Identification of Novel Unspecific Peroxygenase Chimeras and Unusual YfeX Axial Heme Ligand by a Versatile High-Throughput GC-MS Approach

Article abstract of DOI:10.1002/cctc.202000618

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high-throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC-MS technique allows a substrate independent 96-well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC-MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC-technique was additionally applied to evaluate an YfeX library in an E. coli whole-cell system for the carbene-transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan.

Full text of DOI:10.1002/cctc.202000618

The European Society Journal for Catalysis
Supported by
Accepted Article
Title: Identification of novel unspecific peroxygenase chimeras and
unusual YfeX axial heme ligand by a versatile high-throughput
GC-MS approach
Authors: Anja Knorrscheidt, Pascal Püllmann, Eugen Schell, Dominik
Homann, Erik Freier, and Martin J. Weissenborn
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.202000618
Link to VoR: https://doi.org/10.1002/cctc.202000618
01/2020

10.1002/cctc.202000618
ChemCatChem
FULL PAPER
Identification of novel unspecific peroxygenase chimeras and
unusual YfeX axial heme ligand by a versatile high-throughput GC-
MS approach
Anja Knorrscheidt,[a] Pascal Püllmann, [a] Eugen Schell, [a] Dominik Homann,[a] Erik Freier,[b] and Martin J.
Weissenborn*[a, c]
[a]
Anja Knorrscheidt, Pascal Püllmann, Eugen Schell, Dominik Homann, Jun.-Prof. Martin J. Weissenborn
Bioorganic Chemistry
Leibniz Institute of Plant Biochemistry
Weinberg 3, 06120 Halle (Saale), Germany
E-mail: martin.weissenborn@ipb-halle.de
Dr. Erik Freier
[b]
[c]
CARS Microscopy
Leibniz-Institut für Analytische Wissenschaften -ISAS- e.V.
Otto-Hahn-Str. 6b, 4227 Dortmund, Germany
Jun.-Prof. Martin J. Weissenborn
Institute of Chemisty
Martin Luther University Halle-Wittenberg
Kurt-Mothes-Str. 2, 06120 Halle (Saale), Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: Catalyst discovery and development requires the screening
of large reaction sets necessitating analytic methods with the potential
for high-throughput screening. These techniques often suffer from
substrate dependency or the requirement of expert knowledge.
Chromatographic techniques (GC/LC) can overcome these limitations
but are generally hampered by long analysis time or the need for
special equipment. The herein developed multiple injections in a
single experimental run (MISER) GC-MS technique allows a substrate
independent 96-well microtiter plate analysis within 60 min. This
method can be applied to any laboratory equipped with a standard
GC-MS. With this concept novel, unspecific peroxygenase (UPO)
chimeras, could be identified, consisting of subdomains from three
different fungal UPO genes. The GC-technique was additionally
applied to evaluate an YfeX library in an E. coli whole-cell system for
the carbene-transfer reaction on indole, which revealed the thus far
unknown axial heme ligand tryptophan.
allow the screening with the exact substrate of interest, ii) be
highly sensitive, iii) be exceedingly reproducible and iv) require
minimal time-periods for analysis. To be applicable in standard
chemical and biochemical laboratories, the necessary
instrumentation should be sufficiently general and accessible. A
flexible and sensitive analytical technique is provided by
chromatographies such as liquid (LC) or gas chromatography
(GC). While these techniques often provide the necessary
sensitivity and are applicable to a wide range of substrates, they
suffer from long analysis times preventing a high-throughput
screening with several hundreds of samples a day. The time
consuming parts are the separation, washing/heating and
column equilibration. Higher throughput was enabled by method,
column and gradient optimisation yielding total run times of less
than four minutes.[7]
Introduction
In the last decades, highly successful and environmentally
benign catalytic methodologies have been developed in organic
chemistry. The discovery and development of these catalysts
have been primarily based on rational or intuitional approaches
and serendipitous findings.[1] Improving the outcome of
unexpected
findings
necessitates
high-throughput
experimentation that enables the analysis of several thousand
reactions. Several smart strategies were developed like DNA
templating,[2] sandwich immunoassay,[3] MALDI labelling,[4]
fluorescence quenching and UV absorption.[5] These techniques
have their distinct advantages but typically rely on expert
knowledge, specialised laboratories or labelled substrates. For
protein engineering, most setups rely on colourimetric or
fluorescence based assays amongst them also microfluidic-
based ultra-high throughput systems.[6] The ideal assay would i)
Figure 1. MISER-GC-MS and its implementation for the identification of novel
enzymatic activities enabled the investigation of two case studies in different
biological matrices: I) carbene-transfer reaction on 1-methyl indole (E. coli) and
II) hydroxylation of 1,2,3,4-tetrahydronaphthalene (S. cerevisiae).
1
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Figure 2. A) Method development with the analytes ethyl 3-
indoleacetate (m/z 203) and methyl indole-3-carboxylate
(m/z 175) with three different concentrations of ethyl 3-
indoleacetate (20, 50 and 90 µM). B) Transferring the best
conditions into 96-well format using
a biological matrix
(E. coli lysate). An alternative stacked method, including
intermediate heating cycles, was developed with 3-(N-
methyl-indole)acetate (m/z 217).
Intriguing developments have brought substantially shortened
analysis times for LC by ultrahigh performance liquid
chromatography (UHPLC) and GC by flow field thermal gradient
gas chromatography (FF-TG-GC).[8] However, these techniques
are not available in every laboratory (UHPLC), only a few
prototypes are existing (FF-TG-GC), or a higher throughput
cannot be achieved with conventional approaches (GC/LC). The
analysis of multiple, overlapping samples in one chromatogram,
coined multiplexing, has been introduced in 1967 by Izawa for
GC[9] and was extended by Trapp and co-workers for a high-
throughput approach. This technique requires a specifically built
injector system. A method to enhance throughput in standard LC
measurements is the multiple injections in a single experimental
run (MISER) approach.[10] This method does not require special
equipment or expert knowledge. MISER relies on the injection of
several samples under isocratic conditions into one
chromatographical run requiring baseline separation of the
peaks. This enabled the performance of long chromatographic
separations in a high-throughput manner as usually signal and
therefore information free areas in the chromatogram are filled
by peaks of multiple injections.[10-11] The method of MISER-GC-
MS has not yet been employed for large sample numbers,[12] but
seems highly suitable for the application in a high-throughput
screening based on several reasons:
Results and Discussion
Development of a versatile MISER-GC-MS approach for highly
reproducible 96-well analysis in biological matrices
A
standard autosampler was modified for fast injection
application by decoupling of the autosampler from the GC
instrument (Supporting Information). The read-out-signal of the
GC was suppressed, enabling an independent control of the
autosampler. The method development commenced by altering
various conditions at the autosampler. The setup optimisations
like post-cleaning with isopropanol after each injection, as well
as variations in filling speed and the number of filling strokes,
were performed using the analyte ethyl 3-indoleacetate. This led
to significantly decreased standard deviations from initially
51.6 to 6.5 % (Table S2) illustrating the importance of adjusting
the settings of the autosampler for multiple injections set up.
Since the MS-detection allows the simultaneous quantification of
different m/z signals, the system was expanded to an internal
standard (methyl indole-3-carboxylate). The correlation between
the analyte and an internal standard enabled the error
minimisation occurring due to solvent evaporation, extraction
and sample injection and allows the comparability of microtiter
plates. For MISER-GC-MS method verification, three different
ethyl 3-indoleacetate concentrations (20, 50 and 90 µM) were
employed with a constant concentration of internal standard. All
MISER runs ought to be isocratic. The lowest oven temperature
of 150 °C led to peak tailing and poor baseline separation (Figure
2A, left). Utilising a temperature of 190 °C resulted in an
excellent baseline separation and improved peak shapes as well
as a reduced standard deviation of 4.0 % using 20 µM ethyl 3-
indoleacetate (Table S3).
i) MISER allows a distinct throughput enhancement for GC
without requiring special equipment,
ii) the quantification of target substances by GC-MS extracted
from crude mixtures is far less prone to ion suppression by
matrix effects than by LC-MS systems and,[13]
iii) MS-based detection is highly sensitive and allows
quantification of low analyte concentrations in complex
mixtures.
Herein, we report the development of a novel MISER-GC-MS
strategy paving the way for a versatile, assay-independent
platform for catalytic reactions. The developed system is
applicable for any GC-MS equipped with an autosampler.
Thereby, the MISER-GC-MS technique was broadened to
measure several analytes combined with an internal standard to
compensate deviations and enables the quantification of multiple
molecules with overlapping peak areas eliminating the need for
chromatographical separation. To facilitate the data evaluation,
an R-script has been written, allowing rapid analysis and quality
control. The developed technique enabled the screening of two
libraries with different host organisms and reactions.
Further parameters, which were investigated, were the split ratio,
the MS mode (SIM or Scan) and the injection interval (Figure 2,
Figure S6). To avoid overlapping of the injection and analyte
peaks, the injection interval was altered from 67 s to 82 s. This
further lowered the standard deviation and allowed the increase
of the split ratio up to 60, resulting in excellent standard
deviations of 1.0 % for 20 µM ethyl 3-indoleacetate. With these
2
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optimised analytical conditions in hand, we further challenged
the system by using E. coli cell lysates spiked with ethyl 3-
indoleacetate and analysed the resulting samples in 96-well
experiments using methyl indole-3-carboxylate as an internal
standard. As further quality control and for calibration purposes
ethyl 3-indoleacetate (20 µM) extracted from a buffer system was
injected after each microtiter plate row (12 samples) as well as
standards at the end of the run. The system was assessed with
the best conditions of the GC system (190 °C, split ratio 60, and
82 s injection interval) leading to a standard deviation of 4.0 %
for 109 injections including controls (Figure 2B, left). This could
be even further improved to a standard deviation of 2.5 % by
increasing the oven temperature to 230 °C. Due to shorter
retention times on the column, this also allowed a quicker
injection interval of 33 s (Figure 2B, middle, Table S6).
Figure 3. 96-well analysis of the carbene-transfer reaction by the previously
developed stacked method for MISER-GC-MS. As controls, the empty vector
was included in column 6 and the parental YfeX variant in column 7, which
harbours the axial ligand H215.
Since the developed methods shall be applicable for the
screening of non-natural enzyme activities, which in general
suffer from low turnovers and require high substrate loading, the
contamination of the GC column could pose a substantial
problem within 96 injections. Heating intervals can remove such
contaminations.[12b] An additional method was therefore
developed, which includes a heating cycle after every 12th
sample (Figure 2B, right). With a split ratio of 60 a standard
deviation of 3.0 % was reached. These different techniques and
setups were applied to the screening of enzyme libraries (see
below). Since the assessment of hundreds of samples results in
large amounts of data points, an automated R-script was written
to ensure high quality and correct peak integration in each
microtiter plate (see Additional Material). The script assesses the
data of the MISERgram for reproducibility using the internal
standard. Based on the injection interval, the peaks are counted
to verify whether every sample has been correctly injected (GC
hardware) and integrated (GC software). The quotient of the
internal standard and product peak is illustrated as a bar chart
and colour-coded microtiter plate for fast data evaluation.
The new MISER-GC-MS system was now applied to two
different enzyme systems to screen protein libraries in altering
biological systems.
tool[18]—and screened the resulting library for the occurrence of
other functional axial ligands in whole-cell reactions by using the
interval heating method for MISER-GC-MS. To ensure the
quality of the generated library, a quick quality control (QQC) was
conducted, demonstrating the expected codon distribution
(Figure S11). The reaction was performed using the carbene-
transfer reaction on 1-methyl-indole with ethyl diazoacetate as
carbene donor (Supporting Information).
As a control, cells harbouring the empty plasmid (pAGM22082)
and the parental YfeX were included within the 96 well plate,
which could be clearly distinguished by MISER-GC-MS (Figure
3). To our delight, a new variant was identified, which carried a
highly unusual tryptophan residue as axial heme ligand. To
prove that the MISERgram revealed a “true positive” result, the
corresponding plasmid was freshly transformed, expressed, and
the corresponding protein purified by metal affinity
chromatography. These results confirmed the YfeX-H215W
variant, showing only slightly reduced activities compared to the
parental variant (Figure S12). This discovery could broaden the
spectrum of canonical amino acids as axial ligands for non-
natural reactions[17c, 19] as well as represent an interesting target
structure for the further development of non-canonical amino
acids for heme complexing.[17b, 20]
Screening of a focussed enzyme library of the YfeX-
catalysed carbene-transfer reaction revealed tryptophan as
a novel axial heme ligand
Screening of a fungal unspecific peroxygenase (UPO) chimera
library in S. cerevisiae with the substrate tetralin revealing six
novel peroxygenase constructs
The dye-decolourising peroxidase YfeX from E. coli was
previously shown to perform non-natural carbene-transfer
reactions[14] such as carbonyl olefination[15] and C—H
functionalisation.[16] The starting activity for the latter reaction
was previously improved by an alanine scan within the active
site.[16]
To demonstrate that the MISER-GC-MS method can be readily
applied to other reactions and environments, the screening was
applied to fungal unspecific peroxygenases[21] (UPOs) for its
hydroxylation of tetralin. The previous method development
demonstrated that simultaneous quantification of product and an
internal standard is possible. We here wanted to expand the
analysis to three different molecules: tetrahydronaphthol (main
reaction product), α-tetralone (side-product) and 1-naphthol
(internal standard). Method development was based on the
results, as stated above. The two products and the internal
standard were injected in three different concentrations, and the
MS response was compared to the obtained values when
injecting only one analyte at a time and all three simultaneously.
The results revealed minimal deviation comparing single and
We were subsequently interested in the influence of the axial
ligand on the activity of YfeX regarding the C—H
functionalisation reaction. The axial ligand complexes the heme
iron and substantially influences its redox potential and
electrophilicity and hence the overall activity of the occurring
heme-carbenoid complex.[17] The starting point of the
mutagenesis was a variant carrying the mutations D143V,
S234C and F248V (parental variant). To study the influence of
the axial ligand in YfeX, we performed saturation mutagenesis
targeting the axial ligand residue histidine 215—using the
recently developed Golden Mutagenesis protocol and its online
3
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Figure 4. A) Extraction and mass spectrometric analysis of 1,2,3,4-tetrahydronaphthol in presence and absence of the other two compounds within a MISER
experiment, B) MISERgram of the hydroxylation of 1,2,3,4-tetrahydronaphthalene with 96 biological replicates in microtiter plate format with a standard deviation of
9.7 %.
multiple m/z trace analysis (Figure 4A, Figure S4). To confirm
the accuracy of the refined MISER-GC-MS method, we selected
one possible functional variant (chimera I, Figure 4B), which was
The secondary structure consists of 13 helices (42 % of overall
sequence) and 15 beta sheets (6 %). The units were then
grouped based on not disrupting pivotal catalytic motives (PCP;
EGD; E196) and secondary structure elements such as alpha
helices and beta sheets (see Figure S15 for details). These
subunits were randomly shuffled, leading to 35 = 243 possible
combinations. Cultivation, as well as biotransformation, were
previously identified by
a
standard colourimetric assay
(unpublished results). The aim was to validate a MISER-GC-MS
technology enabling the analysis of an entire microtiter plate with
individually expressed variants with an overall standard deviation
of less than 10 %. We were delighted to see that the
measurement of the entire 96 well plate within an analysis time
of 60 minutes showed a standard deviation of only 9.7 % for the
implemented in
a high-throughput manner using 96 well
microtiter plates. Besides the analytics of the product formation,
the peroxygenases were equipped with a C-terminal split-GFP-
tag thus allowing the determination of the protein
concentration.[24] The screening of 672 transformants by MISER-
GC-MS was done in seven hours and revealed 34 hits. Figure 5
depicts a resulting MISERgram of one microtiter plate. The best
performing variants from this library were reproduced in four
individual biological replicates in a microtiter plate, confirming the
accuracy of this method (Figure S14).
formation of tetrahydronaphthol (Figure 4B).
UPOs represent a class of highly promising enzymes, which
show remarkable activities as well as stabilities and solely
consume hydrogen peroxide as co-substrate.
A
major
bottleneck, however, is the heterologous enzyme expression.
Even though several thousands of putative peroxygenases have
been assigned, only very few could be produced heterologously
thus far.[21b, 22] To create structural diversity three (putative) UPO
genes from different fungal origins bearing high sequence
similarity were selected for the construction of a shuffled
peroxygenase library: the yeast secretion variant PaDa-I
originating from Agrocybe aegerita, GmaUPO from Galerina
marginata (72 % identity) and CciUPO from Coprinopsis cinerea
22]
(62 % identity).[21b,
The wildtype enzymes GmaUPO and
CciUPO showed no activity and no expression in S. cerevisiae,
respectively. The secondary structural units were grouped, and
sequence subunits were created by loop cuts yielding five
subunits for each gene (Figure 6). The structural assignment
was done based on the crystal structure of PaDa-I (pdb:
5OXU).[23]
Figure 5. Screening of a fungal unspecific peroxygenase library. A) MISERgram
of microtiter plate 1, B) automated R-script evaluation of plate 1(○=identified hit,
⁎=PaDa-I as control).
Figure 6. Subunits of the shuffled peroxygenase library and repetitive identified
hits which were analysed by MISER-GC-MS and subsequently sequenced.
4
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From the previously identified chimeras I-V (unpublished results), Lysate preparation for extraction experiments. The YfeX WT
only the chimera I, II, III and V showed activity towards tetralin
and could be identified during the MISER-GC-MS screening.
However, a novel construct Chimera VI could be identified, and
the parental variant PaDa-I was rediscovered three times. Based
on the GFP signal, the chimera VI showed a 3.8 fold enhanced
secretion compared to PaDa-I (Supporting Figure S14B) and
gene (cloned into pAGM22082) was transformed into chemically
competent E. coli BL21 (DE3) pLysS cells (Merck Millipore,
Darmstadt, DE) by heat shock. Freshly-plated transformants
were grown overnight in 5 mL TB medium containing kanamycin
and chloramphenicol (50 μg/mL each). 2 mL of the preculture
was then used to inoculate 400 mL of main culture consisting of
TB autoinduction medium containing kanamycin and
demonstrated
a good starting point for a heterologously
expressed UPO, which can be screened on additional substrates. chloramphenicol (50 μg/mL each). Cells were incubated at 37 °C
and 120 rpm. After 4 h of initial cultivation, aqueous solutions of
FeCl3/5-aminolevulinic acid (final concentration: 100 μM) were
added, the temperature was reduced to 30 °C, and the cells were
incubated for further 16.5 h. Cells were harvested by
Conclusion
centrifugation (3000 x g, 20 min, 4 °C). The supernatant was
discarded, and the pellet was resuspended in binding buffer (50
mM KPi, pH 7.0, 200 mM NaCl). Cells were lysed by sonication
(Bandelin Sonoplus HD3100: 6 x 30 s, 70 % amplitude, pulse
mode). The resulting lysate was stored at – 20 °C until further
utilisation for lysate extraction experiments.
The development of MISER-GC-MS methods was successfully
utilised for its identification of novel enzyme activities. Two
methods were developed: one method for the screening of
natural enzymatic reactions, which generally have only a few
side-products and therefore an injection interval of 33 s was
applied for 96 well microtiter plate analysis within 60 min. The
other developed technique was the stacked method with
particular relevance to low activities and large amounts of side-
products, including a heating step after every 12th injection to
improve the quality of the acquired data.
Site-saturation mutagenesis (SSM) and E. coli cultivation in
96-deep-well plates. Mutagenesis was performed using the
Golden Mutagenesis technique[18] combined with the “22c-
trick”[25] for randomisation. The YfeX gene from E.coli was
chosen as a template, targeting amino acid residue position
Histidine 215. The created library was then transformed into
Both systems were employed for the screening of two different
enzyme classes and reactivities within two biological systems.
For the YfeX catalysed carbene-transfer reaction, the screening
of a focused library led to a highly unusual axial heme ligand
(H215W). By screening a chimera library of three shuffled
unspecific peroxygenase genes, MISER-GC-MS aided the
detection of five peroxygenase chimeras. These chimeras were
active and heterologous producible catalysts and hence provide
access to new peroxygenase scaffolds.
chemically competent E. coli BL21 (DE3) pLysS cells.
A
preculture of the transformants was grown in 350 µL TB medium
with added kanamycin and chloramphenicol (50 μg/mL each) in
an EnzyScreen plate at 37 °C and 300 rpm overnight. For the
main culture, 730 µL of TB autoinduction medium per well (plus
50 µg/mL kanamycin and chloramphenicol and 100 µM of
FeCl3/5-aminolevulinic acid) was inoculated with 20 µL of the
respective preculture. In a first phase the cells were cultivated at
37 °C and 300 rpm for 4 h. Afterwards the temperature was
decreased to 25 °C and protein expression was continued
overnight. The cells were harvested by centrifugation (30 min,
3000 g, 4 °C) and the supernatant discarded.
To put the MISER-GC-MS in perspective to the colourimetric
assay based on 4-nitrocatechol formation: the 96-well analysis
of the colourimetric product 4-nitrocatechol takes 2 minutes. It
has a sensitivity in the range of 10-20 µM, which amounts to
2000-4000 pmol (200 µl volume in one microtiter plate well).
MISER-GC-MS revealed an analysis time of 60 minutes with a
detection limit of 5-10 µM corresponding to 0.08-0.17 pmol (1 µL
sample injection and a split ratio of 1:60). The high sensitivity of
MISER-GC-MS could enable the engineering of minute initial
activities for new unnatural reactions in the future.
Whole-cell biotransformation with YfeX. The 96 deep well
plate harbouring the cell pellets was transferred into a glove box
(N2 atmosphere) and incubated on ice for 1 h to remove residual
oxygen from the gas phase. 200 µL of degassed 50 mM KPi
buffer (pH 7.0, 200 mM NaCl, 2 mM MgCl2) was added to each
well, and the cells were resuspended by vortexing for 1 min. 200
µL of a reaction master mix (stock solutions: 50 mM 1-
methylindole, 50 mM ethyl diazoacetate, 100 mM sodium
dithionite) were added to each well (final concentration: 2.5 mM
1-methylindole, 2.5 mM ethyl diazoacetate, 10 mM sodium
dithionite and 20 % ethanol as co-solvent) and the plate was
closed tightly with a cover. The reaction was performed at 30 °C
and 300 rpm for 1 h. The samples were extracted by addition of
1 mL ethyl acetate (EtOAc) containing 100 µM methyl indole-3-
carboxylate as internal standard and shaking at 300 rpm for 20
min at 25 °C. After centrifugation (10 min, 3000 g, 10 °C) 400 µL
of the organic phase were transferred into a glass-coated 96-well
plate for GC-MS analysis.
The herein demonstrated MISER-GC-MS technology can be
implemented into any laboratory with a GC-MS equipped with an
autosampler and has proven potential as a versatile, specific and
cost-effective high-throughput approach.
Experimental Section
Biological procedures
5
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Expression and purification of YfeX variants for hit
verification. The YfeX gene and its corresponding mutants
(plasmid backbone: pAGM22082) were transformed into
chemically competent E.coli BL21(DE3) pLysS cells by heat
shock procedure. Freshly-plated transformants were grown
overnight (160 rpm) as preculture in 5 ml TB medium containing
chloramphenicol and kanamycin (50 μg/mL each). 2 ml of the
precultures were used to respective inoculate 400 ml TB
autoinduction medium (+ kanamycin and chloramphenicol).
Cells were incubated at 37 °C (120 rpm shaking). After 4 hours
of cultivation, aqueous solutions of FeCl3 and 5-aminolevulinic
acid (final concentration: 100 μM) were added and the
temperature reduced to 30 °C. The cells were incubated for
further 16.5 h. Cells were finally harvested by centrifugation
(3000 x g, 20 min, 4 °C). The cultivation supernatant was
discarded, and the pellets were resuspended in binding buffer
(50 mM KPi; pH = 7.4, 200 mM NaCl, 1 mg/ml lysozyme, 100
µg/ml DNAse I). Cells were lysed by sonication (Bandelin
Sonoplus HD3100: 6x30 s, 70% amplitude, pulse mode). The
cell debris was removed by centrifugation for 45 min at 4 °C and
6000 x g. The proteins exhibiting an N-terminal attached
hexahistidine-Tag were purified by IMAC (immobilised metal ion
affinity chromatography) using 1 ml His GraviTrap TALON
columns (GE Healthcare Europe GmbH, Freiburg, DE). After
applying the cleared supernatant to the column, the column was
washed with 10 column volumes (10 ml) of washing buffer (50
mM KPi; pH = 7.4, 200 mM NaCl, 5 mM imidazole). YfeX variants
were finally eluted by the addition of elution buffer (50 mM Kpi;
pH = 7.4, 200 mM NaCl and 250 mM imidazole). For subsequent
buffer exchange of the pooled elution fractions PD-10 desalting
columns (GE Healthcare Europe GmbH, Freiburg, DE) were
used according to the manufacturer’s protocol. YfeX samples
were eluted using 50 mM KPi (pH = 7.0) with addition of 10 %
glycerol (v/v) as storage buffer. Afterwards samples were flash
frozen in liquid nitrogen and stored at -20 °C. Protein
concentrations were determined in duplicates using the Pierce
TM BCA Protein Assay Kit (Thermo Scientific, Rockford, US).
HNIEQQLLSMFGDTDGKRDAMLRFTKPVTGGYYFAPSLDKL
MALSDGGSGGGSTSRDHMVLHEYVNAAGIT
Yeast supernatant preparation for extraction experiments.
The empty plasmid (lacking UPO gene) for yeast expression was
transformed into chemically competent yeast cells (INVSc1
strain) by polyethylene glycol/lithium acetate transformation. For
the preculture 100 ml SC Drop selection Media (lacking Uracil as
supplement; containing 2 % raffinose as carbon source and 25
µg/ml chloramphenicol) were inoculated with a single yeast
colony from a previously grown SC Drop out plate (lacking Uracil)
at 30 °C and 130 rpm for 2 days. For expression rich expression
media (Yeast extract Peptone Galactose) containing 2 % of
galactose as inducer was utilised. The final main culture OD was
adjusted by addition of the preculture to 0.3. Expression was
performed for further 72 h (120 rpm; 30 °C). After 72 h cultivation
time cells were separated from the supernatant by centrifugation
(3400 rpm; 45 min; 4 °C). The supernatant was stored at 4 °C
until further utilisation for supernatant extraction experiments.
Generation of a shuffled peroxygenase gene library and
Yeast cultivation in 96-well plates. The gene of the
peroxygenase yeast secretion mutant PaDa-I,[21b] an annotated
peroxygenase gene from the fungus Galerina marginata and
previously described peroxygenase from Coprinopsis cinerea[22]
were divided into 5 structural subunits and randomly shuffled
together (243 possible combinations). Full length fragments
were then reassembled into
a yeast expression plasmid
(Galactose inducible promoter) with an N-terminal signal peptide
and a C-terminal GFP 11 detection tag within a single Golden
Gate cloning reaction (unpublished results). Corresponding
plasmid mixtures were transformed into yeast cells (INVSc1
strain) by polyethylene glycol/lithium acetate transformation.
Yeast cells were cultivated in liquid culture as described by
Molina Espeja et al.[21b] with slight modifications. 220 µl of
minimal expression medium per well (containing 2 % (w/v)
Galactose final concentration as carbon source and inducer)
were inoculated with a single yeast colony from a previously
grown SC Drop out plate (lacking Uracil). For cultivation
EnzyScreen half-deepwell plates were utilised. Expression was
performed for 72 h (230 rpm; 30 °C). After 72 h cultivation time
cells were separated from the peroxygenase containing
supernatant by centrifugation (3400 rpm; 45 min; 4 °C). For
subsequent screening 20 µL (for split GFP Assay) and 100 µL
(for biotransformation) were transferred to a respective plate
using a multichannel pipet.
Purified enzyme biotransformation. For the purified enzyme
biotransformation the whole cell biotransformation conditions
were adapted and the final enzyme concentration was set to 15
µM. The reaction was performed at 30 °C and 600 rpm for 1 h.
The samples were extracted by addition of 1 mL EtOAc
containing 100 µM methyl indole-3-carboxylate as internal
standard and vortexing. After centrifugation (5 min, 20.000 g)
600 µL of the organic phase were transferred into glass vials for
subsequent GC-MS analysis.
Amino acid sequence of the YfeX parental variant. Plasmid
derived N-terminal hexahistidine-tag + T7 tag indicated in italic.
The C-terminal GFP11 detection tag is underlined. Mutations in
comparison to the YfeX wild type protein sequence (Uniprot ID:
P76536) are indicated as bold letters.
Supernatant biotransformation in S. cerevisiae for
peroxygenases. A volume of 100 µL of the peroxygenase
containing yeast supernatant was transferred to a 96-well
EnzyScreen plate (CR1496), followed by the addition of 240 µl
of 100 mM citrate buffer (pH 6), 40 µl of 1,2,3,4-
tetrahydronaphthalene stock solution (10 mM in acetonitrile, final
concentration: 1 mM) and 20 µl H2O2 stock solution (6 mM in
H2O, final concentration: 300 µM). After a centrifugation step (1
min, 100 x g, 10 °C), the reaction was shaken for 16 h at 230 rpm
and 30 °C. The reaction was stopped by addition of 400 µl freshly
prepared 500 µM internal standard extraction solution (1-
naphthol in EtOAc). For extraction the aqueous and organic
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDGMSQV
QSGILPEHCRAAIWIEANVKGEVDALRAASKTFADKLATFEAK
FPDAHLGAVVAFGNNTWRALSGGVGAEELKDFPGYGKGLAP
TTQFDVLIHILSLRHDVNFSVAQAAMEAFGDCIEVKEEIHGFR
WVEERDLSGFV
VQRWEHNLKQLNRMSVHDQEMVIGRTKEANEEIDGDERPET
SHLTRVDLKEDGKGLKIVRQ LPYGTASGTHGLY CAYCARL
VGTENPAGEETRREVAVIKDGVDAGGSYVF
C
V
6
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10.1002/cctc.202000618
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E.F. gratefully acknowledges the financial support by the
Ministerium für Innovation, Wissenschaft und Forschung des
Landes Nordrhein-Westfalen and M.J.W, A.K., E.S. and E.F
thank the Bundesministerium für Bildung und Forschung
(„Biotechnologie 2020+ Strukturvorhaben: Leibniz Research
Cluster“, 031A360B and 031A360E) for generous funding. P.P.
thanks the Landesgraduiertenförderung Sachsen-Anhalt for a
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and Benjamin Jones for substantial support on Figure 1.
Keywords: High-throughput analytics • Biocatalysis • Carbene
transfer • Unspecific peroxygenase • Hydroxylation
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Entry for the Table of Contents
The developed multiple injections in a single experimental run (MISER) GC-MS technique allows a substrate independent assay for
the 96-well plate analysis. Utilisation of this concept yielded novel unspecific peroxygenase chimeras, produced in S. cerevisiae,
consisting of subdomains from three different fungal UPO genes for the hydroxylation of tetralin. The GC-technique was additionally
applied to evaluate a YfeX library in an E. coli whole-cell system for the carbene-transfer reaction on indole, which revealed an thus far
unknown axial heme ligand: tryptophan.
Institute and/or researcher Twitter usernames: Martin Weissenborn (@Weissenborn_Lab)
8
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