Enzymatic Electrosynthesis of Alkanes by Bioelectrocatalytic Decarbonylation of Fatty Aldehydes

Article abstract of DOI:10.1002/anie.201712890

An enzymatic electrosynthesis system was created by combining an aldehyde deformylating oxygenase (ADO) from cyanobacteria that catalyzes the decarbonylation of fatty aldehydes to alkanes and formic acid with an electrochemical interface. This system is able to produce a range of alkanes (octane to propane) from aldehydes and alcohols. The combination of this bioelectrochemical system with a hydrogenase bioanode yields a H₂/heptanal enzymatic fuel cell (EFC) able to simultaneously generate electrical energy with a maximum current density of 25 μA cm^?2 at 0.6 V and produce hexane with a faradaic efficiency of 24 %.

Full text of DOI:10.1002/anie.201712890

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Enzymatic Electrosynthesis of Alkanes via Bioelectrocatalytic
Decarbonylation of Fatty Aldehydes
Authors: Sofiene Abdellaoui, Florika Macazo, Rong Cai, Antonio de
Lacey, Marcos Pita, and Shelley Minteer
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201712890
Angew. Chem. 10.1002/ange.201712890
Link to VoR: http://dx.doi.org/10.1002/anie.201712890
http://dx.doi.org/10.1002/ange.201712890

10.1002/anie.201712890
Angewandte Chemie International Edition
COMMUNICATION
Enzymatic Electrosynthesis of Alkanes via Bioelectrocatalytic
Decarbonylation of Fatty Aldehydes
Sofiene Abdellaoui,[a] Florika C. Macazo,[a] Rong Cai,[a] Antonio L. De Lacey,[b] Marcos Pita,[b] and Shelley D.
Minteer*[a
]
Abstract: An enzymatic electrosynthesis system is created by
combining an aldehyde deformylating oxygenase (ADO) from
cyanobacteria catalyzing the decarbonylation of fatty aldehydes to
alkanes plus formic acid with an electrochemical interface. This
system is able to produce a range of alkanes (octane to propane) from
aldehydes and alcohols. The combination of this bioelectrochemical
system with a hydrogenase bioanode yields a H2/heptanal enzymatic
fuel cell (EFC) able to simultaneously generate electrical energy with
a maximum current density of 25 µA cm-2 at 0.6 V while producing
hexane with a faradaic efficiency of 24 %.
FDCOOs, the ADOs require an external auxiliary reducing system
to supply four electrons per catalytic cycle, such as NADPH
combined with ferredoxin (Fdx) and a ferredoxin reductase
(Fdr).[8] Artificial chemical reductants can also be used to supply
electrons to ADOs such as the redox couple NADH/PMS
(phenazine methosulfate).[6]
ADO
O
+ HCOO- + H2O
+ O2
R
CH3
R
4e- + 3H+
(PMS/NADH)
or
Renewable biofuels are alternatives to petroleum-based fuels.
However, the large-scale production of biofuels involves a
massive production of food crops requiring the utilization of large
quantities of water, fertilizer, and land.[1] As an alternative,
researchers have tried to exploit fatty acid catabolism pathways
of eukaryotic and prokaryotic organisms to biochemically
synthesize hydrocarbons – the major constituents of gasoline,
diesel, and other fuels.[2] Cyanobacteria, having characteristics of
both plants and prokaryotes and being easy to genetically
manipulate, offer the most advantage for this purpose, especially
for industrial applications.[3] Two key enzymes involved in
cyanobacteria-mediated hydrocarbon biosynthesis include an
acyl carrier protein reductase (AAR), which catalyzes the
reduction of fatty acyl-ACP (acyl carrier protein) to fatty aldehydes,
and an aldehyde decarbonylase (AD), which catalyzes the
decarbonylation of fatty aldehydes to alkanes and formic acid.[4]
However, the main drawbacks of microbial production of “bio-
alkanes” involve complex and costly extraction/purification
techniques, as well as production of side/end products that can
be toxic to the cells. Therefore, an in vitro system working with
isolated enzymes, such as ADs, could overcome these problems.
ADs belong to the ferritin-like diiron-carboxylate oxygenase and
oxidase (FDCOO) protein structural superfamily.[5] In
(Fdx/Frd/NADPH)
Scheme 1. ADO-mediated enzymatic conversion of aldehyde to alkanes and
formic acid.
Numerous oxidoreductases have been interfaced with an
electrode to achieve a bioelectrocatalytic reaction in the context
of biosensors and enzymatic biofuel cells (EFC).[9] The
heterogeneous electron transfer between redox enzymes and
electrodes can take place either directly or via mediated electron
transfer (MET) using redox mediators. Enzyme-modified
electrodes were recently used for enzymatic electrosynthesis of
several molecular targets, whereby the high chemo-, regio- and
stereoselectivity of enzymes are used to circumvent the necessity
for harsh reaction conditions in traditional chemical synthesis.[10]
Milton et al. have demonstrated the coupling of a hydrogenase
bioanode with a nitrogenase biocathode resulting in EFC able to
simultaneously produce electrical energy and NH3.[11]
Herein, we demonstrate the use of toluidine blue O (TBO) as a
redox mediator to achieve the bioelectrocatalytic decarbonylation
of an aldehyde to an alkane using the recombinant ADO from
Prochlorococcus marinus (PmADO). We employ TBO to mediate
the electron transfer between a carbon electrode and the active
site of PmADO to produce alkanes ranging from octane (C8) to
propane (C3). In addition, we combine PmADO with a NAD-
dependent alcohol dehydrogenase to achieve an enzymatic
cascade reaction yielding alkanes from a range of short chain fatty
alcohols. Finally, we construct a H2/heptanal EFC employing a
hydrogenase at the anode and PmADO at the cathode, which is
able to simultaneously generate electrical energy and hexane as
the final product.
cyanobacteria, AD is
a soluble non-heme di-iron protein
catalyzing the production of formic acid and alkane from aldehyde
6]
(Scheme 1).[4a, This enzyme was recently designated as an
aldehyde-deformylating oxygenase (ADO), since it has been
shown that it requires oxygen to catalyze a cryptic redox
oxygenation reaction to form alkanes and formic acid from a broad
range of aldehydes (heptadecanal to butanal).[7] Like most of the
[a]
[b]
Department of Chemistry, University of Utah, 315 S 1400 E, Salt Lake
City, UT, 84112 (USA)
E-mail: minteer@chem.utah.edu
Instituto de Catalisis y Petroleoquimica, CSIC, C/ Marie Curie 2, L10,
28049, Madrid (Spain)
It was shown that ADOs require four electrons per catalytic cycle
to reduce the cofactor Fe2III/III form to the O2-reactive active form
(µ-peroxo-Fe2II/II), which result in the reductive cleavage of an
[7,
intermediate Fe2II/II-peroxyahemiacetal formed with substrates.
12] In order to achieve bioelectrocatalysis with PmADO by MET on
carbon electrodes, we chose TBO as a cofactor regeneration
mediator, since it can easily be chemically reduced by NADH and
Supporting information for this article is given via a link at the end of
the document
This article is protected by copyright. All rights reserved.

10.1002/anie.201712890
Angewandte Chemie International Edition
COMMUNICATION
electrochemically reduced/oxidized at the electrode surface.[13]
For this prospect, we initially evaluated the activity of PmADO
using the redox couple TBO/NADH with a range of different
inhibition processes. These results demonstrate that
a
combination of PmADO, TBO and a carbon electrode can
constitute a system that is able to bioelectrocatalyze the
decarbonylation of aldehydes to alkanes.
aldehyde substrates. The turnover number (kcat
) for each
substrate was calculated by detecting and quantifying the alkanes
formed using gas chromatography (GC) coupled with a flame
ionization detector (FID) (Table 1). The calculated values indicate
that PmADO has slow turnover with the highest activity observed
with heptanal. These values are close to the kcat values reported
for ADO/PMS-mediated assays.[14] Khara and Al reported a kcat
value of 0.0031 min-1 for the decarbonylation of butanal to
propane by PmADO using PMS/NADH, which is close to the value
we obtained with TBO (0.0024±0.0004 min-1).[14] Furthermore, the
kcat for heptanal (1.31±0.17 min-1) is close to the highest value
described in the literature (~ 1 min-1).[7]
Table 1. Comparison of PmADO kcat with NADH/TBO.
Substrate[a]
Nonanal
Octanal
kcat (min-1)
0.13 ± 0.03
Scheme 2. Schematic of the PmADO biocathode.
0.50 ± 0.02
Heptanal
Hexanal
Pentanal
Butanal
1.31 ± 0.17
0.47 ± 0.03
0.0029 ± 0.0003
0.0024 ± 0.0004
A carbon electrode was then used to build a biocathode with
PmADO and TBO as a redox mediator (Scheme 2). This system
was investigated via cyclic voltammetry (CV) using heptanal
substrate. Since TBO is sensitive to O2, all measurements were
carried out in a sealed system prepared under argon atmosphere.
Of note, the stock solution of heptanal was prepared under
aerobic conditions in order to introduce some O2 required for the
enzymatic reaction. The voltammogram in Figure 1 reveals the
reversible redox peaks of TBO (black solid curves), with a
reduction and oxidation peaks at -0.26 V and -0.22 V vs. SCE,
respectively. To demonstrate enzymatic activity, 10 mM heptanal
was added and a bioelectrocatalytic wave was observed,
indicating that electrocatalytic reduction of heptanal has occurred.
A control using denatured PmADO shows no catalytic signal in
the presence of heptanal. Moreover, new redox peaks can be
seen at ~ -0.06 V vs. SCE after the addition of heptanal in the
presence of the native or denaturated enzyme, suggesting that
heptanal is potentially affecting the redox properties of TBO. This
could be attributed to the hydrophobicity of the substrate that
induces the formation of TBO aggregates in the bulk.[15]
To illustrate substrate concentration dependence, we carried out
a series of CVs in the presence of increasing concentrations of
heptanal to 10 mM. The catalytic current densities at -0.50 V vs.
SCE were then extracted and plotted against the concentration of
heptanal (Figure 2). It is evident from Figure 2 that the current
density, and hence, the enzymatic electrocatalytic activity,
increases as the concentration of heptanal increases. After 5 mM
heptanal, the signal decrease due to low solubility and possible
Figure 1. Catalytic reduction of heptanal by TBO-mediated PmADO (scan rate:
10 mV/s-1). (top) Catalytic CV’s obtained for a Toray electrode immersed in
buffer/TBO/PmADO solution (black solid curve) and in the presence of 10 mM
heptanal (blue dashed curve). (bottom) CV obtained in a solution containing
buffer/TBO/Denatured PmADO (black solid curve) and in the presence of 10
mM heptanal (blue dashed curve).
This article is protected by copyright. All rights reserved.

10.1002/anie.201712890
Angewandte Chemie International Edition
COMMUNICATION
reduction of fatty acyl-CoA to aldehydes, will allow for the
production of alkanes from fatty acids.
Table 2. Quantification of alkanes and faradaic efficiencies obtained from
the enzymatic electrosynthesis of alkanes from aldehydes.
Product
Octane
Quantity (nmol h-1)
Faradaic efficiency (%)
1.966
2.18
13.02
7.32
4.28
0.46
1.16
Heptane
Hexane
Pentane
Butane
8.666
29.570
6.549
3.026
Propane
0.6289
Figure 2. Heptanal-induced changes in the catalytic reduction current by TBO-
mediated PmADO with a -0.5V applied potential.
Since we have already established the bioelectrocatalytic activity
of PmADO, we then sought to demonstrate its ability as an
enzymatic electrosynthesis system to produce a range of alkanes
from octane to propane. For that, bulk electrolysis in the presence
and absence of PmADO was performed at -0.5 V vs SCE using
six aldehydes shown in Table 1. These experiments were carried
out in sealed vials prepared anaerobically (O2 < 1 ppm) (Figure
S1) and the electrosynthesis was initiated by the addition of
substrates and 10 mL of air to introduce a small amount of O2
required for the reaction. After an hour of electrolysis, alkanes
were detected and quantified by GC/FID (chromatograms shown
in Figure S2). The quantities of alkanes and the faradaic
efficiencies were calculated and summarized in Table 2. The
faradaic efficiencies are generally low, with the highest value
obtained for the conversion of octanal to heptane (13.02 %). This
can be attributed to the electro-reduction of O2 competing with the
electro-decarbonylation of the aldehydes understudy (Figure S3).
Scheme 3: Illustration of the bioelectrochemical system combining PmADO and
ADH.
Table 3. Rate of production of alkanes from alcohols by enzymatic
electrosynthesis.
The PmADO-modified electrode was then combined with a NAD-
dependent alcohol dehydrogenase (ADH) to achieve an
enzymatic cascade reaction sequentially converting fatty alcohols
to aldehydes by enzymatic oxidation, and aldehydes to alkanes
by bioelectrocatalytic decarbonylation (Scheme 3). We aimed to
produce octane to propane from fatty alcohols, which are more
sustainable than aldehydes for practical applications. Prior to
testing this enzymatic cascade reaction, the ADH activity with
these alcohols was evaluated by UV-Vis (Figure S4.). The results
show significant activities towards butanol, pentanol, hexanol and
heptanol and low activities with octanol and nonanol. These
alcohols were then used as substrates for the enzymatic
electrosynthesis of alkanes monitored by GC/FID (Figure S5).
After electrolysis, the alkanes produced were detected and
quantified (Table 3). The slow conversions of nonanol and octanol
to octane and heptane, respectively, are due to the low activity of
ADH towards those substrates, while the slow production of
propane (0.003mgL-1h-1) is due to the low activity of PmADO with
butanal. However, the production of hexane from heptanol is 50
to 100 times higher, with a rate of 1.353mgL-1h-1. This burst of
production rate could be explained by the combination of the high
activities of PmADO with heptanal and ADH with heptanol. The
combination of ADO with other enzymes, such as an acyl-CoA
synthase and acyl carrier protein reductase, which catalyze the
Substrate → Product
Nonanol Octane
mg L-1 h-1
0.017
→
Octanol → Heptane
Heptanol → Hexane
Hexanol → Pentane
Pentanol → Butane
Butanol → Propane
0.024
1.353
0.014
0.020
0.003
Finally, a H2/heptanal EFC resulting from the combination of a
hydrogenase bioanode with a PmADO biocathode was evaluated.
The Ni-Fe hydrogenase (from Desulfovibrio gigas)[16] at the anode
facilitates the electro-oxidation of H2 to H+ using methyl viologen
(MV2+/MV.+) as a redox mediator and subsequently supplies
electrons for the bioelectrocatalytic decarbonylation of heptanal to
hexane (Figure 3). This EFC operating with H2 as a fuel and
heptanal as an oxidant yielded an OCP of 597±5 mV and
generated a maximum current density (Jmax) and power density
This article is protected by copyright. All rights reserved.

10.1002/anie.201712890
Angewandte Chemie International Edition
COMMUNICATION
(Pmax) of 25±3 µA cm-2 and 4.7±0.5 µ cm-2, respectively. In order
to probe the effect of O2 reduction, a control was performed
without PmADO in the presence of O2 and heptanal, which
yielded a Jmax of 13±2 µAcm-2 and a Pmax of 2.9±0.1 µWcm-2.
These results show that the reduction of oxygen itself contributes
to about half of the current and power density obtained for the
H2/heptanal EFC. A Jmax of 13±3 µAcm-2 and a Pmax of 3.6±0.2 µW
cm-2 were obtained with a second control carried out in the
absence of oxygen and in the presence of PmADO and heptanal.
It was shown previously that PmADO are also able to achieve a
slow decarbonylation of aldehyde in the absence of O2 through an
oxygen-independent hydrolytic mechanism,[6, 17] thus, the signal
obtained with the second control could be induced by this side
enzymatic reaction. Finally, hexane produced from EFCs was
quantified after the passage of 40 mC, yielding 22±1 nmol of
hexane with a faradaic efficiency of 24±4%. These results
demonstrate that the H2/heptanal EFC is able to simultaneously
generate electrical energy and produce hexane from heptanal.
[4]
aA. Schirmer, M. A. Rude, X. Li, E. Popova, S. B. del Cardayre,
Science 2010, 329, 559-562; bD. M. Warui, N. Li, H. Nørgaard, C.
Krebs, J. M. Bollinger, S. J. Booker, Journal of the American
Chemical Society 2011, 133, 3316-3319.
C. Krebs, J. M. Bollinger, S. J. Booker, Current Opinion in
Chemical Biology 2011, 15, 291-303.
D. Das, B. E. Eser, J. Han, A. Sciore, E. N. G. Marsh, Angewandte
Chemie International Edition 2011, 50, 7148-7152.
N. Li, W.-c. Chang, D. M. Warui, S. J. Booker, C. Krebs, J. M.
Bollinger Jr, Biochemistry 2012, 51, 7908-7916.
L. J. Rajakovich, H. Nørgaard, D. M. Warui, W.-c. Chang, N. Li, S.
J. Booker, C. Krebs, J. M. Bollinger Jr, M.-E. Pandelia, Journal of
the American Chemical Society 2015, 137, 11695-11709.
aF. C. Macazo, S. D. Minteer, Current Opinion in Electrochemistry
2017; bS. D. Minteer, B. Y. Liaw, M. J. Cooney, Current opinion in
biotechnology 2007, 18, 228-234; cM. Rasmussen, S. Abdellaoui,
S. D. Minteer, Biosensors and Bioelectronics 2016, 76, 91-102.
aX. Dominguez-Benetton, S. Srikanth, Y. Satyawali, K.
Vanbroekhoven, D. Pant, J Microb Biochem Technol S 2013, 6, 2;
bC. Kohlmann, W. Märkle, S. Lütz, Journal of Molecular Catalysis
B: Enzymatic 2008, 51, 57-72.
R. D. Milton, R. Cai, S. Abdellaoui, D. Leech, A. L. De Lacey, M.
Pita, S. D. Minteer, Angewandte Chemie International Edition
2017, 56, 2680-2683.
aN. Li, H. Nørgaard, D. M. Warui, S. J. Booker, C. Krebs, J. M.
Bollinger Jr, Journal of the American Chemical Society 2011, 133,
6158; bM. W. Waugh, E. N. G. Marsh, Biochemistry 2014, 53,
5537.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
S. Abdellaoui, M. Bekhouche, A. Noiriel, R. Henkens, C.
Bonaventura, L. J. Blum, B. Doumèche, Chemical
Communications 2013, 49, 5781-5783.
B. Khara, N. Menon, C. Levy, D. Mansell, D. Das, E. N. G. Marsh,
D. Leys, N. S. Scrutton, ChemBioChem 2013, 14, 1204-1208.
J. Jebaramya, M. Ilanchelian, S. Prabahar, sensors 2009, 21, 22.
R. Cammack, V. M. Fernandez, E. Claude Hatchikian, in Methods
in Enzymology, Vol. 243, Academic Press, 1994, pp. 43-68.
B. E. Eser, D. Das, J. Han, P. R. Jones, E. N. G. Marsh,
Biochemistry 2011, 50, 10743.
[15]
[16]
[17]
Figure 3: Representative power (red lines) and polarization curves (blue lines)
for H2/heptanal fuel cells (solid lines); a control fuel cell without ADO in the
presence of O2 and heptanal (dotted lines), and a control with ADO in the
presence of heptanal without O2 (dashed lines). The measurements were
performed by linear sweep polarization at 0.2 mV s-1 with vigorous stirring.
Acknowledgements
The authors would like to thank the Army Research Office MURI
grant (#W911NF1410263) and the Spanish Economy Ministry
(MINECO/FEDER CTQ2015-71290-R grant) for funding this work.
Keywords: bioelectrochemical catalysis, aldehyde-
deformylating oxygenase, alkane, enzymatic electrosynthesis
[1]
[2]
[3]
A. K. Agarwal, Progress in Energy and Combustion Science 2007,
33, 233-271.
M. A. Rude, A. Schirmer, Current Opinion in Microbiology 2009,
12, 274-281.
A. Parmar, N. K. Singh, A. Pandey, E. Gnansounou, D.
Madamwar, Bioresource Technology 2011, 102, 10163-10172.
This article is protected by copyright. All rights reserved.

10.1002/anie.201712890
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
An
H2/aldehydes
Sofiene Abdellaoui,[a] Florika C. Macazo,[a] Rong
Cai,[a] Antonio L. De Lacey,[b] Marcos Pita,[b] and
Shelley D. Minteer*[a]
enzymatic fuel cell (EFC)
able to simultaneously
generate
electrical
Page No. – Page No.
energy and produce
alkanes
Enzymatic Electrosynthesis of Alkanes via
Bioelectrocatalytic Decarbonylation of Fatty
Aldehydes
This article is protected by copyright. All rights reserved.
[a]
Department of Chemistry, University of Utah, 315 S 1400 E, Salt Lake
City, UT, 84112 (USA)