Solar-Powered Whole-Cell P450 Catalytic Platform for C-Hydroxylation Reactions

Article abstract of DOI:10.1002/cssc.202100944

Photobiocatalysis is a green platform for driving redox enzymatic reactions using solar energy, not needing high-cost cofactors and redox partners. Here, a visible light-driven whole-cell platform for human cytochrome P450 (CYP) photobiocatalysis was developed using natural flavins as a photosensitizer. Photoexcited flavins mediate NADPH/reductase-free, light-driven biocatalysis by human CYP2E1 both in vitro and in the whole-cell systems. In vitro tests demonstrated that the photobiocatalytic activity of CYP2E1 is dependent on the substrate type, the presence of catalase, and the acid type used as a sacificial electron donor. A protective effect of catalase was found against the inactivation of CYP2E1 heme by H₂O₂ and the direct transfer of photo-induced electrons to the heme iron not by peroxide shunt. Furthermore, the P450 photobiocatalysis in whole cells containing human CYPs 1A1, 1A2, 1B1, and 3A4 demonstrated the general applicability of the solar-powered, flavin-mediated P450 photobiocatalytic system.

Full text of DOI:10.1002/cssc.202100944

Chemistry–Sustainability–Energy–Materials
Accepted Article
Title: Solar-Powered Whole-Cell P450 Catalytic Platform for C-
Hydroxylation Reactions
Authors: Chul-Ho Yun, Thien-Kim Le, Jinhyun Kim, Ngoc Anh Nguyen,
Thi Huong Ha Nguyen, Eun-Gene Sun, Su-Min Yee, Hyung-
Sik Kang, Soo-Jin Yeom, and Chan Beum Park
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To be cited as: ChemSusChem 10.1002/cssc.202100944
Link to VoR: https://doi.org/10.1002/cssc.202100944
01/2020

10.1002/cssc.202100944
ChemSusChem
COMMUNICATION
Solar-Powered Whole-Cell P450 Catalytic Platform for C-
Hydroxylation Reactions
Thien-Kim Le,‡[a] Jinhyun Kim,‡[b] Ngoc Anh Nguyen,[a] Thi Huong Ha Nguyen,[a] Eun-Gene Sun,[a] Su-
Min Yee,[a] Hyung-Sik Kang,[a] Soo-Jin Yeom,[a] Chan Beum Park,*[b] and Chul-Ho Yun*[a]
[a]
[b]
Dr. T-K. Le, Dr. N. A. Nguyen, Dr. T. H. H. Nguyen, Dr. E-G. Sun, S-M. Yee, Prof. Dr. H-S. Kang, Prof. Dr. S-J. Yeom, Prof. Dr. C-H. Yun
School of Biological Sciences and Technology
Chonnam National University, Gwangju 61186 (Republic of Korea)
E-mail: chyun@chonnam.ac.kr
J. Kim, Prof. Dr. C. B. Park
Department of Materials Science and Engineering
Korea Advanced Institute of Science and Technology (KAIST)
335 Science Road, Daejeon 34141 (Republic of Korea)
E-mail: parkcb@kaist.ac.kr
‡These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: Photobiocatalysis is a green platform for driving redox
enzymatic reactions using solar energy, not needing high-cost
cofactors and redox partners. Here, we present a visible light-driven
whole-cell platform for human P450 photobiocatalysis using natural
to the P450 heme irons in whole cells. For the light-driven P450
catalysis not relying on NADPH and reductase, three components
are required: (i) a photosensitizer as a photoexcitable redox
mediator, (ii) a sacrificial electron donor, and (iii) a reductase-free
P450 as a biocatalyst. Flavins, which are vital compounds for
diverse redox reactions in living organisms, undergo reversible
flavins as
a
photosensitizer. Photoexcited flavins mediate
NADPH/reductase-free, light-driven biocatalysis by human CYP2E1
both in vitro and in the whole-cell systems. Our in vitro tests
demonstrate that the photobiocatalytic activity of CYP2E1 is
dependent on the substrate type, the presence of catalase, and the
acid type used as a sacificial electron donor. We found a protective
effect of catalase against the inactivation of CYP2E1 heme by H2O2
and the direct transfer of photo-induced electrons to the heme iron not
by peroxide shunt. Furthermore, the P450 photobiocatalysis in whole
cells containing human CYPs 1A1, 1A2, 1B1, and 3A4 demonstrates
the general applicability of the solar-powered, flavin–mediated P450
photobiocatalytic system.
redox
conversion.
Furthermore,
they
are
excellent
photosensitzers that absorb visible blue light.[5] As a model P450
system, we tested reductase-free CYP2E1 that was expressed in
Escherichia coli.[6] The current study pursued two specific goals.
First, we performed a set of appropriate experiments to
investigate the influence of sacrificial electron donors on the
catalytic activity of light-driven CYP2E1 using in vitro systems.
This information is critical for defining the design prerequisites of
a successful flavin-sensitized P450 system in whole cells and thus
the scope of the concept. Second, on the basis of the gained
knowledge, we applied the solar-powered whole-cell P450
catalytic platform to the synthesis of valuable metabolites using
other human P450s with various substrates, including drugs
(simvastatin and lovastatin)[7] and a steroid (17β-estradiol).[8]
Introduction
Cytochrome P450s (P450 or CYP) are a superfamily of heme-
containing monooxygenases that catalyze direct C-H
functionalization of various endogenous and exogenous
compounds using molecular oxygen and the electrons delivered
by redox partners, such as diflavin-containing NADPH-P450
reductase
(CPR).[1]
Regioselective
and
stereospecific
oxygenation activities of P450s toward a variety of substrates are
of considerable importance in drug development and the
synthesis of fine chemicals.[2] The electron transfer cascade in the
P450 system begins with the CPR that delivers electrons from a
cofactor [reduced nicotinamide adenine dinucleotide (phosphate),
NAD(P)H] to flavin adenine dinucleotide (FAD); then, the reduced
FAD transfers the electrons to flavin mononucleotide (FMN),
which finally supplies the electrons to the heme of the P450.[3]
Thus, efficient and continuous supplementation of electrons to the
P450 heme is critical to sustain the catalytic turnover of P450s.
The dependence on NAD(P)H and the redox partners limits the
potential of P450s as industrial biocatalysts.[2, 4]
Here, we report whole-cell photobiocatalysis of human
P450s (CYPs 2E1, 1A1, 1A2, 1B1, and 3A4) using natural flavins
as a photosensitizer to accomplish direct electron transfer from
the photosensitizer to the P450 heme. Scheme 1 illustrates the
concept of NADPH/reductase-free, light-driven P450 reactions
through photo-induced electron transfer from photoexcited flavins
Scheme 1. A possible mechanism for FMN as a photosensitizer to transfer
electrons to CYP2E1 in the presence of sacrificial electron donor (ED) (EDTA
or TEOA) and light. Direct electron transfer occurs from reduced FMN, which
are made by light and sacrificial electron donors, to P450 heme without
reductase. The heme can directly hydroxylate the substrate (R-H) with yielding
H2O as a side product. On the other hand, the reduced FMN quickly reacts with
O2 to yield H2O2 and superoxide.
1
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10.1002/cssc.202100944
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COMMUNICATION
Results and Discussion
TTNs of ~3.0 at 12 h incubation. These results indicate that the
sensitivity of CYP2E1 heme to H2O2 is dependent on the type of
substrate.
To test the effect of sacrificial electron donors on the light-driven
CYP2E1 biocatalysis, we used FMN as a photosensitizer both in
vitro and in the whole-cell systems. We carried out reactions by
adding catalase or superoxide dismutase (SOD) to verify whether
light-driven biocatalysis occurs via the heme iron (Fe2+) directly
reduced by the photoexcited FMN or via the intrinsic
peroxygenase activity of P450 using H2O2 produced by FMN and
sacrificial electron donor under light. We tested a set of different
sacrificial electron donors for 4-nitrophenol (4-NP) hydroxylation
and chlorzoxazone (CHZ) 6-hydroxylation as marker activities of
CYP2E1 (Fig. S1). 4-Nitrophenolis a typical substrate for CYP2E1.
4-Nitrophenol 2-hydroxylation has been widely used as a
measure of CYP2E1 catalytic activity.[9] CHZ is a centrally acting
muscle relaxant used to treat muscle spasms, which is a typical
substrate for measuring CYP2E1 activity both in vivo (clinic) and
in vitro human studies.[10] According to our results (Table S1),
ethylenediaminetetraacetic acid (EDTA) was the best sacrificial
electron donor to support photobiocatalytic 4-NP hydroxylation by
CYP2E1. The presence of catalase increased the 4-NP
hydroxylation activity with EDTA by 2.2-fold. On the other hand,
triethanolamine (TEOA) was the most efficient donor to drive CHZ
6-hydroxylation by CYP2E1 (Table S2). For all tested acids, the
enhancing effect of catalase on CHZ 6-hydroxylation activity was
lower than that on 4-NP hydroxylation. We attribute the
distinguishing effect of catalase on CHZ 6-hydroxylation
(compared to 4-NP hydroxylation) to the protective effect of CHZ
against the CYP2E1 deactivation by H2O2. According to the
literature,[11] H2O2 produced by FMN/EDTA under light can inhibit
the CYP2E1 activity or inactivate the CYP2E1. The heme
destruction of CYP2E1 during reaction is prevented by catalase
and/or by the substrate.[11] In this study, the CYP2E1 samples
without catalase showed much lower total turnover numbers
(TTNs) of 12-13 at 8 h incubation for 4-NP hydroxylation than the
samples with catalase (TTNs of 37-38) (Fig. 1a). However, the
effect of catalase on CHZ 6-hydroxylation is less pronounced than
4-NP hydroxylation (Fig. 1b). Although the presence of
catalase/SOD shows the highest activity of CHZ 6-hydroxylation
during the reaction with TTN of 4.1 at 12 h incubation, the
enhancing effect of catalase was not apparent when compared to
the case of 4-NP hydroxylation. Other samples showed similar
We examined the effect of substrate concentration on
photobiocatalytic hydroxylation of 4-NP and CHZ by CYP2E1 (Fig.
S2 and S3). The production of 4-nitrocatechol (4-NC) from 4-NP
was enhanced with the increasing substrate concentration up to
500 μM, in which TTNs were 38 and 14 in the presence and
absence of catalase/SOD for 8 h incubation under light,
respectively (Fig. S2). The addition of catalase and SOD to the
reaction mixture increased the TTNs by 2.7-fold. This result
clearly shows that photobiocatalytic 4-NP hydroxylation occurred
via direct electron transfer to the heme iron by reduced FMN, not
by the peroxide shunt. It is well known that H2O2 inhibits P450
activities via heme modification.[12] The CYP2E1 activities in the
absence of catalase should come from either (1) the reduced
heme iron by a direct electron transfer from activated FMN or (2)
the peroxygenase activity of CYP2E1 supported by H2O2 that is
generated by photoexcited FMN/EDTA. The enhancement of the
CYP2E1 activity by catalase indicates that CYP2E1 has very low
or no peroxygenase activity. According to the literature,[12]
cumene hydroperoxide supported the peroxygenase activities of
CYP2E1 toward ethanol and alkyl isonicotinic acid esters.
However, CYP2E1 peroxygenase activity supported by H2O2 has
not been reported yet.
Figure 2. Cytometry analysis to measure the transport of flavins into E. coli
cells. (a) Combined histograms of cytometry showing the transport of each flavin
into the E. coli cells with time course. (b) Comparison of cytometric histograms
with mean fluorescence intensity of each analysis. Cytometry experiments to
check the time course of 1, 5, and 10 min were performed for FMN, riboflavin,
and FAD to enter into E. coli cells. Twenty μM of each flavin was used.
Absorbance of E. coli cells at 660 nm was 1.0.
To validate the photocatalytic activity of CYP2E1 by direct
reduction of heme through FMN-mediated electron transfer from
EDTA, we conducted CO-binding analysis.[14] After light irradiation
to the solutions containing FMN, EDTA, and CYP2E1, the
characteristic Fe2+·CO absorption peak at 450 nm appeared,
which evidences substantial reduction of the heme moiety (Fig.
S4). Photoexcited FMN reduced 21% and 12% of the CYP2E1 in
the presence and absence of catalase, respectively, when
compared to the complete reduction of the CYP2E1 heme by
sodium hydrosulfite. The increase of heme reduction by catalase
shows the protective effect of the catalase on heme destruction.
This result supports that photoexcited FMN induced the reduction
of the CYP2E1 heme iron using EDTA as an electron donor. Note
that the characteristic P450 peak was not observed when any
Figure 1. Time profiles of photobiocatalytic 4-NP hydroxylation (a) and CHZ 6-
hydroxylation (b) catalyzed by human CYP2E1 in the presence and absence of
catalase (CAT) and/or SOD. TTNs of photobiocatalytic CYP2E1 (0.4 μM) by
FMN (200 μM) toward 0.5 mM substrate were measured with catalase (blue),
SOD (purple), and catalase/SOD (green), or without catalase/SOD (red) for 0.5-
12 h incubation. EDTA (50 mM) and TEOA (50 mM) were used as sacrificial
electron donors for 4-NP hydroxylation and CHZ 6-hydroxylation assays,
respectively. Concentrations of catalase and SOD were 1000 and 10 units per
mL, respectively. Only P450 was excluded for negative control experiments
(black). The value presented are means of results of triplicate determinations
with S.E.M.
2
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10.1002/cssc.202100944
ChemSusChem
COMMUNICATION
component (i.e., EDTA, FMN, or light) was missing. Our in vitro
tests demonstrate that the photobiocatalytic activity of CYP2E1 is
dependent on the substrate type, the presence or absence of
catalase, and the acid type used as a sacificial electron donor.
The enhanced product formation of the photoactivated CYP2E1
in the presence of catalase suggests a protective effect of
catalase against the inactivation of CYP2E1 heme by H2O2. The
inconsistent effect of substrate type and catalase on the light-
driven activity of CYP2E1 is attributed to the distinct sensitivity of
each P450 system on H2O2.
and simvastatin)[7] and a steroid (17β-estradiol)[8] as substrates.
The recombinant cells expressing human CYPs 1A1, 1A2, 1B1,
and 3A4 showed apparent product formation toward
corresponding substrates such as lovastatin, simvastatin, and
17β-estradiol (Fig. 4). These results indicate that the
reductase/cofactor-free solar reactions sensitized by flavins are
generally applicable to different types of human P450 enzymes
expressed in whole cells. According to the literature,[16] in vivo
overproduction of riboflavin, FMN, and FAD is possible by
modular engineering of the flavin pathway in bacteria such E. coli
and Bacillus subtilis. Engineered E. coli strain by overexpression
of ribF gene encoding the bifunctional riboflavin kinase/FMN
adenylyltransferase
could
increase
the
intracellular
concentrations of FMN and FAD up to 481 and 213 μM,
respectively.[16c] We envision that the engineered cells may
generate the natural photosensitizer intracellularly for driving
P450 photocatalysis in human P450- expressing recombinant
bacterial cells.
Figure 3. Whole-cell photobiocatalytic reactions by flavins and human CYP2E1.
(a) Time profiles of photobiocatalytic CHZ 6-hydroxylation in whole cells
expressing CYP2E1. The product concentrations were determined with 0.5 mM
CHZ, 200 M flavin, and 50 mM TEOA in whole cells (15 g cells L-1). Negative
control experiments (squares marked with dark) with 200 μM flavin, in which
only light was excluded. (b) Dependence of photobiocatalytic CHZ 6-
hydroxylation on substrate concentrations in whole cells expressing CYP2E1.
The product concentration was determined with 0.05–2 mM CHZ, 200 M FMN,
and 50 mM TEOA in whole cells (15 g cells L-1) after 12 h incubation. Negative
control experiments (black circle) with 200 μM FMN, in which only light was
excluded. The value presented are means of results of triplicate determinations
with S.E.M.
For whole-cell photocatalysis (Table S3, Fig. S5), we
observed the transport of flavins into the E. coli cells producing
CYP2E1 by cytometric analysis (Fig. 2). Utilization of whole-cell
catalysis has more application potential than enzyme catalysis,
because the whole-cell catalysis not only simplifies the
downstream processing, but also enzymes are significantly more
stable in the protected cell environment.[15] The redox system of
E. coli was not omitted but a light-dependent regeneration system
was used for the whole-cell photocatalysis. When each flavin (20
μM) was added to the cells, the florescent intensities at three time
intervals (1, 5, and 10 min) and even at 6 h were similar. The
population of the E. coli cells with EDTA or TEOA was not
apparently changed up to 12 h by analysis of the cell debris using
the flow cytometry. This result indicates that the transport of
flavins is saturated within a minute. The fluorescent intensities of
flavins were in order of FMN ≈ riboflavin > FAD. This result shows
that flavins simultaneously enter into the cytoplasm of E. coli. The
combination of the cellular uptake of flavins and the photo-
induced activation of the CYP2E1 increased the 6-OH CHZ
production of E. coli cells producing CYP2E1 up to 51-53 μM (10-
11% yields) for all three tested flavins (i.e., riboflavin, FMN, and
FAD) with 0.5 mM CHZ at 12 h incubation (Fig. 3a). The
productivity of 6-OH CHZ at 0.2 mM and 0.5 mM CHZ was 32 μM
(16% yield) and 47 μM (9.4% yield), respectively, at 12 h
incubation (Fig. 3b). Decreasing the product formation at 1 and 2
mM substrate is considered as substrate inhibition. The
productivity of 6-OH CHZ increased with the increasing FMN
concentration (Fig. S6).
Figure 4. Whole-cell photobiocatalytic reactions by FMN and human CYPs.
Product concentrations of 6’β-OH lovastatin, 6’β-OH simvastatin, and 4-OH
17β-estradiol obtained from lovastatin, simvastatin, and 17β-estradiol were
shown, respectively. Corresponding human P450 enzymes to catalyze each
hydroxylation reaction were indicated. The product concentrations were
determined with 1 mM (lovastatin, simvastatin) or 0.5 mM (17β-estradiol), 200
M FMN, and 50 mM TEOA in whole cells expressing each P450 (15 g cells L-
1) after 5 h incubation for lovastatin and simvastatin, and after 12 h incubation
for 17β-estradiol. Negative control experiments (filled bar), in which only light
was excluded. The value presented are means of results of triplicate
determinations with S.E.M.
To overcome the dependence of P450 catalysis on high-
cost cofactor NAD(P)H and the redox partners that limited the
employment of P450 enzymes in the industry[2, 4], many attempts
have been made in the past, such as NAD(P)H regeneration and
the use of oxygen surrogates (i.e., H2O2).[17] Industrial-scale
cofactor regeneration is an enzymatic method that uses
secondary enzymes (e.g., formate dehydrogenase, glucose
dehydrogenase).[18] Various other cofactor regeneration methods
include
chemical,[19]
(photo)electrochemical,[20]
and
photocatalytic[21] routes.[22] The electrochemical approach has
some advantages, such as (i) low-cost electricity, (ii) transfer of
quasi mass-free electrons, and (iii) easy control of regeneration
23]
kinetics by tuning electrons’ Fermi level.[18,
However, the
reaction rates and productivities of electrochemical platforms are
generally lower than those of enzymatic platforms.[24]
For industrial fine chemical production, the economic
feasibility of a given enzymatic reactions requires minimal
productivity of 133 (kg product/kg enzyme) and minimal TTN of
26,666. In other words, a minimum space-time yield of 0.1 g L-1 h-
To demonstrate general applicability of solar-powered P450
catalysis in whole cells, we tested other human P450s (CYPs 1A1,
1A2, 1B1, and 3A4) in addition to CYP2E1 using drugs (lovastatin
1
and a minimal final product concentration of 1 g L-1 are
required.[25]
3
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10.1002/cssc.202100944
ChemSusChem
COMMUNICATION
[3]
[4]
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35.
Recently, we have shown that flavins mediate light-driven
catalysis by bacterial CYP102A1 heme domain using in vitro
systems.[26] In the previous study, the flavin-sensitized CYP102A1
activities mainly resulted from H2O2-supported peroxygenase
activity of the CYP102A1 heme domains. In this study, we found
that human CYP2E1 catalyzes light-driven catalysis with the
direct transfer of photo-induced electrons to the heme iron without
apparent peroxygenase activity. The photobiocatalytic activity of
CYP2E1 was also dependent on the substrate type and the acid
type used as a sacificial electron donor. Futhermore, flavins could
mediate light-driven catalysis by human CYP2E1 and other
various human P450s in whole-cell systems. The current study is
a proof of concept, so the level of productivity is lacking for
industrial applications yet. Future follow-up studies are expected
to improve the productivity through de novo synthesis of
photosensitizers (i.e., FMN and FAD),[16] engineering of P450s
through directed evolution,[27] and reaction optimization of
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We have devised a catalytic scheme for whole-cell-based flavin-
sensitized P450 catalytic reactions that are free of NADPH and a
reductase. The current study demonstrates that the need for
reductase (i.e., CPR) and cofactor NADPH (or its complicated
regenerating system) can be avoided using visible light as a
source of energy and EDTA (or TEOA) as an electron donor. We
have shown that natural flavins mediate light-driven catalysis by
human CYP2E1 both in vitro and in the whole-cell systems. The
photoactivation of flavins was productively coupled with the direct
transfer of photo-induced electrons to CYP2E1 heme iron to boost
the photobiocatalytic C-hydroxylation reactions of P450.
Furthermore, the FMN-sensitized P450 biocatalysis in whole cells
expressing human CYPs 1A1, 1A2, 1B1, and 3A4 for the
bioconversion of marketed drugs and a steroid was conducted to
demonstrate general applicability of the photobiocatalytic system.
The light-driven, flavin-sensitized P450 catalysis paves a cost-
effective and eco-friendly way to create a promising discipline of
green and sustainable chemistry with high potential for P450
applications.
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This work was supported by the National Research Foundation of
Korea via the Creative Research Initiative Center (Grant number:
NRF-2015R1A3A2066191) and the Basic Research Lab Program
(Grant number: NRF-2018R1A4A1023882), Republic of Korea.
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Keywords: biocatalysis • C-H activation • cytochrome P450 •
hydroxylation • photocatalysis
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10.1002/cssc.202100944
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Schematic illustration of whole-cell photobiocatalytic reaction by CYP2E1 with FMN as a photosensitizer. The catalytic
turnover of human CYP2E1 is mediated by photosensitized FMN that directly transfers electrons from the sacrificial electron donor to
the heme of CYP2E1. The FMN-sensitized P450 biocatalysis in whole cells expressing human CYPs 1A1, 1A2, 1B1, and 3A4 for the
bioconversion of marketed drugs and a steroid demonstrates general applicability of the photobiocatalytic system.
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