Widely applicable background depletion step enables transaminase evolution through solid-phase screening

Article abstract of DOI:10.1039/c8sc05712e

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.

Full text of DOI:10.1039/c8sc05712e

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Widely applicable background depletion step
enables transaminase evolution through solid-
Cite this: DOI: 10.1039/c8sc05712e
phase screening†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
a
Matteo Planchestainer,
Eimear Hegarty,
Christian M. Heckmann,
*
b
ac
Louise J. Gourlay
and Francesca Paradisi
Directed evolution of transaminases is a widespread technique in the development of highly sought-after
biocatalysts for industrial applications. This process, however, is challenged by the limited availability of
effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and
widely applicable background depletion method for solid-phase screening of transaminase variants,
which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through
rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the
identification of transaminase variants in viable cells with significantly improved activity towards para-
substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates.
Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT
crystal structure presented herein.
Received 20th December 2018
Accepted 26th April 2019
DOI: 10.1039/c8sc05712e
rsc.li/chemical-science
subsequent engineering efforts of many ATAs, resulting in the
production of an array of highly specialised ATAs for chiral
amine synthesis.8,9,14
Introduction
Transaminases have attracted considerable attention for the
production of compounds containing chiral amino groups,
which are widespread in the pharmaceutical, agrochemical and
ne chemical industry.1,2 Of particular interest, in terms of
synthetic applications, are the amine transaminases (ATAs), (R)-
or (S)-selective enzymes that are capable of aminating carbonyls
(both aldehydes and ketones) in the absence of a neighbouring
carboxylic group.3 Despite the enormous potential of ATAs, the
widespread application of these biocatalysts in their wild type
form is hindered by a number of challenges including limited
substrate specicities, substrate and product inhibition, and
unfavourable reaction equilibria.4
Protein engineering by rational design5–7 and directed
evolution8,9 have played a major role in the ability to address
these issues. Evolutionary strategies, in particular, are ideally
suited to the optimisation of the physical properties of enzymes
for large-scale manufacturing processes, as well as enabling
ne-tuning of substrate and stereochemical preferences.10,11
The evolution of ATAs by Codexis and Merck12,13 pioneered
Even though a fully randomised approach to mutagenesis,
targeting the whole length of the protein, has become less
common, directed evolution clearly requires high-throughput
screening methods to evaluate variable size libraries of engi-
neered variants. The generation of smaller, smart libraries
designed by rational targeted approaches such as CASTing,15 or
saturation mutagenesis of specic residues identied in silico,
still yield a sizable number of clones to screen (saturation at
three positions affords 8000 variants). The identication of hits
within live cells is advantageous for library screening because, if
it is efficient, it prevents culturing of large number of clones
followed by assays with cell extracts, which, in the absence of
robotics, is very time-consuming.16 Recently, microuidic
screening,17,18 in addition to the application of desorption
electrospray ionisation mass spectrometry19 on whole colonies,
have emerged as powerful tools for screening large libraries,
however, the equipment required in both cases is still highly
specialised and expensive, therefore they are not yet suitable for
widespread implementation. Solid-phase screening (SPS), on
the other hand, is a simpler approach that facilitates the
screening of a medium-large number of mutant variants based
on colorimetric changes that are easily observed by the naked
eye.20 SPS was successfully applied in the evolution of mono-
amine oxidase (MAO-N) enzymes, using two libraries that were
fully saturated at two positions each (400 variants).21 SPS
methods applicable to ATA mutant libraries, however, are
currently limited to a few strategies to screen for amino donors.
aUniversity of Nottingham, School of Chemistry, University Park, Nottingham NG7
2RD, United Kingdom
b
`
Structural Biology Unit, Dep. Biosciences, Universita degli Studi di Milano, Via
Celoria 26, 20133 Milano, Italy
cUniversity of Bern, Department of Chemistry and Biochemistry, Freiestrasse 3, 3012
Bern, Switzerland. E-mail: francesca.paradisi@dcb.unibe.ch
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8sc05712e
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Wu and co-workers, for example, reported the use of SPS to
Test screening of pre-treated colonies to establish timelines
screen libraries of approximately 100 000 ATA variants for and reaction efficiency, were performed with both pyruvate and
reduced inhibition towards methoxyisopropylamine.22 Later, acetophenone and compared with a control plate not supple-
Bornscheuer and colleagues applied SPS to evolve an ATA mented with any amino acceptor (Fig. S2†). ortho-Xylylenedi-
towards suitable amino donors (screening ca. 4600 colonies), amine was used in the rst instance as an amino donor. The
however this required co-expression of a second enzyme as control plate did not give any colorimetric change up to two
a coupled reporting system, increasing the complexity of the hours, aer which time a slightly brown tinge could be observed
system.23,24 Similarly, Turner and co-workers evolved SPS for likely due to the cell healthy metabolic system producing
ATA library screening, specically to identify either (R)- or (S)- sufficient amounts of pyruvate detectable through the reaction.
selective ATAs (screening ca. 1000 colonies), which also In the absence of the background depletion step, the cells
required multiple reporting enzymes, as well as ash-freezing in almost immediately respond to the amino donor preventing any
liquid nitrogen, to increase permeability.25
signicant discrimination between exogenous amino acceptor
Screening strategies for varying carbonyl acceptors, which and the endogenous pyruvate (Fig. 1), as was also observed by
are key substrates for ATAs to produce chiral amine products, O'Reilly and co-workers.26 Likewise, the addition of pyruvate to
appear to be much more challenging. ortho-Xylylenediamine26 the membrane as the amino acceptor, causes visible darkening
and 4-nitrophenethylamine27 have been recently reported as of the colonies within 5 minutes of incubation. Acetophenone,
amine donors in proof of concept studies to rapidly generate which in liquid-phase assays had shown no detectable activity
coloured by-products upon transamination. Both these strate- over 3 h with wild-tpe HEWT, performs similarly to the control
gies suffer from strong background interference in live cells due plate, with no observable colour formation during total incu-
to endogenous pyruvate which is nearly always an excellent bation (Fig. S2†). The mild treatment, preserved cell viability,
substrate for ATAs; in a mutant library screening, the number of which is essential for the direct growth and plasmid extraction,
false positives is certain to be signicant without an efficient bypassing the need for a replica plate. To further evaluate the
system to deplete background. Pyruvate is a primary metabolite, effectiveness of this strategy, 4-nitrophenethylamine was used
essential for cell growth, therefore knock-out strategies are not as amino donor in the assay.27 Remarkably, a signicant
feasible, making it inherently difficult to minimise the back- reduction in background noise was observed once more, high-
ground noise in SPS.
lighting the transferability of this methodology (should o-xyly-
We here report the development of a simple and reliable SPS lenediamine not be accepted by the investigated TA). Indeed,
method that encapsulates (S)-1-phenylethylamine as a straight- the background was even further suppressed when compared to
forward background depletion agent to minimize high levels of the ortho-xylylenediamine screen, with barely detectable dark-
pyruvate activity. To highlight the power of this methodology, ening of the colonies aer 24 h (Fig. S3†). Liquid nitrogen- and
we successfully applied it to the screening of a fully randomised chloroform-induced permeabilization steps were attempted
mutant library of an (S)-selective ATA from Halomonas elongata also,24,25 but in our case neither method was successful.
(HEWT)28 against a series of prochiral aromatic and cyclic
The use of amines other than (S)-1-phenylethylamine, such
ketones. In addition, the crystal structure of the wild-type as isopropylamine and cadaverine, for the background deple-
HEWT is reported, which facilitated rationalisation of the tion step was also investigated. Cadaverine, previously
mutations introduced.
described as a “smart” amine donor,29 can be used in ATA
reactions ex vivo and may effectively convert endogenous pyru-
vate in whole cells. Isopropylamine is a very inexpensive amine
donor, commonly used in industrial processes;12 the generated
acetone is produced in minimal quantities and expected to be
harmless to colonies. While both amines also resulted in
Results and discussion
Depletion of background activity and test screening
Reliable methodology to quench background activity, which
leads to a high number of false positives, was designed to
specically deplete endogenous pyruvate. Unlike previously
discussed methods, our alternative approach is based on pre-
reacting cellular pyruvate with (S)-1-phenylethylamine. The
reaction is catalysed by the expressed transaminase to produce
alanine and acetophenone with an extremely favourable equi-
librium, effectively removing pyruvate as a non-specic acceptor
from the environment. The screening was initially optimised
with E. coli BL21(DE3) cells expressing wild-type HEWT, which
were spread onto a nitrocellulose membrane deposited on a LB-
agar plate. This permitted easy transfer of the colonies through
the different assay steps (Fig. S1†). Following expression and
dialysis (Experimental in the ESI†), the membrane was le for
30 minutes on lter paper soaked with (S)-1-phenylethylamine
and then washed with 50 mM phosphate buffer, pH 8.
Fig.
1 ortho-Xylylenediamine solid screening control. E. coli
BL21(DE3) cells expressing wild-type HEWT after 1 h of incubation with
ortho-xylylenediamine at RT, following background depletion step (A),
and in the absence of such pre-treatment (B).
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a signicant reduction in background (Fig. S4†), (S)-1-phenyl- template did not provide any signicant hits, an improved
ethylamine appeared to be most effective, possibly due to better variant was isolated from the B1 library. The new mutant,
diffusion of this substrate. However, this demonstrates that identied as B2, afforded comparable molar conversion aer 2
other amines can also be used to quench endogenous pyruvate, hours and a further 1.5-fold increase in turn-over frequency
for example with aminotransferases that do not accept 1- (Table 1). The e.e. measured at 24 h was in all cases >99%
phenylethylamine as a substrate.
(though the overall yields were low due to experimental condi-
tions needed to assess the kinetic behaviour of the mutants). A
third round of mutagenesis, using the B2 variant as a template,
initially failed to identify better variants, as the colour change
on solid screening was too rapid and did not allow discrimi-
nation between parental activity and mutants. Reducing the
concentration of para-nitroacetophenone and ortho-xylylenedi-
amine from 10 mM to 1 mM, with just 5% (v/v) DMSO prolonged
the screening window to ca. 1 h. An additional variant, B3, was
isolated from the screening and while, upon purication, it
displayed comparable reaction velocity and 2 h conversions to
B2, it had 2-fold higher expression levels which justies a more
rapid colour development with respect to the parental variant.
Each variant was also fully characterised in terms of activity and
stability at different temperatures and pHs. In all the cases, the
mutants generated did not show signicant alterations when
compared to the WT enzyme (Fig. S5–S8†).
A second substrate, para-cyanoacetophenone (2a), was also
investigated. HEWT wild-type has negligible activity towards
this molecule, even lower than the nitro substitution, with
a turnover frequency of 4 ꢀ 10ꢁ3 sꢁ1. The B2 variant showed an
impressive 60-fold increase in turn-over frequency (229 ꢀ 10ꢁ3
sꢁ1), and enhanced stereoselectivity, affording >99% of the (S)-
enantiomer (Table 1). Again, B3 showed virtually identical
catalytic properties to B2.
Evolution towards substituted acetophenones
To test the methodology, HEWT was subjected to directed
evolution using error-prone PCR to generate a completely
randomised mutant library with a high number of mutations.
The enzyme has minimal activity towards the aromatic
substrate para-nitroacetophenone (1a) and this was selected as
the amino acceptor for the initial screening, using ortho-xyly-
lenediamine as the amino donor. DMSO (10% (v/v)) was
required for substrate solubility and enhanced cell-
permeability. The discrimination between wild-type and an
improved variant relies on the rapidity with which the enzyme
converts the substrate in vivo. Screening of ca. 15 000 colonies
provided two variants with enhanced catalytic activity towards
para-nitroacetophenone. The two new variants (referred to as A1
and B1) were expressed and puried for further character-
isation. They exhibited a 2-fold increase in turnover frequency
with respect to the wild-type, achieved higher conversion aer 2
hours, and maintained excellent enantioselectivity (Table 1).
Both variants were subjected to a second round of error-prone
PCR and the libraries screened as before with 1a. Interest-
ingly, in this case the positive colonies were isolated aer
a
signicantly shorter incubation period (2–15 minutes
compared to 25 minutes for the parental colony). While the A1
Table 1 HEWT evolution towards substituted acetophenones. Turnover frequencies (TOF) measured at 10 mM carbonyl concentration, 2 hour
molar conversions (m.c.), and enantiomeric excess (e.e.) for wild-type HEWT and isolated variants towards para-nitro- (1a) and para-cyanoa-
cetophenone (2a)a
WT
A1
B1
B2
B3
TOF
m.c.
e.e.
(%)
TOF
m.c.
e.e.
(%)
TOF
m.c.
e.e.
(%)
TOF
m.c.
e.e.
(%)
TOF
m.c.
e.e.
(%)
Substrate
(10ꢁ3 sꢁ1) (%)
(10ꢁ3 sꢁ1) (%)
(10ꢁ3 sꢁ1) (%)
(10ꢁ3 sꢁ1) (%)
(10ꢁ3 sꢁ1) (%)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
36 ꢂ 6
4 ꢂ 1
4
9
66 ꢂ 9
6
65 ꢂ 26
6
92 ꢂ 5
7
84 ꢂ 14
7
>99
(S)
>99
(S)
>99
(S)
87 (S) 50 ꢂ 7
11
94 (S) 127 ꢂ 5 12
229 ꢂ 7 11
246 ꢂ 7 12
a
Biotransformation reactions were performed with 10 mM ketones, 500 mM L-alanine, 0.1 mM PLP, 0.1 mg mLꢁ1 (1.9 mM) enzyme in 50 mM
phosphate buffer pH 8 and 10% (v/v) DMSO at 37 ꢃC (see Experimental in the ESI, Fig. S9). All experiments were conducted in triplicate and the
standard error is reported accordingly. Mutations; A1: W56C, V435A; B1: W56C, L211V, L306M; B2: W56C, L211V, L306M, V361A, Q388R, P453L;
B3: W56C, L211V, A254V, L306M, V361A, Q388R, P453L.
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The C1 variant was subsequently screened against a panel of
representative cyclic ketones (Table 2, 4a, 5a, 6a). The mutant
had improved TOF with both tetrahydrofuan-3-one (4a) and
1-methylpiperidin-3-one (6a) (35% and 90% increase, respectively)
but it was in fact slower with 1-methylpyrrolidin-3-one (5a) (see
Table S1†). Similarly to what was observed with the library evolved
towards para-nitroacetophenone, an additional round of muta-
genesis did not permit the identication of better variants towards
tetrahydrothiophen-3-one (3a) as the colour change on the plates
was too rapid to discriminate further enhanced variants.
This clearly demonstrates that this solid phase screen is able
to identify better variants, both in terms of TOFs and expression
levels, from libraries that are of desirable size for semi-rational
approaches to directed evolution.10 The application of this
methodology was successful even with a fully randomised
approach which can only afford limited improvements of the
mutants, given the size of the library, due to the high probability
of unsuitable mutations.31
Evolution towards small cyclic ketones
To further expand the catalytic scope of HEWT, the original
mutant library was screened towards a structurally different
substrate, tetrahydrothiophen-3-one (3a), a small cyclic ketone
which if successfully aminated yields a chiral amine (3b) that
has potential use as a building block for active pharmaceutical
ingredients.30
In this case, the wild-type enzyme already possesses some
activity towards the target substrate (3a), with a turnover
frequency of 206 ꢀ 10ꢁ3 sꢁ1 and complete molar conversion
(>99%) aer 24 h using equimolar amounts of (S)-1-phenyl-
ethylamine as donor and higher catalyst concentration (1 mg
mLꢁ1 – 19 mM). The optimised solid phase screening with ortho-
xylylenediamine yielded a new variant (C1) which, upon
expression and purication, displayed a 2-fold increase in
turnover frequency (Table 2). On this occasion, the mutant
showed a colour change aer 18 minutes, compared to 30
minutes for the wild-type control. Notably, the variant matched
the excellent conversion displayed by the wild-type enzyme,
affording the (S)-enantiomer of the corresponding amine with
>99% conversion and 66% e.e.
3D structural analysis of wild type HEWT and mutant variants
In order to understand the effect of the various mutations on
wild-type HEWT, the crystal structure was determined at
Table 2 HEWT evolution toward small cyclic ketones. Turnover frequencies (TOF) measured at 10 mM carbonyl concentration, molar
conversions (m.c.), and enantiomeric excess (e.e.) for wild-type HEWT and isolated variants towards tetrahydrothiophen-3-one (3a) tetrahy-
drofuran-3-one (4a), 1-methylpyrrolidin-3-one (5a) and 1-methyl-piperidin-3-one (6a). Final conversion was obtained within 2 hours of
reactiona
WT
C1
Substrate
TOF (10ꢁ3 sꢁ1
)
m.c. (%)
>99
e.e. (%)
TOF (10ꢁ3 sꢁ1
)
m.c. (%)
>99
e.e. (%)
206 ꢂ 5
430 ꢂ 1
66 (S)
460 ꢂ 6
580 ꢂ 3
66 (S)
>99
94
70 (S)
90 (S)
>99
94
70 (S)
90 (S)
18 ꢂ 0.2
10 ꢂ 0.8
11.6 ꢂ 0.3
19 ꢂ 0.8
98
90 (S)
98
90 (S)
a
Biotransformation reactions were performed with 10 mM ketones, 10 mM (S)-1-phenylethylamine, 0.1 mM PLP, 1 mg mLꢁ1 (19 mM) enzyme in
50 mM phosphate buffer pH 8 and 10% (v/v) DMSO at 37 ꢃC (see Experimental in the ESI, Fig. S10). All experiments were conducted in
triplicate and the standard error is reported accordingly. Mutation in C1: D5E.
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˚
a resolution of 2 A, as described in the methods. Atomic coor- contributed from each monomer, with an interface area of 5395
2
˚
dinates and structure factors have been deposited in the Protein A . The main dimerisation interactions include 4 salt bridges
Data Bank (www.rcsb.org) under PDB accession code 6GWI.
and 47 hydrogen bonds, as calculated by PDBsum available
Two identical HEWT monomers (chains A and B; RMSD 0.23 from the European Bioinformatics Institute (www.ebi.ac.uk).
˚
A) were present in the asymmetric unit (Matthews coefficient ¼
Based on a secondary structure-based homology search, the
3
2.14 A Daꢁ1), with an estimated solvent content of 42.5% (see DALI server identied the 4-aminobutyrate transaminase from
˚
Table S2†). This homodimer arrangement is biologically rele- Pseudomonas as the top hit (PDB entry 5KR6 32) with a RMSD of
vant as HEWT is a catalytically active dimer. Electron density 1.3 A over 445/460 aligned main chain residues.33 Sequence and
˚
was present for residues 2 to 451 (chain A) and 1 to 450 (chain structure conservation between HEWT and all other structures
B). The HEWT monomer comprises 3 sub-domains: (i) a prin- deposited in the PDB was assessed using ENDscript 2.0 (http://
ciple PLP-dependent transferase-like domain (residues 82 to endscript.ibcp.fr).34 HEWT was compared with 124 structures
318) that hosts a central mixed b-sheet, comprising 7 strands, with sequence conservation >30%. As expected, the highest
(b9–b8–b10–b11–b12–b13–b7), with b-strand 13 being antipar- conservation (sequence and structural) is located in the PLP-
allel to the rest, surrounded by 9 a-helices and two 310 helices dependent transferase-like domain and active site region
(h1–2) (Fig. 2); (ii) domain 2 (residues 2–79; 320–451) that (Fig. 3A).
contains a small N-terminal sub-domain (residues 2 to 32; a1–
Sequencing of the selected mutants identied two amino
b1–b2–a2–b3–5) which forms the main interactions with the acid changes in the A1 variant, (W56C, V435A), while the B1
opposing monomer. One molecule of pyridoxal-50-phosphate variant displayed three amino acid substitutions (W56C, L211V,
(PLP), produced by the covalent attachment to the 3-amino L306M) (notes in the ESI†). Interestingly, both mutants harbour
group of the catalytic lysine (K284), via an aldimine linkage, is a tryptophan to cysteine change in position 56, which is located
bound to each monomer. Dimerisation involves 100 residues in the active site. This residue has previously been identied as
a hotspot in in silico rational design studies.7,35 In this case,
removing the large indole ring and replacing it with the smaller
cysteine side chain allows for easier substrate binding.
Furthermore, the thiol can promote hydrogen bonding with the
Fig. 3 HEWT 3D structure conservation and mutant residue positions.
Secondary structure of the HEWT monomer shown in sausage
representation, as automatically generated by ENDscript 2.0 (http://
endscript.ibcp.fr).34 Structure conservation between chain A of HEWT
and 124 structure homologs present in the PDB is indicated by ribbon
thickness, with regions of low conservation being thicker than highly
conserved regions (thin regions). Sequence conservation is indicated
by red shading; the redder the residue, the more conserved it is.
Mutated residues in variants A1 (blue), B1 (yellow), B2 (green) and C1
(orange) are shown as sticks. The B3 mutant shares all B2 mutations
and contains an extra A254V mutation (pink sticks). P453 from variants
Fig. 2 The crystal structure of the HEWT WT monomer. Cartoon B2 and B3 is not present in the model. The N- and C-termini are
secondary structure representation of the (A) PLP-dependent trans- indicated and PLP is shown in sticks. This figure was generated using
ferase-like domain (residues 82 to 318), with PLP indicated in sticks. Pymol 2.0.6. Modelling of the active site for the A1 and B1 mutants,
For simplicity, the other domain is not coloured. The N-and C-termini where the bulky Trp (the steric encumbrance of the residue is shown
are labelled. (B) The second domain of HEWT, comprising a subdomain as an orange cloud) (D) is substituted with a cysteine (E), allowing the
(labelled) that mediates dimerisation with the opposing monomer. In aromatic ring substituent (para-nitroacetophenone in the aldimine
this case, the PLP-dependent domain is not shown for clarity. This intermediate complex with the pyridoxamine phosphate (PMP)) to be
figure was generated using Chimera.36
more easily accommodated.
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aromatic substituent and stabilise para-nitroacetophenone enzymes a much less challenging task. The use of 4-nitro-
(Fig. 3B and C). Accordingly, W56 is located in a highly struc- phenethylamine as an alternative reporter, as well as alternative
turally conserved region (Fig. 3A), as is V435 (Fig. 3A). The amine donors for the depletion step, proved successful. The
L306M mutation is peripheral and not conserved with regards limit of this screen is set by the power of discrimination
to sequence, and therefore less conserved than the A1 muta- between the mutant and the parent enzyme; if the substrate
tions with regards to structure, and therefore is somewhat more conversion is too fast, the colonies turn black rapidly, which
difficult to rationalise. The L306M mutation introduces a longer leads to false positives. Tuning the screening conditions to slow
side chain that may form more stabilising hydrophobic inter- the reaction can extend the screening window. In addition,
actions with surrounding hydrophobic residues (M126, V302 through modulation of the parental activity, by regulating the
and F313). From the structure, this hydrophobic cluster may in- expression levels (with the use of a promoter that allows for
turn stabilise regions of the protein that mediate dimerisation stricter control),38 this screening method could be a very
(Fig. S11†). In agreement with this hypothesis, thermal stability powerful tool in further rounds of evolution towards the iden-
analysis of the wild-type and mutant B2 indicates that the latter tication of more and more active mutants.
is more stable (Fig. S12†). The second round of evolution
introduced three additional mutations (V361A, Q388R, P453L)
in the B2 variant, which are also located outside of the active site
Conflicts of interest
and all are exposed to the solvent; their side-chains do not make There are no conicts to declare.
any signicant stabilising intramolecular interactions. B3
introduced mostly silent mutations, apart from a single A254V
mutation neighbouring the D255 residue, forming a hydrogen
Acknowledgements
bond with the pyridine-type nitrogen of the co-factor PLP.
This work was supported by the Biotechnology and Biological
On the other hand, structural analysis of the C1 variant Sciences Research Council [grant number BB/P002536/1 and
revealed a single mutation (D5E) positioned at the N-terminus BB/M008770]. The authors thank the University of Nottingham
of the protein (Fig. 3A). Using the protein structure quasi-rigid through the Green Chemicals Beacon for support. Structural
`
domain decomposition (PiSQRD) web server (http:// studies were supported by Universita degli Studi di Milano
pisqrd.escience-lab.org),37 the HEWT crystal structure (chain Linea 2 funding.
A) was decomposed into four sub-domains that can behave as
independent units during conformational uctuations
(Fig. S13†). D5 is located in domain 4 (residues 2–34 and 41–46)
References
that adopts an a1-helix–loop–a2-helix structure. Based on these
observations, an aspartate to glutamate mutation may alter the
local protein dynamics that in-turn may cause alterations in the
topology of the active site and hence substrate specicity and
activity.
1 I. Slabu, J. L. Galman, R. C. Lloyd and N. J. Turner, ACS
Catal., 2017, 7, 8263–8284.
2 M. Fuchs, J. E. Farnberger and W. Kroutil, Eur. J. Org. Chem.,
2015, 2015, 6965–6982.
3 F. Steffen-Munsberg, C. Vickers, H. Kohls, H. Land,
H. Mallin, A. Nobili, L. Skalden, T. van den Bergh,
¨
H. J. Joosten, P. Berglund, M. Hohne and
Conclusions
U. T. Bornscheuer, Biotechnol. Adv., 2015, 33, 566–604.
4 I. Slabu, J. L. Galman, C. Iglesias, N. J. Weise, R. C. Lloyd and
N. J. Turner, Catal. Today, 2018, 306, 96–101.
5 K. S. Midelfort, R. Kumar, S. Han, M. J. Karmilowicz,
K. McConnell, D. K. Gehlhaar, A. Mistry, J. S. Chang,
M. Anderson, A. Villalobos, J. Minshull, S. Govindarajan
and J. W. Wong, Protein Eng. Des. Sel., 2013, 26, 25–33.
6 L. Skalden, C. Peters, J. Dickerhoff, A. Nobili, H. J. Joosten,
The introduction of an amine donor as a background depletion
agent, which exploits the expressed ATA to quench endogenous
pyruvate proved to be very effective in delivering an efficient
screening platform for aminotransferase directed evolution.
This high-throughput screening strategy was developed on the
basis of the ortho-xylylenediamine assay described by O'Reilly
and co-workers,26 which was a fundamental tool in the evolution
of HEWT towards a diverse set of aromatic and cyclic ketones.
Aer three rounds of directed evolution, a 2.5-fold increase in
activity was obtained for para-nitroacetophenone, while with
para-cyanoacetophenone the identied mutant exhibited a 60-
fold higher turnover frequency coupled with a substantial
improvement of the enantioselectivity with respect to the wild
type. Furthermore, the evolution towards tetrahydrothiophen-3-
one yielded a 2-fold increase in the overall catalytic efficiency.
This enzyme also showed improved catalytic properties when
¨
K. Weisz, M. Hohne and U. T. Bornscheuer, ChemBioChem,
2015, 16, 1041–1045.
7 K. E. Cassimjee, M. S. Humble, H. Land, V. Abedib and
P. Berglund, Org. Biomol. Chem., 2012, 10, 5466–5470.
8 H. Yun, B. Y. Hwang, J. H. Lee and B. G. Kim, Appl. Environ.
Microbiol., 2005, 71, 4220–4224.
9 I. V. Pavlidis, M. S. Weiß, M. Genz, P. Spurr, S. P. Hanlon,
B. Wirz, H. Iding and U. T. Bornscheuer, Nat. Chem., 2016,
1–7.
screened against similarly challenging cyclic ketones. The 10 U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz,
background depletion step is straightforward and reliable, and J. C. Moore and K. Robins, Nature, 2012, 485, 185–194.
can be applied to other screening methods where endogenous 11 H. Renata, Z. J. Wang and F. H. Arnold, Angew. Chem., Int.
pyruvate interferes with detection making the evolution of these
Ed., 2015, 54, 3351–3367.
Chem. Sci.
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Edge Article
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12 C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, 25 S. C. Willies, J. L. Galman, I. Slabu and N. J. Turner, Philos.
W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, Trans. R. Soc. A Math. Phys. Eng. Sci., 2016, 374, 20150084.
P. N. Devine, G. W. Huisman and G. J. Hughes, Science, 2010, 26 A. P. Green, N. J. Turner and E. O'Reilly, Angew. Chem., Int.
329, 305–310. Ed., 2014, 53, 10714–10717.
13 J. Limanto, E. R. Ashley, J. Yin, G. L. Beutner, B. T. Grau, 27 D. Baud, N. Ladkau, T. S. Moody, J. M. Ward and
A. M. Kassim, M. M. Kim, A. Klapars, Z. Liu, H. C. Hailes, Chem. Commun., 2015, 51, 17225–17228.
H. R. Strotman and M. D. Truppo, Org. Lett., 2014, 16, 28 L. Cerioli, M. Planchestainer, J. Cassidy, D. Tessaro and
2716–2719. F. Paradisi, J. Mol. Catal. B Enzym., 2015, 120, 141–150.
14 A. R. Martin, R. DiSanto, I. Plotnikov, S. Kamat, D. Shonnard 29 A. Gomm, W. Lewis, A. P. Green and E. O'Reilly, Chem. – Eur.
and S. Pannuri, Biochem. Eng. J., 2007, 37, 246–255. J., 2016, 22, 12692–12695.
15 M. T. Reetz, J. D. Carballeira, J. Peyralans, H. Hobenreich, 30 X. Chen, J. Adrian, T. Cushing, H. DiMaio, L. Liang,
¨
A. Maichele and A. Vogel, Chem. – Eur. J., 2006, 12, 6031–
6038.
16 H. Xiao, Z. Bao and H. Zhao, Ind. Eng. Chem. Res., 2015, 54,
4011–4020.
17 R. Obexer, A. Godina, X. Garrabou, P. R. E. Mittl, D. Baker,
A. D. Griffiths and D. Hilvert, Nat. Chem., 2017, 9, 50–56.
18 C. K. Longwell, L. Labanieh and J. R. Cochran, Curr. Opin.
Biotechnol., 2017, 48, 196–202.
V. Mayorga, S. Miao, M. G. Peterson, J. P. Powers,
F. Spector, C. Stein, M. Wright, D. Xu, Q. Ye and J. Jaen,
Bioorganic Med. Chem. Lett., 2007, 17, 2188–2192.
31 D. A. Drummond, B. L. Iverson, G. Georgiou and
F. H. Arnold, J. Mol. Biol., 2005, 350, 806–816.
32 M. Wilding, T. S. Peat, S. Kalyaanamoorthy, J. Newman,
C. Scott and L. S. Jermiin, Green Chem., 2017, 19, 5375–5380.
¨
33 L. Holm and P. Rosenstrom, Nucleic Acids Res., 2010, 38, 545–
19 C. Yan, F. Parmeggiani, E. A. Jones, E. Claude, S. A. Hussain,
549.
N. J. Turner, S. L. Flitsch and P. E. Barran, J. Am. Chem. Soc., 34 X. Robert and P. Gouet, Nucleic Acids Res., 2014, 42, W320–
2017, 139, 1408–1411. W324.
20 F. H. Arnold and G. Georgiou, Directed Enzyme Evolution, 35 A. Nobili, Y. Tao, I. V. Pavlidis, T. Van Den Bergh,
Humana Press, New Jersey, 2003, vol. 230.
21 I. Rowles, K. J. Malone, L. L. Etchells, S. C. Willies and
N. J. Turner, ChemCatChem, 2012, 4, 1259–1261.
22 G. Matcham, M. Bhatia, W. Lang, C. Lewis, R. Nelson,
A. Wang and W. Wu, Chim. Int. J. Chem., 1999, 53, 584–589.
H. J. Joosten, T. Tan and U. T. Bornscheuer, ChemBioChem,
2015, 16, 805–810.
36 E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch,
D. M. Greenblatt, E. C. Meng and T. E. Ferrin, J. Comput.
Chem., 2004, 25, 1605–1612.
23 M. S. Weiß, I. V. Pavlidis, P. Spurr, S. P. Hanlon, B. Wirz, 37 T. Aleksiev, R. Potestio, F. Pontiggia, S. Cozzini and
H. Iding and U. T. Bornscheuer, Org. Biomol. Chem., 2016,
14, 10249–10254.
24 M. S. Weiß, I. V. Pavlidis, C. Vickers, M. Hohne and
U. T. Bornscheuer, Anal. Chem., 2014, 86, 11847–11853.
C. Micheletti, Bioinformatics, 2009, 25, 2743–2744.
38 B. C. Joseph, S. Pichaimuthu and S. Srimeenakshi, J. Cell Sci.
Ther., 2015, 6, 221.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci.