Production of enantiopure chiral epoxides with e. Coli expressing styrene monooxygenase

Article abstract of DOI:10.3390/molecules26061514

Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 g_DCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (>95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO.

Full text of DOI:10.3390/molecules26061514

molecules
Article
Production of Enantiopure Chiral Epoxides with E. coli
Expressing Styrene Monooxygenase
Dominika Gyuranová 1, Radka Štadániová 2 , Zuzana Hegyi 1, Róbert Fischer 2 and Martin Rebroš 1,
*
1
Faculty of Chemical and Food Technology, Institute of Biotechnology, Slovak University of Technology
in Bratislava, Radlinského 9, 812 37 Bratislava, Slovakia; dominika.gyuranova@stuba.sk (D.G.);
zuzana.hegyi@stuba.sk (Z.H.)
Faculty of Chemical and Food Technology, Institute of Organic Chemistry, Catalysis and Petrochemistry,
Slovak University of Technology in Bratislava, Radlinského 9, 812 37 Bratislava, Slovakia;
radka.stadaniova@stuba.sk (R.Š.); robert.fischer@stuba.sk (R.F.)
2
*
Correspondence: martin.rebros@stuba.sk; Tel.: +421-2-59-325-480
Abstract: Styrene monooxygenases are a group of highly selective enzymes able to catalyse the
epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds
containing oxirane ring or products of their hydrolysis represent key building blocks and precursors
in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial
scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale
was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density
fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited
excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months
and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess
(>95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-
diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-
cell SMO.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Gyuranová, D.; Štadániová,
R.; Hegyi, Z.; Fischer, R.; Rebroš, M.
Production of Enantiopure Chiral
Epoxides with E. coli Expressing
Styrene Monooxygenase. Molecules
2021, 26, 1514. https://doi.org/
10.3390/molecules26061514
Keywords: styrene monooxygenase; whole-cell biocatalysis; chiral epoxides
1. Introduction
Academic Editor: Maciej Szaleniec
Styrene monooxygenase (SMO) is a flavoprotein monooxygenase that incorporates a
single oxygen atom into a styrene molecule to form styrene oxide [1]. Originally found in
Pseudomonas and Rhodococcus species, SMO is comprised of two subunits—FAD-dependent
monooxygenase (StyA) and NADH-dependent flavin-reductase (StyB)—encoded by styA
Received: 15 February 2021
Accepted: 7 March 2021
Published: 10 March 2021
and styB of styABDE gene cluster responsible for the degradation of styrene [2,3]. Since
the epoxidation of styrene to styrene oxide by SMO is highly enantioselective, styA and
styB are possible candidates for biocatalysis. Initially, wild-type SMOs of Pseudomonas
were widely employed for biotransformation of styrene, but, to date, many other bacterial
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species that share styrene degradation pathways have been described [1]. Such discoveries,
together with the expansion of recombinant technologies, metagenomics, and protein
engineering, have led to the generation of SMOs with higher stability, selectivity, and
activity towards a broad substrate spectrum, usually expressed as recombinant enzymes
in Escherichia coli [
2,4–7]. Significant progress was achieved by Panke et al. in previous
Copyright:
© 2021 by the authors.
studies [ 11]. In 1998, they characterised the styrene degradation metabolic pathways in
8–
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Attribution (CC BY) license (https://
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4.0/).
Pseudomonas sp. and, in 2002, produced 388 g of enantiopure (S)-styrene oxide in a 30 L
scale by the whole-cell SMO expressed in E. coli JM101, which is the highest (S)-styrene
oxide yield achieved so far. However, only a low cell concentration (12 g/L) was reached
during fermentation, despite being operated on a pilot-scale [11]. Although recombinant
production of SMO has been known for several decades, the majority of biotransformations
are performed using whole-cell SMO that may cause a decline in enantiomeric excess (ee) of
Molecules 2021, 26, 1514. https://doi.org/10.3390/molecules26061514
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1514
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chiral products because of possible side reactions [
2,
12
–14]. Few attempts of SMO isolation
have been reported, but only the small-scale expression of StyA and StyB separately was
successful [4,15,16].
Chiral epoxides comprise a group of important building blocks and intermediates
of biologically active pharmaceuticals [17]. To date, many epoxides have been applied
to the synthesis of various drugs, for instance, antiviral agents (Indinavir, Cidofovir),
chemotherapeutics (Paclitaxel, Docetaxel), antidepressants (Tomoxetine, Fluoxetine), or
sleeping disorder treatments (Tesamelteon) [2,17,18]. Among them, styrene oxide, glycidol,
epichlorohydrin, chlorostyrene oxide, and others are produced on a large scale [17]. How-
ever, biocatalysis still suffers from the low availability of enzymes in large quantities and is
often replaced by chemical catalysis on an industrial scale. Sharpless and Jacobsen oxida-
tion that demand extreme reaction conditions and suffer from poor enantioselectivity are
mostly employed in pilot-scale chiral epoxides preparation [14,17]. Further improvement of
SMO production (e.g., fermentation optimisation, enzyme stability improvement, effective
purification development) significantly improves the production cost and enantiopurity
of chiral epoxides. Thanks to their broad substrate scope, further application of SMOs on
an industrial scale could also extend the spectrum of optically active chiral compounds
produced by green chemistry.
In this work, the gene of SMO from Marinobacterium litorale was cloned to a pET28b(+)
vector and expressed in Escherichia coli BL21(DE3) under the control of isopropyl-
thiogalactopyranoside (IPTG) inducible lacT7 promoter. Recombinant SMO originating
from M. litorale has been expressed and characterised in [ ]. However, the substrate
β-D-
2
specificity of the enzyme was evaluated only towards few structurally similar styrene
derivatives. Used SMO encoded by styA and styB was fused by the linker L2 designed
in [16] (StyAL2StyB). Firstly, induction conditions were optimised in the flask, then the
production of SMO was upscaled to a 1.5 L scale for high cell density production. SMO
was further purified by affinity chromatography and characterised. The affinity of SMO
towards 34 various structurally different alkenes was tested. Selected biotransformations
were upscaled for the production of optically pure chiral epoxides.
2. Results and Discussion
2.1. Expression of SMO
SMO expression was optimised by varying media composition, IPTG concentration,
and induction temperature. When cells reached a concentration above 0.6 g/L during
cultivation in LB medium, the formation of a blue pigment was observed. This phenomenon
was described before and is the result of tryptophanase coexpression, which together
with SMO transform tryptophan to indigo in cultivation media [19]. In E. coli, excess
tryptophan induces the expression of tryptophanase that hydrolyses tryptophan to indole.
The primary function of indole is to regulate cellular processes [20], but its structural
similarity to styrene also makes it a substrate of SMO that converts indole to indoxyl.
Further isomerisation of indoxyl then generates indigo. Production of indigoid dyes is a
widely applied high-throughput screening method for monooxygenase directed evolution
library screenings [3,21]. In the present work, however, the presence of indigo was also
observed in noninduced cultures grown on LB agar plates, demonstrating only unregulated
enzyme expression (data not shown). Therefore, mineral–amino acids free M9 medium was
applied for SMO expression. Due to the lack of nutrients, the cell growth in M9 medium
was slower; however, comparable cell concentrations and SMO specific activity were
reached (Figure S1). SDS-PAGE protein profiles confirmed the expression of recombinant
SMO with a molecular size of 66 kDa, consisting of StyA (45 kDa) and StyB (19 kDa) [
which corresponds to the predicted size of the enzyme.
2],
Different concentrations of IPTG resulted in the same SMO expression, as identified
by SDS-PAGE (Figure S2). However, even at IPTG concentrations lower than 1 mM, a
high amount of SMO was formed in inclusion bodies, probably due to the high induction

Molecules 2021, 26, 1514
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temperature [
3
]. Its decrease to 20 ◦C led not only to an enhancement of SMO solubility
(Figure S3), but also to a 70% increase in SMO specific activity (Figure S1).
The final optimised flask conditions in M9 medium were 0.25 mM IPTG and 20 ◦C
induction for 22 h, where a specific activity of 12 U/gDCW of SMO was achieved.
2.2. High Cell Density Fermentation
High cell density (HCD) fermentation is a well-established method for recombinant
enzyme production. However, the process performance and productivity highly depend
on many factors (fermentation media composition, feeding strategy, induction conditions,
expression host, gene of the desired product) and require optimisation [22–24].
To upscale the SMO production, 0.5 L HCD fermentations were performed with
glycerol as a carbon source. As the utilisation of glycerol by E. coli generates organic
acids such as acetate, which significantly decreases biomass growth at a concentration
above 2 g/L [22], fermentation was performed with a fixed pH strategy. The expression
of SMO was initially induced at a low cell concentration of 0.4 gDCW/L, but under these
conditions, the cell growth was inhibited immediately. The concentration of IPTG is one
of the crucial factors that affect the growth of E. coli, the level of expression, and also the
activity of a recombinant enzyme [22,24,25]. As previous studies showed that excessive
IPTG concentration could inhibit or even repress biomass growth [22,23], we assume that
the concentration of 0.4 gDCW/L was too low for the induction. Expression of SMO was
further induced at 15 gDCW/L (OD600 = 50) for 5.5 h (Figure S4), where a rising trend of SMO
activity was demonstrated. After 16 h of fermentation, and complete glycerol utilisation,
31 gDCW/L of biomass and 10.5 U/gDCW of SMO were achieved. HCD fermentation was
then upscaled to 1.5 L (Figure 1, Figure S5). The results are summarised in Table 1.
Figure 1. HCD batch fermentation of E. coli expressing SMO performed on the 1.5 L scale.
Table 1. Results of HCD fermentations.
Cell Concentration
(gDCW/L)
Total Dry Cell Weight
Enzyme Activity
Final Volume (L)
Total Activity (U)
(gDCW
)
(U/gDCW)
0.5 L
1.5 L
31
35
15.5
52.5
10.5
9.6
162.8
504
The final, optimised HCD fermentation protocol for E. coli BL21(DE3) expressing re-
combinant SMO was as follows: M9 inoculation medium and induction with 0.25 mM IPTG

Molecules 2021, 26, 1514
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at 30 ◦C and 15 gDCW/L in a batch mode. Following the protocol, HCD fermentation with
1.5 L provided 35 gDCW/L of overexpressed SMO with a specific activity of 9.6 U/gDCW
.
2.3. Isolation and Characterisation of Recombinant SMO
2.3.1. Purification of Recombinant SMO
Due to the presence of the His tag, recombinant SMO was isolated by immobilised
Ni2+ affinity chromatography according to [26] (Figure S6). Initially, the purification was
optimised on a small-scale, where only a small amount of SMO (Table S1) was recovered
per single run. Upscaled purification achieved a 4-fold higher SMO yield (Table S1), but
in general, only up to 0.2 mg of purified SMO per mL of crude extract were isolated
(approximately 0.01 gSMO per gDCW). Since this is not the first report of SMO isolation
causing an obstacle [21], the low purification yield and substantial decrease in the specific
activity of purified SMO compared to its crude form (discussed below) are probably caused
by the loss of FAD during purification [27].
2.3.2. Determination of Optimal pH, Temperature, and Storage Conditions
As Figure 2 shows, the mechanism of styrene epoxidation by SMO is quite complex
and, due to the StyB dependence on reduced NADH, also requires a cofactor regeneration
system in a cell-free system. Therefore, a glucose dehydrogenase (GDH) regeneration
system was employed [26].
Figure 2. Biocatalytic cascade of styrene epoxidation involving GDH cofactor regeneration system.
SMO exhibited a maximum activity at pH 7.8, but was highly active at the range
7.7–8.3 (Figure 3a). Since gluconic acid was formed in the reaction (Figure 2), a pH of 8 was
applied similar to a previous report [2
]. The SMO reached a maximum activity at 40 ◦C
(Figure 3b). However, evaluation of specific activity during SMO isolation revealed that,
with increasing purity of enzyme, the catalytic activity decreases significantly (Table S2).
Therefore, further application of whole cells and crude enzyme extract was applied, which
slightly shifted the optimal temperature (Figure S7). ◦In its crude form, SMO retained 70%
of its initial activity after 16 months of storage at
with SMO were also suitable for repeated biotransformations (Figure S9).
−
80 C (Figure S8). Moreover, whole cells

Molecules 2021, 26, 1514
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Figure 3. pH (a) and temperature (b) profile of purified SMO.
2.4. Biotransformation of Alkenes
The substrate specificity of SMOs has been investigated for many years [12,13,28,29].
Initially, SMOs were considered as enzymes with a narrow substrate spectrum, especially
those originating from Pseudomonas and Rhodococcus species. However, recent studies de-
scribed novel SMOs from various bacteria or metagenome, which provided novel insights
into the catalytic mechanism of styrene epoxidation and, thereby, enabled the generation of
SMOs with improved biocatalytic activities and a broader substrate spectrum [4,6,14,30].
So far, all studies have confirmed that few structural characteristics of the substrate signifi-
cantly impact the biocatalysis: acyclic double bonds are preferred; an electron-withdrawing
group conjugated to a double bond, α-/β-substitution of styrene/styrene derivatives, as
well as 2-substitution of an aromatic ring decrease activity [5,6,12,13,31].
The affinity of SMO towards a wide range of alkenes (Table 2) was examined. Screened
substrates were divided into the following groups: styrene derivatives (
1), unconjugated
aromatic alkenes ( ), unsaturated aldehydes and ketones ( ), unsaturated carboxylic acid
2
3
and esters (4), linear alkenes (5), and unsaturated amines and imines (6–8).
Table 2. Substrate specificity of recombinant CE of SMO.
Structure
Substituents
Entry
Activity (U/gDCW
)
Conversion (%)
R1, R2, R3, R4 = H
R1, R2, R4 = H; R3 = Br
R1 = CHO, R2, R3, R4 = H
R1 = CHO, R2, R3 = H, R4 = OCH3
R1 = CHO, R2, R3 = H, R4 = NO2
R1, R2, R3 = H, R4 = Cl
1a
1b
1c
1d
1e
1f
7
-
-
-
-
99
-
-
-
-
12
90
R1 = H
2a
23
-
87
-
R1 = OH
2b
R1, R2, R3, R4 = H
R1, R3, R4 = H; R2 = CH3
R1 = CH3; R2, R3, R4 = H
R1, R2, R3 = H; R4 = (CH2)2CH3
R1 = Cl; R2, R3, R4 = H
3a
3b
3c
3d
3e
-
13
-
-
-
-
100
-
-
-

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Table 2. Cont.
Structure
Substituents
Entry
Activity (U/gDCW
)
Conversion (%)
R1 = OH; R2, R3, R4 = H
R1 = CH3; R2, R3, R4 = H
R1, R2 = CH3; R3, R4 = H
4a
4b
4c
4d
4e
4f
-
-
-
-
-
-
-
-
-
-
-
-
R1, R3 = CH3; R2, R4 = H
R1 = (CH2)3CH3; R2, R3, R4 = H
R1 = CH2CH3; R2, R3 = H; R4 = CHO
R1 = H, R2 = CH2OH
R1 = CH3, R2 = CH2OH
R1 = H, R2 = (CH2)3OH
R1 = H, R2 = (CH2)4OH
R1 = H, R2 = CH(OH)(CH2)3CH3
R1 = H, R2 = CH(OH)(CH2)2
R1 = H, R2 =
5a
5b
5c
5d
5e
5f
-
-
-
25
-
-
-
-
-
99
-
-
5g
5
99
ꢀ
ꢁ
(CH2)2CH(O)CH2
R1 = H, R2 =
5h
3
67
ꢀ
ꢁ
(CH2)4CH(O)CH2
R1 = Br, R2 = CH3
R1 = H, R2 = (CH2)3Br
R1 = H, R2 = CN
5i
5j
5k
5l
-
7
-
-
99
-
R1 = H, R2 = CH(OCH2CH3)2
-
-
6
7
8
-
-
-
-
-
-
The substitution of a styrene derivatives’ double bond seemed to be unfavourable for
epoxidation, as none of the 1b alkenes were converted to epoxide. Both double bond
–
e
and aromatic ring substitution presumably acted as a steric barrier to the active site of
the enzyme. On the other hand, the bioconversion of 4-Cl-substituted styrene derivative
(1f) and unconjugated allylbenzene (2a) was high. In both cases, a higher activity of SMO
compared to styrene was achieved. Both 4-chlorostyrene oxide and allylbenzene oxide
were formed in the upscaled process, with excellent conversion (more than 99%), ee, and
yield (Table 3).
2-hydroxyl substitution of the aromatic ring also seemed unfavourable (2b). SMO
showed no affinity towards 5a–c, e, and f, but was highly active in the epoxidation of
5d, implying a trend of rising activity with increasing distance between the double bond
and the hydroxyl group. A similar trend was confirmed compared to the epoxidation of
bromo-alkenes 5i and 5j. On the contrary, the activity of SMO decreased with increasing
distance of the oxirane ring from the double bond (5g, 5h). Eventually, both epoxides
were converted to diepoxides, but the conversion of longer 5h and ee of the corresponding
diepoxide was lower compared to 5g.

Molecules 2021, 26, 1514
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Table 3. Summary of upscaled biotransformations by whole-cell SMO.
Reaction
Volume/Yield
(mL/mg)
Entry
1f
Substrate
Product
Configuration
ee (%)
Conversion (%)
Yield (mg)
S
>99%
99
60
1.27
2a
5g
S
>95%
>97%
99
99
26
2.88
1.28
2-R,5-R
157
ND 1
S
>99% 2
>99% 3
5j
93
99
173
76
1.73
1.32
5d
1
2
Not determined due to low stability of epoxide. Determined by chiral GC-FID. 3 Based on optical rotation value.
From the group of unsaturated aldehydes and ketones, only 3b was transformed to
epoxide with excellent enantiopurity (ee > 99%). Interestingly, 3b only differs from 3a by
one methyl- substitution on C2 carbon, which supports the previous finding of SMO activity
enhancement by double bond methylation [14]. On the other hand, no activity towards
4c was observed, suggesting that the combination of methyl- and carbonyl- substituents
was crucial. Unfortunately, SMO showed no affinity to any tested unsaturated ketone,
carboxylic acid, ester, amine, or imine.
2.5. Upscale Production of Chiral Epoxides
To evaluate the scope of SMO applicability, upscaled biotransformations were per-
formed. It was found that the crude extract of SMO was not suitable for upscale epox-
idations, as many undesired compounds were repeatedly isolated from crude reaction
mixtures besides epoxides (data not shown), since additional purification steps provided
only trace final amounts of epoxides. Therefore, whole-cell SMO was employed, provid-
ing the same enantioselectivity and even higher conversion compared to crude extracts
(Table 3). As Table 3 shows, almost all epoxides were produced in (S) configuration, similar
to previous reports [28,29,32]. As far as we know, this is the first report of (2R,5R)-1,2:5,6-
diepoxyhexane and 2-(3-bromopropyl)oxirane enzymatic production by SMO in such a
configuration and/or enantiopurity.
Evaluation of Chiral Epoxides
The application of chiral epoxides prepared in this work has not been reported so far.
However, structurally similar (R)-1f epoxide is a possible intermediate in the synthesis of
neuroprotective agent Eliprodil. Similarly, (R)-3-1f epoxide serves as an intermediate of an-
tidiabetic and antiobesity agents and is produced on an industrial scale (100–4000 L) [17
,33].
2-methyl-2a epoxide is an intermediate for the synthesis of (R)-( )-mevalonolactone, a key
−
intermediate of cellular biological processes and regulation [34]. The possible retrosynthetic
pathway of these epoxides is shown in Scheme S1.
Despite the rising trend of biocatalysis or chemo-biocatalysis involvement in indus-
trial production, chiral epoxides are still preferably produced by the manner of chemical
catalysis. The ability of SMOs to operate at mild reaction conditions, together with their
tremendous enantioselectivity, thereby emphasise their potential for industrial applications.

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3. Materials and Methods
3.1. Chemicals and Media
Kanamycin was purchased from Gibco® (Life Technologies, Glasgow, UK). NAD+
was purchased from Prozomix (Haltwhistle, UK). N-hexadecane was purchased from
Alfa Aesar (Haverhill, MA, USA). Ethyl acetate was purchased from Merck (Darmstadt,
Germany). Styrene, styrene oxide, and 4-chlorostyrene oxide were purchased from Sigma
Aldrich (St. Louis, MO, USA). 4-chlorostyrene was purchased from Carbosynth (San Diego,
CA, USA). Other alkenes and standards of epoxides were provided by the Department of
Organic Chemistry of the Slovak University of Technology.
Lysogeny broth (LB) was prepared according to [35]. Semidefined medium contained
90 g/L glycerol, 10 g/L tryptone, 5 g/L (NH4)2SO4, 3.64 g/L NaH2PO4.2H2O, 4.53 g/L
K2HPO4, 4 g/L citric acid, 1% (v/v) trace element solution, 1 g/L MgSO4, 7H2O. Mineral
M9 medium contained 5 g/L glycerol, 0.5 g/L MgSO4.7H2O, 0.11 g/L CaCl2, 0.01 g/L
thiamine-HCl, and 5% (v/v) M9 salts (85 g/L Na2HPO4.12H2O, 15 g/L KH2PO4, 2.5 g/L
NaCl, 5 g/L NH4Cl). All media were supplemented with kanamycin to a final concentration
of 30 µg/mL.
3.2. Construction of Expression Vectors
Codon-optimised genes styA and styB [2] encoding SMO from Marinobacterium litorale
were linked with linker WYHHHH according to [16] and were purchased from Generay
Biotech Co., Ltd. (Shanghai, China). Genes were inserted into a pET28b vector system.
Plasmids were then transformed into competent cells of Escherichia coli BL21(DE3).
3.3. Expression of SMO
A single colony of E. coli BL21(DE3) with pET28b(+)SMO was transferred to 3 mL
of LB/M9 medium supplemented with kanamycin (30 µ
g/mL) and cultivated at 37 ◦C
and 200 rpm overnight. A total of 100 mL of LB/M9 medium in a 500 mL shaking flask
were inoculated with 1% (v/v) of overnight culture and cultivated◦at 37 ◦C and 200 rpm
until OD600 reached 0.5–0.7. The temperature was reduced to 20/30 C and the expression
of SMO was induced by the addition of 0.25/0.5/1 mM of IPTG. To determine OD600
and level of SMO expression performed by denaturing polyacrylamide gel (SDS-PAGE)
electrophoresis (Mini PROTEAN® Tetra Cell, Bio-Rad, Hercules, CA, USA), 1 mL samples
were taken regularly after induction, which took 22 h. The cells were then harvested by
centrifugation (16,639× g, 6 ◦C, 30 min) and applied for biotransformation.
3.4. High Cell Density Fermentation
Fermentations were performed in 1.3/3 L New Brunswick BioFlo Bioreactors (Eppendorf,
Hamburg, Germany) with an initial fermentation medium volume of 500 mL/1.5 L.
Preinoculum was prepared in a glass tube as described above. The inoculum was
prepared by inoculation of 100 mL of M9 medium in a 500 mL shaking flask with 1%
◦
(v/v) of the preinoculum culture and cultivation at 37 C, 200 rpm until OD600 reached 2.5.
Fermentation was started by the inoculation of a bioreactor containing the semidefined
medium with 2% (v/v) of inoculum culture. Temperature and pH were set to 37 ◦C and
7, maintained by the addition of 26% (v/v) ammonia and 3.1 M phosphoric acid. Air
saturation of fermentation medium was maintained at dissolved oxygen (DO) = 30% by
aeration (1 vvm) and agitation speed cascade (200–1200 rpm). When agitation reached
◦
1200 rpm, the temperature was reduced to 30 C and the expression of SMO was induced by
the addition of 0.25 mM IPTG. To determine OD600, glycerol and acetic acid concentration,
and specific activity and level of SMO expression, samples of fermentation medium were
withdrawn regularly. Samples were processed as described above, except the supernatant
was transferred to a fresh tube, diluted, and analysed by high-performance liquid chro-
matography (HPLC). Biomass for SMO activity determination was sampled separately and
◦
kept at 4 C until the activity assay. Fermentation was terminated after complete substrate

Molecules 2021, 26, 1514
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utilisation. Cells were harvested by centrifugation (16,639
×
g, 6 ◦C, 30 min) and stored at
−80 ◦C.
3.5. Activity Assay
Harvested cells were resuspended in 100 mM potassium phosphate buffer (pH 8)
◦
to OD600 = 10 and disrupted by high-pressure homogeniser (276 MPa, 4 C, 2 cycles)
(CF Range, Constant Systems Ltd., Daventry, UK). Crude cell extract (CE) was clarified
from cell debris by centrifugation (16,639
×
g, 6 ◦C, 30 min). The reaction mixture (1 mL)
containing CE, glucose (100 mM), GDH (0.1 mg/mL), and NAD+ (1 mM) was prepared
into 4 mL screw-neck vials and tempered in a shaking incubator at 30 ◦C and 200 rpm for
3 min. Biotransformation was started by the addition of 10 µL of styrene from 150 mM
stock in methanol. To obtain a styrene oxide linear concentration increase, samples were
withdrawn regularly and analysed by gas chromatography (GC). Each biotransformation
was performed in two parallel runs, and given values represent mean values. One unit (U)
was defined as the amount of SMO that catalyses the formation of 1 µM of styrene oxide in
1 min. The specific SMO activity was calculated as U per g of dry cell weight (U/gDCW).
3.6. Purification of Recombinant SMO
CE of SMO (biomass diluted to OD600 = 50) were prepared as described above. CE was
filtered by vacuum filtration (Fisherbrand 0.45
µm; Fisher Scientific, PA, USA) or microfil-
tration (0.4
µ
m TangenXTM PRO PDn Casette; Repligen, MA, USA) performed on an ÄKTA
flux filtration unit (GE Healthcare, Chicago, IL, USA). Recombinant SMO was purified
by fast protein liquid chromatography (FPLC) performed on an ÄKTA purifier/ÄKTA
pilot unit equipped with 5 mL His Trap FF (GE Healthcare, Chicago, IL, USA)/50 mL
Ni-NTA (Merck, Darmstadt, Germany) affinity column according to [26]. In brief, the
column was equilibrated with binding buffer (10 mM imidazole, 300 mM NaCl, 0.3 mM
sodium phosphate buffer pH 5.8), then the CE was loaded while the continual addition of
binding buffer was remained to elute unbound proteins. Next, elution buffer (B) (500 mM
imidazole, 300 mM NaCl, 0.3 mM sodium phosphate buffer, pH 7.4) was loaded into the
column by 3-step concentration increase (25, 80, 100% of B) to elute nonspecific bounded
proteins (at 25%) and His-tagged SMO (at 80%). Then, 50 µL of each eluted fraction was
sampled to determine the presence of SMO by SDS-PAGE electrophoresis (Figure S6).
Pooled SMO was concentrated and desalted in Amicon® ultracentrifuge membrane (15 mL,
10 kDa; Merck, Darmstadt, Germany) or ultrafiltration cassette (10 kDa, TangenXTM PRO
PDn Casette, Repligen, MA, USA). The concentration of purified SMO was measured by an
Eppendorf µCuvette® (Eppendorf, Hamburg, Germany). The molar extinction coefficient
of SMO was determined using ProtParam tool.
pH Profile and Temperature Profile
To determine the optimal pH, purified SMO was employed in the biotransformation
of styrene. Reaction mixtures (1 mL) containing glucose (100 mM), GDH (0.3 mg/mL),
SMO (1 mg/mL), NAD+ (1 mM), and potassium phosphate buffer (100 mM; pH 6.1, 6.4, 7.8,
8.7) were prepared. Biotransformations were performed as described above. To determine
the optimal temperature, the same biotransformations were performed in a water bath
(T = 20, 25, 30, 35, 40, and 45 ◦C) instead of a shaking incubator.
The storage stability of CE of SMO was determined, when 1 mL aliquots (100 mM
potassium phosphate buffer, pH 8; OD600 = 50) were stored at 4,
−
20, and
−
80 ◦C. Reac-
tion mixtures contained glucose (100 mM), GDH (0.3 mg/mL), CE (OD600 = 25), NAD+
(1 mM), and potassium phosphate buffer (100 mM, pH 8) or glucose (100 mM) and cell
suspension (OD600 = 25) in potassium phosphate buffer (100 mM, pH 8) were prepared.
Biotransformations were performed as described previously.

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3.7. Biotransformation of Alkenes
Small-scale (1 mL) biotransformations were performed in 4 mL screw-neck vials
employing CE of SMO (OD600 = 50) as described above. Additional biotransformations
of 5, 10, and 20 mM 1f
, 2a, 3b, 5d, 5g, 5h, 5j alkenes were performed and allowed to
proceed for 2–5 h. Specific SMO activity was expressed in U/gDCW. One unit (U) was
defined as the amount of SMO that catalyses the formation /depletion (for 3b) of 1 µmol of
epoxide/alkene in 1 min.
Upscale (75–300 mL, specified in Table 3) of biotransformations was performed in
a 500 mL baffled Erlenmeyer flask employing whole-cell SMO. The reaction mixture
containing a cell suspension (OD600 = 50; 100 mM potassium phosphate buffer, pH 8) and
glucose (100 mM) was tempered for 5 min in a shaking incubator at 30 ◦C and 200 rpm,
then 1 mL of 1f
biotransformation. Biotransformations were allowed to proceed for 9 h. To determine the
conversion of alkenes and ee of corresponding epoxides, 100 L samples were withdrawn
, 2a, 5d, 5g, 5j from a 2 M stock solution in methanol was added to start the
µ
for GC analysis. The rest of the reaction mixture was used for isolation and purification of
corresponding epoxides performed immediately after biotransformation termination. All
small-scale and upscale biotransformations were performed in two parallel runs.
3.8. Analytics
3.8.1. High-Performance Liquid Chromatography
To determine glycerol and acetic acid concentrations, samples of cell-free fermentation
medium were diluted with deionised water into a glass vial and analysed by an Agilent
Infinity 1220 LC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a
WATREX polymer IEX H+ column (250 mm
detector (Agilent Technologies, Santa Clara, CA, USA), and 9 mM H2SO4 as a mobile
phase under the following conditions: injection volume 20
0.8 mL/min.
×
8 mm
×
8 µm), an Agilent Infinity 1260 RI
◦
µ
L, temperature 50 C, and flow
3.8.2. Gas Chromatography
During all biotransformations, 100
and extracted to 200 L of ethyl acetate containing 0.25 mM of n-hexadecane as an internal
standard by 30 s vortexing. The organic phase, containing the substrate and product, was
separated from the water phase by centrifugation (14,000 g, 1 min) and transferred to
µL of the reaction mixture was sampled regularly
µ
×
a glass vial. To determine the conversion of biotransformation, samples were analysed
by an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA)
equipped with a DB5 capillary column (Agilent J&W, 30 m
×
0.25 mm
×
0.25 µm), a
flame-ionisation detector (FID), and H2 as a carrier gas◦under the following conditio◦ns:
◦
injection volume 1
µL, split 50:1, temperature gradient 90 C (3.5 min), 30 C/min to 280 C;
H2 flow 1.2 mL/min. Temperature gradients were adjusted according to the molecular
weight of alkenes during substrate specificity screening. Conversion of biotransformation
was established according to (1).
(peak area of epoxide)
(peak area of alkene + peak area of epoxide)
conversion =
× 100%
(1)
To determine the ee of the products, samples were analysed by an Agilent 7890N
gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a CP-
Chirasil-Dex CB capillary column (Agilent J&W, 25 m
×
0.25 mm
×
0.25
µ
m), FID detector,
and H2 as a carrier gas under the following conditions: injection volume 1
µL; split 50:1;
temperature gradient 100 ◦C (0 min), 2 ◦C/min to 120 ◦C (1 min), 30 ◦C/min to 280 ◦C
(1 min); H2 flow 1.2 mL/min.

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3.9. Isolation and Characterisation of Chiral Epoxides
3.9.1. General Information
Flash column chromatography (FCC) was carried out with a Büchi system (Pump
Manager C-615 and Fraction Collector C-660) using Normasil 60 silica gel (0.040–0.063 mm;
VWR). Thin layer chromatography (TLC) analysis was carried out using TLC silica gel
60 F254 (aluminium sheets, Merck), and plates were visualised with UV light or by treatment
with permanganate solution followed by heating. Optical rotations were measured with a
JASCO P-2000 digital polarimeter with a Na-D lamp (10 cm cell length). Concentrations
(c) are given in grams per 100 mL. NMR spectra were recorded with a Varian INOVA-300
spectrometer (1H, 299.95 MHz) in CDCl3 using tetramethylsilane as the internal standard.
Data are presented as follows: chemical shift (in ppm), multiplicity (bs = broad singlet,
t = triplet, dd = doublet of doublets, m = multiplet), integration and coupling constants
(J/Hz). All solvents used were dried and distilled according to conventional methods.
3.9.2. General Procedure for the Isolation of Chiral Epoxides. Isolation of
(S)-4-Chlorostyrene Oxide
The crude biotransformation mixture (100 mL) was vigorously stirred with ethyl
acetate (150 mL) for 20 min. Then, the slurry was filtered through a pad of Celite, the
organic phase was separated, and an aqueous layer was extracted with ethyl acetate
(2 × 30 mL). The combined organic layers were dried over anhydrous MgSO4, and the
solvent was evaporated under reduced pressure (max 120 mbar in a 25 ◦C water bath or
max 50 mbar in a 35 ◦C water bath for long-chain aliphatic epoxides). The crude product
was purified by FCC using hexanes as the eluent to give (S)-4-chlorostyrene (60 mg, 34% of
theoretical yield) oxide as a colourless liquid with spectroscopic data in good agreement
with those reported in the literature [36,37].
(S)-4-Chorostyrene Oxide
1H-NMR (300 MHz, CDCl3):
δ = 2.75 (dd, 1H, J = 5.5, 2.5 Hz, OCH2), 3.14 (dd, 1H,
J = 5.5, 4.1 Hz, OCH2), 3.83 (dd, 1H, J = 4.1, 2.5 Hz, OCH), 7.18–7.23 (m, 2H, H-Ar), 7.29–7.34
(m, 2H, H-Ar).
[α]2D0 = +27.3 (c 1.35, CHCl3).
Rf = 0.15 (n-hexane/ethyl acetate, 50:1).
Reported rotation for (S)-4-chlorostyrene oxide [α]2D0 = +26 (c 1.29, CHCl3) for 99%
ee [36], [α]2D0 = +26 (c 1.2, CHCl3) for 99% ee [37].
(S)-Allylbenzene Oxide
Yield: 26 mg (13% of theoretical yield), colourless liquid with spectroscopic data in
good agreement with those reported in the literature [38].
1H-NMR (300 MHz, CDCl3):
δ = 2.55 (dd, 1H, J = 5.0, 2.7 Hz, OCH2), 2.78–2.85 (m, 2H,
PhCH2, OCH2), 2.93 (dd, 1H, J = 14.5, 5.6 Hz, PhCH2), 3.13–3.19 (m, 1H, OCH), 7.21–7.34
(m, 5H, H-Ar).
[α]2D0= –17.1 (c 1.3, EtOH).
Rf= 0.30 (n-hexane/ethyl acetate, 9:1).
Reported rotation for (S)-allylbenzene oxide [α]D25
ee [39], [α]2D0 = −17.3 (c 1.0, EtOH) for 98% ee [40].
(2R,5R)-1,2:5,6-Diepoxyhexane
=
−
18.37 (c 2.01, EtOH) for 99%

Molecules 2021, 26, 1514
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Yield: 157 mg (purified by FCC, hexanes/ethyl acetate, 9:1, 34% of theoretical yield),
colourless liquid with spectroscopic data in good agreement with those reported in the
literature [32].
1H-NMR (300 MHz, CDCl3):
δ = 1.57–1.85 (m, 4H, CH2CH2), 2.51 (dd, 2H, J = 4.9,
2.7 Hz, OCH2), 2.78 (dd, 2H, J = 4.9, 4.0 Hz, OCH2), 2.91–3.01 (m, 2H, OCH).
[α]26= +25.7 (c 1.2, CHCl3).
D
Rf= 0.18 (n-hexane/ethyl acetate, 7:3).
Reported rotation for (2R,5R)-1,2:5,6-diepoxyhexane [α]2D6 = +26.8 (c 5.03, CHCl3) [41],
[α]2D0 = +20.4 (c 1.3, CHCl3) [42].
(S)-4-(Oxiran-2-yl)butan-1-ol
Yield: 76 mg (purified by FCC, hexanes/ethyl acetate, 4:1, 33% of theoretical yield),
colourless liquid with spectroscopic data in good agreement with those reported in the
literature [32].
1H-NMR (300 MHz, CDCl3):
δ = 1.51–1.68 (m, 6H, CH2CH2CH2), 2.49 (dd, 1H, J = 5.0,
2.7 Hz, OCH2), 2.76 (dd, 1H, J = 5.0, 4.0 Hz, OCH2), 2.90–2.96 (m, 1H, OCH), 3.67 (t, 2H,
J = 6.2 Hz, CH2OH).
[α]25= −11.2 (c 1.0, CHCl3).
D
Rf= 0.15 (n-hexane/ethyl acetate, 1:1).
Reported rotation for (S)-4-(oxiran-2-yl)butan-1-ol [α]D24.7
>99.9% ee [32].
=
−
12.8 (c 1.0, CHCl3) for
2-(3-Bromopropyl)oxirane
Yield: 173 mg (26% of theoretical yield) of the crude product (could not be purified by
FCC because of its instability on silica gel), orange liquid with spectroscopic data in good
agreement with those reported in the literature [43,44].
1H-NMR (300 MHz, CDCl3):
δ = 1.53–1.66 (m, 1H, CH2CH2), 1.76–1.88 (m, 1H,
CH2CH2), 1.98–2.08 (m, 2H, CH2CH2), 2.51 (dd, 1H, J = 4.9, 2.7 Hz, OCH2), 2.77 (dd, 1H,
J = 4.9, 4.0 Hz, OCH2), 2.92–2.98 (m, 1H, OCH), 3.41–3.53 (m, 2H, CH2Br).
Rf = 0.19 (n-hexane/ethyl acetate, 7:3).
4. Conclusions
Herein, the production of recombinant SMO by HCD fermentation that yielded
35 gDCW/L was optimised. It was found that SMO loses stability in the isolation pro-
cess and was not suitable for biotransformations in a purified form. The highest specific
activity, stability, enantioselectivity, and purity of desired products was achieved by appli-
cation of whole-cell SMO, which was proven to be the most suitable form for epoxidation
of alkenes. In its crude form, SMO exhibited the highest activity at pH 8 and 35 ◦C and
retained 70% of its initial activity within 16 months of storage. The enzyme transformed
7 out of 34 tested alkenes to corresponding epoxides, many of them with higher specific
activity than styrene. Eventually, (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-
1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were
produced with excellent enantiopurity (ee > 99%, 95%, 97%, 99%, and 99%, respectively).

Molecules 2021, 26, 1514
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Supplementary Materials: The following are available online, Figure S1: Cell growth after induction
of SMO expression (0.25 mM IPTG) in LB and M9 medium and final specific activity of SMO, Figure S2:
SDS-PAGE electrophoresis of induction of SMO expression by different concentration of IPTG. OD600
= 0.7–0.8. Lane 1–protein ladder; Lane 2, 3–0.25 mM IPTG 0, 4 h; Lane 4, 5–0.5 mM IPTG 0, 4 h;
Lanes 6, 7, 8, 9–1 mM IPTG 0, 2, 3, 4 h; Lane 10—cell pellet (inclusion bodies); Lane 11–cell extract
(soluble SMO); Lane 12—protein ladder, Figure S3: SDS-PAGE electrophoresis of induction of SMO
◦
◦
expression at 20 and 30 C in LB and M9 medium. OD600 = 0.4–0.5. Lanes 1, 2, 3—LB, 20 C whole
◦
cells, crude extract, pellet; Lanes 4, 5, 6—LB, 30 C whole cells, crude extract, pellet; Lane 7–protein
◦
◦
ladder, Lanes 8, 9, 10—M9, 20 C whole cells, crude extract, pellet; Lanes 11, 12, 13—M9, 30 C whole
cells, crude extract, pellet, Figure S4: HCD batch fermentation of E. coli expressing SMO performed
on the 0.5 L scale, Figure S5: Protein profile of E. coli after induction of SMO expression during
HCD fermentation. Lane 1: protein ladder, Lane 2: 0 h, Lane 3: 2 h, Lane 4: 4 h, Lane 5: 6 h, Lane
6: 7.5 h after induction. Figure S6: Isolation of SMO by immobilised Ni2+ affinity chromatography,
Figure S7: Temperature profile of SMO in form of crude extract, Figure S8: Storage of SMO in form of
whole cells (a) and crude extract (b), Figure S9: Repeated biotransformation of styrene by whole-cell
SMO, Scheme S1. Retrosynthesis of (R)-4-chlorostyrene oxide (
a
) [37], (R)-3-chlorostyrene oxide
1
(
b
) [35], and 2-benzyl-2-methyloxirane (
c
) [35], Figure S10: H-NMR spectrum of (S)-4-chlorostyrene
oxide, Figure S11: 1H-NMR spectrum of (S)-allylbenzene oxide, Figure S12: 1H-NMR spectrum
of (2R,5R)-1,2:5,6-diepoxyhexane, Figure S13: 1H-NMR spectrum of (S)-4-(oxiran-2-yl)butan-1-ol,
1
Figure S14: H-NMR spectrum of 2-(3-bromopropyl)oxirane, Table S1: Summarised results of SMO
purification, Table S2. The evaluation of SMO specific activity during purification.
Author Contributions: Conceptualisation, M.R. and R.F.; methodology, M.R. and R.F.; validation,
D.G., R.Š. and R.F.; investigation, D.G., Z.H. and R.Š.; resources, M.R. and R.F.; data curation, D.G.
and R.Š.; writing—original draft preparation, D.G.; writing—review and editing, M.R. and R.F.;
supervision, M.R.; funding acquisition, M.R. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by the Slovak Research and Development Agency under the
Contract no. PP-COVID-20-0056. This work was supported by the Slovak Grant Agency for Science
VEGA (project no. 1/0552/18). This work was created thanks to support under the Operational
Program Integrated Infrastructure for a major project Improving University Capacities and Compe-
tences in Research, Development, and Innovation–Accord, ITMS code: 313021X329, co-financed by
the European Regional Development Fund.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of enzymes and epoxides are available, contact corresponding author.
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