Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering

Article abstract of DOI:10.1002/chem.202002077

Protein design is limited by the diversity of functional groups provided by the canonical protein ?building blocks“. Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.

Full text of DOI:10.1002/chem.202002077

Accepted Article
Accepted Article
Title: Folding assessment of incorporation of non-canonical amino
acids facilitates expansion of functional group diversity for
enzyme engineering
Authors: Ivana Drienovská, Matúš Gajdoš, Alexia Kindler, Mahsa
Takhtehchian, Barbara Darnhofer, Ruth Birner-Gruenberger,
Mark Dörr, Uwe T. Bornscheuer, and Robert Kourist
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
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: Chem. Eur. J. 10.1002/chem.202002077
Link to VoR: https://doi.org/10.1002/chem.202002077
01/2020

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
Folding assessment of incorporation of non-canonical amino
acids facilitates expansion of functional group diversity for
enzyme engineering
Ivana Drienovská[a], Matúš Gajdoš[a], Alexia Kindler[a], Mahsa Takhtehchian[a], Barbara Darnhofer[b] [d]
Ruth Birner-Gruenberger[b] [c] [d], Mark Dörr[e], Uwe T. Bornscheuer[e] and Robert Kourist*[a]
,
[a]
Dr. I. Drienovská, M. Gajdoš, A. Kindler, M. Takhtehchian, Prof. Dr. R. Kourist
Institute of Molecular Biotechnology
Graz University of Technology
Petersgasse 14, 8010 Graz, Austria
E-mail: kourist@tugraz.at
[b]
[c]
[d]
[e]
Barbara Darnhofer, Prof. Dr. R. Birner-Gruenberger
Diagnostic and Research Institute of Patholoy
Diagnostic and Research Center of Molecular Medicine, Medical University of Graz
Neue Stiftingtalstraße 6, 8010 Graz, Austria
Prof. Dr. R. Birner-Gruenberger
Institute of Chemical Technologies and Analytics
Vienna University of Technology
Getreidemarkt 9/164, 1060 Wien, Austria
Barbara Darnhofer, Prof. Dr. R. Birner-Gruenberger
Omics Center Graz
BioTechMed-Graz
Stiftingtalstraße 24, 8010 Graz, Austria
Dr. Mark Dörr, Prof. U. T. Bornscheuer
Biotechnology & Enzyme Catalysis, Institute of Biochemistry
Greifswald University
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: Protein design is limited by the diversity of functional
groups provided by the canonical protein “building blocks”.
Incorporating non-canonical amino acids (ncAAs) into enzymes
enables a dramatic expansion of their catalytic features. For this,
quick identification of fully translated and correctly folded variants is
crucial. Herein, we report the engineering of the enantioselectivity of
an esterase utilizing several ncAAs. Key for the identification of active
and soluble protein variants was the use of the split-GFP method,
which is crucial as it allows simple determination of the expression
levels of enzyme variants with ncAA incorporations by fluorescence.
Several identified variants led to improved enantioselectivity or even
inverted enantiopreference in the kinetic resolution of ethyl 3-
phenylbutyrate.
Additionally, this technology can be used to enhance/improve
enzyme performance in hostile environments.[8] Despite of these
very promising examples, the biocatalytic use of enzymes
containing non-canonical amino acids (further referred to as
allozymes) lacks behind the use of ncAAs as tools for therapy and
research. In particular, ncAA incorporation is far from being a
standard method for enzyme engineering.[1c,9] Furthermore, it
should be noted that ncAA incorporation poses several different
challenges. For de novo enzyme generation, usually spacious
scaffolds with large pockets or cavities are chosen, and ncAA
incorporation focuses on one site where the practicability of SCS
has been proven. The use of ncAA for classical rational protein
design, however, needs to incorporate novel functional groups
into a tightly packed protein scaffold and to embed the ncAAs in
the complex network of interactions between the amino acid
residues. As the success of SCS is highly context-dependent,[10]
application of ncAAs for enzyme engineering creates the demand
for rapid methods to successfully determine ncAA incorporation
and soluble production of the resulting allozymes (Fig. 1).
While a diversification of the limited set of functional groups in
enzymes would greatly expand the possibilities of enzyme
engineering, available methods for the incorporation of chemical
entities to proteins are very limited.[1] Posttranslational
modifications of proteins typically rely on enzymatic steps with
narrow substrate spectra and often require purified enzymes, that
is usually too expensive for industrial biocatalytic applications.[2]
In contrast, incorporation of functional groups via the ribosomal
protein machinery is, in principle, scalable. In general, ncAAs
incorporation can be categorized in the modification of the protein
backbone and the introduction of new functionalities in side-
chains.[3] The in vivo incorporation of ncAAs either by sense-
codon reassignment (SCR) or stop-codon suppression (SCS) has
already been applied for the stabilization of enzymes,[4] the
modification of the enzymatic catalytic properties,[5] and even the
generation of new catalytic activities.[6] In terms of biocatalysis,
ncAAs can also be utilized for site-specific immobilization.[7]
Figure 1. Technical hurdles of enzyme expression with ncAAs, which need to
be addressed for the identification of soluble, active and functional enzymes.
1
This article is protected by copyright. All rights reserved.

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
The reason lies in the insufficient efficiency of the
incorporation on the one hand (resulting in protein yields that are
We expected that a split-GFP assay coupled to an activity
screen would allow discriminating between misfolded variants
too low for application), and the limited flexibility on the other hand. that showed neither fluorescence nor enzymatic activity, and
Incorporation via SCS bears the risk of an abortion of the
translation, leading to a high concentration of inactive enzyme
fragments. The incomplete translation is controlled by active
processes such as the recruitment of the release factor by the
amber codon[11] and depends on the position in the peptide chain
(incorporations closer to the N-terminal part being most
successful), the ncAA and the surroundings of its position in the
three-dimensional structure. SCR leads to global substitution of a
non-canonical amino acid into all positions of a chosen canonical
amino acid. Although examples of successful engineering utilizing
this method has been showed, the need to substitute all residues
poses some limitations for the engineering of proteins with a large
number of the target residue[12] Finally, the SCR and SCS were
successfully combined to produce one enzyme, however this
approach also did not lead to eliminations of given drawbacks.[13]
Protein purification using a C-terminal affinity tag is often used
to remove fragmented proteins and to detect incorporation
problems. Yet, affinity purification is rather unpractical for high-
throughput screens and severely reduces the throughput of the
assay. The split-GFP technique was developed by Waldo et al.[14]
and constitutes an easy way to verify production and successful
folding of a protein. The technique was successfully adapted by
some of us for rapid detection of soluble enzyme production levels
for directed enzyme evolution.[15] As incomplete translation is one
of the main obstacles for the engineering of allozymes, we
envisioned that this method might be highly practical to determine
the amount of fully translated enzyme variants in high-throughput
screens with ncAAs. Prescreens and selection assays relying
solely on activity of the enzyme would sort out misfolded variants,
but usually overlook correctly folded variants with low specific
activity. These variants are often of high interest for properties
such as selectivity and can be valuable to increase knowledge
and to provide starting points for further engineering. This is
further complicated by the generally lower expression levels of
proteins with ncAAs.
correctly folded variants that gave a signal with split-GFP, but
showed little activity. Control cultivations without addition of the
ncAA can easily highlight promiscuous incorporation of canonical
amino acids. To demonstrate the feasibility of the assay, we
investigated the rational engineering of the enantioselectivity of a
Pseudomonas fluorescence esterase (PFE)[16] in the kinetic
resolution of rac-ethyl 3-phenylbutyrate by incorporation of a set
of five different ncAAs. Using the split-GFP assay in combination
with an activity test allows the quick identification of folded and
active variants with interesting properties and decreases the need
for time-consuming screenings of all prepared variants (Fig. 2).
Ten residues in the active site region of PFE were selected as
positions for ncAA incorporation. Enantioselectivity strongly
depends on the difference of transition state energy in the
conversion of both enantiomers of the substrate, which is
influenced by steric hindrances, hydrogen bond formation, charge
and π-π interactions. We believe that ncAAs can provide these
effects beyond their canonical counterparts. Therefore, the
residues surrounding the catalytic triad, or forming the acyl or
alcohol binding pocket were selected (Fig. 3).
Figure 3. Surface view of the PFE protein (PDB: 1VA4) and a close-up of the
active site pocket. In both representations, the positions that were used for the
introduction of ncAAs are highlighted in color (green for the alcohol-binding site,
blue for the acyl-binding site and magenta for surrounding residues) and the
catalytic triad is highlighted in orange. The same color coding was used in the
list of chosen positions shown in the Figure.
The selection of residues for incorporation was led mainly by
the structure of PFE, with previous reported studies on the
engineering of this enzyme taken into consideration.[17] The side
chains of Trp28 and Val225 lie on either side of the acyl binding
region, while the side chains of Val121 and Phe198 lie above and
behind the acyl binding region. Residues F93 and F162 form an
alcohol-binding pocket. The substrate-binding site of PFE is
almost exclusively hydrophobic with numerous aromatic residues,
therefore the hydrophobic character was also kept in
incorporation of ncAA. However, we decided to incorporate
additional features, in terms of side chain bulkiness as well as
additional interactions to pursue novel binding modes and
subsequent favoritism of one substrate stereoisomer via size
restrictions and aromatic systems π-π stacking between the ncAA
and the substrate. The chosen ncAAs are summarized in Figure
2. The amber stop codon suppression methodology was used to
introduce this set of ncAAs into all the selected position
Figure 2. Schematic representation of the envisioned work-flow of the proposed
methodology comprising the detection of esterase activity by the colorimetric
pNPA assay and determination of PFE protein content using the split-GFP
method. From these data the specific activity can be calculated and normalized.
Furthermore, the chosen ncAA are shown.
2
This article is protected by copyright. All rights reserved.

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
(Supplementary Table 1).[18] To successfully apply the split-GFP
method for the screening of protein expression, the protein of
interest must be expressed with the GFP11 fragment as a fusion
tag. The cloning of the PFE gene with the split-GFP tag as well as
incorporation of the amber stop codon was performed as
described in the method section of the Supplementary material.
Ten variants and wild-type PFE and the corresponding plasmids
containing the orthogonal translation system for the introduction
of the ncAAs were transformed into E. coli and coexpressed in
deep-well plates; all experiments were performed in triplicates.
Negative controls were grown identically, without addition of the
ncAAs. Hence, if present, the unspecific incorporation of
canonical AAs could be observed and screened for. Selection of
active variants of PFE was done based on the pNPA activity
assay monitoring the hydrolysis of the acyclic ester p-nitrophenyl
acetate 1 (Scheme 1a). The activity determined in the cell free
extract (CFE) was related to the protein content determined by
fluorescence measurement employing the GFP-split assay (see
SI for more details) and compared to the activity of the wild-type
PFE (wtPFE). Results of the split-GFP assay and the activity tests
are summarized in Figs. 4 and 5 and in more details in the
Supporting Information.
pULTRA_pCNF was used for incorporation of pCNF. In the
conditions used for the incorporation of pCNF, undesired
background incorporation was significantly higher compared to
the other systems described in this study (i.e. the pEVOL-based
plasmids). Interestingly, in the case of these two ncAAs (pBF,
pCNF), lower levels of expression/amount of soluble protein of
wtPFE were also observed, with almost 10x reduction in
expression for pBF suggesting that these systems may have
significant effects on the general “healthiness” of the expression
machinery. PFE variants with pAzF at position F162, F198, I224
and V225 were well expressed, with F198_pAzF and I224_pAzF
having specific enzyme activities higher than the wtPFE. The pAF
incorporation was achieved by the reduction of pAzF after
expression, therefore similar trends are observed for
incorporation levels, while allowing us to study effects of different
ncAAs on reaction rates and specific activity. Out of the ten PFE
variants, the NapA substitution at positions F158, F198 and V225
gave significant expression levels, with high specific esterase
activity in case of F198_NapA, albeit slightly lower than the wt (Fig.
4 and Fig. 5).
Scheme 1. (a) pNPA assay, hydrolysis of p-nitrophenylacetate 1 to p-
nitrophenolate 2 and acetic acid. The progress of the reaction was monitored
following the increase in absorbance at 410 nm due to the formation of 2. (b)
Kinetic resolution of rac-ethyl 3-phenylbutyrate 3. The progress of the reaction
was monitored by gas chromatography analysis using a chiral column. X in
PFE_X represents different mutant variants of PFE.
The overall results of these experiments (Figure 4a) show that
the incorporation of different ncAAs is not well tolerated in the PFE
scaffold at multiple positions, underlining the need for an efficient
method for the identification of well-produced variants. Out of the
ten positions selected, four (W28, F93, A120 and F253) gave no
or minimal levels of soluble protein, independent of the studied
ncAA. Positions V121, F158 and F162 resulted in low expression,
depending on the studied ncAA, and positions F198, I224 and
V225 were generally well tolerant for ncAA incorporation. When
looking at the specific ncAAs, three of them (pAF, pAzF and NapA,
see Fig. 2) were well incorporated, while limited amounts of
expressed, soluble enzyme were observed for the remaining two
(pBF, pCNF). The expression levels of most pBF-carrying PFE
variants were not significantly higher than the background
incorporation and showed no detectable activity. pBF has a
significantly bulkier side chain compared to pAzF or NapA, which
may cause the inability of the enzyme to fold correctly, or the
active site accessibility is too restricted. Secondly, the minimal
levels of soluble protein were also observed in case of
Figure 4. Graphic 3D representation of a) expression levels of PFE variants
measured in cell-free extracts (in μM) using the split-GFP assay and b)
corresponding reaction rates measured for pNPA assay (in μM/min). Conditions
with added ncAA (pAzF, pBF, pCNF, NapA) during expression are compared
with control experiments, where ncAA was not added (no_pAzF, no_pBF,
no_pCNF, no_NapA). For details on expression and reaction conditions see
Supporting Information.
incorporation of pCNF. Herein,
a different vector system
3
This article is protected by copyright. All rights reserved.

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
of 1 for (S)-4 in the kinetic resolution of 3. In our study, the variant
I224pAzF_PFE enabled almost a doubling in the enantiomeric
ratio. A direct influence of I224 on the enantioselectivity was also
strongly expected since its side chain is in direct contact with the
acyl moiety of the substrate on the opposite side to F198.
Substitution for aromatic side chains possibly enables
π
interaction that leads to the different binding mode and
subsequent preference of the (S)-enantiomer. This supports the
previous claims that bulky and hydrophobic amino acids, which
block the entrance to the active site of the PFE have effect on
selectivity. Overall, our data showcases that utilizing ncAAs
provides another means to redesign the active site next to
standard directed evolution techniques, where a switch of
enantioselectivity can also be achieved.[19]
Table 1. Enantiomeric ratios obtained for selected PFE variants. The calculated
enantiomeric ratios (E) are shown, calculated from all measurements with
conversion in between 10-80%, with enantiomeric excesses of products (eep)
and the respective conversion (c) given for 4 hours measurement.
Figure 5. Comparison of specific enzyme activities calculated using the pNPA
assay for PFE variants with three different non-canonical amino acids at three
different positions compared to the wtPFE. Error-bars stem from three
independent cultivations.
PFE
Conv. (%)[a]
eep (%)[a]
E[b]
wt
72
5
27
35
47
68
58
47
21
(R) 2.3
(R) 2.6
(R) 3.0
(R) 5.8
(R) 4.4
(R) 2.7
(S) 1.9
All variants selected for further studies were produced in E.
coli and purified by metal affinity chromatography (see SI, Fig. S1
and S2). Typical purification yields were in the range of 28-72 mg
L-1, generally lower than the expression yield of wild-type PFE
(105-120 mg L-1). The expression levels of purified proteins were
consistent with the results from the small-scale experiments using
the split-GFP methodology, in-between 25-64 % in comparison to
the wild type (100%). The level of expression was strongly
depended on the ncAA itself, as well as on the position in the
sequence. The incorporation of ncAAs in the proteins were
confirmed with electrospray ionization mass spectrometry (ESI-
MS), at different studied positions in at least one variant per
unnatural amino acid. No peaks corresponding to alternative
amino acid incorporation were observed (Fig. S3). The split-GFP
method is therefore straightforward in identifying any variants
where promiscuous incorporation is a potential problem. These
results, together with our negative controls for small-scale
screening suggests no (or minimal) background incorporation of
natural amino acids.
The effect of the ncAAs on the activity and enantioselectivity
were further studied using the kinetic resolution of ethyl 3-
phenylbutyrate (3, Scheme 1b). Results are summarized in Table
1. In several cases, significant effects on enantioselectivity were
observed. A significant increase in optical purity (% ee) was
achieved by substituting F198 by pAzF and F162 by NapA. The
substitution of F198 with pAzF nearly doubled the enantiomeric
ratio in the kinetic resolution of 3 showing preference for the (R)-
enantiomer. The enantioselectivity increase was even stronger
when F162 was substituted by NapA. Residue F198 had already
been reported in literature to be a hot-spot for enantioselectivity
of PFE.[17bd] This residue forms the acyl-binding pocket of the
enzyme and is in direct proximity with the substrate, around 3.5 Å
from the aromatic ring of the 3. On the other hand, a mutation of
the F162 residue, based on our knowledge, has not been reported
yet. This residue is part of the alcohol-binding pocket. The
mechanism, whose consequence is improvement of
enantioselectivity, is probably indirect through a rearrangement of
neighboring amino acid side chains. A substitution at position I224
has already yielded a switch in enantioselectivity in a previous
report.[17d] In the reported study, the substitution I224F led to an E
F158_pAzF
F158_NapA
F162_NapA
F198_pAzF
F198_NapA
I224_pAzF
14
23
26
9
18
[a] Determined by GC-FID analysis [b] calculated according to Chen et al. [20]
In summary, we report the engineering of the aryl esterase
from Pseudomonas fluorescens utilizing a pool of ncAAs.
Altogether five different aromatic and bulky ncAAs were used to
remodel the binding pocket of PFE, at ten different sites. The
resulting 50 PFE variants were expressed and their activity and
foldability were assessed. The PFE variants, which were selected
as attractive based on their activity and foldability, underwent
enantioselectivity screening using the ethyl ester of 3-
phenylbutyrate in a kinetic resolution. Substitution of F198 for
pAzF and F162 for NapA led to improved enantioselectivity of the
enzyme towards (R)-3. Substitution of I224 for pAzF swapped the
enantiopreference of the enzyme for (S)-3 and outperformed the
canonical amino acid substitution of I224Y, in enantiomeric ratio
by a factor of two. Our data demonstrate that the split-GFP self-
assembly method facilitates high-throughput screening of
production levels of an ncAA-incorporated enzyme. The speed
and simplicity of the method allow for easy screening of
expression levels, reliable elimination of misfolded variants and
those with incomplete translation, as well as estimation of ncAA-
carrying enzyme concentration in cell-free extracts. We expect
that this method will widely extend the capacity to test ncAA-
carrying enzyme variants in a short time and thus to overcome
obstacles for wide application of ncAA for enzyme engineering.
Ultimately, we showcase that the power of the method expands
beyond traditional directed evolution screenings to libraries of
ncAA-carrying enzymes.
4
This article is protected by copyright. All rights reserved.

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
[17]
a) J. D. Cheeseman, A. Tocilj, S. Park, J. D. Schrag, R. J. Kazlauskas,
Acta Crystallogr. Sect. D Biol. Crystallogr. 2004, 60, 1237–1243; b)
S. Park, K. L. Morley, G. P. Horsman, M. Holmquist, K. Hult, R. J.
Kazlauskas, Chem. Biol. 2005, 12, 45–54; c) A. Schließmann, A.
Hidalgo, J. Berenguer, U. T. Bornscheuer, ChemBioChem 2009, 10,
2920–2923; d) H. Jochens, U. T. Bornscheuer, ChemBioChem 2010,
11, 1861–1866.
Acknowledgements
The authors acknowledge financial support by the Competitive
Initial Funding Programme (TU Graz). Matúš Gajdoš and Alexia
Kindler acknowledge Erasmus+ of the European Union.
Keywords: biocatalysis • enzyme expression • non-canonical
amino acids • protein engineering • Pseudomonas fluorescens
esterase
[18]
a) T. S. Young, I. Ahmad, J. A. Yin, P. G. Schultz, J. Mol. Biol. 2010,
395, 361–374; b) A. Chatterjee, S. B. Sun, J. L. Furman, H. Xiao, P.
G. Schultz, Biochemistry 2013, 52, 1828–1837.
[19]
[20]
M. T. Reetz, Proc. Natl. Acad. Sci. USA. 2004, 101, 5716–5722.
C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc.
1982, 104, 7294–7299.
[1]
a) O. Boutureira, G. J. L. Bernardes, Chem. Rev. 2015, 115, 2174–
2195; b) A. Dumas, L. Lercher, C. D. Spicer, B. G. Davis, Chem. Sci.
2015, 6, 50–69; c) F. Agostini, J. S. Völler, B. Koksch, C. G. Acevedo-
Rocha, V. Kubyshkin, N. Budisa, Angew. Chem. Int. Ed. 2017, 56,
9680–9703, Angew. Chem. 2017, 129, 9810– 9835.
[2]
[3]
[4]
N. M. Okeley, W. A. van der Donk, Chem. Biol. 2000, 7, 159–171.
V. Kubyshkin, N. Budisa, Curr. Opin. Biotechnol. 2019, 60, 242–249.
a) K. Deepankumar, M. Shon, S. P. Nadarajan, G. Shin, S. Mathew,
N. Ayyadurai, B. G. Kim, S. H. Choi, S. H. Lee, H. Yun, Adv. Synth.
Catal. 2014, 356, 993–998; b) K. Deepankumar, S. P. Nadarajan, S.
Mathew, S. G. Lee, T. H. Yoo, E. Y. Hong, B. G. Kim, H. Yun,
ChemCatChem 2015, 7, 417–421; c) K. Ohtake, A. Yamaguchi, T.
Mukai, H. Kashimura, N. Hirano, M. Haruki, S. Kohashi, K. Yamagishi,
K. Murayama, Y. Tomabechi, et al., Sci. Rep. 2015, 5, 9762.
a) J. C. Jackson, S. P. Duffy, K. R. Hess, R. A. Mehl, J. Am. Chem.
Soc. 2006, 128, 11124–11127; b) J. N. Kolev, J. M. Zaengle, R.
Ravikumar, R. Fasan, ChemBioChem 2014, 15, 1001–1010; c) A. P.
Green, T. Hayashi, P. R. E. Mittl, D. Hilvert, J. Am. Chem. Soc. 2016,
138, 11344–11352.
[5]
[6]
a) I. Drienovská, C. Mayer, C. Dulson, G. Roelfes, Nat. Chem. 2018,
10, 946–952; b) M. Pott, T. Hayashi, T. Mori, P. R. E. Mittl, A. P.
Green, D. Hilvert, J. Am. Chem. Soc. 2018, 140, 1535–1543; c) C.
Mayer, C. Dulson, E. Reddem, A.-M. W. H. Thunnissen, G. Roelfes,
Angew. Chem. Int. Ed. 2019, 58, 2083–2087, Angew. Chem. 2019,
131, 2105-2109; d) A.J. Burke, S.L. Lovelock, A. Frese, R.
Crawshaw, M. Ortmayer, M. Dunstan, C. Levy, A.P. Green, Nature,
2019, 570, 219–223.
[7]
[8]
a) S. I. Lim, Y. Mizuta, A. Takasu, Y. H. Kim, I. Kwon, PLoS ONE
2014, 9, e98403; b) J. C. Y. Wu, C. H. Hutchings, M. J. Lindsay, C.
J. Werner, B. C. Bundy, J. Biotechnol. 2015, 193, 83–90; c) A. I.
Benítez-Mateos, I. Llarena, A. Sánchez-Iglesias, F. López-Gallego,
2018, ACS Synth. Biol. 2018, 7, 875–884.
C. G. Acevedo-Rocha, M. G. Hoesl, S. Nehring, M. Royter, C.
Wolschner, B. Wiltschi, G. Antranikian, N. Budisa, Catal. Sci. Technol.
2013, 3, 1198–1201.
[9]
I. Drienovská, G. Roelfes, Nat. Catal. 2020, 3, 193–202.
D. D. Young, P. G. Schultz, ACS Chem. Biol. 2018, 13, 854–870.
K. Wals, H. Ovaa, Front. Chem. 2014, 2, 15.
[10]
[11]
[12]
M. G. Hoesl, C. G. Acevedo-Rocha, S. Nehring, M. Royter, C.
Wolschner, B. Wiltschi, N. Budisa, G. Antranikian, ChemCatChem
2011, 3, 213–221.
[13]
[14]
[15]
M. G. Hoesl, N. Budisa, ChemBioChem 2011, 12, 552–555.
S. Cabantous, G. S. Waldo, Nat. Methods 2006, 3, 845–854.
J. Santos-Aberturas, M. Dörr, G. S. Waldo, U. T. Bornscheuer, Chem.
Biol. 2015, 22, 1406–1414.
[16]
N. Krebsfänger, K. Schierholz, U. T. Bornscheuer, J. Biotechnol.
1998, 60, 105–111.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202002077
Chemistry - A European Journal
COMMUNICATION
Entry for the Table of Contents
In enzyme engineering using an expanded genetical code, quick identification of fully translated and correctly folded variants is crucial.
Herein, we report the engineering of the aryl esterase from Pseudomonas fluorescens utilizing a pool of ncAAs. Key for the identification
of active and soluble protein variants was the use of the split-GFP method, allowing for quick identification of interesting variants.
Institute and/or researcher Twitter usernames: @IvanaDrienovska
6
This article is protected by copyright. All rights reserved.