Enhancing the Catalytic Performance of a CYP116B Monooxygenase by Transdomain Combination Mutagenesis

Article abstract of DOI:10.1002/cctc.201800054

The cytochrome P450 monooxygenase discovered in Labrenzia aggregata (P450_LaMO) is a self-sufficient redox system with versatile oxygenation functions. However, its catalytic performance is severely hindered by a low reaction rate, poor electron coupling efficiency (CE) and fragile thermostability. Herein, a simple transdomain combination mutation strategy was proposed for engineering this multi-domain P450 enzyme with redox partners fused to the heme domain. After focused mutagenesis on the heme domain, a triple mutant H3 (N119C/V264A/V437G) was hit, that improved the turnover frequency (TOF) and CE of P450_LaMO by about 7.8-fold and 3.0-fold, respectively. A redox domain-based mutant with higher cytochrome c reduction activity, MR1 (M612L/K774Y), mediated more efficient electron transfer, elevated the TOF by 4.9-fold, and the coupling efficiency by 4.2-fold. The beneficial effect was further enhanced by combining the mutation sites from different domains, resulting in a combinatorial mutant (N119C/V264A/V437G/M612L/N694D) with a 9.1-fold increase in coupling efficiency, 10-fold in TOF, as well as +3.8 °C in thermostability (T₅₀¹⁰). Meanwhile, for series of tetrahydronaphthalene derivatives, this combinator showed higher hydroxylation activity. This work suggested that employing this combinatorial strategy targeting on both the redox and heme domains is efficient to improve holoenzyme activity, CE and stability of a CYP116B subfamily member from the low starting point.

Full text of DOI:10.1002/cctc.201800054

Accepted Article
Title: Enhancing the catalytic performance of a CYP116B
monooxygenase by transdomain combination mutagenesis
Authors: Ren-Jie Li, Jian-He Xu, Qi Chen, Jing Zhao, Ai-Tao Li, and
Hui-Lei Yu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201800054
Link to VoR: http://dx.doi.org/10.1002/cctc.201800054
A Journal of
www.chemcatchem.org

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
Enhancing the catalytic performance of a CYP116B
monooxygenase by transdomain combination mutagenesis
Ren-Jie Li,[a] Jian-He Xu,[a]* Qi Chen,[a] Jing Zhao,[b] Ai-Tao Li,[c] Hui-Lei Yu[a]*
Abstract: The cytochrome P450 monooxygenase discovered in
Labrenzia aggregata (P450LaMO) is a self-sufficient redox system with
versatile oxygenation functions. However, its catalytic performance
is severely hindered by a low reaction rate, poor electron coupling
NADPH, through the specific FMN and Fe2S2 domains, to their
acceptor, the heme domain.[5]
Until now, there have been a few typical members in the
CYP116B subfamily, including P450RhF (CYP116B2),[6]
efficiency (CE) and fragile thermostability. Herein,
a
simple
CYP116B3,[7]
CYP116B1,[8] CYP116B5,[9]
CYP116B4,[10]
as well as the
[11]
[12]
transdomain combination mutation strategy was proposed for
engineering this multi-domain P450 enzyme with redox partners
fused to the heme domain. After focused mutagenesis on the heme
domain, a triple mutant H3 (N119C/V264A/V437G) was hit, that
improved the turnover frequency (TOF) and CE of P450LaMO by
about 7.8-fold and 3.0-fold, respectively. A redox domain-based
P450SMO
,
P450RpMO, P450ArMO, P450CtMO
latest reported CYP116B29, CYP116B46, CYP116B63,
CYP116B64 and CYP116B65.[13] Moreover, various reaction
types have been reported, such as aromatic hydroxylation, O-
dealkylation, epoxidation and asymmetric sulfoxidation by
P450RhF; N-dealkylation by CYP116B1; benzene derivative
dealkylation by CYP116B3; medium- and long-chain alkane (C14,
C16, C24 and C36) hydroxylation by CYP116B5. However, the
poor activity and low efficiency of electron utilization of the
members in this CYP116B subfamily, made it overshadowed
compared to another self-sufficient redox family,[14] CYP102,
such as the well-known P450BM3. Therefore, it is highly desired
to improve the catalytic properties and to release its
oxyfunctionalization potential.
mutant with higher cytochrome
c
reduction activity, MR1
(M612L/K774Y), mediated more efficient electron transfer, elevated
the TOF by 4.9-fold, and the coupling efficiency by 4.2-fold. The
beneficial effect was further enhanced by combining the mutation
sites from different domains, resulting in a combinatorial mutant
(N119C/V264A/V437G/M612L/N694D) with a 9.1-fold increase in
coupling efficiency, 10-fold in TOF, as well as +3.8℃ in
thermostability
(T5010).
Meanwhile,
for
series
of
tetrahydronaphthalene derivatives, this combinator showed higher
hydroxylation activity. This work suggested that employing this
combinatorial strategy targeting on both the redox and heme
domains is efficient to improve holoenzyme activity, CE and stability
of a CYP116B subfamily member from the low starting point.
Protein engineering provides an efficient approach to
improve P450 monooxygenases’ catalytic efficiency. Many
efforts have been made in terms of local segment mutagenesis,
such as the random mutagenesis for heme domain of
[10b]
P450LA1
,
which improved the efficiency of anti-Markvonikov
addition of styrene and the domain-based engineering method
[15]
for P450BM3.
In particular, P450BM3 mutants achieved the
highest electron coupling efficiency for propane by introducing
mutagenesis into the heme, FMN and FAD domains followed by
mutation fusion. Moreover, high-throughput screening was
focused on pseudosubstrate-based colorimetric assays.
However, there are only very few high-throughput screening
methods available for the CYP116B subfamily. For example, Liu
Introduction
Cytochrome P450 monooxygenases are a superfamily of heme-
containing catalysts in nature,[1] performing various reactions
such as inert C-H bond hydroxylation,[2] O-dealkylation[3] and
sulfoxidation.[4] Self-sufficient CYP116B subfamily members fuse
the heme and redox domains into a single polypeptide chain,
and the redox partners shuttle electrons from the reductant
et al.[3c] established
a heme domain mutation library of
CYP116B3 to enhance its dealkylation activity using a 7-
ethoxycoumarin-based fluorescence method, which is limited to
this specific type of substrate. Another report by Yan et al.[16]
employed desorption electrospray ionization coupled with ion
mobility mass spectrometry imaging (DiBT-IMMS) to screen for
higher hydroxylation activity for diclofenac transformation within
live bacterial colonies, but it seems that it is still far from
generalization due to the high cost of special facilities for
screening. Latest report by Hammer et al[10c] used a Purpald-
based method to efficiently screen error prone randomly libraries
to improve the productivity of CYP116B4, however, this method
was aldehyde-specific could not be used to other reactions such
as hydroxylation.
[a]
R. J. Li, Prof. J. H. Xu, Dr. Qi Chen, Prof. H. L. Yu
Laboratory of Biocatalysis and Synthetic Biotechnology
State Key Laboratory of Bioreactor Engineering
East China University of Science and Technology
130 Meilong Road, Shanghai 200237 (China)
E-mail: jianhexu@ecust.edu.cn; huileiyu@ecust.edu.cn.
Dr. J. Zhao
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences
Tianjin 300308 (China)
[b]
[c]
Prof. A. T. Li
Hubei Collaborative Innovation Center for Green Transformation of
Bio-resources
Hubei Key Laboratory of Industrial Biotechnology
College of Life Sciences, Hubei University, Wuhan 430062 (China)
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
It was reported that the redox domain plays an irreplaceable role
in the catalytic cycle of P450s, especially important for
accelerating the catalytic efficiency of the heme domain.[18] In
this work, introduction of mutations into the redox domain,
through random mutagenesis using 7-ethoxycoumarin as the
initial screening substrate, resulted in four single-site mutants
(R1~R4) with significant improvements in cytochrome
c
reduction activity (Table 1: R1, 319 U/mg, 41-fold; R3, 139 U/mg,
18-fold; R4, 80 U/mg, 10.4-fold), which are also helpful for
increasing the turnover frequency (R1, 0.028 min-1, 1.6-fold; R3,
0.029 min-1, 1.6-fold; R4, 0.031 min-1, 1.7-fold) and improving the
coupling efficiency (R1, 1.3%, 2.0-fold; R3, 0.85%, 1.3-fold; R4,
0.96%, 1.5-fold) of the holoenzyme (Table 1 and Figure S3).
When combining these sites, it is found that a double-site mutant,
MR1 (M612L/K774Y), had 4.9-fold and 4.2-fold improvements in
TOF and coupling efficiency for tetralin hydroxylation,
respectively.
Scheme 1. High-throughput screening methods constructed for the
engineering of P450LaMO. A) The high-throughput screening of redox domain
libraries was based on 7-ethoxycoumarin-based fluorescence method. B) The
high-throughput screening of the heme domain libraries was based on the
NBT-PMS colorimetric assay.
Table 1. Catalytic properties of the redox domain-based mutants of
P450LaMO
The P450LaMO discovered from Labrenzia aggregate, as
reported in our previous work,[10a] showed hydroxylation activity
for the core structure of tetralin substrates, albeit with a low
reaction rate (0.018 min-1, Table 1) and an extremely low
electron coupling efficiency (0.7%, Table 1). In the catalytic cycle
of P450s, obtaining a better electron coupling efficiency seems
to be more important towards utilizing this catalyst in
bioconversions. There are two aspects leading to the low
coupling efficiency:[17] a) an unnatural substrate is difficult to
form an effective intermediate within the heme domain, resulting
in the formation of H2O2 and other oxygen species; b) the redox
domain cannot efficiently accept the electrons from NADPH and
transfer the electrons to the heme domain. Therefore, to improve
the electron coupling efficiency and holoenzyme’s activity of
P450LaMO, we used a transdomain combination mutation in this
work. Redox domain-based mutants with higher electron transfer
Cytochrome c
reduction activity
(U/mg protein)[b]
TOFtetralin
(min-1)[c]
CEtetralin
(%)[d]
Mutant
Mutation
WT
R1[a]
R2[a]
R3[a]
R4[a]
─
7.7 ± 0.3
319 ± 39
26 ± 3
0.018 ± 0.002
0.028 ± 0.002
0.036 ± 0.004
0.029 ± 0.002
0.031 ± 0.008
0.7 ± 0.1
1.3 ± 0.3
M612L
Q684F
N694D
K774Y
1.1 ± 0.03
0.85 ± 0.02
0.96 ± 0.05
139 ± 4
80 ± 6
M612L/K
774Y
MR1
MR2
298 ± 7
0.088 ± 0.002
0.078 ± 0.004
2.8 ± 0.3
2.7 ± 0.6
M612L/N
694D
112 ± 10
efficiency were screened by
a high-throughput screening
method using a 7-ethoxycoumarin-based fluorescence substrate
(Scheme 1A). On the other hand, higher electron utilizing heme-
domain based mutants, forming more amounts of hydroxylation
product, were selected by an NBT-PMS (nitro blue tetrazolium-
phenazine methosulfate) assay that is correlated to changes in
the concentration of NADPH produced by the dehydrogenation
of sequential product alcohol.[21](Scheme 1B) We expected that
both the coupling efficiency and the activity of this CYP116B
member could be promoted by this transdomain combination
mutation strategy (TDCM) with the aid of appropriate high-
throughput screening methods for the screening of different
domain-based mutants. We also tried to find some key clues to
the factors limiting both the electron utilization and the electron
transfer efficiency using this simple combination strategy.
[a] Redox domain-based mutants was obtained based on 7-
ethoxycoumarin fluorescence initial screening method;
[b] Cytochrome c reduction activity was measured with spectrophotometric
method, as described in the Supporting Information, where 1.0 U = 1
µmolcyt c min-1;
[c] TOF: Turnover frequency (TOF) was defined as the number of product
molecules generated by each P450 enzyme molecule per minute, with the
unit of nmol tetral-1-ol nmol P450-1 min-1;
[d] CE: Coupling efficiency (CE) was defined as the ratio of tetral-1-ol
formation rate to NADPH consumption rate.
To characterize the electron transfer capability of the redox
domain-based mutants (MR1 and MR2), we measured their
kinetic parameters of cytochrome c (Cyt c) reduction (Table 2
and Figure S1, Supporting Information).[19] The kcat of MR1 for
cytochrome c was improved to 128 s-1 from 58.7 ± 1.4 s-1 of the
wild type and kcat of MR2 for cyt c was to 187 s-1 (2.2-fold and
3.2-fold enhancement over WT, respectively, see Table 2).[20] All
of the single and double-site mutations in the redox domain
contributed to an improvement in turnover frequency of the
holoenzyme, suggesting that these variants offer a more efficient
Results and Discussion
Mutagenesis in the Redox Domain of P450LaMO
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
Via site-directed overlap extension,[21] we combined the
beneficial mutations from the heme and redox domains. Among
the resulting mutants, the best mutant HR1 exhibited significant
improvements in TOF(0.18 min-1) by 10-fold and in coupling
efficiency (6%) by 9.1-fold compared to P450LaMO wild type
redox partner, which is helpful for achieving higher activity of the
holoenzyme. Meanwhile, the results of homologous modeling
showed that three substitutions (Q684, N694 and K774) were
clustered in the Fe2S2 domain, while M612 was located in the
FMN domain. The Q684 situated mainly on the surface between
the FMN and Fe2S2 subdomains (Figure 1A). In particular,
residues N694 and K774 were close to the heme domain, and
they may affect local changes in protein conformation to bridge
the electron transfer between two distinct domains.
Table 2. Cytochrome c reduction kinetics of some redox domain-based
mutants of P450LaMO
.
KM
kcat
kcat/KM
Folds
Mutant
(cyt c, mM)
(s-1)[a]
(s-1mM-1)
improvement[b]
WT
MR1
MR2
0.031 ± 0.03
0.21 ± 0.03
0.63 ± 0.04
58.7 ± 1.4
128 ± 9.0
187 ± 7.1
189
610
297
1.0
3.2
1.6
[a] Kinetic parameters determined with different concentrations of cytochrome
c, 0.5 mM NADPH, P450s 20 nM, 100 mM KPi buffer pH 8.0, by measuring
the absorbance at 550 nm;
[b] Folds improvement was based on kcat/KM.
Mutagenesis in the Heme Domain of P450LaMO
The redox domain beneficial mutations improved the coupling
efficiency and turnover efficiency of holoenzyme (Table 1).
However, the turnover frequency for the target substrate tetralin
was still low (e.g., only 0.088 min-1 for MR1, Table 1). Therefore,
we tried to perform directed evolution of the heme domain in
order to accelerate both the electron accepting and substrate
conversion processes.
As a result, the heme domain-based mutagenesis also
helped us improve the TOF value and coupling efficiency of the
holoenzyme by about 7.8-fold and 3.0-fold, respectively (Table
3). The conversion by three mutants (H1 (V264A), H2
(V264A/V437G) and H3 (N119C/V264A/V437G)) was improved;
in particular, H3 could efficiently transform the substrate tetralin.
It is shown that mutations in the heme domain effectively
promote the catalytic efficiency of the whole P450 enzyme.
Based on the molecular docking results, we could elucidate
the approximate locations of the three mutation sites in the
heme domain structure (Figure 1B), showing that site 264 was
near the heme, site 119 was close to the entrance channel of
substrate and the site 437 was on the surface of the protein.
Moreover, the mutation at sites 264 and 119, might decrease the
interaction with surrounding amino acid residues because of the
reduced number of hydrogen bonds. However, the mechanism
on the site G437 influence was still unclear.
Figure 1. Mutation sites in the heme and redox domains of P450LaMO. A) All
beneficial sites were showned in the whole protein of P450LaMO. Individual
domains are shown with different colors: green for FMN domain, gray for
Fe2S2 domain and purple for heme domain. B) The important residues in heme
domain of P450LaMO, C119 (yellow), A264 (green) and G437 (cyan) and
surrounding interacting sites S271 (yellow), M260 (green) and V170 (cyan).
(0.018 min-1, 0.7%, Table 3). These results indicated that
simultaneously introducing mutagenesis into the heme and
redox domains[14, 22] could efficiently improve the global activity
and electron coupling efficiency of the holoenzyme. Additionally,
mutations within the heme domain (based on M612L/K774Y and
M612L/N694D) caused obvious improvements in thermostability,
especially when considering the outstanding contribution of the
single mutation K774Y. The resultant variants HR1 (+3.8°C) and
HR2 (+5.3°C) enhanced the thermostability (Figure S2, as
measured by T5010, Supporting Information) of the holoenzyme
by 3.8°C and 5.3°C, respectively, as compared with the wild-
type.
By performing transdomain combinatorial mutation of
P450LaMO, we were able to excavate the holoenzyme’s catalytic
efficiency. Some literatures have shown that P450 systems have
Transdomain combination evolution of P450LaMO
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
different rate limiting steps.[25] Our results showed that the redox
domain laid the foundation for electron transfer and acceptance,
even affected the holoenzyme’s catalytic performance,[26] but the
heme segment is more important in electron utilization and the
holoenzyme’s activity.
To test the performance of the best mutant HR1 as a
biocatalyst, in the 4-hydroxylation of various 1,2,3,4-
tetrahydronaphthalene derivatives 1a-e (Table 4) were
conducted.[23] The hydroxylation products 2a-e were identified by
mass spectra and compared with those of standards (Scheme
S1, Table S3, Supporting Information). The results show that the
TOF (0.18 min-1) of HR1 for 1a was improved by 4.2 folds over
the wild type (0.043 min-1). When halogen atoms were
introduced into the substrates (1b, F-; 1c, Cl-; 1d, Br-; 1e, I-), the
HR1’s TOF values (0.16 min-1, 0.15 min-1, 0.25 min-1 and 0.24
min-1) are higher than the wild type’s (0.062 min-1, 0.06 min-1,
0.042 min-1 and 0.067min-1), with 2.6-fold, 2.5-fold, 6.0-fold and
3.6-fold improvements, respectively. Significant improvements
were also observed for the conversion of tetralin derivatives, e.g.,
4.1-fold (1a), 2.7-fold (1b), 2.4-fold (1c), 5.7-fold (1d) and 3.7-
fold (1e). Moreover, these hydroxylated products may be used to
synthesize drugs for the treatments of cancer or dermatosis.[24]
Based on combinatorial mutation, the resulting mutant HR1
did enhance the coupling efficiency, TOF value and
thermostability of the P450 monooxygenase. Moreover, it is of
improved high spin content, which showed that this enzyme
formed more efficient intermediates (Figure S5, Table S1,
Supporting Information). From the view of electrochemistry, the
redox potential was lowered from -27 mV of wild-type to -90 mV
of HR1 (Figure S4), illustrating that the efficiency of the driving
force for this P450’s catalytic cycle is much more powerful than
that of the native protein. Although the coupling efficiency of this
monooxygenase was improved to varied extents, questions such
as the destiny of the remaining electrons are not yet clear.
Therefore, further exploration on the mechanism of the electron
leakage in every process of catalytic cycle would be intriguing to
improve the coupling efficiency.
Table 3. Catalytic properties of P450LaMO mutants toward tetralin 1-
monooxygenation.
TOF
CE
Conv.
(%)[c]
Mutant
Mutation
(min-1)[a]
(%)[b]
WT
H1[a]
H2[a]
─
0.018 ± 0.002
0.030 ± 0.002
0.049 ± 0.007
0.7 ± 0.1
0.8 ± 0.2
1.4 ± 0.03
10
13
22
V264A
V264A/V437G
N119C/V264A/
V437G
H3[a]
0.140 ± 0.020
0.180 ± 0.010
2.0 ± 0.2
6.0 ± 0.2
94
Table 4. Comparison of P450LaMO-HR1 with its wild-type in enzymatic 4-
hydroxylation of various tetrahydronaphthalene derivatives.
N119C/V264A/
V437G/M612L/
N694D
Conv
TOF
HR1
>99
Entry
Substrate
Product
Enzyme
.
(min-1)[a]
(%)[b]
0.043 ±
0.005
0.180 ±
0.030
0.062 ±
0.011
0.160 ±
0.010
0.060 ±
0.002
0.150 ±
0.070
0.042 ±
0.006
0.205 ±
0.010
N119C/V264A/
V437G/
M612L/K774Y
1
2
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
2a
2a
2b
2b
2c
2c
2d
2d
2e
2e
WT
HR1
WT
8.7
36
12
32
12
29
8.6
49
13
48
HR2
0.130 ± 0.010
5.8 ± 0.2
98
[a] Heme domain-based mutants was initially obtained based on NBT-PMS
colorimetric screening method.
3
4
HR1
WT
[b] TOF: Turnover frequency (TOF) was defined as the number of tetral-1-ol
molecules generated from tetralin per each P450 enzyme molecule per
minute, with a unit of nmol tetral-1-ol nmol P450-1 min-1;
5
[c] CE: Coupling efficiency (CE) was defiined as the ratio of tetral-1-ol
formation rate to NADPH consumption rate;
6
HR1
WT
[d] Conversion was measured after 24 h reaction.
7
8
HR1
WT
In terms of the redox domain, the kcat of MR1 (for
cytochrome c) was improved to 128 s-1 from 58.7 ± 1.4 s-1 of the
wild type. It is suggested that engineering of redox domain could
further strengthen the electron transfer and elevate the catalytic
performance, which can be adopted not only for enhancing the
productivity of other reactions catalyzed by P450LaMO such as
anti-Markovnikov addition of styrene, but also helping other
CYP116B members to achieve higher electron transfer
0.067 ±
0.001
0.240 ±
0.010
9
10
HR1
[a] TOF: turnover frequency [nmol product nmol P450-1 min-1];
[b] Conversion was measured after 24 h reaction.
efficiency.[6c,7,8,10a,13]
.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
fragments were hybridized in a PCR cycler (80 °C for 3 min, 60 °C for 10
min, 20 °C for 20 min, ramp 0.1 °C/s, 0.1 °C/cycle, 4 °C for 10 min).
Finally, 10 µL of each hybridization sample was transformed into
competent cells E. coli BL21 (DE3).[28]
Conclusions
In conclusion, the best mutant of P450LaMO
(N119C/V264A/V437G/M612L/N694D),
,
HR1
by
was
formed
introducing the mutated sites from redox domain into mutated
heme domain, yielding 9.1-fold improvement of coupling
efficiency, 10-fold enhancement of turnover frequency and 3.8°C
increase in thermostability. This resulting HR1 also showed
higher hydroxylation activity for series of tetrahydronaphthalene
derivatives, which can be used as important building blocks to
synthesize biologically active molecules. Moreover, the
transdomain combination evolution strategy is expected to be
useful for engineering other members of the CYP116B subfamily,
especially for improving the low NADPH coupling efficiency and
the total reaction rate of unnatural substrates. Meanwhile,
engineering of the redox domain could further strengthen the
electron transfer and elevate the catalytic performance, which
can be adopted not only for enhancing the productivity of other
P450LaMO-catalyzed reactions, such as anti-Markovnikov addition
of styrene, O-dealkylation and thioether sulfoxidation.
Screening of the Heme Domain Mutation Library Based on the NBT-
PMS Assay
Single colonies were picked with toothpicks, inoculated and grown at
37 °C in 96-deep well plates with 300 µL LB medium per well, containing
50 mg/L kanamycin and using P450LaMO (WT) as the positive control. The
screening was performed with the full length enzyme. The expression
culture was inoculated by transferring 50 µL overnight culture into 850 µL
Terrific Broth (TB) expression medium supplemented with 50 mg/L
kanamycin. When the OD600 reached ~1.0, 0.2 mM IPTG (isopropyl-β-D-
thiogalactopyranoside) and 0.2 mM ALA were added to each well and the
temperature was decreased to 16 °C for another 24 h. The cells were
harvested by centrifugation (3220 g, 10 min). The cell pellets were then
resuspended in 190 µL potassium phosphate (KPi) buffer (100 mM, pH
8.0, saturated with oxygen) and transferred into 96-well microplates.
The reaction (200 µL) containing whole cells expressing P450LaMO
(1.6 g CDW/L), 0.5 mM 1,2,3,4-tetrahydronaphthalene, and 100 mM KPi
buffer (pH 8.0) in 96-deep well plates was performed at 220 rpm for 4 h.
Then, the mixture was separated by centrifugation (3220 g, 15 min) and
80 µL supernatant was taken to carry out the further dehydrogenation of
the hydroxylation product into an aldehyde. This dehydrogenation was
catalyzed by the reductase SsCR from Scheffersomyces stipitis CBS
6045 with high activity but low enantioselectivity (Scheme 1B, Table S2,
Figure S6, Figure S7, Supporting Information) toward the hydroxylation
product. The dehydrogenation was started by adding 10 µL SsCR (0.5 U)
and 10 µL mixed NBT-PMS solution (NBT 2 mg/mL, PMS 0.1 mg/mL and
NADP+ 0.3 mM), and then the change in absorbance at 580 nm was
monitored after the reaction was kept at 30 °C for 1 h away from light.
Experimental Section
For full experimental details please refer to the Supporting Information.
Random Mutagenesis of the Heme or Redox Domain
Modified error-prone PCR was used for random mutagenesis of the
heme and redox domains. The phosphatethioate-based gene cloning
method was used for high positive ligation rates.[27] The MnCl2
concentration was set at 75~100 µM for controlling the mutagenesis rate
at 1~2 amino acid residues for both the heme and redox domains. Heme
domain mutagenesis (1201 bp; 51% of the holoenzyme): modified PCR
primers were as follows: 5’-CGGATCTT*CCTTGAAG*AGATG-3’ as the
forward primer (forward, designated as Lamo-1201-F, where ‘*’ denotes
Screening of the Redox Domain Mutation Library with the 7-
Ethoxycoumarin-based Fluorescence Method
The redox domain of P450LaMO as an electron partner affects the electron
transfer capability and the total catalytic efficiency of the holoenzyme. A
fluorogenic substrate, 7-ethoxycoumarin, was used for preliminary high-
throughput screening of positive mutants from the redox domain
mutagenesis library. The screening was performed with holoenzyme.
After collecting the wet cells, 200 µL 100 mM KPi buffer (pH 8.0) was
added to resuspend them. The preliminary screening mixture, containing
190 µL cell suspension (final conc., 1.6 g CDW/L) and 10 µL 7-
ethoxycoumarin in DMSO (10 mM) stock solution (final conc., 0.5 mM),
was incubated at 30 °C for 4 h. After centrifugation (3220 g, 10 min), the
supernatant was transferred into a new microplate to measure the
fluorescence intensity of the indicating product (7-hydroxycoumarin,
excitation at 398 nm, emission at 458 nm, Scheme 1A). The
measurements were performed for nine cycles with 90 s intervals. After
the initial screening using whole cells and fluorogenic substrate, all
potential hits were purified to measure the specific activity and coupling
efficiency using the target substrate 1,2,3,4-tetrahydronaphthalene.
the
phosphorothioate
modified
position)
and
5’-
ATGCGGCC*GCAAGCTT*TTACAGCGCCAGCT-3’ as the reverse
primer (reverse, Lamo-PRSTduet1 R). The PCR mixture (50 µL) for
amplifying the heme gene fragment was composed of KOD-neo-plus (1
U), 1×buffer for KOD-plus-Neo, 0.2 mM dNTP mix, 1 mM MgSO4, 100
nM primers and 50 ng template (wild type, plasmid named prsfduet-
p450lamo). PCR cycling conditions were as follows: 94 °C for 2 min, 30
cycles [98 °C for 10 s, 56 °C for 30 s, 68 °C for 160 s], and 68 °C for 10
min. Redox domain mutagenesis (1148 bp; 49% of the holoenzyme):
modified
PCR
primers
were
as
follows:
5’-
TCCGAATT*CGAGCTCG*ATGGAACGTACTGC-3’ as the forward
primer (designated as PRSFduet1-Lamo-F, where ‘*’ denotes the
phosphorothioate
modified
position)
and
5’-
CTTCAAGG*AAGATCCG*CATTTCCATGCGGGCG-3’ as the reverse
primer (Lamo-1201-R). The PCR mixture contained 1× rTaq buffer, 0.2
mM dNTP mix, 100 nM forward and reverse primers, 75 µM Mn2+, 1 U
rTaq DNA polymerase, and 50 ng plasmid template (plasmid prsfduet-
p450lamo). PCR cycling conditions were as follows: 95 °C for 5 min, 30
cycles [95 °C for 30 s, 58 °C for 30 s, 72 °C for 90 s] and 72 °C for 10
min. The PCR products were digested using DpnI and purified using a
DNA purification kit (Macherey-Nagel, Dueren, Germany). Iodine was
used to treat the fragments at 70 °C for 10 min. The mixture (25 µL) was
composed of 15 µL DNA (8 pmol), 5 µL cleavage buffer (0.5 M Tris-HCl,
pH 9.0), 3 µL iodine stock solution (100 mM iodine in absolute ethanol),
and 2 µL Milli-Q water. Subsequently, the purified iodine-treated DNA
Cell Cultivation and Protein Purification
Single colonies of E. coli BL21 (DE3) were picked and inoculated into 4
mL LB medium (50 mg/L kanamycin) and cultivated at 37 °C for 12 h.
Then, 1 mL of the pre-culture was inoculated into 80 mL TB medium
containing 50 mg/L kanamycin. The cells were grown at 37 °C and 180
rpm until an OD600 of approx. 1.0, and then the enzyme was induced by
adding 0.2 mM IPTG and 0.2 mM ALA. The culture was continued by
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
incubation at 16 °C and 180 rpm for another 24 h. More details of the
protein purification can be found in a previous work.[12] The purified
protein was concentrated via ultrafiltration tubes for further use (30 kDa,
Millipore, MA).
222201514039), and the Science and Technology Commission
of Shanghai Municipality (No. 15JC1400403).
Keywords: biocatalysis • CYP116B • cytochromes P450 •
directed evolution • electron transfer
Assay of Enzyme Activity and Coupling Efficiency
[1]
a) R. Bernhardt, J Biotechnol. 2006, 124, 128-145; b) Z. Li , JBV Beilen,
W. A. Duetz, A. Schmid, A. D. Raadt, H. Grieng, B. Witholt, Curr. Opin.
Chem. Biol. 2002, 6, 136-144;.c) D. W. Nebert, F. J. Gonzalez, Annu
Rev Biochem 1987, 56, 945-993.
All of the enzyme catalytic reaction analysis was performed in 1-mL
reaction tubes on a mini-shaker (HLCBioTech, MHR23) at 600 rpm and
20°C. The reaction mixture (1 mL) was composed of 4.5 µM P450s
(P450 concentration was measured by CO-difference spectra,
Supporting Information),[29] 0.3 mM NADPH and 0.3 mM tetralin. Ethyl
acetate was used to extract the reaction product and the resulting
organic extract was analyzed by GC-MS.
The electron coupling efficiency of P450LaMO was calculated as the
ratio of the tetral-1-ol formation rate to the NADPH consumption rate. The
P450 enzyme sample (10 µL, 11 µM) was mixed with 930 µL of KPi
buffer (0.1 M, pH 8.0) containing 10 µL of substrate stock solution
(ethanol, 50 mM). After standing for 5 min, 50 µL of 10 mM NADPH
solution was added, followed by standing at 30 °C for 10 min to detect
the absorbance of NADPH at 340 nm within a linear range of variation.
Then, 500 µL ethyl acetate was used (with 0.5 mM guaiacol as an
internal standard) to extract the sample twice, and the organics were
dried over anhydrous Na2SO4. Subsequently, the extract was
concentrated to 50 µL to perform GC-MS analysis for quantitation of the
reaction product.
[2] a) M. W. Peters, P. Meinhold, A. Glieder, F. H. Arnold, J. Am. Chem. Soc.
2003, 125, 13442-13450; b) F. W. Strӧhle, E. Kranen, J. Schrader, R.
Maas, D. Holtmann, Biotechnol. Bioeng. 2016, 113:1225-1233.
[3] a) A. Celik, G. A. Roberts, J. H. White, S. K. Chpman, N. J. Turner, S. L.
Flitsch, Chem. Commun. 2006, 43, 4492-4494; b) J. C. Lewis, S. M.
Mantovani, Y. Fu, C. D. Snow, R. S. Komor, C. H. Wong, F. H. Arnold,
ChemBioChem 2010, 11, 2502-2505; c) L. Liu, R. D. Schmid, V. B.
Urlacher, Biotechnol. Lett. 2010, 32, 841-845; d) K. D. Zhang, S. E.
Damaty, R. Fasan. J. Am. Chem. Soc. 2011, 133, 3242-3245; e) S. H.
Lee, Y. C. Kwon, D. M. Kim, C. B. Park, Biotechnol. Bioeng. 2013, 110,
383-390.
[4]
a) J. D. Zhang, A. T. Li, Y. Yang, J. H. Xu, Appl. Microbiol. Biotechnol.
2010, 85, 615-624; b) J. D. Zhang, A. T. Li, J. H. Xu, Bioprocess.
Biosyst. Eng. 2010, 33, 1043-1049.
[5]
a) D. J. B. Hunter, G. A. Roberts, T. W. B. Ost, J. H. White, S. Muller, N.
J. Turner, S. L. Flitsch, S. K. Chapman, FEBS Lett. 2005, 579, 2215-
2220; b) G. P. Kurzban, H. W. Strobel, J. Biol. Chem. 1986, 261, 7824-
7830; c) J. L. Vermilion, D. P. Ballou, V. Massey, M. J. Coon, J. Biol.
Chem. 1981, 256, 266-277.
GC-MS Analysis
Product analysis was carried out by GC/MS (Shimadzu GCMS-QP2010,
HP-5 MS, column length 30 m, internal diameter 0.25 mm, film thickness
0.25 mm) with helium as the carrier gas. Mass spectra were collected
after 4 min (solvent cut time) by electron ionization with the scan mass
range of 40-300 (m/z). The GC-MS retention times for tetralin, tetralol
and the internal standard were 10.5 min, 13.5 min, and 9.3 min,
respectively.
[6]
a) J. M. Klenk, B. A. Nebel, J. L. Porter, J. K. Kulig, S. A. Hussain, S. M.
Richter, M. Tavanti, N. J. Turner, M. A. Hayes, B. Hauer, S. L. Flitsh,
Biotechnol. J. 2017, 12, 1600520. DOI: 10.1002/biot.201600520; b) G.
A. Roberts, G. Grogan, A. Greter, S. L. Flitsch, N. J. Turner, J. Bacteriol.
2002, 184, 3898-3908; c) G. A. Roberts, A. Celik, D. J. B. Hunter, T. W.
B. Ost, J. H. White, S. K. Chapman, N. J. Turner, S. L. Flitsch, J. Biol.
Chem. 2003, 278, 48914-48920.
[7]
[8]
L. Liu, R. D. Schmid, V. B. Urlacher, Appl. Microbiol. Biotechnol. 2006,
72, 876-882.
Molecular Docking and Mutation Site Exhibition
A. J. Warman, J. W. Robinson, D. Luciakova, A. D. Lawrence, K. R.
Marshall, M. J. Warren, M. R. Cheesman, S. E. J. Rigby, A. W. Munro,
K. J. McLean, FEBS J. 2012, 279, 1675-1693.
A structural model of P450LaMO wild-type was constructed based on the
crystal structures of its homologue enzymes (PDB IDs: 1CPT, 1Z8O,
1JIP and 2UUQ). The on-line SWISS-MODEL web server
(http://www.swissmodel.expasy.org/) was used to perform the homology
modeling. The 3D structure of substrate tetralin was generated by energy
minimization. Then the substrate structure was docked into the binding
pocket of the constructed P450LaMO model (AutoDock 4.0). The best
scoring results were selected for structural comparison and catalytic
mechanism analysis. After introducing mutagenesis, the mutation sites
were shown by the visual software Pymol 2.5.
[9]
D. Minerdi, S. J. Sadeghi, G. D. Nardo, F. Rua, S. Castrignsno, P.
Allegra, G. Gilardi, Mol. Microbiol. 2015, 95, 539-554.
[10] a) Y. C. Yin, H. L. Yu, Z. J. Luan, R. J. Li, P. F. Ouyang, J. Liu, J. H. Xu,
ChemBioChem 2014, 15, 2443-2449; b) S. C. Hammer, G. Kubik, E.
Watkins, S. Huang, H. Minges, F. H. Arnold, Science 2017, 358, 215-
218.
[11]
A. T. Li, J. D. Zhang, J. H. Xu, W. Y. Lu, G. Q. Lin, Appl. Environ.
Microbiol. 2009, 75, 551-556.
[12] R. J. Li, J. H. Xu, Y. C. Yin, N. Wirth, B. B. Zeng, H. L. Yu, New J.
Chem. 2016, 40, 8928-8934.
[13] M. Tavanti, J. L. Porter, S. Sabatini, N. J. Turner, S. L. Flitsch,
ChenCatChem 10.1002/cctc.201701510
Acknowledgements
[14] a) M. T. Lundemo, J. M. Woodley. Appl. Microbiol. Biotechnol. 2015, 99,
2465-2483; b) E. O’Reilly, V. Kohler, S. L. Flitsch, N. J. Turner, Chem.
Commun. 2011, 47, 2490-2501.
We are sincerely grateful to Professor Manfred T. Reetz (Max-
Planck-Institut für Kohlenforschung and Fachbereich Chemie
der Philipps-Universität, Germany) for his helpful discussions.
Thanks to Prof. Dr. R. Hong from Shanghai Organic Institute of
Chinese Academy of Sciences for revising the manuscript. This
work was financially supported by the National Natural Science
[15] R. Fasan, M. M. Chen, N.C. Crook, F. H. Arnold, Angew. Chem. Int. Ed.
2007, 46, 8414-8418.
[16] Y. C. Yan, F. Parmeggiani, E. A. Jones, E. Claude, S. A. Hussain, N. J.
Turner, S. L. Flitsh, P. E. Barran, J. Am. Chem. Soc. 2017, 139, 1408-
1411.
Foundation of China (Nos. 21672063
& 21536004), the
Fundamental Research Funds for the Central Universities (No.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
[17] a) K. Y. Choi, E. Jung, H. Yun, Y. H. Yang, B. G. Kim, Appl. Microbiol.
Biotechnol. 2014, 98, 8191-8200; Bb) A. W. Munro, H. M. Girvan, K. J.
McLean, Nat. Prod. Rep. 2007, 24, 585-609.
[23] a) S. Hayakawa, H. Matsumura, N. Nakamura, M. Yohda, H. Ohno,
FEBS J. 2014, 281, 1409-1416; b) J. B. Lim, K. A. Barker, K. A. Eller, L.
Jiang, V. Molina, J. F. Saifee, H. D. Sikes, Protein Science 2015, 24,
1874-1883.
[18] a) R. Woodyer, W. A. van der Donk, H. Zhao, Biochemistry 2003, 42,
11604-11614; b) W. L. Tang, Z. Li, H. Zhao, Chem. Commun. 2010, 46,
5461-5463; c) Z. Li, L. Butikofer, B. Witholt, Angew. Chem. Int. Ed.
2004, 43, 1698 – 1702; d) Y. Z. Chen, W. L. Tang, J. Mou, Z. Li, Angew.
Chem. Int. Ed. 2010, 49, 5278 – 5283.
[24] a) D. Batabyal, L. S. Richards, T. L. Poulos, J. Am. Chem. Soc.
DOI:10.1021/jacs.7b07656; b) S. L. Freeman, A. Martel, E. L. Raven, G.
C.K. Roberts, Scientific Reports, DOI:10.1038/s41598-017-09840-8.
[25] a) J. Ren, L. Lu, J. Xu, T. Yu, B. B. Zeng, Synthesis 2015, 47, 2270-
2280; b) A. Wu, Y. Duan, D. Xu, R. G. Harvey, Tetrahedron 2010, 66,
2111-2118; c) K. C. Nicolaou, D. L. F. Gray, T. Montagnon, S. T.
Harrison, Angew. Chem. Int. Ed. 2002, 41, 996-1000.
[19] M. C. U. Gustafsson, O. Roitel, K. R. Marshall, M. A. Noble, S. K.
Chapman, A. Pessegueiro, A. J.Fulco, M. R. Cheesman, C. Von
Wachenfeldt, A. W. Munro, Biochemistry 2004, 43, 5474–5487.
[20] a) J. Y. Kang, S. H. Ryu, S. H. Park, G. S. Cha, D. H. Kim, K. H. Kim, A.
W. Hong, T. Ahn, J. G. Pan, Y. H. Joung, H. S. Kang, C. H. Yun,
Biotechnol. Bioeng. 2014, 111, 1313-1322; b) D. F. Estrada, J. S.
Laurence, E. E. Scott, J. Biol. Chem. 2016, 291, 3990-4003; c) M.
Sugishima, H. Sato, Y. Higashimoto, J. Harada, K. Wada, K. Fukuyama,
M. Noguchi, PNAS 2014, 111, 2524-2529.
[26] a) I. Bichlmaier, A. Siiskonen, M. Finel, J. Yli-Kauhaluoma, J. Med.
Chem. 2006, 49, 1818–1827; b) G. B. afGennas, V. Talman, O. Aitio, E.
Ekokoski, M. Finel, R. K. Tuominen, J. Y. Kauhaluoma, J. Med. Chem.
2009, 52, 3969–3981; c) D. G. Barrett, J. G. Catalano, D. N. Deaton, S.
T. Long, R. B. McFadyen, A. B. Miller, L. R. Miller, K. J. WellsKnecht, L.
L. Wright, Bioorg. Med. Chem. Lett. 2005, 15, 2209–2213; d) Tibotec
Pharmaceuticals Ltd, WO2008/99019 A1, 2008.
[21] a) J. C. Lewis, S. M. Mantovani, Y. Fu, C. D. Snow, R. S. Komor, C. H.
Wong, F. H. Arnold, ChemBioChem 2010, 11, 2502-2505; b) S.
Simionatto, S. B. Marchioro, V. Galli, T. D. Luerce, D. D. Hartwig, A. N.
Moreira, O. A. Dellagostin, J Microbiol. Meth. 2009, 79, 101-105; c) A.
Urban, S. Neukirchen, K. E. Jaeger, Nucleic Acids Res. 1997, 25, 2227-
2228.
[27] a) N. S. Berrow, D. Alderton, S. Sainsbury, J. Nettleship, R. Assenberg,
N. Rahman, D. I. Stuart, R. J. Owens, Nucleic Acids Res. 2007, 35, 1-
12; b) M. Blanusa, A. Schenk, H. Sadeghi, J. Marienhagen, U.
Schwaneberg, Anal. Biochem. 2010, 406, 141-146; c) R. Zou, K. Zhou,
G. Stephanopoulos, H. P. Too, PLoS ONE 2013, 8, e79557.
[22] a) O. Salazar, P. C. Cirino, F. H. Arnold, ChemBioChem 2003, 4, 891-
893; b) Y. T. Chang, G. Loew, Biochemistry 2000, 39, 2484-2498; c) M.
A. McLean, S. A. Maves, K. E. Weiss, S. Krepich, S. G. Sligar, Biochem.
Biophys. Res. Commun. 1998, 252, 166-172.
[28] a) D. Hanahan, J. Jessee, F. R. Bloom, Method Enzymol.1991, 204,
63-113; b) H. Y. Liu, A. Rashidbaigi, Biotechniques 1990, 8, 24-25.
[29] T. Omura, R. Sato, J. Biol. Chem. 1964, 239, 2370-2378.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201800054
ChemCatChem
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 2:
FULL PAPER
CYP116B4 monooxygen-
ase was hindered by poor
reaction rate and electron
coupling efficiency. Via
transdomain combination
mutagenesis, the resulting
variant improves TOF and
CE for 1-hydroxylation of
tetralin and other deriva-
tives by up to 10- and 9.1-
folds.
R. J. Li, J. H. Xu, Q. Chen,
J. Zhao, A. T. Li, H. L. Yu*
Page No. – Page No.
Enhancing the catalytic
performance of a
CYP116B monooxygen-
ase by transdomain
combination mutagenesis
This article is protected by copyright. All rights reserved.