A metagenomics approach for new biocatalyst discovery: Application to transaminases and the synthesis of allylic amines

Article abstract of DOI:10.1039/c6gc02769e

Transaminase enzymes have significant potential for the sustainable synthesis of amines using mild aqueous reaction conditions. Here a metagenomics mining strategy has been used for new transaminase enzyme discovery. Starting from oral cavity microbiome samples, DNA sequencing and bioinformatics analyses were performed. Subsequent in silico mining of a library of contiguous reads built from the sequencing data identified 11 putative Class III transaminases which were cloned and overexpressed. Several screening protocols were used and three enzymes selected of interest due to activities towards substrates covering a wide structural diversity. Transamination of functionalized cinnamaldehydes was then investigated for the production of valuable amine building blocks.

Full text of DOI:10.1039/c6gc02769e

View Article Online
View Journal
Green
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Hailes, D. Baud,
J. Jeffries, T. Moody and J. M. Ward, Green Chem., 2017, DOI: 10.1039/C6GC02769E.
This is an Accepted Manuscript, which has been through the
Volume 18 Number
7 7 April 2016 Pages 1821–2242
Royal Society of Chemistry peer review process and has been
accepted for publication.
Green
Chemistry
Accepted Manuscripts are published online shortly after
Cutting-edge research for a greener sustainable future
www.rsc.org/greenchem
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1463-9262
CRITICAL REVIEW
G. Chatel et al.
Heterogeneous catalytic oxidation for lignin valorization into valuable
chemicals: what results? What limitations? What trends?
rsc.li/green-chem

Please do not adjust margins
Green Chemistry
Page 1 of 11
View Article Online
DOI: 10.1039/C6GC02769E
Journal Name
ARTICLE
A metagenomics approach for new biocatalyst discovery:
Application to transaminases and the synthesis of allylic amines
Damien Baud,a§ Jack W. E. Jeffries,b§ Thomas S. Moody,c John M. Ward,b* and Helen C. Hailesa*
Received 00th January 20xx,
Accepted 00th January 20xx
Transaminase enzymes have significant potential for the sustainable synthesis of amines using mild aqueous reaction
conditions. Here a metagenomics mining strategy has been used for new transaminase enzyme discovery. Starting from
oral cavity microbiome samples, DNA sequencing and bioinformatics analyses were performed. Subsequent in silico mining
of a library of contiguous reads built from the sequencing data identified 11 putative Class III transaminases which were
cloned and overexpressed. Several screening protocols were used and three enzymes selected of interest due to activities
towards substrates covering a wide structural diversity. Transamination of functionalized cinnamaldehydes were then
investigated for the production of valuable amine building blocks.
DOI: 10.1039/x0xx00000x
www.rsc.org/
unique starting point has the potential to ensure more
diverse enzymes are available. In addition new TAms not
encumbered by the current patent space would be very
Introduction
The use of biocatalytic strategies holds significant potential
for the sustainable synthesis of fine chemicals and
pharmaceuticals. One important class of enzymes is the
transaminases (TAms, EC 2.6.1) that can convert ketones
and aldehydes into the corresponding amines in high yields
and selectivities under mild aqueous reaction conditions.1,2
TAms catalyse the amino group transfer from an amine
donor to a ketone, or aldehyde amine acceptor, using
pyridoxal-5’-phosphate (PLP) as co-factor.3 The first step of
the reaction is the transfer of the amino group of the amine
donor resulting in the formation of pyridoxamine-5’-
phosphate and release of the amine donor as a ketone or
aldehyde. The second step is the regeneration of PLP with
the transfer of the amino group to the amine acceptor
which is released as an amine.1,4 Notably, TAms are an
interesting alternative to chemical synthetic methods from
an environmental and economic point of view and have
been reported in the synthesis of a number of (R)- or (S)-
pharmaceutical ingredients or biologically active
compounds.5
valuable. Here our approach has been to use
metagenomics mining strategy for new TAm discovery.
a
Metagenomics is the concept of processing and
analysing DNA extracted from an environment as if it were
a single large genome.6 A frequently quoted statistic
suggests that on average only 0.1-1% of bacteria are
culturable from any given niche, depending on the
complexity and the understanding of said niche.7,8 Attempts
have been made to address this shortfall between
cultivatable bacteria and the diversity known to be present.
Extracted DNA can be fragmented and ligated into large
Bacterial Artificial Chromosome (BAC) clone libraries. Once
the environmental DNA is in a suitable host such as E. coli
the BACs can be screened for desired activity using plate
screens or amplified to a level compatible with Sanger
sequencing methods of analysis. Such BAC and Fosmid
libraries are currently used to interrogate metagenomic
samples to discover new enzyme activities or new examples
of enzymes with existing activities, although this is a
strategy that requires suitable high-throughput screens.9,10
With the advent of high throughput sequencing
technologies, raw DNA extracted from the environment can
be used for sequence based analysis without the need for
amplification in BAC libraries. Most of the research using
such sequence based analysis has been aimed towards
using microbial 16S based taxonomy to understand the
range and diversity in a microbiome. More recently it has
been used to observe how the microbiome changes with
different inputs or host alterations, interactions between
organisms and between organisms and the host, with
protein annotations being used primarily to infer nutrient
use and nutrient flow through the niche.11-13
Currently TAms have significant potential for the
synthesis of an increasing number of amine products.
Although a number of TAms have been reported and are
now available commercially there is a need to identify new
TAms with variation in the primary sequence for future
applications including enzyme engineering projects: a
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 2 of 11
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/C6GC02769E
Figure 1. SDS gels of induced TAms. Two samples for each enzyme was loaded onto the gels, total protein fraction and soluble fraction respectively (1= total protein
fraction and 2= soluble fraction). 5 µL of sample was loaded into each well (from a mixture of 10 µL of protein sample and 30 µL of loading dye). Protein markers 10-
250 kDa (broad range ladder NEB). Enzyme molecular weights were calculated using the online ExpPASy ProtParam tool and are given in kDa after the plasmid name.
A) Empty vector B) pQR1108 – 52; C) pQR1109 - 46.2; D) pQR1110 - 50.4; E) pQR1111 - 47.5; F) pQR1112 - 44.6; G) pQR1113 - 52.2; H) pQR1114 – 54; I) pQR1115 -
47.4; J) pQR1116 - 47.9; K) pQR1117 - 48.6; L) pQR1118 - 48.4.
More recently it has been used to observe how the
microbiome changes with different inputs or host
alterations, interactions between organisms and between
organisms and the host, with protein annotations being
used primarily to infer nutrient use and nutrient flow
through the niche.11-13
Sequence data is used less frequently as an aid to
enzyme discovery, most often by providing consensus
sequences for primer design.14,15 The strategy applied here
sufficient depth to create 39,971 contigs containing 76.8
Mbase pairs with the largest contig being 42 kbp and 2,347
contigs were over 5 kb giving a large library of contigs
containing several full length open reading frames (ORFs).11
The contig library was analysed with the Genemark
software to identify and mark ORFs. These putative protein-
coding stretches of DNA were then scanned by the Pfam
standalone tool. This process marked the ORFs with a Pfam
ID linked to a specific protein family or enzyme class.
Because of this all ORFs of a desired enzyme class, provided
they have a Pfam ID, within the contig library could be
extracted.
The Pfam ID for Class III TAms was used to extract all
ORFs marked from the contig library. 53 ORFs were
identified of which 15 were full length and non-redundant.
Enzymes identified from the metagenome were to be
cloned using conventional restriction cloning techniques. To
this end the sequences for retrieval were scanned for
internal restriction sites. Primer pairs where designed for
enzymes including an Nde1 restriction site in the forward
primer and various restriction enzyme sites in the reverse
primer depending on any endogenous restriction sites. 11
out of 15 sequences were successfully retrieved from the
metagenome. Blunt end PCR product was ligated into the
PCR capture vector PCR-Blunt and through a series of
restrictions and ligation was cloned into the pET29a
expression vector. The pET29a vector containing the
enzyme DNA sequences was transformed into the
BL21*DE3 pLysS strain of E. coli for expression. The 11
putative enzymes were named with pQR numbers (See SI)
and analysed by SDS-PAGE. All of the enzymes had a good
level of expression except pQ1108 with a lower level of
total expression of soluble and insoluble protein (figure 1,
B) and pQR1117 (figure 1, K) with very little total
expression. pQR1112 was completely insoluble (Figure 1, F1
(total protein), F2 (soluble fraction)) and pQR1114 was
partially insoluble (Figure 1, H1 (total protein), H2 (soluble
fraction).
uses
a recently reported sequence-directed enzyme
retrieval method involving the identification of individual
open reading frames (ORFs) with subsequent specific
primer design to each non redundant example of a desired
enzymes class.16 From amino acid sequence alignment
there are six groups of TAms.1 The Class III TAms are
characterized by broad substrate acceptance and high
regio- and stereoselectivities and have been used in a wide
range of applications for the synthesis of single isomer
chiral amines.1,2 Here, the interrogation of an in silico
metagenomic library identified 15 full length non
redundant Class III transaminases of which 11 were
successfully expressed and used in screens to identify
substrate profiles.
Results and discussion
Protein selection and production
DNA, extracted from combined oral tongue scrapings from
nine volunteers, was sequenced using Roche 454 Titanium
platform.16 Sequencing returned 1.182 million reads with a
mean sequence length of 385 base pairs (bp). This raw data
was submitted to bioinformatics analysis for quality control
to remove corrupted sequences, trim corrupted ends and
filter out human sequences. After this the data set
contained 1,048 million reads with a mean length of 410
bp. This reduced data set was assembled using MIRA for
the creation of a contiguous (contig) read library, where
individual reads were joined into longer stretches of DNA
with Spoiler detection in MIRA to prevent chimeric contigs
from joining. Sequencing of the DNA sample was of
Screening of 11 potential TAms
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 11
Green Chemistry
Please do not adjust margins
Journal Name
ARTICLE
The 11 potential TAms were then screened as crude cell
lysates using three different methods (Scheme 1): an (S)-α-
methylbenzylamine ((S)-MBA 1) amine donor assay against
21 aldehydes and ketones (Figure 2A) with detection of
acetophenone 2 formation by HPLC at 250 nm (Scheme 1A);
a benzaldehyde 3 amine acceptor screening method based
on benzylamine 4 formation detected by HPLC at 210 nm
(Scheme 1B) with five classical amine donors (Figure 2B),
and a copper sulfate assay based on the detection of
alanine 6 by formation of a complex with copper,17
monitored with a spectrophotometer at 595 nm using
sodium pyruvate 5 as amine acceptor (Scheme 1C) and nine
amine donors (Figure 2C).
Three of the 11 TAm enzymes, pQR1108, pQR1113 and
pQR1114 were found to accept a wide range of structurally
diverse aldehydes and ketones 7-25 from the (S)-MBA
screening, and selected results are shown in Figure 3.
pQR1108 showed the highest activity towards both linear
and cyclic substrates. For the cyclic substrates, the best
conversion yields were obtained with benzaldehyde 3 (61%)
and cinnamaldehyde 15 (44%). Modification or absence of
the aromatic ring led to significantly lower conversions with
densitometry to calculate protein loading, specific activities
View Article Online
for pQR1108, pQR1113 and pQR1114DwOeI:r1e0.1in03v9e/sCt6igGaCt0e2d76f9oEr
two substrates 3 and 10.
O
NH2
A
B
C
TAm, PLP
R
H
R
H
or
O
or
O
NH2
1a
2
NH2
Ph
HPLC
detection at
250 nm
Ph
R1
R2
R1
R2
O
NH2
TAm, PLP
R
H
R
H
or
O
or
NH2
O
NH2
3
4
Ph
HPLC
Ph
H
R1
R2
R1
R2
detection at
210 nm
O
NH2
TAm, PLP
CO2Na
R
H
R
H
NH2
or
O
or
O
5
6
NH2
CO2H
R1
R2
R1
R2
CuSO4
MeOH
monitor at 595 nm
Scheme 1. Screening assays performed with the TAms. A (S)-MBA 1a
screening reaction conditions: 5 mM aldehyde or ketone, 25 mM (S)-MBA
1a, 1 mM, KPi pH 7.5 100 mM, enzyme as crude cell lysate 0.2-0.4 mg/mL, 18
h, 30 °C, 500 rpm. B Benzaldehyde screening reaction conditions: 5 mM
benzaldehyde 3, 25 mM amine donor, 1 mM, KPi pH 7.5 100 mM, enzyme as
crude cell lysate 0.2-0.4 mg/mL, 18 h, 30 °C, 500 rpm. C Copper sulfate
screening reaction conditions: 15 mM sodium pyruvate 5, 20 mM amine
donor, 1 mM KPi pH 7.5 100 mM, enzyme as crude lysate 0.2-0.4 mg/mL, 1 h,
30 °C, 500 rpm.
for
example
4-hydroxybenzaldehyde
13
and
cyclohexanecarboxaldehyde 10 (22% and 24% conversion
yields, respectively). More hindered aromatic substrates
like 1-indanone 14 resulted in lower conversion yields (8%).
Cyclohexanone 7 was also well accepted by pQR1108 (27%)
and modification of the six-membered ring led to smaller
activity for 2-methylcyclohexanone 8 (4%) or no activity for
the enone cyclohex-2-en-1-one 9. When using linear
substrates, the length of the carbon chain seemed to be
important: a 42% conversion yield was obtained with
pQR1108 and butanal 25 but 11% for octanal 24.
Functionalized aldehydes or ketones were also accepted,
for example when using L-erythrulose 20 (9% yield) and
glycoaldehyde 21 (9% yield) (Figure 3). Linear ketones such
as 2-heptanone 22 and 2-butanone 23 were not accepted.
In order to confirm that the activity was due to the
overexpressed TAm an assay for each (positive) substrate
was performed in triplicate with E. coli cells containing an
empty vector: no activity was observed. The benzaldehyde
screening with five classical amine donors ((S)-MBA 1a, (R)-
MBA 1b, (S)-alanine 6a, (R)-alanine 6b and isopropylamine
26) did not give rise to any new activities: benzylamine
formation was only observed when using (S)-MBA. This
highlighted that all three enzymes exhibited (S)-
stereoselectivity. For the copper sulfate screening, with
sodium pyruvate as amine acceptor and nine different
amine donors 4, 27-34, no activity was observed, consistent
with the (S)-MBA screen where pyruvate 5 was not
accepted as a substrate. The remaining 8 enzymes showed
negligible activity against the substrates screened. This
reflected for pQR1117 poor enzyme expression, pQR1112
low levels of soluble protein formed, and for pQR1109,
pQR1110, pQR1111, pQR1115, pQR1116, pQR1118 most
likely that preferred donors/acceptors were not screened.
Using a combination of the Bradford assay and SDS page
A
H
O
O
O
O
O
O
8
9
11
12
7
10
O
O
O
O
H
Ph
H
H
Ph
O
13
14
15
3
HO
HO
O
O
O
O
O
O
OH
ONa
OH
5
18
19
16
17
OH
OH
O
O
O
O
O
O
20
21
22
OH
HO
HO
H
OH
O
O
O
H
H
5
24
25
23
O
B
C
O
1a
1b
6a
6b
26
Ph
Ph
OH
OH
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OMe
H2N
NH2
H2N
3
30
NH2
27
O
28
31
29
OH
Ph
H2N
4
NH2
NH2
33
32
4
34
O
NH2
Figure 2. Substrates screened with assays A ((S)-MBA 1 and HPLC detection
of 2), B (benzaldehyde 3 and HPLC detection of 4), and C (pyruvate 5 and
spectrophotometric detection of alanine 6 after treatment with CuSO4).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 4 of 11
View Article Online
DOI: 10.1039/C6GC02769E
Journal Name
70%
ARTICLE
60%
50%
40%
30%
20%
10%
0%
pQR1108
pQR1113
pQR1114
Figure 3. Screening results of three of the TAms against 12 selected substrates. Screening conditions: 5 mM aldehyde or ketone, 25 mM (S)-MBA 1a, 1 mM KPi (pH
7.5, 100 mM), enzyme as crude cell lysate 0.2-0.4 mg.mL-1, 18 h, 30 °C, 500 rpm. Conversion yields (Scheme 1A assay via detection of 2) were obtained from three
independent experiments and varied by <± 4%.
that are closely related to each other and are likely to have
other donor specificity.
pQR1108 had the highest specific activities of 1.7 µmol min-
1 mg-1, and 0.4 µmol min-1 mg-1 for 3 and 10 respectively.
pQR1113 had an activity of 0.1 µmol min-1 mg-1 for 10. It
was not possible to calculate specific activities for pQR1113
with 3 and pQR1114 with 3 and 10 due to low activity over
the course of the reaction.
Sequence homology of the 11 potential TAms was
compared18,19 against 4 reported TAms from Vibrio fluvialis
(Vf-TAm)20, Chromobacterium violaceum DSM30191 (Cv-
TAm)21, Pseudomonas putida KT2440 (Pp-TAm)22 and
Klebsiella pneumonia JS2F (Kp-TAm)23. Interestingly,
pQR1113 and pQR1114 were found to have high sequence
homology (93% identity) in agreement with the screening
results. pQR1108 was found to have reasonably high
sequence homology with Kp-TAm (55% identity plus 23%
similarity) (Figure 4). The three enzymes had less than 30%
identity to Cv-TAm and Vf-TAm. The remaining 8 enzymes
which had shown negligible activity had less than 30%
identity with the 4 reported TAms (see Table 1 SI for
sequence homology comparison to Cv-TAm). pQR1109,
pQR1110, pQR1111, pQR1115, pQR1116 and pQR1118 had
between 70-95% homology with each other and it is likely
that these TAms require a different amine donor. Similarly
pQR1113, pQR1114 and pQR1117 form a cluster of TAms
Figure 4. Amino acid sequences of the metagenomics TAms and 4 TAms from
identified species (Kp-TAm, Pp-TAm, Cv-TAm and Vf-TAm) were aligned and
used to generate a maximum likelihood phylogenetic tree (bootstrap values
calculated from 1000 replicates).19,24
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 4
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 5 of 11
View Article Online
DOI: 10.1039/C6GC02769E
Journal Name
ARTICLE
Figure 5. Mulitple alignment of the active metagenomics enzymes with Kp-TAm, Pp-TAm, Cv-TAm and Vf-TAm. Residues important for substrate and PLP binding in
the Cv-TAm dimer are shown in red for the key residues in the first monomer and in green for the key residues in the second monomer.26 Lysine 288 crucial for Schiff
base formation is represented with a red star.
The important active site residues of pQR1108, pQR1113
and pQR1114 were also compared with the same 4
reported TAms. Nine residues are considered as very
important in the Cv-TAm dimer and are colored in red for
the key residues of the first monomer and in green for the
key residues of the second monomer (Figure 5).25 Most
residues were conserved in the 3 new enzymes. For
example, Lys288 (using numbering of amino acids based on
the sequence of Cv-TAm) is involved in the formation of a
covalent bond with the PLP. Asp259, Ser121 and Tyr153
which are known to be important for the coordination of
the PLP in the active site are also all fully conserved in our 3
enzymes. The residue Asp259 is involved in hydrogen
bonding with the pyridine ring of the PLP and Ser121 and
Tyr153 have roles in the phosphate group coordination of
the PLP.
factor concentrations, pH, co-solvents,) were studied with
enzyme pQR1108, pQR1113 and pQR1114 using (S)-MBA 1a
with cyclohexanecarboxaldehyde 10 (5 mM) as amine
acceptor in order to identify the preferred reaction
conditions. The results are shown in Figure 6. Regarding the
buffer, a slightly higher conversion was obtained with
pQR1108 and PIPES, while HEPES gave a much lower
conversion: due to the low cost of potassium phosphate
buffer this was used in subsequent experiments. For (S)-
MBA 1a concentration, with pQR1108 no inhibition was
observed when using up to 50 mM, and for the 3 enzymes
25 mM of 1a was preferred so was used. A slight increase in
conversion yield was also observed for pQR1108 when
lowering the potassium phosphate (KPi) buffer
concentration to 50 mM (27% yield compared to 24% yield
at 100 mM) and by operating at pH 8.0 (30% yield against
24% yield at pH 7.5). For pQR1113 and pQR1114 a pH of 7.5
and 50 mM KPi was preferred.
Reaction optimisation
The main parameters influencing the rate of a biocatalytic
transamination (temperature, buffer, substrate and co-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 6 of 11
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/C6GC02769E
Figure 6. Effect of various parameters for the transamination of cyclohexanecarboxaldehyde 10. Conversion yields (Scheme 1A assay via detection of 2), were
obtained from experiments in triplicate and varied by <± 4%.
perhaps reflecting increased substrate solubility. The best
Using these preferred conditions it was established that the
3 enzymes were not affected by addition of a co-solvent
(10% v/v). Interestingly, the conversion with pQR1108
increased with addition of co-solvents DMSO or MeOH
(30% and 29% yields compared to 24% without cosolvent)
reaction temperature observed was 30 °C for the 3
enzymes, and a PLP concentration in the range of 1-1.5 mM
was required (data not shown). With the following
improved conditions, (S)-MBA 1a 25 mM, amine acceptor
10 5 mM, PLP 1 mM, KPi buffer pH 8 and 50 mM, and
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 11
Green Chemistry
Please do not adjust margins
Journal Name
ARTICLE
O
DMSO as co-solvent (10% v/v) and pQR1108 as cell lysate
(0.3 mg/mL) for 18 hours gave a 41% conversion yield.
View Article Online
DOI: 10.1039/C6GC02769E
pQR1108, PLP
R
NH2
R
H
O
NH2
NH2.TFA
Synthesis of allylic amines
Ph
Ph
OMe
48
The transamination of conjugated aldehydes including
cinnamaldehyde and functionalised derivatives could give
access to allylamine building blocks for the synthesis of
analogues of drugs such as the antifungal Naftin®
(naftifine).26,27 They could also be used for the synthesis of
cinnamylamine derivatives: indeed several related
compounds such as cinnamamides have shown promising
activities against breast cancer cells.28,29
Transaminases have been reported for the synthesis of
a wide range of amines, however little has been described
on the acceptance of conjugated aldehydes/ketones other
than the use of cinnamaldehyde 15 as an amine acceptor
with Cv-TAm and Vf-Tam.21,30,31 Since pQR1108
demonstrated good activity towards cinnamaldehyde 15 in
initial screens it was used with a range of conjugated
aromatic aldehydes 35-41, 3-phenylpropanal 42 to
investigate the acceptance of a non-conjugated analogues,
and several conjugated aliphatic analogues 43-47 to
generate allylic amines (Table 1). The previously optimized
reaction conditions were used with (S)-MBA 1a as the
amine donor. Cinnamaldehyde 15 was most readily
accepted in comparable conversion yields to those reported
with Cv-TAm.21 Other cinnamaldehyde analogues were
Table 1. Conjugated aldehydes screened.
Aldehyde
Conversiona
H
15
72%
75%b
Ph
O
H
35
36
39%
48%
O
MeO
H
O
Br
H
37
38
49%
0%
O
H
O
Me2N
H
39
58%
57%b
O
84%c of 48
OMe
H
40
41
57%
54%
accepted
other
than
(E)-3-(4-
Ph
O
(dimethylamino)phenyl)acrylaldehyde 38 probably due the
dimethylamine electron donating group reducing the
electrophilicity of the aldehyde. Modification to the
aromatic ring with other electron donating groups including
ortho- and para-methoxy and para-methyl moieties, 35, 39
and 37 respectively, gave rise to lower yields (39%-58%)
than with cinnamaldehyde again reflecting the lower
H
Ph
O
Br
H
42
43
44
56%
58%b
Ph
O
H
35%
electrophilicity
of
the
aldehyde.
(E)-3-(4-
O
H
Bromophenyl)acrylaldehyde 36 was also readily accepted in
48% conversion yield. Increasing the steric bulk of the
amine acceptor and addition of a methyl group or bromine
at the alkene C-2 position led to a slight decrease in
conversion yield compared to cinnamaldehyde: (2E)-2-
49%
O
H
45
46
47
45%
37%
37%
O
H
methyl-3-phenylpropenal
40
and
(2Z)-2-bromo-3-
O
phenylpropenal 41 were accepted in 57% and 54% yield.
The non-conjugated aldehyde 42 gave a conversion yield
similar to the substituted cinnamaldehydes (56%). For the
linear conjugated aldehydes 43-47, acrolein 43 was
accepted although the use of crotonaldehyde 44 gave rise
to higher yields. 3-Methylcrotonaldehyde 45, 2-pentenal 46
and 2-methyl-2-pentenal 47 were also accepted but in
slightly lower conversion yields than for crotonaldehyde 44.
The ease of generating such allylic amines using
transaminases is extremely useful and has not been
reported to date: it provides a sustainable synthetic
approach avoiding the use of metal catalysts or toxic
reducing agents.
H
O
Reaction conditions: (S)-MBA 25 mM, amine acceptor 5 mM, PLP 1 mM, KPi
buffer 50 mM at pH 8, DMSO as co-solvent (10% v/v) and pQR1108 cell lysate
(1.2 mg/mL), 500 rpm for 18 hours. aConversions (Scheme 1A assay via
detection of 2) were obtained from three independent experiments and
varied by <± 4%; bQuantified using the amine product and HPLC; Isolated
c
yield after scale-up.
To demonstrate the applicability of using pQR1108 further
a preparative scale reaction was carried out with the
optimised
reaction
conditions
and
2-
methoxycinnamaldehyde 39. After 18 hours a quantitative
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 8 of 11
ARTICLE
Journal Name
conversion based on acetophenone formation was
observed possibly due to an improved reaction when
stirring on the larger scale. After purification, amine 48 was
isolated in 84% yield.
Contigs that contained ORFs marked wiVthiewtAhrteiclePOfnalimne
identifier for Class III TAms were coDllOecI:t1e0d.10a3n9d/C6vGisCu0a2l7is6e9dE
using the Artemis DNA viewer. ORFS that were truncated by
being at the start or end of the contig were discarded. DNA
corresponding to full length ORFs was excised. Due to
inaccuracies in the in silico sequences stemming from the
sequencing technology, ORFs comprising the whole
sequence were often fragmented across the three reading
frames. Due to this DNA sequence downstream of the
suspected stop codon was collected to ensure the full
length of the coding sequence was taken forward for
analysis. Collected DNA for the ORFs was aligned and
visualised using the MEGA software. Multiple sequences
were highly similar at the amino acid level, for these
matching sequences one example was chosen to take
forward for primer design.
PCR primers were designed for the 15 identified
enzymes. Due to inconsistencies in the in silico DNA
sequences, terminal primers were designed beyond the
suspected 3’ end of the enzyme. Primers were designed to
contain an NdeI restriction site in the forward primer and
an NcoI, XhoI or BglII site in the terminal primer depending
on restriction sites within the coding sequence. PCR was
carried out on the metagenomics DNA sample using NEB
Phusion PCR kit. All reactions were carried out following the
standard protocol given in the kit with 52 ng of
metagenomic DNA per reaction and a primer concentration
of 5 pmol per reaction. Successful PCR was followed by gel
electrophoresis with PCR products cut out of the gel and
DNA recovered using Qiagen Gel extraction columns. Blunt
ended PCR products were ligated into a PCR-Blunt capture
vector and transformed into Top10 cloning cells. Top10
cells harbouring the plasmid were grown overnight in 5 mL
of Terrific Broth with kanamycin 50 mg/mL. Plasmids were
extracted using Qiagen miniprep kits and sent for
sequencing to confirm the 3’ end of the coding sequence of
the enzymes with confidence. A second round of terminal
primers were designed to remove the stop codon and
introduce a restriction site. PCR was carried out using these
primers and the PCR capture vector containing the PCR
product from the first round of PCR. The same process of
cloning as described above was carried out on the 2nd
round of PCR products. Plasmid carrying the PCR product
from this second round of cloning was purified and digested
with Nde1 and the requisite restriction enzymes to
generate cohesive ends. The digested plasmid was run on a
gel and the cohesive ended insert cut out and extracted
from the gel. The digestion fragments were ligated into
pET29a, digested to generate matching cohesive ends, and
transformed into BL21* DE3 pLysS cells.
Conclusions
The present study demonstrates the efficiency of
combining metagenomics for new enzyme discovery and
screening to identify new transaminases. Metagenomics
allows the mining of enzymes based on their protein
sequence from in silico libraries of enzymes. It can thus
uncover diverse enzymes that may have not been detected
in microwell or agar plate screens. Eleven putative
transaminases have been screened against several
substrates and three new transaminases were identified as
being active towards a broad range substrates. The
biocatalytic potential of the new TAms was illustrated with
the sustainable synthesis of allylic amines using pQR1108:
such amine building blocks can be used as precursors for
the synthesis of analogues of several drugs. We are
currently expanding the enzyme discovery approach using
other metagenomic samples and a range of important
biocatalytic enzyme families.
Experimental
General information. The solvents and chemicals were
purchased from Sigma Aldrich or Fluorochem and were
used as supplied. NMR spectra were recorded on a Bruker
Spectrometer AMX300. Chemical shifts (in ppm) are quoted
relative to tetramethylsilane and referenced to residual
pronated solvent. Coupling constants (J) are measured in
Hertz (Hz) and multiplicities for 1H NMR coupling are shown
as s (singlet), d (doublet), t (triplet), and m (multiplet). HPLC
analyses were performed on an Agilent 1260 Infinity
provided with a 1260 VWD detector. Mass spectrometry
analyses were performed at the UCL Chemistry Mass
Spectrometry Facility using a Finnigan MAT 900XP mass
spectrometer. Accurate mass determination was performed
by the peak-matching method.
Enzyme selection and production. Sequences generated by
Roche 454 titanium platform were passed to the online
platform MG-Rast, redundant sequences were removed,
sequences filtered against human reference sets and
dereplicated. The filtered output of MG-Rast was further
analysed using the Galaxy platform to assign a Phred quality
score to each nucleotide base. All sequence fragments that
had a minimum score of 20 and length of 50 nucleotides
were carried on for further analysis. The MIRA assembler
software was used to take these individual sequences reads
and assemble them into larger contiguous reads. These
ORFS within these contigs were marked using the
Genemark software and subsequently marked with a Pfam
ID using the Pfam standalone tool.
Small scale enzyme expression and preparation. The TAms in
E. coli BL21(DE3)pLysS were added to terrific broth medium
(5 mL) containing kanamycin (50 mg/mL) and
chloramphenicol (25 mg/mL) and incubated overnight at 37
°C: 1 mL of the overnight culture was then used to inoculate
terrific broth (100 mL containing the same concentration of
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 11
Green Chemistry
Please do not adjust margins
Journal Name
ARTICLE
antibiotic) which was grown at 37 °C until an OD600 of 0.6
was reached at which point they were induced with 1 mM
IPTG and grown for a further 5 h at 25 °C. The cells were
harvested by centrifugation, the supernatant removed, and
the cell pellet resuspended in potassium phosphate buffer
(KPi; 5 mL of 0.1 M KPi pH 7.5, 1 mM PLP) and flash frozen
in liquid nitrogen. The frozen pellet was place on a freeze
dryer. 25 mg of freeze dried cell was resuspended in the
same 0.1 M KPi buffer pH 7.5 (1 mL) as before and
sonicated. The lysed freeze dried cells were spun down and
the enzyme concentration was determined using the
Bradford method and then diluted if necessary to obtain an
buffer (pH 7.5, 100 mM) and the substrate (5 mM in water)
View Article Online
were added in a 96 well plate to giveDaOtI:o1t0a.1l0v3o9l/uCm6GeCo02f716890E
µL. The reaction was started by the addition of 20 µL of
clarified cell extract (final concentration of 0.2-0.4 mg.mL-1).
After incubation for 1 h at 30 °C and 500 rpm, 40 µL of the
prepared staining solution was added and the reaction
mixture was then centrifuged (1 minute at 9000 rpm) to
remove the insoluble phosphate-copper complex. The
supernatant was then added to a 96 well-plate and
absorbance was measured at 595 nm with a spectrometer
SpectraMax Plus 384 from Molecular Devices. DOD was
then calculated between the assay and a negative control
without amine acceptor. This value was compared with
DOD calculated for CV2025 and benzylamine as an amine
donor and used as a reference (for 100% of relative
activity). The limit of sensitivity was established to be 10%
of relative activity.
enzyme concentration of between 2.0 and 4.0 mg.mL-1
.
Initial (S)-MBA screening method. (S)-MBA 1a (25 mM) and
PLP (1 mM) in KPi buffer (pH 7.5, 100 mM), and the
substrate (5 mM in water other than for cinnamaldehyde
15 and 1-indanone 14 when DMSO was used) were added
in a 96 well-plate to give a total volume of 180 µL. The
reaction was started by the addition of 20 µL of clarified cell
extract (final concentration of 0.2-0.4 mg.mL-1). After
incubation for 18 h at 30 °C and 500 rpm, the reaction was
quenched with 1 µL of TFA and denatured protein was
removed by centrifugation. The supernatant (180 µL) was
diluted with water (540 µL) and analysed by HPLC using an
ACE 5-C18 300 column (150 x 4.6 mm) with UV detection at
250 nm. The concentration of acetophenone produced was
determined using a linear gradient 15% - 72% over 10
minutes at 1 mL.min-1 (A = water with 0.1% of TFA and B =
acetonitrile) with subtraction of a negative control without
amine acceptor for all substrates. The acetophenone
produced eluted at a retention time of 8.8 minutes. Results
were verified in triplicate against a negative control without
amine acceptor following the same procedure.
(S)-MBA screening with preferred conditions for
conjugated aldehydes. All substrates were solubilized in
DMSO to give a final concentration of 10% v/v. (S)-MBA (25
mM) and PLP (1 mM) in KPi (pH 8.0, 50 mM) and the
aldehyde (5 mM in water) were added to a 96 well plate to
give a total volume of 140 µL. The reaction was started by
the addition of 60 µL of clarified cell extract of pQR1108
(final concentration of 1.2 mg.mL-1 ). After an incubation for
18 h at 30 °C and 500 rpm, the reaction was quenched with
TFA (1 µL, 0.5% v/v) and denatured protein was removed by
centrifugation. The supernatant (180 µL) was diluted with
water (540 µL) and analysed by HPLC using a ACE 5-C18 300
column (150 x 4.6 mm) with UV detection at 250 nm.
Concentrations of acetophenone were determined using a
linear gradient 15% - 72% B over 10 minutes at 1 mL.min-1
(A = water with 0.1% of TFA and B = acetonitrile) with
subtraction of a negative control without amine acceptor
for all the substrates. The acetophenone produced eluted
at a retention time of 8.8 minutes.
Benzaldehyde screening method. The amine donor (25
mM) and PLP (1 mM) in KPi buffer (pH 7.5, 100 mM), and
benzaldehyde (5 mM in water) were added in a 96 well
plate to give a total volume of 180 µL. The reaction was
started by the addition of 20 µL of clarified cell extract (final
concentration of 0.2-0.4 mg.mL-1). After incubation for 18 h
at 30 °C and 500 rpm, the reaction was quenched with 1 µL
of TFA and denatured protein was removed by
centrifugation. The supernatant (180 µL) was diluted with
water (540 µL) and analysed by HPLC using a ACE 5-C18 300
column (150 x 4.6 mm) with UV detection at 210 nm. The
concentration of benzylamine was determined using a
gradient 15% - 72% B over 10 minutes at 1 mL.min-1 (A =
water with 0.1% of TFA and B = acetonitrile). The
benzylamine produced eluted at a retention time of 3.4
minutes.
Preparative scale reaction. The enzymatic reaction was
performed using a total volume of 100 mL, containing (S)-
MBA 1 (25 mM, 2.5 mmol, 302 mg) as amine donor, (E)-3-
(2-methoxyphenyl)acrylaldehyde 39 (5 mM, 0.5 mmol, 82
mg) as amine acceptor, PLP (1 mM), KPi buffer pH 8.0 (50
mM), cell lysate of pQR1108 (1.2 mg.mL-1) and DMSO as co-
sovent (10% v/v) at 30 °C and 500 rpm for 18 h. The
reaction mixture was acidified and centrifuged. The
supernatant was extracted with Et2O (3 x 100 mL) then
basified with NaOH (2 M) and re-extracted with Et2O (3 x
100 mL). The combined organic extracts were concentrated
in vacuo. The product was added to water with 0.1% TFA (8
mL) and purified by preparative HPLC (Varian ProStar model
210) using a Dr. Maisch GmbH C8 Reprosil Gold 200 column
(150 x 10 mm, 5µm) with UV detection at 210 nm with a
linear gradient 5% - 50% B over 10 minutes at at 6 mL.min-1
(A = water with 0.1% of TFA and B = acetonitrile with 0.1%
of TFA). Compound 48 eluted with a retention time of 5.4
minutes to afford compound 48 as a white solid (116 mg,
CuSO4/MeOH screening method. Preparation of the
CuSO4/MeOH solution: 300 mg of copper(II) sulfate
pentahydrate ws dissolved in 500 µL of water and 30 mL of
MeOH. Sodium pyruvate (25 mM) and PLP (1 mM) in KPi
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 10 of 11
ARTICLE
Journal Name
84%). M.p. 217 °C (decomposed); 1H NMR (300 MHz; D2O) δ
7.52 (1H, d, J = 7.5 Hz, 6-H), 7.32-7.43 (1H, m, 4-H), 6.98-
7.09 (3H, m, 3-H, 5-H, =CHPh), 6.30 (1H, dt, J = 15.6 and 6.9
Hz, CH=CHPh), 3.86 (s, 3H, OCH3), 3.76 (2H, d, J = 6.9 Hz,
CH2N); 13C (75 MHz; D2O with MeOH as a standard, TFA salt
signals not described) δ 157.0, 131.5, 130.6, 127.9, 125.0,
121.8 (signals superimposed), 112.6 (CH), 56.2 (CH3), 42.2
(CH2); m/z (CI+) 164 ([M]+, 60%), 147 (100); HRMS (CI+)
found [M]+ 164.1071; C16H15N3O4 requires 164.1070.
11 V. Lazarevic, K. Whiteson, S. Huse, D. Hernandez, L.
View Article Online
Farinelli, M. Østerås, J. Schrenzel and P. François, J.
Microbiol. Methods, 2009, 79, 266.DOI: 10.1039/C6GC02769E
12 S. Jacquiod, S. Demanèche, L. Franqueville, L. Ausec, Z.
Xu, T. O. Delmont, V. Dunon, C. Cagnon, I. Mandic-Mulec,
T. M. Vogel and P. Simonet, J. Biotechnol., 2014, 190, 18.
13 J. S. McLean, Front. Cell. Infect. Microbiol., 2014, 4, 98.
14 N. Itoh, K. Isotani, Y. Makino, M. Kato, K. Kitayama and T.
Ishimota, Enzyme Microb. Technol., 2014, 55, 140.
15 J. G. Owen, B. V. B. Reddy, M. Ternei, Z. Charlop-Powers,
P. Y. Calle, J. H. Kim and S. F. Brady, Proc. Natl. Acad. Sci.
U.S.A. 2013, 110, 11797.
16 J. W. E. Jeffries, N. Dawson, C. Orengo, T. S. Moody, D. J.
Quinn, H. C. Hailes and J. M. Ward, ChemistrySelect, 2016,
1, 2217.
17 B. Y. Hwang and B. G. Kim, Enzyme Microb. Technol.,
2004, 34, 429.
18 C. Notredame, D. G. Higgins and J. Heringa, J. Mol. Biol.,
2000, 302, 205.
19 S. Guindon, J. Dufayard, V. Lefort, M. Anisimova, W.
Hordijk and O. Gascuel, Syst. Biol., 2010, 59, 307.
20 J. S. Shin, J. W. Wang and B. G. Kim, Appl. Microbiol.
Biotechnol. 2003, 61, 463.
21 U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes and
J. M. Ward, Enzyme Microb. Technol., 2007, 41, 628.
22 M. Espinosa-Urgel and J. L. Ramos, Appl. Environ.
Microbiol., 2001, 11, 5219.
23 J. S. Shin and B. G. Kim, Biosci. Biotechnol. Biochem.,
2001, 65, 1782.
24 D. C. Higgins and J. Heringa, J. Mol. Biol., 2000, 302, 205.
25 M. S. Humble, K. E. Cassimjee, M. Håkansson, Y. R.
Kimbung, B. Walse, V. Abedi, H. J. Federsel, P. Berglund
and D. T. Logan, FEBS J., 2012, 279, 779.
26 E. G. Evans, I. G. James, R. A. Seaman and M. D.
Richardson, Br. J. Dermatol., 1993, 129, 437.
27 C. T. Lee, Y. C. Giam and T. Tan, Singapore Med. J., 1987,
28, 429.
28 A. R. Germain, L. C. Carmody, P. P. Nag, B. Morgan, L.
Verplank, C. Fernandez, E. Donckele, Y. Feng, J. R. Perez,
S. Dandapani, M. Palmer, E. S. Lander, P. B. Gupta, S. L.
Schreiber and B. Munoz, Bioorg. Med. Chem. Lett., 2013,
23, 1834.
29 Y. Xiong, T. Ye, M. Wang, Y. Xia, N. Wang, X. Song, F.
Wang, L. Liu, Y. Zhu, F. Yang, Y. Wei and L. Yu, Cellular
Phys. And. Biochem., 2014, 34, 1863.
30 J. S. Shin and B. G. Kim, J. Org. Chem., 2002, 67, 2848.
31 J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J.
Pfeffer, T. Haas and W. Kroutil, Angew. Chem. Int. Ed.,
2012, 51, 9156.
Acknowledgements
Funding from the Biotechnology and Biosciences Research
Council (BBSRC) (BB/L007444/1) for D. B. and J. W. E. J. and
ALMAC is gratefully acknowledged. Furthermore, we thank
the UCL Mass Spectrometry and NMR Facilities in the
Department of Chemistry.
References
1
2
J. M. Ward and R. Wohlgemuth, Curr. Org. Chem., 2010,
14, 1914.
For reviews: a) D. Koszelewski, K. Tauber, K. Faber and W.
Kroutil, Trends Biotechnol., 2010, 28, 324; b) M. S. Malik,
E. S. Park and J. S. Shin, Appl. Microbiol. Biotechnol., 2012,
94, 1163; c) S. Mathew and H. Yun, ACS Catal., 2012, 2,
993. d) M. Fuchs, J. E. Farnberger and W. Kroutil, Eur. J.
Org. Chem., 2015, 32, 6965.
3
4
R. A. John, Biochim. Biophys. Acta, 1995, 1248, 81.
C. Sayer, R. J. Martinez-Torres, N. Richter, M. N. Isupov, H.
C. Hailes, J. A. Littlechild and J. M. Ward, FEBS J., 2014,
281, 2240.
5
For example: a) C. K. Savile, J. M. Janey, E. C. Mundorff, J.
C. Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F.
J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman and G. J.
Hughes, Science, 2010, 329, 305; b) T. Sehl, H. C. Hailes, J.
M. Ward, R. Wardenga, E. von Lieres, H. Offermann, R.
Westphal, M. Pohl and D. Rother, Angew. Chem. Int. Ed.,
2013, 52, 6772; c) N. Richter, R. C. Simon, W. Kroutil, J. M.
Ward and H. C. Hailes, Chem. Commun., 2014, 50, 6098;
d) C. K. Chung, P. G. Bulger, B. Kosjek, K. M. Belyk, N.
Rivera, M. E. Scott, G. R. Humphrey, J. Limanto, D. C.
Bachert and K. M. Emerson, Org. Process Res. Dev., 2014,
18, 215; e) E. Busto, R. C. Simon, B. Grischek, V. Gotor-
Fernandez and W. Kroutil, Adv. Synth. Catal., 2014, 356,
1937; f) B. R. Lichman, E. D. Lamming, T. Pesnot, J. M.
Smith, H. C. Hailes and J. M. Ward, Green Chem., 2015,
17, 852; g) M. T. Gundersen, P. Tufvesson, E. J. Rackham,
R. C. Lloyd and J. M. Woodley, Org. Proc. Res. Dev., 2016,
20, 602.
6
7
8
9
J. Handelsman, M. R. Rondon, S. F. Brady, J.Clardy, and R.
M. Goodman, Chem. Biol., 1998, 5, 245.
B. Temperton and S. J. Giovannoni, Curr. Opin. Microbiol.,
2012, 15, 605.
V. Torsvik, L. Øvreås, T. F. Thingstad, Science, 2002, 296,
1064.
P.-Y. Colin, B. Kintses, F. Gielen, C. M. Miton, G. Fischer,
M. F. Mohamed, M. Hyvönen, D. P. Morgavi, D. B. Janssen
and F. Hollfelder, Nat. Commun., 2015, 6, 10008.
10 T. Narihiro, A. Suzuki, K. Yoshimune, T. Hori, T. Hoshino, I.
Yumoto, A. Yokota, N. Kimura and Y. Kamagata, Microbes
Environ., 2014, 29, 154.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 11
Green Chemistry
View Article Online
DOI: 10.1039/C6GC02769E
ToC Entry
A metagenomics mining strategy was used to identify new transaminases that were utilised in the
synthesis of allylic amines