Whole-Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

Article abstract of DOI:10.1002/anie.201804924

An Escherichia coli whole-cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild-type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non-native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N-heptyl-l-prolyl-l-phenylalanine (C7-Pro-Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non-native substrates to intracellular P450BM3.

Full text of DOI:10.1002/anie.201804924

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Accepted Article
Title: Whole-Cell Biotransformation of Benzene to Phenol Catalysed
by Intracellular Cytochrome P450BM3 Activated by External
Additives
Authors: Masayuki Karasawa, Joshua Kyle Stanfield, Sota
Yanagisawa, Osami Shoji, and Yoshihito Watanabe
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804924
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10.1002/anie.201804924
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Whole-Cell Biotransformation of Benzene to Phenol Catalysed
by Intracellular Cytochrome P450BM3 Activated by External
Additives
Masayuki Karasawa,[b] Joshua Kyle Stanfield,[b] Sota Yanagisawa,[b] Osami Shoji,[b], [c]* and Yoshihito
Watanabe [a]*
Abstract: An Escherichia coli whole-cell biocatalyst for the direct
hydroxylation of benzene to phenol has been developed. By adding
amino acid derivatives as decoy molecules to culture medium, wild-
type cytochrome P450BM3 (P450BM3) expressed in E.coli can be
activated and non-native substrates hydroxylated, without
supplementing NADPH. The yield of phenol reached 44% when N-
heptyl-L-prolyl-L-phenylalanine (C7-Pro-Phe) was employed as the
decoy molecule. It was shown that decoy molecules, especially
those lacking fluorination, reached the cytosol of E. coli, thus
imparting in vivo catalytic activities for oxyfunctionalisation of non-
native substrates onto intracellular P450BM3.
reaction system utilising the substrate misrecognition of
P450BM3. Herein inert mimics of native substrates (decoy
molecules) stimulate P450BM3 into generating the active
oxygen species (Compound I, Figure 1b),[6] culminating in the
hydroxylation of non-native substrates.[7] Since decoy molecules,
such as perfluorinated carboxylic acids (PFCs), are
misrecognised as substrates and accommodated by P450BM3,
they can remodel the active site of P450BM3, permitting the
hydroxylation of small organic molecules.[8] We recently reported
that 'second-generation decoy molecules', PFCs modified with L-
amino acids, significantly improved the efficacy of P450BM3
activation for the hydroxylation of gaseous alkanes.[9] More
recently, we demonstrated that this strategy enables even non-
Direct oxidation of unactivated carbon-hydrogen bonds has been
a
long-standing
challenge
in
synthetic
chemistry.[1]
variety of
Monooxygenases, in nature, catalyse
a
oxyfunctionalization reactions under very mild conditions and
have thus been at the focal point of attention as promising
candidates for green catalysts to accomplish the aforementioned
task.[2] Unfortunately, monooxygenases, such as cytochrome
P450s, require a stoichiometric amount of expensive cofactors,
e.g. NAD(P)H, as electron donors,[3] which has hindered further
in vitro application of these enzymes. Hence, the development of
whole-cell reaction systems, wherein monooxygenase reactions
are supported by the intracellular production of NAD(P)H, is a
powerful approaches to overcome this limitation.[4] CYP102A1
(P450BM3), an NADPH-dependent monooxygenase isolated
from Bacillus megaterium, has been regarded as an attractive
target enzyme for application in whole-cell biocatalysis, due to
its exceptionally high monooxygenase activities.[5] P450BM3
efficiently catalyses the sub-terminal hydroxylation of long-chain
fatty acids (Figure 1a), owing to its well-designed substrate
binding site for native substrates. Conversely, the high substrate
specificity of P450BM3 resulted in very low catalytic activities
towards non-native substrates, such as gaseous alkanes and
benzene. To expand the potential substrate scope for
hydroxylation by wild-type P450BM3, we developed a unique
[a]
Prof. Dr. Y. Watanabe
Research Center for Materials Science, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81) 52-789-2953
E-mail: p47298a@nucc.cc.nagoya-u.ac.jp
M. Karasawa, J. K. Stanfield, S. Yanagisawa, Dr. O. Shoji
Department of Chemistry, Graduate School of Science
Nagoya University
[b]
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81) 52-789-3557
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
Dr. O. Shoji
Core Research for Evolutional Science and Technology, Japan
Figure 1. a) General catalytic cycle of P450BM3. b) Plausible catalytic cycle
for benzene hydroxylation catalysed by P450BM3 with perfluorononanoic acid
(PFC9) as a decoy molecule. c) Modelled active site of P450BM3 with Z-Pro-
Phe and benzene. Benzene was docked into the crystal structure of Z-Pro-Phe
bound-form of P450BM3 (PDB: 5XA3).[10] d) Concept of this study; whole-cell
biocatalyst for aromatic hydroxylation activated by decoy molecules.
[c]
Science and Technology Agency,
Tokyo, 102-0075, Japan
5 Sanbancho, Chiyoda-ku,
Supporting information for this article is available on the WWW
under http://www.angewandte.org.
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10.1002/anie.201804924
Table 1. Product formations and yields of benzene hydroxylation[a]
Phenol
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fluorinated carboxylic acids, collectively termed 'third-generation
decoy molecules’, to function as activators of P450BM3.
Moreover, these third-generation decoy molecules were reported
to strongly enhance the hydroxylation of benzene to phenol.[10]
Furthermore, the stereoselectivity of benzylic hydroxylation can
be controlled by tweaking the structure of third-generation decoy
molecules.[11] These results clearly showed that the substrate
misrecognition system possesses the potential to be applied to
the oxidation of a broad range of small organic molecules.
Regardless, it remains imperative that the problem of costly
NADPH consumption be solved, ensuring the decoy molecule
system becomes practical for use in synthetic chemistry. NADPH
is produced and regenerated cost-efficiently in living organisms,
such as E. coli, requiring only inexpensive growth medium as
sustenance. Thus, if both decoy molecules and substrates were
to be taken up into E. coli cells expressing P450BM3, non-native
substrates would be hydroxylated by intracellular P450BM3,
sustained solely by the cell's NADPH pool (Figure 1d).
Endeavouring to expand the substrate misrecognition system to
encompass whole-cell reactions, we investigated the efficacy of
whole-cell reactions in the presence of various decoy molecules.
We report herein the facile direct hydroxylation of benzene to
yield phenol in E. coli cells expressing wild-type P450BM3 by
simply adding a small amount of decoy molecules to the culture
medium.
GC-yield
in vitro Rate[d]
Decoy molecule
concentration
[μM][b]
[%][c]
[min-1 P450-1]
None
PFC9
40 ± 10
60 ± 3
0.4
0.6
1.9
1.7
7.9
9.2
4.4
20
trace
120 ± 5
157 ± 4
169 ± 10
192 ± 8
201 ± 11
175 ± 7
225 ± 9
236 ± 4
229 ± 9
259 ± 12
PFC9-Phe
C9-Trp
190 ± 30
170 ± 20
790 ± 80
920 ± 50
440 ± 20
1960 ± 20
3760 ± 110
300 ± 50
3810 ± 150
C9-Phe
C10-Phe
C11-Phe
R-Ibu-Phe
S-Ibu-Phe
Z-Pro-Phe
C7-Pro-Phe
38
3.0
38
[a] Reaction conditions: E. coli BL21(DE3) expressing Wild-type P450BM3
(OD600 = 6.3), decoy molecule (100 μM), benzene (10 mM), Glucose (40 mM),
at 25 ºC for 5 h in 110 mM phosphate buffer, pH 7.4. [b] The uncertainty is
given as the standard deviation of at least three measurements using different
batches of cell cultures. Phenol formation was determined by GC. [c] [Phenol]/
[Initial benzene]
× 100. [d] Catalytic turnover rate of in vitro benzene
hydroxylation by P450BM3 as reported in the literature.[8a, 10]
the cell membrane, thus activating intracellular P450BM3.
Yet, addition of PFC9 did not affect the whole-cell
biotransformation (phenol yield 0.6%). However, a second-
generation decoy molecule, PFC9 modified with L-phenylalanine
(PFC9-Phe), yielded a marginal increase in phenol formation
(1.9% yield). Nevertheless, this was lower than expected, as
PFC9-Phe has been shown to be effective for in vitro reactions.
Intriguingly, phenol formation was significantly increased by the
addition of PFC9-Phe's non-fluorinated analogue C9-Phe (7.9%
yield), indicating that fluorinated decoy molecules are most likely
ill-suited for application in whole-cell biotransformations.
Interestingly, phenol formation drastically varied depending upon
the structure of the decoy molecule employed, but no
appreciable correlation between in vivo and in vitro hydroxylation
activities could be observed (Table 1). Amongst a series of non-
fluorinated decoy molecules (Figure 2), (S)-Ibuprophen modified
with L-phenylalanine (S-Ibu-Phe) and N-heptyl-L-prolyl-L-
phenylalanine (C7-Pro-Phe) induced a substantial increase in
product yields (38%). It is noteworthy that R-Ibu-Phe and Z-Pro-
Phe were not as effective, despite in vitro catalytic turnover rates
being comparable to those of S-Ibu-Phe and C7-Pro-Phe. The
cytotoxicity of decoy molecules was examined by monitoring
mortality of E. coli in the presence of 100 μM of each decoy
molecule. Surprisingly, all examined decoy molecules exerted
no visible effect upon survival (Figure S2, see also Figure 4),
indicating that the observed differences in phenol formation were
not caused by the potential cytotoxicity of the decoy molecules.
Dissociation constants (Kd) of third-generation decoy molecules
to the haem domain of P450BM3 have been tentatively
estimated from UV-Vis titration experiments, but no marked
difference in their respective Kds (8 – 41 μM) was observed
amongst all examined third-generation decoy molecules.[10]
Considering the negligible cytotoxicity and similar in vitro
hydroxylation activities of the third-generation decoy molecules
employed in this study, we deduced that differences in cell-
permeability between the respective decoy molecules present
Figure 2. Decoy molecules employed in this study.
As a whole-cell biocatalyst, E. coli BL21(DE3) harbouring a
pET28a(+) vector coding for wild-type P450BM3 was employed
(See supporting information). Whole-cell biotransformations
were performed in a phosphate buffer by adding 10 mM benzene
and 100 μM decoy molecules. The concentration of both
benzene and respective decoy molecule were the same as
those used in in vitro reactions as previously reported.[8a,
10]
Decoy molecules possessing a high potency for in vitro benzene
hydroxylation were selected for screening (Figure 2).[10] In the
absence of any decoy molecule, P450BM3 scarcely catalysed
the hydroxylation of benzene to phenol with a product yield of
0.4% after 5 h reaction time (Table 1). As PFCs are mimics of
fatty acids, known to be actively transported into the cell,[12] we
initially investigated whether PFC9 was also able to permeate
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10.1002/anie.201804924
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the most likely explanation for this discrepancy. To gain further
insight into the cell membrane permeability of decoy molecules,
the dependence of decoy molecule concentration upon the
hydroxylation activity was examined (Figure 3a). As expected,
C7-Pro-Phe worked efficiently even at very low concentrations,
with 5 μM being sufficient to elicit the maximum catalytic activity,
44% yield for a 12 h reaction (Figure 3b). Even 0.5 μM of C7-
Pro-Phe was effective, yielding 23% product. These results
indicated that, even at submicromolar concentrations, sufficient
C7-Pro-Phe for the activation of intracellular P450BM3 is
accumulated within the cell, owing to the potentially superior cell
membrane permeability of C7-Pro-Phe. Unexpectedly, S-Ibu-
Phe and R-Ibu-Phe, although possessing similar in vitro
activities, delivered significantly different yields at equal
concentrations in vivo. These observations further support the
hypothesis of cell-permeability being a decisive factor governing
decoy molecule efficacy in whole-cell biotransformations.
explanation for the observed reduced product yields (Figure 4,
see also Figures S3 and S4).
Table 2. Product yields and regioselectivities of the hydroxylation of
monosubstituted benzenes using C7-Pro-Phe as the decoy molecule[a]
ortho Selectivity
GC-yields
[%][b]
Substrate
[%]
Toluene
(Ph-CH3)
97
91
26
23
Anisole
(Ph-OCH3)
Fluorobenzene
(Ph-F)
61
91
16
Chlorobenzene
(Ph-Cl)
2.7
[a] Reaction conditions: E. coli BL21(DE3) expressing wild-type P450BM3
(OD600 = 6.3), C7-Pro-Phe (100 μM), substrates (10 mM), glucose (40 mM), at
25ºC for 5 h. [b] [Product formation]/ [Initial substrate] × 100. The yield is given
as the average of at least three measurements using different batches of cell
cultures. Product formation was determined by GC.
Figure 4. Fluorescence microscopy images of Escherichia coli BL21(DE3)
treated with 10 mM substrates or 100 μM C7-Pro-Phe for 5 h. Images are
shown as an overlay of propidium iodide (red; dead cells) and SYTO-9 (green;
living cells) fluorescence. Scale bar represents 3 μm.
According to the time-course measurement of benzene
hydroxylation no clear increase in phenol concentration could be
observed after 5 h (Figure S5). We speculated that accumulation
of phenol may trigger further oxidation by the whole-cell
biocatalyst. To identify the highly hydrophilic overoxidation
products, the supernatant of whole-cell reaction mixture
following a 5 h reaction was directly evaluated by GC/MS,
revealing hydroquinone as the main overoxidation product,
accompanied by a small amount of catechol (Figure S7). In vitro
control experiments using 10 mM phenol as a substrate similarly
yielded hydroquinone and catechol, lending support to the
stimulation of overoxidation in the presence of excess phenol
(Figure S6a).[8a] In our previous studies on the in vitro reaction of
benzene, to estimate the turnover frequency (TOF), detectable
overoxidation of phenol had not been observed, even at
extended reaction times (12 h), possibly due to the
comparatively low phenol concentration of 1 mM formed.[10]
In order to optimise the reaction conditions, whole-cell
benzene hydroxylations were carried out by varying the cell
density. As expected, increased cell density resulted in improved
phenol yields (Figure S8). In the case that OD600 = 12.6 of
Figure 3. a) Concentration-dependent effect of decoy molecules on the phenol
formation. Other conditions were the same as Table 1, except for the reaction
time of 12 h. The uncertainty is given as the standard deviation of at least
three measurements. The phenol yield was calculated by [Phenol]/ [Initial
benzene]
× 100. b) GC analysis of extract of the whole-cell benzene
hydroxylation catalysed by P450BM3 with 5 μM C7-Pro-Phe as the decoy
molecule for 12 h. Indane was used as the internal standard.
Whole-cell
biotransformations
of
monosubstituted
benzenes (Ph-CH3, Ph-OCH3, Ph-F, and Ph-Cl, see Table 2)
were also accelerated by the addition of C7-Pro-Phe. Akin to in
vitro reactions,[8a, 10] the ortho position of their aromatic rings was
selectively hydroxylated. Whilst the in vitro catalytic activities for
the hydroxylation of substrates anisole and toluene exceeded
those of benzene (623 min-1 and 641 min-1, respectively),[10] they
were significantly lower using the whole-cell biocatalyst. The
cytotoxicity of substrates was evaluated by fluorescence-based
live-dead staining. Fluorescence microscopy of E. coli
BL21(DE3) treated with 10 mM of each substrate for 5 h showed
enhanced cytotoxicity of anisole and toluene, offering an
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Green Chem. 2011, 13, 226–265; b) J. C. Lewis, P. S. Coelho, F. H.
Arnold, Chem. Soc. Rev. 2011, 40, 2003–2021; c) R. Fasan, ACS Catal.
2012, 2, 647–666; d) F. Nastri, M. Chino, O. Maglio, A. Bhagi-
Damodaran, Y. Lu, A. Lombardi, Chem. Soc. Rev. 2016, 45, 5020–5054.
[3] E. Romero, J. R. Gómez Castellanos, G. Gadda, M. W. Fraaije, A.
Mattevi, Chem. Rev. 2018, DOI 10.1021/acs.chemrev.7b00650.
[4] a) T. Hudlicky, J. W. Reed, Chem. Soc. Rev. 2009, 38, 3117–3132; b) M.
Schrewe, M. K. Julsing, B. Bühler, A. Schmid, Chem. Soc. Rev. 2013, 42,
6346.
[5] a) C. J. C. Whitehouse, S. G. Bell, L.-L. Wong, Chem. Soc. Rev. 2012,
41, 1218–1260; b) V. B. Urlacher, M. Girhard, Trends Biotechnol. 2012,
30, 26–36; c) R. Fasan, M. M. Chen, N. C. Crook, F. H. Arnold, Angew.
Chemie. 2007, 119, 8566–8570; d) H. Schewe, B. A. Kaup, J. Schrader,
Appl. Microbiol. Biotechnol. 2008, 78, 55–65; e) A. Li, A. Ilie, Z. Sun, R.
Lonsdale, J. H. Xu, M. T. Reetz, Angew. Chem. Int. Ed. 2016, 55, 12026–
12029; f) A. J. Ruff, M. Arlt, M. van Ohlen, T. Kardashliev, M.
Konarzycka-Bessler, M. Bocola, A. Dennig, V. B. Urlacher, U.
Schwaneberg, J. Mol. Catal. B Enzym. 2016, 134, 285–294; g) S.
Dezvarei, J. H. Z. Lee, S. G. Bell, Enzyme Microb. Technol. 2018, 111,
29–37.
whole-cell biocatalyst, the phenol yield reached 59% with 75% of
benzene being converted to phenol or hydroquinone, when 100
μM of the decoy molecule C7-Pro-Phe and 10 mM of the
substrate benzene were added (phenol selectivity 78%, Figure
S8b). Moreover, phenol selectivity can be controlled by altering
the reaction time (Table S1). A shorter reaction time afforded a
higher phenol selectivity of 93% (1 h reaction). Finally, “cell
recycling” was investigated, wherein the supernatant of the cell
suspension after
a
2
hour reaction was removed by
centrifugation, and the collected cell pellet was resuspended
with fresh reaction medium. The next reaction cycle was initiated
by adding 10 μM of C7-Pro-Phe and 10 mM of benzene. The GC-
yield of phenol for the 1st cycle (39±0.5%), 2nd cycle (41±1%),
and 3rd cycle (40±1%) was almost the same, indicating that the
whole-cell biocatalyst can be recycled. Even though the phenol
yield gradually decreased in subsequent cycles (4th cycle:
27±9%, 5th cycle: 20±4%, and 6th cycle: 15±2%), there was still
activity up to the 6th cycle, and possibly beyond.
[6] J. Rittle, M. T. Green, Science. 2010, 330, 933–937.
[7] a) F. E. Zilly, J. P. Acevedo, W. Augustyniak, A. Deege, U. W. Häusig, M.
T. Reetz, Angew. Chem. 2011, 123, 2772–2776; b) N. Kawakami, O.
Shoji, Y. Watanabe, Angew. Chem. Int. Ed. 2011, 50, 5315–5318.
[8] a) O. Shoji, T. Kunimatsu, N. Kawakami, Y. Watanabe, Angew. Chem. Int.
Ed. 2013, 52, 6606–6610; b) N. Kawakami, O. Shoji, Y. Watanabe,
Chem. Sci. 2013, 4, 2344; c) O. Shoji, Y. Watanabe, Isr. J. Chem. 2015,
55, 32–39; d) O. Shoji, Y. Watanabe, Chem. Lett. 2017, 46, 278–288.
[9] Z. Cong, O. Shoji, C. Kasai, N. Kawakami, H. Sugimoto, Y. Shiro, Y.
Watanabe, ACS Catal. 2015, 5, 150–156.
[10] O. Shoji, S. Yanagisawa, J. K. Stanfield, K. Suzuki, Z. Cong, H.
Sugimoto, Y. Shiro, Y. Watanabe, Angew. Chem. Int. Ed. 2017, 56,
10324–10329.
[11] K. Suzuki, J. K. Stanfield, O. Shoji, S. Yanagisawa, H. Sugimoto, Y.
Shiro, Y. Watanabe, Catal. Sci. Technol. 2017, 7, 3332–3338.
In conclusion, a facile whole-cell biocatalysis system for
the direct hydroxylation of benzene has been developed,
wherein wild-type P450BM3 expressed in E. coli could be
activated by the straightforward addition of decoy molecules.
Furthermore, this system did not require the application of
laborious mutagenesis techniques to function. Decoy molecules
can permeate into the cell, thus strongly activating intracellular
P450BM3 for the hydroxylation of benzene to phenol; this is a
demonstration of a new reaction concept, wherein external
additives impart another catalytic function upon the enzyme in
vivo. We believe that P450BM3 catalysing the bioconversion of
benzene to phenol with the assistance of decoy molecules, as
presented here, presents a promising green alternative to the
conventional high energy-consuming cumene process. Whereas
there exist a few examples of whole-cell biocatalysts for
benzene hydroxylation, to the best of our knowledge, this is the
first report of a P450-based whole-cell biotransformation of
benzene to phenol. Furthermore, our system reported here
yielded the highest phenol concentration from direct
hydroxylation of benzene using a whole-cell biocatalyst in a
single aqueous phase.[13] Although the number of decoy
molecules available for use with whole-cell biocatalysis reactions
is still limited, further screening of decoy molecules coupled with
mutagenesis is expected to improve the catalytic activity of
P450BM3,[14] realising a versatile whole-cell biocatalyst for the
hydroxylation of small organic molecules.
[12]
P. N. Black, C. C. DiRusso, Biochim. Biophys. Acta 1994, 1210, 123–
145.
[13] a) Y. Tao, A. Fishman, W. E. Bentley, T. K. Wood, Appl. Environ.
Microbiol. 2004, 70, 3814–3820; b) Y. Tao, W. E. Bentley, T. K. Wood,
Appl. Microbiol. Biotechnol. 2005, 68, 614–621; c) F. Ma, S. N. Shi, T. H.
Sun, A. Li, J. T. Zhou, Y. Y. Qu, Appl. Microbiol. Biotechnol. 2013, 97,
5097–5103.
[14] S. D. Munday, O. Shoji, Y. Watanabe, L.-L. Wong, S. G. Bell, Chem.
Commun. 2016, 52, 1036–1039.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research (S) to Y. W. (24225004) from the Ministry of
Education, Culture, Sports, Science, and Technology (Japan)
and JST CREST Grant Number JPMJCR15P3, Japan. This work
was also supported by JSPS KAKENHI Grant Number
JP15H05806 in Precisely Designed Catalysts with Customized
Scaffolding to O. S.
Keywords: whole-cell biotransformation • benzene hydroxylation
• cytochrome P450 • decoy molecule • N-acyl amino acids
Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))
[1] A. S. Borovik, Chem. Soc. Rev. 2011, 40, 1870–1874.
[2] a) F. Hollmann, I. W. C. E. Arends, K. Buehler, A. Schallmey, B. Bühler,
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By adding amino acid derivatives
as decoy molecules to culture
medium, wild-type cytochrome
P450BM3 expressed in E.coli can
be activated and non-native
substrates were hydroxylated
without supplementation of
NADPH.
Masayuki Karasawa,[b] Joshua
Kyle Stanfeld,[b] Sota
Yanagisawa,[b] Osami Shoji,* [b], [c]
and Yoshihito Watanabe [a]
*
Page No. – Page No.
Whole-Cell Biotransformation of
Benzene to Phenol Catalysed by
Intracellular Cytochrome P450BM3
Activated by External Additives
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