Manipulating the stereoselectivity of the thermostable Baeyer-Villiger monooxygenase TmCHMO by directed evolution

Article abstract of DOI:10.1039/c7ob02692g

Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability o

Full text of DOI:10.1039/c7ob02692g

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Manipulating the stereoselectivity of the
thermostable Baeyer–Villiger monooxygenase
TmCHMO by directed evolution†
Cite this: DOI: 10.1039/c7ob02692g
d
Guangyue Li,a,b Maximilian J. L. J. Fürst, c Hamid Reza Mansouri,
Anna K. Ressmann, d Adriana Ilie,a,b Florian Rudroff, d Marko D. Mihovilovic,
*
d
Marco W. Fraaije *c and Manfred T. Reetz
*
a,b
Baeyer–Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent bioca-
talysts in many stereoselective Baeyer–Villiger transformations, but industrial applications are rare which is
partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study,
the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum
municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety
of structurally different ketones with notable activity and enantioselectivity, including the desymmetriza-
tion of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this
reaction as well as the transformations of other substrates, directed evolution based on iterative saturation
mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of
the biocatalyst.
Received 1st November 2017,
Accepted 6th November 2017
DOI: 10.1039/c7ob02692g
rsc.li/obc
the carbonyl function leading to the so-called Criegee inter-
mediate. In the final step, one of the alkyl or aryl groups next
Introduction
The Baeyer–Villiger oxidation of ketones or aldehydes is a valu- to the original carbonyl function migrates. The product is then
able transformation in synthetic organic chemistry in which a released, after which NADP+ is also released. Being NADPH-
single oxygen atom is inserted into the respective C–C bond dependent, BVMOs require a cofactor regeneration system
adjacent to a carbonyl moiety with formation of esters or lac- such as glucose dehydrogenase or a whole-cell system.
tones.1 Many different reagents such as peracids or hydroper-
By using BVMOs from different sources and numerous
oxides have been used as stoichiometric oxidants, sometimes structurally different ketones as non-native substrates, high
in the presence of chiral transition metal catalysts2 or organo- stereoselectivity has been observed numerous times.4
catalysts3 for obtaining enantiomerically enriched or pure pro- Moreover, directed evolution5 can be applied in those cases in
ducts. The C–C activating process can also be catalyzed by which stereoselectivity proves to be moderate to poor, or if
Baeyer–Villiger monooxygenases (BVMOs),4 which require only inversion of the enantioselectivity is required.6 For example,
dioxygen from air, thereby avoiding the use of potentially cyclohexanone monooxygenase from Acinetobacter calcoaceticus
explosive reagents. Mechanistically, O2 reacts with the reduced NCIMB 9871 (AcCHMO) has been subjected to protein engin-
form of the enzyme-bound flavin (FAD), brought into this eering a number of times.7 Nevertheless, a major obstacle for
redox state by NADPH, with formation of an intermediate wide industrial applications appears to persist, namely the
hydroperoxide. This intermediate in the anionic form adds to lack of robustness of most BVMOs under operating con-
ditions.8 The discovery of the thermostable phenylacetone
monooxygenase from Thermobifida fusca (PAMO) set a mile-
aMax-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim
stone in BVMO research, although the respective substrate
an der Ruhr, Germany
scope proved to be limited.9 Protein engineering of PAMO,
especially by means of directed evolution, was applied several
times with formation of variants that show moderate to excel-
lent activity and stereoselectivity, depending upon the nature
of the substrate.10
bFachbereich Chemie, Philipps-Universität, Hans-Meerwein-Strasse, 35032 Marburg,
Germany. E-mail: reetz@mpi-muelheim.mpg.de
cMolecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands. E-mail: m.w.fraaije@rug.nl
dInstitute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163-OC, 1060
Vienna, Austria. E-mail: marko.mihovilovic@tuwien.ac.at
A different approach would be to apply protein engineering
for achieving significant thermostability of AcCHMO without a
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7ob02692g
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
trade-off in its broad substrate scope. Alternatively, the recently the case of AcCHMO, lacks sufficient thermostability and
discovered CHMO from Thermocrispum municipale DSM 44069 robustness under operating conditions to be of practical sig-
(TmCHMO) offers yet another option.11 This BVMO is charac- nificance. A PAMO variant was also shown to be (R)-selective
terized by good thermostability, and it has been shown to (98% ee), but activity proved to be poor.10b
accept ketones such as cyclohexanone, acetophenone, phenyl-
We started our investigations with directed evolution based
acetone and 2-octanone, but stereoselectivity has not been on saturation mutagenesis at sites surrounding the binding
studied to date.11 Furthermore, the crystal structure of pocket (CAST-sites; Combinatorial Active-site Saturation Test)14
TmCHMO has been solved, providing beneficial information and iterative saturation mutagenesis (ISM).14 Utilizing the
for guiding enzyme engineering and for developing a theory X-ray structure of TmCHMO (pdb accession code 5M10),11 we
for explaining the evolved catalytic profiles. Therefore, we con- docked the substrate 1a in the active site. Subsequently, 11
sider TmCHMO to be an attractive starting point for exploring amino acids within 5 Å from the docked substrate were chosen
and possibly engineering such properties as regio- and stereo- for saturation mutagenesis (L145, L146, F248, F279, R329,
selectivity as well as substrate acceptance.
F434, T435, N436, L437, W492 and F507) (Fig. 1). These posi-
tions were subjected individually to NNK-based randomization
in which all 20 canonical amino acids are used as building
blocks, requiring in each case the screening of about 96 trans-
formants for 95% library coverage (one microtiter plate).14
Such exploratory experiments are fast and deliver valuable
information for subsequent mutagenesis steps.
Results and discussion
As the model reaction for assessing the enantioselectivity of
TmCHMO, we chose the BV-oxidation of 4-methyl-
cyclohexanone (1a) with formation of (R)- and (S)-2a
(Scheme 1). We discovered that in whole-cell reactions, WT
TmCHMO shows excellent enantioselectivity and conversion in
favour of (S)-2a (99% ee; conversion 100%/24 h). Thus, in this
particular transformation, the result is comparable to the use
of AcCHMO, the most prominent prototype enzyme of this
family, which is likewise (S)-selective (>98% ee). However,
TmCHMO has the advantage that it is more thermally stable,
the melting temperature (Tm) of the wildtype being 49.8 °C as
determined here and reported earlier,11 compared to AcCHMO
(Tm = 37 °C).
We then set out to invert stereoselectivity by protein engin-
eering. Lactone (R)-2a is a chiral compound of particular inter-
est because it is a precursor of (R)-β-methyl-substituted adipic
acid, which in turn is an important intermediate in the syn-
thesis of an effective inhibitor of acetylcholinesterase (AChE)12
as shown in Scheme 2. In earlier work, a mutant of cyclopenta-
none monooxygenase from Comamonas sp. was shown to be
(R)-selective (96% ee) in this reaction,13 but this BVMO, as in
In the mini-libraries generated by saturation mutagenesis
at positions 146, 434, 435, 437 and 507, several mutants were
discovered showing decreased (S)-selectivity, namely L146E,
F434I, T435F, T435Y, T435 W, L437G, L437T, L437A and
F507W, thereby pointing the way towards reversal of enantio-
selectivity. The libraries created at the other six positions
failed to harbor any positive variants (Table S1†). At this stage
we explored two mutagenesis strategies in parallel. In one
approach, saturation mutagenesis at a relatively large 5-residue
randomization site defined by the above hot spots was per-
formed. This time, appropriately reduced amino acid alpha-
bets, individually designed for each of the five residues were
used.14 The respective design was based on the information
that the initial positive mutants provide (Table S2†).
Accordingly, 146E/L, 434I/F, 435F/Y/W/T, 437T/A/G/L and
507W/F, including the amino acid alphabets encoding wild-
type and positive mutations at individual positions, were
chosen for creating a 5-residue saturation mutagenesis library.
Scheme 1 Baeyer–Villiger oxidation of 4-methycyclohexanone (1a)
catalyzed by TmCHMO.
Fig. 1 TmCHMO structure model showing docked 4-methyl-
cyclohexanone (1a) (in cyan) based on the crystal structure of wild-type
TmCHMO (5M10),11 which served as a guide for choosing amino acids
for saturation mutagenesis (in purple).
Scheme 2 Synthesis of (R)-β-methyl-substituted adipic acid, the pre-
cursor of an AChE-inhibitor.12
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
A total of only 384 transformants were screened for 95% mated. For the LGY3-4-D11 mutant, a Km could not be accu-
library coverage, and three (R)-selective variants were indeed rately determined, because the activities with concentrations
identified (Table S3†) showing moderately reversed enantio- lower than the apparent Km were almost undistinguishable
selectivity (50%–66% ee).
from the uncoupling rate (0.16 s−1). The Km of the LGY3-4-
According to the second strategy, the best hit identified in E5 mutant is approximately 5.5 mM and a lowered uncoupling
the exploratory NNK-based mini-libraries, mutant L437A, was rate in 5% methanol of 0.05 s−1 was observed. The kcat was
chosen as a template for performing iterative saturation muta- determined to be 8 times lower than WT for the LGY3-4-
genesis (ISM) at the remaining four hot spot residues (L146, D11 mutant, while the LGY3-4-E5 mutant was close to WT with
F434, T435 and F507), which were grouped into two randomiz- 1.23 s−1. It is striking to see that all four mutants that show a
ation sites: A (F434, T435) and B (L146, F507). Thereafter, high enantioselectivity (Fig. 2) have a mutation of F507 in
several ISM pathways were explored using the respective best common, while L146 is only mutated in two of these mutants.
variants as templates and NDT codon degeneracy encoding This suggests that the F507 mutations play a dominant role in
only 12 amino acids (Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, equipping the enzyme with high enantioselectivity for the
Arg, Ser, and Gly) (Fig. 2). Two excellent (R)-selective mutants, target substrate while the L146 mutations may only have a
which could not have been found in the above described marginal effect and may even harm the kinetic performance as
5-residue saturation mutagenesis library, were identified, observed in the LGY3-4-D11 mutant.
LGY3-4-E5 (F434I/T435L/L437A/F507V) (91% ee) and LGY3-4-
Some degree of substrate inhibition, which is not unusual
D11 (L146F/F434I/T435L/L437A/F507C) (94% ee). All results for BVMOs,4 was observed in all cases. The WT is half-
are summarized in Fig. 2 and in the ESI (Table S4†). In this maximal inhibited at a concentration of 13 mM. In the LGY3-
ISM strategy, a total of only 768 transformants had to be 4-E5 mutant, inhibition is less strong, with a Ki roughly 3
screened for 95% library coverage. This is more than the first times higher than that of WT. The fitting for the LGY3-4-
approach in which the 5-residue site was randomized by a D11 mutant suggests a 20 times higher Ki than WT, but has to
single saturation mutagenesis experiment using optimally be considered unreliable for the same reasons as the Km deter-
designed codons at each individual position (384 transfor- mination (Table 1, Fig. S1†).
mants). However, the screening effort involves only 8 microtiter
The melting temperatures (Tm) of WT and both mutants
plates which can easily be handled by automated GC. The were determined with a fluorescence-based thermal shift assay
superiority of the ISM-based approach has to do with the (ThermoFAD).16 It was found that the thermostability of LGY3-
greater structural diversity at protein level.
4-D11 and LGY3-4-E5 mutants is maintained as compared to
By measuring the consumption of NADPH in the presence WT TmCHMO (Table 1).
of varying amounts of 4-methylcyclohexanone (1a), the enzyme
In order to further explore the potential of WT TmCHMO
kinetic parameters were determined using a spectrophoto- and the two best mutants as catalysts in organic chemistry, we
meter.11,15 Under the experimental conditions (150 µM tested the BV-reaction of a number of structurally diverse
NADPH, 5% methanol, pH 7.4), WT showed an uncoupling ketones using recombinant whole cells. The reaction mixture
rate of 0.22 s−1. The maximum turnover rate kcat was deter- was shaken at 30 °C (220 rpm) for 24 h in a 25 mL flask with
mined to be 1.9 s−1
, while the affinity for 4-methyl- sufficient oxygen, containing recombinant E. coli expressed
cyclohexanone was so high that a Km < 1 µM could only be esti- cells (OD600 = 35) and 10 mM substrates in 1 mL PBS buffer
(pH 7.4, 50 mM). As shown in Table 2, considerable improve-
ment in stereoselectivity towards compound 1b was achieved
with both mutants relative to WT. Enantioselectivity was
boosted from 18% to 99% ee, although reversal was not
observed (Scheme 3 and Table 2). A similar trend was also
found for substrate 5a, the two mutants inducing pronounced
enhancement of stereoselectivity from 49% ee (R) to 98–99%
ee (R). It is also interesting to note that 4-tert-butylcyclohexa-
none (1c) was converted completely by WT TmCHMO (93% ee,
R), but this sterically bulky substrate is hardly accepted by the
two variants LGY3-4-D11 and LGY3-4-E5. A similar tendency
was found for compound 1d. In the case of substrate 3, WT
TmCHMO and both mutants showed essentially identical
behaviour with excellent (4S,6R)-selectivity (99% ee), as in the
case of the more sensitive AcCHMO.17 Of special interest is the
enzymatic oxidation product of 3, which enables the ready syn-
thesis of the C11–C16 subunit of ionomycin, a naturally occur-
ring polyether antibiotic.18 Importantly, variants LGY3-4-D11
Fig. 2 Iterative saturation mutagenesis (ISM) exploration of TmCHMO
and LGY3-4-E5 lead to reversed stereoselectivity in the reaction
as catalyst in the model reaction of ketone 1a (Scheme 1) with focus on
reversal of enantioselectivity.
of compound 5b (94%–95%, R) relative to WT TmCHMO,
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Table 1 Kinetic and thermostability data of WT TmCHMO, and mutants LGY3-4-D11 and LGY3-4-E5. 4-Methylcyclohexanone (1a) was used as sub-
strate for the kinetic analysis
Entry
Mutations
kcat (s−1
)
Km (µM)
Ki (mM)
kunc (s−1
)
Tm (°C)b
WT
LGY3-4-D11
LGY3-4-E5
1.93 0.03
0.2a
1.23 0.09
< 1
13
3
8
0.22 0.08
0.16 0.01
0.053 0.002
49.8
53.0
51.5
L146F/F434I/T435L/L437A/F507C
F434I/T435L/L437A/F507V
n.d.a
n.d.a
47
5500 750
a n.d. = not determined, due to unreliable fitting only a maximal observed rate is given. All experiments were done in triplicates. b The standard
deviation (triplicates) was <0.5 °C.
Table 2 Substrate scope of WT TmCHMO and variants LGY3-4-D11 and LGY3-4-E5
WT
LGY3-4-D11
Conversion
LGY3-4-E5
Conversion
Substrate
Conversion
ee [%]
ee [%]
ee [%]
1a
1b
1c
1d
3
5a
5b
7
+++
+++
+++
++
99(S)
18(R)
93(S)
88(−)
99(4S,6R)
49(R)
95(S)
N : ABN = 50 : 50
ee% >99(−), >99(−)
N : ABN = 55 : 45
ee% 79(−), 98(−)
P : D = 45 : 55
ee% 99(−), 99(−)
97(R)
+++
+++
n. c.
+
++
+++
++
94(R)
99(R)
n. d.
16(−)
99(4S,6R)
99(R)
94(R)
55 : 45
83(−), 99(−)
48 : 52
97(−), 97(−)
35 : 65
28(−), 44(−)
75(R)
66(R)
+++
+++
n. c.
+
91(R)
99(R)
n.d.
30(+)
99(4S,6R)
98(R)
95(R)
50 : 50
98(−), 99(−)
48 : 52
98(−), 98(−)
30 : 70
86(−), 55(−)
96(R)
++
++
+++
+++
+++
+++
+++
+++
+++
9
+++
+++
+++
+++
+++
+++
11
13
15
++
+
+
+
+
+
98(R)
58(R)
Conversion: +++ >80%, ++ 50–80%, + <50%; P: proximal, D: distal; N: normal, ABN: abnormal.; n.d. not determined, n.c. not converted.
Scheme 3 Baeyer–Villiger oxidation of a series of structurally diverse substrates catalyzed by TmCHMO.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Organic & Biomolecular Chemistry
Paper
which is (S)-selective (95% ee) (Scheme 3 and Table 2). This
reversal is a synthetically significant result because the stan-
Acknowledgements
dard BVMO, AcCHMO, is (S)-selective (81% ee),19 while the (R)- Support from the Max-Planck-Society and the LOEWE
product constitutes a chiral precursor in the synthesis of the Research Cluster SynBioChem is gratefully acknowledged. The
therapeutically useful γ-aminobutyric acid receptor (GABAB) research for this work also received funding from the
agonist (R)-baclofen.20 As shown in Table 2, all three enzymes European Union (EU) project ROBOX (grant agreement
convert substrate 7 with a fast reaction rate and almost the 635734) under EU’s Horizon 2020 Programme Research and
same ratio of normal lactone to abnormal lactone (N : ABN %) Innovation actions H2020-LEIT BIO-2014-1. Financial support
and high ee value, which is similar for AcCHMO [N : ABN = by FWF (P28477-B21), COST-Action CM1303 (Systems
51 : 49, 95(−), 95(−)].21 The expected “normal” lactone can be Biocatalysis) and TU Wien ABC-Top Anschubfinanzierung are
used as an intermediate in the synthesis of the Corey lacto- gratefully acknowledged. Open Access funding provided by the
ne,4h a versatile building block in the synthesis of prostaglan- Max Planck Society.
dins. Furthermore, the “abnormal” lactone was used as a start-
ing material in the chemoenzymatic total synthesis of various
brown algae pheromones.22 The conversion results of
3-methyl-cyclohexanone (11) indicate that the mutations
Notes and references
improve the tendency of the enzyme to produce more of the
distal product. Protein engineering of other BVMOs resulting
in a change of the normal/abnormal lactone ratio has been
reported earlier.23 However, the introduced mutations have
little effect in reactions of compound 13 and a negative effect
in the reaction of 15 (Scheme 3 and Table 2).
1 Reviews of Baeyer–Villiger reactions: (a) G. R. Krow, Org.
React., 1993, 43, 251; (b) M. Renz and B. Meunier,
Eur. J. Org. Chem., 1999, 737; (c) G.-J. ten Brink, I. W. C.
E. Arends and R. A. Sheldon, Chem. Rev., 2004, 104, 4105;
(d) M. D. Mihovilovic, F. Rudroff and B. Grötzl, Curr. Org.
Chem., 2004, 8, 1057; (e) C. Jiménez-Sanchidrián and
J. R. Ruiz, Tetrahedron, 2008, 64, 2011; (f) M. Uyanik and
K. Ishihara, ACS Catal., 2013, 3, 513.
2 Asymmetric BV reactions using synthetic transition metal
catalysts: (a) C. Bolm, G. Schlingloff and K. Weickhardt,
Angew. Chem., Int. Ed. Engl., 1994, 33, 1848; (b) K. Ito,
A. Ishii, T. Kuroda and T. Katsuki, Synlett, 2003, 643;
(c) G. Strukul, Angew. Chem., Int. Ed., 1998, 37, 1198;
(d) J.-C. Frison, C. Palazzi and C. Bolm, Tetrahedron, 2006,
62, 6700; (e) A. V. Malkov, F. Friscourt, M. Bell,
M. E. Swarbrick and P. Kocovský, J. Org. Chem., 2008, 73,
3996; (f) L. Zhou, X. Liu, J. Ji, Y. Zhang, X. Hu, L. Lin and
X. Feng, J. Am. Chem. Soc., 2012, 134, 17023; (g) L. Zhou,
X. Liu, J. Ji, Y. Zhang, W. Wu, Y. Liu, L. Lin and X. Feng,
Org. Lett., 2014, 16, 3938; (h) P. P. Poudel, K. Arimitsu and
K. Yamamoto, Chem. Commun., 2016, 52, 4163; (i) A. Russo,
C. De Fusco and A. Lattanzi, ChemCatChem, 2012, 4, 901.
3 Asymmetric BV reactions using chiral organocatalysts:
(a) S. Murahashi, S. Ono and Y. Imada, Angew. Chem., Int.
Ed., 2002, 41, 2366; (b) Y. Imada, H. Iida, S. I. Murahashi
and T. Naota, Angew. Chem., Int. Ed., 2005, 44, 1704;
(c) B. Wang, Y.-M. Shen and Y. Shi, J. Org. Chem., 2006, 71,
9519; (d) G. Peris and S. J. Miller, Org. Lett., 2008, 10, 3049;
(e) S. Xu, Z. Wang, X. Zhang, X. Zhang and K. Ding, Angew.
Chem., Int. Ed., 2008, 47, 2840.
4 Reviews of BVMOs as catalysts in organic chemistry:
(a) M. M. Kayser, Tetrahedron, 2009, 65, 947;
(b) J. D. Stewart, Curr. Org. Chem., 1998, 2, 195;
(c) M. D. Mihovilovic, in Enzyme Catalysis in Organic
Synthesis, ed. K. Drauz, H. Gröger and O. May, Wiley-VCH,
Weinheim, 3rd edn, 2012, vol. I–III; (d) V. Alphand,
G. Carrea, R. Wohlgemuth, R. Furstoss and J. M. Woodley,
Trends Biotechnol., 2003, 21, 318; (e) M. D. Mihovilovic,
Curr. Org. Chem., 2006, 10, 1265; (f) R. Wohlgemuth, Eng.
Life Sci., 2006, 6, 577; (g) D. E. Torres Pazmino,
Conclusions
This study shows that the recently discovered thermally stable
Baeyer–Villiger monooxygenase TmCHMO, and the two var-
iants LGY3-4-D11 and LGY3-4-E5, are excellent biocatalysts in
the asymmetric transformation of a variety of structurally
different ketones. The two mutants were generated by directed
evolution utilizing a special version of iterative saturation
mutagenesis (ISM). As demonstrated by the determination of
the Tm values, the thermostability of the two TmCHMO
mutants is retained relative to WT. In some cases the high
enantioselectivity in desymmetrization reactions is similar to
those reported for the prototype CHMO, AcCHMO, but this
well-known BVMO is only moderately stable. Moreover, reversal
of enantioselectivity as reported herein allows ready access to
several chiral compounds of particular synthetic value. The
overall results of this study show that TmCHMO is an attractive
scaffold for future mutagenesis. It will be interesting to see
how TmCHMO and the two generated variants perform in BV-
reactions of other ketones, and as biocatalysts in enantio-
selective sulfoxidations. Further ISM studies starting from WT
or the two mutants may also be a rewarding task in future
work. Finally, a complete theoretical study, hopefully unveiling
the molecular basis of stereoselectivity, would not only be of
fundamental interest, it could also guide future directed evol-
ution of this promising BVMO.
Conflicts of interest
There are no conflicts of interest to declare.
This journal is © The Royal Society of Chemistry 2017
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
H. M. Dudek and M. W. Fraaije, Curr. Opin. Chem. Biol.,
2010, 14, 138; (h) H. Leisch, K. Morley and P. Lau, Chem.
19, 197; (d) E. Malito, A. Alfieri, M. W. Fraaije and
A. Mattevi, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 13157.
Rev., 2011, 111, 4165; (i) F. Hollmann, I. W. Arends, 10 (a) M. T. Reetz and S. Wu, J. Am. Chem. Soc., 2009, 131,
K. Buehler, A. Schallmey and B. Bühler, Green Chem., 2011,
13, 226; ( j) C. T. Walsh and Y. C. J. Chen, Angew. Chem., Int.
Ed. Engl., 1988, 27, 333; (k) K. Balke, M. Kadow, H. Mallin,
S. Saß and U. T. Bornscheuer, Org. Biomol. Chem., 2012, 10,
6249; (l) M. Bučko, P. Gemeiner, A. Schenkmayerová,
T. Krajčovič, F. Rudroff and M. D. Mihovilovič, Appl.
Microbiol. Biotechnol., 2016, 100, 6585; (m) G. de Gonzalo,
M. D. Mihovilovic and M. W. Fraaije, ChemBioChem, 2010,
11, 2208; (n) F. M. Ferroni, C. Tolmie, M. S. Smit and
D. J. Opperman, PLoS One, 2016, 11(7), e0160186;
(o) M. D. Mihovilovic, B. Müller and P. Stanetty, Eur. J. Org.
Chem., 2002, 3711.
15424; (b) S. Wu, J. P. Acevedo and M. T. Reetz, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 2775; (c) M. T. Reetz and
S. Wu, Chem. Commun., 2008, 5499; (d) Z.-G. Zhang,
R. Lonsdale, J. Sanchis and M. T. Reetz, J. Am. Chem. Soc.,
2014, 136, 17262; (e) H. M. Dudek, D. E. T. Pazmiño,
C. Rodríguez, G. de Gonzalo, V. Gotor and M. W. Fraaije,
Appl. Microbiol. Biotechnol., 2010, 88, 1135; (f) H. L. van
Beek, G. de Gonzalo and M. W. Fraaije, Chem. Commun.,
2012, 48, 3288; (g) H. M. Dudek, M. J. Fink, A. V. Shivange,
A. Dennig, M. D. Mihovilovic, U. Schwaneberg and
M. W. Fraaije, Appl. Microbiol. Biotechnol., 2014, 98, 4009;
(h) L. P. Parra, J. P. Acevedo and M. T. Reetz, Biotechnol.
Bioeng., 2015, 112, 1354.
5 Recent reviews of directed evolution: (a) A. S. Bommarius,
Annu. Rev. Chem. Biomol. Eng., 2015, 6, 319; (b) A. Currin, 11 E. Romero, J. R. G. Castellanos, A. Mattevi and
N. Swainston, P. J. Day and D. B. Kell, Chem. Soc. Rev., M. W. Fraaije, Angew. Chem., Int. Ed., 2016, 55, 15852.
2015, 44, 1172; (c) C. A. Denard, H. Ren and H. Zhao, Curr. 12 D. dos Santos Pisoni, J. S. da Costa, D. Gamba, C. L. Petzhold,
Opin. Chem. Biol., 2015, 25, 55; (d) E. M. J. Gillam,
J. N. Copp and D. F. Ackerley, in Methods in Molecular
A. C. de Amorim Borges, M. A. Ceschi, P. Lunardi and
C. A. S. Gonçalves, Eur. J. Med. Chem., 2010, 45, 526.
Biology, Humana Press, Totowa, NJ, 2014, vol. 1179; 13 C. M. Clouthier, M. M. Kayser and M. T. Reetz, J. Org.
(e) Directed Enzyme Evolution: Advances and Applications, ed. Chem., 2006, 71, 8431.
M. Alcalde, Springer, Stuttgart, 2017; (f) H. Renata, 14 Reviews of saturation mutagenesis in the directed evolution
Z. J. Wang and F. H. Arnold, Angew. Chem., Int. Ed., 2015,
54, 3351; (g) M. T. Reetz, Directed Evolution of Selective
Enzymes: Catalysts for Organic Chemistry and Biotechnology,
Wiley-VCH, Weinheim, 2016.
of stereoselective enzymes: (a) M. T. Reetz, Angew. Chem.,
Int. Ed., 2011, 50, 138; (b) Z. Sun, Y. Wikmark, J.-E. Bäckvall
and M. T. Reetz, Chem.
– Eur. J., 2016, 22, 5046;
(c) C. G. Acevedo-Rocha, S. Kille and M. T. Reetz, Methods
Mol. Biol., 2014, 1179, 189.
6 Review of protein engineering of Baeyer–Villiger monooxy-
genases: Z. G. Zhang, L. P. Parra and M. T. Reetz, 15 F. P. B. Brondani, H. M. Dudek, C. Martinoli, A. Mattevi
Eur. J. Org. Chem., 2012, 10160. and M. W. Fraaije, J. Am. Chem. Soc., 2014, 136, 16966.
7 (a) M. T. Reetz, B. Brunner, T. Schneider, F. Schulz, 16 F. Forneris, R. Orru, D. Bonivento, L. R. Chiarelli and
C. M. Clouthier and M. M. Kayser, Angew. Chem., Int. Ed., A. Mattevi, FEBS J., 2009, 276, 2833.
2004, 43, 4075; (b) M. Reetz, F. Daligault, B. Brunner, 17 M. D. Mihovilovic, F. Rudroff, A. Winninger, T. Schneider,
H. Hinrichs and A. Deege, Angew. Chem., Int. Ed., 2004, 43, F. Schulz and M. T. Reetz, Org. Lett., 2006, 8, 1221.
4078; (c) H. L. van Beek, E. Romero and M. W. Fraaije, ACS 18 M. J. Taschner and Q.-Z. Chen, Bioorg. Med. Chem. Lett.,
Chem. Biol., 2016, 12, 291; (d) Z. G. Zhang, G. D. Roiban, 1991, 1, 535.
J. P. Acevedo, I. Polyak and M. T. Reetz, Adv. Synth. Catal., 19 M. J. Fink, D. V. Rial, P. Kapitanova, A. Lengar, J. Rehdorf,
2013, 355, 99; (e) D. J. Opperman and M. T. Reetz,
ChemBioChem, 2010, 11, 2589; (f) S. Schmidt, M. Genz,
Q. Cheng, F. Rudroff and M. D. Mihovilovic, Adv. Synth.
Catal., 2012, 354, 3491.
K. Balke and U. T. Bornscheuer, J. Biotechnol., 2015, 214, 20 C. Mazzini, J. Lebreton, V. Alphand and R. Furstoss,
199; (g) H. L. van Beek, H. J. Wijma, L. Fromont, Tetrahedron Lett., 1997, 38, 1195.
D. B. Janssen and M. W. Fraaije, FEBS Open Bio, 2014, 4, 21 D. V. Rial, P. Cernuchova, J. B. van Beilen and
168. M. D. Mihovilovic, J. Mol. Catal. B: Enzym., 2008, 50, 61.
8 L. Goncalves, D. Kracher, S. Milker, F. Rudroff, M. J. Fink, 22 (a) J. Lebreton, V. Alphand and R. Furstoss, Tetrahedron
R. Ludwig, A. Bommarius and M. Mihovilovic, Adv. Synth.
Catal., 2017, 359, 2121.
Lett., 1996, 37, 1011; (b) J. Lebreton, V. Alphand and
R. Furstoss, Tetrahedron, 1997, 53, 145.
9 (a) M. W. Fraaije, J. Wu, D. P. Heuts, E. W. Van Hellemond, 23 (a) K. Balke, S. Schmidt, M. Genz and U. T. Bornscheuer,
J. H. L. Spelberg and D. B. Janssen, Appl. Microbiol.
Biotechnol., 2005, 66, 393; (b) F. Zambianchi, M. W. Fraaije,
G. Carrea, G. de Gonzalo, C. Rodríguez, V. Gotor and
G. Ottolina, Adv. Synth. Catal., 2007, 349, 1327;
(c) C. Rodríguez, G. de Gonzalo, D. E. T. Pazmino,
M. W. Fraaije and V. Gotor, Tetrahedron: Asymmetry, 2008,
ACS Chem. Biol., 2015, 11, 38; (b) K. Balke, M. Bäumgen
and U. Bornscheuer, ChemBioChem, 2017, 18, 1627;
(c) M. D. Mihovilovic, F. Rudroff, A. Winninger,
T. Schneider, F. Schulz and M. T. Reetz, Org. Lett., 2006, 8,
1221; (d) H. L. van Beek, E. Romero and M. W. Fraaije, ACS
Chem. Biol., 2016, 12, 291.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2017