The identification and use of robust transaminases from a domestic drain metagenome

Article abstract of DOI:10.1039/c8gc02986e

Transaminases remain one of the most promising biocatalysts for use in chiral amine synthesis, however their industrial implementation has been hampered by their general instability towards, for example, high amine donor concentrations and organic solvent content. Herein we describe the identification, cloning and screening of 29 novel transaminases from a household drain metagenome. The most promising enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unprecedented robustness for a wild-type transaminase. One enzyme in particular readily accepted IPA as an amine donor giving the same conversion with 2-50 equivalents, as well as being tolerant to a number of co-solvents, and operational in up to 50% DMSO-a characteristic as yet unobserved in a wild-type transaminase. This work highlights the value of using metagenomics for biocatalyst discovery from niche environments, and here has led to the identification of one of the most robust native transaminases described to date, with respect to IPA and DMSO tolerance.

Full text of DOI:10.1039/c8gc02986e

Volume 21 Number 1 7 January 2019 Pages 1–188
Green
Chemistry
Cutting-edge research for a greener sustainable future
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ISSN 1463-9262
PAPER
John M. Ward, Helen C. Hailes et al.
The identification and use of robust transaminases from a
domestic drain metagenome

Green Chemistry
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The identification and use of robust transaminases
from a domestic drain metagenome†
Cite this: Green Chem., 2019, 21, 75
a
b
b
b
Leona Leipold, Dragana Dobrijevic, Jack W. E. Jeffries, Maria Bawn,
c
b
a
Thomas S. Moody, John M. Ward * and Helen C. Hailes
*
Transaminases remain one of the most promising biocatalysts for use in chiral amine synthesis, however
their industrial implementation has been hampered by their general instability towards, for example, high
amine donor concentrations and organic solvent content. Herein we describe the identification, cloning
and screening of 29 novel transaminases from a household drain metagenome. The most promising
enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-
solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unpre-
cedented robustness for a wild-type transaminase. One enzyme in particular readily accepted IPA as an
amine donor giving the same conversion with 2–50 equivalents, as well as being tolerant to a number of
co-solvents, and operational in up to 50% DMSO – a characteristic as yet unobserved in a wild-type trans-
aminase. This work highlights the value of using metagenomics for biocatalyst discovery from niche
environments, and here has led to the identification of one of the most robust native transaminases
described to date, with respect to IPA and DMSO tolerance.
Received 21st September 2018,
Accepted 14th November 2018
DOI: 10.1039/c8gc02986e
rsc.li/greenchem
Over the past decade, transaminases have garnered a huge
Introduction
amount of interest in the field of biocatalysis, and remain one
5
Amine-containing drugs represent an important class of thera- of the most promising enzymes for chiral amine synthesis.
peutics, with a recent analysis of the FDA database finding They are a class of pyridoxal 5′-phosphate (PLP) dependent
that 84% of approved small molecule drugs contain at least enzymes that catalyse the reversible transfer of an amino
1
one nitrogen. In recent years, the use of biocatalysts for single group from an amine donor to a ketone or aldehyde. The sim-
isomer chiral amine synthesis has gained significant traction, plicity and broad availability of the starting materials, as well
with the pharmaceutical industry identifying the sustainable as the excellent stereoselectivity associated with these
2
13
production of chiral amines as a key research priority.
enzymes, make them valuable reagents for further research.
Traditional chiral amine synthesis often requires the use of Transaminases have been employed in the synthesis of a
1
4
toxic transition metal catalysts, protecting groups, multi-step number of pharmaceutical compounds, both on a small and
3
procedures and harsh conditions. These factors mean, that as industrial scale, as demonstrated in the manufacture of the
4
15
well as offering the advantages attributed to biocatalysis, bio- antidiabetic drug, Sitagliptin. Recent work has also demon-
catalytic aminations in particular are significantly more sus- strated the benefit of using transaminases in the synthesis of
tainable than their chemical counterpart. A number of small molecule amines, which contain functional groups that
1
6
different enzymes catalyse the formation of chiral amines, are susceptible to reductive chemical reaction conditions.
5
,6
7
including transaminases (TAms),
monoamine oxidases,
Despite the promise that transaminases hold in biocatalytic
syntheses, progress has been hampered by the unfavourable
reaction equilibrium and poor enzyme stability.
8
,9
10
11,12
imine reductases, lipases and amine dehydrogenases.
1
7,18
Researchers have tried to shift the equilibrium towards the
desired amine product, either by removal of the ketone by-
a
Department of Chemistry, University College London, 20 Gordon Street,
London WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk
1
3,19–24
b
product or by using a large excess of amine donor.
In
The Advanced Centre for Biochemical Engineering, Department of Biochemical
Engineering, University College London, Bernard Katz Building, Gower Street,
London WC1E 6BT, UK. E-mail: j.ward@ucl.ac.uk
this regard, isopropylamine (IPA) is often described as the
ideal amine donor for industrial scale up; it is relatively cheap
c
Department of Biocatalysis and Isotope Chemistry, Almac,
25,26
and acetone, the ketone by-product, is highly volatile.
However, IPA is not broadly accepted as an amine donor,
20 Seagoe Industrial Estate, Craigavon, Northern Ireland, UK
26,27
†
Electronic supplementary information (ESI) available: Taxonomical assign-
and the high equivalents required to shift the equilibrium
ments and sequence information, protein gels, analytical methods and traces,
calibration curves, kinetic data and NMR spectra. See DOI: 10.1039/c8gc02986e
1
8
often leads to denaturation of the enzyme. Even in the case
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of Sitagliptin, the (R)-selective transaminase was subjected to Fig. S1†) and percentage identities ranged from 17–54% for
multiple rounds of evolution to increase its tolerance towards Cv-TAm, 16–36% for Vf-TAm and identities to Mv-TAm were the
1
5
IPA. The general instability of transaminases to high amine lowest between 13–20%. Only three of the new drain TAms had
donor concentrations and solvent content are a major hurdle identities over 45% with Cv-TAm (pQR2189 – 48%, pQR2200 –
for their industrial application, thus novel, robust trans- 54% and pQR2208 – 48%). This showed that the drain meta-
1
8,28
aminases are highly desirable.
genome is a source of novel and distantly related Class III
In this work, a metagenomic approach was used to identify TAms.
6 putative Class III transaminases from a domestic drain
3
Initial screening of 29 cloned transaminases
metagenome. Twenty-nine were successfully cloned and over-
expressed in E. coli and their activity compared using a The 29 successfully cloned Class III transaminases were
number of assays. The most active transaminases were then initially screened against four substrates (1–4) (Fig. 1) to ident-
characterised and their value in preparative scale reactions was ify active enzymes. Representative substrates 1–4 were chosen
investigated.
to assess the activity towards cyclic ketones, α-keto acids,
linear and aromatic aldehydes or ketones respectively. A
number of amine donors – 2-(4-nitrophenyl)ethan-1-amine
hydrochloride 5 (Fig. 1a), xylylenediamine 6 (Fig. 1b), (S)- and
Results and discussion
Transaminase identification, cloning and expression from a
(
R)-α-methylbenzylamine (S)/(R)-7 (Fig. 1c) – were also tested
against each substrate to establish activities in these quantitat-
ive or qualitative assays (Fig. 1a–c). Amine donors 5 and 6 are
drain metagenome
Metagenomics is a culture independent method to study the from previously published colorimetric assays and both rely on
2
9,30
collective genomes in an environmental sample,
and the the formation of a coloured product following conversion of
1
3,39
adoption of a sequence based approach allows us to access a the amine donor.
Using MBA 7 as an amine donor relies
As on the formation of acetophenone and subsequent HPLC
microbial lifeforms are found in virtually every ecological detection at 254 nm, allowing for quantitative analysis.
niche, the analysis of their microbiomes could lead to the dis- No conversion was observed with ten of the transaminases,
covery of enzymes with wider operating parameters as well as pQR2192, pQR2194, pQR2195, pQR2196, pQR2197, pQR2198,
an excellent starting point for mutagenesis. pQR2199, pQR2203, pQR2210 and pQR2212 (data not shown).
3
1
largely untapped resource of unculturable bacteria.
Previous work has employed metagenomic approaches in However, 19 of the assayed enzymes displayed some activity
the search for novel transaminases, using either a functional with at least one of the amine donors tested and Fig. 1 shows
3
2
33,34
based or a sequence-based approach.
In this work, trans- results from these assays. Pyruvate 2, a natural substrate for
aminases have been retrieved from a domestic household many transaminases, was the most broadly accepted substrate,
drain metagenome. DNA was extracted from the drain and displaying activity with most of the transaminases. The linear
sequenced using the Illumina MiSeq platform, as opposed to ketone 3 showed low levels or no activity towards the enzymes
the Roche 454 Titanium platform which had been used in pre- assayed. Cyclohexanone 1 and benzaldehyde 4 were more
3
3,34
vious work.
This generated 10 million individual sequence widely accepted and enzymes that accepted one tended to also
reads – 10 times the number generated using the Roche 454 show activity with the other; pQR2205 was a notable exception
3
5
Titanium platform for a tongue metagenome – due to the and activity was only observed with benzaldehyde 4 and amine
larger number of reads giving a much greater sequence depth donor 6.
which allows the recovery of novel genes.
(S)- and (R)-MBA were also used to determine the selectivity
Thirty-six full-length, non-redundant novel Class III trans- of the enzymes. Class III transaminases are known to be (S)-
4
0,41
aminase candidates were identified in the drain metagenome. selective,
Multiple sequence alignments showed an average identity of observed with (S)-MBA. Overall, conversions with (S)-7 (Fig. 1c)
8% (ESI, Fig. S1†) indicating the enzyme diversity. In a BLAST correlated well with donor 5 (Fig. 1a), with pQR2188, pQR2189,
and assay results confirmed that activity was only
2
search against the NCBI database, amino acid sequences of pQR2191, pQR2200, pQR2208 and pQR2214 giving the best
these 36 putative transaminases showed an average of 87% activities. Enzymes pQR2193, pQR2201, pQR2202, pQR2207,
identity (range 64–100%) with proteins in sequence databases pQR2211 and pQR2216 also showed some potentially interest-
(ESI, Table S1†). Of these, 29 genes were successfully cloned ing activities towards one or more substrates. Transaminase
and expressed in E. coli BL21 (DE3). All enzymes were well pQR2190, pQR2204, pQR2205, pQR2206, pQR2209, pQR2213
expressed except for pQR2204, which showed low levels of and pQR2215 showed low conversion levels across the assays
total expression. Most of the enzymes showed good solubility performed so were not used in further assays.
and only pQR2196 and pQR2198 were completely insoluble
Probing the substrate scope
(ESI, Fig. S2–9†).
The homologies of the expressed enzymes, compared to the Enzymes that showed the highest or potentially interesting
known and extensively studied (S)-TAms Chromobacterium vio- activities in the first round of screening were assayed against a
3
6
37
laceum (Cv-TAm), and Vibrio fluvialis (Vf-TAm), and (R)-TAm wider set of substrates (8–17) (Fig. 2). Substrates were selected
3
8
Mycobacterium vanbaalenii (Mv-TAm), were assessed (ESI, to increase the range of aromatic (8–11), aliphatic (12–14) and
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Fig. 1 Colorimetric assays with amine donors 5 and 6 and conversions with (S)-MBA (S)-7. Using 5 as amine donor: 5 (25 mM), amino acceptor
buffer pH 7.5 (100 mM) and clarified cell extract (0.2–0.4 mg mL− ), 30 °C, 400 rpm, 18 h. Water was used as a negative
1
(
10 mM), PLP (0.5 mM), KP
control (N) in place of a substrate, red precipitate is indicative of activity. Using 6 as an amine donor: 6 (5.5 mM), amino acceptor (5 mM), PLP (1 mM),
KP
buffer pH 7.5 (100 mM) and clarified cell extract (0.2–0.4 mg mL−1), 30 °C, 400 rpm, 18 h. Water was used as a negative control (N) in place of a
i
i
substrate, a black precipitate indicates transamination has occurred. All colorimetric assays were performed in duplicate. For both colorimetric
assays, water was used in place of an enzyme extract as a negative control (results not shown). (S)- and (R)-MBA were used to determine selectivity:
buffer pH 7.5 (100 mM) and clarified cell extract (0.2–0.4 mg mL− ), 30 °C, 400 rpm,
1
(
S)/(R)-7 (25 mM), amino acceptor (10 mM), PLP (0.5 mM), KP
i
1
8 h. Acetophenone was detected at 254 nm by HPLC and used to determine percentage conversions. Water was used as a negative control, and
any background levels of acetophenone production were subtracted from the reactions. All reactions were performed in triplicate, and standard
deviations were <4%.
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Fig. 2 shows the results of the expanded substrate screen
with the 12 most active enzymes from the first screen.
Heterocycles 8 and 10 were widely accepted, giving a strong
coloration for a number of enzymes (pQR2188, pQR2189,
pQR2200, pQR2208 and pQR2214), whilst activity was predo-
minantly seen with pQR2189 and pQR2208 for the pyrrole 9.
Cinnamaldehyde 11 and all of the straight chain aldehydes
(
12–14) were readily accepted, showing some activity for all
active enzymes. There appeared to be a slight preference for
hexanal 12, with octanal 14 giving rise to lower conversions.
Interestingly, only four enzymes (pQR2189, pQR2191,
pQR2200 and pQR2208) showed activity towards the cyclic
compounds 1, 15–17. Generally, better conversions were
observed for the 6-membered rings (1 and 17) than their
5-membered counterparts (15 and 16), and α-methyl substi-
tution led to a drop in activity. No activity was observed with
more complex substituted cyclic compounds (ESI, Fig. S10†).
Notably, pQR2189 showed activity with almost every substrate.
In addition, pQR2191 and pQR2208 performed well with many
of the substrates tested including the cyclic ketones, so these
were explored further as detailed below.
Pharmaceutically relevant substrates
Substituted aminopyrrolidines, aminopiperidines and amino-
azepanes are common motifs found in a number of pharma-
4
2–44
ceutically active compounds.
Previous studies have probed
the feasibility of introducing the amine functionality of these
products with transaminases, demonstrating that they are rela-
tively challenging substrates requiring either a large excess of
amine donor (e.g. with Vibrio fluvialis (Vf-TAm) up to 27%
yield) or use of a second enzyme for ketone co-product
removal (e.g. with Vf-TAm up to 80% yield) to give moderate to
2
2,38,45
good yields.
Scale-up strategies have also been used to
generate an aminopiperidine using a pure transaminase
4
6
enzyme in 70% yield.
Compounds 18a–26a were selected to assay against the
three most promising enzymes: pQR2189, pQR2191 and
pQR2208. The activities of the three metagenomic-derived
enzymes were investigated with three amine donors: phenethyl-
Fig. 2 Colorimetric assay with 2-(4-nitrophenyl)ethan-1-amine 5 as
amine donor using substrates 1, 8–17: 5 (25 mM), amino acceptor 1,
amine 5, (S)-MBA (S)-7 and IPA 27 (Table 1). The well-studied
8
i
–17 (10 mM), PLP (0.5 mM), KP buffer pH 7.5 (100 mM) and clarified
−1
36
cell extract (0.2–0.4 mg mL ), 30 °C, 400 rpm, 18 h. Water was used as (S)-selective transaminase Cv-TAm was screened in parallel
a negative control (N), and a red precipitate was indicative of activity. with amine donors 5 and (S)-7 for comparison purposes.
Assays were performed in duplicate.
On the whole, the benzyl-protected pyrrolidone and piperi-
done substrates were less well accepted than their Boc-protected
equivalents. For example, the unsymmetrical piperidone 21a
cyclic ketones and aldehydes (15–17) tolerated. Heterocycles gave low to no conversion with (S)-7 and 27 as donors, while
8
–10 were chosen based on the recently published work detail- the Boc-piperidone 20a gave yields of up to 60% of 20b with
1
6
ing the transamination of various furfural analogues. To the 27. The colorimetric assay also indicated higher reactivities
best of our knowledge, this work represents the first time that towards 20a compared to 21a. Similarly, the Boc-pyrrolidine
thiophene and pyrrole analogues have been used with trans- 18b was formed in higher yields than 19b using (S)-7 and 27 as
aminases. Transaminases from an oral cavity metagenome donors, indeed pQR2189 gave 18b in 72% and 19b in 20%
were previously shown to accept a number of unsaturated alde- yield with 27. However, the colorimetric assay suggested that
hydes/ketones, and therefore cinnamaldehyde 11 was included 19a was more readily converted by the metagenomics trans-
3
4
for comparison. Substituted cyclopentanones and cyclohexa- aminases. Overall, moderate to good conversions were
nones were also selected, as substitution of cyclic compounds observed with pyrrolidinone 18a and piperidone 20a, with a
2
6
often leads to a drop in conversion.
universal preference for the five-membered ring. A similar ring
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Table 1 Results for TAm catalysed reactions of 18a–26a using phenylethanamine 5, (S)-MBA 7 and IPA 27 as amine donors for pQR2189, pQR2191,
a,b,c
pQR2208 and Cv-TAm
pQR2189
pQR2191
pQR2208
Cv-TAm
5
MBA (S)-7
IPA 27
5
MBA (S)-7
IPA 27
5
MBA (S)-7
IPA 27
5
MBA (S)-7
18a
19a
20a
21a
22a
23a
24a
25a
26a
43%
72%
8%
25%
45%
67%
30%
23%
30%
4%
20%
5%
4%
9%
10%
55%
1%
6%
60%
11%
19%
1%
30%
2%
27%
0%
d
2%
68%
e
e
e
e
41%
30%
31%
35%
13%
Quant.
39%
11%
36%
3%
Quant.
46%
Quant.
25%
19%
39%
12%
3%
51%
30%
70%
27%
34%
37%
75%
Quant.
f
f
f
g
75%
13%
32%
n.d.
g
g
g
g
n.d.
7%
n.d.
n.d.
n.d.
a
Reaction conditions: Amine donor 5 (25 mM), amino acceptor (10 mM), PLP (0.5 mM), KPi buffer pH 7.5 (100 mM) and clarified cell extract
−1
(0.4 mg mL ), 30 °C, 400 rpm, 18 h. Water was used as a negative control (not shown), red precipitate is indicative of activity. Assay was per-
b
formed in duplicate. Reaction conditions: Amine donor (S)-7 (25 mM), amino acceptor (5 mM), PLP (1 mM), KPi buffer pH 7.5 (100 mM) and
clarified cell extract (0.4 mg mL− ), 30 °C, 400 rpm, 18 h. Acetophenone was detected at 254 nm by HPLC and used to determine percentage con-
versions. Water was used as a negative control, and any background levels of acetophenone production were subtracted from the reactions. All
1
c
reactions were performed in triplicate, and standard deviations were <7%. Reaction conditions: Amine donor 27 (100 mM), amino acceptor
10 mM), PLP (1 mM), KPi buffer pH 7.5 (100 mM) and clarified cell extract (0.4 mg mL− ), 35 °C, 400 rpm, 18 h. Yields were determined using
1
(
d
HPLC against product standards. All reactions were performed in triplicate, and standard deviations were <5%. By acetophenone production,
e
f
product detection confirmed negligible amounts of 21b. Quantitative conversion. Yields were determined by 25a depletion, but product pres-
ence was confirmed against a product standard. Not determined.
g
size preference has been noted when using 18a and 20a in Vf-
TAm mediated kinetic resolutions.
The 3-oxo-azepane 25a was accepted by all of the meta-
genomics transaminases, being converted in 75% yield with
4
5
The use of compounds with Boc or benzylic groups at the pQR2189 and IPA 27. For the 4-oxo-azepane 26a, reduced con-
-position gave greatly improved yields, with the amine donor versions were noted with (S)-MBA, so activities were not
4
having a significant effect on the reaction. The use of IPA con- explored further. To the best of our knowledge, this is the first
sistently gave higher yields. Piperidones 22a and 24a were time the seven-membered N-Boc-protected azepane structures
more readily accepted than 23a by all the TAms, and quantitat- 25a and 26a have been used with transaminases. It is note-
ive yields were observed using 22a and 24a with pQR2189 and worthy, that for almost all substrates, higher conversions were
pQR2191. Enzyme pQR2189 performed best with 23a and IPA, observed with at least one of the metagenomic transaminases
producing 23b in 75% yield.
compared with Cv-TAm.
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The stereoselectivities were determined for all reactions unchanged at 40% for pQR2191, but a decline could be seen
giving a chiral product in yields >50% by chiral GC analysis. above this. Remarkably, pQR2189 was operating at 80% of the
Both pQR2189 and pQR2208 showed excellent selectivity with relative activity even at 50% DMSO concentrations.
1
8a and 20a, giving the (S)-isomer exclusively for 18b and 20b An improvement in the yield was also observed above 20%
with ee values >99%.
levels of DMSO, although this may reflect improved substrate
solubility. An example of another wild-type transaminase
reported to show activities with DMSO concentrations above
Characterisation of pQR2189, pQR2191 and pQR2208
1
0% is Polaromonas sp. JS666 (ω-TAPo), however a marked
In order to determine the scope of the operating conditions of
pQR2189, pQR2191 and pQR2208, the effect of temperature,
pH, co-solvent and buffer concentration were investigated
using pyrrolidinone 18a as an amine acceptor and IPA as the
donor (Fig. 3). Varying the temperature between 20 °C and
47
decrease in yield was observed above 20% levels.
The
(
R)-selective transaminase ArRMut11 is also known to be oper-
ational in 50% DMSO however, 11 rounds of evolution were
required to increase its stability towards these conditions.
1
5
PEG200 was also explored as an alternative co-solvent, and
although there was a slight fall in activity for pQR2189 and
pQR2208, the activity was similar across all concentrations
tested (Fig. 3d). The buffer concentration was reduced from
5
0 °C proved to have little effect on the activity for pQR2189
and pQR2191, both maintained a similar activity. The optimal
activity for pQR2208 was observed at 25 °C, after which there
was a steady decline up until 40 °C, where above this the
activity dropped markedly. For both pQR2191 and pQR2208,
optimal activity was observed between pH 9–10, after which
there was a significant reduction. Conversely, pQR2189 was
stable across all pH values tested, pH 6–11. DMSO is often
used as a co-solvent in enzymatic transformations for substrate
100 mM to 50 mM and no effect on yield was observed
(
results not shown); subsequent reactions were run at the
lower concentration.
Further investigation of pQR2189
solubilisation, so DMSO concentrations (v/v) of 5–50% were Due to the remarkable DMSO tolerance of pQR2189, the effect
investigated. All enzymes were tolerant of up to 30% DMSO, of the concentration of IPA, enzyme and substrate as well
after which there was a significant drop in yields for pQR2208 kinetic parameters were investigated further (Fig. 4).
with almost total loss of activity at 50%. Activity was Thermoactivity up to 70 °C was also investigated to determine
i
Fig. 3 Effects of (a) temperature 20–50 °C, (b) KP buffer pH 6–11 (100 mM), (c) 5–50% (v/v) DMSO, and (d) 5–50% (v/v) PEG200 on the yield of 18b
i
using IPA 27 as the amine donor. General reaction conditions: Amine donor 27 (100 mM), 18a (10 mM), PLP (1 mM), KP buffer pH 8 (100 mM) and
clarified cell extract (0.4 mg mL− ), 20% DMSO, 35 °C, 400 rpm, 18 h. Yields were determined using HPLC against product standards. All reactions
1
were performed at least in duplicate, and standard deviations were <8%.
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Fig. 4 Effects of temperature (a), enzyme concentration (b), IPA equivalents (c) and substrate concentration (d) on yield of 18b using IPA 27 as an
i
amine donor for pQR2189. General reaction conditions: Amine donor 27 (100 mM), amino acceptor (10 mM), PLP (1 mM), KP buffer pH 8 (100 mM)
and clarified cell extract (0.4 mg mL− ), 20% DMSO, 35 °C, 400 rpm, 18 h. Yields were determined using HPLC against product standards. All reac-
1
tions were performed in triplicate, and standard deviations were <8%. Reaction condition variations: (a) Temperature 20–70 °C, (b) clarified cell
−1
lysate 0.04–4 mg mL , (c) 1–50 equivalents of IPA 27 and (d) 10–200 mM amino acceptor 18a.
its active temperature range. Although activity was seen to Fig. S48†). Using purified pQR2189, kinetic parameters were
drop above 50 °C, 70% relative activity was still observed at determined for the conversion of pyrrolidinone 18a to amine
6
0 °C with lower yields observed at 70 °C (Fig. 4a), demonstrat- 18b with donor 27, as well as the more general reaction
4
8,49
ing a similar temperature profile to two recently published between pyruvate 2 and (S)-7.
transaminases from thermophilic sources. Different concen- the Michaelis–Menten kinetics, the apparent K
trations of cell-free extract were also used, and similar yields
Based on the assumptions of
, kcat and
m
cat/K values for 2, (S)-7, 18a and 27 were calculated, and
3
3
m
k
−
1
were observed with 0.1 mg mL total protein, compared to have been summarised in Table 2. The apparent specific
higher concentrations of protein, and 80% relative activity activity of pQR2189 with pyruvate 2 and donor (S)-7 are two to
compared to higher concentrations with only 0.04 mg mL−
1
three orders of magnitude higher than that of Cv-TAm.
50,51
total protein (Fig. 4b). To determine both the minimum
amount of IPA required to drive the equilibrium towards
amine formation, as well as the maximum amount of IPA toler-
ated, reactions with 1–50 equivalents of IPA were investigated.
As little as one equivalent of IPA was required to achieve
almost 80% activity relative to the maximum yield, increasing
to over 90% with only two equivalents. Additionally, there was
no decrease in yield observed with 50 equivalents of IPA
Preparative scale reaction pQR2189 and pQR2208
As the overall yield of 18b could not be increased with
pQR2191, preparative scale reactions were carried out with
Table 2 Kinetic parameters for pQR2189 for the reaction between 2
(500 mM final concentration) (Fig. 4c). Although increasing
a
and (S)-7, as well as the conversion of 18a with donor 27
the substrate concentration led to a decrease in yield, the
overall amount of 18b formed continued to increase,
suggesting that this is not due to substrate inhibition (Fig. 4d).
Further kinetic studies showed that no substrate inhibition (S)-7
was observed up to 200 mM (ESI, Fig. S45†).
m
kcat/K )
(s−1 M−1
K
(mM)
k
cat (s−
1
)
m
2
0.29 ± 0.08
14.8 ± 3.2
18.9 ± 3.1
10.6 ± 1.6
25
58
4.5
2.9
87 069
3942
237
1
2
8a
7
275
Kinetic studies with crude cell lysate showed rapid reaction
rates, with full conversion being observed after 2 hours (ESI,
a
Reaction conditions detailed in the experimental.
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Conclusions
In this work, a metagenomic approach has been used to dis-
cover and clone 29 putative transaminases from a household
drain metagenome. It was believed that the habitat of the
organisms in this microbiome might lead to the identification
of niche organisms and their enzymes. The location in the
waste pipe of the domestic shower with periods of drought
and high water temperatures with surfactants and cleaning
chemicals could be considered as an extreme environment.
Twelve transaminases showed good activity towards a wide
range of substrates. Three transaminases, pQR2189, pQR2191
and pQR2208 were selected for further analysis due to their
promising performance toward heterocyclic ketones and the
effect of temperature, pH and co-solvents established. Of
these, reactions with pQR2189 and pQR2208 were performed
Fig. 5 Effect on yield of 18b with various co-solvents (10% final con-
centration). The reaction conditions used were the same as those indi-
cated in Fig. 4, all reactions were performed in triplicate and standard
deviations were <8%.
on
a preparative scale. Transaminase pQR2189 showed
remarkable robustness, displaying characteristics often only
observed in highly engineered enzymes. Up to 50% DMSO was
used in reactions without a significant drop in activity. Whilst
coming from a non-thermophilic environment, pQR2189 was
also operational at a wide range of temperatures (20–60 °C) as
well as pH values (6–11), good relative activity was observed
with as little as one equivalent of IPA and as many as 50
equivalents of IPA. These attributes have been described as
highly desirable for novel transaminases; pQR2189 shows
unique properties for a wild type enzyme, making it an excel-
lent scaffold for enzyme engineering. This work highlights not
only the value of metagenomics in biocatalyst discovery, but
also its unrivalled ability to identify novel enzymes specifically
adapted to operate in harsh conditions.
pQR2189 and pQR2208. Given the known difficulty of using
DMSO in large-scale reactions (due to its high boiling point)
and the apparent robustness of the enzyme, alternative co-sol-
vents were investigated. Whilst pQR2208 showed reduced
stability in most of the co-solvents selected, CPME, TBME,
acetonitrile and methanol all had limited effect on the activity
for pQR2189. Methanol was therefore selected as it was well
tolerated (other than DMSO) by both pQR2189 and pQR2208
(Fig. 5).
Reactions were then performed on a 20 mM scale (50 mL)
using 10 equivalents of IPA. Whilst scale-up to a 50 mL reac-
tion volume had little effect on the yield for pQR2189, a signifi-
cant drop in yield was observed for pQR2208 from 67% to
3
4%. Enantiopure amine (S)-18b (by chiral GC-analysis) was
isolated in 64% and 31% for pQR2189 and pQR2208 respect-
ively (Table 3). Additionally, a preparative scale reaction was
carried out with pQR2189 on a 50 mM scale (50 mL) with only
Experimental
General information
2
equivalents of IPA to demonstrate the usefulness of this
enzyme. Amine (S)-18b was isolated in 57% yield (ee > 99%), All chemicals were obtained from chemical suppliers with no
and the reaction was complete after eight hours.
further purification.
During kinetic experiments, a 100% conversion yield of 18a
Column chromatography was carried out using Biotage®
was observed with purified pQR2189, therefore a 50 mM scale Isolute 1 with Biotage® Snap Cartridge KP-NH and analytical
50 mL) reaction was repeated with the purified enzyme. HPLC thin layer chromatography was carried out using Biotage®
analysis showed a product yield of 86%, and 18b was isolated KP-NH TLC plates. Compounds were visualised by ultra-violet
(
in 82% yield.
light, potassium and phosphomolybdic acid stains. Infrared
a,b
Table 3 Yields for preparative scale reaction with 18a and pQR2189 and pQR2208
Conc. 18a
mM)
Equivalents
IPA 27
HPLC yield c
HPLC yield c
Isolated yield
18b (50 mL)
c
(
18b (200 μL)
18b (50 mL)
ee (%) (S)
a
a
b
d
pQR2189
pQR2208
pQR2189
pQR2189
20
20
50
50
10
10
2
69%
67%
55%
100%
65%
34%
57%
86%
64%
31%
57%
82%
>99%
>99%
>99%
e
2
nd
a
Reaction conditions for 20 mM scale: Amine donor 27 (200 mM), amino acceptor (20 mM), PLP (1 mM), KPi buffer pH 10 (50 mM) and clarified
−
1
b
cell extract (0.4 mg mL ), 35 °C, 250 rpm, 24 h. Reaction conditions for 50 mM scale: Amine donor 27 (100 mM), amino acceptor (50 mM), PLP
−
1
c
d
(
1 mM), KPi buffer pH 10 (50 mM) and clarified cell extract (1 mg mL ), 30 °C, 250 rpm, 24 h. Total reaction volume. Reaction conditions
−1 e
same as for (b) but using purified pQR2189 (130 μg mL ). Not determined.
82 | Green Chem., 2019, 21, 75–86
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(
IR) spectra were recorded using a Bruker Alpha Platiunum- 4 months. To prepare the clarified cell lysate, freeze dried cells
1
ATR. H NMR spectra were recorded at 600 MHz on a Bruker (40 mg) were re-suspended in potassium phosphate buffer
Avance 600 spectrometer in deuterated chloroform (residual (100 mM, pH 7.5, 2 mM PLP) and disrupted by sonication (5 ×
protic solvent δ 7.26 ppm, s). Chemical shifts are quoted in 45 s), and centrifuged (12 000 rpm, 20 min, 4 °C). Clarified cell
1
ppm relative to tetramethylsilane, with multiplicities for
H
lysate was prepared fresh for each use and kept on ice at all
spectra shown as s (singlet) and m (multiplet). The coupling times. Total protein concentration was determined using a
1
3
constants (J) are measured in hertz. C NMR spectra were standard Bradford assay. Clarified cell lysate was diluted to a
recorded at 150 MHz on a Bruker Avance 600 spectrometer in final concentration of 4 mg mL− with KP buffer (100 mM, pH
1
i
deuterated chloroform (δ 77.00 ppm, t). Chemical shifts are 7.5, 2 mM PLP).
reported to the nearest 0.1 ppm. Mass spectra were obtained
using a Waters LCT Premier XE ESI Q-TOF mass spectrometer
at the Department of Chemistry, UCL. HPLC analysis was The enzymatic reaction was carried out in 96-well plates
2
-(4-Nitrophenyl)ethan-1-amine colorimetric assay
−1
carried out on an Agilent 1260 Infinity HPLC and Dionex (200 μL system) containing clarified cell lysate (0.2–0.4 mg mL ),
Ultimate 3000 with an Ace 5 C18 150 × 4.6 mm column. KP buffer (100 mM, pH 7.5), PLP (0.5 mM), 2-(4-nitrophenyl)
i
Sterilisation of waste and media was carried out in a Priorclave ethan-1-amine 5 (25 mM) as amine donor and the substrate
autoclave at 121 °C for 30 min. The following centrifuges were (10 mM), with 20% DMSO to aid substrate solubility, at 30 °C
used: Beckman Coulter Allegras x-15R centrifuge, Eppendorf and 400 rpm for 18 h. A red precipitate indicated a positive
3
9
Centrifuge 5415R, Eppendorf Centrifuge 5810R, Eppendorf result.
Centrifuge 5430R. A Kuhner ShakerX ClimoShaker ISFI-X, New
ortho-Xylylenediamine colorimetric assay
Brunswick Scientific Innova 44 or BIOER Mixing Block MB-102
incubating shaker was used.
The enzymatic reaction was carried out in 96-well plates
−1
(200 μL system) containing clarified cell lysate (0.2–0.4 mg mL ),
Enzyme selection and cloning
KP buffer (100 mM, pH 7.5), PLP (1 mM), xylylenediamine
i
DNA was extracted from a domestic drain metagenome and dihydrochloride 6 (5.5 mM) as amine donor and the substrate
5
2
sequenced using the Illumina MiSeq platform. Predicted (5 mM), with 10% DMSO to aid substrate solubility, at 30 °C
protein sequences from the drain metagenome were previously and 400 rpm for 18 h. A black precipitate indicated a positive
1
3
functionally annotated by scanning the sequences against result.
Pfam28.0 libraries of domain families (pfam.xfam.org). To
Initial (S)-MBA assay
identify putative ω-transaminases, sequences annotated with
the Pfam domain family: aminotransferase class-III (PF00202) The enzymatic reaction was carried out in 96-well plates
−
1
were retrieved. Of 77 sequences, 36 were judged to be full- (200 μL system) containing clarified cell lysate (0.4 mg mL ),
length, non-redundant proteins by methods previously KP buffer (100 mM, pH 7.5), PLP (1 mM), (S)/(R)-MBA 7
i
3
4,35
described.
The genes of interest were amplified by PCR (25 mM) as amine donor, and substrate (10 mM) with 20%
from the metagenome DNA using NEB Phusion PCR kit or DMSO to aid substrate solubility, at 30 °C and 400 rpm for
NEB Q5 High-Fidelity kit. PCR products were visualised by gel 18 h. To stop the reaction, 10% TFA (10 μL) was added to each
electrophoresis, isolated via Qiagen Gel extraction columns well and denatured protein removed via centrifugation
and cloned into pET-28a(+) with the C- and N-terminal His6- (20 min, 12 000 rpm, 4 °C). The supernatant was diluted and
tag for pQR2188–pQR2199 using the restriction enzymes NdeI analysed by analytical HPLC.
and XhoI and pET-29a(+) with the C-terminal His6-tag for
pQR2200–pQR2216 (ESI, section 4†). In all cases, recombinant
Subsequent (S)-MBA assay
plasmids were transformed into E. coli TOP10, then isolated The enzymatic reaction was carried out in 96-well plates or
and verified by sequencing before transformation into E. coli Eppendorf tubes (200 μL system) containing clarified cell
lysate (0.4 mg mL− ), KP
1
(100 mM, pH 7.5), PLP (1 mM),
BL21 (DE3).
i
(
S)/(R)-MBA 7 (25 mM) as amine donor, and substrate (5 mM)
Enzyme expression and preparation
with 10% DMSO to aid substrate solubility, at 30 °C and 400
For each expression strain, the overnight starter culture (4 mL) rpm for 18 h. To stop the reaction, 10% TFA (10 μL) was added
was used to inoculate 400 mL Terrific Broth (TB) sup- to each well, and denatured protein removed via centrifugation
−
1
plemented with kanamycin (50 μg mL ). The culture was (20 min, 12 000 rpm, 4 °C). The supernatant was diluted and
incubated at 37 °C with shaking (200 rpm) until the OD600 analysed by analytical HPLC.
reached 0.6–0.8, at which point the cells were induced by
Isopropylamine assay
addition of IPTG to a final concentration of 1 mM. Post induc-
tion, cells were incubated at 25 °C with shaking (200 rpm) The enzymatic reaction was carried out in 96-well plates or
overnight. The cells were harvested by centrifugation (8000 Eppendorf tubes (200 μL system) containing clarified cell
lysate (0.4 mg mL− ), KP
1
buffer (100 mM, pH 8), PLP (1 mM),
rpm, 20 min, 4 °C) and the cell pellet re-suspended in KP
i
i
buffer (100 mM, pH 7.5, 2 mM PLP) and freeze dried. The IPA 27 (100 mM, pH 8) as amine donor and the substrate
freeze-dried cells were used fresh or stored at −20 °C for up to (10 mM) with 20% DMSO to aid substrate solubility, at 35 °C
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and 400 rpm for 18 h. To stop the reaction, 10% TFA (10 μL) further by size exclusion chromatography using a Superdex
was added to each well, and denatured protein removed via 200 10/300 GL 24 mL column (GE Healthcare). The fractions
centrifugation (20 min, 12 000 rpm, 4 °C). The supernatant were pooled and concentrated to 24 mg mL− and stored at
1
was diluted and analysed by analytical HPLC.
−80 °C.
The buffer concentration (KP ) was also tested at a final con-
centration of 50 mM. The pH was varied by changing the pH
i
Kinetics assay with pyruvate and (S)-MBA
of potassium phosphate buffer (pH 6–11) and IPA (pH 6–11). To determine the linear range of enzymatic activity, pyruvate 2
The temperature range was investigated by leaving the reaction (2.5 mM), (S)-7 (5 mM), PLP (0.1 mM), KP buffer pH 7.5
i
at the specified temperature and 400 rpm for 18 h. DMSO (50 mM), 1% DMSO and varying amounts pQR2189
−
1
assays used 5–50% (v/v). Final IPA concentrations of 10, 20, 50, (2.6–130 μg mL ) were mixed in a cuvette at 30 °C for 180 s
00 and 500 mM were also examined, with the substrate and absorbance at 300 nm continuously followed.
10 mM) to give 1–50 equivalents of IPA. The substrate concen-
The enzymatic reaction was carried out in cuvettes (1 mL
tration was investigated by varying the amine acceptor system) containing amine donor (S)-7 (5 mM) and varying
1
(
(10–200 mM) using 2 equivalents of amine donor, IPA 27 amounts of pyruvate 2 (0.25–5 mM) or pyruvate 2 (1 mM) and
(
20–400 mM). Enzyme concentrations of 0.04, 0.1, 0.2, 0.4, 1, 2 varying (S)-7 (3–15 mM), PLP (0.1 mM), KP buffer pH 7.5
i
−
1
−1
and 4 mg mL were also examined.
(50 mM) and pQR2189 (6.5 μg mL ), 1% DMSO, 30 °C, 180 s.
Yields were determined by constant monitoring of UV absorp-
tion at 300 nm. All reactions were performed in triplicate.
Determination of ee for pQR2189 and pQR2207
48
The enantiomeric excess of the amine products 1-Boc-3-amino-
pyrrolidinone 18b and 1-Boc-3-aminopiperidine 20b were
determined by chiral-GC analysis. Reactions were carried out
as described above, using either (S)-MBA 7 (5 mM) or IPA 27
Kinetics assay with 1-Boc-3-pyrrolidinone 18a and IPA
To determine the linear range of enzymatic activity, amino
acceptor 18a (10 mM), donor 27 (100 mM), PLP (1 mM), KP
i
(
10 mM) as an amine donor on a 200 μL scale reaction in
Eppendorf tubes. The reaction mixture was extracted with
ethyl acetate (1 mL), dried (Na SO ) and concentrated to
buffer pH 8 (50 mM), 20% DMSO and varying amounts
pQR2189 (6.5–130 μg mL− ) were mixed in an Eppendorf at
1
2
4
3
(
5 °C for 20 min. Reactions were stopped by adding aliquots
100 μL) to 1% TFA (400 μL) at specified time points and
dryness. The product was then either re-dissolved in ethyl
acetate (18b) and analysed by chiral-GC or derivatized for
chiral-GC analysis (20b). The retention time for (S)-1-Boc-
denatured protein removed via centrifugation (10 min, 12 000
rpm, 4 °C). The supernatant was analysed by analytical HPLC.
The enzymatic reaction was carried out in Eppendorf tubes
3
-aminopyrrolidinone 18b was 7.4 min and 7.3 min for the
(
R)-enantiomer.
(
1 mL system) containing amine donor 27 (200 mM) and
varying amounts of amino acceptor 18a (10–200 mM) or amino
acceptor 18a (10 mM) and varying amounts of amine donor 27
Derivatisation of 1-Boc-3-aminopiperidine
The residue 20b in dichloromethane (50 μL) was derivatized
with trifluoroacetic anhydride (7 μL for the reaction with
(
i
10–500 mM), PLP (1 mM), KP buffer pH 8 (50 mM) and
−
1
pQR2189 (26 μg mL ), 20% DMSO, 35 °C, 400 rpm, 20 min.
Reactions were stopped by adding aliquots (100 μL) to 1% TFA
(
S)-MBA 7 and 14 μL for the reaction with IPA 27) and shaken
overnight at room temperature. The reaction mixture was then
analysed by chiral-GC without further dilution. The retention
time for the N-TFA-derivatized (S)-enantiomer was 7.4 min and
(
400 μL) at specified time points and denatured protein
removed via centrifugation (10 min, 12 000 rpm, 4 °C). The
supernatant was analysed by analytical HPLC. All reactions
were performed in triplicate.
4
9
7
.6 min for the (R)-enantiomer.
Purification of pQR2189
Calculating Km and Vmax
Transaminase pQR2189 was expressed on a 400 mL scale as
previous described. After centrifugation cells were resus-
pended in 5 mL buffer (Na HPO 50 mM, NaCl 500 mM, imid-
Km and Vmax were determined by a Michaelis–Menten non-
linear regression of initial velocity vs. substrate concentration
using Prism 7. kcat is given in s− and defined as:
1
2
4
azole 10 mM, pH 7.4, 0.5 mM PLP) and lysed by sonication
4 × 45 s). Cell debris was removed via centrifugation (45 min,
0 500 rpm, 4 °C) and the cell lysate loaded into an open tubal
Vmax
(
1
kcat
¼
Eꢀmol
½
mini column containing Chelating Sepharose™ Fast Flow where [E]mol is defined as:
resin, and the column washed with 50 mL of start buffer.
½
proteinꢀ
Proteins were eluted with elution buffer (Na HPO 50 mM,
NaCl 500 mM, imidazole 50–500 mM, pH 7.4) with increasing
½Eꢀmol ¼
2
4
MW
concentrations of imidazole. The eluents with the highest con- and [protein] is in mg mL− and MW is in Daltons.
1
centrations of protein were pooled and dialysed overnight at
Preparative scale reaction (20 mM)
4
0
°C into dialysis buffer (Tris 20 mM, NaCl 150 mM, PLP
.5 mM, pH 7.5). Protein was concentrated using a 10 kDa cut The reaction was scaled up to 50 mL with 18a (186 mg,
off Vivaspin concentrator (Sartorius, Germany), and purified 1.00 mmol), isopropylamine (860 μL, 10.5 mmol), PLP (1 mM),
84 | Green Chem., 2019, 21, 75–86
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buffer (50 mM, pH 10), clarified cell lysate (0.4 mg mL−
1
)
KP
and incubated at 35 °C and 250 rpm for 24 h. To determine
reaction yields, 10% TFA (2.5 μL) was added to an aliquot There are no conflicts to declare.
i
Conflicts of interest
(50 μL) and denatured protein removed via centrifugation
(10 min, 13 000 rpm, 4 °C), then the supernatant was
diluted and analysed by analytical HPLC. To stop the reaction,
methanol (150 mL) was added to the reaction mixture,
Acknowledgements
and denatured protein removed via centrifugation (20 min, Funding from the Biotechnology and Biosciences Research
000 rpm, 4 °C). Methanol was removed in vacuo, the Council (BBSRC) and ALMAC for D. D. (BB/L007444/1),
4
aqueous layer acidified with sat. ammonium chloride and L. L. (BB/L016168/1) and J. W. E. J. (BB/J013005/1) is gratefully
any remaining starting material extracted with ethyl acetate acknowledged. We also thank the UK Engineering and
(3 × 100 mL).
Physical Sciences Research Council (EPSRC) for financial
pQR2189. The pH was then adjusted to pH 13 and amine support of this work (EP/K014897/1) for M. B. Finally, we
8b extracted with ethyl acetate (11 × 100 mL) and the com- thank the UCL NMR and Mass Spectrometry facilities in the
1
2 4
bined organic extracts were dried (Na SO ), filtered and con- Department of Chemistry.
centrated in vacuo. The crude oil was purified by column
chromatography (ethyl acetate in petroleum ether 40–60 °C,
2
0–100% gradient) to give 18b (119 mg, 64%) as a clear oil;
Notes and references
f
R 0.27 (20% ethyl acetate in petroleum ether 40–60); νmax
film/cm− ) 3335, 3092, 2961, 2932, 2869, 1689; H NMR
CDCl ; 600 MHz) δ 1.45 (9H, s, C(CH ), 1.59–1.69 (1H, m,
1
1
1 E. Vitaku, D. T. Smith and J. T. Njardarson, J. Med. Chem.,
2014, 57, 10257–10274.
(
(
3
3 3
)
CHH), 2.00–2.07 (1H, m, CHH), 2.97–3.08 (1H, m, CHH)
2 D. J. C. Constable, P. J. Dunn, J. D. Hayler, G. R. Humphrey,
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3
3
1
5
.30–3.58 (4H, m, CHNH , CHH, CH ); C NMR (CDCl3;
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4.6, 79.3, 154.8 (two peaks seen for each carbon in pyrrolidine
2 2
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+
(
ES+) m/z 187 (80%, M ), 145 (100%), 131 (100%), 113 (63%).
4
5
Spectroscopic data was in agreement with the literature.
pQR2208. The pH was then adjusted to pH 13 and amine
8b extracted with ethyl acetate (5 × 100 mL) and the com-
1
bined organic extracts were dried (Na
centrated in vacuo to give 18b (57 mg, 31%) as a clear oil with
spectroscopic data identical to that above.
2
SO
4
), filtered and con-
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Preparative scale reaction (50 mM) with pQR2189
8
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2
.53 mmol), containing isopropylamine (430 μL, 5.25 mmol),
PLP (1 mM), potassium phosphate buffer (50 mM, pH 10),
9 M. Lenz, N. Borlinghaus, L. Weinmann and B. M. Nestl,
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−
1
clarified cell lysate (1 mg mL
)
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ing starting material extracted with ethyl acetate (3 × 100 mL).
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4
1
8b (266 mg, 57%) for clarified cell lysate and 18b (377 mg,
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2%) with purified pQR2189 as a clear oil with spectroscopic
data as recorded above.
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