Efficient Stereoselective Synthesis of Structurally Diverse γ- and δ-Lactones Using an Engineered Carbonyl Reductase

Article abstract of DOI:10.1002/cctc.201900382

Structurally diverse γ- and δ-lactones were efficiently synthesized stereoselectively using an engineered carbonyl reductase from Serratia marcescens (SmCR_V4). SmCR_V4 exhibited improved activity (up to 500-fold) and thermostability toward 14 γ-/δ-keto acids and esters, compared with the wild-type enzyme, with 110-fold enhancement in catalytic efficiency (k_cat/K_m) toward methyl 4-oxodecanoate. The preparative synthesis of alkyl and aromatic γ- and δ-lactones with 95 %–>99 % ee and 78 %–90 % yields was demonstrated. The highest space-time yield, 1175 g L^?1 d^?1, was achieved for (R)-γ-decalactone.

Full text of DOI:10.1002/cctc.201900382

Accepted Article
Title: Efficient Stereoselective Synthesis of Structurally Diverse γ- and
δ-Lactones Using an Engineered Carbonyl Reductase
Authors: Meng Chen, Xiao-Yan Zhang, Chen-Guang Xing, Chao
Zhang, Yu-Cong Zheng, Jiang Pan, Jian-He Xu, and Yunpeng
Bai
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To be cited as: ChemCatChem 10.1002/cctc.201900382
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10.1002/cctc.201900382
ChemCatChem
COMMUNICATION
Efficient Stereoselective Synthesis of Structurally Diverse γ- and
δ-Lactones Using an Engineered Carbonyl Reductase
Meng Chen,[a] Xiao-Yan Zhang,[a] Chen-Guang Xing,[b] Chao Zhang,[a] Yu-Cong Zheng,[a] Jiang Pan,[a]
Jian-He Xu*[a] and Yun-Peng Bai*[a]
Abstract: Structurally diverse γ- and δ-lactones were efficiently
synthesized stereoselectively using an engineered carbonyl
reductase from Serratia marcescens (SmCRV4). SmCRV4 exhibited
improved activity (up to 500-fold) and thermostability toward 14 γ-/δ-
keto acids and esters, compared with the wild-type enzyme, with 110-
fold enhancement in catalytic efficiency (kcat/Km) toward methyl 4-
oxodecanoate. The preparative synthesis of alkyl and aromatic γ- and
δ-lactones with 95%–99% ee and 78%-90% yields was demonstrated.
The highest space-time yield, 1175 g L−1 d−1, was achieved for (R)-γ-
decalactone.
Chiral γ- and δ-hydroxy acid derivatives are of importance in
synthetic chemistry as key building blocks for constructing natural
products and pharmaceutically active compounds.[1] The
Scheme 1 Stereoselective synthesis of optically pure γ- and δ-lactones.
asymmetric reduction of γ- and δ-keto acids and esters is
regarded as an atom-economic method to prepare such active
compounds. However, the position of the γ-/δ-carbonyls in the
carbon chain is distant from the terminal carboxyl group, which
decreases the reactivity, and makes it difficult to accurately
control the stereoconfiguration of the hydroxy group.
In particular, an environmentally friendly preparation process
is imperative for the synthesis of chiral γ- and δ-lactones, which
are used for a variety of artificial flavors in the food and cosmetic
industries.[7] To date, the whole-cell biotransformation of γ- and δ-
keto acids has been studied using wild-type yeasts, such as
Saccharomyces cerevisiae and Yarrowia lipolytica.[8] However, in
The use of ruthenium catalyts to catalyze the asymmetric
hydrogenation of keto esters to chiral hydroxy esters is well-
established.[2] Yang and co-workers have studied the use of
this method a large amount of fermentation by-products are
iridium complexes for the synthesis of chiral diols.[3] Recently, Arai
produced, and the volumetric productivity is low because of the
toxicity of the lactones to the growth of yeast cells.[9] To solve
these problems, it is necessary to find and engineer the functional
enzymes responsible for the highly enantioselective reduction of
γ- and δ-keto acids in yeast. Unfortunately, although many
carbonyl reductases have been found in Saccharomyces
cerevisiae[10a], these enzymes are still unknown up to now.[10]
Although several enzymatic processes have been exploited
for the synthesis of chiral γ- and δ-lactones,[11] the
stereoselectivity and productivity of these processes are not yet
satisfactory. Lavandera and co-workers[1a] made a breakthrough
et al. have developed a flexible method to prepare either chiral
lactones or diols depending on the reaction conditions (Scheme
1a).[4] Zhao et al. have demonstrated the potential of this method
for keto amide hydrogenation.[5a] Lin et al. have developed a
surfactant-type rhodium complex to catalyze the asymmetric
reduction of long-chain aliphatic keto esters (Scheme 1b).[5b] In
addition to using the organometallic catalyst, Meninno and co-
workers have developed a quinine-catalyzed route for preparing
chiral keto esters (Scheme 1c).[6] Although noble metals and
organocatalysts are efficient for the stereoselective synthesis of
hydroxy esters, either high pressure of H2 (normally 8–70 atm)[3,4]
in enantioselective synthesis of short-chain γ- or δ‑alkyl and
or low temperature (–20 °C)[6] are required.
aromatic hydroxy esters and lactones. Recently, the first carbonyl
reductase, which could convert linear long-chain γ/δ-keto acids
into the corresponding γ-/δ-lactones with high enantioselectivity
up to >99% ee, such as (R)-γ-/δ-decalactones (3c, 3d), was
discovered from Serratia marcescens (SmCR).[12] SmCR
displayed catalytic activity toward long-chain γ-/δ-keto acids and
esters, in contrast to the majority of reported ketoreductases,
which are mainly active toward the α- or β-carbonyls in short-
chain alkyl and aromatic keto acids.[13] However, the activity of
SmCR was too low for practical application. In this report we
describe the engineering of SmCR for improved catalytic
performance. The effectiveness of this enzyme was
demonstrated in the synthesis of structurally diverse γ- and δ-
[a]
[b]
M. Chen, Dr. X.-Y. Zhang, C. Zhang, Y.-C. Zheng, Dr. J. Pan, Prof.
Dr. J.-H. Xu, Assoc. Prof. Dr. Y.-P. Bai
State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology, 130 Meilong
Road, Shanghai 200237, P.R. China.
E-mail: jianhexu@ecust.edu.cn; ybai@ecust.edu.cn.
C.-G. Xing
Xiamen Oamic Biotech. Co. Ltd, 36 Longmen Road, Xiamen
361026, P.R. China
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201900382
ChemCatChem
COMMUNICATION
Table 1 Kinetic parameters of purified SmCRwt and engineered proteins toward substrate methyl 4-oxodecanoate
Entry
Evolutionary strategy
starting enzyme
epPCR
Enzyme
SmCRwt
SmCRV1
SmCRV2
SmCRV3
SmCRV4
Mutation
-
kcat (min-1)
5.4 ± 0.3
Km (mM)
kcat/Km (min-1mM-1)
Folda
1.0
1
2
3
4
5
2.38 ± 0.34
1.59 ± 0.34
0.87 ± 0.15
1.35 ± 0.12
0.79 ± 0.16
2.3 ± 0.3
4.5 ± 1.0
101 ± 19
114 ± 11
249 ± 53
R123C
7.5 ± 0.5
2.0
epPCR
R123C/L209P
R123C/L209P/F183Y
R123C/L209P/F183Y/V61K
88.0 ± 5.3
153 ± 5.0
197 ± 14.0
44.8
62.3
110
ISM
ISM
a Fold change enhancement in kcat/Km compared to the wild-type enzyme
lactones with space-time yields up to 1175 g L-1 d-1 and ee’s up to
>99% (Scheme 1d).
Notably, the stereoselectivity of SmCRV2 was also improved as
the ee value slightly increased from 91% to 93%.
Subsequently, the active pocket of SmCRV2 was finely tuned
to further enhance the catalytic efficiency by iterative saturation
mutagenesis.[15] Using the homologous model, we targeted 19
potential residues lining the substrate binding pocket of SmCRV2
and screened the library constructed by the combinatorial
saturation mutagenesis at these sites (Figure S3). This endeavor
led to the discovery of the important residues F183 and V61. The
introduction of the mutation F183Y yielded SmCRV3 with an
activity of 3.51 ± 0.22 U/mgprotein, affording methyl (R)-4-
hydroxydecanoate (2g) with 95% ee (Figure 1b and Table S3).
The further incorporation of V61K into SmCRV3 led to the mutant
SmCRV4, which showed the highest specific activity of all the
engineered enzymes (7.66 ± 0.92 U/mgprotein), an 86-fold increase
compared with SmCRwt (Table S3). Reaction kinetics analysis
was performed for the wild-type enzyme and the four variants
(Table 1). Clearly, directed evolution increased the binding affinity
of the substrate with the enzyme as the Km of SmCRV4 toward 1g
was decreased to 0.79 ± 0.16 mM compared with 2.38 ± 0.34 mM
for SmCRwt. In particular, the turnover number kcat was gradually
increased by the iterative accumulation of beneficial mutations,
with the kcat value for SmCRV4 reaching 197 ± 14 min-1, which was
36.5-fold higher than that of SmCRwt (5.4 ± 0.3 min-1). The total
catalytic efficiency (kcat/Km) of SmCRV4 was increased by 110-fold
compared with SmCRwt. Notably, the stereoselectivity of SmCRV4
was also improved from 91% for the wild-type enzyme to 97%
(Figure 1b).
SmCRV4 could be heterologously expressed in a soluble form
in Escherichia coli cells and was purified for characterization
(Figure S4). SmCRV4 displayed the highest activity at 45 °C and
pH 7.0 in a phosphate buffer (Figure S5 and S6). The enzyme
activity was unchanged, or enhanced slightly, in most frequently
used organic solvents except THF, acetonitrile, and isopropanol
(Figure S7). SmCRV4 showed enhanced thermostability with half-
lives of 124 and 72 h at 30 and 40 °C (Figure S8 and Table S4),
respectively, which were higher than 83 h (30 °C) and 52 (40 °C)
h for SmCRwt (Figure S9 and Table S5). To investigate the
Figure 1. Directed evolution of SmCRwt. (a) Homology model of SmCRwt binding
with NADPH in the active site. Ser138, Tyr151 and Lys155 are catalytic triad.
(b) The specific activity and stereoselectivity of SmCR variants toward 1g.
Initially, SmCR displayed a specific activity of 3.3 and 89.0
mU/mgprotein toward 4-oxodecanoic acid (1c) and methyl 4-
oxodecanoate (1g) with >99% and 90% ee, respectively. To
improve the activity, we performed directed evolution of the wild-
type enzyme, with 1g as a model substrate. The homology model
of SmCRwt was constructed based on the known structure of a β-
ketoacyl-acyl carrier protein reductase (PDB ID: 1Q7B) in
complex with NADPH form Escherichia coli using the Modeller
program (Figure 1a). The template protein possesses 2.05 Å
resolution with 87.3% sequence identity of SmCRwt. First, error-
prone polymerase chain reaction (epPCR) was used to construct
a library of variants covering random changes in the whole
nucleotide sequence of the gene for the enzyme.[14] After first-
round mutagenesis and screening, the variant SmCRV1 R123C
with 1.6-fold increased activity was identified (Figure 1b and Table
S1). The catalytic efficiency (kcat/Km) was also improved by 2-fold
(Table 1). Using SmCRV1 R123C as the parent, we performed an
additional round of directed evolution, and, out of 6000 clones,
obtained the variant SmCRV2 R123C/L209P with a dramatic 28.4-
fold increase in the specific activity (Figure 1b and Table S2).
Compared with the R123C mutation which occurred at a distant
helix in the homology model of SmCRwt, the model suggests that
mutation at residue 209 is near to the active site, which is
essential for increased activity (Figure 1a). Thus, further
mutations at this position were investigated by site-saturation
mutagenesis. The activity assays for the mutants showed that
proline was indeed the best residue (Figure S1). Due to this
mutation, kcat/Km was improved dramatically by 44.8-fold, which
can be attributed to the smaller Km and larger kcat (Table 1).
substrate scope of SmCRV4
,
the specific activity and
stereoselectivity of the purified enzyme toward a panel of
structurally diverse aliphatic and aromatic keto acids and esters
were measured (Table 2). SmCRwt showed only marginal activity
toward the majority of the substrates tested (1a–1n). The activity
of SmCRV4 toward all the substrates was significantly enhanced
by directed evolution with improvements from 3.8-fold (1l) to 500-
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10.1002/cctc.201900382
ChemCatChem
COMMUNICATION
fold (1f). Specifically, SmCRV4 showed similar activity toward
linear medium- and long-chain γ/δ-keto acids (8–12 carbons, 1a–
1f). In particular, the stereoselectivity toward these substrates
was high, with ee values for the corresponding (R)-hydroxy acids
>99% for 1c, 1e, and 1f. SmCRV4 was more active toward γ-/δ-
keto esters than acids. For example, the activity toward 1g (7.66
U/mgprotein) and methyl 5-oxodecanoate (1h, 2.51 U/mgprotein) was
higher than toward 1c and 1d, respectively. The position of the
carbonyl group in the aliphatic keto acids and esters also affected
the activity of SmCRV4. Normally, SmCRV4 showed higher activity
toward γ- (1c, 1g, and 1j) compared with δ-keto acids and esters
(1d, 1h, and 1k). In addition to the aliphatic keto acids, SmCRV4
also showed measurable activity and high stereoselectivity
toward the aromatic γ/δ-keto acids, 1l and 1m, which are two
important chiral building blocks for the synthesis of various
pharmaceuticals.
substrates were dissolved in a sodium phosphate buffer (100 mM,
pH 7.0) containing 0.2 mM NADP+, 5% v/v DMSO, and the
necessary amount of E. coli cells expressing SmCRV4. The
reaction was performed at 30 °C with the addition of glucose and
cells containing glucose dehydrogenase from Bacillus
megaterium (BmGDH) to recycle of the cofactor nicotinamide
adenine dinucleotide phosphate (NADPH). Under these
conditions, the time-course for the asymmetric reduction of 1c and
1d by SmCRwt and SmCRV4 was explored (Figure S10). Ten mM
1c was completely converted in 3 h by the variant, while only 10%
was converted by the wild-type enzyme. For 1d, 10 mM substrate
was completely converted by SmCRV4 after 10 h, which was
superior to the conversion using the wild-type enzyme. Next, we
prepared eight optically pure γ-/δ-lactones (Table 3).[16] SmCRV4
displayed a powerful ability for the synthesis of (R)-γ-decalactone
(3c). After work-up, using 50 mM 1c as the substrate gave 99%
conversion after 2 h, yielding 3c (82%) with >99% ee and a
Table 2 Substrate scope of SmCRwt and SmCRV4
[ ]30
specific rotation of 훼 +45.0° (Table S7). The space-time yield
퐷
(STY) reached 83 g L-1d-1, which was nearly 10-fold greater than
the previously reported value (8.8 g L-1d-1). The STY was
increased up to 1175 g L-1d-1 for 3c, the highest value reported to
date, when 1.0 M 1g was 98% converted after 3 h, yielding 3c
(88%) with an ee of 97%. SmCRV4 was also very effective in the
synthesis of (R)-δ-decalactone (3d). After 3 h, 99% of 50 mM 1d
was converted, affording an 83% isolated yield of 3d with the
same ee value of 95% (R) as previously reported; however, the
catalyst consumption was greatly reduced from 150 to 20 g L-1
and the STY was increased to 56 g L-1 d-1. (R)-γ-undecalactone
(3e) and (R)-γ-dodecalactone (3f) are difficult to synthesize
because of the long carbon chains (11 and 12 carbons,
respectively) that decrease the reactivity of aliphatic keto acids.
Herein, both 3e and 3f were obtained with >99% ee in yields of
87% and 83%, respectively. The reactions were completed with
99% and 98% conversion with STY values of 24 and 12 g L-1 d-1,
respectively. Additionally, (R)-γ-lactones with medium chain
lengths, such as (R)-γ-octalactone (3a) and (R)-γ-nonalactone
(3b), were obtained with good efficiency. Furthermore, SmCRV4
was effective in the asymmetric synthesis of 3l and 3m, which are
used for preparing many pharmaceuticals. Previously, 3l and 3m
have been prepared using Ru complexes at high pressures of H2
(8–70 atm).[3,4] Herein, SmCRV4 also showed high
stereoselectivity for aromatic γ- and δ-keto esters and efficiency
comparable with the Ru-catalyzed asymmetric hydrogenation. In
10 or 6 h, 50 mM substrates were >98% converted giving 3l and
3m in 85% and 78% yield, respectively. In particular, the reactions
proceeded at room temperature and normal pressure.
In conclusion, the efficient enantioselective synthesis of
diverse aliphatic and aromatic γ- and δ-lactones using the
engineered carbonyl reductase SmCRV4 was achieved. The
variant was identified using directed evolution and showed
enhanced activity, stability, and stereoselectivity toward a panel
of γ-/δ-keto acids and esters, compared with the wild-type enzyme
and other variants. Under the optimized conditions, the γ-/δ-keto
acids and esters were efficiently reduced to the corresponding γ-
and δ-hydroxy acids and esters, followed by simple intramolecular
cyclization yielding γ- and δ-lactones with ee values up to >99%.
The volumetric productivity for the preparation of the (R)-γ/δ-
decalactones was the highest reported to date, and lactones
Entry
Substrate
Specific activity (mU/mg)
eep (%)
SmCRwt
SmCRwt
SmCRV4
210
Folda
SmCRV4
96 (R)
99 (R)
>99 (R)
95c (R)
>99 (R)
>99 (R)
97 (R)
70c (S)
7 (R)
1
2
1a
1b
1c
1d
1e
1f
3.7
17.0
3.3
56.8
5.1
61 (R)
97 (R)
>99 (R)
95c (R)
>99 (R)
>99 (R)
91 (R)
31c (S)
n.d.b
86.0
640
3
194
157
186
500
86.1
49.2
126
49
4
2.1
330
5
2.2
410
6
0.8
400
7
1g
1h
1i
89.0
51.0
12.0
69.0
51.0
5.3
7660
2510
1510
3380
2510
20
8
9
10
11
12
13
14
1j
75 (R)
40c (S)
n.d.
80 (R)
71c (S)
98 (S)
97 (S)
45 (S)
1k
1l
49.2
3.8
1m
1n
1.7
130
76.5
54.3
57 (S)
n.d.
9.4
510
a Fold change improvement in the activity of SmCRV4 over SmCRwt. b n.d. = not
detected. c These numbers are estimated values.
With the best variant SmCRV4 in hand, we investigated the
synthesis of a series of valuable optically pure γ-/δ-lactones with
diverse structures at a preparative scale. In general, the
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10.1002/cctc.201900382
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COMMUNICATION
Table 3 Preparative synthesis of optically pure γ- and δ-lactones
Entry
1
Product
Substrate
(mM)
Catalyst
(g L-1)
20b
Time
(h)
Conv.
(%)
Isolated
STY
ee
Ref.
yield (%)
(g L-1 d-1)
(%)
50
50
50
4
98
>99
95
82
90
73
34
13
96 (R)
99 (R)
this work
(3a)
2
3
20b
12
16
this work
12
(3b)
150a
8.8
>99 (R)
4
5
50
20b
75c
2
3
>99
98
82
88
83
>99 (R)
97 (R)
this work
this work
(3c)
1000
1175
6
7
50
50
150a
20b
16
3
92
75
83
8.8
56
95 (R)
95 (R)
12
>99
this work
(3d)
8
9
50
50
20b
20b
8
>99
98
87
83
24
12
>99 (R)
>99 (R)
this work
this work
(3e)
16
(3f)
10
11
50
50
20b
10
>99
98
85
78
18
25
98 (S)
97 (S)
this work
this work
(3l)
20b
6
(3m)
a Glucose (5.0 equiv), NADP+ (0.5 mM), lyophilized E. coli/BmGDH (10 g L-1). b Glucose (1.5 equiv), NADP+ (0.2 mM), lyophilized E. coli/BmGDH (2 g L-1). c Glucose
(1.5 equiv), NADP+ (0.2 mM), wet cells of E. coli/SmCRV4 (75 g L-1), lyophilized E. coli/BmGDH (10 g L-1). d Space-time yield. e Determined by chiral GC.
all obtained in CDCl3. High resolution mass spectra (HRMS) were obtained
by electro spray ionization (ESI) and the errors between observed and
theoretical monoisotopic molecular masses were less than 5 ppm. Column
chromatography was carried out by using silica gel of the selected particle
size of 200-300 mesh. n-Dodecane was used as internal standard. A
Rudolph Research Analytical Autopol I automatic polarimeter was used for
optical rotation measurement.
substituted with alkyl chains of 11 and 12 carbons were also
prepared for the first time using this enzyme-catalyzed route. This
enzyme-catalyzed synthesis exhibited high efficiency and was
environmentally friendly, thus making this a promising method for
the industrial preparation of optically pure lactones for various
purposes.
Directed evolution ： The recombination plasmid containing SmCRwt
gene was used as the template for random mutagenesis. 0.15 mM MnCl2
was used to obtain the desired level of mutagenesis rate (about 1 to 2
amino acid substitutions). The amplified PCR products were extracted,
digested with EcoR I and Hind III, and ligated into the EcoR I and Hind III
sites of pET-28a, then transformed into chemical competent E. coli BL21
(DE3) cells. The recombination plasmid containing SmCRV2 gene used
was amplified by PCR with NNK codon degeneracy. The resulting PCR
products were digested with Dpn I (20 U) at 37°C for 1 h and was next
transformed into the chemical competent E. coli BL21 (DE3) cells and
plated on LB agar plate containing 50 μg/ml kanamycin. The colonies were
Experimental Section
General information：All chemicals were purchased from TCI (Japan),
Aladdin and Shaoyuan (Shanghai, China) without further treatment unless
otherwise indicated. NADPH and NADP+ were purchased from Bontac
Bioengineering (Shenzhen, China). Luria-Bertani media was used for the
culture of Escherichia coli cells.
A
Shimadzu GC-2014 gas
chromatography was used for GC analysis with a CP-Chirasil-Dex CB
column (25 m × 0.25 mm × 0.39 mm, Varian) and a flame ionization
detector (FID). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
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10.1002/cctc.201900382
ChemCatChem
COMMUNICATION
picked with sterile toothpicks to inoculate in 200 mL LB media containing
50 μg/mL kanamycin in 96-well plates. The cultures were grown overnight
at 37°C prior to inoculate 600 mL LB in new 96-well plates. The plates
were incubated at 37°C for 3 h, and protein expression was induced with
the addition of IPTG (0.1 mM) at 16°C for another 24 h. Cells were lysed
by adding 200 mL buffer containing lysozyme (0.75 mg/mL) and DNase I
(0.01 mg/mL) at 37°C for 2 h. The plates were centrifuged at 3420 × g for
20 min at 4°C. A volume of 50 mL sample from each well was transferred
to a microtiter plate, and then the reduction reaction was initiated by adding
a reaction mixture of 150 mL 100 mM phosphate buffer (pH 7.0), 0.2 mM
NADPH and 4 mM substrate 1g. The activity of the mutants was
determined by measuring the absorbance change of NADPH at 340 nm
for 10 min at 30°C using a microplate spectrophotometer (BioTek, USA).
Mutants with higher acitivity were chosen for re-screening in an 96 deep-
well plate, and the top hits were further grown on a 100 mL scale. The best
mutants were selected for sequencing and purified by nickel affinity
chromatography using N-terminal His-tagged for further characterization.
This work was financially sponsored by the National Key
Research
and
Development
Program
of
China
(2016YFA0204300), National Natural Science Foundation of
China (Nos. 21536004, 21776085 & 21871085), Natural Science
Foundation of Shanghai (18ZR1409900 & 18DZ1112703) and the
Fundamental Research Funds for the Central Universities
(22221818014, 222201714026 & WF1714026).
Keywords: Asymmetric reduction• directed evolution• chiral
lactones• carbonyl reductase• biocatalysis
[1]
a) A. Díaz-Rodríguez, W. Borzęcka, I. Lavandera, V. Gotor, ACS Catal.
2014, 4, 386-393; b) K. Aplander, O. Hidestål, K. Katebzadeh, U. M.
Lindström, Green Chem. 2006, 8, 22-24; c) M. Seitz, O. Reiser, Curr.
Opin. Chem. Biol. 2005, 9, 285-292; d) R. R. A. Kitson, A. Millemaggi, R.
J. K. Taylor, Angew. Chem. Int. Ed. 2009, 48, 9426-9451; e) H. K. Grover,
M. R. Emmett, M. A. Kerr, Org. Lett. 2013, 15, 4838-3841.
a) R. Noyori, T. Ohkuma, M. Kitamura, H. Takaya, N. Sayo, H.
Kumobayashi, S. Akutagawa, J. Am. Chem. Soc. 1987, 109, 5856-5658;
b) R. Noyori, Angew. Chem. Int. Ed. 2002, 41, 2008-2022.
X. H. Yang, J. H. Xie, W. P. Liu, Q. L. Zhou, Angew. Chem. Int. Ed. 2013,
52, 1-5.
Enzyme assay and kinetic analysis: The reductase activity was assayed
at 30°C by monitoring the decrease in the absorbance of NADPH at 340
nm on a UV spectrophotometer (Beckman DU730). The standard assay
mixture (1 mL) consists of 970 μL of sodium phosphate buffer (100 mM,
pH 7.0), 10 μL of methyl 4-oxodecanoate(200 mM), 10 μL NADPH (10 mM),
and 10 μL of pure enzyme with an appropriate concentration. One unit of
enzyme activity (U) is defined as the amount of enzyme that can catalyze
the oxidation of 1 μmol of NADPH per minute under above conditions. The
substrates were dissolved to different final concentrations with DMSO, and
then the specific activity was measured by a UV spectrophotometer. The
data were processed using Origin 9.0 based on the Michaelis–Menten
equation.
[2]
[3]
[4]
[5]
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Acknowledgements
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This article is protected by copyright. All rights reserved.

10.1002/cctc.201900382
ChemCatChem
COMMUNICATION
In this work, structurally diverse γ- and
Meng Chen, Xiao-Yan Zhang, Chen-
Guang Xing, Chao Zhang, Yu-Cong
Zheng, Jiang Pan, Jian-He Xu* and Yun-
Peng Bai*
δ-lactones
were
efficiently
stereoselectively synthesized using an
engineered carbonyl reductase from
Serratia
marcescens
(SmCRV4).
Page No. – Page No.
SmCRV4 exhibited improved activity
(up to 500-fold) and thermostability
toward 14 γ-/δ-keto acids and esters
compared with the wild-type enzyme,
with 110-fold enhancement in catalytic
efficiency (kcat/Km) toward methyl 4-
Efficient Stereoselective Synthesis of
Structurally Diverse γ- and δ-Lactones
Using an Engineered Carbonyl
Reductase
oxodecanoate.
The
preparative
synthesis of alkyl and aromatic γ- and
δ-lactones with 95.0%–99.9% ee was
demonstrated. The highest space-time
yield, 1175 g L−1 d−1, was achieved for
(R)-γ-decalactone.
This article is protected by copyright. All rights reserved.