Novel Old Yellow Enzyme Subclasses

Article abstract of DOI:10.1002/cbic.201800770

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal- and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic-like group, their substrate scope more closely resembles the latter subclass.

Full text of DOI:10.1002/cbic.201800770

Accepted Article
Title: Novel Old Yellow Enzyme Subclasses
Authors: Christin Peters, David Frasson, Martin Sievers, and Rebecca
Maria Ursula Buller
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ChemBioChem
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Novel Old Yellow Enzyme Subclasses
Christin Peters, David Frasson, Martin Sievers and Rebecca Buller*[a]
[
a]
[b]
[b]
Abstract: Many drug candidate molecules contain at least one chiral
centre and consequently, the development of biocatalytic strategies
to complement existing metal- and organocatalytic approaches is of
high interest. However, time is a critical factor in chemical process
development and thus, the introduction of biocatalytic steps, even if
more suitable, is often prevented by the limited availability of off-the-
shelf enzyme libraries. To expand the biocatalytic toolbox with
additional ene reductases, we screened 19 bacterial strains for
double bond reduction activity using the model substrates
cyclohexanone and carvone. Overall, we identified 47 genes coding
for putative ene reductases. Bioinformatic analysis of all genes and
the biochemical characterization of four representative novel ene
reductases led us to propose the existence of two new Old Yellow
Enzyme subclasses, which we named OYE class III and class IV.
Our results demonstrate that while on a DNA level each new OYE
respect to their industrial potential.[6] Members of the Old Yellow
Enzyme family are NAD(P)H-dependent oxidoreductases which
catalyze the stereo- and enantioselective reduction of α,β-
unsaturated ketones, aldehydes, nitro alkenes, and carboxylic
acids.[1a] The first enzyme of this family was discovered by
Christian and Warburg in bottom-fermented yeast who named it
“yellow enzyme”.[7] Subsequently, several other yellow enzymes
were found, and the first ene reductase was redefined as the
“old yellow enzyme”(OYE1).[8] In many cases the enzymes are
important for the detoxification of electrophilic substances, in
some cases like enzyme YqjM from Bacillus subtilis they play an
important role in response to the oxidative stress.[9] Nevertheless,
for most enzymes of the OYE family the natural substrates and
physiological roles remain unknown.
subclass features
a distinct combination of sequence motifs
previously known from the classical and the thermophilic-like group,
their substrate scope more closely resembles the latter subclass.
Introduction
Members of the enzyme families collectively called ene
reductases (ER) are effective biocatalysts for stereo-selective
trans-hydrogenation[1] and - in rare cases - cis-hydrogenation[2]
of activated alkenes complementing the respective chemical cis-
hydrogenations via chiral rhodium or ruthenium phosphines
(
Knowles and Noyori, Nobel prize in chemistry 2001).[3]
Recently, the canonical function of ene reductases was further
expanded via enzyme engineering to now also include
asymmetric
cyclopropanes.
reductive
carbocyclization
yielding
chiral
Figure 1. Schematic representation of an asymmetric double bond reduction
by Old Yellow Enzymes and an overview of the different ene reductase
families.
[4]
Today, members of the ene reductase family are assigned to
five enzyme subfamilies.[1b] Most biocatalytic transformations are
carried out by members of the Old Yellow Enzyme family of ene
reductases. However, the Fe-S cluster depending enoate
reductases, the medium-chain dehydrogenases, the short-chain
The catalytic mechanism, which all enzymes of the OYE family
follow, is well understood.[10] The enzymes use a bi-bi ping-pong
mechanism which can be divided into the reductive half-reaction,
where the FMN is reduced through hydride transfer from
NAD(P)H, and in the oxidative half-reaction, where the hydride is
transferred from the reduced flavin to the Cβ-atom of the
activated alkene. To complete the reaction the missing proton
for the Cα is provided by a tyrosine residue.[10-11] This specific
mechanism leads to an anti-addition hydrogenation.[12] To date,
the enzymes of OYE family have been divided into two main
subclasses, called “classical OYEs” and “thermophilic-like
OYEs”. All known enzymes of the OYE family have a typical
TIM-barrel as basic structure,[13] however the two classes can be
distinguished by sequence homology and structural features.
The more recently identified thermophilic-like OYE (Class II) was
introduced 2010 by the group of N. Scrutton.[1a, 14] The main
difference to the classical OYEs is that the thermophilic like
OYEs form a functional dimer-dimer interface. An arginine at the
dehydrogenases and the recently identified quinone reductase-
like ene reductases[5] (Figure 1) are also being investigated with
[
[
a]
b]
Dr. C. Peters, Prof. Dr. R. Buller
Competence Center for Biocatalysis, Institute of Chemistry and
Biotechnology, School of Life Sciences and Facility Management
Zurich University of Applied Sciences
Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland
E-mail: Rebecca.Buller@zhaw.ch
D. Frasson, Prof. Dr. M. Sievers
Molecular Biology, Institute of Chemistry and Biotechnology, School
of Life Sciences and Facility Management
Zurich University of Applied Sciences
Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland
Supporting information for this article is given via a link at the end of
the document.
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ChemBioChem
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C-terminus of class II OYEs assist in substrate binding of the
adjacent monomer[15] leading to the formation of dimers whereas
the classical OYEs are found as monomers or dimers.[16]
stereopreference can be changed in some cases by point
mutations making available stereocomplementary pairs of ene
reductases,[5, 27] however, more catalysts of complementing
enantioselectivity are needed. In addition, the industrial utility of
enzymes is also defined by factors such as enzyme availability,
substrate scope, productivity, enzyme stability under process
conditions and ultimately enzyme cost.[6]
The classical OYE subgroup contains yeasts enzyme such as
OYE1 from Saccaromyces carlbergensis[7] as well as variants
from proteobacteria (NCR[17], MR[18], PETNR[19]), flavobacteria
(
Chr-OYE2[20] and plants (LeOPR1-3[21]). Recently, it was
)
proposed to further subdivide the classical OYEs into two
groups: classical OYEs from plants and bacteria and classical
OYEs from fungal sources.[22] In addition, a new OYE subclass
containing fungal enzymes was proposed by the group of Verma
based on the phylogenetic analysis of a range of fungal
strains.[23]
Thus, to enhance the competitiveness of ene reductase
catalyzed reactions we set out to expand the biocatalytic toolbox.
By screening a collection of selected bacterial strains with two
widely accepted model substrates, we identified strains
harboring enzymes capable of double bond reductions. Using a
bioinformatic approach, more than 40 genes encoding putative
new ene reductases were sourced. Further phylogenetic and
biochemical analysis led us to propose two new subclasses in
the OYE family. We provide details of this study including our
analysis of sequence similarities between the four OYE
subclasses, the biochemical characterization of representative
members of the novel groups and an assessment of their
substrate scope and scalability.
Applications of ene reductases are manifold and especially Old
Yellow Enzymes have been investigated for their synthetic
potential. They have been employed, for example, to produce
intermediates (R)-levodione for the synthesis of carotenoids[24] or
methyl (S)-2-bromobutanoate which is used for the synthesis of
drugs to treat noninsulin-dependent type-2 diabetes mellitus.[25]
A remaining challenge for the industrial use of the OYE enzyme
family is that their natural stereospecificity is nearly always the
same.[26] Initial engineering examples show that the
Results and Discussion
Identification of novel Old Yellow Enzymes
Rebecca Buller (née Blomberg)
obtained her Ph.D. (2011) from ETH
Zurich, Switzerland (Prof. Dr. D.
Given the broad applicability of ene reductases for synthetic
purposes, we set out to identify new members belonging to
these enzyme families from microbial sources. In total, 19
bacterial wild type strains from the Culture Collection of
Switzerland were screened for their ability to reduce the model
substrates carvone (1) and 2-cyclohexen-1-one (2). As positive
controls Bacillus subtilis and Gluconobacter were used, which
Hilvert) before she accepted
position as laboratory head at
Firmenich, flavor and fragrance
company. Since 2015, she is
Professor for Chemical and
a
a
are known to harbor ene reductases YqjM[9, 28] and GOX[29]
respectively.
,
Biotechnological Methods, Systems
and Processes at the Zurich
Nine of the screened strains exhibited activity against both
substrates while six strains were found to be active on only one
of the substrates (Table S1). Using the published genome data
available for 14 of the positive strains, we searched for the
responsible ene reductases using a reported fingerprint motif.[
This allowed us to identify 47 genes with putative ER activity.
Phylogenetic analysis revealed that 22 genes (47%) encoded
members of the Old Yellow Enzyme family. As this ER subclass
figures most prominently in practical applications, we then
concentrated on the characterization of this sub-set of genes. A
closer phylogenetic analysis of these 22 sequences revealed
that six genes belong to the classic OYE class and six are
member of the thermophilic like class (Figure 2). Interestingly,
ten of the putative ER genes cluster apart of the two described
OYE branches (classical and thermophilic) and build up two
distinct new branches in the OYE family phylogenetic tree
together with enzymes previously assigned as ‘lost proteins’.[
This finding is in line with the domain family hierarchy of the
University of Applied Sciences and
leads the Swiss Competence Center for Biocatalysis (CCBIO). Her research
interests include the expansion of the biocatalytic toolbox by sourcing and
engineering enzymes for synthetic applications.
30]
Christin Peters (born 1987)
studied biochemistry at the
University of Greifswald and
obtained her PhD from University
Greifswald (U. T. Bornscheuer)
with focus on the establishment
of enzyme cascades using ene
reductases
in
whole
cell
biocatalysis. Then she joined the
Biocatalysis group of Prof. Buller
22]
at the Zürich University of Applied
Science as a postdoc. Since August 2018, she is leading the Biosystems
Technology group at the Zürich University of Applied Science which
concentrates on enzyme cascade reactions and process optimisation of
biotransformations in large scale.
OYE-like FMN family as classified by NCBI’s Conserved Domain
Database (CDD)[31] which includes several subfamilies beyond
class
I and class II enzymes. Using this databank, the
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ChemBioChem
10.1002/cbic.201800770
FULL PAPER
sequences of the here-proposed “class III” and “class IV”
Sequence Analysis
enzymes
OYE_like_4_FMN”
uniquely
cluster
and
into
“cd04747
subfamily
“cd04735
On the sequence level, the proposed new OYE subclasses are
characterized by a combination of sequence motifs from the
previously described classical and thermophilic groups (Table 1
and Figure S1). Especially in context of the FMN binding
environment strictly conserved sequence motifs have been
reported for class I and II. These interactions between the
protein and the flavin are important as they control the redox
potential of the cofactor. Our alignments highlight that in all four
OYE classes a glutamine residue (Gln 102 in YqjM) and an
arginine residue (Arg 215 in YqjM) known to interact with the
pyrimidine ring of the cofactor FMN[15] are strictly conserved.
OYE_like_5_FMN,
respectively. Supported by the CDD classification and based on
our phylogenetic analysis and the biocatalytic, structural and
sequence characterization of the newly discovered ERs we thus
suggest an extension of the current OYE classification to
henceforth include also class III and IV. Our phylogenetic
analysis also gives a first indication that the reported ene
reductase Nox[32] and the newly detected Sma-ER1 may build up
a
putative subclass
V together with a set of previously
[23]
discovered fungal enzymes.
Figure 2. The phylogenetic tree illustrates the integration of the putative novel OYEs (underlined) into the existing OYE family. The maximum likelihood distance
[33]
[34]
tree was constructed using Mega 7. The corresponding alignment was produced via Clustal W.
table S4.
The GenBank codes of the used sequences are deposited in
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In addition to that class II and class IV enzymes possess a
conserved histidine pair (His 167, His 164 in YqjM), which in
YqjM form hydrogen bonds to the N1 and N3 atom of flavin.[15]
Class I and class III enzymes, on the other hand, are commonly
characterized by a replacement of the second histidine with an
asparagine, for example Asn195 in OYE I.[35] In class I, III and IV
affinity chromatography (Ni-NTA) and, in the case of Lla-ER and
Rer-ER7, the cofactor FMN was reconstituted before further
Table 1. Sequence motifs specific for the four Old Yellow Enzyme subclasses.
Predicted interactions between FMN and residues of class III and class IV
enzymes are based on sequence alignments and docking studies using
homology models of of YqiG and Ppo-ER3 as representative members of
class III and class, respectively (Figure S11 and S112). All residue numbers
refer to an arbitrarily selected example enzyme of the respective class. The
enzyme name is given in brackets.
a further interaction to the cofactor FMN is provided by a
conserved threonine (Thr 37 in OYE1)[36-37] whereas a unique
feature of class II enzymes is the presence of a corresponding
cysteine residue (Cys 26 in YqjM) for interaction with the flavin
O4 atom.[15, 36] The conserved methionine, leucine and arginine
residues (Met 25, Leu 311 and Arg 301) in YqjM),[15, 22] which
form hydrophobic interactions to the dimethylbenzene moiety of
the flavin isoalloaxazine ring and were previously uniquely
reported for class II enzymes, are also conserved in the here
reported OYE classes III and IV. In class I ERs, on the other
hand, a combination of leucine, isoleucine and arginine/tyrosine
has been described for this function.[15] A major difference in
sequence between all OYE classes can be found in the C-
terminal region of their sequence. Class II enzymes feature a
combination of two arginines, a glutamine and a tyrosine (Arg
Associated
role of
sequence
motif
Class I
(OYE I)
Class II
(YqjM)
Class III
(YqiG)
Class IV
(Ppo-ER3)
Interaction
with
Gln 115
Arg 244
Gln 102
Arg 215
Gln 104
Arg 226
Gln 122
Arg 244
pyrimidine
ring of
[15]
FMN
Interaction to
N1 and N3 of
His 164
His 167
His 191
His 194
[15, 35]
FMN
His 192
Asn195
His 169
Asn172
3
12, Gln 333, Tyr 334 and Arg 336 in YqjM) which are involved
in monomer-monomer interactions.[14] This sequence stretch is
not present in the other classes. However, we found that class
IV enzymes contain a distinct additional amino acid stretch
consisting of 17-25 residues which putatively extend the loop
between helix 7 and β-sheet 7 based on a comparison with the
YqjM sequence and crystal structure.[15] Overall, the sequence
alignment highlights that the proposed class III and IV Old
Yellow Enzyme feature sequence motifs of both class I and
class III enzymes, positioning them in between the classical and
thermophilic-like OYEs.
Interaction
with
Thr 38
Thr 30
Thr 48
isoalloxazine
ring O4 atom
Cys 26
[36]
of FMN
Interaction
with the
Met 25
Leu 311
Arg 308
Met 29
Leu 322
Arg 319
Met 47
Leu 366
Arg 363
dimethyl
benzene
moiety of
Leu 37
Ile 352
Arg 348
[15, 22]
FMN
Biochemical characterization of class III and IV enzymes
To understand not only the sequence-based differences of class
III and IV enzymes but also obtain an insight into their potential
biocatalytic applicability, we set out to biochemically characterize
selected candidates. For this study we cloned a total of four
genes, two from each new subclass. Our study enzyme set was
comprised of YqiG from Bacillus subtilis (class III), Rer-ER7 from
Rhodococcus erythropolis (class III), Ppo-ER3 from
Paenibacillus polymyxa (class IV) and Lla-ER from Lactococcus
lactis (class IV). The gene product from yqig (Bacillus subtilis)
has been recently reported for the first time[38] and was assigned
C-terminal
sequence for
monomer-
monomer
Arg 312
Gln 333
Tyr 334
Arg 336
[14]
interaction
analysis.
All four purified ene reductases were obtained as active
enzymes, capable of converting cyclohexenone 2. As is the case
for class I and class II OYEs,[22] we found that NADPH is the
preferred physiological coenzyme of the novel ERs (Table S3).
This cofactor is commercially expensive, however, the
dependency on stoichiometric amounts of this compound can be
circumvented by established recycling systems such as the
as a ‘lost protein’[22]
.
Expression and characterization of the recombinant OYEs
After isolation of the genomic DNA from the selected strains, all
genes were successfully amplified and cloned into pET28b by
Fastcloning[39] or restriction site cloning and ligation. All plasmids
[41]
[42]
coupled enzyme approach
or alternative hydride sources.
Further relevant parameters for application such as pH optimum,
temperature optimum and long-term temperature-stability were
determined for YqiG (class III) and Ppo-ER3 (class IV) using
substrate 2 or 7.
were constructed with
6
a C-terminal His -tag for protein
purification via affinity chromatography. Soluble recombinant
expression in E. coli was achieved in all cases. Lla-ER was
expressed in E. coli Rosetta 2 (DE3), while YqiG, Ppo-ER3 and
Rre-ER7 were produced in E. coli BL21 (DE3). Rre-ER7 was
cultivated in the presence of chaperones GroEL and GroES
(
Gro7 chaperone system).[40] Target proteins were purified by
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Thus, temperature optima of the investigated class III and IV
enzymes are more similar to those of their mesophilic
counterparts from class I, e.g. 25-40 °C for OYE2p[47] or 30-
5
0 °C for NemA.[44] In contrast, thermophilic-like class II OYEs,
which have an average increased thermal stability compared to
class I enzymes, partly exhibit higher temperature optima as
observed for CrS (Topt = 65°C)[48] GeoER (Topt = 70°C)[49] and
FOYE1 (Topt = 50°C).[50] However, it must be noted that the
thermophilic-like subclass displays a wide variety of temperature
profiles and also includes non-thermostable OYE relatives such
as YqjM [45] and XenA.[51]
Substrate scope
To obtain an impression of the substrate scope, activity of all
four enzymes toward a series of structurally diverse aliphatic
and cyclic alkenes bearing ketone, aldehyde, carboxylic acid or
ester function as electron-withdrawing groups was investigated
(Table 2). In class III, YqiG showed medium to good conversions
for most substrates within one hour and accepted both cyclic
and linear structures. Lla-ER, on the other hand, demonstrated
to be a poorer catalyst for the investigated substrates and
incubation times of 24 hours were required to obtain meaningful
results. Lla-ER preferentially accepted unsubstituted cyclic
substrates or small aldehydes. In class IV, Ppo-ER3 readily
accepted most substrates showing very good conversion
especially for substrate 2, 7, 8 and 9 while Rer-ER7 only
reduced linear aldehydes 9 and 10 with conversions > 10 %
while all other substrates were not or only very poorly accepted.
Interestingly, class III and IV enzymes were found to have no or
extremely low activity for cyclic β-methylated substrates such as
Figure 3. Activity profile of YqiG and Ppo-ER3 correlated to pH, measured
with NADPH-Assay in different buffers and with 1 mM substrate 2 in case of
YqiG and with 7 in case of Ppo-ER3.
3
-methylcyclopentenone 4 and 3-methylcyclohexenone 5 while α
As reported, enzyme YqiG exhibits a broad pH optimum,
-substituted substrates (R)-carvone and 2-
1
[38]
preferably operating between pH 6 and 9 (Figure 3A). YqiG’s
pH profile is thus similar to the one of LacER[43] (pHopt = 7 - 9)
which, judging from our phylogenetic analysis, also classifies as
a group III enzyme. The pH profile of Ppo-ER3 is more bell-
shaped with the pH-optimum at 7.5 (Figure 3B). Overall, the
observed pH profiles for the class III and IV OYEs are in line
with reported values for class I and II enzymes, which mostly
exhibit pH optima between 6 - 7.5 (class I examples: XenB[
and NemA[44]) and 6 - 8 (class II: YqjM[45] and Chr-OYE3[46]).
methylcyclohexenone 6 were accepted. This result clusters the
new ER classes with class II enzymes which are similarly
reported to not accept cyclic β -methylated unsaturated
substrates, while class
I enzymes typically convert these
[22]
compounds. Also for the aliphatic dienal substrate citral, class
III and IV enzymes exhibit a similar conversion behavior as class
II enzymes and show only low to modest conversions (class III:
YqiG 8.7% conversion, Lla-ER 1.3 % conversion; class IV: Ppo-
ER 3 12.4 % conversion, Rer-ER 7 12.3 % conversion). Class I
enzymes, on the other hand, are known to accept citral well
44]
In terms of thermal robustness, class III enzyme YqiG was
shown by us and others to possess interesting long-term stability
up to 30°C. After 26 h incubation at 20 °C, the enzyme activity
leading to conversion yields of up to 67 % for OYE 2.6 and 69 %
for NemA.[52] Based on the results obtained with our substrate
series, the investigated class III and IV enzymes seem to display
a similar stereo-preference as class I and II enzymes.[22]
toward substrate
2 remained virtually unchanged while a
residual activity of 90 % was detected after an identical
incubation at 30 °C consistent with the results of Sheng et. al.[
Furthermore, short-term exposure (up to 6 hours) of YqiG to
Enzymes from the new groups preferentially form the expected
38]
(R)-product in the reduction of α-substituted cyclohexenone 6
and yield the (S)-product for conversion of the β-substituted
cyclohexenone 5 (conversions for all four enzymes were < 1 %).
The diastereoselectivity for the conversion of substrate 1 by
YqiG and Ppo-ER3 reached 89% de and 91% de, respectively.
Thus, the two enzyme operate in the same range as other well-
40°C led to an only marginal activity loss of 10 % opening the
door to applications that require elevated temperatures (Figure
S6).
Class IV enzyme Ppo-ER3 exhibited similar long-term stabilities
at 20°C and 30°C as YqiG and residual activities for the
conversion of substrate 7 after 24 h of incubation were 95 %
and 83 %, respectively (Figure S7). Ppo-ER3’s temperature
optimum for biocatalysis reactions was shown to be between
performing OYEs such as for example LacER (class III) from
Lactococcus casei (86 % de)[43] or OYE1 (class I) from
Sacchromyces cerevisiae (88 % de).[53]
25 °C and 40 °C as determined with substrate 7 (Figure S8).
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ChemBioChem
10.1002/cbic.201800770
FULL PAPER
Table 2. Conversion and enantiomeric excess of various substrates with purified enzymes as determined by GC after 1h [a]
Substrate
Product
YqiG
Lla-Er [b]
Ppo-Er3
Rer-Er7[b]
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
ee [%]
2
2.8 ± 3.1
89
2.6 ± 0.1
>99
24.3 ± 1.2
91
1.7 ± 0.1
>99
5
1
4.9 ± 6.1
0.3 ± 0.8
34.9 ± 4.2
3.2 ± 0.8
85.8 ± 6.8
51.4 ± 2.9
2.5 ± 0.3
n.d.
n.d.
n.d.
< 1
n.d.
<1
n.d.
<1
<
1
> 99
> 99
>99
86
>99
16
8
.7 ± 0.3
83
2.1 ± 0.2
11
21.4 ± 0.2
2 ± 0.1
3
7.0 ± 0.2
52.4 ± 0.9
68.2 ± 6.0
3.0 ± 0.1
5
8.2 ± 2.4
3.3 ± 2.4
65.8 ± 4.1
14.9 ± 0.6
72.9 ± 2.6
74.5 ± 0.1
n.d.
5
33
5
66
13.5 ± 0.6
1
8
.7 ± 0.3
1.4 ± 0.2
12.4 ± 0.1
12.3 ± 0.1
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ChemBioChem
10.1002/cbic.201800770
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n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4
.5 ± 0.3
n.d.
15.0 ± 1.2
<1
n.d.
n.d.
n.d.
n.d.
1
0.5 ± 1.3
c]
<1 [c]
n.d [c]
n.d. [c]
[
[
a] All reactions were performed in triplicates with 5 mM substrate, 6 mM NADPH and 100 µg ml-1 purified enzyme. In all cases, biotransformation results were
verified by control reactions omitting the enzyme. [b] t = 24 h. [c] in a plastic Eppendorf tube.
Only very few Old Yellow Enzymes (e.g LyngbyaER1[54] or
XenB[44]) have been reported to yield a better diastereomeric
excess for the conversion of substrate 1.
value compounds. Enzymatic reactions at an increased scale
give insights into process robustness and therefore produce
valuable information for further process optimization work. We
decided for the reduction of cinnamaldehyde in a substrate
-1
The only weakly activated acid and ester substrates 11, 12
and 14 were not converted by any of the four enzymes. This
is in line with results obtained for other OYE members such
as OYE1 (class I),[55] PETNR (class I)[56] and YqjM (class
II),[45] which also do not reduce monocarboxylic acid or esters
like 12. Interestingly, class III enzymes Ppo-ER3 and Lla-ER
reduce the bulky substrate 15. Ppo-ER3 even achieves
conversions of up to 10% in one hour, thus outperforming all
members of a library of 23 ene reductases reported in a
recent substrate scope evaluation by Reβ et al.[57] In this
study, only enzyme DBVPG from Kazachstania lodderae and
OPR I from Lycopersicon escultentum showed activity for this
substrate and conversions of 3% and 4%, respectively, were
detected after 24 hours.
concentration of 5.6 mM (0.75 g L , total substrate load 150
mg) using the previously well-performing Ppo-ER3 as the
biocatalyst. In the preparative-scale experiment the starting
compound 8 was converted by the class IV enzyme to the
desired product in 1 h and the resulting product 8a was
isolated in 88% yield. Notably, this reaction proceeded with
complete conversion underlining the compatibility of the ene
reductase Ppo-ER3 with higher substrate loadings.
Conclusions
We successfully screened 19 bacterial wild type strains to
identify novel, synthetically useful ene reductases. Genome
analysis of active strains allowed us to identify 47 genes with
putative ER activity, 42 of which had to our knowledge not
been described in literature before. Our phylogenetic and
Preparative biocatalysis
Finally, we set out to demonstrate the practical applicability of
the novel Old Yellow Enzymes for the synthesis of added-
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ChemBioChem
10.1002/cbic.201800770
FULL PAPER
sequence analysis of the 22 genes belonging to the Old
Yellow Enzyme family led us to propose the introduction of
two new OYE subclasses, named OYE class III and IV,
complementing the existing classical and the thermophilic like
OYE groups. Both new subclasses contain a mixture of
sequence motifs previously associated uniquely with class I
or class II enzymes.
autoclaving at 121°C for 20 min. Aqueous stock solutions were
sterilized by filtration through 0.20 μm syringe filters. Agar plates were
prepared using LB medium supplemented with 1.5% (w/v) agar.
Screening for ene reductase activity in bacterial strains: The
different bacterial strains were cultivated in triplicates in 48 deep well
plates using 1.5 ml TSB media without glucose for 24 h at 25 °C with
6
00 rpm in a Eppendorf Thermomixer C. All screening plates
To investigate the biochemical characteristics of the novel
groups, we solubly produced two enzymes for each class,
YqiG and Lla-ER for class III and Rre-ER7 and Ppo-ER3 for
class IV in E. coli. Cofactor preference, pH and temperature
optimum were found to be similar to known OYEs. Substrate
scope for the four enzymes was investigated with a panel of
contained two positive control strains and blanks with no strain. After
harvesting by centrifugation (15 min 3500 g HERAEUS Multifuge 4KR
centrifuge), the cell pellets were resuspended in 1 ml lysis buffer (50
mM sodium phosphate buffer pH 7.5, 1 mg ml-1 lysozyme) and
incubated for 1.5 h at 30 °C and 600 rpm. The supernatant of the
crude cell extract was used after 20 min centrifugation (3500 g) for a
NADPH-assay and biocatalysis reactions with substrates 1 and 2. The
NADPH-assay was performed during 3 min using 20 µl or 100 µl
supernatant in the presence of 0.6 mM NADPH and 2 mM substrate in
15 compounds and especially enzymes YqiG and Ppo-ER3
accepted a wide range of structures ranging from aliphatic
aldehydes and ketones, to cyclic ketones and even a bulky
ester (compound 15, only accepted by YqiG and Lla-ER).
Elucidated substrate preference of class III and IV enzymes
based on the tested compound panel showed a higher
similarity to the substrate scope of class II OYE as β -
substituted cyclic alkenes were not accepted and citral
conversion was low.
2
00 µl 50 mM sodium phosphate buffer (pH 7.5) monitoring at 340 nm
with the Tecan Spark 10M plate reader. For the 500 µl biocatalysis
reactions in sodium phosphate buffer (50 mM, pH 7.5) with 2 mM
substrate, 3 mM NADPH and 491 µl supernatant were used and
incubated 16 h at 30 °C and 600 rpm. 500 μl sample volume was
extracted twice with 200 μl and 100 µl methyl tert-butyl ether and
dried over sodium sulphate. The extracted solution was then
subjected to GC analysis.
Finally, applicability of the here-introduced enzymes to
preparative scale synthesis was demonstrated using class IV
enzyme Ppo-ER3 for the conversion of cinnamaldehyde at an
elevated substrate concentration.
Cloning: Total genomic DNA from all strains was extracted by using
the DNeasy Blood and Tissue Kit from Qiagen (Hilden, Germany).
Plasmid isolation, PCR purification, and gel extraction (Macherery
Nagel, Düren, Germany) were performed according to the
manufacturers’ instructions. All genes were cloned into pet28b with a
C-terminal His-tag.
Experimental Section
Cloning of the lla-er and yqig gene into pET28b(+) was performed
applying the FastCloning method[39]. Primers for amplification of the
lla-er gene from genomic DNA were: Fw-5’-GAG TGC GGC CGC
AAG CTT GAT ATC ATA CTT TCC TG-3’, Rev-5’-GAA GGA GAT
ATA CCA TGC CAA ATC AAT TAA C-3’
Materials: All chemicals of the highest available purity or of analytical
grade were purchased from Merck (Darmstadt, Germany), VWR
(Hannover, Germany), or Carl Roth (Karlsruhe, Germany) and were
used without further purification unless otherwise specified. NADPH
tetrasodium salt was ordered from Oriental Yeast Co. Ltd (Tokyo,
Japan). Restriction enzymes, polymerases and T4 ligase were
obtained from New England Biolabs GmbH (Beverly, MA, USA) and
primers were ordered from Microsynth (Balgach, Switzerland). The
sequencing was performed by Eurofins Genomics GmbH (Ebersberg,
Germany) or Microsynth (Balgach, Switzerland). The His-Trap FF and
the HiTrap desalting columns were ordered from GE Healthcare
Corresponding primers for amplification of the plasmid pET28b(+):
Fw-5’-CAG GAA AGT ATG ATA TCA AGC TTG CGG CCG C-3’; Rev-
5
’-GTT AAT TGA TTT GGC ATG GTA TAT CTC CTT CTT AAA GTT
AAA C-3’
Primers for amplification of the yqig gene from genomic DNA: Fw-5’-
GAA GGA GAT ATA CCA TGA ATC CTA AGT ATA AGC C-3’; Rev-
(Uppsala, Sweden).
5
’- GTG CGG CCG CAA GCT TAT CTT TAT AAG GCA CCC AG-3’;
Bacterial strains, culture conditions, and plasmids: All bacterial
screening strains were purchased from the Culture Collection of
Switzerland (CCOS, Wädenswil, Switzerland). E. coli BL21 (DE3)
Corresponding primers for amplification of the plasmid pET28b(+):
Fw-5’- GCT TAT ACT TAG GAT TCA TGG TAT ATC TCC TTC TTA
AAG-3’; Rev-5’- GTG CCT TAT AAA GAT AAG CTT GCG GCC GCA
CTC-3’
[fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS] was purchased from New
England Biolabs (Beverly, MA, USA), E. coli XL-1 Blue [recA1 endA1
gyrA96 thi-1 hsdR17 supE44 relA1 lac F
́
proAB lacIq ZΔM15 Tn10
r
(
Tet )] was ordered from Agilent Technologies (Stevens Creek Blvd.,
Cloning of the ppo-er3 gene and rer-er7 gene was done by PCR
amplification, restriction enzyme digestion and ligation into the pet28b
plasmid. For Rer-ER7 the NcoI and HindIII restriction sites were used
and the following primers were used for amplification: Fw-5’-GGG
CCC CCA TGG GCT TAA TAG TTA ATG CAC-3’ and Rev-5’-GGG
CCC AAG CTT GTT CAG GTG CGT GCG ATG TTC-3’
-
B B B
CA, US). The E.coli strain Rosetta 2 (DE3) [F ompT hsdS (r m
-)
-
gal dcm (DE3) pLysSRARE2 (CamR)] and the empty plasmid
pet28b(+) were obtained from Merck (Darmstadt, Germany). E. coli
strains were cultured routinely in LB or TB medium and if necessary
-
1
were supplemented with kanamycin (50 mg mL ). Bacterial cultures
were incubated in baffled Erlenmeyer flasks in a New Brunswick™
Innova® 42 orbital shaker at 200 rpm at 37 °C. Bacteria on agar
plates were incubated in a HERATherm Thermo Scientific incubator in
air. All materials and biotransformation media were sterilized by
For Ppo-ER3 the NcoI and XhoI restriction sites were used and the
following primers were utilized for amplification: Fw-5’- GGG CCC
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ChemBioChem
10.1002/cbic.201800770
FULL PAPER
CCA TGG GCG AAT TGT ATA ACA GAA -3’ and Rev-5’- GGG CCC
CTC GAG ATT CAA CGT TTT TAA TAC TTC AT-3’
profile was generated using appropriate buffer systems at a
concentration of 50 mM each (Tris-HCl, sodium phosphate, glycine-
NaOH or acetate-NaOH buffer).
Sequences have the following GenBank accession numbers YqiG
(
MK257766), Ppo-ER3 (MK257767), Lla-ER (MK257768) and Rer-
Biocatalysis reaction: For in vitro biocatalysis reactions desalted
-
1
ER7 (MK257769).
enzyme solution (final concentration of 100 µg mL ), 5 mM substrate
in DMF) supplemented with 6 mM NADPH were combined in a
(
sodium phosphate buffer (50 mM, pH 7.5) and adjusted to a final
volume of 1 mL. Reactions were incubated in closed GC-vials at 30°C
and 1000 rpm. All biocatalysis reactions were done in triplicates and
samples for analysis (500 μl) were taken after 1 h and 24 h. In all
cases, biotransformation results were verified by control reactions
omitting the enzyme.
Expression: Expression of Ppo-ER3 and YqiG in E. coli BL21 (DE3)
was performed by inoculation of LB media (400 mL) supplemented
with kanamycin (100 μgꢀmL−1) with an overnight culture (4 mL; 1:100)
of the recombinant strains. The main culture was incubated at 37ꢀ°C
and 180 rpm until OD600 = 0.6–0.8 was reached. At this point enzyme
expression was induced with 0.1 mM IPTG, and incubation was
continued for 16 h at 25ꢀ°C. The cultivation of Lla-ER in E.coli Rosetta
2
(DE3) was performed in the same way except that TB medium was
Preparative scale biocatalysis reaction: For the preparative
biocatalysis purified and desalted Ppo-ER3 (final concentration 75 µg
mL-1 enzyme solutions) in 200 ml sodium phosphate buffer (50 mM,
pH 7.5) with 150 mg substrate (5.6 mM; 1.12 mmol) 8, 400 mg
glucose, 1 mM NADPH and 3 U/ml GDH were used. After 1h an
analytical sample was taken for GC analysis (full conversion) before
the product was extracted three times with diethyl ether, dried over
used and temperature after induction was lowered to 20°C. Rer-ER7
was produced in an identical manner as YqiG except that the E. coli
BL21 (DE3) strain co-expressed chaperones GroEL and GroES from
plasmid Gro7 (Takara Bio Inc., Tokyo, Japan). Consequently,
chaperone production was induced previous to enzyme production at
OD600 = 0.4 with 0.1 mg ml-1 L-arabinose. Cells were harvested by
centrifugation at 4500×g for 10 min at 4ꢀ°C and used directly or cell
pellets were stored at -20 °C.
2 4
Na SO and the solvent was evaporated to obtain the final product.
Yield: 132 mg (0.98 mmol, 88%) Purity: 95 % (GC).
Enzyme purification: For cell disruption, the cell pellet was
NMR data of the isolated product:
resuspended in 25 mL buffer (100 mM sodium phosphate buffer pH
1H NMR (500 MHz, chloroform-d): δ [ppm] 9.86-9.85 (m, 1H), 7.37 –
7.31 (m, 3H), 7.26 – 7.21 (m, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.84 – 2.80
(m, 2H).
7
.5, 300 mM NaCl) supplemented with 30 mM imidazole. Cell
disruption was performed by a single passage through a French
pressure cell at 2000 psi. By centrifugation at 4500×ꢀg for 30 min cell
debris was separated from the crude cell extract. Purification was
performed by affinity chromatography by the C-terminal His-Tag with
an automated Äkta purifier system. A 5 mL HisTrap FF column was
equilibrated with buffer. After the crude cell extract was applied on the
column, unbound protein was eluted with five column volumes of
buffer supplied with 39 mM of imidazole. The elution of the desired
enzyme was accomplished by three column volumes of buffer
containing 300 mM imidazole. Washing, flow through, and elution
fractions were analyzed by SDS-PAGE. Desired fractions were
collected for desalting using the Äkta purifier to remove the imidazole
and to change the buffer to 50 mM sodium phosphate (pH 7.5). This
step was performed by using three coupled 5 mL HiTrap desalting
columns. After the column was equilibrated with sodium phosphate
buffer (50 mM, pH 7.5), the collected enzyme solution was loaded.
Protein fractions were analyzed using the Äkta purifier system by
online absorption measurement at 280 nm. In the case of enzyme
Rer-ER7 Tris-HCl buffer at pH 9.0 supplemented with 0.1 mM FMN
was used instead to avoid precipitation. The protein content of the
crude cell extracts as well as of the purified and desalted fractions
was determined by using the BCA kit (Thermo Fisher Scientific,
Waltham, Massachusetts, USA). Standard curves were recorded with
bovine serum albumin (BSA) at 0.02–2 mgꢀmL−1. Samples were
measured in triplicate at suitable dilutions. The NanoDrop was used to
determine the protein content of purified protein samples based on the
corresponding extinction coefficients.
13C-NMR (500 MHz, chloroform-d): δ [ppm] = 28.14 (t, 1-C); 45.30 (t,
1-C); 126,32 (d, 1-C); 128.29 (d, 2-C); 128,61 (d, 2-C); 140.33 (s, 1-
C); 201.55 (d, 1-C)
GC-Analysis: 500 μl samples were extracted twice with 200 μl and
1
00 µl methyl tert-butyl ether and dried over sodium sulfate. The
extract was then subjected to GC analysis (see supplement for
detailed GC methods).
Keywords: ene reductase • biocatalytic toolbox • novel Old
Yellow Enzyme subclasses • phylogenetic analysis • enzyme
sourcing
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To expand the biocatalytic toolbox of
available ene reductases we
Christin Peters, David Frasson, Martin
Sievers and Rebecca Buller
screened selected bacterial strains
for double bond reduction activity.
The genomes of strains exhibiting
activity were analysed bio-
Page No. – Page No.
Novel Old Yellow Enzyme
subclasses
informatically and 47 genes encoding
putative ene reductases were
identified. Interestingly, our combi-
natorial approach of phylogenetic and
biochemical analysis allowed us to
identify two novel Old Yellow Enzyme
subclasses.
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Author(s), Corresponding Author(s)*
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Title
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