Carbon as a Simple Support for Redox Biocatalysis in Continuous Flow

Article abstract of DOI:10.1021/acs.oprd.9b00410

A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also considered as optimization targets, such as low E factors between 2.5-11 and space-time yields of up to 22.9 g L-1 h-1. The long-term stability of the biocatalytic reactor was also demonstrated, with full conversion maintained over more than 30 h of continuous operation.

Full text of DOI:10.1021/acs.oprd.9b00410

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Carbon as a Simple Support for Redox Biocatalysis in Continuous
Flow
Barnabas Poznansky,§ Lisa A. Thompson,§ Sarah A. Warren, Holly A. Reeve,* and Kylie A. Vincent*
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ABSTRACT: A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple
carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by
co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also
considered as optimization targets, such as low E factors between 2.5−11 and space-time yields of up to 22.9 g L−1 h−1. The long-
term stability of the biocatalytic reactor was also demonstrated, with full conversion maintained over more than 30 h of continuous
operation.
KEYWORDS: biocatalysis, flow chemistry, cofactor recycling, immobilized enzyme, packed bed
and can cause a significant loss of activity compared with the
free enzyme in solution.
INTRODUCTION
■
The push toward greener, more efficient methods for chemical
production is currently a major focus of the pharmaceutical
and fine-chemical industries, driven by economic, environ-
mental, and regulatory factors.1−4 Biocatalysts are known for
their excellent selectivity (removing the need for protection/
deprotection steps) under mild reaction conditions and are
widely seen as a greener alternative to traditional chemical
synthesis methods,5,6 particularly for chiral products. Protein
evolution techniques are commonly used to expand the
substrate scope and improve the stability of biocatalysts in
order to increase their industrial viability.
Many useful redox enzymes are dependent on nicotinamide
adenine dinucleotide cofactors (NAD+/NADH or NADP+/
NADPH) for operation. The reduced forms, NAD(P)H,
function as hydride donors to the large family of alcohol
dehydrogenases (ketoreductases), which are well-established
for the introduction of chiral alcohol moieties in pharmaceut-
ical synthesis,7 as well as imine reductases for the synthesis of
chiral amines8 and ene reductases for CC bond reductions.9
The high cost of these cofactors means that cofactor recycling
systems are required. The most commonly used systems
employ additional enzymes (glucose/formate/alcohol dehy-
drogenases) and sacrificial reagents (glucose/formate/isopro-
panol, respectively), which generate superstoichiometric
quantities of carbon-based waste and lead to the requirement
for continuous pH monitoring and adjustment in batch
reactions.
Flow chemistry is another powerful technique for greener,
more efficient chemical production that has gained in
importance in recent years.17−19 Flow reactors can offer
improved heat and mass transfer and a smaller footprint
compared with batch reactors for the same product yield. Flow
chemistry also makes it easier to design and optimize scalable
reaction methods and intensify reaction conditions, opening up
a wider chemical space than is possible in batch reactions.20,21
Immobilization of biocatalysts makes them amenable to use in
continuous flow packed bed reactors, wherein the solid catalyst
is contained within a tubular reactor and the reaction solution
is continuously pumped through it. The high ratio of catalyst
to substrate achievable in packed bed reactors also often
enables shorter reaction times and increased yields. Addition-
ally, the constant removal of reaction solution can help to
overcome substrate or product inhibition of the enzyme, which
can be a common problem. Consequently, the number of
examples of supported biocatalysis in flow is increasing.22,23
Existing examples of cofactor-dependent redox biocatalysis
in flow often employ the cofactor recycling enzymes in
solution,24,25 contributing to increased waste and cost for the
process. However, there are a number of reports in which the
enzyme responsible for the biotransformation and the cofactor
recycling enzyme have been coimmobilized in flow reac-
tors.15,16,26−29 Common problems include significant loss of
activity, complex immobilization strategies, and low productiv-
ities.
Immobilization of biocatalysts can lead to improved stability
and increase enzyme lifetimes.10−12 Recent advances in
enzyme immobilization have exploited techniques such as
HaloTag13 and histidine tagging of proteins as well as the use
of a range of support materials, including resins,14 agarose,15
and microbeads.16 However, many of these enzyme immobi-
lization techniques require expensive supports, modification of
the support and/or enzyme, or lengthy immobilization times
Special Issue: Flow Chemistry Enabling Efficient
Synthesis
Received: September 24, 2019
Published: January 31, 2020
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© XXXX American Chemical Society
A

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Communication
Here we demonstrate heterogeneous biocatalytic ketone
reduction with in situ biocatalytic cofactor recycling using a
dual-enzyme system supported on simple carbon particles in a
continuous packed bed flow reactor. We chose carbon as a
cheap and commercially available support, facilitating simple
one-step enzyme immobilization via adsorption.30 There are a
limited number of examples of the use of carbon as a support
for biocatalysis, although it has been demonstrated as an
electronic linker between enzymes for oxidation and reduction
reactions using a range of carbon materials.31−33 Carbon is the
standard support for heterogeneous metal-nanoparticle catal-
ysis in both batch and flow reactors. The use of similar carbon
supports could offer a route to implementing biocatalysis in
industry-standard flow reactors.
CO2 using formate dehydrogenase (FDH), an enzyme that has
been used extensively for NADH recycling, for example, in
biocatalytic synthesis of pharmaceutical molecules.37 Existing
examples of FDH cofactor recycling in flow mainly employ
FDH in solution.24 The few examples of immobilized FDH in
flow suffer from lengthy immobilizations, the use of non-
commercial support materials, or the requirement of directed
evolution of FDH for sufficient immobilization.38,39 Here we
investigate simple one-step immobilization of FDH and LDH
on carbon particles for application in a packed bed reactor.
The heterogeneous biocatalyst was prepared by co-
immobilization of the two enzymes on Black Pearls 2000
particles via simple adsorption at 4 °C with no further
modification of the carbon support or the enzymes. After 4 h
the enzyme-modified particles were collected by centrifugation
and removal of the supernatant solution containing any
unadsorbed enzyme.
The enzyme immobilization efficiency and activity on the
carbon support were determined in batch for the individual
enzymes (see the Supporting Information (SI)). For LDH,
>99% immobilization on the carbon particles was achieved
with an activity of 19 units mg−1 in a heterogeneous batch
reaction. For FDH, 82% immobilization on the carbon
particles was achieved with an activity of 0.015 units mg−1 in
a heterogeneous batch reaction.
RESULTS AND DISCUSSION
■
In this study we chose Black Pearls 2000 particles (Cabot
Corporation) as the enzyme support material for the
continuous flow process. This is an amorphous carbon black
material with particles on the nanometer scale that agglomerate
to form larger aggregates with high nanoscale porosity.34 They
present a very high surface area (>1000 m2 g−1) for enzyme
adsorption, and we have found that this carbon type functions
well as a support material for many different enzymes under
batch conditions.33,35,36
For the flow reactor setup (Scheme 1B), biocatalyst particles
were mixed with glass beads (1 mm) and manually packed into
an Omnifit glass column (6.6 mm i.d., variable bed length).
The enzyme-modified particles were mixed with glass beads to
increase the volume of the solid support in the reactor and
therefore allow a larger reactor column to be used. This was
necessary to achieve the desired residence time (tRes) of 10−
30 min within the flow rate capability of the available pumps.
Additionally, the use of a matrix material such as glass beads
mixed with a heterogeneous catalyst in packed bed reactors is a
common method to reduce the pressure drop across the
reactor bed, particularly when very small catalyst particles are
used. The reaction solution was pumped through the column
using a continuous syringe pump (Asia, Syrris), and the
components were connected using PTFE tubing (0.5 mm i.d.)
The conversion of pyruvate to lactate using NADH-
dependent lactate dehydrogenase (LDH) was chosen as the
test reaction (Scheme 1A). Cofactor regeneration was
provided by the NAD+-dependent conversion of formate to
Scheme 1. (A) NADH-Dependent Biocatalytic Conversion
a
of Pyruvate to Lactate; (B) Flow Reactor Setup
1
and standard /4-28 PTFE flangeless fittings. The reaction
solution was collected at the reactor outlet using a BioRad
model 2110 fraction collector.
Initially the effect of a flow stream on the enzyme
immobilization was determined by pumping Tris-HCl buffer
(50 mM, pH 8.0) through the packed column and monitoring
the outlet solution by UV−vis spectroscopy for the presence of
leached enzyme (see the SI for calibration curves and leaching
data). Only 0.2% leaching of LDH was observed over 6 h,
demonstrating good retention of this enzyme on the carbon
support. This is particularly significant because no chemical
transformation or enzyme engineering was required for
immobilization, only a simple adsorption procedure. For
FDH, a higher rate of leaching was observed: 6% leaching
over 6 h and a total of 10% over 28 h. In all of the reactions,
the packed column was flushed with buffer prior to the start of
the reaction in order to ensure that this initial amount of
leached material was removed before the start of the reaction.
The packed bed reactor was tested for the conversion of
pyruvate to lactate with in situ cofactor recycling. The reactor
was set up with a single liquid feed containing different
concentrations of sodium pyruvate, NAD+, and sodium
formate in Tris-HCl buffer (50 mM, pH 8.0). For each
a
Legend for flow reactor setup: (i) reaction solution vessel; (ii)
syringe pump; (iii) packed bed reactor; (iv) fraction collector; LDH =
lactate dehydrogenase; FDH = formate dehydrogenase; ● = enzyme-
modified carbon particle; ○ = glass bead.
B
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Figure 1. Effect of varying (A) tRes, (B) the NAD+ mol %, (C) the number of equivalents of formate, and (D) the Tris-HCl buffer concentration
on the conversion of pyruvate to lactate in the continuous packed bed reactor. Unless otherwise stated, all experiments were performed with 50
mM pyruvate, 5 equiv of formate, 1 mol % NAD+, 50 mM Tris-HCl buffer (pH 8.0), 3 mg of LDH, 30 mg of FDH, and 30 mg of carbon at room
temperature. Symbols: green circles, tRes = 10 min; blue squares, tRes = 20 min; red triangles, tRes = 30 min.
experiment, the reactor was run until a steady state was
amount of NAD+ from 0.01 to 1 mol % led to an increase in
conversion up to full conversion at the highest cofactor loading
at a tRes of 30 min (Figure 1B). At 0.5 mol % NAD+, 88%
conversion was achieved, corresponding to a cofactor turnover
number of 176. Increasing the number of equivalents of
formate from 1 to 2.5 had little effect on the conversion
(approximately 70%), but more than 90% conversion was
achieved with 3 and 5 equiv (Figure 1C) at a tRes of 20 min.
This dependence appeared to be consistent across all of the
different tRes tested. Lowering the buffer concentration from
50 to 12.5 mM (pH 8.0) and then using unbuffered H2O had a
negligible effect on the outcome of the reaction (Figure 1D).
Being able to run the reaction in pure H2O may overcome any
enzyme compatibility issues with pH or buffer and would
reduce waste and simplify the process. From these studies, the
operating conditions to achieve optimum conversion were tRes
= 30 min, 1 mol % NAD+, and 3 equiv of formate.
The results in Figure 1 detail optimization of the system for
conversion. When additional responses such as productivity
(space-time yield, STY) and waste (measured as the E factor)
are considered,40 more complete process optimization can be
achieved.
The data points were used to generate predictive models
employing statistical design software (JMP 14, available from
SAS), with the reaction parameters (tRes, NAD+ mol %, equiv
of formate, and buffer concentration) used as inputs. Models
were created for predicted conversion, STY, and E factor. The
reached (approximately 3−5 reactor volumes), and then
1
multiple samples were collected for H NMR analysis. The
value quoted for the conversion was an average across the
samples analyzed at steady state under a specific set of reactor
conditions (minimum of three data points). Initial testing was
carried out in the flow reactor for the conversion of 10 mM
pyruvate to lactate in order to determine an appropriate
LDH:FDH:carbon ratio. This provided loadings of 3 mg of
LDH, 30 mg of FDH, and 30 mg of carbon, which were used
for all further experiments.
Optimization of the reaction conditions in flow was carried
out by variation of tRes, the number of equivalents of formate,
and the mole percent of NAD+ in order to maximize the
conversion of 50 mM pyruvate (Figure 1). The operational
window for the variables was determined by a set of desired
characteristics: tRes to be no longer than 30 min (for
reasonable productivities), the number of equivalents of
formate to be no greater than 5 (to limit the waste
contribution), and the NAD+ loading to be no greater than
1 mol % (for viable cofactor turnover numbers and to limit the
cofactor contribution to waste). The results are summarized in
Figure 1.
Variation of tRes from 10 to 30 min showed an increase in
conversion up to >99% at the longest residence time (Figure
1A), consistent with the increased contact time between the
reaction solution and the catalyst. As expected, increasing the
C
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Figure 2. Contour profiles showing the combination of predicted responses for conversion (red), STY (green), and E factor (blue) for the
continuous biocatalytic conversion of pyruvate to lactate for various operating conditions: (A) tRes vs NAD+; (B) tRes vs formate; (C) NAD+ vs
formate. Colored sections denote the operating conditions where the desired response limit is not achieved, and the white sections denote the
optimum operating windows in which all of the desired response limits are achieved. The profiles were generated using the statistical design
software JMP 14.
full models generated are presented and discussed in SI section
S7. From the models, the predicted responses for conversion
(red), STY (green), and E factor (blue) were combined, and
contour profiles were generated across the parameter ranges
explored (Figure 2). The desired response limits were set to
conversion > 80%, STY > 10 g L−1 h−1, and E factor < 4 in
order to determine the predicted operating window within
which all of these selected criteria are met (white areas of the
contour profiles). Generally, to optimize for STY, a shorter
tRes would have been more favorable, as more material is
processed in a shorter time. In contrast, to optimize for E
factor and conversion a longer tRes is favorable, as the longer
exposure to the catalyst leads to higher conversion, and
therefore, less unreacted material is left over as waste. All three
responses would have been improved by the higher driving
force provided by higher NAD+ mol % and more equivalents of
formate, as this leads to higher conversion, higher productivity
per unit time, and less waste due to unreacted starting material.
However, use of formate in too high an excess would begin to
negatively impact the E factor. From the contour profiles it can
be determined that the reaction parameters required for
operation in the optimum window for all three responses are
tRes = 25−30 min, 0.8−1 mol % NAD+, and 2.5−5 equiv of
formate. These ranges encompass the optimized parameter
values for conversion while suggesting that lower values could
be used without negatively impacting the process. Additionally,
if the main optimization focus for the process were shifted to
another specific response (e.g., the E factor), this information
could be used to reassess the optimum operating parameters
and provide new ranges.
The longer-term stability of the biocatalytic reactor was also
investigated using the optimal conditions for high conversion:
50 mM pyruvate, 5 equiv of formate, 1 mol % NAD+, tRes = 30
min, and 50 mM Tris-HCl buffer (pH 8.0). In this case,
enzymes were immobilized on carbon within the column to
prevent loss of catalyst during transfer into the reactor column
and for ease of use, allowing for catalyst preparation and
reaction to take place in the same reactor column. The results
in Figure 3 show that full conversion was maintained for 30 h,
demonstrating the viability of the biocatalyst over extended
operating periods. During this 30 h of operation, a STY, E
factor, and productivity of 11.1 glactate L−1 h−1, 4.4, and 34.9
μmollactate min−1 gsupport were maintained, respectively. The
−1
effective activities of the coupled FDH and LDH in this flow
D
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30 min, 1 mol % NAD+, 5 equiv of formate). Further
interrogation of the results aided by statistical modeling
generated an optimum operating window for the process in
terms of additional process metrics (STY and E factor) in
combination with conversion. The longer-term stability of the
biocatalytic reactor was demonstrated, with high conversion
and productivity achieved in the flow reactor for more than 30
h. The methodology investigated here displays significant
advantages over existing methods in terms of the simple, cheap,
and readily available carbon support and the one-step
immobilization strategy, which requires no modification of
the support or the enzymes. Therefore, this biocatalytic system
overcomes some of the key challenges associated with the
adoption of biocatalysis for greener chemical production,
namely, enzyme stability and cofactor recycling, and shows that
by considering multiple targets for optimization, the
biocatalytic reactor conditions can be tailored to the desired
process outcome.
Figure 3. Long-term operation of the continuous biocatalytic packed
bed reactor for the conversion of pyruvate to lactate. Conditions: 50
mM pyruvate, 5 equiv of formate, 1 mol % NAD+, 50 mM Tris-HCl
buffer, pH 8.0, room temperature, tRes = 30 min, 16 μL min−1,
reactor volume = 0.48 mL.
EXPERIMENTAL SECTION
■
experiment were 0.03 and 0.25 units mg−1, respectively. These
values can be compared with the activities achieved in the
single-enzyme immobilization studies (0.015 and 19 units
mg−1 for FDH and LDH, respectively; SI section S3). FDH
had a higher effective activity when coupled to LDH in the
flow system, but LDH was significantly less active than
expected. To determine whether the overall reaction was
limited by FDH, the LDH loading was decreased from 3 mg to
0.1 mg at constant FDH loading (30 mg). The activity of LDH
in the resulting catalyst was determined to be 10.7 units mg−1,
which is much closer to that observed in the immobilization
studies, showing that the flow process could be further
optimized with respect to the enzyme loading.
General. Sodium pyruvate (Sigma), sodium formate
(Sigma), NAD+ (Prozomix), carbon black particles (Black
Pearls 2000, Cabot Corporation), and Trizma-base (Sigma)
were used as received without further purification. All solutions
were prepared with Milli-Q water (Millipore, 18 MΩ cm).
Lactate dehydrogenase (from rabbit muscle, Merck) and
formate dehydrogenase (FDH-102, Johnson Matthey) were
used as supplied. UV−vis spectra were recorded using an
1
Agilent Cary 60 spectrophotometer. H NMR spectroscopy
was performed using a Bruker Avance III HD nanobay
spectrometer (400 MHz), with the addition of 10% D2O to all
samples. Reaction products were determined by comparison to
analytical standards.
At the end of a separate 24 h run, the packed column reactor
was flushed with Tris-HCl buffer (50 mM, pH 8.0) and stored
at 4 °C overnight. The reactor was then reused and achieved
72% conversion under the same reaction conditions (data are
provided in the SI). This demonstrates the potential for
reusability and stability of the catalyst column upon further
optimization of the storage conditions.
A comparative batch reaction (24 mL, 24 h) was carried out
using the same catalyst loading as in the flow reactor (Figure
S10), and key parameters are compared with those for the flow
process (Table S5). After 24 h in batch, 96% conversion was
achieved, giving a comparable activity (mglactate h−1) and the
same E factor (4.4) as the flow process. The STY was 0.23 g
L−1 h−1 for the batch process compared with 11.2 g L−1 h−1 for
the flow process, demonstrating the higher productivity that
can be achieved using the low flow reactor volume (0.48 mL vs
24 mL in batch). The enzyme activities recorded were very
similar, with slightly higher initial activities recorded in the
batch reaction.
Preparation of the Supported Biocatalyst. Carbon
particles (30 mg, Black Pearls 2000, Cabot Corporation) were
suspended in 1.5 mL of Tris-HCl buffer (50 mM, pH 8.0) and
sonicated for 75 min. A mixture of LDH (3 mg) and FDH (30
mg) was added to the 1.5 mL carbon suspension, and the
enzymes allowed to adsorb at 4 °C. After 4 h, the mixture was
centrifuged, and the supernatant solution was removed to yield
the biocatalytic particles.
Enzyme Immobilization Efficiency. The immobilization
efficiency was determined by analysis of the supernatant
recovered from the enzyme immobilization procedure by
measurement of the UV−vis absorbance at 280 nm and
comparison to calibration curves (see the SI) to determine the
protein concentration relative to the amount of added protein.
Analysis of Immobilized Enzyme Activity. The enzyme
activity was determined by monitoring the conversion of
NADH to NAD+ over time by UV−vis spectroscopy (see the
SI). The catalyst particles were added to a cuvette (1 mL,
Hellma) containing NADH (0.1 mM) and either sodium
pyruvate (0.1 mM) or sodium formate (0.5 mM) in Tris-HCl
buffer (50 mM, pH 8.0, 1 mL total volume). The ratio of peaks
at 260 and 340 nm was then used to calculate the enzyme
activities after comparison to calibration curves.33
CONCLUSIONS
■
We have presented the use of a simple carbon support for
straightforward co-immobilization of NADH-dependent LDH
with FDH to facilitate ketone reduction with in situ cofactor
recycling in a continuous packed bed reactor. FDH is well-
established as a cofactor recycling system for NADH-
dependent enzymes, suggesting that it should be possible to
translate many other biotransformations requiring NADH into
flow. Variation of reaction parameters generated a set of
optimum reactor conditions to maximize conversion (tRes =
Continuous Packed Bed Reactor Setup. An Omnifit
glass column (6.6 mm i.d., variable bed length) was manually
packed with the catalyst particles and glass beads (1 mm). For
all experiments, the Omnifit column bed length was adjusted
to give a total bed volume of 1.02 mL. The actual reactor
volume was then calculated using the void fraction method,41
where the reactor volume is the total bed volume minus the
E
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space filled by the catalyst, as calculated by the difference in
column mass before and after filling with water (see the SI).
The column was connected to the syringe pump (Asia, Syrris)
ACKNOWLEDGMENTS
■
Dr. Beatriz Dominguez (Johnson Matthey) is gratefully
acknowledged for providing FDH.
1
using PTFE tubing (0.5 mm i.d.) and /4-28 PTFE flangeless
fittings. Samples were collected at the reactor outlet using a
BioRad model 2110 fraction collector.
ABBREVIATIONS
■
LDH, lactate dehydrogenase; FDH, formate dehydrogenase;
i.d., inner diameter; PTFE, polytetrafluoroethylene; tRes,
residence time; STY, space-time yield
Biocatalytic Production of Lactate in the Continuous
Packed Bed Reactor. Sodium pyruvate (50 mM), NAD+
(0.05−0.5 mM), and sodium formate (50−250 mM) were
dissolved in Tris-HCl buffer (50 mM, pH 8.0), and the
reaction solution was pumped through the packed bed reactor
at flow rates between 17.2 and 51.6 μL min−1 (tRes = 10−30
min). Once the reactor reached a steady state, samples were
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.9b00410.
Immobilization efficiency data and calibration curves,
enzyme activity data, enzyme leaching data, packed
column reuse data, JMP models, and NMR spectra
(PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
Holly A. Reeve − Department of Chemistry, University of
Oxford, Oxford OX1 3QR, U.K.; orcid.org/0000-0001-
5855-651X; Email: holly.reeve@chem.ox.ac.uk
Kylie A. Vincent − Department of Chemistry, University of
Oxford, Oxford OX1 3QR, U.K.; orcid.org/0000-0001-
6444-9382; Email: kylie.vincent@chem.ox.ac.uk
Authors
Barnabas Poznansky − Department of Chemistry, University of
Oxford, Oxford OX1 3QR, U.K.
Lisa A. Thompson − Department of Chemistry, University of
Oxford, Oxford OX1 3QR, U.K.; orcid.org/0000-0003-
1128-7927
Sarah A. Warren − Dr. Reddy’s Laboratories Ltd., Cambridge
CB4 0PE, U.K.
́
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Fernandez-Lafuente, R. Modifying enzyme activity and selectivity by
immobilization. Chem. Soc. Rev. 2013, 42, 6290−6307.
(12) Liese, A.; Hilterhaus, L. Evaluation of immobilized enzymes for
industrial applications. Chem. Soc. Rev. 2013, 42, 6236−6249.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.9b00410
(13) Dobber, J.; Pohl, M. HaloTagTM: Evaluation of a covalent one-
̈
step immobilization for biocatalysis. J. Biotechnol. 2017, 241, 170−
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Author Contributions
(14) Contente, M. L.; Dall’Oglio, F.; Tamborini, L.; Molinari, F.;
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§B.P. and L.A.T. contributed equally. All of the authors helped
to design the experiments. B.P. performed the experiments.
B.P. and L.A.T. analyzed the data. All of the authors
contributed to writing and approved the final version of the
manuscript.
Funding
The research of K.A.V., H.A.R., and L.A.T. was supported by
Engineering and Physical Sciences Research Council (EPSRC)
IB Catalyst Award EP/N013514/1. B.P. was supported by
iCASE funding from the Biotechnology and Biological
Sciences Research Council (BBSRC,Grant BB/M011224/1),
with support from Dr. Reddy’s Laboratories.
Notes
The authors declare no competing financial interest.
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