Identification of a Robust Carbonyl Reductase for Diastereoselectively Building syn-3,5-Dihydroxy Hexanoate: A Bulky Side Chain of Atorvastatin

Article abstract of DOI:10.1021/acs.oprd.7b00194

t-Butyl-6-cyano-(3R,5R)-dihydroxyhexanoate is an advanced chiral precursor for the synthesis of the side chain pharmacophore of cholesterol-lowering drug atorvastatin. Herein, a robust carbonyl reductase (LbCR) was newly identified from Lactobacillus brevis, which displays high activity and excellent diastereoselectivity toward bulky t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate (7). The engineered Escherichia coli cells harboring LbCR and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of substrate 7. As a result, as much as 300 g L^-1 of water-insoluble substrate was completely converted to the corresponding chiral diol with >99.5% de in a space-time yield of 351 g L^-1 d^-1, indicating a great potential of LbCR for practical synthesis of the very bulky and bi-chiral 3,5-dihydroxy carboxylate side chain of best-selling statin drugs.

Full text of DOI:10.1021/acs.oprd.7b00194

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Full Paper
Identification of a Robust Carbonyl Reductase for Diastereoselectively
Building syn-3,5-Dihydroxy Hexanoate: a Bulky Side Chain of Atorvastatin
xumin gong, Gao-Wei Zheng, you yan Liu, and Jian-He Xu
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00194 • Publication Date (Web): 05 Jul 2017
Downloaded from http://pubs.acs.org on July 5, 2017
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Identification of a Robust Carbonyl
Reductase for Diastereoselectively Building
syn-3,5-Dihydroxy Hexanoate: a Bulky Side
Chain of Atorvastatin
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XuꢀMin Gong a, GaoꢀWei Zheng a,*, YouꢀYan Liu b,c and JianꢀHe Xu a,*
a State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation
Center for Biomanufacturing, East China University of Science and Technology, 130
Meilong Road, Shanghai, 200237, P.R. China, Fax: (+86)ꢀ21ꢀ64250840; eꢀmail:
gaoweizheng@ecust.edu.cn; jianhexu@ecust.edu.cn
b
School of Chemistry and Chemical Engineering, Guangxi University, Nanning
530004, Guangxi, P.R. China
c
Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning
530003, Guangxi, P.R. China
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OH
O
O
OH OH
O
E. coli/pLbCR
NC
NC
O
O
8
300 g L-1
7
NADP+
NADPH
351 g L-1 d-1 space-time yield
Glucose
Gluconic acid
78% yield, >99.5% de
E. coli/pBmGDH
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Abstract
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tꢀButyl 6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate is an advanced chiral precursor for the
synthesis of the side chain pharmacophore of cholesterolꢀlowering drug Atorvastatin.
Herein, a robust carbonyl reductase (LbCR) was newly identified from Lactobacillus
brevis, which displays high activity and excellent diastereoselectivity toward bulky
tꢀbutyl 6ꢀcyanoꢀ(5R)ꢀhydroxyꢀ3ꢀoxoꢀhexanoate (7). The engineered Escherichia coli
cells haboring LbCR and glucose dehydrogenase (for cofactor regeneration) were
employed as biocatalysts for the asymmetric reduction of substrate 7. As a result, as
much as 300 g L−1 of waterꢀinsoluble substrate was completely converted to the
corresponding chiral diol with >99.5% de in a space−time yield of 351 g L−1 d−1,
indicating a great potential of LbCR for practical synthesis of the very bulky and
biꢀchiral 3,5ꢀdihydroxy carboxylate side chain of bestꢀselling statin drugs.
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Keywords: asymmetric reduction; biocatalysis; carbonyl reductase; tꢀbutyl
6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate; statin side chain; enzymatic catalysis
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Introduction
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Statins are an important class of cholesterolꢀlowering drugs, which hinder the
synthesis of cholesterol by inhibiting 3ꢀhydroxyꢀ3ꢀmethylglutaryl−CoA reductase, a
rateꢀlimiting enzyme in the biosynthesis of cholesterol. Among the statin family,
Atorvastatin calcium, under the brand name Lipitor®, was the first drug to have
annual sales exceeding 10 billion US dollars. Like all statins, Atorvastatin contains a
synꢀ3,5ꢀdihydroxy hexanoate side chain pharmacophore with two chiral centers
(Scheme 1), which is required with >99.5% ee and >99.5% de.
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In terms of sustainable synthesis, environmentally benign enzymatic catalysis has
attracted increasing attention as an alternative to traditional chemical catalysis in the
synthesis of chiral intermediates from cheap achiral materials1. Numerous elegant
enzymatic strategies have been developed, such as the sequential aldol condensation
catalyzed by 2ꢀdeoxyriboseꢀ5ꢀphosphate aldolase (DERA) to give chiral intermediate
3 from the cheap bulk chemicals chloroacetaldehyde 1 and acetaldehyde 2 (Scheme 1,
Route I)2. Desymmetrization of 3ꢀhydroxyglutaronitrile 5 catalyzed by nitrilase
affords chiral 6 (Route II)3. Asymmetric reduction of ethyl 4ꢀchloroacetoacetate 9
accesses chiral alcohol 10 using ketoreductase (KRED)4, and replacement of the
chlorine by a cyano group using halohydrin dehalogenase (HHDH)5 enables the
synthesis of chiral 11 (Route III).
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Scheme 1. Chemoenzymatic synthetic routes for Atorvastatin chiral intermediates.
KRED: Ketoreductase; GDH: Glucose Dehydrogenase; HHDH: Halohydrin
Dehalogenase.
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Although these enzymatic protocols are economically viable and environmentally
attractive, they have mainly been used to synthesize primary intermediates with a
single stereocenter. Indeed, a major challenge is the formation of second stereocenter
to give advanced intermediates in high diastereoselectivity and productivity, such as
tꢀbutyl 6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate 8, a more advanced precursor for the
manufacture of Atorvastatin. The homoꢀchiral intermediate 8 was traditionally
synthesized by diastereoselective chemical reduction of 7, derived from 11, using
NaBH4 and Et2BOMe6. To obtain high diastereoselectivity (>99.5% de), extremely
low temperature (−90°C) and pyrophoric triethyl borane are necessary, resulting in
extensive energy consumption and a substantial amount of waste formation. An
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alternative chemical route was developed to replace the traditional reduction using
chlororuthenium (II) arene/βꢀamino alcohol. However, diastereoselectivity was
insufficient (80% de)7. Hence, it is highly desirable to develop green and sustainable
processes for the synthesis of 8.
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Biocatalytic asymmetric reduction provided a straightforward and environmentally
attractive process for the synthesis of 8. Although several ketoreductases for the
asymmetric synthesis of 8 have been reported8, most of them showed insufficient
activity towards nonꢀnatural substrate 7 at high substrate loading. Only an engineered
KRED gave 98% conversion at 300 g L−1 substrate concentration but with only 0.84
s–1 mM–1 of catalytic efficiency (kcat/Km)9. Therefore, it is still highly desirable to
develop more robust biocatalysts to reduce the difficultꢀtoꢀreduce ketone 7. Herein,
we describe a newly discovered carbonyl reductase from Lactobacillus brevis (LbCR)
capable of efficiently reducing 7 to 8 on a preparative scale.
Results and discussion
Screening of recombinant reductases
Initially, carbonyl reductases RhCR10 and CpAR211 possessing high activity
towards ethyl 8ꢀchloroꢀ6ꢀoxooctanoate S16 were trialed in the reduction of target
substrate 7, which is structurally similar to S16. However, none was found to be
active against 7. Subsequently, a ketoreductase toolbox containing 100 enzymes
developed by our laboratory was evaluated. As a result, 17 enzymes displayed
activities for substrate 7 (Figure 1), however, activities of majority of the enzymes
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were extremely low (<10% relative activity compared with LbCR, Table S1). To our
delight, LbCR (GenBank accession no.: NC_008497.1) displayed a relatively high
activity toward the difficultꢀtoꢀreduce ketone 7, and was thus chosen for further
investigation.
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Figure 1. The screening results of a home reductase library for reduction of a bulky
substrate 7.
A multiple sequence alignment shown in Figure S1 revealed that the amino acids of
LbCR have merely 46% and 49% identities respectively with the reported LbADH
from Lactobacillus brevis (GenBank accession no.: CAD66648.1)12 and LkADH from
Lactobacillus kefir (GenBank accession no.: WP_054768785.1)13. LbADH and
LkADH exhibited exclusively catalytic activities toward the Cꢀ5 carbonyl group of
tertꢀbutyl 6ꢀchloroꢀ3,5ꢀdioxohexanoate (CDOH). However, only LbCR showed a
significnat activity toward the Cꢀ3 keto group of substrate 7, indicating its unique
feature of which is distinct from majority of wellꢀknown ketoreductases.
Characterization of LbCR
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The intrinsic catalytic properties of LbCR were then investigated using the enzyme
purified by nickelꢀaffinity chromatography. A single band with an apparent molecular
size of 29 kDa corresponds to LbCR (Figure S2); this confirms the calculated
molecular mass of 26 kDa derived from the amino acid sequence. The specific
activity of the pure protein was 24.8 U mg−1 protein under standard conditions.
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The optimum temperature and pH were determined to be 40°C (Figure S3B) and
6.0 (Figure S3A), respectively. The halfꢀlife of LbCR was 2 h at 30°C and 1.2 min at
40°C (Figure S3C). The effect of various metal ions and EDTA on the activity of
LbCR were also investigated. The results (Table S2) showed that EDTA and most of
the tested metal ions had no significant effect on the activity of LbCR except Cu2+,
which inhibited the activity strongly.
The kinetic parameters of LbCR were also determined (Table S3). The KM and kcat
of LbCR toward substrate 7 are 0.58 mM and 24.6 s−1, while those toward NADPH
are 0.026 mM and 25.5 s−1. Compared with other reported ketoreductases (Table 1),
LbCR afforded the lowest Km and the highest catalytic efficiency constant kcat/KM
(42.4 s−1 mM−1), indicating a high binding affinity with ketoester 7.
Table 1. Kinetic parameters of LbCR and other reported enzymes toward substrate 7.
Enzyme
KM
kcat
kcat/KM
Ref.
(mM)
10.9 ± 2.3
1.91
(s–1)
(s–1 mM–1)
KREDa
9.12 ± 0.78 0.84
3.64 1.91
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CaAKR
8h
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KlAKR
KlAKRb
LbCR
4.4
11.6
2.64
12.4
42.4
8g
3.4
42.2
8i
0.58 ± 0.13
24.6 ± 6.7
This work
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KRED was synthesized according to SEQ ID No. 316, which was used for the
b
preparation of tꢀbutyl 6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate at 300 g L−1. KlAKR
mutant (Y295W/W296L).
In order to understand the high binding affinity of LbCR toward 7, we performed
homology modeling and docking calculation. As shown in Figure 2, LbCR displays a
typical “Rossmannꢀfold” of shortꢀchain dehydrogenases/reductases (SDRs) family,
while KlAKR and KRED have a classical (α/β)8 barrel of the aldoꢀketo reductases
(AKRs). According to the proposed catalytic mechanism of SDR, the conserved Y158
and S145 form hydrogen bonds with the carbonyl oxygen atom of the substrate and a
hydrogen from NADPH attacks the carbonyl carbon atom of the substrate at the
siꢀface to generate corresponding (R)ꢀalcohol. The orientation of substrate 7 docked in
the substrateꢀbinding pocket of LbCR showed that the tꢀBu of the substrate was
surrounded by more hydrophobic residues (V96, L152, A155, A201, I197 and V198)
than the corresponding residues of KlAKR and KRED, and the CNꢀgroup pointed
toward Q247, facilitating the binding of substrate. The substrate binding pocket of
LbCR (Figure 2D) exhibited apparently different geometry from KlAKR (Figure 2E)
and KRED (Figure 2F). A tunnel through LbCR was buried inside, and the size of this
tunnel was more suitable to accommodate 7 than the other enzymes. The special
geometry and polarity of the substrate binding pocket of LbCR may contribute to its
high binding affinity toward 7.
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Figure 2. Docking of substrate 7 into the active sites of LbCR, KlAKR and KRED.
(A, D) LbCR; (B, E) KlAKR; (C, F) KRED. Interactions were highlighted by blue
dashed lines. The active sites, NADP+ and 7 were shown as yellow sticks, green sticks
and cyan sticks. Other residues surrounding the substrate were shown as magenta
lines. Tunnels of LbCR calculated by Caver 3.0 were shown as magenta mesh.
Substrate specificity
The substrate specificity of LbCR was investigated using various ketoesters and
ketones (Table S4). As shown in Figure 3, LbCR displayed higher activity toward
substrates bearing long side chains adjacent to the carbonyl function group (S2, S3,
S9, and S13–15). In addition, LbCR showed excellent activity towards chlorinated
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compounds (S11, S16 and S18), probably because chlorine leads to a higher
electrophilicity of the carbonyl group. LbCR showed higher activity for aryl
ketoesters compared with alkyl ketoesters, and gave the highest activity towards S20,
the important precursor of angiotensinꢀconverting enzymes (ACE) inhibitors14.
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Figure 3. Substrate profile of LbCR.
To explore the substrate scope of this new enzyme, the kinetic parameters of LbCR
towards several representative substrates were measured. As shown in Table 2, LbCR
exhibited markedly high catalytic efficiency towards aliphatic esters (S13, S15, S16)
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and ketones (S3) with long chains, mainly attributing to their lower Km values.
However, LbCR showed higher KM values towards aliphatic esters with short chains.
7
8
9
Table 2. Kinetic parameters of LbCR toward several ketones and ketoesters.
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Substrate
KM
kcat
kcat/KM
(s–1 mM–1)
28.9
(mM)
(s–1)
S2
0.56 ± 0.02
0.09 ± 0.01
13.1 ± 1.5
5.5 ± 1.3
16.2 ± 0.2
12.3 ± 0.3
92.4 ± 2.9
14.3 ± 1.3
22.4 ± 0.7
49.7 ± 4.3
15.6 ± 1.0
13.4 ± 0.5
25.7 ± 0.9
26.1 ± 4.2
21.0 ± 1.0
56.6 ± 1.2
42.5 ± 2.6
136.7
40.0
S3
S5
2.6
S7
1.2 ± 0.1
18.6
S9
S11
S13
S15
S16
S18
S19
S20
S22
0.31 ± 0.06
0.075 ± 0.030
0.035 ± 0.008
0.136 ± 0.020
0.22 ± 0.10
1.2 ± 0.2
160.5
207.4
382.6
189.3
118.6
17.5
0.26 ± 0.03
1.4 ± 0.3
217.5
30.4
The substrates S5 and S15 were docked into the active site of LbCR to understand
the substrate specificity. As shown in Figure 4, the catalytic residue Y158 was
proximal to the carbonyl oxygen of substrates 7 and S15, but a bit distal to S5.
Otherwise, the substrate tunnels seemed too large for S5 with short chain, and several
substrate molecules may bind in the active site simultaneously, leading to
nonꢀproductive binding and lowering the binding affinity.
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Figure 4. Docking of substrates 7, S15 and S5 into the active sites of LbCR. The
active sites, NADP+ and substrate were shown as yellow sticks, green sticks and cyan
sticks. Tunnels calculated by Caver 3.0 were shown as magenta mesh and orange
mesh.
In general, LbCR is able to accept a broad range of substrates, particularly bulky
substrates, suggesting its great potential as a biocatalyst for the asymmetric reduction
of carbonyl compounds.
Preparative Synthesis of 8
Finally, to demonstrate the utility of LbCR for practical applications, the
asymmetric reduction of 7 was performed on a gram scale using lyophilized cells of
E. coli/pLbCR coupled with glucose dehydrogenase (GDH) for the regeneration of
NADPH. Initial experiments were performed in 10 mL of potassium phosphate buffer
(100 mM, pH 6.0) with a substrate concentration of 23 g L−1. The pH of the reaction
mixture was maintained at pH 6.0 by titrating 2 M K2CO3. It was found that the
reaction was complete within 1 h with >99% conversion (Table 3, entry 1). When the
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substrate loading was raised to 100 g L−1 without changing any of the other
conditions, the reaction was completed smoothly within 3 h (entry 2). Moreover, the
substrate was almost totally converted in 4 h at the same substrate loading when the
amount of external addition of NADP+ was reduced from 0.5 mM to 0.1 mM (entry
3). This prompted us to increase the substrate concentration further to 200 g L−1,
resulting in >99% conversion and a productivity of 522 g L−1 d−1 (entry 4). The
reaction proceeded smoothly, even with a substrate concentration of 300 g L−1 (35.5%
v/v). After a reaction time of 16 h (entry 5), the product was harvested in >99.5% de
(Figure S6B) and 351 g L−1 d−1. These results indicate that LbCR is able to tolerate
high substrate concentrations, making it promising for practical applications.
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Table 3. Asymmetric reduction of 7 with lyophilized E. coli cells of LbCR and
BmGDHa.
Substrate loading Cell loading NADP+
Time
(h)
Conv.
(%)b
de
Entry
(%)b
(g L–1)
(g L–1)
(mM)
1
2
3
4
5
23
16
16
16
16
16
0.5
0.5
0.1
0.1
0.1
1
>99
>99
>99
>99
>99
>99.5
>99.5
>99.5
>99.5
>99.5
100
100
200
300
3
4
8
16
a
Reaction conditions: substrate 7 (1.22–3.66 g substrate), glucose (1.2 equiv.),
lyophilized E. coli/pLbCR (0.16 g), lyophilized E. coli/pBmGDH (0.055 g), NADP+
(0.1–0.5 mM), potassium phosphate buffer (100 mM, pH 6.0) to 10 mL at 30°C, and
pH was kept at 6.0 with 2 M K2CO3.
b Determined by HPLC.
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In conclusion, we have identified a new carbonyl reductase, LbCR, from
Lactobacillus brevis, which displayed very high specific activity, diastereoselectivity,
and
substrate
tolerance
in
the
asymmetric
synthesis
of
tꢀbutyl
6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate 8. As much as 300 g L−1 of substrate 7 was
reduced to give diastereomerically pure tꢀbutyl 6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate
8 in >99.5% de and 351 g L−1 d−1 under the optimized conditions. The enzyme should
therefore be suitable for the synthesis of an advanced statin side chain intermediate.
Further studies to improve the catalytic activity and stability by protein engineering
are now in progress to further reduce the enzyme loading, and make it a more efficient
tool for industrial manufacturing.
Experimental
General Information
tꢀButyl
6ꢀcyanoꢀ(3R,5R)ꢀdihydroxyhexanoate
and
tꢀbutyl
6ꢀcyanoꢀ(3S,5R)ꢀdihydroxy hexanoate were obtained from J&K (Shanghai, China).
S13 and S15 were obtained from Xiamen Bestally Biotechnology Co., Ltd. (Fujian,
China). All others chemicals and reagents were purchased from commercial suppliers.
E. coli DH5α and E. coli BL21 (DE3) were used as the cloning and expression hosts,
1
respectively. Both cells were incubated in LuriaꢀBertani (LB) medium. H NMR was
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measured on a Bruker Avance 400 MHz spectrometer using tetramethylsilane (TMS)
as internal standard. Optical rotation measurements were taken on a Rudolph
Research Analytical Autopol I automatic polarimeter at 25°C.
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Expression of ketoredutase in E. coli
E. coli BL21/pET28a−LbCR and other recombinant cells were cultivated in
Luria−Bertani (LB) medium containing 50 mg mL−1 kanamycin at 37°C and 180 rpm
until OD600
reached 0.6–0.8, then IPTG was added to the culture (final
nm
concentration 0.2 mM) to induce enzyme expression at 16°C for further 20–24 h.
Cells were harvested by centrifugation (7000 × g, 10 min, 4°C) and washed with
normal saline.
Enzyme assay
The reductase activity was assayed at 30°C by monitoring the decrease in the
absorbance of NADPH at 340 nm on a UVꢀspectrophotometer (Beckman DU730).
The standard assay mixture (1 mL) was composed of 970 ꢁL potassium phosphate
buffer (100 mM, pH 6.0), 10 ꢁL tꢀbutyl 6ꢀcyanoꢀ(5R)ꢀhydroxyꢀ3ꢀoxohexanoate (200
mM), 10 ꢁL NADPH (10 mM), and 10 ꢁL enzyme with an appropriate concentration.
One unit of enzyme activity (U) was defined as the amount of enzyme that can
catalyze the oxidation of 1 ꢁmol NADPH per minute at above conditions.
Asymmetric bioreduction of ketoester 7
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The reaction mixture (10 mL) was prepared by dissolving substrate 7 (1−13.2
mmol, 0.28−3.66 g), glucose (1.2 equiv. of substrate), lyophilized cells of LbCR (10
kU, 0.16 g), lyophilized cells of GDH (10 kU, 0.055 g), 5% DMSO (v/v) and NADP+
(1−5 ꢁmol, 0.1−0.5 mM) in potassium phosphate buffer (100 mM, pH 6.0). The pH
value of the mixture was monitored and adjusted to 6.0 using 2 M K2CO3 during the
reaction. Products were extracted twice with ethyl acetate. The combined organic
layers were washed twice with brine solution, dried over anhydrous Na2SO4, filtered,
and evaporated under vacuum, giving crude 8 as yellow oil. The crude product was
purified by column chromatography eluting with a mixture of ethyl acetate and
petroleum ether (1:5) to provide pure 8 (2.4 g, HPLC purity, 92%). [α]2D5 = −41.3
(c=1.0, CHCl3), >99.5% de(8g, 9). 1H NMR (CDCl3, 400 MHz), δ/ppm: 1.47 (s, 9H),
1.71~1.75 (m, 2H), 2.42ꢀ2.44 (m, 2H), 2.54ꢀ2.56 (m, 2H), 3.71 (brs, 2H), 4.18ꢀ4.22
(m, 1H), 4.26ꢀ4.31 (m, 1H).
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ASSOCIATED CONTENT
Supporting Information
Complete experimental procedures and characterization of the enzymes and new
products; NMR spectra and HPLC chromatograms.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: gaoweizheng@ecust.edu.cn; Fax: (+86)ꢀ21ꢀ64250840.
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*Eꢀmail: jianhexu@ecust.edu.cn; Fax: (+86)ꢀ21ꢀ64252498.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENT
This work was financially supported by the National Natural Science Foundation of
China (No. 21472045 and 21536004) and the Fundamental Research Funds for the
Central Universities (22A201514043).
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