Biocatalytic reduction of α,β-unsaturated carboxylic acids to allylic alcohols

Article abstract of DOI:10.1039/d0gc00867b

We have developed robust in vivo and in vitro biocatalytic systems that enable reduction of α,β-unsaturated carboxylic acids to allylic alcohols and their saturated analogues. These compounds are prevalent scaffolds in many industrial chemicals and pharmaceuticals. A substrate profiling study of a carboxylic acid reductase (CAR) investigating unexplored substrate space, such as benzo-fused (hetero)aromatic carboxylic acids and α,β-unsaturated carboxylic acids, revealed broad substrate tolerance and provided information on the reactivity patterns of these substrates. E. coli cells expressing a heterologous CAR were employed as a multi-step hydrogenation catalyst to convert a variety of α,β-unsaturated carboxylic acids to the corresponding saturated primary alcohols, affording up to >99percent conversion. This was supported by the broad substrate scope of E. coli endogenous alcohol dehydrogenase (ADH), as well as the unexpected CC bond reducing activity of E. coli cells. In addition, a broad range of benzofused (hetero)aromatic carboxylic acids were converted to the corresponding primary alcohols by the recombinant E. coli cells. An alternative one-pot in vitro two-enzyme system, consisting of CAR and glucose dehydrogenase (GDH), demonstrates promiscuous carbonyl reductase activity of GDH towards a wide range of unsaturated aldehydes. Hence, coupling CAR with a GDH-driven NADP(H) recycling system provides access to a variety of (hetero)aromatic primary alcohols and allylic alcohols from the parent carboxylates, in up to >99percent conversion. To demonstrate the applicability of these systems in preparative synthesis, we performed 100 mg scale biotransformations for the preparation of indole-3-aldehyde and 3-(naphthalen-1-yl)propan-1-ol using the whole-cell system, and cinnamyl alcohol using the in vitro system, affording up to 85percent isolated yield.

Full text of DOI:10.1039/d0gc00867b

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Biocatalytic reduction of α,β-unsaturated
carboxylic acids to allylic alcohols†
Cite this: DOI: 10.1039/d0gc00867b
Godwin A. Aleku,
George W. Roberts and David Leys
*
We have developed robust in vivo and in vitro biocatalytic systems that enable reduction of
α,β-unsaturated carboxylic acids to allylic alcohols and their saturated analogues. These compounds are
prevalent scaffolds in many industrial chemicals and pharmaceuticals. A substrate profiling study of a car-
boxylic acid reductase (CAR) investigating unexplored substrate space, such as benzo-fused (hetero)aro-
matic carboxylic acids and α,β-unsaturated carboxylic acids, revealed broad substrate tolerance and pro-
vided information on the reactivity patterns of these substrates. E. coli cells expressing a heterologous
CAR were employed as a multi-step hydrogenation catalyst to convert a variety of α,β-unsaturated car-
boxylic acids to the corresponding saturated primary alcohols, affording up to >99% conversion. This was
supported by the broad substrate scope of E. coli endogenous alcohol dehydrogenase (ADH), as well as
the unexpected CvC bond reducing activity of E. coli cells. In addition, a broad range of benzofused
(hetero)aromatic carboxylic acids were converted to the corresponding primary alcohols by the recombi-
nant E. coli cells. An alternative one-pot in vitro two-enzyme system, consisting of CAR and glucose
dehydrogenase (GDH), demonstrates promiscuous carbonyl reductase activity of GDH towards a wide
range of unsaturated aldehydes. Hence, coupling CAR with a GDH-driven NADP(H) recycling system pro-
vides access to a variety of (hetero)aromatic primary alcohols and allylic alcohols from the parent carbox-
ylates, in up to >99% conversion. To demonstrate the applicability of these systems in preparative syn-
thesis, we performed 100 mg scale biotransformations for the preparation of indole-3-aldehyde and 3-
(naphthalen-1-yl)propan-1-ol using the whole-cell system, and cinnamyl alcohol using the in vitro
system, affording up to 85% isolated yield.
Received 10th March 2020,
Accepted 24th April 2020
DOI: 10.1039/d0gc00867b
rsc.li/greenchem
entities such as aldehydes, alcohols, esters, acyl chlorides and
amides under mild reaction conditions are highly desired.
Introduction
Devising sustainable, cost-effective and environmentally
The role of biocatalysis is crucial to the development of
friendly catalytic methods is pivotal to achieving green manu- clean manufacturing technologies for today and tomorrow. A
facturing goals. An important strategy is to harness abundant number of biocatalytic synthetic routes are emerging as
and replenishable resources such as non-food lignocellulosic methods of choice in many industrial processes.5,6 This is
biomass to derive raw materials for industrial chemical pro- largely a result of significant progress achieved in the identifi-
duction. Carboxylic acids readily occur in biomass and recent cation and development of biocatalysts for various biocatalytic
efforts towards developing biomass utilisation technologies functional group interconversions,7–12 including those acting
can further enhance their availability.1–3 Given the versatility on carboxylic acids and their derivatives.13–18 One such class
of carboxylic acids as substrates in organic synthesis,4 there is of enzymes are the carboxylic acid reductases (CARs), which
an attractive opportunity to harness biomass-derived carboxy- catalyse the selective one-step (two-electron) reduction of car-
lates as renewable raw materials for sustainable chemical pro- boxylic acids to the corresponding aldehydes at the expense of
duction. Hence, synthetic methods for the derivatization of ATP and NADPH cofactors.13,16 These multi-domain enzymes
these carboxylates to provide access to a variety of chemical (comprising the adenylation domain, PCP phosphopan-
tetheine linker and the terminal reductase domain) mediate
carboxylate reduction via a multi-step process. At the adenyla-
tion domain, an ATP-dependent activation of the carboxylate
occurs, generating the corresponding acyl adenylate, followed
by the transfer of the acyl group onto the PCP phosphopan-
tetheine linker. The reduction of the acyl-thioester occurs at
Manchester Institute of Biotechnology, School of Chemistry, University of
Manchester, Manchester, UK. E-mail: David.Leys@manchester.ac.uk
†Electronic supplementary information (ESI) available: Preliminary biotrans-
formation data and sample chromatograms of biotransformation reactions. See
the terminal reductase domain to yield the final aldehyde
DOI: 10.1039/d0gc00867b
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Fig. 1 Biocatalytic conversion of carboxylic acids to alcohols applying carboxylic acid reductases (CARs) for carboxylate reduction. (a) Route for
conversion of fatty acids to fatty alcohol employing recombinant aldehyde reductase (AHR) for the carbonyl reduction step.27 (b) Route for conver-
sion of hydroxyl or dicarboxylic acids to diols employing lactaldehyde reductase (YahK) or aldo-keto reductases (AKR) for the carbonyl reduction
step.28
product. Recent structural insights reveal domain dynamics (hetero)aromatic alcohols and allylic alcohols. These com-
underpins strict selective two electron reduction.13,19
pounds are prevalent scaffolds in industrial chemicals and
The use of CAR enzymes in synthetic processes is impeded pharmaceuticals, Fig. 1c–f. We envisage that development of
by requirement to supply stoichiometric quantities of expen- such methods can provide an alternative green synthetic route
sive cofactors NADPH and ATP.20 To circumvent this, appli- to the traditional chemical methods employing metal hydrides
cation in whole-cell preparations has been considered, as they (e.g. LiAlH4 or Zn(BH4)2), borane-reducing agents (e.g.
afford in situ cofactor regeneration system in place. However, BH3·SMe2),29,30 and transition metal homogeneous and hetero-
in vitro CAR biotransformation is preferred where conversion geneous catalysts.31,32 More so, as metal hydrides are required
exclusively to the aldehyde is desired. In this regard, co-factor in stoichiometric amounts, prone to inactivation by air and
recycling systems have been coupled to the CAR reaction.21–25
moisture, generate inorganic waste and are poorly selective. In
There are limited examples demonstrating biocatalytic car- addition, homogeneous and heterogeneous metal catalysts are
boxylate reduction to the corresponding alcohols.26–28 Akhtar expensive and require harsh reaction conditions including
et al. employed E. coli cells expressing Mycobacterium marinum elevated temperatures and pressures.
CAR and a native aldehyde reductase (AHR) to convert fatty
acids to the corresponding alcohols, Fig. 1a.27 Similarly,
Kramer et al. have recently developed a whole-cell CAR-based
pathway for the conversion of a panel of short-chain dicar-
boxylic acids and hydroxy acids to the corresponding diols,
employing yahK-encoded aldehyde reductase for carbonyl
Results and discussion
Profiling unexplored substrate space: CAR activity on
benzofused (hetero)aromatic and α,β-unsaturated acids
reduction,28 Fig. 1b. In this work, we aimed to develop CAR- To assess the activity of CARs towards benzo-fused (hetero)aro-
based in vivo and in vitro systems for the conversion of as yet matic carboxylic acids and acrylic acid derivatives, we selected
unexplored substrate space: benzo-fused (hetero)aromatic car- two CAR enzymes: Tsukamurella paurometabola carboxylic acid
boxylic acids and acrylic acid derivatives to provide access to reductase (TpCAR) which was identified as a substrate pro-
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miscuous CAR,33 and the structurally characterised For example, SrCAR displayed a ∼2-fold lower activity towards
Segniliparus rugosus CAR (SrCAR).34 The CAR enzymes were cinnamic acids bearing bulkier p-substituents such as Me, Br,
each co-expressed with Bacillus subtilis phosphopantetheinyl Ph, carboxy (28a–31a, group II, 0.6–1.0 U mg−1) when com-
transferase (sfp) in E. coli to ensure posttranslational modifi- pared to a small para substituent (p-fluorocinnamic acid 27a,
cation and activation. We then set out to investigate CAR specific activity 1.7 U mg−1). Acrylic acids bearing heteroaro-
activity towards a variety of bulky benzo-fused (hetero)aromatic matic systems at the β-carbon (43a–45a) were reactive, albeit
carboxylic acids and acrylic acids, initially using whole-cell with lower rates (group III), whereas α,β-unsaturated monocyc-
systems. Preliminary profiling for TpCAR and SrCAR revealed lic carboxylic acids 41a, 42a (group II) showed good reactivity
similar substrate spectrum and comparable conversion values (specific activity, 0.8–1.1 U mg−1).
for the two enzymes, ESI Fig. S1.† Therefore, we selected
While previous substrate profiling studies of CARs have
SrCAR for further study since significant structural and examined scope for monocyclic aromatic carboxylic acids, fatty
in silico information are available for this enzyme34,35 which acids and linear aliphatic carboxylates,26–28,33 this work rep-
may guide future engineering efforts.
resents the first systematic investigation into the scope of CAR
To assess the substrate scope and the relative reactivity of enzymes towards bulky benzo-fused (hetero)aromatic carboxy-
the substrates, we determined specific activity of isolated lates as well α,β-unsaturated carboxylic acids. In general, we
SrCAR from initial rate of carboxylate reduction. Two distinct find SrCAR an efficient catalyst for the reduction of a broad
compound libraries were constructed containing structurally range of benzo-fused (hetero)aromatic carboxylic acids.
diverse benzo-fused (hetero)aromatic carboxylic acids, and Similarly, a large collection of previously un-investigated
α,β-unsaturated carboxylic acids bearing a wide range of substi- acrylic acids bearing structurally diverse groups at α- or
tuents at the β- and α-carbons.
β-carbon to the carboxylate were efficiently reduced by SrCAR.
An assessment of the reactivity pattern for benzo-fused In view of the relatively small acid substrate binding pocket of
(hetero)aromatic acids reveals that benzo-fused S-, O-, and the SrCAR A-domain,34 the broad substrate tolerance towards
N-heteroaromatic carboxylates (1a, 2a, 5a & 6a) and naphthoic these relatively bulky benzo-fused aromatic compounds
acids 3a & 4a (group I) displayed superior specific activity suggests that significant intradomain and interdomain
(0.9–1.4 U mg−1) compared to the corresponding 2-carboxy- motions occur during catalysis to accommodate and orientate
lates, 7a, 8a & 9a and isoquinoline derivate 10a, (group II, substrates for catalysis. In addition, the efficiency of the CAR
specific activity = 0.4–0.8 U mg−1). Further down the specific enzymes towards these benzofused (hetero)aromatic carboxy-
activity scale are quinolone-2-carboxylic acid 11a and lates suggests a role for the benzo component in facilitating
2-naphthoic acid derivatives bearing a hydroxyl or an amino productive interaction between the substrate and substrate
group at 3- or 6-position of the aromatic system (13a–16a), dis- binding site residues of SrCAR A-domain (and by extension
playing specific activity between 0.20 and 0.40 U mg−1 with a other CARs) which are mostly hydrophobic.22,34 Taken
significantly slower rate observed for a 1-hydroxy substituent together, the results from Fig. 2 reveal the following substrate
(22a). The presence of an additional heteroatom significantly structure–activity determinants: (i) for benzo-fused heteroaro-
decreases activity; for example, the least reactive group IV matic carboxylic acids, the reactivity differs with the position
(specific activity <0.2 U mg−1) features benzo-fused heteroaro- of the carboxylate on the benzofused heteroaromatic ring
matic carboxylates containing two heteroatoms (17a–19a). system; substrates bearing 2-carboxylate displayed lower reac-
Similarly, 1-naphthoic acid derivatives bearing a hydroxyl or an tivity when compared with the 3-carboxylate regio-isomer. (ii)
amino group at 2- or 6-position of the aromatic system (20a– The type of heteroatom (O, N, S) in the benzo-fused 5-meme-
22a) exhibited weak reactivity. Marked decrease in reactivity bered heteroaromatic system influenced reactivity in line with
was also observed with diheteroatom-containing carboxylates the degree of aromatic stability of the systems. For example,
(vs. the mono-heteroatom substrates).
SrCAR displayed highest activity towards benzothiophene
SrCAR also displayed broad biocatalytic scope for derivatives whereas indole derivatives where least reactive. (iii)
α,β-unsaturated carboxylic acids, exhibiting high specific Steric hindrance resulting from bulky substituents was the
activity for acrylic acids bearing a naphthyl group (24a and most important factor contributing to weak reactivity,
25a), as well as cinnamic acid 26a and derivatives bearing especially when such substituents are adjacent to the carboxy-
weakly e−-donating group at the α or β-carbon to the carboxy- late group. This is consistent with previously established trend
late (e.g. α-Me 36a, β-Me 37a), Fig. 2b, (group I, specific activity with benzoic acid derivatives bearing sterically demanding
1.50–2.30 U mg−1). In comparison, cinnamic acid derivatives ortho-substituents.33,36
bearing small weak e−-withdrawing group as α-F 38a (group
III), or bulky substituents at the α-carbon such as α-Ph,
α-NHCOMe 39a, 40a (group IV) displayed an order of a magni-
Whole cell biotransformation for the conversion of unexplored
carboxylic acid substrates to alcohols
tude lower reactivity. This trend highlights the importance of To assess the biocatalytic scope of recombinant E. coli whole
both steric and electronic effects of substituents attached to cells containing SrCAR for the conversion of sterically demand-
the α-carbon. The effect of substituents on the phenyl ring of ing benzo-fused (hetero)aromatic carboxylic acids and acrylic
cinnamic acid was also investigated, revealing that the steric acid derivatives to the corresponding alcohols, we set out to
effect of para-substituted bulky substituents was less profound. investigate CAR in vivo activity towards a variety of these acids.
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Fig. 2 Substrate profiling studies for Segniliparus rugosus CAR (SrCAR) against unexplored substrate groups: (a) benzo-fused (hetero)aromatic car-
boxylic acids; (b) α,β-unsaturated carboxylic acids. SA = specific activity determined from initial rates. Assays were performed in triplicate and the
error bars represent standard deviation from the mean specific activity. A unit of activity = the amount of pure enzyme required to consume 1 µmol
NADPH per min.
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Simultaneously, carbonyl reductase activity of E. coli endogen- structural,22,34 computational35,37 and directed evolution36
ous alcohol dehydrogenase (EcADH) on corresponding alde- studies.
hydes to generate the corresponding primary alcohols was
monitored. First, we performed biotransformation for the
reduction of benzothiophene-2-carboxylic acid 1a using E. coli
Scope for conversion of acrylic acids to corresponding alcohols
using recombinant E. coli cells
whole-cells containing SrCAR, with glucose added to enable Allylic alcohols or their saturated analogues are important
in situ cofactor recycling. Conversion of >99% to the corres- building blocks for organic synthesis; hence we sought to
ponding alcohol was obtained, indicating that the carboxylate explore the CAR-route for the conversion of acrylic acid deriva-
and the corresponding aldehyde are good substrates for the tives which are abundant in biomass to the corresponding
SrCAR and EcADH respectively. Encouraged by this initial alcohols. Cinnamic acid 26a and derivatives bearing p-substi-
result, we investigated the two-step reduction of a wide range tuents on the aromatic ring such as weakly e−-withdrawing
of carboxylic acids to the corresponding alcohols using E. coli groups p-halogens (27a, 29a) and weakly e−-donating groups
whole-cells expressing SrCAR.
such as p-Me (28a), as well as strong e−-donating groups p-OH
48a were tolerated by SrCAR and endogenous EcADH, generat-
ing the corresponding alcohols from the parent carboxylates in
good to excellent conversion values (60–92%, Table 2, entries
1–6). Interestingly, while we had anticipated the formation of
Scope for conversion of benzofused (hetero)aromatic
carboxylic acids to corresponding alcohols
We found a structurally diverse set of (hetero)aromatic car- the corresponding allylic alcohols (cinnamyl alcohol deriva-
boxylic acids were converted to the corresponding alcohols, tives) as the final reduction product, GCMS analysis of the bio-
affording moderate to excellent conversion in most cases, with transformation reactions revealed the formation of corres-
no requirement for recombinant overexpression of a carbonyl ponding saturated alcohols (hydrocinnamyl alcohols) as the
reductase. Benzo-fused furan and thiophene 3-carboxylates 1a, major product in most cases, indicating a novel CvC reducing
2a or 2-carboxylates 7a, 8a were converted to the corresponding activity of E. coli whole-cell biocatalysts, Table 2. Substrate 30a
alcohols with excellent conversion values (96–>99%) and no which carries two carboxyl groups was reduced to the corres-
accumulated aldehyde intermediate; Table 1, entries 1–7. ponding diol with high conversion, again with the acrylic car-
However, lower conversion values were achieved with indole-2- boxylate moiety reduced to the corresponding hydrocinnamyl
carboxylate 9a, yielding the corresponding alcohol at 48% con- alcohol (72%).
version. Interestingly, the regio-isomer indole-3-carboxylate 6a
Bulky substituents on the aromatic moiety such as p-Ph
was an excellent CAR substrate, but yielded indole-3-carboxyal- group 31a were tolerated, affording high conversion, yielding
dehyde (96% conversion) as the latter was not converted by predominantly the corresponding hydrocinnamyl alcohol
EcADH. Indazole-3-carboxylic acid 5a yielded the corres- (92%). Similarly, di-, and penta-functionalised cinnamic acid
ponding alcohol in up to 96% conversion. The system also dis- derivatives 34a, 35a, 49a were converted to the corresponding
played tolerance towards six-membered benzo-fused hydrocinnamyl alcohols as final products (Table 2, entries
N-heteroaromatic carboxylates (such as quinoxaline, quinolone 8–10). Furthermore, cinnamic acid derivatives bearing small
and isoquinoline derivatives 10a–12a), yielding the corres- substituents at the α-carbon to the carboxylate (α-Me 36a) as
ponding alcohols in a range between 15 to >99%. In particular, well as bulky substituents (α-Ph 39a) were accepted, yielding
isoquinoline-3-carboxylate 10a was an excellent substrate. We the corresponding hydrocinnamyl alcohols as major products,
then examined a range of 1-naphthoic and 2-napthoic acid while β-Me substitution (37a) generated a mixture of the
derivatives Table 1, entries 12–19. Unsubstituted naphthoic corresponding allylic alcohol and the saturated analogue.
acids 3a & 4a were good substrates, generating the corres- Interestingly, reduction of α-F cinnamic acid 38a yielded the
ponding alcohol in high conversion of up >99%. Substitution corresponding allylic alcohol (70%) as the sole alcohol
at different positions of the aromatic ring system with strong product, while the saturated aldehyde, α-F hydrocinnamalde-
e−-donating such as –OH, –NH2 were tolerated by both CAR hyde 38e was also detected (28%).
and EcADH. However, 1-hydroxy-2-naphthoic acid 22a yielded
the aldehyde (83%), indicating that the corresponding alde- (24a, 25a) were excellent substrates, yielding the corresponding
hyde was unreactive with EcADH. 1-propanol in up to >99% conversion. Similarly, acrylic acids
Bicyclic aromatic substituents at β-carbon such as naphthyl
Despite the weak activity of SrCAR towards structurally chal- bearing heteroaromatic systems at β-carbon (44a, 45a), as well
lenging (hetero)aromatic carboxylic acids (e.g. substrates as α,β-unsaturated cyclic carboxylic acids 41a, 42a were
bearing steric bulk at adjacent carbon to the carboxylate) accepted, affording the corresponding 1-propanols and satu-
observed from our initial rate study (Fig. 2), moderate conver- rated cyclic methanols respectively (57–90% conversion).
sion values were achieved upon longer biotransformation incu-
The presence of intermediates suggest CvC reducing activity
of E. coli whole-cells on α,β-unsaturated acids and aldehydes
bation (18 h, Table 1) indicating potential for application of
CAR enzymes for valorization of these substrates. In addition,
there is also an opportunity to further improve the efficiency of The CvC reducing activity of E. coli whole-cells enabled an
CAR enzymes, for example via (muti)site saturation of crucial unexpected 3-step hydrogenation route for the conversion
active site residues that have recently been identified from α,β-unsaturated carboxylic acids to the corresponding satu-
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Table 1 (Contd.)
Table 1 Conversion of benzo-fused (hetero)aromatic carboxylic acids
to the corresponding primary alcohols by E. coli whole-cells expressing
SrCAR
Conversion [%]
Conversion [%]
Entry
16
Substrates
Entry
1
Substrates
0
0
98
78
0
>99
17
2
3
<1
1
>98
96
18
19
0
82
0
83
4
96
0
Reaction contained 5 mM substrate, 20 mM D-glucose, 10 mM MgCl2,
2% v/v DMSO and fresh resting E. coli cells containing overexpressed
SrCAR at OD600 = 30, all in NaPi (50 mM, pH 7.5), incubated at 30 °C,
250 rpm for 18 h. SrCAR = Segniliparus rugosus carboxylic acid
reductase. Conversion values were determined from HPLC/GC-MS
analyses.
5
6
0
0
>99
98
7
8
0
0
48
rated alcohols. An in vitro system applying SrCAR as purified
enzyme preparation and supplying stoichiometric amounts of
NADPH and ATP for the reduction of α,β-unsaturated acid 26a
or the corresponding aldehyde lacked CvC reducing activity.
This suggests the presence of (likely oxygen-stable) E. coli CvC
reducing enzymes, perhaps of the ene/enoate reductases
(EREDs) or short chain dehydrogenases/reductases (SDRs)
enzyme families.38,39 GCMS analysis of intermediates detected
from biotransformation reactions applying SrCAR as whole-
cell catalyst highlights two plausible routes to the saturated
alcohol derivatives. In the first route, the α,β-unsaturated alde-
hyde intermediate generated from the CAR step can undergo
CvC hydrogenation to generate the corresponding propanal
derivative prior the carbonyl reduction step. Evidence for this
route is supported by detection of α-F hydrocinnamaldehyde
38e from analysis of biotransformation for reduction of α-F
cinnamic acid 38a, ESI, Fig. S13.† In this case, the propanal
derivative was unreactive with EcADH and therefore accumu-
lates up to 28%. A complementary route to the saturated
alcohol via CvC reducing activity on α,β-unsaturated car-
boxylic acids can occur prior to carboxylate and carbonyl
reduction steps (see reaction scheme, Table 2). Detection of
the corresponding saturated carboxylic acid analogues from
α,β-unsaturated carboxylates for substrates 27a, 28a, 29a, 36a,
42a and 48a supports the latter, ESI, Fig. S14–S16.†
>99
9
0
93
10
11
0
15
63
37
12
13
0
0
>99
98
14
15
0
0
87
73
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Table 2 (Contd.)
Table 2 Conversion of acrylic acid derivatives to the corresponding
primary alcohols by E. coli whole-cells expressing SrCAR
Conversions [%]
Substrates
Conversions [%]
Substrates
Entry
19
Entry
1
6
92
20
2
3
4
5
6
27a (R = p-F)
<1
<1
<1
4
60
71
82
92
90
28a (R = p-Me)
29a (R = p-Br)
31a (R = p-Ph)
48a (R = p-OH)
Reaction contained 5 mM substrate, 20 mM D-glucose, 10 mM MgCl2,
2% v/v DMSO and fresh resting E. coli cells containing overexpressed
SrCAR at OD600 = 30, all in NaPi (50 mM, pH 7.5), incubated at 30 °C,
250 rpm for 18 h. Conversion values were determined from HPLC/
GC-MS analyses. a Enantiomeric excess (ee) and absolute configuration
for chiral products not determined. NB: 3–15% of saturated acid ana-
logues of the α,β-unsaturated acids were detected with substrates 27a,
28a, 29a, 35a, 36a, 42a, 48a and 49a. 4–8% of decarboxylated product
detected with substrate 48a & 49a. <1–10% of α,β-unsaturated aldehyde
was detected in most reactions and 28% of propanal derivative was
detected with substrate 38a.
<1
7
8
<1
87
9
6
75
68
Our whole-cell CAR system demonstrates for the first time
(to the best of our knowledge) the use of E. coli expressing CAR
as an efficient biocatalytic multi-step hydrogenation system
enabling conversion of a wide range of acrylic acids to the
corresponding saturated alcohols. This self-sufficient system
provides the 6e− required for the three step reduction process
(i.e. carboxylate reduction, α,β CvC bond reduction and CvO
reduction) at the expense of exogenously added glucose. A
similar trend has been previously observed with the fungus
Mucor sp. A-73 cells, in this case the reduction of C6
α,β-unsaturated carboxylic acids (e.g. hexenoic acid, sorbic
acid) to the corresponding α,β saturated alcohols was
achieved40 suggesting that recombinant fungal cells contain-
ing CAR can further be developed for this multi-step hydrogen-
ation process. In addition, a wide range benzo-fused bulky
(hetero)aromatic alcohols can be accessed via this simple
recombinant E. coli whole-cell system harboring CAR.
10
<1
11
12
13
10
70
0
74a
0
84a
14
15
58
<1
43a
>99
In vitro biotransformation to access benzofused hetero
16
<1
87
(aromatic) alcohols and allylic alcohols
We sought an alternative route to access allylic alcohols from
the α,β-unsaturated carboxylates as these are highly versatile
building blocks for organic synthesis as they allow further
functionalisation of the allylic moiety.41 Hence, we next aimed
to develop a simple in vitro approach for the conversion of a
variety of carboxylic acids to the corresponding alcohols; with
17
18
11
0
80
90
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selective access to allylic alcohols of particular interest. To cir- substituent at the β-carbon such as in 45a underwent a one-
cumvent the need to use a stoichiometric amount of NADPH step reduction to the corresponding aldehydes (97% conver-
in the two NADPH-dependent reduction steps, we employed sion, Table 3, entry 33) indicating that BsGDH lacked carbonyl
glucose dehydrogenase (GDH)-based NADPH-recycling system. reducing activity towards the corresponding aldehyde.
Next, we attempted to identify a carbonyl reductase for the However, cinnamic acid derivatives bearing small groups at
second step. Inspired by recent report on the promiscuous car- the α- or β-Me carbon to the carboxylate moiety (α-Me 36a,
bonyl reductase activity of GDH on naphthoquinones,42,43 we β-Me 37a, and α-F 38a) were accepted by the CAR–GDH system,
envisioned a system where GDH is applied as a bifunctional affording the corresponding allylic alcohols in up to 99% con-
catalyst; to recycle NADPH and as carbonyl reductase. In this version, Table 3. Similarly, α,β-unsaturated carboxylates
system, GDH catalyses the oxidation of glucose to recycle bearing bulky bicyclic aromatic substituents at the β-carbon
NADPH while concurrently reducing the aldehyde intermedi- such as naphthyl (24a) was converted to the allylic alcohols
ates generated from the CAR step to the corresponding (>99% conversion) and the cyclic carboxylate afforded 70% of
alcohols.
the corresponding α,β-unsaturated alcohol whereas cinnamic
acid derivative bearing a bulky substituent at the α-carbon
such as α-Ph (39a) only afforded 7% of the allylic alcohol with
the aldehyde intermediate accumulating. Analysis of biotrans-
Bifunctional glucose dehydrogenase as promiscuous carbonyl
reductase
We first examined the performance of isolated Bacillus subtilis formation reactions for selected substrates (8a, 26a, 38a) incu-
GDH (BsGDH, GenBank reference: AFQ56330.1) as a bifunc- bated for 1 h showed that the carbonyl reduction rate (vs. CAR-
tional catalyst for glucose oxidation and promiscuous carbonyl catalysed carboxylate reduction) is significantly slower, and
reduction of benzo-fused (hetero)aromatics aldehydes to the differs depending on the intermediate aldehydes (ESI
corresponding alcohols. We attempted a one-pot biotransform- Fig. S2†). Hence, this leads to the observed differences in the
ation reaction containing a carboxylic acid substrate 8a, a stoi- amount of aldehyde intermediates accumulating for biotrans-
chiometric amount of ATP, a catalytic amount of NADP+, formation of different carboxylic acids (Table 3).
D-glucose (4 equivalents), purified SrCAR and BsGDH. This Biotransformation reactions performed for substrates (e.g. 26a,
system yielded the corresponding alcohol in up to 77% conver- 27a & 28a) under identical conditions, but supplying 2 equiva-
sion. Having established the substrate scope for the CAR-cata- lents of NADPH while excluding GDH and glucose, led to the
lysed carboxylate reduction, we next aimed to investigate the accumulation of the corresponding aldehydes.
substrate scope of GDH-carbonyl reduction step. Surprisingly,
The results from Table 3 clearly demonstrate that our
GDH displayed a broad substrate scope and was capable of simple novel in vitro system provides a green, potentially
reducing a wide range of benzo-fused (hetero)aromatic alde- cheaper biocatalytic route for the synthesis of a wide variety
hydes to generate the corresponding primary alcohols in mod- of synthetically useful allylic alcohols.41 In particular, this
erate to excellent conversion values (37–98%), Table 3. system allows indirect screening of promiscuous carbonyl
N-heteroaromatic carboxylates 6a, 9a and sterically demanding reductase activity of GDH against a wide range of hard-to-
naphthoic acid derivatives, 14a, 21a & 22a only underwent a access aldehydes, uncovering the versatility of GDH as carbo-
one-step reduction to the corresponding aldehydes, indicating nyl reductase for structurally diverse carbonyl compounds.
that the aldehydes formed were unreactive with BsGDH.
The bifuntionality of GDH (for NADPH regeneration and car-
bonyl reduction) ensured a significant reduction of reaction
components and simplified optimisation process. We
suggest that our indirect approach to monitor promiscuous
In vitro one-pot CAR–GDH cascade enables selective access to
allylic alcohols
Finally, the CAR–GDH in vitro system was tested against enzymatic activity can be extended to explore catalytic prom-
α,β-unsaturated carboxylic acids to allow selective access to the iscuity of SDRs.44,45 To avoid ambiguity in the indirect profil-
corresponding allylic alcohols. Indeed, the one-pot CAR–GDH ing of promiscuous carbonyl activity of GDH, we relied on
system was capable of converting α,β-unsaturated carboxylic the use of a stoichiometric amount of ATP to demonstrate
acids to the corresponding allylic alcohols with 14 to >99% the potential for the in vitro CAR–GDH system as a proof of
conversion. Saturated alcohol products were not detected with concept. However, for future economically viable application,
this system. Cinnamic acid derivatives bearing weak or strong we suggest that a simple ATP co-factor regeneration system
e−-withdrawing p-substituents on the aromatic ring such as can be coupled to the CAR–GDH in vitro system. For
p-halogens and p-NO2 (27a, 29a, 33a) as well as weakly e−- example,
a family-2 polyphosphate kinase (PPK2, e.g.
donating groups such as p-Me (28a) and bulky p-Ph 30a CHU0107) which catalyses two-step phosphorylation of AMP
yielded the corresponding allylic alcohols albeit in low conver- to ATP (via ADP) to ensure a straight-forward ATP regener-
sion values (14–41%), Table 3, entries 21–25. BsGDH was inert ation for CAR-based cascades22,46 is suitable for the CAR–
towards p-hydroxycinnamaldehydes as p-hydroxycinnamic acid GDH system.
derivatives such as p-coumaric acid 48a and ferulic acid 49a
only underwent a one-step reduction to the corresponding
Preparative scale reactions
aldehydes with >99% conversion (Table 3, entries 26 & 27). Our analytical whole-cell indirect substrate profiling of EcADH
Similarly, α,β-unsaturated carboxylates bearing heteroaromatic highlights aldehydes that are non-reactive with the EcADH, e.g.
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Table 3 (Contd.)
Table 3 In vitro CAR–GDH system for conversion of carboxylic acids to
the corresponding primary alcohols with GDH employed as bifunctional
glucose oxidant and carbonyl reductase
Conversion [%]
Conversion [%]
Entry
14
Substrates
>99
0
0
Entry
1
Substrates
4
91a
15
92a
2
3
2
96a
68
16
17
0
96
37
10
18
4
79
0
18
19
55
40
0
0
5
6
0
0
>99
77
7
8
43
7
0
20
21
22
23
24
25
26
26a (R = H)
11
66
25
56
36
48
>99
87a
14
22
41
30
16
0
27a (R = p-F)
29a (R = p-Br)
28a (R = p-Me)
31a (R = p-Ph)
33a (R = p-NO2)
48a (R = p-OH)
>92
9
6
>93
27
>99
0
10
11
14
13
>85
85
28
56
44
29
30
31
<1
7
99
94a
7
12
13
0
0
>99
92
83
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Table 3 (Contd.)
Finally, by applying the in vitro one-pot two enzyme CAR–
GDH system and using catalytic amount of NADP+, 100 mg cin-
namic acid 1a at 10 mM substrate loading was converted to
cinnamyl alcohol 1c, affording 75% conversion and 62% iso-
lated yield.
Conclusion
Conversion [%]
In summary, we have developed simple and efficient in vivo and
in vitro biocatalytic systems that enable access to a variety of
benzo-fused (hetero)aromatic alcohols, allylic alcohols and their
saturated analogues from the corresponding carboxylic acids.
We reveal the broad substrate scope of E. coli endogenous ADH,
as well as novel CvC bond reducing activity of E. coli cells.
Hence, recombinant E. coli cells expressing SrCAR can be
applied as efficient multi-step hydrogenation catalysts to convert
a variety of α,β-unsaturated carboxylic acids to the corresponding
saturated primary alcohols. Where allylic alcohols are required,
an alternative in vitro system can yield these compounds. The
promiscuous carbonyl reductase activity of BsGDH towards a
wide range of benzo-fused (hetero)aromatic aldehydes and
α,β-unsaturated aldehydes supports a simple in vitro system
coupling CAR-catalysed carboxylate reduction and GDH-cata-
lysed carbonyl reduction to access a variety of (hetero)aromatic
alcohols and allylic alcohols from the parent carboxylates. The
application of GDH as a bifunctional enzyme in this system (i.e.
as glucose oxidant and carbonyl reductase), enabled develop-
ment of a two-enzyme system, which efficiently couples NADP
(H) recycling system to the two hydrogenation steps from carbox-
ylate to alcohol. The development of these simple whole-cell and
in vitro systems provide alternative green hydrogenation catalytic
routes that can potentially replace harsh abiotic reducing agents
for the preparation industrial alcohols from biomass derived car-
boxylic acids. Altogether, we present novel biocatalytic routes
that enable the conversion of a wide range of biomass-derived
carboxylic acids to a variety of primary alcohols.
Entry
32
Substrates
0
>99a
33
34
97
20
0
70
In vitro reaction contained 5 mM carboxylic acid substrate, 20 mM
D-glucose, 10 mM MgCl2, 2% v/v DMSO, purified SrCAR (0.5 mg mL−1),
purified BsGDH (0.6 mg mL−1). Reaction performed in NaPi (50 mM,
pH 7.5), incubated at 30 °C, 250 rpm for 18 h. a 1 mg mL−1 purified
GDH was used. SrCAR = Segniliparus rugosus carboxylic acid reductase;
BsGDH = Bacillus subtilis glucose dehydrogenase. Conversion values
were determined from HPLC/GC-MS analyses.
indole-3-carboxyaldehyde 6b. This can be advantageous if
selective access to the aldehyde product is desired via a CAR-
whole-cell process and in synthetic cascades for which the
aldehyde serves as an intermediate for a subsequent step (e.g.
reductive amination). Hence, we sought to demonstrate the
applicability of CAR expressing recombinant E. coli cells for
selective preparative synthesis of indole-3-carboxyaldehye 6b,
an important precursor for the preparation of many biologi-
cally active compounds including indole alkaloids. We
attempted a 100 mg scale whole-cell biotransformation at
10 mM substrate concentration, using 100 mM sodium phos-
phate buffer. However, we observed a steady drop in the reac-
tion pH, from an initial pH of 8 to pH < 6 within 6 h of the
incubation. Increasing buffer strength to 150 mM, or changing
the buffer to Tris-HCl did not stabilise the reaction pH,
neither did decreasing the substrate concentration to 5 mM.
Hence, we resorted to hourly adjustment of the reaction pH
during the incubation period. With this strategy, 100 mg
indole-3-carboxylic acid 6a was exclusively converted to the
corresponding aldehyde 6b by the recombinant resting E. coli
cells, affording 95% conversion and an isolated yield of 85%
Experimental section
Chemicals
Commercially available chemicals and reagents of highest purity
were purchased from Sigma-Aldrich (Poole, Dorset, UK), or
Fluorochem (Hadfield, Derbyshire, UK) unless stated otherwise.
HPLC solvents were obtained from Sigma-Aldrich (Poole, Dorset,
UK) or ROMIL (Waterbeach, Cambridge, UK) and GC gases from
BOC gases (Guildford, UK). Enzyme nicotinamide cofactors
NADP+ and NADPH were purchased from Bio Basic (Markham,
Ontario, Canada), and Adenosine triphosphate (ATP) was
sourced from Sigma-Aldrich (Poole, Dorset, UK). LB auto induc-
tion media was purchased from Formedium (Hunstanton, UK).
after
7 h. Similarly, we explored the unexpected CvC
reduction activity observed with recombinant E. coli cells.
Using the same reaction conditions, 100 mg of (E)-3-(naphtha-
len-1-yl)acrylic acid 24a was converted to 3-(naphthalen-1-yl)
Preparation of CARSfp whole-cell biocatalyst
propan-1-ol 24f, in 75% conversion and 54% isolated yield, Plasmids of carboxylic acid reductases TpCAR, SrCAR, and
after 7 h.
plasmid of phosphopantetheine transferase (Sfp) from Bacillus
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subtilis were sourced from in-house plasmid collections and Hz), 123.94, 123.27, 121.93, 120.65, 117.99, 112.23. HRMS
cloned as previously reported.12 E. coli cells were transformed calcd for C9H8NO+ for 146.06 [M + H]+, found 146.0602.
with CAR and Sfp plasmids. A single colony of recombinant
3-(Naphthalen-1-yl)propan-1-ol 24f was prepared from 3-
E. coli BL21 (DE3) containing pET28-b-FL-SrCAR and pCDF1b- (naphthalen-1-yl)acrylic acid 24a, brown solid was isolated,
Sfp was inoculated into 10 mL lysogeny broth (LB) (1% tryp- 54% yield. 1H NMR (400 MHz, DMSO) δ 8.09 (d, J = 8.1 Hz,
tone, 0.5% yeast extract, 1% NaCl) and incubated overnight at 1H), 7.91 (d, J = 7.6, 1.7 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H),
37 °C in an orbital shaker with 200 rpm shaking. This starter 7.57–7.49 (m, 2H), 7.45–7.35 (m, 2H), 4.57 (s, 1H), 3.51 (t, J =
culture was used as inoculum. A 2 L flask containing 500 mL 6.3 Hz, 2H), 3.08 (t, 2H), 1.87–1.75 (m, 2H). 13C NMR
LB autoinduction media, supplemented with kanamycin (101 MHz, DMSO-d6) δ 138.58, 133.68, 131.61, 128.78, 126.49,
(30 μg mL−1) and streptomycin (50 μg mL−1) was inoculated 126.08 (d, J = 2.6 Hz), 125.77 (d, J = 9.6 Hz), 123.97, 60.59,
with 5 mL of the starter culture. Cultivation was performed at 34.05, 28.99. HRMS calcd for C13H15O+ for 187.11 [M + H]+,
37 °C in an orbital shaker with 200 rpm shaking, initially for found 187.5821.
6 h. Incubation was continued at 24 °C and 200 rpm for a
further 42 h. Cells from a 500 mL culture were harvested by
Analytical scale in vitro biotransformation
centrifugation at 6000 rpm for 15 min, and washed in sodium For the one-pot, two-step in vitro conversion of benzofused
phosphate buffer (sodium phosphate, 50 mM, pH 7.5). (hetero)aromatic acids or acrylic acids derivatives respectively
Harvested cells containing CARSfp were used as fresh resting to the corresponding (hetero)aromatic primary alcohols or
whole cells or purified according to a previously reported allylic alcohols, purified enzymes were used. A typical 500 μL
method.12
reaction contained 5 mM carboxylic acid substrate, 2% v/v
DMSO, 7.5 mM ATP, 10 mM MgCl2, 20 mM D-glucose, 0.4 mM
NADP+, purified SrCAR (0.4 mg mL−1), and purified BsGDH
(0.5–1 mg mL−1) in 50 mM sodium phosphate buffer, pH 7.5.
Reaction mixtures in 2 mL tightly closed Eppendorf’s were
incubated at 30 °C with 250 rpm shaking for 18 h, after which
the samples were prepared for RP-HPLC or GC-MS analysis.
Analytical scale whole-cell biotransformation
Recombinant E. coli cells containing SrCARsfp were applied as
fresh resting cells for the conversion of carboxylic acids to the
corresponding alcohols via the aldehyde intermediate. Glucose
was added to enable in situ regeneration of cofactors.
Reactions were performed in 2 mL Eppendorf tubes. A typical
500 μL reaction mixture contained 5 mM carboxylic acid sub-
strate, 2% v/v DMSO, 20 mM glucose, 10 mM MgCl2, E. coli
resting whole-cells containing over-expressed CARsfp to the
final OD600 of 30 in NaPi buffer (50 mM, pH 7.5). Reaction
mixtures were incubated at 30 °C with 250 rpm shaking for
18 h, after which the samples were prepared for RP-HPLC or
GC-MS analysis.
Preparative in vitro biotransformation
In vitro preparative scale biotransformation reactions were per-
formed in 250 mL glass flask. Purified enzyme biotransform-
ation reactions contained 4 mM MgCl2, 30 mM D-glucose
1 mM NADP+, 12 mM ATP, 100 mg of 26a (10 mM), 2% v/v
DMSO, 0.8 mg mL−1 purified SrCAR and 2 mg mL−1 purified
GDH in 100 mM sodium phosphate buffer (supplemented
with 50 mM NaCl, pH 8.0) to a total volume of 60 mL. The
reaction was incubated at 30 °C with 180 rpm shaking. After
27 h of incubation, the reaction was basified to pH ∼12 with
Preparative whole-cell biotransformation
Preparative scale biotransformation reactions were performed 10 M NaOH, extracted twice into EtOAc with intermediate cen-
in a 250 mL glass flask sealed with breathable membrane to trifugation (4000 r.p.m, 5 min, 10 °C). The organic layers were
ensure adequate aeration. Biotransformation reactions con- combined, dried with anhydrous MgSO4 and solvent was care-
tained 4 mM MgCl2, 120 mM D-glucose, 100 mg of carboxylic fully concentrated.
acid 6a or 24a (at 5 mM or 10 mM) and 125 g L−1 of fresh
Cinnamyl alcohol 26c was prepared from cinnamic acid
resting recombinant E. coli cells expressing SrCAR in 100 mM 26a. Pale yellow liquid was isolated, 62% yield. 1H NMR
sodium phosphate buffer supplemented with 50 mM NaCl, pH (400 MHz, DMSO-d6) δ 7.44–7.26 (m, 5H), 6.56 (d, J = 15.9, 2.0
8.0. Following addition of all components, reaction pH was Hz, 1H), 6.38 (dt, J = 16.0, 5.0 Hz, 1H), 4.87 (s, 1H), 4.12 (t, J =
adjusted to pH 8, incubated at 30 °C with 180 rpm shaking, 5.3, 1.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 136.89,
and hourly pH adjustments to pH ∼8.0 was performed. After 130.80, 128.59, 128.39, 127.18, 126.11, 61.47.
7 h incubation, the reaction was basified to pH ∼12 with 10 M
Initial rate measurements
NaOH, extracted twice into EtOAc with centrifugation (4000 r.
p.m, 5 min, 10 °C). The organic layers were combined, dried Specific activities where determined from initial rates of CAR-
with anhydrous MgSO4 and solution was carefully catalysed carboxylate reduction. Unless stated otherwise,
concentrated.
measurements were performed in triplicate at 370 nm (ε =
Indole-3-carboxyladehyde 6b was prepared from indole-3-car- 2.216 mM−1 cm−1) using a Tecan infinite M200 microplate
1
boxylic acid 6a, yellow solid was isolated, 85% yield. H NMR reader (Tecan Group, Switzerland). The samples were prepared
(400 MHz, DMSO-D6) δ 12.14 (s, 1H), 9.96 (s, 1H), 8.29 (s, 1H), in 96-well microtitre plates. A typical reaction mixture in a well
8.15–8.08 (dt, 1H), 7.53 (dt, J = 8.2, 0.9 Hz, 1H), 7.30–7.15 (m, contained 1–2 mM carboxylic acid substrate, 2 mM MgCl2,
2H). 13C NMR (101 MHz, DMSO) δ 184.77, 137.55 (d, J = 137.6 1 mM ATP, 0.5 mM NADPH, 1% (v/v) DMSO and 20–100 µg of
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purified SrCAR in a total volume of 200 µl (50 mM sodium the clear supernatant was collected and centrifuged further.
phosphate, pH 7.5). A unit of SrCAR was equal to the amount The clarified solution was transferred to HPLC vials for analysis.
of the pure enzyme required to consume 1 µmol NADPH per
Where analysis of biotransformation was performed on the
min. Activity measurement for substrates 24a, 25a, 31a, 34a GC-MS, an equal volume of EtOAc (containing a known con-
and 45a were determined from initial reaction rates measured centration of an internal standard where necessary) was added
by RP-HPLC using the same reaction conditions. In this case, to biotransformation mixture, vigorously mixed, centrifuged
reactions were stopped after 5–10 min incubation and the (15 °C, 13 000 rpm, 10 min) and the organic layer was
samples were prepared for analysis on RP-HPLC. Specific extracted. The aqueous fraction was then acidified to a pH of
activities for these substrates were determined from HPLC ∼2 and further extracted into EtOAc. Where the substrates con-
activity measurements relative to reference activities for sub- tained an amino group, a further extraction step was per-
strates 1a and 36a (which have been standardised using both formed, the aqueous fraction was basified to pH ∼12 and
microplate reader and RP-HPLC measurements) under the extracted into EtOAc. The organic fractions were combined
same reaction conditions.
and dried over anhydrous MgSO4 and the samples were trans-
ferred to vials for analysis on GC-MS.
Analysis: chromatography columns and conditions and
sample preparation
Reverse phase HPLC was performed on an Agilent system Conflicts of interest
(Santa Clara, CA, USA) equipped with a G1379A degasser,
The authors declare that they have no conflict of interest with
G1312A binary pump, a G1367A well plate autosampler unit, a
G1316A temperature-controlled column compartment and a
G1315C diode array detector. Syncronis C18 column, 250 mm
length, 4.6 mm diameter, 5 μm particle size (Thermo Scientific;
Waltham, MA USA) was used. Substrate standards and product
markers, and the resulting biotransformation products were
analysed by reverse phase HPLC using isocratic methods with
different solvent ratios of acetonitrile and water, supplemented
with 0.1% triflouroacetic acid. The flow rate was maintained at
1 mL min−1 and elutes were detected by the U.V. detector (λ =
245 nm). To account for the variation in UV response between
the starting material and the product, relative response factors
were experimentally determined. Correction factors were calcu-
lated from the ratio of the slopes of standard curves plotted for
varying concentrations of both the acid and the corresponding
products at the UV detection wavelength of 245 nm.
the contents of this article.
Acknowledgements
This work was supported by the grants BBSRC BB/K017802
and ERC pre-FAB 695013. The Authors wish to thank Rachel
Heath (Manchester) for providing the GDH plasmid,
Deepankar Gahloth and Sasha Derrington (Manchester) for
useful discussion, and Reynard Spiess (Manchester) for assist-
ance with high resolution mass spectroscopy. D. L. is a Royal
Society Wolfson Merit Award holder.
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