A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation

Article abstract of DOI:10.1016/j.mcat.2021.111404

We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient C[dbnd]C double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h^?1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L^?1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 μM) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.

Full text of DOI:10.1016/j.mcat.2021.111404

Molecular Catalysis 502 (2021) 111404
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.journals.elsevier.com/molecular-catalysis
A robust and stereocomplementary panel of ene-reductase variants for
gram-scale asymmetric hydrogenation
a
,
a
a
a
b
Nathalie Nett *, Sabine Duewel , Luca Schmermund , Gerrit E. Benary , Kara Ranaghan ,
b
c
a
Adrian Mulholland , Diederik J. Opperman , Sabrina Hoebenreich
a
Fachbereich Chemie, Philipps-Universit a¨ t Marburg, 35032 Marburg, Germany
b
Center for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK
c
Department of Biotechnology, University of the Free State, Bloemfontein 9300, South Africa
A R T I C L E I N F O
A B S T R A C T
We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines
Keywords:
Biocatalysis
control over facial selectivity in the reduction of electron deficient C–
–
C double bonds with thermostability (up to
◦
C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to
Biphasic
7
0
Stereoselective
Old yellow enzyme
Protein-ligand docking
literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystal-
lisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess
(
ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihy-
ꢀ 1
drocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h were achieved, which are com-
parable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also
demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for
ꢀ 1
product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L R-levodione
from a 45 mL aqueous reaction, containing less than 17 mg (10 M) enzyme and the reaction only stopping
μ
because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base
for further exploitation as a tool in preparative organic synthesis.
1
. Introduction
large-scale syntheses [8–13], the core problem of controlling the facial
selectivity of the reduction remains. Excellent stereoselectivities for both
Significant progress has been made over the past decade in bio-
catalytic reduction reactions [1,2]. One of these important reactions is
isomers are only accessible for a handful of substrates [8,14,15], as
almost all wild-type ERs show the same stereopreference [16]. Alter-
natively, substrate-based stereocontrol, starting from either the E- or
Z-alkene, may give access to both stereoisomers [17]. While protein
engineering of ERs has created pairs of stereocomplementary variants
for specific compounds [18–23], little is known about their synthetic
usefulness, i.e. if they are generally stereocomplementary, about their
substrate scope, solvent and thermal stability, or productivity in large
scale reactions.
–
the asymmetric transfer hydrogenation of activated C
is catalysed by ene-reductases (ERs) from the Old Yellow Enzyme family
OYE, EC 1.6.99.1). ERs are highly chemo-, regio- and stereoselective
–
C bonds, which
(
due to precise positioning of the substrate via hydrogen bonds and
van-der-Waals interactions, allowing an overall trans addition of a hy-
dride and a proton, employing NAD(P)H as hydride source via a flavin
cofactor [3–6].
Asymmetric alkene hydrogenation is one of the most widely used
industrial reactions, which is still dominated by transition metal catal-
ysis [7]. Access to both stereoisomers with chemical catalysis is achieved
using the mirror image of the ligand. This is in stark contrast to enzyme
catalysis, where access to both stereoisomers is a major challenge in
catalyst development. Although wild types (wt) of recombinant ERs
have been intensively studied and include some impressive examples of
We have previously described stereocomplementary pairs of engi-
neered variants for three compounds from the thermostable ER of
Thermus scotoductus SA-01 (TsER/TsOYE). Opposite stereoselectivity, or
facial selection, was achieved during the reduction of 3-methylcyclohex-
enone and methyl-2-(hydroxymethyl)-acrylate with TsER variant C25D/
I67T and (S)-carvone with TsER variant C25G/I67T [24]. Here we
expand on these variants and present a small panel of engineered TsERs,
*
Corresponding author.
E-mail address: nathalie.nett@gmail.com (N. Nett).
https://doi.org/10.1016/j.mcat.2021.111404
Received 13 December 2020; Received in revised form 1 January 2021; Accepted 4 January 2021
Available online 21 January 2021
2
468-8231/© 2021 Elsevier B.V. All rights reserved.

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
with convenient catalytic characteristics for organic synthesis. This not
only includes high activity and stereoselectivity, but also easy produc-
tion, handling and storage of the catalyst as well as a simple but flexible
reaction procedure. Benchmarking tests reveal that this panel combines
a broad substrate scope, tolerance to organic solvents and high tem-
perature with convenient catalyst handling. We demonstrate that this
combination of properties enables convenient gram-scale synthesis with
excellent yields for poorly water-soluble compounds. Importantly,
exceptional control over facial selectivity was achieved for half of the
substrates.
tribasic dihydrate pH 5.0, 10 % w/v polyethylene glycol 10,000) at 289
K. Crystals were soaked in reservoir solution containing 30 % glycerol
prior to cryocooling. X-ray diffraction data were collected at Diamond
(UK) on beamline i03. Data was processed using MOSFLM [28] and
POINTLESS, with intensities scaled and merged using SCALA [29].
Molecular replacement was performed using Phaser [30] with TsER wt
(PDB 3HF3) as search model. Refinement was performed through iter-
ative cycles of manual model building in COOT [31] and restrained
refinement using Refmac [32]. The structure was validated using pro-
grams within the CCP4 suite [33] and coordinates and structure factors
have been deposited in the Protein Data Bank (PDB) under accession
code 5NUX (Table S1).
2
3
. Experimental
The crystal structures of TsER (wt) in complex with p-hydrox-
ybenzaldehyde (PDB: 3HGJ) [34] was used in conjunction with the TsER
C25D/I67T variant for docking analysis. The protein was prepared using
the Protein Preparation Wizard from Maestro, version 11.0, Schr o¨ dinger
LLC. For all calculations the dimer structure was retained. Protonation
states for titratable amino acids were assigned based on the most
favourable interactions with neighbouring residues and the PROPKA
program. The His172 and His175 residues were protonated at both
epsilon and delta nitrogen atoms, to enable hydrogen bond formation to
the substrate. All water molecules and co-crystallized ligands were
removed, with the exception of the oxidized FMN cofactors. The
oxidized FMN cofactor was converted to the reduced FMNH2 using
Antechamber. Substrate 1a was built in Maestro and prepared for
docking using the LigPrep program, version 4.0, Schr o¨ dinger LLC and
docked into the active site of reduced TsER wt and C25D/I67T using the
Glide docking protocol for Rigid body docking (RGB) with Standard
Precision settings or using the Induced fit docking (IFD) protocol within
the Schr o¨ dinger suite.
Compounds 1a, 2a, 4a-10a, 12a-14a,16a, 19a, 20a, 24-26, 30-32,
4-36 were obtained from Sigma Aldrich, Alfa Aesar, TCI or Acros.
Compounds 3a, 15a, 29 and 33 were received as gifts and synthesis is
described by the original authors. Compounds 11a, 17a, 18a, 21a - 23a,
2
7 and 28 have been synthesized according to the literature, with details
given in SI.
2
.1. TsER variant creation and biocatalysts production
TsER variants were created using the megaprimer PCR method as
′
reported before [24] using either TsER_C25D (5 -GTCCCCCA
′
′
TGGACCAGTACTCC-3 ) or TsER_C25G (5 -GTCCCCCATGGGTCAGTA
′
′
CTCC-3 ) as forward primers and TsER_I67T (5 - CATA-
′
′
AGGGCTGGTACGACCCAAAG-3 ) TsER_I67V (5 -CATAAGGGCTCAC
′
′
ACGACCCAAAG-3 ) TsER_I67C (5 -CATAAGGGCTGCAACGACCCA
′
AAG-3 ) as reverse primers. Heterologous expression and purification by
heat treatment was performed as previously reported [24]. Briefly TsER
variants were heterologous expressed in E. coli BL21(DE3)ΔnemA [25]
using pET22b(+) based gene constructs. After heat purification, TsER
2.3. Biotransformations: screening scale
variants were incubated with an excess of FMN for 90 min in the dark at
◦
2
7
2 C, prior to dialysis against 100 mM potassium phosphate buffer (pH
All reactants were mixed in 1.5 mL reaction tubes containing (final
◦
.4) at 4 C. Purity of proteins was confirmed by SDS-PAGE (Fig. S1) and
μ
2
concentrations) 100 mM glucose, 10 M enzyme, 0.1 mM CoCl , 0.25
◦
+
ꢀ 1
enzymes were lyophilized and stored at ꢀ 20 C until further use. The
amount of ER were determined after dissolving the lyophilized powder
before a reaction absorption at 455 nm using the reported extinction
mM NADP , GDH-60 (2.2 U mL ) and 10 mM substrate (from 1 M stock
in EtOH). The reactions were performed at least in triplicates in total
◦
reaction volume of 200
μ
L at 30 C and 700 rpm in a Thermomixer
ꢀ
1
ꢀ 1
coefficient of YqjM (
ε
= 11 600 M cm ) [26].
(Eppendorf). To stop the reaction, the aqueous solution was extracted
with 200 L ethyl acetate. Conversion and enantiomeric excess was
For BsGDH, the gene construct pACYC-gdh (Bacillus subtilis 168
glucose dehydrogenase variant E170K/Q252L) [27] was transformed
into E. coli BL21-Gold DE3. Expression was performed using TB medium
μ
determined by GC or HPLC. Peaks were assigned based on available
authentic standards, by mass analysis or full characterisation. Precise ee
determination for low conversion levels was performed as previously
described [24]. The optical rotation of 2-methylcyclopentan-1-one
(13b) was determined to assign chirality. All reactants were mixed in
a 50 mL reaction containing 45 mL KPi buffer (100 mM, pH 7.4), glucose
ꢀ
1
◦
containing 35 mg L chloramphenicol in baffled flasks at 37 C (200
rpm) until the OD600 reached 0.4ꢀ 0.6 IPTG was added to a final con-
◦
centration of 1 mM and the temperature was reduced to 30 C. The
◦
cultures were incubated overnight, harvested (4480 xg, 4 C, 15 min)
+
ꢀ 1
and resuspended in KPi buffer (100 mM, pH 7.4). Cells were lysed by
sonication on ice (5 × 30 s, 30 s intervals, 5 × 10 cycles, 40 % power,
Bandelin Sonoplus HD 2070 with a SGH 213G booster horn and a tita-
nium flat tip (TT13)) and the supernatant was recovered by centrifu-
(87.5 mM), NADP (0.25 mM), BsGDH (2.2 U mL ) and TsER
C25G/I69T (20 M). Reaction started upon addition of 50 mM 2-meth-
μ
ylcyclopenten-1-one (13a) with 5 mL n-pentane and was performed at
◦
30 C and 200 rpm. After completion the whole reaction mixture was
◦
gation (29 819 xg, 4 C, 45 min). The cleared lysate was purified by
extracted with n-pentane and the solvent was carefully evaporated
◦
◦
ꢀ 1
incubation at 60 C for 1 h. After centrifugation (29 819 xg, 4 C, 45 min)
under vacuum. The optical rotation was measured with a 20 mg mL
◦
the supernatant was lyophilised and stored at ꢀ 20 C. Glucose dehy-
solution in chloroform in a 50 mm cuvette with a polarimeter P800-T,
KRÜSS with λ =589 nm at 25 ◦C and result in [∝]
25
-81 indicating
◦
drogenase (GDH-60) was obtained from Evocatal.
D
that 2R-methylcyclopentanone is formed according to Shimoda et al.
2
.2. Protein crystallization, X-ray structure determination and docking
[35].
analysis
2
.4. Biotransformations: 20–500 mL scale
Heat purified TsER C25D/I67T was concentrated through ultrafil-
tration (30 kDa NMWL, Amicon) with additional FMN added to ensure
full occupancy. The protein was further purified using size exclusion
chromatography [Sephacryl S100HR (GE Healthcare)] and eluted in 10
mM MOPS buffer (pH 7.4) containing 20 mM NaCl. Crystals of TsER
3-Methyl-cyclohexanone (1b): All reactants were mixed in a 100 mL
Schott bottle (50 mL reaction volume) with 45 mL KPi buffer (100 mM,
+
pH 7.4) containing glucose (100 mM), NADP (0.25 mM), BsGDH (2.2 U
ꢀ
1
mL ), 0.1 mM CoCl
2
, TsER variant (10 M), 3-methylcyclohexenone
μ
C25D/I67T were grown using sitting-drop vapour-diffusion in 1
μ
L
(1a, 50 mM) and 10 % (v/v) n-pentane. The reaction was performed
ꢀ 1
◦
drops consisting of equal volumes of 8 mg mL TsER C25D/I67T and
at 30 C and shaking (150 rpm). To monitor conversion, 20
μL samples
crystallization solution (15 % v/v 2-propanol, 0.1 M sodium citrate
were taken from the n-pentane phase and diluted with 200
μ
L n-pentane
2

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
for analysing via GC. Upon complete conversion, the whole reaction
mixture was extracted three times with 20 mL DEE. The combined
culture. We found that shock-frozen catalyst solutions maintained their
activity when stored at ꢀ 20 ◦C for over a year, with only repetitive
thawing and freezing having negative effects. To overcome this limita-
tion, we produced freeze-dried powders, which offer advantages upon
storage and handling.
4
organic phases were washed with brine, dried over MgSO and the
solvent was carefully evaporated under vacuum. Compound S-1b was
further purified using flash column chromatography (n-pentane/EtOAc
2
5:1).
R)-Levodione (8b): All reactants were mixed in a 100 mL Erlen-
meyer flask (20 mL reaction volume) containing KPi buffer (100 mM, pH
(
3.1. Substrate scope
+
ꢀ 1
7
.4): glucose (500 mM), NADP (0.41 mM), BsGDH (2.2 U mL ), TsER
To expand on our earlier work uncovering the stereo-
complementarity of variants C25D_I67T and C25G of TsER [24], we
decided to further explore the active site architecture by modifying
residue I67. A panel of TsER variants was created by mutating I67 to
either a Val, Thr or Cys into variants C25D and C25G. The engineered
TsER variants and the wild-type enzyme (wt) were tested against a total
of 38 electron-poor alkenes, which included 3-methylcylcohexenone
(1a), (S)-carvone (9a) and methyl-2-(hydroxymethyl)-acrylate (17a)
previously reported on [24]. This benchmarking set of substrates con-
C25D/I67T (10
μ
M), ketoisophorone (8a, 125 mM) and 10 % (v/v)
◦
diisopropylether (DIPE). The reaction was performed at 30 C and
shaking (150 rpm). 100
L EtOAc for analysing via GC. After completion (91 % conversion) the
whole reaction mixture was extracted twice with 20 mL EtOAc. The
μ
L samples were taken and extracted with 100
μ
combined organic phases were washed with brine, dried over MgSO
and the solvent was evaporated under vacuum.
4
2
-Methyl-5-(prop-1-en-2-yl)cyclohexan-1-one (9b): All reactants
were mixed in a 300 mL Erlenmeyer flask (100 mL reaction volume)
tained cyclohexenones and cyclopentenones,
α,β-unsaturated linear
+
containing KPi buffer (100 mM, pH 7.4), glucose (82.8 mM), NADP (2
alkenones and alkenals, acrylesters, nitroalkenes, a maleimide, cit-
ronellylnitrile, two alkynals and an alkynone (Schemes 1 and S1).
A total number of 23 compounds were reduced at the C–
ꢀ
1
mM), GDH-60 (2.2 U mL ), TsER variant (3.4
μ
M), carvone (9a, 69
◦
mM). The reaction was performed at 30 C with shaking (110 rpm). 100
–
C bond
μ
L samples were taken and extracted with 200
μ
L EtOAc for analysing
(Scheme 1), introducing new stereocenters with high stereoselectivity
(Table 1). Access to both stereoisomers was observed for 11 substrates
(1a-4a, 6-7a, 9a, 14-15a, 17a, 22a) with stereoselectivities ranging
from 17 to >99 % ee. This represents a broad control over the facial
selectivity for a single set of ER variants. In eight additional cases (8a,
10-13a, 18-19a, 21a) high chiral purity (88 to >99 % ee) for one ste-
reoisomer was obtained. Comparable conversions were observed with
the wt as previously reported [2], but the differences in reaction con-
ditions lead to a 40 % increase in citral reduction. Also, the use of 10 %
v/v organic solvents suppressed background racemisation of stereo-
centers, especially for 13b. The stereochemical outcome of 13a reduc-
tion using TsER, previously reported to be S [15,36,37], was
unambiguously corrected to be R by optical rotation.
via GC. After completion the whole reaction mixture was extracted twice
with 100 mL EtOAc. The combined organic phases were washed with
brine, dried over MgSO
-Methyl-5-(prop-1-en-2-yl)cyclohexan-1-one (9b): All reactants
were mixed in a 500 mL Schott bottle (500 mL reaction volume) with
50 mL KPi buffer (100 mM, pH 7.4) containing glucose (100 mM),
4
and the solvent was evaporated under vacuum.
2
4
+
ꢀ 1
NADP (0.25 mM), BsGDH (2.2 U mL ), 0.1 mM CoCl
2
, TsER variant (5
μ
M), 9a (51.2 mM) and 10 % (v/v) n-pentane. The reaction was per-
◦
formed at 30 C with shaking 150 rpm. 20
the n-pentane phase and diluted with 200
μ
L samples were taken from
μ
L n-pentane for analysing via
GC. After completion the whole reaction mixture was extracted three
times with 100 mL DEE. The combined organic phases were washed
with brine, dried over MgSO
vacuum.
4
and the solvent was evaporated under
In addition, excellent conversions up to >99 % were achieved under
screening conditions (Table 1). Originally, these mutations were evolved
for cyclohexanone derivatives [24,38], therefore it is particularly
interesting that structures such as 17-19a as well as derivatives of cin-
namic acid (20-23a) are accepted with excellent conversions. These
compounds are valuable building blocks of industrial synthons,
fragrance and flavour substances. Besides typical ER substrates, several
new compounds for ERs were tested of which three were transformed
(4b, 16b, 21b). We noted that kinetic resolution of 4a occurred and
produced a compound with >99:1 dr. C25D based variants consume the
opposite enantiomer of 4a than C25G based variants. Nevertheless, due
to the C2-symmetry of 4b, the same meso isomer is formed (Scheme S2).
Reduction of compound 16a creates two new stereocenters with >99:1
dr.
Ethyl 2-benzyl-3-oxobutanoate (21b): All reactants were mixed in a
1
00 mL Erlenmeyer flask (20 mL reaction volume) in KPi buffer (100
+
mM, pH 7.4): glucose (500 mM), NADP (0.41 mM), GDH-60 (2.2 U
ꢀ 1
mL ), TsER C25D/I67T (10 M), ethyl (Z)-2-benzylidene-3-oxobuta-
μ
noate (21a, 50 mM) in 10 % (v/v) diisopropylether (DIPE). The reaction
◦
was performed at 30 C and 100 rpm. 200
μ
L samples were taken and
extracted with 200
conversion) the whole reaction mixture was extracted twice with 20
mL EtOAc. The combined organic phases were washed with brine, dried
over MgSO and the solvent was evaporated under vacuum. The product
was cleaned via column chromatography (n-pentane/EtOAc 10:1).
μL EtOAc for analysing via GC. After completion (81
%
4
2
.5. Sequential biphasic batch reaction
3
.2. Structural characterization
All reactants were mixed in a 50 mL reaction tube with 45 mL KPi
+
buffer (200 mM, pH 7.4) containing NADP (0.27 mM), C25D/I67T (10
One of the most promising stereocomplementary variants (C25D/
ꢀ 1
μ
M), glucose (100 mM) and BsGDH (2.2 U mL ). 5 mL n-pentane was
I67T) was selected for structural investigation. No significant differences
could be observed in the main chain conformation in the active site as
compared with the wt structure (Fig. S2). The side chain of D25 adopts a
conformation that is rotated away from the oxidized FMN and increases
marginally the active site volume together with the I67T replacement.
Numerous attempts however to co-crystallize C25D/I67T with different
substrates to evaluate binding orientations were unsuccessful. We
therefore turned to in silico experiments and analysed the interactions
for 1a with the wt and C25D/I67T variant using both rigid body docking
added containing 0.5 M 8a, to yield a final concentration of 50 mM 8a
◦
within the biphasic system (50 mL). The reaction was incubated at 30 C
and 110 rpm. The reaction was monitored by GC. When the reaction
completed or stopped, phases were separated by centrifugation (4000
◦
rpm, 15 C) and a fresh amount of glucose and organic solvent con-
taining 8a was added.
3
. Results and discussion
(
RBD) and induced-fit docking (IFD). 1a was selected as it not only
All enzyme variants reported in this study are easily separated from
showed a reversal of facial selection, but also a dramatic increase in
activity [24]. The structures were prepared with a reduced FMN cofactor
to mimic the active state and the potential substrate anchors (H172 and
H175) were fully protonated, to enable hydrogen bonding. Fig. 1 shows
◦
E. coli host proteins by heat treatment for 90 min at 70 C [24,36].
Enzyme preparations were reconstituted with FMN followed by dialysis,
yielding approximately 110ꢀ 130 mg purified enzyme per litre of E. coli
3

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
Scheme 1. Substrate scope of the TsER panel. rac: racemic; s.m.: starting material.
an overlay of the best RBD and IFD pose for the wt (A) and C25D/I67T
correct orientation for proton transfer, as shown in the work of Lonsdale
◦
(
B). Binding poses were considered as productive when the hydride
et al. [41] and the hydride attack angle has improved from 81.4 to
◦
◦
transfer angle ranges from 80 to 120 and the distance (H to Cβ) shorter
than 4.85 Å [19,39]. The hydride attack angles and distances improve in
both cases with IFD. Now, productive poses in the wt unambiguously
predict the experimentally observed S-selectivity (76 % ee) and
R-selectivity (93 % ee) for the C25D/I67T variant, due to a “flipped”
binding mode (Tables S2 and S3). The improved predictions from IFD
originate from a side chain flip of residue 67 and rearrangements of side
93.8 . We predict that the more hydrophobic pocket created in the C25G
based mutants, allows sufficient space and van-der Waals interactions to
host smaller substituents on the Cβ, allowing “normal” binding poses to
occur, to give the experimentally determined S-selectivity.
3
.3. Physicochemical characterization
◦
chains Y27, H172 and Y177. The imidazole ring of H172 rotates by 90
Encouraged by our initial screening, we investigated the physico-
in the wt, an effect that was also observed in crystallo for OYE1-W116I
upon substrate binding [40]. Notably, in C25D/I67T, the phenolic
proton of Y177 changes position in the IFD structure, adopting the
chemical limits of our reaction system. The reaction system consists of
two enzymes, the ER and a NADPH recycling enzyme (glucose dehy-
drogenase, GDH) supplying the consumed hydride from glucose.
4

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
Table 1
Substrate scope and stereoselectivities of the TsER variant panel.
wt
C25D/I67C
conv/%
ee/%
C25D/I67T
conv/%
ee/%
C25D/I67V
conv/%
ee/%
C25G
C25G/I67C
conv/%
ee/%
C25G/I67T
conv/%
ee/%
C25G/I67V
conv/%
ee/%
Product
conv/%
ee/%
conv/%
ee/%
a
1
63
95
98
6
6
49
97
1
2
3
b
7
6S
94R
22
93R
29
92R
32
>99S
1
57S
6
74S
36
62S
34
n.c
b*
b*
9
9R
99R
>99
>99R
51
99R
>99
>99R
33
n.d.
>99
>99S
10
99S
>99
>99S
4
99S
>99
88S
53
99S
>99
>99S
21
>
>
1
99
99R
>99
>99R
46
4
5
6
b
rac
99
88
>99
99
>99
99
>99
99
>99
85
n.d.
99
>99
99
>99
99
b*
*
,b
68
84
74
38
8
25
6
b
8
3(2)
49(2)
>99
13S
27 (2)
>99
5R
64 (2)
>99
5R
57(2)
>99
72R
72
24(1)
>99
18S
25
17(1)
>99
14S
88
17(1)
>99
24S
71
>
99
7
8
b*
9
3R
>99
0R
99
91
c
>99
92R
>99
92
>99
92R
>99
92
>99
92R
>99
93
b
9
86R
86
91R
87
88R
87
86R
90
>
d
89
91
88
86
9
1
b
(
2R,5S)
(2R,5S)
cis
(2R,5S)
cis
(2R,5S)
cis
(2R,5S)
cis
(2S,5S)
trans
86
(2S,5S)
trans
98
(2S,5S)
trans
58
cis
>
99
>99
96
>99
96
>99
96
97
d
96
98
95
98
99
0b
(
2R,5R)
(2R,5R)
trans
>99
>99R
99
(2R,5R)
trans
>99
>99R
>99
>99R
91
(2R,5R)
trans
(2R,5R)
trans
38
(2R,5R)
trans
25
(2R,5R)
trans
97
(2R,5R)
trans
34
trans
d
>
99
>99
1
1b*
2b*
>
99R
>99R
99
>99R
>99
>99R
90
>99R
>99
>99R
>99
84R
96R
>99
>99R
99
54R
>99
>99R
96
9
7
1
>
99R
>99R
99
>99R
99
9
9
1
3b*
79R
66R
57R
69R
87R
81R
85R
e
e
e
e
e
e
e
e
9
1
9
6R
71R
88R
88R
88R
82R
80R
84R
4
8
9
10
nc
nc
nc
nc
1
1
1
1
1
1
2
4b
4S
47R
>99
>99R
7
57R
>99
>99R
2
37R
>99
>99R
17
>
>
2
99
>99
87S
<1
>99
96S
<1
n.d.
2
>99
90S
2
>99
99S
1
5b*
6b
99R
n.d.
n.d.
96
n.d.
98
n.d.
83
n.d.
nc
n.d.
11
n.d.
nc
9
7
9
4
6
9
9
4
7b*
8b
6R
4
>99S
35
97S
51
>99S
27
nd
79R
37
7
12
36
6S
0
26R
52
5R
47R
81
51R
31
99R
5
92R
49
>99R
14
70
9b
9S
2
99S
85
99S
47
99S
57
87S
97
86S
96
94S
94
88S
98
0b
rac
rac
58
rac
43
rac
rac
38
rac
45
rac
48
rac
37
4
0
60
e
e
e
e
e
e
e
e
2
1b*
91
88
88
96
39
42
90
37
>
99
>99
27
>99
28
>99
33
>99
62
>99
51
>99
64
>99
63
1
3
2
2
2b
3b
>
4
99S
>99S
1
>99S
4
>99S
10
>99R
1
>99R
4
>99R
3
>99R
7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
All reactions were done in triplicates; standard deviation for conversion is ± 5 %. The reaction was stopped after 24 h unless otherwise specified. The numbers in
parenthesis for 6b indicate the major diastereomer based on retention times.
a
5
2
h.
h.
b
c
2
.5 h.
0 min.
0 % (v/v) MTBE.
d
e
*
9
1
Confirmed by GC/MS analysis; s.m. = starting material; rac. = racemic; nc = no conversion, nd = not determined. Control reactions without ER showed no
–
–
O bonds.
background reduction of C
–
C or C
–
First, we investigated the effect of pH on our reaction system (Fig. 2).
C25D/I67T is less affected by a change in pH and still shows 8b-pro-
duction at pH 10. Remarkably, it is still 40 % active at a pH of 5,
We found that up to 200 mM potassium phosphate buffer can be used,
especially when aiming for higher substrate loadings (>100 mM, vide
infra). An alternative would be process engineering to compensate for
the pH decrease.
+
especially considering that NADPH/NADP rapidly degrades below pH
7
[42]. The glucose based NADPH recycling irreversibly produces glu-
Enzymes with high thermal stability are of particular interest to
biotechnology as increased stability and life-times enables more flexi-
bility in process design. Before studying the impact of temperature on
our reaction system, we assessed both the ERs and GDHs individually.
The commercially available recycling system (GDH-60, Evocatal) was
conic acid as side product, leading to acidification of the reaction [43].
Our 100 mM buffer capacity is ten-times higher than the amount of
possible acid equivalents under screening conditions, and ensures a
constant pH, even when considering uncoupling through side reactions.
5

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
Fig. 1. Comparison of RBD (cyan) and IFD (grey) docking of 1a in TsER wt (A) and TsER C25D/I67T (B).
thermolabile cofactors are used, increased reaction temperatures may
negatively impact yields for reaction times longer than a few hours,
despite the benefits of increased reaction rate and enhanced solubility of
organic molecules at higher temperatures.
R-levodione racemizes in buffered aqueous solution approximately 3
%
ee per hour at ambient temperatures [47,48]. We expected the effect
to be more dominant at increased temperature and found that incuba-
◦
tion of R-8b at 65 C and above for 1 h results in a loss of enantiopurity
yielding an ee of 8 %. This temperature accelerated racemization is
observed when levodione is incubated in buffer, in the presence of the
recycling system or in presence of a TsER variant. When all components
◦
of the reaction system are present, R-8b is produced at 70 C with an ee
of 37 % (Fig. 3B). Thus, an increased reaction temperature seems only
beneficial for non-racemizable or achiral compounds, but the panel is
robust enough to tolerate exposure to elevated temperatures.
Fig. 2. Effect of pH on productivity. pH profile of 8b-production with C25D/
I67T or C25G/I67T and GDH-60.
◦
found to be active up to 50 C (Table S6). Thus, we switched to an
3
.4. Organic solvent tolerance
engineered GDH from Bacillus subtilis (BsGDH E170 K/Q252 L) [27,44],
◦
which still shows 60 % specific activity at 70 C under our reaction
We also investigated the effect of organic solvents as a process
conditions.
parameter on the bioreduction. Low water-solubility of most ER sub-
strates represent a considerable challenge, often resulting in low sub-
strate loadings and significant amounts of waste water [1]. Addition of
organic co-solvents and use of biphasic reaction systems therefore offer
significant advantages, often increasing the biocatalyst performance in
synthetic chemistry. We found that water-miscible solvents are tolerated
up to 10 % (v/v) without loss of productivity (Fig. 4A,B). Polar solvents
like C1 to C3 alcohols, DMSO or acetonitrile, common co-solvents in
biotransformation, reduce productivity at 20 % v/v and above. Other
ERs have likewise shown a similar intolerance against such co-solvents
Thermal inactivation of variant C25G/I67T and C25D/I67T was
assessed by incubating purified enzymes in buffer at temperatures be-
◦
◦
tween 4 and 70 C for 14.5 h prior to a reaction at 30 C (Fig. 3A). Both
TsER variants retain full activity up to an incubation temperature of 60
◦
◦
C. At 70 C incubation, C25D/I67T lost 3 % and C25G/I67T 14 % of its
initial activity. This thermal stability is comparable to that of the wt
[
45]. Notable is the activity increase of C25G/I67T after pre-incubation
◦
at room temperature (22 C) and above, while the activation effect
might be masked for C25D/I67T, since full conversion was reached.
After ensuring thermal stability of both enzymes, the reaction system
was tested for levodione (8b) production at reaction temperatures be-
[
11,49,50], but no clear trend is apparent. In contrast,
water-immiscible solvents are tolerated without loss of productivity up
to a phase ratio of 1:4 (20 % v/v of total reaction volume). In general, a
small volumetric amount of organic solvents (5–10 % v/v) already
increased 21b productivity up to 2-fold, enabling full conversion of this
poorly water-soluble substrate.
◦
◦
tween 30 C and 75 C. Formation of 8b completes in 150 min under
screening conditions. Increasing the temperature accelerated the reac-
tion, now reaching full conversion in less than 20 min (C25D/I67T) or
◦
4
0 min (C25G/I67T) at 55 C (Fig. 3C,D). Productivity was not affected
◦
◦
up to 65 C, while both variants start losing activity above 65 C. In
Addition of n-pentane, MTBE, diisopropylether (DIPE) or toluene
proved to be particularly effective. Due to the low water miscibility of n-
◦
general, working above 75 C reduced the lifetime of the reaction system
below 20 min. Not only the enzymes, but also the nicotinamide cofactor
ꢀ 1
pentane (39 mg L ), the enzymes stayed active in all tested phase ratios
+
(
NADPH/NADP ) is labile at higher temperatures. The half-life of dis-
(
max. 2:3 n-pentane: buffer) and recovery of a clean product by simple
◦
solved NADPH at 70 C and above is less than 10 min [46]. Therefore, it
is impossible to distinguish between cofactor or enzyme degradation as
source for lost activity at high temperatures. Consequently, as long as
phase separation was possible. Our findings that immiscible solvents are
more compatible and enhance conversion are in-line with observations
made for other wild-type ERs [49–51].
6

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
◦
Fig. 3. Thermostability assessment of the reaction system. A) Residual activity at 30 C of TsER C25G/I67T and C25D/I67T after 14.5 h incubation at different
temperatures. B) Effect of temperature on the final enantiopurity of 8b. Values are obtained with three different enzyme batches, ±0.8 % s.d. of ee. C and D)
◦
Temperature-time dependent formation of 8b using the engineered BsGDH and C25G/I67T (C) and C25D/I67T (D) at reaction temperatures between 30 and 75 C.
Fig. 4. Effect of organic solvents on TsER variants C25G/I67T (A) and C25D/I67T (B). The total reaction volume was kept constant; percentages correspond to phase
ratios of 1:19, 1:9, 1:4 and 2:3 in biphasic systems.
7

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
Table 2
Preparative scale bioreduction using TsER variants and engineered BsGDH.
a)
b)
c)
d)
ꢀ 1
Product
TsER
V
(mL)
C (mM)
Cat. Loading (mol%)
T
(h)
Conv.(%GC)
er/dr
Isolated Yield (%)
TON
TOF (h )
e)
h)
1
1
8
9
9
9
2
b
b
b
b
b
b
C25G/I67T
C25D/I67T
C25D/I67T
C25G/I67T
C25D/I67T
C25D/I67T
C25D/I67T
50
50
20
50
50
125
69
69
51
50
10
10
10
3.4
3.4
μ
μ
μ
M (0.02)
M (0.02)
M (0.008)
8.0
9.0
1.5
4.5
6.8
7.0
2.0
78
76:24 S
>99:1 R
99:1 R
72 (0.2 g)
h)
3,900
1,986
3,911
7,583
5,958
4,928
40,634
2,025
e)
f)
i)
99
91 (0.26 g)
4,970
91
65 (0.25 g)
90 (0.94 g)
78 (0.83 g)
11,375
20,294
20,294
10,189
4,050
g)
100
100
500
20
μ
M (0.005)
M (0.005)
>99
>99
>99
81
94:6 trans
i)
g)
μ
98:2 cis
e)
g)
h)
5
μM (0.01)
98:2 cis
93 (3.6 g)
f i)
1b ,
10
μM (0.02)
>99:1
76 (0.17 g)
a)
b)
c)
d)
Volume: Total reaction including both phases.
Time until completion of reaction.
Turnover numbers (TONs) are defined as mol product (as per GC conversion) formed per mol enzyme upon completion of the reaction.
Turnover frequency (TOF) is defined as the mol product (as per GC conversion) formed per mol enzyme per hour. TOFs were determined after 30 min, with the
exception of 8b and 21b, calculated after 1.5 and 2 h, respectively (time of completion of reaction).
e)
9
:1 buffer: n-pentane.
f)
9
:1 buffer: DIPE.
g)
h)
i)
Starting material contains 2 % enantiomer.
Extraction with diethyl ether increased yield.
Performed with GDH-60 (Evocatal) without second phase.
3
.5. Preparative scale reactions
To ultimately prove the high potential of the panel, we performed
bioreductions at a preparative scale of between 20–500 mL with selected
◦
variants for compounds 1a, 8a, 9a and 21a at 30 C (Table 1). Scale-up
successfully reproduced conversion and selectivity values observed
during screening experiments. We were also able to reduce the amount
of glucose from a 10-fold excess to 1.75 molar equivalents. To minimize
2
side reactions, we also limited the available O by reactor design (closed
bottles with varying head space). The use of a bi-phasic reaction system
increased the enantiopurity of 8b. The beneficial effect of a biphasic
system on enantiopurity has been previously described for compounds
prone to racemization, such as 13b and 8b [47,52].
Excellent yields of up to 93 % were achieved, demonstrating that our
reaction system is easily scaled to gram quantities. Substrate loadings up
ꢀ 1
to 125 mM (19 g L ) are demonstrated. Catalyst loadings were 100-fold
reduced from 0.1 mol%, used during screening and our previous reports
on TsER [24], to 0.02ꢀ 0.008 mol% resulting in excellent TONs (3,900 to
2
0,294) with turnover frequencies (TOF) between 1,986 and 40,634
ꢀ 1
h
. All reactions were complete in less than 9 h, whereas other pre-
Fig. 5. Consecutive biphasic batch reactions to evaluate reusability of TsER
C25D/I67T for conversion of 8a. Four consecutive batches in 50 mL volume
parative scale bioreductions with ERs are reported to run between
0ꢀ 96 h [9–11] or at 10–40 times higher catalyst loadings [53]. A 500
mL scale bioreduction of 9a resulted in an isolated yield of 93 % of 2R,
S-9b (3.63 g). Overall, these examples demonstrate the efficiency of
our TsER variant panel for organic synthesis with space time yields
2
(
1:9, n-pentane:buffer) were performed.
5
transfer hydrogenation of a wide range of substrates. The increased
substrate scope, broad stereocomplementarity and high enantiose-
lectivities of the panel complements the already established reactions
with wild type TsER [24,36,45].
ꢀ 1
ꢀ 1
(
STYs) ranging between 0.5–8.3 g L
h (Table 2).
3
.6. Sequential biphasic batch reaction
Our reaction system, consisting of two engineered enzymes, is
convenient to prepare and handle and also allows for long term storage
As biphasic systems enables enzyme reusability, we performed
ꢀ 1
as freeze-dried powders. TOF of up to 40,000 h , which are comparable
to hydrogenation rates by heterogeneous and homogeneous transition
metal catalysts were demonstrated at preparative scale. The observed
tolerance to organic solvents not only allows working at high substrate
loadings, particularly for poorly water-soluble substrates, but also sim-
plifies product recovery and decreases racemization. Non-miscible sol-
vents also allows for sequential batch reactions, whereby product can be
removed and the reaction repeated using the same enzymes.
The high activity, robustness and stereocomplementarity demon-
strated by the TsER variant collection, provides a base to further exploit
these enzymes as a tool in preparative organic synthesis, especially for
cascade and chemo-enzymatic one-pot reactions [54,55]. Moreover, the
use of variants of the same ER would reduce efforts during reaction
optimization.
consecutive batch experiments in which the organic phase containing
the substrate (0.5 M 8b in 5 mL n-pentane) was repeatedly replaced after
each transformation. This allowed consecutive substrate loadings of 50
mM 8b to the biphasic system (50 mL) while leaving the enzyme and
recycling system in the aqueous phase (45 mL). We achieved four cycles
(
Fig. 5), with the first two cycles at 90 min each, cycle 3 at 180 min and
ꢀ 1
the final cycle at 205 min. Relative to the aqueous phase, ca. 27 g L 8b
were produced before the reactions stopped, with a TTN of more than 17
5
00. We identified the build-up of stoichiometric amounts of gluconate
and a pH drop to 5.2 as cause, also for the reduced productivity over the
later batch cycles.
4
. Conclusions
We present here a robust panel of TsER variants for asymmetric
8

N. Nett et al.
Molecular Catalysis 502 (2021) 111404
Credit author statement
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This work was supported by the LOEWE Research Cluster Syn-
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Declaration of Competing Interest
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We thank Manfred T. Reetz for access to instruments, Val ´e rie Alezra
and Timo V o¨ lker for providing compounds 29 and 33, Monika Ballmann,
Alexandra Richter, Saskia D o¨ hring and the mass department for excel-
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2021.111404.
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