Enzyme Cascades in Whole Cells for the Synthesis of Chiral Cyclic Amines

Article abstract of DOI:10.1021/acscatal.7b00513

The increasing diversity of reactions mediated by biocatalysts has led to development of multistep in vitro enzyme cascades, taking advantage of generally compatible reaction conditions. The construction of pathways within single whole cell systems is much less explored, yet has many advantages. Herein we report the generation of a successful whole cell de novo enzyme cascade for the diastereoselective and/or enantioselective conversion of simple, linear keto acids into valuable cyclic amine products. The pathway starts with carboxylic acid reduction that triggers a transamination, imine formation, and subsequent imine reduction. Construction and optimization of the system was achieved by standard genetic manipulation and the cascade required only starting material, amine donor, and whole cell catalyst with cofactors provided internally by glucose metabolism. A panel of synthetic keto acids provided access to piperidines in high conversions (up to 93%) and enantiomeric excess (up to 93%).

Full text of DOI:10.1021/acscatal.7b00513

Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI
Article
De Novo Enzyme Cascades in Whole Cells
for the Synthesis of Chiral Cyclic Amines.
Lorna J Hepworth, Scott P. France, Shahed Hussain, Peter Both, Nicholas J Turner, and Sabine L. Flitsch
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b00513 • Publication Date (Web): 14 Mar 2017
Downloaded from http://pubs.acs.org on March 14, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
De Novo Enzyme Cascades in Whole Cells for
the Synthesis of Chiral Cyclic Amines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
a
a,b
a
a
Lorna J. Hepworth , Scott P. France , Shahed Hussain , Peter Both , Nicholas J. Turner ,
a,
Sabine L. Flitsch *.
a. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology,
1
31 Princess Street, Manchester M1ꢀ7DN, United Kingdom.
b. present address: Dr. Reddy’s Laboratories, Chirotech Technology Centre, 410 Cambridge
Science Park, Milton Road, Cambridge, CB4 0PE, United Kingdom.
ABSTRACT
The increasing diversity of reactions mediated by biocatalysts has led to development of
multiꢁstep in vitro enzyme cascades, taking advantage of generally compatible reaction
conditions. The construction of pathways within single whole cell systems is much less
explored, yet has many advantages. Herein we report the generation of a successful whole
cell de novo enzyme cascade for the diastereoselective and/or enantioselective conversion of
simple, linear keto acids into valuable cyclic amine products. The pathway starts with
carboxylic acid reduction that triggers a transamination, imine formation and subsequent
imine reduction. Construction and optimization of the system was achieved by standard
genetic manipulation and the cascade required only starting material, amine donor and whole
cell catalyst with cofactors provided internally by glucose metabolism. A panel of synthetic
keto acids provided access to piperidines in high conversions (up to 93%) and ee (up to 93%).
ACS Paragon Plus Environment

ACS Catalysis
Page 2 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
KEYWORDS: biocatalysis, chiral piperidines, enzyme cascades, gene duplication, whole
cell biotransformation
The exploitation of synthetic biology methodologies to produce pharmaceutically and
industrially relevant synthetic products, using the design principles of biosynthetic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1–3
retrosynthesis, is a highly promising method for chemical synthesis. The everꢁexpanding
biocatalytic toolbox presents an opportunity to design and build cascades that have no
biological origin for the production of high value chemicals. A number of enzymatic cascades
4–13
have been described during the past several years
although very few have been used as
1
4–20
single whole cell biocatalysts, until recently
. Whereas natural biosynthetic pathways have
evolved to be biocompatible, de novo synthetic cascades can potentially raise problems in
whole cells, such as generation of bioreactive intermediates, metabolic degradation, transport
issues through membranes, heavy cofactor requirements and control of activity of the
individual biocatalysts. Although these issues make development of whole cell systems more
complex, the overall benefit of having a single (whole cell) biocatalyst that potentially
provides biochemical cofactors by glucose metabolism makes cascades in single cells very
attractive. Here, we describe the development of a fourꢁenzyme, fourꢁstep cascade that was
chosen as it allows for the synthesis of high value chiral amines from easily accessible keto
acids. The cascade also challenges existing whole cell systems by generating a reactive
aldehyde intermediate and by (multi)ꢁstoichiometric requirements for intracellular cofactors
ATP and NAD(P)H.
2
1
The DesignꢁBuildꢁTestꢁAnalyze (DBTA) cycle was adapted to the generation of novel
whole cell in vivo biocatalytic cascades as outlined in Figure 1: (1) designing of the pathway
via biocatalytic retrosynthetic analysis, (2) selection of homologs from each enzyme class to
test (e.g. based on substrate scope), (3) generation of a library of plasmids containing various
gene combinations, (4) introduction of a single plasmid into a suitable chassis and expression
ACS Paragon Plus Environment

Page 3 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
of cascade proteins and (5) testing the system by screening of each whole cell system against
appropriate substrate(s). Using this workflow, a variety of pathway scenarios can be quickly
tested without need for enzyme isolation and purification and the most successful whole cells
taken through to process optimization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Overview of the DesignꢁBuildꢁTest workflow for the generation of de novo enzyme
cascades in whole cell systems. The design follows the principles of biocatalytic
retrosynthesis with the catalyst then built at the genetic level and directly tested as a whole
cell system. Results from the test phase are fed back to the design and the cycle is repeated
until the desired whole cell catalyst is generated.
Chiral secondary amines are ubiquitous functionalities found in biologically active
compounds but are not readily accessible through natural biosynthetic pathways. Chemical
synthesis of cyclic chiral amines is also challenging and often involves the use of precious
2
2,23
transition metal catalysts requiring increased downstream processing,
the use of high
ACS Paragon Plus Environment

ACS Catalysis
Page 4 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
pressures or undesirable solvent systems, and frequently results in the production of
24
unwanted intermediate racemates. As such, a facile enzymatic route to these scaffolds, in
23,24
particular chiral piperidine derivatives, is of great interest.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Disconnecting the target piperidine structures through a biocatalytic retrosynthetic approach
suggested keto acids 1 as starting materials for a cascade consisting of carboxylic acid
25,26
27,28
29,30
reductase (CAR),
ωꢁtransaminase (ωꢁTA)
and imine reductase (IRED)
enzymes
5
(
Figure 1). Keto acids 1 are stable compounds which are easily obtainable through scalable
chemical routes but are potentially also accessible through biosynthetic pathways.
The enzyme cascade is initiated by the ATPꢁ and NADPHꢁdependent reduction of linear keto
acids 1 to the corresponding keto aldehydes 2, catalyzed by CAR. CARs require
posttranslational modification with coenzyme A through the actions of a 4’ꢁ
31
phosphopantetheinyl transferase (Sfp), which needs to be included in the whole cell system.
The products 2 are substrates for transaminases, with the less hindered aldehyde group
selectively undergoing transformation. The resulting aminoketone products then
spontaneously cyclize to imines 3 that are substrates for imine reductases.
The design and construction of the cascade started with generating suitable expression
vectors that would programme the host cell to express the chosen biocatalysts. Based on our
1
9
previous work we started with pPB01, a plasmid devoid of regulatory elements such that
each gene could be regulated independently through a monocistronic operon design.
TM
32
BioBrick cloning strategies were used in the combinatorial assembly of the expression
constructs pLH01ꢁ08 [pPB01/ATA + CAR + IRED + BsSfp] (Figure 2) using different
homologs, which were then used to transform E. coli BL21 (DE3) cells. Protein expression
from the new plasmid systems harbored within the cells was induced with isopropylꢁβꢁDꢁ1ꢁ
thiogalactopyranoside (IPTG) and SDSꢁPAGE analysis confirmed soluble expression of all
enzymes (see Supporting Information, SI).
ACS Paragon Plus Environment

Page 5 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Plasmids pLH01ꢁ08 and pLH09ꢁ10 were generated using homologs in a
combinatorial fashion for the first and second DesignꢁBuildꢁTest cycles, respectively.
Introduction of the plasmids into E. coli resulted in transformation of 1aꢁe to 4aꢁe via the
28
reaction cascade shown. (ATAꢁ117, ωꢁtransaminase from Arthrobacter sp. ; SitATA,
28
33
mutated version of ATAꢁ117 ; NCAR, carboxylic acid reductase from Nocardia sp. ;
26
MCAR, carboxylic acid reductase from Mycobacterium marinum ; (S)ꢁIRED, imine
reductase from Streptomyces sp. GF3546 ; (R)ꢁIRED, imine reductase from Streptomyces sp.
GF3587 ; BsSfp, 4’ꢁphosphopantetheinyl transferase from Bacillus subtilis .)
3
4
35
31
To assess the activity range of this in vivo cascade several commercially available, or readily
synthesized, keto acid substrates (1aꢁe) were screened against the cells harboring pLH01ꢁ08.
It was envisaged that the use of a single whole cell acting as a biocatalyst would eliminate the
+
need to supplement the reactions with expensive exogenous cofactors (NAD(P) , PLP),
5
cofactor regeneration systems, or an external driving force for the ωꢁTA reaction (lactate
36
dehydrogenase, LDH). Although glucose is present in all respiring cells, it was expected
that supplying additional glucose would be required to relieve any metabolic burden on the
ACS Paragon Plus Environment

ACS Catalysis
Page 6 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cells and enable the cascade reaction to proceed successfully. An excess of racemic D/Lꢁ
alanine or isopropylamine (IPA) was also supplied to the whole cell in order to drive the
reductive amination to completion. Encouragingly, GC analysis of the reaction mixtures
revealed the formation of chiral secondary amine for all keto acids tested. In fact, subsequent
analysis indicated that the amine product freely diffused out of the cell into the reaction
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
buffer, which also offers several advantages with regards to biocatalyst recycling and product
37,38
extraction.
Comparison of the different vectors pLH01ꢁ08 showed that the best
conversions were obtained using plasmid pLH02 (Table 1). For further details regarding
pLH01 and pLH03ꢁpLH08, see Supporting Information. The ee and de values of amine
5
,29
products 4 are consistent with known IRED selectivities for the reduction of imine 3.
Notably, the product of rac-1e with the reported cascade affords rac-4e with high cisꢁ
diastereoselectivity, revealing that CAR and TA can convert both enantiomers of their
respective substrates in this case during the course of the reaction. Furthermore, The (R)ꢁ
IRED has previously been shown to be sensitive to preꢁexisting chirality in a cyclic imine
substrate, possibly due to steric factors between the incoming NADPH cofactor and substrate
5
ring substituents. The stereogenic centre of imine 3e is capable of overriding the inherent
selectivity of the IRED and both enantiomers are converted to afford the cisꢁdiastereomers.
ACS Paragon Plus Environment

Page 7 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
Table 1. Synthesis of Monoꢁ and Disubstituted Piperidines Through the Use of Single Whole
Cell Expressing Plasmid pLH02
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Entry
Substrate
Consumption
of 1/%
Conv.
imine 3/%
to
Conv. to
keto alcohol/%
Conv. to
4/%
4
ee/% (abs.
4 de/% (cis
or trans)
config.)
30 (S)
90 (S)
93 (S)
30 (S)
-
1
2
3
4
5
1a
1b
1c
1d
1e
>99
>99
>99
>99
>99
-
-
47
50
7
-
3
47
42
46
19
-
51
n.d.
24
-
54
57
>98 (cis)
>98 (cis)
ꢁ
1
Reaction conditions: 3 mM substrate, 40 mg mL wet E. coli BL21 (DE3) cells expressing plasmid pLH02,
2
50 mM ꢁalanine, 50 mM glucose, 500 mM pH 7 NaP buffer. Reaction incubated at 30 °C (250 rpm) for 24
D
/
L
i
h to allow full conversion of keto acid substrate, therefore conversion is based on imine:amine:keto alcohol
product ratio (absolute conversion to 4a was calculated against a calibration curve using decane as internal
standard, conversion to imine and keto alcohol was therefore not calculated for this example). n.d., not detected.
A variety of parameters concerning both protein expression and reaction conditions were
investigated, with the following achieving the highest conversion to amine product 4 when
expressing plasmid pLH02 in E. coli BL21 (DE3) cells: 16 h protein expression at 20 °C after
induction with IPTG (0.8 mM) at OD600 0.6 in LB growth media, followed by whole cell
biotransformations (24 h, 30 °C) using 3 mM substrate in 500 mM sodium phosphate buffer
containing glucose (50 mM) and either D/Lꢁalanine (250 mM) or IPA (100 mM).
ACS Paragon Plus Environment

ACS Catalysis
Page 8 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In order to gain further insight into the factors affecting the yield of amine 4 production, a
number of optimization experiments were performed using 1a as substrate (for more details
see SI). One sideꢁreaction observed was metabolism of the keto aldehydes 2 to the
corresponding keto alcohols, which occurred for all substrates using this cascade system
(Table 1). The mismatch of the kinetics of the first two steps of the cascade appeared to lead
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to accumulation of the reactive aldehydes 2, an issue that may have been resolved in natural
38,39
pathways through evolution.
The design cycle of the whole cell system provided us with a
number of options to increase ωꢁTA activity, including change of promoter or finding more
active transaminases, both of which would require extensive screening.
An alternative way in which Nature amplifies the dosage of a given protein in response to
4
0,41
environmental stimuli is through gene duplication.
Since gene duplication could boost the
slow step, ωꢁTA conversion of keto aldehyde, this approach was investigated further. A
duplicate ATAꢁ117 gene was introduced into the original construct to give an alternate 5ꢁ
gene plasmid (pLH09) (Figure 2) in the second DBTA cycle. Gratifyingly,
biotransformations of 1a using cells harboring pLH09 showed a 14% increase in amine
production compared with pLH02 (Table 2). This gene duplication strategy was also used to
circumvent accumulation of the imine intermediate; a duplicate (R)ꢁIRED gene was cloned
into the original construct to give a new plasmid pLH10 (Figure 2). Increasing the expression
level of (R)ꢁIRED resulted in even greater improvements with respect to conversion to amine
4
product (up to 93% conversion when keto acid 1e was used) (Table 2). Interestingly, the
addition of a duplicate (S)ꢁIRED gene into constructs pLH01 and pLH03 also drastically
improved the conversion to amine 4a seen for these cascades, increasing the conversion from
<
5 % to conversions comparable to those seen for construct pLH02 (See SI). It is thought
that an improvement in the IRED step moderates accumulation of the imine intermediate 3,
whilst also driving the ωꢁTA reaction towards amine 4 production. These results show that
ACS Paragon Plus Environment

Page 9 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
using gene duplication, varying expression levels for bottleneck proteins could be rapidly
assessed, which is an attractive alternative to testing promoter/terminator pairs of varying
42
strengths.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Effect of Gene Duplication on Piperidine Production
Substrate
Plasmid
Conv. to
imine 3/%
Conv. to
keto
Conv. to
[a]
4/%
alcohol/%
1
1
a
a
pLH09
pLH10
pLH09
pLH10
pLH09
pLH10
pLH09
pLH10
pLH09
pLH10
-
-
61
67
77
87
9
-
-
1b
n.d.
n.d.
67
21
n.d.
11
21
n.d.
23
13
24
12
25
5
1
b
1c
1
1
1
1
1
c
d
d
e
e
67
75
84
71
93
8
7
ꢁ
1
Reaction conditions: 3 mM substrate, 40 mg mL wet E. coli BL21 (DE3) cells expressing plasmid pLH09 or
pLH10, 250 mM ꢁalanine, 50 mM glucose, 500 mM pH 7 NaP buffer. Reactions incubated at 30 °C (250
D/
L
i
rpm) for 24 h to allow full conversion of keto acid substrate, therefore conversion is based on imine:amine:keto
alcohol product ratio (absolute conversion to 4a was calculated against a calibration curve using decane as
internal standard, conversion to imine and keto alcohol was therefore not calculated for this example). n.d., not
detected.
A hybrid cascade system comprised of two separate whole cell catalysts containing
MCAR/BsSfp and (R)ꢁIRED, respectively, and a commercial crude cell lysate containing the
transaminase ATAꢁ113 was previously identified as a route to chiral piperidines in high
5
yields. Due to the in vitro application of the transaminase it was necessary to provide this
system with external cofactor regeneration (GDH, glucose) and a driving force for the
ACS Paragon Plus Environment

ACS Catalysis
Page 10 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reductive amination reaction (LDH, D/Lꢁalanine), as well as the addition of expensive
+
cofactors (NAD , PLP) (Figure 3a). Implementing the cascade in a single whole cell
abolished the need for this exogenous supplementation but necessitated the use of a different
transaminase, as the nucleotide sequence for the ATAꢁ113 gene was not available (Figure
3b).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Comparison of the requirements of a. multiꢁcomponent one pot hybrid cascade and
b. single whole cell in vivo cascade. Items highlighted in blue represent reaction components
added to facilitate the cascade reaction.
ATAꢁ117 was selected as a suitable alternative to ATAꢁ113 due to previous studies where
43
both transaminases were shown to act on related diketone substrates. In an effort to assess
the performance of ATAꢁ117 against ATAꢁ113 in the cascade reaction, keto acid 1a was
tested with hybrid cascade conditions using either ATAꢁ117 or ATAꢁ113, with ATAꢁ113
resulting in significantly higher conversions to 4a than when ATAꢁ117 was used (see SI).
This difference in activity between the two transaminase homologs suggests that the lower
conversions seen for the in vivo cascade reported here compared with the hybrid cascade
ACS Paragon Plus Environment

Page 11 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
described previously can be attributed to the use of ATAꢁ117 rather than an inherent
inferiority with using a single whole cell system containing several biocatalysts.
The use of a single whole cell system also allowed for the rapid screening of further, more
complex, cascade substrates due to the relative ease of implementing the in vivo system
compared with the hybrid cascade reaction. E. coli BL21 (DE3) cells harboring pLH10 were
used for the production of bicyclic amine 4f and thiomorpholine 4g from keto acids 1f and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1g, and were subsequently compared with hybrid cascade reactions using either ATAꢁ117 or
ATAꢁ113 for the transaminase step (Table 3). Reactions using ATAꢁ113 again resulted in
higher conversions to product amine than their ATAꢁ117 counterparts, whilst systems
utilizing ATAꢁ117 (both the in vivo system and the hybrid system) displayed comparable
conversions.
Table 3. Synthesis of Amines 4f and 4g Through Use of in vivo or Hybrid Cascade Systems
[a]
[a]
Cascade System
Conv. to 4f/%
Conv. to 4g/%
[
a]
in vivo (ATA-117 + MCAR + (R)-IRED)
Hybrid (ATA-117 + MCAR + (R)-IRED)
Hybrid (ATA-113 + MCAR + (R)-IRED)
14
13
30
8
[b]
8
[
b]
30
ACS Paragon Plus Environment

ACS Catalysis
Page 12 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
a]
ꢁ1
Reaction conditions: 5 mM substrate, 40 mg mL wet E. coli BL21 (DE3) cells expressing plasmid pLH10,
[
b]
ꢁ1
2
50 mM
D/
L
ꢁalanine, 50 mM glucose, 500 mM pH 7 NaP
i
buffer. 5 mM substrate, 75 mg mL E. coli BL21
ꢁ
1
ꢁ1
(
DE3) cells expressing MCAR, 50 mg mL E. coli BL21 (DE3) cells expressions (R)ꢁIRED, 2.5 mg mL ATAꢁ
ꢁ1
ꢁ1
+
1
13 or ATAꢁ117, 1 mg mL GDH, 0.5 mg mL LDH, 250 mM D/Lꢁalanine, 100 mM glucose, 1.5 mM NAD , 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mM PLP, 500 mM pH 7 NaP
i
buffer. Reactions incubated at 30 °C (250 rpm) for 24 h to allow full conversion
of keto acid substrate, absolute conversion to 4f and 4g was calculated against a calibration curve using decane
as internal standard.
Finally, optimized reaction conditions using plasmid pLH10 were used in the preparativeꢁ
scale synthesis of (S)ꢁ4a and (±)ꢁcisꢁ4e, affording 70 mg of 4a (58 % isolated yield, 30 % ee)
and 50 mg of 4e (59 % isolated yield, >98 % de) (Scheme 1), thereby demonstrating the
utility and future potential of this in vivo system for the production of chiral amine building
blocks at scale (see SI).
Scheme 1. Isolated yields from preparativeꢁscale biotransformations using E. coli cells
harboring pLH10
To summarize, ten novel plasmids pLH01ꢁ10 were designed and built to express CAR, Sfp,
ωꢁTA and IRED in a single cell for the conversion of achiral keto acid substrates into their
ACS Paragon Plus Environment

Page 13 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
valuable chiral amine counterparts, at the expense of D/Lꢁalanine or IPA, and glucose. A
panel of keto acids 1aꢁg were screened against these new constructs, with the expected
amines 4aꢁg produced in all cases. Expression levels of ATAꢁ117 and (R)ꢁIRED proteins
were increased through the introduction of gene duplication, leading to increased yields of
amine products.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
This work demonstrates that it is possible to design and build entirely de novo biosynthetic
cascades into E. coli host cells, with the cells able to meet all cofactor requirements and act as
microbial factories for the production of pharmaceutically and industrially relevant chemical
scaffolds.
ASSOCIATED CONTENT
Supporting Information
Nucleotide sequences for plasmid pPB01 and all enzymes used in the study, cloning strategy,
general biotransformation protocols, analytical methods, process optimization details and
characterization data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Sabine.Flitsch@manchester.ac.uk
Funding Sources
The research was supported by Innovative Medicines Initiative Joint Undertaking under grant
agreement no 115360, (FP7/2007ꢁ2013; and EFPIA companies’ in kind contribution);
BBSRC (sLoLa BB/L502005/1 to LJH), (CASE/Pfizer to SPF); EPSRC (CASE/AstraZeneca
to SH). NJT and SLF are grateful to the Royal Society for Wolfson Research Merit Awards.
ACS Paragon Plus Environment

ACS Catalysis
Page 14 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
REFERENCES
(1)
(2)
(3)
(4)
(5)
Turner, N. J.; O’Reilly, E. Nat. Chem. Biol. 2013, 9, 285–288.
Bachmann, B. O. Nat. Chem. Biol. 2010, 6, 390–393.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Green, A. P.; Turner, N. J. Perspect. Sci. 2016, 9, 42–48.
Simon, R. C.; Richter, N.; Busto, E.; Kroutil, W. ACS Catal. 2014, 4, 129–143.
France, S. P.; Hussain, S.; Hill, A. M.; Hepworth, L. J.; Howard, R. M.; Mulholland,
K. R.; Flitsch, S. L.; Turner, N. J. ACS Catal. 2016, 6, 3753–3759.
(
6)
7)
Muschiol, J.; Peters, C.; Oberleitner, N.; Mihovilovic, M. D.; Bornscheuer, U. T.;
Rudroff, F. Chem. Commun. 2015, 51, 5798–5811.
(
Scism, R. A.; Bachmann, B. O. ChemBioChem 2010, 11, 67–70.
(8)
Sehl, T.; Hailes, H. C.; Ward, J. M.; Wardenga, R.; Von Lieres, E.; Offermann, H.;
Westphal, R.; Pohl, M.; Rother, D. Angew. Chem., Int. Ed. 2013, 52, 6772–6775.
(9)
Fessner, W.ꢁD.; Heyl, D.; Rale, M. Catal. Sci. Technol. 2012, 2, 1596.
(10) Schmidt, S.; Scherkus, C.; Muschiol, J.; Menyes, U.; Winkler, T.; Hummel, W.;
Gröger, H.; Liese, A.; Herz, H. G.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2015,
5
4, 2784–2787.
(
11) Suljić, S.; Pietruszka, J.; Worgull, D. Adv. Synth. Catal. 2015, 357, 1822–1830.
12) Mutti, F. G.; Knaus, T.; Scrutton, N. S.; Breuer, M.; Turner, N. J. Science 2015, 349,
(
1
525–1529.
(13) France, S. P.; Hepworth, L. J.; Turner, N. J.; Flitsch, S. L. ACS Catal. 2017, 7, 710–
24.
7
(14) Wu, S.; Zhou, Y.; Wang, T.; Too, H.ꢁP.; Wang, D. I. C.; Li, Z. Nat. Commun. 2016, 7,
–13.
1
(15) Oberleitner, N.; Peters, C.; Muschiol, J.; Kadow, M.; Saß, S.; Bayer, T.; Schaaf, P.;
Iqbal, N.; Rudroff, F.; Mihovilovic, M. D.; Bornscheuer, U. T. ChemCatChem 2013, 5,
3
524–3528.
ACS Paragon Plus Environment

Page 15 of 16
ACS Catalysis
1
2
3
4
5
6
7
8
9
(16) Schrewe, M.; Ladkau, N.; Bühler, B.; Schmid, A. Adv. Synth. Catal. 2013, 355, 1693–
1
697.
(
(
(
(
17) Otte, K. B.; Kittelberger, J.; Kirtz, M.; Nestl, B. M.; Hauer, B. ChemCatChem 2014, 6,
1
003–1009.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18) Oberleitner, N.; Ressmann, A. K.; Bica, K.; Gärtner, P.; Fraaije, M. W.; Bornscheuer,
U.; Rudroff, F.; Mihovilovic, M. Green Chem. 2017, 19, 367–371.
19) Both, P.; Busch, H.; Kelly, P. P.; Mutti, F. G.; Turner, N. J.; Flitsch, S. L. Angew.
Chem., Int. Ed. 2016, 55, 1511–1513.
20) Li, A.; Ilie, A.; Sun, Z.; Lonsdale, R.; Xu, J.ꢁH.; Reetz, M. T. Angew. Chem., Int. Ed.
2
016, 55, 12026–12029.
(21) Bailey, J. E. Science 1991, 252, 1668–1675.
(
22) Ferraboschi, P.; Mieri, M. De; Grisenti, P.; Lotz, M.; Nettekoven, U. Tetrahedron:
Asymmetry 2011, 22, 1626–1631.
(
(
(
(
23) Nakamura, I.; Yamamoto, Y. Chem. Rev. 2004, 104, 2127–2198.
24) Legault, C. Y.; Charette, B. J. Am. Chem. Soc. 2005, 127, 8966–8967.
25) Naporaꢁwijata, K.; Strohmeier, G. A.; Winkler, M. Biotechnol. J. 2014, 9, 822–843.
26) Akhtar, M. K.; Turner, N. J.; Jones, P. R. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,
8
7–92.
(27) Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol. 2010, 28, 324–
32.
3
(28) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.;
Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.;
Hughes, G. J. Science 2010, 329, 305–309.
(29) Hussain, S.; Leipold, F.; Man, H.; Wells, E.; France, S. P.; Mulholland, K. R.; Grogan,
G.; Turner, N. J. ChemCatChem 2015, 7, 579–583.
(30) Mitsukura, K.; Kuramoto, T.; Yoshida, T.; Kimoto, N.; Yamamoto, H.; Nagasawa, T.
Appl Microbiol Biotechnol 2013, 97, 8079–8086.
ACS Paragon Plus Environment

ACS Catalysis
Page 16 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(31) Quadri, L. E. N.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.; Walsh, C. T.
Biochemistry 1998, 2960, 1585–1595.
(
(
(
(
(
32) Knight, T. Idempotent Vector Design for Standard Assembly of Biobricks, 2003,
th
Retrieved from http://hdl.handle.net/1721.1/21168 (accession date 14 March 2017).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33) He, A.; Li, T.; Daniels, L.; Fotheringham, I.; Rosazza, J. P. N. Appli 2004, 70, 1874–
1881.
34) Leipold, F.; Hussain, S.; Ghislieri, D.; Turner, N. J. ChemCatChem 2013, 5, 3505–
3508.
35) Mitsukura, K.; Suzuki, M.; Tada, K.; Yoshida, T.; Nagasawa, T. Org. Biomol. Chem.
2
010, 8, 4533–4535.
36) Koszelewski, D.; Lavandera, I.; Clay, D.; Rozzell, D.; Kroutil, W. Adv. Synth. Catal.
008, 350, 2761–2766.
2
(37) Chen, R. R. Appl. Microbiol. Biotechnol. 2007, 74, 730–738.
(
(
(
(
(
38) Khalil, A. S.; Collins, J. J. Nat. Rev. 2010, 11, 367–379.
39) Khosla, C.; Keasling, J. D. Nat. Rev. 2003, 2, 1019–1025.
40) Kondrashov, F. A. Proc. R. Soc. B 2012, 279, 5048–5057.
41) Elliott, K. T.; Cuff, L. E.; Neidle, E. L. Future Microbiol. 2013, 8, 887–899.
42) Alper, H.; Fischer, C.; Nevoigt, E.; Stephanopoulos, G. Proc. Natl. Acad. Sci. U. S. A.
2005, 102, 12678–12683.
(43) O’Reilly, E.; Iglesias, C.; Ghislieri, D.; Hopwood, J.; Galman, J. L.; Lloyd, R. C.;
Turner, N. J. Angew. Chem., Int. Ed. 2014, 53, 2447–2450.
INSERT TABLE OF CONTENTS GRAPHIC
ACS Paragon Plus Environment