Transition Metal-Free Reduction of Activated Alkenes Using a Living Microorganism

Article abstract of DOI:10.1002/anie.201903973

Microorganisms can be programmed to perform chemical synthesis via metabolic engineering. However, despite an increasing interest in the use of de novo metabolic pathways and designer whole-cells for small molecule synthesis, the inherent synthetic capabilities of native microorganisms remain underexplored. Herein, we report the use of unmodified E. coli BL21(DE3) cells for the reduction of keto-acrylic compounds and apply this whole-cell biotransformation to the synthesis of aminolevulinic acid from a lignin-derived feedstock. The reduction reaction is rapid, chemo-, and enantioselective, occurs under mild conditions (37 °C, aqueous media), and requires no toxic transition metals or external reductants. This study demonstrates the remarkable promiscuity of central metabolism in bacterial cells and how these processes can be leveraged for synthetic chemistry without the need for genetic manipulation.

Full text of DOI:10.1002/anie.201903973

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Accepted Article
Title: Transition Metal-Free Reduction of Activated Alkenes using a
Living Microorganism
Authors: Stephen Wallace, Richard Brewster, Jack Suitor, and Adam
Bennett
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903973
Angew. Chem. 10.1002/ange.201903973
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Transition Metal-Free Reduction of Activated Alkenes using a
Living Microorganism
Richard C. Brewster, Jack T. Suitor, Adam W. Bennett and Stephen Wallace*
of the reductase NemA.[3] Furthermore, native Old Yellow
Abstract: Microorganisms can be programmed to perform chemical
Enzymes in the yeast Saccharomyces cerevisiae have also been
synthesis via metabolic engineering. However, despite an increasing
used to reduce -substituted, ,-unsaturated carbonyl
interest in the use of de novo metabolic pathways and designer whole-
compounds in a stereospecific manner.[4]
cells for small molecule synthesis, the inherent synthetic capabilities
of native microorganisms remain underexplored. Herein we report the
use of unmodified E. coli BL21(DE3) cells for the reduction of keto-
acrylic compounds and apply this whole-cell biotransformation to the
synthesis of aminolevulinic acid from a lignin-derived feedstock. The
reduction reaction is rapid, chemo- and enantioselective, occurs
under mild conditions (37 ˚C, aqueous media) and requires no toxic
transition metals or external reductants. This study demonstrates the
remarkable promiscuity of central metabolism in bacterial cells and
how these processes can be leveraged for synthetic chemistry without
the need for genetic manipulation.
Synthetic biology is revolutionizing the field of chemical synthesis.
This approach uses recombinant DNA to enable the synthesis of
a small molecule target directly from renewable feedstocks via
fermentation.[1] Despite the elegance of this approach, microbial
cells are frequently used in forward engineering endeavors as a
biosynthetic starting-point, where synthetic capabilities are added
via the introduction of new genetic parts. The background native
chemical capabilities of these organisms remain underexplored.
Research into the synthetic potential of native microbial cells
could reveal new reactivity for use in synthetic chemistry and
would also yield reactions that are inherently linked to cellular
metabolism for use in metabolic pathway design. Furthermore,
these reactions can also be interfaced with transition metal
catalysis for the renewable synthesis of non-natural chemicals.[2]
In a seminal example, Balskus et al. used H2(g) generated via
engineered metabolism in E. coli DD-2 to hydrogenate a range of
alkene substrates using the biocompatible Royer Pd catalyst.[2d]
However, alkene reduction is also an inherent metabolic
process of many native microorganisms. For example, facultative
anaerobes use small organic molecules to respire via a reductive
process. A classic example is the reduction of fumarate by
fumarate reductase. This flavin-dependent C=C bond reduction
can be used to support growth and replication under anaerobic
conditions (Figure 1A). Other native enzymes can also be used to
reduce C=C bonds. For example, E. coli BL21(DE3) and DH5
cells have been shown to reduce conjugated nitroalkenes in
Figure 1. Cellular approaches to alkene reduction. A) Native respiratory
enzymes reduce activated alkenes under anaerobic conditions. B) Metabolic
H2(g) production can be interfaced with a biocompatible Pd catalyst for the
reduction of coumaric acids. C) Exploring the native reductive capability of
moderate yields and this has been attributed, in part, to the activity
unmodified microorganisms for transition metal-free keto-acrylate reduction.
Inspired by these studies, we set out to investigate the reduction
of substituted keto-acrylic compounds (KACs) by microbial cells
[*]
Dr R. C. Brewster, J. T. Suitor and Dr S. Wallace
Institute for Quantitative Biology, Biochemistry and Biotechnology,
School of Biological Sciences, University of Edinburgh, King’s
Buildings, Alexander Crum Brown Road, Edinburgh, EH9 3FF, UK
A. W. Bennett
School of Chemistry, University of Edinburgh, Joseph Black
Building, David Brewster Road, King’s Buildings, Edinburgh, EH9
3FJ
(Figure 1C). KACs feature in a range of pharmaceutical and fine
chemical syntheses and are predominantly reduced in organic
solvent using transition metals under an atmosphere of H2(g).
Biocatalytic approaches to KAC reduction have been limited to in
vitro studies using purified enzymes. In 2014, Pietruszka et al.
reported the use of the enoate reductase YqjM from Bacillus
subtilis for the reduction of short-chain KACs in vitro and coupled
this with an alcohol dehydrogenase (ADHLK) to synthesize chiral
-butyrolactones.[5] Despite the elegance of this approach, the
E-mail: stephen.wallace@ed.ac.uk
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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strict cofactor dependence of YqjM and ADHLK requires the use
of exogenous NADPH and FMN, which must also be regenerated
in situ using additional enzymes. The reaction also requires
constant buffering using a pH-stat. There are currently no
methods to achieve this reaction using green synthetic methods
or microbial whole cells.
cell density by 77% (Figure 3A, columns 2-4). Interestingly, using
5 mM (ca. 1.2 g/L) of the benzyl ester 1-Bn increased the reaction
yield to 79% and increased the cell density to 118% of control
samples containing no substrate (Figure 3A, columns 5-7).
We reasoned that the similarity of the KAC motif to fumarate
suggested that native microbial cells could facilitate this
transformation without the need for genetic manipulation. This
hypothesis was inspired by our previous studies in biocompatible
chemistry where reduction of the unnatural compound diethyl
maleate was observed during a cyclopropanation reaction and
attributed to the metabolic activity of E. coli NST74.[2b]
Furthermore, E. coli possesses a series of YqjM homologues in
its genome ( ≤ 33% identity, Table S10) and can generate
reducing equivalents in situ via oxidative glucose metabolism.
Therefore, we set out to investigate whether keto-acrylate
reduction could be performed by living E. coli cells and, if so,
whether this could be used in the synthesis of an industrially-
relevant small molecule. Herein we report the discovery that the
common laboratory strain of bacteria E. coli BL21(DE3) can
reduce a range of keto-acrylates without the need for genetic
modification. This whole-cell reaction is mild, efficient,
enantioselective and represents an operationally simple and
green alternative to current transition metal-based methods that
utilize H2(g).
We began our studies by investigating whether E. coli could
reduce butenyl-substituted keto-acrylate esters 1 in cell culture
(Figure 2A). The butenyl side chain was included to assess the
chemoselectivity of any observed reduction. The benzyl ester 1-
Bn was included to localize the substrate to the cell membrane,
and the carboxylic acid 1-H was used as a hydrophilic control.
Substrates were synthesized in 4 steps from commercially
available glyoxal dimethyl acetal via a Horner-Wadsworth-
Emmons olefination, acetal hydrolysis, Grignard addition and
oxidation (see S2.1). The laboratory strain E. coli BL21(DE3) was
chosen due to its widespread availability in molecular biology labs
and its well-annotated genome. We were pleased to find that C=C
reduction occurred for all the compounds tested, as confirmed by
1H NMR (Figure 2B, C). The methyl and ethyl esters 1-Me and 1-
Et were reduced in 39% and 43% yield, respectively, and were
isolated alongside significant quantities of hydrolyzed substrate
and product (Table S1). Hydrolysis was reduced to 8% using the
bulkier benzyl ester 1-Bn, which increased the product yield to
69%. However, use of the carboxylic acid substrate 1-H
eliminated this side-reaction and provided the desired reduced
product 2-H in quantitative yield (Figure 2C).
To determine whether the reaction was biocompatible we
analyzed its effect on E. coli cells (Figure 2D and Table S2). The
methyl and ethyl esters 1-Me/1-Et were highly toxic and the
benzyl ester reduced cell viability by 400-fold (Figure 2D, columns
2-4). The most biocompatible substrate was the carboxylic acid 1-
H, which caused only a 2-fold drop in cell viability after 48 h
(Figure 2D, column 5). This observation suggested that the
toxicity of 1-Me/1-Et may be due to the release of MeOH/EtOH
into the cell. However, when we cultured cells in the presence of
1-H and an equimolar volume of EtOH, no toxicity was observed.
Instead, cell viability increased by 3-fold (P<0.001, Figure 2D,
column 6 and Table S8). The addition of EtOH is known to mimic
the heat shock response in E. coli and increases the rigidity of the
cell membrane, an effect that could account for this observation.[6]
At this point, due to poor product conversions, competing
hydrolysis and the high toxicity of reactions involving 1-Me and 1-
Et we decided to focus the remainder of this study on the benzyl
ester and carboxylic acid 1-Bn and 1-H.
Figure 2. A) Initial KAC ester and carboxylic acid screen. B) Product
identification via comparison of culture extracts with a synthetic standard by 1H-
NMR. C) Product yields for various substrate esters and carboxylic acid. D)
Serial dilution and plate count assays. All substrates were added at 1 mM
concentration. Reactions were performed in sealed Hungate tubes under an
atmosphere of air for 48 h. E. coli BL21(DE3) cells transformed with an empty
pEdinBrick plasmid (pSB1A2 derived, OD600=0.6) were used to ensure the
presence of a monoculture. All cultures were grown in M9 media containing D-
glucose (4 g/L) and ampicillin (100 mg/L). Product concentrations in crude
culture extracts were determined by 1H NMR spectroscopy relative to an internal
standard of 1,3,5-trimethoxybenzene. All data is shown as an average of three
independent experiments to one standard deviation. **P<0.005
Differences between 1-H and 1-Bn were also observed when
analyzing the rate of product formation over time. Firstly, we found
that both reactions were complete in <6 h. Reduction of 1-H was
complete after 3 h, whereas the reduction of 1-Bn was rapid
during the first 1.5 h (65% conversion) and then plateaued before
Next, we tested the reaction at higher substrate concentration and
investigated the rate of product formation during the reaction.
Increasing the concentration of carboxylic acid 1-H to 5 mM (ca.
0.8 g/L) decreased the yield of the reaction to 66% and reduced
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reaching completion after 6 h (Figure 3B and Table S3). One
explanation for this involves consideration of the effect(s) and
relative location of 1-Bn/1-H in the cell interior. Hydrophobic
compounds are known to partition in the membrane of E. coli
(logPoctanol/water = 3.7 for 1-Bn vs –1.7 for 1-H at pH 7.4, Table S7)
and this in-turn has been shown to accelerate glucose
consumption via increased growth/maintenance demands.[7,8]
NemA, FadH and PpsA are homologs of the known KAC-
reductase YqjM from B. subtillus (Table S12). To our surprise, the
reduction of 1-H was unaffected by the absence of the frdB, nemA
or ppsA genes (77% ± 21%, 82% ± 4.6% and 91% ± 7.3% yield,
respectively, P>0.05 relative to the parent Keio strain), whereas
deletion of fadH reduced the product yield to 65% ± 6.0% (P<0.05,
Figure 3D and Table S11). This partial yet significant effect has
been observed whilst studying
the reduction of nitroalkenes by
E. coli BL21∆nemA.[3] Together,
this suggests the presence of
either
a
multifaceted KAC
reduction pathway in the cell
interior or a potentially novel
C=C keto-acrylate reductase in
the genome of Escherichia sp.
Having shown that KAC
reduction occurs rapidly under
mild conditions using E. coli
BL21(DE3) cells we next
investigated
the
enantioselectivity
of
this
process using ,-substituted
substrates. We synthesized the
- or -methyl substituted
carboxylic acid and benzyl
ester substrates 3-H-Me, 3-H-
Me, 3-Bn-Me and 3-Bn-Me
from either glyoxal dimethyl
acetal or pyruvic aldehyde
dimethyl acetal in
a similar
manner to 1-Bn/H (see S2.1).
Under our optimized conditions,
the -methylated carboxylic
acid 3-H-Me was reduced in
79% and the corresponding
benzyl ester 3-Bn-Me was
Figure 3. Investigating the effect of the reaction on E. coli. A) The effect of increasing substrate concentration on culture
density and product yield. B) Time-course analysis of product formation and glucose levels during the reaction. C) The
effect of varying cell density on the initial rate of product formation using suspended whole-cells. D) Control experiments
investigating the effect of the components of the reaction on the reduction of 1-H. Numbers indicated within the graphs
represent the yield of the reaction. Substrates were added at 1 mM unless stated otherwise. All data is shown as an
average of three independent experiments to one standard deviation. *P<0.05, ***P<0.0005, n/s = P>0.05
reduced
in
98%
yield.
Conversely, the -methylated
isomers were both reduced in
Indeed, we found that a three-fold increase in cell density
<5% yield (Figure 4A). Furthermore, both the - and -methyl
benzyl ester substrates reduced cell viability by 1,500-fold,
whereas the carboxylic acid congeners were biocompatible
(Table S10). Analysis of the product of the reaction by chiral
HPLC confirmed that the reduction of 3-Bn-Me was
enantioselective, affording the reduced product in 87% e.r. The
carboxylic acid 3-H-Me was reduced in 51% e.r. A cyclic -
substituted KAC substrate 6 was also reduced under the reaction
conditions, affording the corresponding chiral cyclohexanone in
44% yield and 62% e.r. Moving the benzyl group to the -position
accelerated the reduction of 1-H after 1 h (72% yield at OD600=0.6
vs. 92% yield at OD600=1.8, P<0.05) but decreased the
conversion of 1-Bn (69% yield at OD600=0.6 vs. 36% yield at
OD600=1.8, P<0.0005) (Figure 3C and Table S6). Furthermore, a
prolonged elongation during cell division as a result of membrane
stress can also increase the number of viable but non-culturable
(VBNC) cells via rpoS-mediated pathways leading to higher cell
density and reduced cell viability (Figure 3A and Table S9).[9]
Together, these observations support the hypothesis that the
decreased reduction of 1-Bn after 1.5 h results from the
cumulative effects of an altered cell physiology leading to
depleted glucose levels (Figure 3B).
We next examined the effect of the reaction components on the
reduction of 1-H. Product formation was reduced to <5% yield in
the absence of cells or the presence of dead cells (heated to 95
˚C for 15 min). No reduction was observed in blank media, spent
supernatant or in media containing excess NADH (10 equiv., 24
h). Conversely, 1-H was reduced in >98% yield using E. coli
TOP10 or DH5 cells, and in 94% yield using BL21(DE3) cells
containing no pEdinBrick plasmid (Figure 3D and Table S11).
Together, this data supports the hypothesis that 1 is reduced in
the cell by an enzyme that is native to E. coli. To explore this
further we examined the reaction using the knock-out strains E.
coli JW4114(K-12)∆frdB, JW1642(K-12)∆nemA, JW3052(K-
12)∆fadH and JW1692-2∆ppsA. FrdB encodes for the catalytic
subunit of fumarate reductase, whereas nemA, fadH and ppsA
in
7 abolished any enantioselectivity. The simplified KAC
substrate 5 was reduced in 100% yield and 77% e.r, whereas its
Z-isomer was reduced in 69% yield and 79% e.r. In contrast,
dimethyl maleate 8-Z was reduced in 78% yield, whereas
dimethyl fumarate 8-E was reduced in 3% yield. Citraconate and
itaconate di-esters (9 and Figure S3) and acrylate 10 were
unreactive under the reaction conditions.
Having confirmed the enantioselectivity of the reduction we
moved on to apply the reaction to the synthesis of a target small
molecule. Aminolevulinic acid (ALA, Levulan®, 13) is a naturally-
occurring non-proteogenic amino acid that is used as an additive
in photodynamic therapy during the treatment of Paget’s disease
and cancer.[10] Although access to ALA via engineered
metabolism has been achieved, this has not translated into a
viable industrial bio-process.[11] ALA is therefore currently
produced on an industrial scale via the oxidation of
petrochemically-derived furfurylamine followed by a Pd-catalyzed
hydrogenation using H2(g). We reasoned that our E. coli-
encode for
a N-ethylmaleimide reductase, 2,4-dienoyl-CoA
reductase and phosphoenolpyruvate synthase, respectively.
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mediated C=C reduction could achieve this same transformation
without the need for toxic Pd salts or pressurized H2(g). To this
end, we synthesized the butenolide hemiacetal 12 from lignin-
derived furan imide 11. Upon addition to E. coli BL21(DE3) cells,
12 underwent ring-opening and C=C reduction, affording the
reduced product in 81% isolated yield. Finally, the phthalimide
was removed under acidic conditions to afford ALA in quantitative
yield (Figure 4B).
stands in stark contrast to modern abiotic methods for transition
metal-free alkene reduction in synthetic organic chemistry, recent
examples of which include: (i) transfer hydrogenation using
diphenylamine, triethylsilane and the electrophilic cationic
phosphonium
catalyst
[(C6F5)3PF)]+[B(C6F5)4]–,
thiourea
organocatalysis using a Hantzsch ester in refluxing H2O and (ii)
the use of NaAlH4/C nanocomposites and 100 bar H2(g) in organic
solvent at 150 ˚C for 48 h.[12] The biocompatibility of whole-cell
reactions is also important, yet is rarely considered in the field of
biocatalysis. Beyond substrate localization effects, biocompatible
reactions can also be readily integrated into de novo metabolic
pathways for renewable chemical production in microbial cells.
In summary, we have shown that unmodified laboratory
strains of E. coli can be used for the reduction of keto-acrylic acids,
esters and -substituted variants thereof. The reaction is efficient,
occurs under mild conditions and uses no toxic transition metals
or external reductants. The remarkable biocompatibility of this
process led us to discover unique facets of the reaction in a
cellular setting that will be used to inform the design of new whole-
cell reactions. This includes, to the best of our knowledge, the first
observation that reactivity effects vary when substrates are
localized to different regions of the cell. Finally, we applied this
reaction to the synthesis of the valuable small molecule
aminolevulinic acid, eliminating the requirement for the use of
Pd/C and H2(g). Future studies will focus on identifying the
enzyme(s) responsible for this reaction using comparative
transcript-/proteomics
and
integration
of
this
novel
biotransformation into metabolic pathways for renewable small
molecule synthesis via fermentation.
Acknowledgements
This work was supported by the University of Edinburgh and an
ISSF grant from the Wellcome Trust. J.T.S. acknowledges a PhD
Scholarship from the Carnegie Trust. We acknowledge Mr Alan
Taylor and the Scottish Instrumentation and Resource Centre for
Advanced Mass Spectrometry (SIRCAMS) for their assistance
during the preparation of this manuscript.
Keywords: biotransformation • green chemistry • reduction •
whole cell • biotechnology
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Entry for the Table of Contents (Please choose one layout)
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Richard C. Brewster, Jack T. Suitor,
Adam W. Bennett, Stephen Wallace*
Page No. – Page No.
Transition Metal-Free Reduction of
Activated Alkenes using a Living
Microorganism
Alkene-Reducing Bugs: The microorganism Escherichia coli BL21(DE3) was found
to reduce a range of keto-acrylic alkene substrates without the need for genetic
modification. The reaction is mild, enantioselective and provides a green alternative
to transition metal-based methods that utilize H2(g). The biocompatibility of the
reaction was examined in a cellular setting and used to inform the metal-free
synthesis of aminolevulinic acid from a lignin-derived substrate.
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