Bioelectrocatalytic Conversion from N2 to Chiral Amino Acids in a H2/α-Keto Acid Enzymatic Fuel Cell

Article abstract of DOI:10.1021/jacs.9b13968

Enzymatic electrosynthesis is a promising approach to produce useful chemicals with the requirement of external electrical energy input. Enzymatic fuel cells (EFCs) are devices to convert chemical energy to electrical energy via the oxidation of fuel at the anode and usually the reduction of oxygen or peroxide at the cathode. The integration of enzymatic electrosynthesis with EFC architectures can simultaneously result in self-powered enzymatic electrosynthesis with more valuable usage of electrons to produce high-value-added chemicals. In this study, a H₂/α-keto acid EFC was developed for the conversion from chemically inert nitrogen gas to chiral amino acids, powered by H₂ oxidation. A highly efficient cathodic reaction cascade was first designed and constructed. Powered by an applied voltage, the cathode supplied enough reducing equivalents to support the NH₃ production and NADH recycling catalyzed by nitrogenase and diaphorase. The produced NH₃ and NADH were reacted in situ with leucine dehydrogenase (LeuDH) to generate l-norleucine with 2-ketohexanoic acid as the NH₃ acceptor. A 92% NH₃ conversion ratio and 87.1% Faradaic efficiency were achieved. On this basis, a H₂-powered fuel cell with hyper-thermostable hydrogenase (SHI) as the anodic catalyst was combined with the cathodic reaction cascade to form the H₂/α-keto acid EFC. After 10 h of reaction, the concentration of l-norleucine achieved 0.36 mM with >99% enantiomeric excess and 82% Faradaic efficiency. From the broad substrate scope and the high enzymatic enantioselectivity of LeuDH, the H₂/α-keto acid EFC is an energy-efficient alternative to electrochemically produce chiral amino acids for biotechnology applications.

Full text of DOI:10.1021/jacs.9b13968

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Article
Bioelectrocatalytic Conversion from N2 to Chiral
Amino Acids in a H2/#-keto Acid Enzymatic Fuel Cell
Hui Chen, Matthew B. Prater, Rong Cai, Fangyuan Dong, Hsiaonung Chen, and Shelley D. Minteer
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 04 Feb 2020
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Bioelectrocatalytic Conversion from N2 to Chiral Amino Acids in a
H2/α‐keto Acid Enzymatic Fuel Cell
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Hui Chen, Matthew B. Prater, Rong Cai, Fangyuan Dong, Hsiaonung Chen, and Shelley D.
Minteer*,†
† Department of Chemistry, University of Utah, 315 South 1400 East, RM 2020, Salt Lake City, Utah 84010, United
States
* Corresponding Author: Shelley D. Minteer (minteer@chem.utah.edu)
KEYWORDS: N2 conversion, enzymatic electrosynthesis, enzymatic fuel cell, chiral amino acid, self‐powered
ABSTRACT: Enzymatic electrosynthesis is a promising approach to produce useful chemicals with the requirement of ex‐
ternal electrical energy input. Enzymatic fuel cells (EFCs) are devices to convert chemical energy to electrical energy via the
oxidation of fuel at the anode and usually the reduction of oxygen or peroxide at the cathode. The integration of enzymatic
electrosynthesis with EFC architectures can simultaneously result in the self‐powered enzymatic electrosynthesis with more
valuable usage of electrons to produce high value‐added chemicals. In this study, a H2/α‐keto acid EFC was developed for
the conversion from chemically inert nitrogen gas to chiral amino acids powered by H2 oxidation. A highly efficient cathodic
reaction cascade was first designed and constructed. Powered by an applied voltage, the cathode supplied enough reducing
equivalents to support the NH3 production and NADH recycling catalyzed by nitrogenase and diaphorase. The produced
NH3 and NADH were in situ reacted with leucine dehydrogenase (LeuDH) to generate L‐norleucine with 2‐ketohexanoic
acid as the NH3 acceptor. 92% NH3 conversion ratio and 87.1% Faradaic efficiency were achieved. On this basis, a H2 powered
fuel cell with hyper thermostable hydrogenase (SHI) as the anodic catalyst was combined with the cathodic reaction cascade
to form the H2/α‐keto acid EFC. After 10 hours of reaction, the concentration of L‐norleucine achieved 0.36 mM with > 99%
enantiomeric excess (eep) and 82% Faradaic efficiency. From the broad substrate scope and the high enzymatic enantiose‐
lectivity of LeuDH, the H2/α‐keto acid EFC is an energy‐efficient alternative to electrochemically produce chiral amino acids
for biotechnology applications.
into the EFC architecture to form a self‐powered bioelec‐
trocatalytic system. In this system, the enzymatic electro‐
synthetic reactions in the cathode compartment consume
the electrons generated at the anode to enable the synthe‐
sis of valuable chemicals instead of low value‐added wa‐
ter.6‐7 This integration simultaneously realizes the self‐
powered enzymatic electrosynthesis without external elec‐
trical energy input and the more valuable usage of elec‐
trons to produce chemicals with high added‐value. For in‐
stance, Minteer’s group reported the bioelectrocatalytic re‐
duction of N2 to NH3 at the biocathode of a hydrogen EFC,8
as well as a H2/heptanal EFC to produce alkanes from alde‐
hydes and alcohols.9 EFCs have also been combined with
enzymatic electrosynthesis cell to realize the production of
l‐3,4‐dihydroxyphenylalanine powered by glucose oxida‐
tion.2 Besides the issue of electron utilization, another
problem which limits the application of EFCs is the poor
operational stability due to the deactivation of enzymes re‐
sulting in short lifetimes and higher costs.6 The enzyme
stability in EFCs can be significantly improved by using en‐
zyme immobilization via physical adsorption, entrapment,
covalent binding or cross‐linking. Once immobilized, en‐
zymes usually obtained extended lifetime compared to the
INTRODUCTION
Enzymatic electrosynthesis (EES) is an interdisciplinary re‐
search area that combines enzymatic catalysis with elec‐
trochemical techniques for the production of value‐added
chemicals.1 EES has been studied recently, because it com‐
bines the advantages of high activity and selectivity of en‐
zymatic catalysis and the utilization of clean and renewa‐
ble electricity as the power source of electrochemical tech‐
niques. However, most enzymatic electrosynthesis systems
require an external electrical energy input.2 Enzymatic fuel
cells (EFCs) are devices that incorporated enzymes as elec‐
trocatalysts at either the anode and/or cathode of a fuel
cell, which is further used to convert the chemical energy
from a fuel (such as glucose, formic acid, ethanol, and hy‐
drogen) into electrical energy in the presence of oxygen or
peroxide, under mild conditions (near neutral pH and tem‐
peratures from room temperature to 45oC).2‐4 The gener‐
ated electrons at the anode flow through the external elec‐
tric circuit to the cathode and are simply used to reduce
the oxidants, oxygen or peroxides in most cases, to water.5‐
6
The reactions in the cathodic compartment are only
needed to ensure the generation of current. In recent years,
enzymatic electrosynthetic reactions have been integrated
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Figure 1. The schematic representation of the H2/α‐keto acid EFC
free enzymes.10 The use of thermoenzymes or protein engi‐
neering enzymes for better stability is another easy way to
improve the operation stability of EFCs.6, 11‐13 The thermo‐
enzymes with high structural stability are able to stay sta‐
ble and remain active for a long time and finally can be
used to improve the operational stability of EFCs. Com‐
pared with immobilization, the use of thermoenzyme has
greater application potential as it does not require the
modification process of the electrode and avoids the loss of
enzyme activity. The scale of EFCs is also a factor affecting
their practical applications. Scale up of EFCs is expected to
obtain higher power density output and more target prod‐
ucts. However, there are considerable challenges to de‐
velop EFCs to be a large scale power source including the
cost of enzyme preparation, the modification of electrode
and the construction of reaction system.14
as a building block for pharmaceutical or other biotechno‐
logical chemical production. In our recent research, we es‐
tablished an upgraded bioelectrocatalytic N2 fixation sys‐
tem in which the chemically inert N2 was captured and re‐
duced by nitrogenase to produce NH3, followed by transfer
to alanine by L‐alanine dehydrogenase and finally utilized
by ω‐transaminase to produce chiral amines at mild con‐
dition without precious metal catalysts.22 Although the re‐
sult has proved that it is feasible for the product of N2 fixa‐
tion to surpass NH3, there were still three deficiencies
which need to be further improved. 1. The low utilization
ratio of generated NH3 (approximately 40%); 2. The low
faradaic efficiency (approximately 27.6%); and 3. The sys‐
tem relies on the external electrical energy input.
In order to realize the comprehensive conversion and
application of N2, the efficient in situ utilization of NH3
generated from N2 reduction is a critical step. Amino acid
dehydrogenase (AADH, EC 1.4.1.9) is a group of enzymes
Dinitrogen (N2) is the most abundant natural gas and
also the ultimate source of nitrogen for nitrogenated in‐
dustrial and natural compounds.15 However, the fixation
and conversion of N2 to useful nitrogenous compounds is
difficult due to the inertness of N2.16 So far, the fully hydro‐
+
which are able to utilize NH3/NH4 as a substrate to cata‐
lyze asymmetric reductive amination of α‐keto acids to
produce a variety of chiral amino acids with fast reaction
rates, excellent enantioselectivity, and high atom econ‐
omy.23‐27 This is interesting to the field, because chiral
amino acids are used as precursors in the food, agricul‐
tural, and pharmaceutical industries, as well as used as chi‐
ral directing auxiliaries and chiral synthons in organic syn‐
thesis.23, 26 Since the reactions catalyzed by AADH consume
reduced equivalents, the efficient supply and regeneration
of reduced cofactor is essential to ensure a smooth reac‐
tion.28 The traditional enzymatic approach for coenzyme
regeneration is to add a second enzymatic reaction which
involves a second enzyme and a second substrate. Through
the oxidation of the second substrate, the reduced cofactor
can be regenerated. In comparison to enzyme‐coupled co‐
factor regeneration, the bioelectrocatalytic approach for
cofactor regeneration uses the electrode to electrochemi‐
cal regenerate the cofactor. This method has long been
acknowledged as potentially powerful, since it directly uses
low‐cost electricity as a regenerating reagent without the
need of a second enzymatic system, the reaction process is
genated product, ammonia (NH3), is the most common
16‐17
product of N2 fixation
This NH3 product is produced
industrially by the Haber‐Bosch process which produces
the NH3 from hydrogen (H2) and N2 at the expense of 2%
of global energy use and 3% of global CO2 emission.18‐19
Minteer’s group constructed a H2/N2 EFC capable of pro‐
ducing NH3 from N2 and H2 at ambient conditions.8, 20 In
this H2/N2 EFC, the electrons generated at the hydrogenase
anode were utilized at the nitrogenase cathode to perform
the N2 reduction and NH3 production with 26.4 % Faradaic
efficiency. A noteworthy issue herein is NH3, the end‐prod‐
uct of both the Haber‐Bosch process and the H2/N2 EFC, is
a commodity chemical. The conversion of NH3 to nitroge‐
nous chemicals with high added‐value and complicated
structure still requires further tedious chemical synthesis
with the use of precious metal catalysts.21 Therefore, it is
necessary to develop a new system in which the generated
NH3 could be in situ converted by a cascade of reaction
steps to a useful intermediate chemical that could be used
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substrate and methyl viologen as the electron mediator to
easy to monitor, and the potential can be controlled to en‐
sure high Faradaic efficiency.29‐30 The bioelectrocatalytic
cofactor regeneration has been widely used in the CO2 fix‐
ation,31‐33 N2 fixation,8, 20 electrosynthesis of biofuels,9, 34 and
perform the N2 reduction. For SHI in the anodic chamber,
it can be immobilized via physical entrapment in the vio‐
logen‐polymer redox hydrogel to prevent the hydrogenase
from the O2 damage as the electrons generated from H2 ox‐
idation catalyzed by hydrogenase can induce viologen cat‐
alyzed O2 reduction at the surface of the polymer.40 How‐
ever, the target of this study is to obtain a high concentra‐
tion of amino acids with high Faradaic efficiency. The ad‐
dition of FeSII protein can only preserve 40% nitrogenase
in the presence of O2. Moreover, the existence of O2 can
also oxidize the reduced methyl viologen in both the an‐
odic and cathodic chambers, which further significantly
decrease the Faradaic efficiency. Based on the above con‐
siderations, the H2/α‐keto acid EFC was opeated under an‐
aerobic conditions.
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value‐added chemicals,2, 35‐36 especially chiral chemicals.22,
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In this study, we constructed an H2/α‐keto acid EFC to
perform the conversion from chemically inert N2 to chiral
amino acids without an external electrical energy input.
The electrons derived from the oxidation of H2 were uti‐
lized to support the production of chiral amino acids in‐
stead of low value‐added H2O. The reaction cascade in the
cathode chamber contained a nitrogenase (EC 1.18.6.1)
from Azotobacter vinelandii, a diaphorase (DI, EC1.6.99.3)
from Geobacillus stearothermophilus, and a L‐leucine de‐
hydrogenase (LeuDH, EC 1.4.1.9) from Bacillus cereus to
perform the conversion from N2 to a chiral amino acid. Me‐
thyl viologen (MV) was employed to mediate both the elec‐
tron transfer between the carbon cathode and nitrogenase
and the active site of diaphorase to realize the regeneration
of NH3 and NADH. The generated NH3 and NADH were in
situ utilized by LeuDH to perform the asymmetric amina‐
tion of a variety of α‐keto acid substrates and the genera‐
tion of chiral amino acids. Finally, by employing a soluble
[NiFe] hydrogenase I (SHI, EC1.12.1.3) from the hyperther‐
mophilic archaeon, Pyrococcus furiosus, at the bioanode
and a N2 conversion cascade at the biocathode, we success‐
fully constructed an H2/α‐keto acid EFC which was able to
efficiently utilize the electrons generated from H2 oxida‐
tion to realize the self‐powered bioelectrocatalytic N2 con‐
version with chiral amino acids as the final product.
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In order to address the problems of low NH3 utilization
ratio (approximately 40%) and low Faradaic efficiency (ap‐
proximately 27.6%) of the previous upgraded N2 fixation
system,22 the reaction and transfer pathway of NH3 in the
cathodic chamber was simplified to improve the transfer
and capture efficiency. In detail, the reduced MV.+ gener‐
ated at the cathode was employed as the electron donor for
the N2 reduction and NH3 generation. Then, the generated
NH3 was captured and in situ utilized by LeuDH to produce
L‐norleucine and a variety of other chiral amino acids with
different α‐keto acids as the NH3 acceptor. Meanwhile, the
consumed NADH could be regenerated by DI and reduced
MV.+. In comparison to the previous upgraded N2 fixation
system,22 the further transformation process of generated
amino acid, such as the reactions catalyzed by ω‐transam‐
inase with amino acids as amino group donor, was not in‐
tegrated into the current system to simplify the transfer
path of NH3 and further improve the conversion efficiency
from NH3 to final products. On the basis of the highly ef‐
ficient cathodic reaction cascade, the hydrogenase (SHI)
anode was integrated into the system to form the H2/α‐
keto acid EFC with H2 as fuel. The electrons generated from
H2 oxidation were transported to the anode via reduced
MV.+ and finally moved to the cathode to support the reac‐
tions occurring in the cathodic chamber.
RESULTS AND DISCUSSION
Design of the biocathode and EFC. As shown in Fig‐
ure 1, a U‐shaped dual‐chamber electrochemical cell was
used and separated by a Nafion proton exchange mem‐
brane (PEM). The protons generated from oxidation of H2
catalyzed by SHI in the anodic chamber passed through
Nafion into the cathodic chamber. The electrons moved
from the anode to cathode and reduced the electron medi‐
ator, MV2+, at the surface of the cathode. The reduced MV.+
was further utilized as reducing power for the reduction of
N2 catalyzed by nitrogenase and the regeneration of NADH
catalyzed by DI. Due to the oxygen sensitivity of MV.‐, SHI,
and nitrogenase, a H2/α‐keto acid EFC requires anaerobic
operation condition which can be easily constructed. In ac‐
tual use, the ultra‐high purity H2 and N2 need to be filled
into the headspace of the anodic and cathodic chamber as
they are the substrates of hydrogenase and nitrogenase, re‐
spectively. While filling ultra‐high purity H2 and N2, anaer‐
obic conditions can be obtained. Meanwhile, it is also pos‐
sible for nitrogenase to catalyze N2 reduction with air as
the substrate. Previous research indicated that a “confor‐
mational switch” protein (FeSII or “Shethna”) was able to
protect nitrogenase upon exposure to O2. Upon exposure
to air in the presence of FeSII protein, 40% N2 reduction
activity can be preserved.39 If the FeSII protein is supple‐
mented into the cathodic chamber of the H2/α‐keto acid
EFC, the nitrogenase would also be able to use air as the
It is important to note that the same mediator is being
used in both the anodic and cathodic chambers for sim‐
plicity. This means there is no standard potential differ‐
ence between the two chambers of the EFC, but the poten‐
tial of the MV2+/MV.+ redox couple at each electrode
changes upon a shift in the equilibrium between MV2+ and
MV.+ as per the Nernst equation owing to their respective
enzymatic activities. The theoretical maximum open cir‐
cuit voltage (OCV) for this system is 250 mV.8
Optimization of the nitrogenase concentration. As
NH3 is the substrate of LeuDH, a higher concentration of
NH3 is beneficial to the production and accumulation of
chiral amino acids. In order to obtain high NH3 concentra‐
tions, the concentration of nitrogenase was first optimized.
As shown in Figure 2, the highest accumulation concen‐
tration of NH3 depended on the concentration of nitrogen‐
ase. Increasing the nitrogenase concentration from 0.125
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U/mL (0.2 mg/mL MoFe protein and 0.9 mg/mL Fe pro‐
utilized by DI and finally was transferred to NAD+ to real‐
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tein) to 0.250 U/mL (0.4 mg/mL MoFe protein and 1.8
mg/mL Fe protein) can make the final concentration of
NH3 increase from 1.3 mM to 2.1 mM. Further increase in
nitrogenase concentration to 0.375 U/mL (0.6 mg/mL
MoFe protein and 2.7 mg/mL Fe protein) led to the reduc‐
tion of final NH3 concentration (1.4 mM). Some previous
studies also indicated that with the total protein concen‐
tration of nitrogenase increased (with the fixed MoFe pro‐
tein/Fe protein molar ratio), the specific activity of nitro‐
genase decreased.41‐42 This inhibition is due to the mass‐ac‐
tion effect on the equilibrium between (MgADP)2 bound
oxidized Fe protein and reduced MoFe protein. The disso‐
ciation of the complex (oxidized Fe protein)‐(MgADP)2‐
(reduced MoFe protein) is rate‐limiting at high protein
concentrations. The high total concentration of nitrogen‐
ase (0.6 mg/mL MoFe protein and 2.7 mg/mL Fe protein,
3.3 mg/mL total protein) is not conducive to the dissocia‐
tion of oxidized Fe protein from the reduced MoFe protein
and the re‐association of the complex of reduced Fe pro‐
tein and oxidized MoFe protein. Consequently, 0.25 U/mL
nitrogenase (0.4 mg/mL MoFe protein coupled with Fe
protein in a 1:16 mol/mol ratio) was the optimum concen‐
tration for NH3 production.
ize the recycling of NADH. To maintain a high current den‐
sity for high conversion rates, the working electrode poten‐
tial was set at ‐0.85 V vs. SCE for all other bulk electrolysis
experiments.
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Figure 3. Amperometric i‐t curve trace of the cathodic reac‐
tion. The MV2+ (1 mM) mediated N2 reduction with and with‐
out nitrogenase was sequentially supplemented with compo‐
nents of NADH recycling (1st injection) and L‐norleucine
generation (2nd injection). In the 1st injection, 0.25 U/mL DI
and 200 µM NAD+ were added. In the 2nd injection, 2.5 mM 2‐
ketohexanoic acid (final concentration) and 0.45 U/mL
LeuDH (final concentration) were added to the reaction mix‐
ture.
After determining the working electrode potential, the
bulk electrolysis analysis was employed to trace the reac‐
tion process and further analyze the feasibility of the sys‐
tem design in this research (Figure 3). In the current ver‐
sus time curve, there are three processes of interest that are
separated by two injections. The first portion of the curve
is the N2 reduction and NH3 generation catalyzed by nitro‐
genase with 1 mM MV2+ as the electron mediator. For the
reaction without nitrogenase, only the MV2+ was reduced,
since no NH3 could be generated. After 5 hours of reaction,
the system achieved steady‐state and the current stabi‐
lized. Upon the addition of 0.25 U/mL DI and 200 µM
NAD+ to the electrolyte (1st injection), both the reaction
with and without nitrogenase exhibited a dramatic in‐
crease in the reductive current. This current response re‐
flects the reduction of NAD+ catalyzed by DI with the re‐
duced MV.+ as the electron donor. Upon the addition of 2.5
mM 2‐ketohexanoic acid (final concentration) and 0.45
U/mL LeuDH (apparent activity, final concentration) (2nd
injection), only the reaction with nitrogenase exhibited a
statistically significant current response. This current re‐
sponse was probably the reflection of the formation of the
final product, L‐norleucine. The enzymatic consumption
of NH3, reduced MV.+ and regenerated NADH further ac‐
celerated the electron transfer from cathode to electrolyte
and resulted in an enhanced current response. For the re‐
action without nitrogenase, no L‐norleucine could be gen‐
erated as there was no NH3 in the electrolyte. Therefore,
the current response could not be observed. The genera‐
tion of L‐norleucine in the reaction with nitrogenase was
Figure 2. Optimization of nitrogenase concentration based on
the concentration of generated NH3. The data shown repre‐
sent the means with standard deviations from triplicate exper‐
iments.
Potentiostatic electrolysis analysis of the reaction
process. First, cyclic voltammetric (CV) analysis was used
to show that MV2+ can act as the electron mediator be‐
tween the cathode and both nitrogenase and DI (Figure
S2). The CV of 100 µM MV.+ indicated that the oxidized
MV2+ could be reduced to MV.+ at a potential of ‐0.75 V vs
SCE. After the addition of 0.15 U/mL nitrogenase (MoFe/Fe
protein ratio of 1:16), a current response was observed due
to the turnover of nitrogenase at ‐0.75 V vs SCE. This cur‐
rent response demonstrated the electron transfer between
the cathode and nitrogenase was mediated by MV2+. After
further adding 0.15 U/mL DI and 100 µM NAD+, a larger
current response was obtained at the same potential. This
result demonstrated that the electrons mediated by MV2+,
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confirmed by UPLC‐MS and GC‐MS (Figure S3 and Figure equilibrium to the side of amine product. A low concentra‐
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S4).
tion of pyruvate is conducive to the generation of the
amine. In the previous research, the concentration of py‐
ruvate was only 50 µM, which was well below the Km value
of L‐alanine dehydrogenase towards pyruvate (880 µM).44
In this study, the reaction catalyzed by ω‐transaminase has
not been integrated into the reaction system to simplify the
transfer path of NH3. More importantly, the concentration
of NH3 acceptor, 2‐ketohexanoic acid, was 2.5 mM, which
was higher than the Km value of LeuDH towards 2‐ketohex‐
anoic acid (1.2 mM).45 The generated NH3 was more effi‐
ciently captured and utilized. Finally, the concentration of
product (1.93 mM) was approximately 3.4 times higher
than that of previous research (0.56 mM).
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EFC performance. The soluble [NiFe] hydrogenase I
(SHI, EC1.12.1.3) from the hyperthermophilic archaeon, Py‐
rococcus furiosus, is a hyper thermostable hydrogenase
which can stay stable and continuously work at 70oC, or
even 80oC, for dozens of hours.46‐47 In addition to hydrogen
production, the reversible nature of the SHI reaction also
allows it to perform the oxidation of hydrogen and the re‐
duction of the electron mediator.48 These properties make
SHI is a good candidate to construct a H2 fuel cell. Since it
was the first time for SHI to be used in an EFC, it is neces‐
sary to determine its electrochemical properties and eval‐
uate the performance of the newly constructed EFC. From
the CV in Figure 5a, the decreased reductive current re‐
sponse at ‐0.73 V vs SCE and the increased oxidative cur‐
rent response at ‐0.65 V vs SCE indicated the oxidative
MV2+ was reduced to MV.+ by SHI via the oxidation of H2.
This result in comparison to the control with BSA and the
blank without hydrogen provide evidence of mediated bi‐
oelectrocatalysis. Figure 5b further demonstrated the elec‐
trochemical oxidation of reduced MV.+ that is produced by
SHI via H2 oxidation by using potentiostatic bulk electrol‐
ysis analysis. After the bubbling of H2, the oxidative MV2+
was reduced by SHI with H2 as an electron donor. Then,
the reduced MV.+ was oxidized at the working electrode to
produce the oxidative current. Figure 5C shows the evolu‐
tion of open‐circuit voltage (OCV) for the H2/α‐keto acid
EFC, where the anodic chamber contained 10 mM oxidized
MV2+ and 100% H2 0.1 mg/mL SHI was injected into anodic
chamber at 300s The cathodic chamber contained 0.25
U/mL nitrogenase, 0.25 U/mL DI, 0.45 U/mL LeuDH (ap‐
parent activity), 1 mM reduced MV.+, 2.5 mM 2‐ketohexa‐
noic acid, and 100% N2. MgCl2 (6.7 mM) and NAD+ (200
µM) were added into the cathodic chamber at 1500s to ini‐
tiate nitrogenase and DI turnover, as it is required for bio‐
catalytic function. After allowing the OCV to evolve for
2500s, the OCV stabilized at approximately 248 ± 6 mV
which was close to the theoretical maximum potential.8 Af‐
ter the determination of the OCV, the performance of the
EFC was evaluated by slow linear polarization (0.5 mV/s)
from OCV to short circuit (Figure 5d). The maximum cur‐
rent and power densities were 42 ± 3 µA/cm2 and 1.45 ± 0.18
µW/cm2. This result also proves that SHI can be used to
construct EFC. When the EFC had a maximum power out‐
put (1.45 µW/cm2), the corresponding potential is 75 mV.
Figure 4. The concentration of L‐norleucine generated in the
bioelectrocatalytic N2 conversion system powered by an
applied voltage (‐0.85 V vs SCE) and corresponding faradaic
efficiency during the 12 hours time course. The concentrations
represent the means from three separate individual U‐shaped
dual‐chamber electrochemical cells. For every single cell, the
concentrations were obtained based on triplicate amino acid
derivatization.
Bioelectrocatalytic L‐norleucine production pow‐
ered by an applied voltage. In order to evaluate the effi‐
ciency of the reaction cascade in the cathodic chamber, the
bioelectrocatalytic L‐norleucine production powered by a
potentiostat at ‐0.85 V vs SCE with 2.5 mM 2‐ketohexanoic
acid as substrate was performed. As shown in Figure 4, the
concentration of generated L‐norleucine achieved the
highest level, 1.93 mM, after 10 hours of reaction. At this
product concentration, the utilization ratio of NH3 was up
to 92% (the highest accumulation concentration is 2.1 mM,
see Figure 2), which is approximately 2.3 times higher than
that in our previous research (~ 40% NH3 utilization ra‐
tio).22 The highest Faradaic efficiency (87.1%) was observed
with the 8 hour run. In comparison with our previous re‐
search (27.6% Faradaic efficiency),22 the faradaic efficiency
was increased approximately 3.4 times. Moreover, the Fa‐
radic efficiency of this study could be stably maintained
above 75% from the 2nd hour to the 12th hour. The above
results indicated that the generated NH3 and electrons
were more efficiently utilized and the performance of the
cathodic reaction cascade was significantly improved in
comparison with the previous research. In the previous re‐
search,22 the generated NH3 was transferred to pyruvate by
L‐alanine dehydrogenase to produce L‐alanine. Then, the
generated L‐alanine utilized by ω‐transaminase as an
amino group donor to produce a chiral amine and py‐
ruvate. For the reaction catalyzed by ω‐transaminase, the
equilibrium is on the side of the substrate (ketone and ala‐
nine) but not on the side of the products (amine and py‐
ruvate).43 The co‐product, pyruvate, cannot be accumu‐
lated and needs to be promptly converted to alanine with
the consumption of generated NH3 to push the reaction
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Consequently, the working potential of the EFC was held the power density output of the H2/α‐keto acid EFC. As a
result, the concentration of reduced MV.‐ in the cathodic
chamber was not high enough to fully support the N2 re‐
duction catalyzed by nitrogenase and the NADH regener‐
ation catalyzed by DI. That was the reason why the final L‐
norleucine concentration of the reaction powered by the
H2/α‐keto acid EFC was lower than that of reaction pow‐
ered by the potentiostat. One the other hand, the MV2+
with low reductive potential (‐0.75V vs SCE) was consid‐
ered to be the best electron mediator of nitrogenase for the
N2 reduction.49 It is difficult to find another electron medi‐
ator with lower reductive potential used in the anodic
chamber to construct the H2/α‐keto acid EFC and generate
high OCV. Although the use of the same mediator in both
the anodic and cathodic chamber only generated 250 mV
OCV, it still successfully drove the H2/α‐keto acid EFC and
generate L‐norleucine with high Faradaic efficiency. Fu‐
ture work could explore other mediator systems to increase
produced potential for improved efficiency.
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Figure 5. The performance of H2/α‐keto acid EFC. (a) Repre‐
sentative CVs of the oxidation of H2 catalyzed by SHI with ox‐
idized MV2+ as the electron acceptor. (b) Bulk electrolysis cur‐
rent versus time curve for the MV2+ mediated oxidation of H2
by SHI. (c) Representative OCV stabilization of the H2/α‐keto
acid EFC. 0.1 mg/mL SHI was added into the anodic chamber
at 300 s (1st injection). 6.7 mM MgCl2 and 200 µM NAD+ were
added into the cathodic chamber at 1500 s to initiate nitrogen‐
ase and DI turnover (2nd injection). (d) Representative polari‐
zation and power curves of the H2/α‐keto acid EFC.
Fuel cell‐powered bioelectrosynthetic L‐norleucine
production. After getting the highly efficient cathodic re‐
action cascade, the H2 fuel cell was integrated into the bi‐
oelectrocatalytic system to construct the H2/α‐keto acid
EFC which was further used to perform the bioelectrocat‐
alytic conversion from N2 to L‐norleucine. As shown in Fig‐
ure 6, the L‐norleucine concentration of the reaction pow‐
ered by the EFC achieved the highest level, 0.36 mM, after
10 hours of reaction. Although the concentration of L‐nor‐
leucine of the reaction powered by EFC was decreased
compared with that of reaction powered by an applied volt‐
age, the Faradaic efficiency still remained at a high level.
The Faradaic efficiency of the reaction powered by the EFC
was maintained at approximately 82% from the 2nd hour to
the 6th hour. After 6th hour, the Faradaic efficiency exhib‐
ited a downward trend. When the reaction was over, the
Faradaic efficiency was 64% which was still 2.5 times
higher than the highest Faradaic efficiency (27.6%) of the
previous research.22 The above results indicate that the
H2/α‐keto acid EFC is able to work without external elec‐
trical energy input. The electrons generated from the oxi‐
dation of H2 were utilized by the cathodic reaction cascade
for the conversion from chemically inert N2 to L‐norleucine
with high Faradaic efficiency. As the same electron media‐
tor is being used in both the anodic and cathodic chamber,
the limited maximum OCV (250 mV, Figure 5c) limited
Figure 6. The concentration of L‐norleucine generated in the
H2/α‐keto acid EFC and corresponding faradaic efficiency at
12 hour time course. The concentrations represent the means
from three separate individual U‐shaped dual‐chamber elec‐
trochemical cells. For every single cell, the concentrations
were obtained based on triplicate amino acid derivatization.
Bioelectrosynthetic reductive amination of differ‐
ent α‐keto acid substrates. LeuDH shows activity toward
straight and branched chain α‐keto acids as well as some
alicyclic α‐keto acids.50 From the wide substrate scope of
LeuDH, the H2/α‐keto acid EFC is able to produce a variety
of chiral amino acids. Herein, 6 prochiral α‐keto acids with
different structures (Figure 7) were employed as a sub‐
strate in the H2/α‐keto acid EFC to produce corresponding
chiral amino acids (Figure S5). After 10 hours of electroly‐
sis, most α‐keto acid substrates were converted to the cor‐
responding amino acids (Table 1). The concentration of
produced L‐norleucine (1b, 0.36 mM), L‐norvaline (2b, 0.38
mM), L‐valine (3b, 0.4 mM), and L‐tert‐leucine (4b, 0.4
mM) were almost at the same level. The concentration of
the generated L‐cyclopropyl glycine (6b) was 0.28 mM,
which was lower than the concentration of other products.
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Figure 7. Prochiral α‐keto acid as substrates of H2/α‐keto acid EFC used to produce chiral amino acids
Table 1. Asymmetric amination of various α‐keto acid by the H2/α‐keto acid EFCa
substrate
product
product concentration (mM)
eep (%)b
yield rate (µmol∙L‐1∙h‐1)
1a
2a
3a
4a
5a
6a
1b
2b
3b
4b
5b
6b
0.36 ± 0.036
0.38 ± 0.014
0.40 ± 0.016
0.40 ± 0.022
n.d.c
>99 (L)
>99 (L)
95.2 (L)
>99 (L)
n.d.c
36
38
40
40
n.d.c
0.28 ± 0.012
>99 (L)
28
[a] Values represent the means from three separate individual U‐shaped dual‐chamber electrochemical cells. For every single cell,
the values were obtained based on triplicate amino acid derivatization.
[b] In [%]. Determined by SFC after derivatization.
[c] n.d. Not determined because of too low a conversion
The amination product of substrate 5a (2‐keto‐4‐methyl‐
thiobutyric acid) was not determined due to too low con‐
centration. Previous research indicated that the reduced
amination activity of LeuDH from Bacillus cereus towards
2‐keto‐4‐methylthiobutyric acid was much lower than that
towards regular aliphatic α‐keto acids.51 As the crystal
structure of LeuDH from Bacillus cereus has not been ob‐
tained, it can only be speculated that the larger atomic ra‐
dius and stronger polarity of the sulfur atom compared
with carbon atom cause the 2‐keto‐4‐methylthiobutyric
acid cannot be well‐positioned in the active pocket of
LeuDH and finally lead to the low activity of LeuDH to‐
wards 2‐keto‐4‐methylthiobutyric acid. Additionally, all of
the generated products had excellent optical purity (Ta‐
ble1 and SFC result in supporting information). Except for
the generated L‐valine (3b, eep = 95.2%), the eep value of all
other products were > 99%. The obtained chiral amino ac‐
ids are important intermediates of various high value‐
added chemicals, especially pharmaceutical intermediates.
The hydroxylated product of L‐norleucine, L‐6‐hydroxy‐
norleucine, is a chiral intermediate for the production of
omapatrilat, indospicines, siderophores, and peptide hor‐
mone analogs.50, 52 L‐norvaline is a vital intermediate in the
chemical synthesis of an antihypertensive drug, Perin‐
dopril.53 L‐valine is an essential amino acid for verte‐
brates.54 L‐tert‐leucine is a valuable precursor for a variety
of active pharmaceuticals, such as hepatitis C antiviral NS1
protease inhibitors (telaprevir, bocepravir), NS3/4 protease
inhibitors (asunaprevir, faldaprevir, and vedroprevir), and
HIV protease inhibitor (atazanavir).50 L‐cyclopropylglycine
is an intermediate for the synthesis of a corticotropin‐re‐
leasing factor‐1 receptor antagonist.55
It can be expected that the bioelectrocatalytic N2 conver‐
sion system can act as a platform to produce a variety of
chiral amino acids with the integration of different
AADHs. So far, 26 different subclasses for AADHs are listed
in the Enzyme Nomenclature database. More than 24,770
amino acid sequences of AADHs are stored in the Uniport
database, and 521 structures of AADHs are available in the
Protein Data Bank. A few AADHs, such as glutamate dehy‐
drogenase, phenylalanine dehydrogenase, meso‐dia‐
minopimelate dehydrogenase and leucine dehydrogenase
have good potential as industrial biocatalysts for the reduc‐
tive amination process.50 Building on these research ad‐
vances in AADHs, the H2/α‐keto acid EFC has an attractive
possibility for the production of chiral amino acid with
wide product scope. Compared with the traditional biocat‐
alytic chiral amino acids production, the chiral amino acids
in a H2/α‐keto acid EFC is a proof of concept research as
some amino acids have been successfully produced on an
industrial scale by using traditional biocatalytic methods.50
However, the H2/α‐keto acid EFC exhibited an attractive
possibility that the chemically inert N2 could directly be
converted to high value‐added chiral amino acids via one‐
pot multi‐enzymatic bioelectrosynthesis. Compared with
regular EFC, the electrons were more valuable used instead
of simply being reduced to water. The electrons generated
from H2 oxidation were utilized to realize the reduction of
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(#W911NF1410263) and the National Science Foundation Cen‐
ter for Synthetic Organic Electrosynthesis for funding (CHE‐
1740656).
N2 and reductive amination α‐keto acids to realize the pro‐
duction of chiral amino acids without the external electri‐
cal energy input or the addition of sacrificial agents and
extra dehydrogenase for the regeneration of reduced
equivalent. This H2/α‐keto acid EFC has the potential to
realize the green and distributed manufacturing of chiral
amino acids.
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CONCLUSION
9
In this research, we demonstrated the feasibility of using
a H2/α‐keto acid EFC to perform the bioelectrocatalytic
conversion from chemically inert N2 to chiral amino acids
with high Faradaic efficiency. The hyper thermostable hy‐
drogenase, SHI, was shown for the first time to be applica‐
ble in the construction of a H2 fuel cell. In this H2/α‐keto
acid EFC, the electrons generated from oxidation of H2
were utilized to support the N2 conversion. Moreover, the
product of N2 conversion went beyond ammonia and
reached chiral amino acids at mild conditions without the
requirement of precious metal catalysts or an external elec‐
trical energy input. Based on the solid research foundation
of AADHs, the H2/α‐keto acid EFC is able to act as a plat‐
form to prepare a variety of amino acids with the integra‐
tion of different AADHs.
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ASSOCIATED CONTENT
Supporting Information. The supporting information is
available free of charge via the Internet at
http://pubs.acs.org.”
The DNA and amino acid sequences of the proteins, material
and methods, the SDS‐PAGE gel of enzymes used in this
study, representative cyclic voltammograms of the nitrogen‐
ase bioelectrocatalysis mediated by MV2+, the UPLC‐MS char‐
acterization of generated L‐norleucine, the GC‐MS character‐
ization of generated L‐norleucine, and the SFC analysis of the
enaniomeric excess analysis of the generated amino acids in
this study (PDF).
AUTHOR INFORMATION
Corresponding Author
* Shelley D. Minteer: minteer@chem.utah.edu
Present Addresses
ǂ R.C.: Department of Chemistry, University of California,
Berkeley, California 94720, USA
ORDID
Hui Chen: 0000‐0002‐8944‐0090
Shelley D. Minteer: 0000‐0002‐5788‐2249
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Prof. Chun You (Tianjin Institute of Industrial Bio‐
technology, Chinese Academy of Science, China) and Prof.
Thomas J. Santangelo (Department of Biochemistry and Mo‐
lecular Biology, Colorado State University, USA) for the sup‐
plying pTE5 plasmid and Thermococcus kodakarensis KOD1
strain for the preparation of hydrogenase SHI. This study was
supported by the Army Research Office MURI grant
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