution at γ’ carbon atom. It is
presumed that the β’-substitution has bigger impact on the positioning of
chloromethyl group than γ’-substitution. Most substitutions at this site were, however,
negative. The reason might be that site 144 is next to the catalytic tetrad and amino
acid variations at this site influence the enzyme function.
The third mutation, Phe156Gln, was on the outer surface of the enzyme (Figure
4d), which is associated with the stability of RtSCR9. When Phe156 bound by
substrate was mutated with the uncharged polar amino acids Gly, Ser, Thr, Cys, Asn,
Tyr, and Gln, substitutions of Ser, Thr, Cys, Asn, and Gln exhibited improved enzyme
activity. Gly156 had no activity, which might be due to the tremendous difference in
both the structure and properties of the two amino acids. Substitutions of Phe156 with
nonpolar amino acids Ala, Vla, Leu, Ile, Pro, Trp, and Met lost activity completely
except for Phe156Trp. Since Phe, Tyr and Trp are all aromatic amino acids sharing
close conformations and physicochemical characteristics, Phe156Tyr and Phe156Trp
could retain the original activity of the wild enzyme. In addition, substitutions with
negatively charged amino acids, Phe156Asp and Phe156Glu, also weakened the
enzyme activity. Oppositely, positively charged amino acids Lys and Arg enhanced
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430
the catalytic activity of the enzyme. Thermostability tests (Figure 3b) and B-factor
analysis (Table S4) of Phe156Gln also confirmed that it displayed more stability than
the other enzymes.44-46 Pro157 and Val158 linked to the Phe156, as well as several
other amino acids, constituted the inner surface of the substrate pocket (Figure 4 h).
Substitution at site 156 also influenced the conformations of sites 157 and 158, thus
further changing the shape of the inner surface.
Whole cell asymmetric reduction of (S)-CHOH in a single batch
reaction. To verify the potential application of mut-Ile144Lys, a time course
bioconversion process was conducted to verify its catalytic efficiency in single batch
reactions. GDH was used as a coupled enzyme to complete the cofactor regeneration
(Figure S7).47-49 Glucose was added as co-substrate. The reaction pH was controlled at
approximately 7.0 by continuous titration with 8.0 M NaOH. Both RtSCR9 and
mut-Ile144Lys displayed outstanding catalytic ability towards (S)-CHOH (Figure 5).
During the first 2 h, the yield of product grew slowly because of mass transfer
resistance, and then the catalytic efficiency increased rapidly. When the yield of
(3R,5S)-CDHH was over 90%, the reaction rate slowed down gradually and finally
leveled at >95% after 12-h biotransformation. In comparison, the asymmetric
reduction process for mut-Ile144Lys presented a higher product yield, reaching 98.9%
after an 8-h reaction. The highest space-time yield of (3
R,5S)-CDHH increased from
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
WCW by mut-Ile144Lys. After isolation, 7.84 g (3R,5S)-CDHH was finally harvested,
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448
449
450
451
452
total yield of product reached 95.1%. These results demonstrated the high catalytic
efficiency and the potential for industrial application of mut-Ile144Lys.
In conclusion, compared with wild type enzyme, the mut-Ile144Lys displayed
3.03- and 3.15-fold improvement of specific activity and kcat/Km respectively.
Potential mechanisms for the improvements in catalytic efficiency were studied
through homology modeling and molecular docking, indicating that there is still
opportunity for further evolution on catalytic activity and stabilities. Mut-Ile144Lys
catalyzed 542.83 mmol L-1 h-1 g-1 WCW during a single batch reaction, demonstrating
its great potential for industrial application.
ASSOCIATED CONTENT
{ꢀꢁꢁꢂꢃꢄꢅꢆ Lꢆꢇꢂꢃꢈꢉꢄꢅꢂꢆ
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
Figure S1: HPLC detection of (3
R,5
S
)-CDHH and (
S)-CHOH. Figure S2:
enantiomeric excess detection of (3 ,5 )-CDHH and (S
R
S
)-CHOH. Figure S3: agarose
gel electrophoresis of error-prone PCR and megaprimer PCR. Figure S4: screening of
clones in a 96-well plate by measuring the absorbance of NADPH at OD340. Figure S5:
ramachandran plot of RtSCR9 chain A. Figure S6: homology modeled structure of
RtSCR9 monomer and the location of mutant sites. Figure S7: asymmetric reduction
of (S)-CHOH by recombinant E. coli BL21(DE3)/pET28a-RtSCR9 coupled with E.
coli BL21(DE3)/pET28a-GDH. Table S1: primers for PCR amplification. Table S2:
specific activities of positive mutants obtained from random mutagenesis. Table S3:
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457
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459
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463
464
465
relative activity and enantioselectivity towards (3R,5S)-CDHH of mutants generated
by saturation mutagenesis. Table S4: the 20 amino acids of RtSCR9 chain A with the
highest B-values.
AUTHOR INFORMATION
/ꢂꢃꢃꢊꢋꢁꢂꢆꢌꢅꢆ !ꢀꢄꢍꢂꢃ
*Phone: +86-571-88320630, E-mail: zhengyg@zjut.edu.cn
Cꢀꢆꢌꢅꢆ
This study was financially supported by the National Natural Science Foundation of
China (no. 21672190) and the Program of Science and Technology of Zhejiang
Province (no. 2015C33137).
bꢂꢄꢊ
The authors declare no competing financial interest.
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