A new clade of styrene monooxygenases for (R)-selective epoxidation

Article abstract of DOI:10.1039/d0cy02312d

Styrene monooxygenases (SMOs) are excellent enzymes for the production of (S)-enantiopure epoxides, but so far, only one (R)-selective SMO has been identified with a narrow substrate spectrum. Mining the NCBI non-redundant protein sequences returned a new distinct clade of (R)-selective SMOs. Among them,SeStyA fromStreptomyces exfoliatus,AaStyA fromAmycolatopsis albispora, andPbStyA fromPseudonocardiaceaewere carefully characterized and found to convert a spectrum of styrene analogues into the corresponding (R)-epoxides with up to >99% ee. Moreover, site 46 (AaStyA numbering) was identified as a critical residue that affects the enantioselectivity of SMOs. Phenylalanine at site 46 was required for the (R)-selective SMO to endow excellent enantioselectivity. The identification of new (R)-selective SMOs would add a valuable green alternative to the synthetic tool box for the synthesis of enantiopure (R)-epoxides.

Full text of DOI:10.1039/d0cy02312d

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A new clade of styrene monooxygenases for (R)-
selective epoxidation†
Cite this: Catal. Sci. Technol., 2021,
11, 2195
Hu Xiao,abd Shuang Dong,c Yan Liu,a Xiao-Qiong Pei,a
c
a
*
*
Hui Lin
and Zhong-Liu Wu
Styrene monooxygenases (SMOs) are excellent enzymes for the production of (S)-enantiopure epoxides, but
so far, only one (R)-selective SMO has been identified with a narrow substrate spectrum. Mining the NCBI
non-redundant protein sequences returned a new distinct clade of (R)-selective SMOs. Among them, SeStyA
from Streptomyces exfoliatus, AaStyA from Amycolatopsis albispora, and PbStyA from Pseudonocardiaceae
were carefully characterized and found to convert a spectrum of styrene analogues into the corresponding
(R)-epoxides with up to >99% ee. Moreover, site 46 (AaStyA numbering) was identified as a critical residue that
affects the enantioselectivity of SMOs. Phenylalanine at site 46 was required for the (R)-selective SMO to
endow excellent enantioselectivity. The identification of new (R)-selective SMOs would add a valuable green
alternative to the synthetic tool box for the synthesis of enantiopure (R)-epoxides.
Received 1st December 2020,
Accepted 16th January 2021
DOI: 10.1039/d0cy02312d
rsc.li/catalysis
to serve as remarkable biocatalysts for (substituted) styrenes
and analogues such as secondary allylic alcohols, and allyl
Introduction
Both (R)- and (S)-enantiomers of epoxides are essential inter-
mediates in organic synthesis for active pharmaceuticals, nat-
ural products, fine chemicals, and advanced polymeric mate-
rials.1,2 Therefore, significant effort has been devoted to
developing efficient asymmetric epoxidation methods.2,3
Stereoselective chemo-catalyzed epoxidations are well-
established for internal alkenes or alkenes bearing functional
groups, but suffer from insufficient enantioselectivity for ter-
minal alkenes.4–8
As a complementary synthetic method, biocatalytic epoxi-
dation has proven efficient to produce highly enantiopure ep-
oxides from terminal alkenes.2,3 Styrene monooxygnases,9,10
cytochrome P450s,11–13 chloroperoxidases,14 and alkene
monooxygenases15 are reported to catalyze the epoxidation of
terminal alkenes with moderate to excellent enantio-
selectivity. Among them, styrene monooxygenases (SMOs) are
known to display almost exclusively epoxygenase activity with
outstanding enantioselectivity. As the first enzyme in the sty-
rene catabolism cascade, SMOs have shown natural capacity
benzenes, and often yield the corresponding (S)-epoxides
with >99% ee.16–28 Recent studies on cascade reaction de-
sign, cosubstrate generation, and new SMO identification
have further increased the potential of SMOs in synthetic
applications.25,29–31
SMOs are classified as group E monooxygenases that cata-
lyze epoxidation and sulfoxidation.10,27,32 They are two-
component flavoproteins that contain
a FAD-dependent
monooxygenase unit (StyA) and a NADH-dependent flavin oxi-
doreductase unit (StyB) for the regeneration of reduced
FAD.33,34 Functionally characterized SMOs display either (R)-
or (S)-selectivity in sulfoxidation.35 However, in epoxidation,
they are well-known to only lead to enantiopure (S)-styrene
oxide.35 Until very recently, we identified the first (R)-selective
SMO from Streptomyces sp. NRRL S-31 (StStyA), which cata-
lyzed the epoxidation of styrene and four analogues, yielding
the corresponding (R)-epoxides with 91–99% ee.29
However, compared to known (S)-selective SMOs, the
enantioselectivity of StStyA toward styrene appeared signifi-
cantly lower (91% ee vs. >99% ee), and several para- and
meta-substituted styrenes, known as substrates for (S)-SMOs,
were not accepted by StStyA.29 Nevertheless, the finding of
StStyA would certainly facilitate the search for more (R)-selec-
tive SMOs, and we expect that some of them may have im-
proved characteristics and complementary substrate scope.
Herein, we report the identification of 8 new (R)-SMOs
and 2 new (S)-SMOs from sequence databases. They are clus-
tered in distinct groups in phylogenetic analysis of known
and postulated SMOs. Three of the new (R)-SMOs were
a CAS Key Laboratory of Environmental and Applied Microbiology, Environmental
Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology,
Chinese Academy of Sciences, Chengdu 610041, China. E-mail: wuzhl@cib.ac.cn
b College of Life Sciences, Sichuan University, No. 29 Wangjiang Road, Chengdu
610064, China
c College of Life Sciences, Henan Agricultural University, 95 Wenhua Road,
Zhengzhou 450002, China. E-mail: huilin@henau.edu.cn
d University of Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cy02312d
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characterized in detail for the epoxidation of 13 alkene sub-
strates in presence of the StyB from Pseudomonas sp. LQ26 to
generate reduced FAD. They displayed improved activity and
enantioselectivity, as well as expanded substrate scope. A resi-
due conserved in (R)-SMOs was also investigated for its role
in stereo-control.
they could also be regarded as SMO-like monooxygenases
(Fig. 1).
The two new (S)-selective SMOs (GenBank No.
WP_125743710.1 and WP_141293187.1) both belong to group
III. They afforded (S)-styrene oxide with good enantioselectivity
(88–90% ee), but the selectivity is significantly lower than
known (S)-SMOs, which often provide (S)-styrene oxide with
>99% ee.10,18,20,25,37 The results indicated that the putative
SMOs in the group III are (S)-selective SMOs.
All the eight newly identified (R)-SMOs belong to group IV.
They converted styrene into (R)-styrene epoxide as the only
product, and with good to excellent enantioselectivity (85% to
>99% ee). Among them, StyA from Pseudonocardiaceae
Results and discussion
Exploring (R)-selective styrene monooxygenases
To explore more (R)-selective SMOs, the amino acid sequence
of StStyA was subjected to BlastP search against the NCBI
non-redundant protein database, which returned 100 se-
quences with E value <3e–89 and identities of >40%. Then,
phylogenetic analysis was conducted for 63 proteins, includ-
ing 34 returned sequences of >49% identities to StStyA, 28
previously known (S)-selective SMOs, and StStyA.
Interestingly, the neighbour joining phylogenetic analysis
demonstrated that those 63 proteins can be divided into
three clades (Fig. 1), in which, all the known (S)-selective
SMOs are divided into two clusters (group I and II). Group I
consists of classic E1-type SMOs with several of them having
established role in styrene degradation.36 Group II consists of
the E2-type, also known as indole monooxygenases, which ap-
pear not related to styrene degradation clusters.34 Group II
also includes some self-sufficient fusion proteins with an as-
sociated monooxygenase such as IndA1/A2B from Variovorax
paradoxus (ADU39062.1 and ADU39063.1, Fig. 1) and
Rhodococcus opacus (ACR43973.1 and ACR43974.1, Fig. 1).35
All the 34 putative SMOs are clustered together with StStyA in
one clade, which is further divided into two clusters (group
III and IV). StStyA appears in group IV, which is located far
away from group I and group II of (S)-selective SMOs, while
group III is located close to the (S)-selective SMOs (Fig. 1).
Accordingly, further study was focused on group IV in
hope of discovering more new (R)-selective SMOs. The coding
sequences of 19 of 25 (76%) putative SMOs in group IV and 2
of 9 (22%) in group III (Table 1) were synthesized and
expressed in Escherichia coli for further functional analysis.
Because all the putative SMOs investigated here contained
only the monooxygenase unit StyA, which require FADH2 to
function, each of the StyA was co-expressed with the NADH-
dependent flavin oxidoreductase PsStyB (GenBank No.
ADE62391.1) to achieve a functional catalytic system.29 The
resulting E. coli whole cells harbouring one putative SMO
and PsStyB were used as the catalyst for the epoxidation of
styrene (Table 1).
Fig. 1 Phylogenetic analysis of known (S)-selective SMOs, StStyA, and
34 putative (R)-selective SMOs. Phylogenetic tree was constructed in
MEGA X and modified by ITOL. The evolutionary history was inferred
using the neighbor-joining method. The tree is divided into four
groups based on the clade of the tree. The alignment of residue 46
(AaStyA numbering) in SMOs is showed on the right side. Group I and
group II contain previously reported (S)-SMOs (GAB22407.1;33
ABV24041.1;18
BAL04132.1;37
ASR05591.1;36
BAL04129.1;37
ANS32444.1;38
WP_039579272.1;39
WP_022977994.1;35
WP_027855270.1;25 GAC06215.1;25 ADE62390.1;20 CAB06823.1;35
The results showed that 11 putative SMOs did not exhibit
catalytic activity, while 10 SMOs exhibited activity in styrene
epoxidation. Among them, eight were (R)-selective, and two
were (S)-selective (Table 1). The adjacent genomic informa-
tion of the 10 active enzymes were also analyzed, but the re-
sults revealed the lack of styrene degradation cluster for all of
them (data not shown), which reflected a significant differ-
ence of those new enzymes from classic SMOs. Therefore,
ABX24519.1;19
ACR43974.1;38 EJJ03822.1;35 WP_028028710.1;35 ABM07034.1;35
BAD56093.1;35 ACR43973.1;38 CAG69430.1;33,40 EEA04565.1;35
AAC23718.1;10
ABM10099.1;35
BAD56094.1;35
CAJ94554.1;35 ADU39062.1;41 ABX34433.1;35 ADU39063.1 (ref. 41)).
Different sequences from the same organism in group II are self-
sufficient fusion proteins IndA2B with an associated monooxygenase
IndA1.35,38,41 Group III and group IV contain 34 putative SMOs, and 21
of them were experimentally investigated in this work, including two
(S)-SMOs (blue), eight (R)-SMOs (red), and non-active SMOs (purple).
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Table 1 Bioepoxidation of styrene by recombinant SMOsa
Source strain
Accession no.
Conv.b (%)
eeb (%)
Group III
Streptomyces gardneri strain NBRC 12865
Streptomyces sp. WAC01280
WP_141293187.1
WP_125743710.1
15
48
88(S)
90(S)
Group IV
Streptomyces achromogenes subsp. strain
Streptomyces noursei ATCC 11455
Streptomyces yunnanensis strain CGMCC
Streptomyces griseocarneus strain 132
Streptomyces eurocidicus strain ATCC
Pseudonocardiaceae bacterium YIM PH 21723
Pseudonocardiaceae bacterium YIM PH 21723
Nocardia altamirensis NBRC 108246
Nocardia pseudobrasiliensis NBRC 108224
Nocardia alba DSM 44684
Streptomyces sp. ZFG47
Streptomyces sp. NRRL S-31
Streptomyces NRRL B-2120
Streptomyces exfoliates strain A1013Y
Amycolatopsis albispora strain WP1
Kutzneria sp. 744
Streptomyces noursei ATCC 11455
Streptomyces sp. 769
WP_051753842.1
WP 067345356.1
WP_073449384.1
WP_121802755.1
WP_102918856.1
WP_120086312.1
WP_120086314.1
WP_069162359.1
WP_067993035.1
WP 067452867.1
WP_112440363.1
WP_030740546.1
WP_030605949.1
WP_137992763.1
WP_113694022.1
WP_043716969.1
WP 067345360.1
WP_052286622.1
WP_073449381.1
WP 102925532.1
0
0
0
0
0
28
0
64
0
13
0
94
15
100
66
29
0
8
0
0
—
—
—
—
—
>99(R)
—
91(R)
—
90 (R)
—
91(R)c
85(R)
95(R)
94(R)
90(R)
—
90(R)
—
—
Streptomyces yunnanensis strain
Streptomyces noursei strain JCM4701
a
The reaction mixture contained 0.1 g CDW E. coli cells and 4 mM styrene in 5 mL potassium phosphate buffer (0.1 M, pH 7.0). The reactions
b
were carried out at 30 °C and 220 rpm for 2 h. Conversion of styrene to styrene oxide and enantiomeric excess of product were determined
using Chiral HPLC. The results were from previous work.29
c
Table 2 Biotransformation of aromatic alkenes by (R)-SMOsa
SeStyA
Conv.b (%)
100
AaStyA
PbStyA
Entry
Substrate
Product
eeb (%)
95(R)
Conv.b (%)
eeb (%)
94(R)
Conv.b (%)
eeb (%)
>99(R)
1
2
3
4
5
6
7
8
R = H, 1a
1b
2b
3b
4b
5b
6b
7b
8b
66
34 1b
28
17 1b
2
28
5
10
0
29
9
3
1
2
R = o-Br, 2a
R = m-Br, 3a
R = p-Br, 4a
R = o-Cl, 5a
R = m-Cl, 6a,
R = p-Cl, 7a
R = o-Me, 8a
R = m-Me, 9a
R = p-Me, 10a
R = m-OMe, 11a
R = p-OMe, 12a
12
93
16
23
1
3
2
3
94(R)
93(R)
94(S)
94(R)
94(R)
88(R)
88(R)
>99(S)
88(R)
90(R)
90(S)
1
>99(R)
>99(R)
—
50(R)
>99(R)
—
>99(R)
>99(R)
—
>99(R)
—
3
39
38
3
2
34 2b
1
14
93
94
3
2
5
4
86(S)
>99(R)
78(R)
>99(S)
91(R)
>99(S)
98(1R,2S)
11 1c
0
87
19
5
5
2
83(R)
74(R)
>99(S)
81(R)
>99(S)
>99(1R,2S)
34
46
0
2
4
9
9b
10
11
12
13
10b
11b
12b
1
76
3
3
1
18
3
28
2
3
18
0
3
1
100
15 1d
>99(1R,2S)
, 13a
, 13b
a
The reaction mixture containing 0.1 g CDW E. coli cells, 5 mL potassium phosphate buffer (0.1 M, pH 7.0), and 4 mM substrate was
b
c
d
incubated at 30 °C for 2 h. Determined using Chiral HPLC or GC. Reaction time was extended to 4 h. Substrate loading was reduced to 1
mM.
(GenBank No. WP_120086312.1, designated as PbStyA)
showed the best enantioselectivity (>99% ee), while StyA
from Streptomyces exfoliatus (GenBank No. WP_137992763.1,
designated as SeStyA) afforded the highest conversion (100%)
and good enantioselectivity (93% ee). The SMO from
Amycolatopsis albispora (GenBank No. WP_113694022.1,
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designated as AaStyA) also exhibited good activity (66% con-
version) and enantioselectivity (94% ee). Therefore, those
three new SMOs, SeStyA, PbStyA, and AaStyA were selected for
further characterization.
The electron effect certainly played a role. In average,
SeStyA showed higher catalytic activities toward substrates
with
electron-donating
substituents
than
electron-
withdrawing ones (8a–11a vs. 2a–7a), which was consistent
with the previously identified (S)-selective SMOs.28 However,
m-bromo-styrene (3a) with an electron-withdrawing group
appeared to an excellent substrate for SeStyA.
SeStyA, AaStyA and PbStyA-catalyzed epoxidation of styrenes
All the three (R)-selective SMOs could catalyze the epoxida-
tion of a variety of styrenes, and yielded the corresponding
oxides with good to excellent enantioselectivity (up to 99%
ee) with no by-product observed (Table 2). Among them,
SeStyA, AaStyA showed activity to all 13 substrates tested,
AaStyA had similar characteristics to SeStyA in epoxidation,
but its whole-cell activities were generally lower than SeStyA ex-
cept for o-chloro- and o-bromo-styrene (2a and 5a). Although
AaStyA showed lower enantioselectivity toward several sub-
strates compared to SeStyA (2a, 3a, 9a, and 11a), it provided ab-
solute selectivity with substrates 4a, 10a, 12a, and 13a.
while PbStyA showed activity to
9 of them, leaving 4
para-substituted styrenes unreacted. Gratifyingly, the ortho-
and para-substituted styrenes (such as 5a, 7a, 12a) that were
not accepted by the first (R)-selective SMO (StStyA),29 were
successfully epoxidized by the newly identified (R)-SMOs.
As expected, most products were of the R configuration,
but surprisingly, all four para-substituted styrenes resulted in
(S)-oxides (Table 2, entry 4, 7, 10 and 12). The enantiomeric
excesses of the (S)-oxides were good to excellent, ranging
from 86–>99% ee. Such drastically reversed enantioselectivity
for a particular set of substrates has not reported for other
group E flavin-dependent monooxygenases. Presumably, the
steric effect, rather than the electron effect of the
p-substituents may play a critical role in determining the sub-
strate orientation in the active cavity, as both electron-
withdrawing (4a and 7a) and -donating (10a and 12a) groups
led to (S)-oxides as long as they are located at the para
position.
SeStyA afforded higher conversions than AaStyA and
PbStyA for 6 substrates (Table 2, entries 1, 3, 8, 9, 11). It
achieved complete conversion for styrene (1a) within 2 h, but
showed lower activity toward substituted styrenes. AaStyA and
PbStyA, on the other hand, each achieved the best conversion
toward substituted styrenes, 8a and 9a, respectively. The re-
sults indicate that the active site of SeStyA might be more
sensitive to steric hindrance effect, especially when the sub-
stituent is located at the ortho-, or para-position.
PbStyA delivered the lowest conversion among the three
SMOs for the majority of substrates, but it had excellent
enantioselectivity. Among the
9 substrates accepted by
PbStyA, only 2-chlorostyrene (5a) yieled (R)-oxide with 50% ee,
while all the others yielded (R)-oxides with >99% ee. In par-
ticular, PbStyA appeared to be the only SMO that produce
enantiopure (R)-styrene oxide (1b) of >99% ee; the selectivity
was comparable to all the previously known (S)-
SMOs.10,18,25,28,37 PbStyA also provided the best enantio-
selectivity for substrate 2a (>99% ee) among all the known
(R)-SMOs.
Enzyme kinetic analysis
To determine the steady-state kinetic parameters of SeStyA,
AaStyA, and PbStyA, each StyA was first purified by Ni2+-NTA
agarose resin (Fig. S3†). The yields from one liter of culture
were 200 mg, 60 mg and 15 mg for SeStyA, AaStyA and
PbStyA, respectively. Then each StyA was mixed with purified
PsStyB and ChkRED20 (ref. 42) to form a reconstituted reac-
tion system. A mixture of 20 μM StyA, 10 μM PsStyB, and
8 μM ChKRED20 was used for the enzymatic assay.29 And the
styrene epoxidation reactions were performed at 30 °C and
pH 7.0 with varied styrene concentration ranging from 1–40
mM (Fig. S4†).
The steady-state kinetic estimation (Table 3) showed that
SeStyA and AaStyA had similar Km values of 1.54 and 2.10
mM, respectively, while PbStyA had much higher value of
17.3 mM, indicating weak binding affinity to styrene. On the
other hand, all the three (R)-SMOs appear to have higher Km
than the (S)-SMOs from Pseudomonas sp. LQ26 (ref. 20) and
Pseudomonas taiwanensis VLB120,10 which were reported to
The bicyclic alkene 1,2-dihydronaphthalene (13a) served
as a good substrate for SeStyA with 100% conversion and
98% ee. It was found to inhibit the activity of PbStyA at 4 mM
loading, but could be epoxidized at a lower substrate loading
of 1 mM with excellent enantioselectivity (>99% ee) (Table 2,
entry 13).
Table 3 The activities of StyAs on styrenea
Enzyme
Km (mM)
kcat (min−1
)
kcat/Km (mM−1 min−1
)
Specific activity (U mg−1
)
SeStyA
AaStyA
PbStyA
1.54 0.29
2.10 0.23
17.3 2.64
16.36 0.75
3.26 0.10
1.63 0.11
10.62 1.51
1.55 0.12
0.092 0.015
0.32
0.053
0.027
a
The reaction mixture containing 20 μM of purified StStyA, 10 μM of purified PsStyB, 8 μM of purified ChKRED20, 260 mM iso-propanol, 0.1
mM NAD+, 50 μM FAD, 650 U catalase, and varying concentrations of styrene (from 1 M stocking solution in iso-propanol) in potassium phos-
phate buffer (0.1 M, pH 7.0) was incubated at 30 °C for 10 min.
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be 0.41 and 0.45 mM. The turnover frequency (kcat) of PbStyA
was 1.63 min−1 toward styrene, similar to the (S)-SMO from
Pseudomonas sp. LQ26 (1.5 min−1),20 but SeStyA and AaStyA
were more active, displaying 10-fold and 2-fold of kcat values
of PbStyA, respectively (Table 3).
Role of residue 46 in the enantioselectivity of SMOs
The phylogenetic analysis and multiple-sequence alignments
revealed that residue 46 (AaStyA numbering) is highly con-
served in each clade (Fig. 1 and S2†). The SMOs in group I, II
and III, catalyze (S)-selective epoxidation. They have Thr, Ser,
and Leu at this position, respectively. However, in group IV,
SMOs catalyze (R)-selective epoxidation, and they have Phe in-
stead. One exception in group IV is a putative SMO from
Pseudonocardiaceae bacterium YIM PH 21723 (Genbank No.
WP_120086314.1), which has Thr at the corresponding posi-
tion (Fig. 1), but this enzyme did not show activity toward
styrene.
Those interesting results indicated that residue 46 might
play an important role in the enantioselectivity of SMOs. To ex-
plore such an effect, we constructed three point mutants
containing the Phe to Thr mutation, each from the (R)-selective
AaStyA, SeStyA and PbStyA, resulting in mutants AaStyA-F46T,
SeStyA-F57T and PbStyA-F46T. In addition, we also constructed
one reverse mutant, PsStyA-T47F, from the (S)-selective PsStyA
(Genbank No. ADE62390.1) for comparison.
Fig.
3
Enantioselectivity of AaStyA variants at site 46 in the
epoxidation of styrene.
yielded racemic styrene epoxide. At the same time, the
enantioselectivity of the reverse mutant PsStyA-T47F, was
switched from S to R, affording (R)-styrene epoxide with 6%
ee and 4% conversion (Fig. 2).
To further enlighten the role of residue 46 in stereo-con-
trol, the eighteen other amino acid substitutions at this site
of AaStyA was generated using site-directed mutagenesis, and
all the mutants were analysed with styrene as the substrate.
Of the collectively 19 variants, four mutants with electrically
charged side chains (F46E, F46K, F46R, and F46D) completely
lost activity, while the others were active, but with reduced
activity (Table S3†). Eleven mutants yielded (R)-styrene epox-
ide with 24–88% ee, lower than the wild-type (94% ee), and
four mutants (F46H, F46N, F46T, and F46P) exhibited re-
versed stereoselectivity, producing (S)-epoxide with 5–65% ee
(Fig. 3). Interestingly, all the mutants with a typical hydro-
phobic side chain (Phe, Ile, Met, Leu, Trp, Ala, Tyr and Val)
displayed R-selectivity (Fig. 3), while those with uncharged
polar residues were divided. Medium-sized residues (His, Asn
and Thr) led to S-selectivity, but others (Gly, Ser, Cys, and
Gln) led to R-selectivity (Fig. 3).
After analysing the activity and enantioselectivity of the
four mutants toward the epoxidation of styrene, we found
that PbStyA-F46T was inactive, but the other two mutants
from (R)-selective SMOs were active (Fig. 2), albeit with signif-
icantly decreased activity. Excitingly, their enantioselectivity
was more or less switched from R to S (Fig. 2). AaStyA-F46T
yielded (S)-styrene epoxide with 22% ee, and SeStyA-F57T
The results clearly supported the hypothesis that residue
46 is a critical residue controlling the enantioselectivity of
SMO. We propose that both the electronic and steric factors
of the residue 46 contribute to the shape of the substrate-
binding site, and consequently, the orientation of the sub-
strate in the catalytic centre. However, it's noteworthy that
this single mutation was not able to completely reverse the
enantioselectivity, indicating the influence of other residues
around the active cavity. Further investigation will require
structural information of (R)-SMOs to fully understand the
mechanism of the stereo-control.
Conclusions
Styrene monooxygenases were previously considered (S)-spe-
cific epoxygenases. Here we identified a distinct clade of (R)-
selective SMOs that clustered together in phylogenetic analy-
sis. Eight of them were functionally verified to catalyze the
Fig. 2 The wild type of SeSMO, AaSMO, and PsSMO, and the mutant
SeStyA F57T, AaStyA F46T, and PsStyA T47F catalyzed the epoxidation
of styrene.
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(R)-selective epoxidation of styrene, yielding (R)-styrene oxide
with 85–>99% ee. SeStyA, AaStyA and PbStyA, in particular,
accepted a spectrum of styrene derivatives, with R-selectivity
in most cases, which makes them competitive candidates for
(R)-epoxide preparation. Our findings confirmed the stereo-
complementary nature of SMOs, and expanded their applica-
tion scope in asymmetric synthesis.
and PsStyA as the template. Specific primers were designed
for each mutation (Table S4†). The successful incorporation
of mutation was confirmed by sequencing (Sangon Biotech,
Shanghai, China).
Biotransformation of styrene and derivatives
Freshly prepared E. coli cells (0.1
g CDW) containing
expressed StyAs and PsStyB (0.5 g) were resuspended in 5 ml
potassium phosphate buffer (100 mM, pH 7.0). Then, 4 mM
substrate (from 1 M stocking solution in iso-propanol) was
added to the mixture. The reaction was carried out at 30 °C
for 2 h with gyratory shaking at 200 rpm and terminated by
extraction with 5 mL ether for twice. The combined organic
phase was dried with anhydrous sodium sulfate, and concen-
trated. The samples were subject to standard GC (Agilent
Technologies 7890B) and HPLC analysis (Shimadzu Promi-
nence LC-20 AD) to determine the conversion ratio of sub-
strates and the optical purity of products.20,28,29 For NMR
analysis, the products were purified by silica gel column
chromatography, and eluted with petroleum ether/ethyl ace-
tate (200 : 1).
Purified enzymes were used in the determination of
steady-state kinetic parameters and specific activity. The reac-
tion mixture contained 20 μM of purified StyA, 10 μM of puri-
fied PsStyB, 8 μM of purified ChKRED20, 0.1 mM NAD+, 50
μM FAD, 650 U catalase (Sangon Biotech, Shanghai), and
varying concentrations of styrene in 1 mL potassium phos-
phate buffer (100 mM, pH 7.0). After incubated at 30 °C for 5
min, the mixture was extracted with ether, and the organic
phase was analysed with gas chromatography or high perfor-
mance liquid chromatography (see ESI† for details). All mea-
surements were conducted in triplicates. Kinetic parameters
were estimated using non-linear regression with Prism
(Graphpad, San Diego, CA, USA). Specific activity was assayed
under the same conditions using 5 mM styrene. One unit (U)
of enzyme activity was defined as the amount of the enzyme
that converts 1 μmol substrate per minute at 30 °C.
Experimental
Homology search and phylogenetic analysis
The amino acid sequence of StStyA (accession No.
WP_030740546.1) was used as a query to conduct BlastP
search against NCBI database. The returned 100 sequences
from BlastP yielded the representatives for further analysis.
Among them, 34 sequences sharing a minimum of 49% iden-
tity with StStyA were aligned with StStyA and the StyAs of 28
known (S)-selective SMOs using the ClustalW algorithm.43
The alignment result was modified by GeneDoc.
The same 63 sequences were used to generate a phylogenetic
tree applying MEGA X software based on neighbor-joining
method,44 and the tree was modified by online tool Interactive
Tree Of Life (https://itol.embl.de/itol.cgi).45 Putative (R)-SMOs for
further characterization were selected from group III and group
IV based on phylogenetic analysis.
Plasmid construction, and protein expression and
purification
The DNA sequences encoding 21 putative StyAs were synthe-
sized by GenScript Biotech Co. (Nanjing, China). Each frag-
ment was inserted in plasmid pETB, which was assembled
from pET28(+) and the fragment encoding PsStyB from
Pseudomonas sp. LQ26.20 The resulting plasmids were trans-
formed into E. coli BL21 (DE3). Single colonies were cultured
in LB medium (2 mL) with 50 μg mL−1 kanamycin for 12 h at
37 °C. The resulting starter culture was transferred into 200
mL terrific broth (TB medium) containing kanamycin (50 μg
ml−1), and incubated at 37 °C with shaking at 180 rpm until
the OD600 value reached 0.8. Then 0.05 mM IPTG was added
and the incubation continued at 20 °C for 18 h with shaking
at 180 rpm. Cells were harvested by centrifugation (5 min,
4700 g) at 4 °C.
Conflicts of interest
There are no conflicts to declare.
To purify the proteins, cell pellets were resuspended in
phosphate buffer (0.1 M, pH 7.0) and lysed by sonication (3 s
on/3 s off, 200 W) for 10 min on ice followed by centrifuga-
tion at 4 °C. The proteins were purified by Ni2+-NTA agarose
resin (Qiagen, Valencia, CA) and eluted with wash buffer (10
mM Tris–HCl, 300 mM NaCl, 250 mM imidazole, pH 7.0) at 1
mL min−1 flow rate. Then, the purified proteins were ana-
lyzed with SDS-PAGE (Fig. S2†).
Acknowledgements
We are grateful to the National Natural Science Foundation of
China (21708038 and 22071237), the Key Laboratory of Envi-
ronmental and Applied Microbiology of the Chinese Academy
of Sciences (KLCAS-2018-1), and the Natural Science Founda-
tion of Henan Province (202300410218) for financial support.
Notes and references
Site-directed mutagenesis
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2200 | Catal. Sci. Technol., 2021, 11, 2195–2201
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