Chemoenzymatic Buta-1,3-diene Synthesis from Syngas Using Biological Decarboxylative Claisen Condensation and Zeolite-Based Dehydration

Article abstract of DOI:10.1002/cbic.202000565

A method for producing buta-1,3-diene (1,3-BD) by an amalgamation of chemical and biological approaches with syngas as the carbon source is proposed. Syngas is converted to the central intermediate, acetyl-CoA, by microorganisms through a tetrahydrofolate metabolism pathway. Acetyl-CoA is subsequently converted to malonyl-CoA using a carbonyl donor in the presence of a carboxylase enzyme. A decarboxylative Claisen condensation of malonyl-CoA and acetaldehyde ensues in the presence of acyltransferases to form 3-hydroxybutyryl-CoA, which is subsequently reduced by aldehyde reductase to give butane-1,3-diol (1,3-BDO). An ensuing dehydration step converts 1,3-BDO to 1,3-BD in the presence of a chemical dehydrating reagent.

Full text of DOI:10.1002/cbic.202000565

Combining Chemistry and Biology
Accepted Article
Title: Chemoenzymatic 1,3-butadiene synthesis from syngas using
biological decarboxylative Claisen condensation and zeolite-
based dehydration
Authors: Sivaraman Balasubramaniam, Sneh S. Badle, Swati
Badgujar, Vinod P. Veetil, and Vidhya Rangaswamy
This manuscript has been accepted after peer review and appears as an
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using the Digital Object Identifier (DOI) given below. The VoR will be
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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: ChemBioChem 10.1002/cbic.202000565
Link to VoR: https://doi.org/10.1002/cbic.202000565
01/2020

10.1002/cbic.202000565
ChemBioChem
FULL PAPER
Chemoenzymatic 1,3-butadiene synthesis from syngas using
biological decarboxylative Claisen condensation and zeolite-
based dehydration
Sivaraman Balasubramaniam, [a] Sneh Badle, [a] Swati Badgujar, [a] Vinod P. Veetil, [a] and Vidhya
Rangaswamy*[a]
[a]
Dr. B. Sivaraman, Dr. S. S. Badle, Dr. S. Badgujar, Dr. V. P. Veetil, Dr. V. Rangaswamy
High Value Chemicals group, Reliance Industries Limited,
Ghansoli, Navi Mumbai, India. Pin code: 400701
E-mail: Vidhya.Rangaswamy@ril.com
Supporting information for this article is given via a link at the end of the document.
Abstract: A method for producing 1,3-butadiene (1,3-BD) by an
amalgamation of chemical and biological approach involving syngas
as the carbon source is proposed here. Syngas is converted to the
central intermediate, acetyl-CoA, through microorganisms having a
tetrahydrofolate metabolism pathway. Acetyl-CoA is subsequently
converted to malonyl-CoA using a carbonyl donor in the presence of
a carboxylase enzyme. A decarboxylative Claisen condensation of the
malonyl-CoA and acetaldehyde ensues in the presence of
acyltransferases to form 3-hydroxybutyryl-CoA that is subsequently
reduced by aldehyde reductase to obtain 1,3-butanediol (1,3-BDO).
An ensuing dehydration step converts 1,3-BDO to 1,3-BD in the
presence of a chemical dehydrating reagent.
synthesis of 1,3-BD from the key intermediate erythritol obtained
from xylose via biomass deconstruction.[17]
In the current study, another unique approach for the
production of 1,3-BD using combination of enzymatic and
chemical reactions is described. A novel approach, where in vitro
sequential enzymatic reactions to produce 1,3-butanediol (1,3-
BDO) from common intracellular metabolites viz. acetaldehyde
and malonyl-CoA has been demonstrated. The 1,3-BDO formed
was successfully dehydrated chemically to produce 1,3-BD.
Results and Discussion
Proposed novel pathway
An alternative, environmentally-friendly and sustainable process
for the production of 1,3-BD 1, would mandate either a purely
biological approach or a combination of biological and chemical
routes. Several methodologies have been proposed for 1,3-BD
production including (i) via enzymatic dehydration of
butanediols[18] (ii) enzymatic dehydrogenation and dehydration of
butanols[18] (iii) enzymatic hydroxylation and dehydration of
butenes[18] (iv) from 2,4-pentadienoate[19] (v) from acetyl-CoA via
crotonyl-CoA and crotyl alcohol.[20] Demonstration of in vitro
production of 1,3-BD from crotyl alcohol using mutants of linalool
dehydratase and alkenol dehydratase was reported by
Genomatica and Braskem recently.[21] Most of these processes
involves extensive genetic engineering to demonstrate proof of
concept.
Introduction
Synthesis gas (syngas) a mixture of hydrogen (H2), carbon
dioxide (CO2) and carbon monoxide (CO), is a waste gas product
in the chemical and biorefining industries that has found a vast
number of applications.[1,2] Besides being used as a fuel, syngas
can be converted into value added chemicals viz. alkanes,[3]
olefins,[4] oxygenates, and alcohols[5] Carbon is fixed in anaerobic
microorganisms from gaseous substrates like CO or CO2 via the
Wood-Ljungdahl (WL) pathway.[6] CO from syngas is converted to
the central intermediate, acetyl-CoA, through the WL pathway
utilizing tetrahydrofolate metabolism. Acetyl-CoA serves as the
key building block to synthesize several products like ethanol and
isopropanol.[7]
Evidently, improved versions of existing enzymes having
promiscuous activity, suitable microbial host and efficient
protocols forms the basis for producing 1,3-BD from various
renewable feedstocks as well as C1 feedstocks such as syngas.
The syngas utilising bacteria employ the WL pathway for
assimilation of CO/CO2 as carbon source along with H2. The key
central intermediate in the WL pathway is acetyl-CoA which can
then be diverted via multitudes of routes to make various products
of interest.
Towards this end, a novel route for 1,3-BD from syngas has
been proposed in the current study using malonyl-CoA 3 and
acetaldehyde 4 as starting materials, both of which can be derived
from acetyl-CoA 2, the key metabolite in the WL pathway.[6]
Acetyl-CoA can be converted to malonyl-CoA using a carbonyl
donor in the presence of a carboxylase enzyme. Decarboxylative
Claisen condensation (DCC) of the malonyl-CoA and
acetaldehyde will form 3-hydroxybutyryl-CoA in the presence of
1,3-butadiene (1,3-BD) is an important industrial chemical
obtained from the steam cracking process of petrochemical-
based feedstocks and purified via extractive distillation.[8,9] The
global annual production of 1,3-BD is about 10 million metric
tons[10] and is applied in the manufacturing of polymers such as
synthetic rubber,[11] ABS resins,[12] and chemicals such as
hexamethylenediamine.[13] However, no biological process using
microorganisms exists in nature for the production of this major
petrochemical intermediate.[14] Given the vagaries of oil price,[15]
dependency on the petrochemical feedstocks along with the
increasing environmental concerns, there is
a need for
alternative, environmentally-friendly and sustainable process[16]
for the production of 1,3-BD.[8] A partial mitigation to these issues
would be to use an alternative approach utilizing bio-based
feedstocks. Recently, by combining the usefulness of chemical
and biological techniques, we demonstrated the successful
1
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10.1002/cbic.202000565
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FULL PAPER
Scheme 1. Novel route for synthesizing 1,3-butadiene from syngas
acyltransferases which can be reduced using a carboxylate
reductase to obtain 1,3-BDO 5. The generated 1,3-BDO can then
be converted to 1,3-BD via a chemical process. The genesis of
the envisaged pathway is based on the DCC reaction, hitherto not
reported, between malonyl-CoA and acetaldehyde (Scheme 1).
Activity of FabH
Enzyme activity of purified FabH protein was performed using the
substrates malonyl-CoA and acetaldehyde in the presence of
purified ACP, FabH and cerulenin which inhibits any undesired
chain elongation. Figure 1 depicts the HPLC analysis of the
reaction mixture indicating a distinct peak of 3-hydroxybutyryl-
CoA that com-pared well with the retention time of standard 3-
hydroxybutyryl-CoA. Demonstration of the evidence that the E.
coli FabH along with ACP can also carry out the DCC reaction
with uncommon substrates malonyl-CoA and acetaldehyde is a
major finding in this study. Successful proof of this activity opens
up tremendous opportunity for FabH to be explored for
condensation activities with other uncommon substrates leading
to novel products besides its classical role.
Construction of recombinant plasmids and strains
As the DCC reaction between malonyl-CoA and acetaldehyde is
the fundamental premise of our proposed strategy, exploring for
enzymes having this promiscuous activity was crucial. One of the
enzymes in the fatty acid pathway, FabH, which already catalyzes
the condensation reaction between malonyl-CoA and acetyl-CoA
appeared to be a probable candidate.[22] Therefore, exploiting this
strategy, FabH encoded by the fabH gene along with the gene for
the acyl carrier protein (ACP) was sourced from E. coli. For the
subsequent aldehyde reductase (AdhE) step, multiple sources
were evaluated. Subsequently, genes coding for AdhE’s from C.
acetobutylicum and C. beijerinckii were chosen.
The fabH and acp genes having a size of about 955 and 237
bp respectively were amplified from genomic DNA of E. coli K12.
Similarly, different aldehyde-alcohol dehydrogenase (adhE) were
amplified using C. acetobutylicum (Ca_adhE) and C. beijerinckii
(Cbei_adhE) genomic DNA using One Taq polymerase with Tm
55 ˚C. PCR amplifications of fabH, acp, and different adhE’s
(Cbei_adhE) from C. beijerinckii are shown in Figure S1a, S2a,
S3a, and S4a respectively. The genes were cloned into pET30a
(+) vector with respective restriction enzymes mentioned in Table
S1. Putative positives were analyzed by colony PCR as shown in
Figure S1b, S2b, S3b, S4b for respective genes. The recombinant
plasmid carrying the fabH, and acp genes were validated by the
digesting with same restriction enzymes pairs i.e. BamHI/HindIII
and KpnI/XhoI respectively. The different adhE genes carrying
pET30a (+) recombinant vectors were also confirmed by
restriction digestion using same pair of enzymes as mentioned in
Table S1. E. coli DH5α strain was used for plasmid propagations
and confirmed plasmid were transformed into E. coli BL21 (DE3)
cells for expression analysis.
Figure 1. HPLC spectrum of 3-hydroxybutyryl-CoA obtained from FabH enzyme
assay and overlaid with standard.
Cascade reaction with AdhE
Once the DCC reaction was successful between malonyl-CoA
and acetaldehyde, the next step was to convert the product 3-
hydroxybutyryl-CoA to 1,3-BDO. It is a well-known fact that CoA
derivatives of compounds are highly unstable and not easy to
isolate. Therefore, we relied on telescoping of the obtained 3-
hydroxybutyryl-CoA for the subsequent reaction, to monitor the
formation of the 1,3-BDO. The activities of FabH/ACP and AdhE
were coupled together in presence of NADH in a cascade
reaction. The production of 1,3-BDO was confirmed using HPLC
(Figure 2 and Figure S6) and GC-MS (Figure S7). The mass
Expression and purification of His-tagged protein
The FabH, ACP and AdhE proteins expressed in E. coli BL21
(DE3) were purified on Ni-NTA and the SDS-PAGE of the
samples confirmed >90 % purity. Purified protein bands of 14
kDa, 52 kDa and 35 kDa size corresponding to the enzymes ACP,
AdhE and FabH respectively are shown in Figure S5.
2
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10.1002/cbic.202000565
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spectra showed the typical fragmentation pattern of 1,3-BDO with
peaks at 43, 45 and 73 m/z values (Figure S8).
corresponding to 1,3-BD; along with trace amount of other
impurities (cis and trans butene) (Figure 3).
Figure 2. HPLC spectrum of 1,3-BDO (retention time ~18.05 min) obtained from
cascade reaction (FabH/ACP and AdhE) and over-laid with standard 1,3-BDO.
Figure 3. Mass spectrum of 1,3 BD obtained by reacting 1,3-BDO with H3PO4
and Zeolite NaY.
Chemical conversion of 1,3-BDO to 1,3-BD
In the absence of any existing enzyme that can dehydrate a diol
to diene, it was decided to convert the 1,3-BDO formed from the
enzymatic reactions to 1,3-BD using chemical catalysis.
Optimization of reaction conditions for the conversion of 1,3-BDO
to 1,3-BD was carried out using commercially available 1,3-BDO.
Double dehydration reaction of 1,3-BDO was carried out under
heating condition in a sealed vial and head space was monitored
for the formation of 1,3-BD by GC. Initial screening using mineral
acids viz. HCl, HI, H2SO4, H3PO4 and zeolites viz. ZSM5 and NaY
(faujasite Y) revealed that dehydration using 35% HCl, HI and
ZSM5 were not effective as they converted only a small fraction
of 1,3-BDO and produced 1,3-BD in miniscule quantities (1-2 %
yield). Whereas, H2SO4, H3PO4 and NaY, produced 1,3-BD in
measurable quantities of around 10 % yield (Scheme 2).
Cognizant of the fact that the 1,3-BDO produced from the
fermentation will be in aqueous layer, use of H2SO4 was avoided
and reaction with aqueous compatible mineral acid H3PO4 and
zeolite NaY was screened further. Taking into account, the
volume of 1,3-BD that will have to be produced at commercial
scale, conditions were optimized by varying several parameters
including equivalence, temperature, and reaction time (Figure
S9).
Based on the m/z results, it was speculated that the peak
corresponding to the RT 5.5 min on GC could be propene. To
confirm, sample from dehydration reaction of propanol to propene
under similar conditions was analysed by GC. The RT and
molecular weights obtained from this reaction matched perfectly
with that of the peak obtained during the dehydration reaction of
1,3-BDO indicating presence of propene. The residual 1,3-BDO
was analyzed using HPLC to determine the conversion rate. The
results of the screening studies are given in supporting
information (Figure S9a, b, c).
Based on the optimization studies, the best conversion was
achieved by reacting 1,3-BDO (1 g) with 1 ml of H3PO4 at 105 °C
for 24 h. The yields of 1,3-BD and propene were found to be 35%
and 4% respectively based on standard graphs (Figure S11 and
Figure S12).
Screening using Zeolite NaY
Synthetic zeolites are widely used as catalysts in the
petrochemical industry for fluid catalytic cracking and
hydrocracking. The acidic forms of zeolites facilitates reactions,
such as isomerisation, alkyla-tion, and cracking.[23-25]
NaY (Faujasite Y), an acidic catalyst was calcinated prior to
the reaction at 540 °C for 16 h to remove moisture and ligand and
allowed to cool to ambient temperature under nitrogen flow in a
desiccator. The free-flowing solid NaY was used as such for initial
screening and was later stored in a desiccator. Initial screening
was performed using 1.0 g of 1,3-BDO and NaY under heated
condition in a sealed vial. The progress of the reaction was
monitored using GC by injecting gas sample from the headspace
of the vials. Similar to the results with H3PO4, two peaks were
observed on GC corresponding to RT= 5.5 min (propene) and
RT= 5.9 min (1,3-BD). Optimization of the reaction temperature,
time and loading (Figure S9d,e and f), revealed that reacting 1,3-
BDO with 500 mg of zeolite NaY at 135 °C for 24 h gave best
conversion resulting in a yield of 24 % of 1,3-BD. Reaction yields
were derived from the standard graphs (Figure S10 and Figure
S11).
Scheme 2. Reaction scheme for synthesizing 1,3-BD from 1,3-BDO.
Screening using H3PO4
For screening, 1.0 g of 1,3-BDO was subjected to double
dehydration reaction in a sealed vial and the progress of the
reaction was monitored by analyzing the headspace gas using
GC. The GC chromatogram indicated the presence of only two
peaks, one appearing at RT of 5.5 min and the other at 5.9 min.
Comparing the GC chromatogram with that of standard 1,3-BD
revealed that the peak corresponding to RT of 5.9 min matched
with that of the standard 1,3-BD. However, the peak at 5.5 min
required further probing. GC-MS analysis revealed the presence
of two major peaks, one of m/z= 42 and other of m/z=54, the latter
Using mixture of H3PO4 and Zeolite NaY
Despite the optimization, the conversion of 1,3-BDO to 1,3-BD did
not exceed beyond 35 % and 24 % using H3PO4 and NaY
3
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10.1002/cbic.202000565
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respectively. Therefore, a different strategy was attempted where
a combination of H3PO4 and NaY was used. The stability of NaY
under highly acidic conditions is not known, as there are no
reports on the combination of both mineral acids and zeolites.
Nevertheless, the best conditions obtained during screening
studies were employed. Temperature screening experiment
revealed that the conversion was extremely low at 105 °C. Hence,
all further reactions were carried out at 135 °C and effect of
varying the loadings of NaY and H3PO4 was evaluated.
onto hot, fluidized catalyst and large gasoline molecules are
broken into smaller gasoline molecules and olefins. The vapor-
phase products are separated from the catalyst using nitrogen
flow. To translate our results, we used fixed bed reactor for the
conversion.
Scale up of the 1,3-BDO to 1,3-BD reaction described above
was met with two major challenges: (i) very high viscosity of feed
1,3-BDO (98.3 cP) and (ii) preparing the extrude having the crush
strength in the range of 1.8-2.0 Pa from the hitherto combination
of zeolite NaY and H3PO4 to form the fixed bed. To circumvent the
viscosity issues, 1,3-BDO was diluted with water and 20-30%
solution was found to be optimum for pumping. To fix the crush
strength, series of extrudes were prepared by mixing NaY and
H3PO4 in H2O to keep the NaY complex intact and using alumina
as a binder. The concentration of H3PO4 was varied from 20 to
40 % and binder was varied from 10 to 50 % to improve the crush
strength. A mixture of 40 % H3PO4 and 20 % binder had the
required crush strength of 1.8 Pa.
Table 1 shows the yield of 1,3-BD at varying concentration
of H3PO4 in a reaction containing 1,3-BDO (1 g) along with 500
mg of NaY at 135 °C for 8 h. The highest yield of 16 % 1,3-BD
was obtained with 0.5 ml of H3PO4.
Table 1. Effect of different loadings of H3PO4 for reaction conducted at 135 °C
for 8 h.
GC observation
Loading of
Reaction
H3PO4 (mL)
1,3-BD (5.9
min)
Propene (5.5 min)
For initial studies, the feed containing 20 % 1,3-BDO was
pumped in to the reactor using peristaltic pump at a rate of 5-10
mL h-1. The mixture was heated in the preheater between 240-
270 °C, followed by pushing the stream into the reactor column
containing the bed at 290-320 °C using nitrogen flow of 40-60 mL
min-1. Initial reaction was carried out for 4 h. The vent coming out
of the reactor was connected to a liquid gas separator which was
maintained at a temperature of 5-10 °C. The head space was
analyzed using GC for the production of 1,3-BD and the liquid
collected in the gas liquid separator was analyzed using HPLC for
residual 1,3-BDO at hourly intervals. After the 1st hour of reaction,
GC analysis of the sample revealed the formation of 1,3-BD (RT
6.0 min) along with propene (RT 5.5 min). The presence of 1,3-
BD was also confirmed by GC-MS. The AUC values for 1,3-BD,
propene and the 1,3-BDO was constant during the hourly analysis
indicating a steady state. Post this, higher concentration of 1,3-
BDO (30 %), preheated at 270 °C, was pumped into the reactor
using peristaltic pump using nitrogen flow. The reactor bed was
maintained at 310 °C and the reaction was carried out for 5.5 h.
The outlet of the reactor was connected to the gas liquid separator
which collects the liquid and the gas coming out was connected
to the hood. The vent coming out of the reactor was analyzed for
1,3-BD every 100 min using GC by collecting the gas in a bladder
for 10 min. The liquid collected in the gas liquid separator was
analyzed using HPLC. The yields were calculated based on the
observations from 3 trial runs. It was observed that, 3.5 g of 1,3-
BDO was consumed in 100 min and 26.5 g of unreacted 1,3-BDO
was recovered from gas liquid separator. Yield of 1,3-BD was
found to be 88% and that of propene was 8.0% from the
consumed 1,3-BDO as revealed by GC. The product was
0.25
0.5
0.75
1.0
~1%
~4%
~5%
~7%
12%
16%
14%
12%
1 g 1,3-
BDO +
500 mg
NaY
Further, effect of varying NaY loading was also tested in a
reaction containing 1,3-BDO (1 g) along with 0.5 ml of H3PO4 at
135 °C for 8 h. The highest yield of 15 % 1,3-BD was obtained
with 200 mg of NaY (Table 2).
Based on the results obtained from loading optimization
studies, it was found that reacting 1,3-BDO (1 g) with a mixture of
0.5 ml of H3PO4 and 200 mg of zeolite NaY at 135 °C for 24 h
gave best conversion of 64 % 1,3-BD. Reaction yields were
derived from the standard graph of 1,3-BD (Figure S11).
Table 2. Effect of different loadings of NaY for reaction conducted at 135 °C for
8 h.
GC observation
Loading of
Reaction
NaY (mg)
Propene (5.5 min)
1,3-BD (5.9 min)
100
200
300
400
~1%
~1%
3%
7%
1 g 1,3-
BDO + 0.5
ml H3PO4
15%
12%
14%
5%
There are several reports in the literature on dehydration of
all types of butanediols to 1,3-BD by chemical catalysis. One
important criteria cited is that although complete dehydration of
these butanediols into BD is possible, the process needs higher
reaction temperatures and results in the production of more
byproducts.[26] A temperature of 200 °C or more is typical of these
reactions with BD yields of < 42 %. With respect to conversion of
1,3-BDO to 1,3-BD per se, a maximum 1,3-BD yield of 60% was
achieved at 300 °C over ZSM5 with a SiO2/Al2O3 ratio of 260 with
the simultaneous formation of propylene at a BD/propylene
selectivity ratio of 2.5.[27] In the current study, at the batch scale,
it was possible to get a 1,3-BD yield of 64 % by operating at
135 °C for 24 h using a combination of H3PO4 and zeolite NaY.
1
characterized and confirmed using H-NMR (Figure S13). Thus,
the catalytic double dehydration of 1,3-BDO to 1,3-BD was
successfully demonstrated in a fixed bed reactor.
After the reaction, the catalyst was regenerated by purging
air to remove the deposited coke, a byproduct in the cracking
process from the surface of the catalyst. The regenerated catalyst
is then circulated back to the reactor to complete its cycle.
Thus far, there is only one other study wherein a fixed bed
reactor has been used for dehydration of 2,3-butanediol to 1,3-BD
supported by a simulated one-dimensional heterogenous reactor
model.[29]
Scale up reaction based on fixed bed reactor
The next step was to translate the feasibility of the reaction
carried out in sealed vials to a higher scale. One of the industrially
scalable process is based on catalytic cracking.[28] Catalytic
cracking uses reactor and a regenerator where feed is injected
4
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8.0. Equal volume of purified proteins were mixed with 2 X SDS loading
buffer and boiled for 10 min to prepare samples for SDS-PAGE. The
samples for FabH and AdhE was run on 12 % SDS-PAGE gel and ACP
was run on 16 % Tricine gel and the gels were visualized by Coomassie
Brilliant Blue R-250 staining. The target proteins were detected by
comparison with protein standard markers. The proteins were buffer
exchanged into the assay buffer 50 mM NaH2PO4 (pH 8.0) using a
prepacked PD-10 Sephadex G-25 desalting column.
Conclusion
A novel sustainable process for producing the commodity
chemical 1, 3-BD using a combination of biological and chemical
method is reported in the current study. The process involves
producing 1,3-BDO using FabH enzyme for DCC using
uncommon substrates viz. malonyl-CoA and acetaldehyde (both
derived from the key metabolic intermediate acetyl-CoA in the WL
pathway). 1,3-BDO is then converted into butadiene by a
chemical dehydration step using a unique combination of mineral
acid and zeolites. The biological process can easily be
engineered into syngas fermenting organisms and hence
provides for a sustainable method for production of 1,3-BD.
Acyl carrier protein was also purified and used in the enzyme
reaction mixture. The total protein content as estimated by bicinchoninic
acid assay (BCA) method for FabH and AdhE was 200 mg L-1 and 15 mg
L-1 respectively
FabH/ACP enzyme activity: Typical reaction mixture for determining
FabH/ACP activity constituted of malonyl-CoA (200 μM), acetaldehyde
(250 μM), purified ACP protein (200 μg) which was mixed thoroughly
followed by addition of cerulenin (250 μM) (solution made in ethanol) and
purified FabH (50 μg) was added which resulted in a 300 μL reaction
mixture. Cerulenin was added to inhibit any undesirable chain elongation.
The enzyme mixture was incubated 37 °C for 2.5 h. The mixture was
centrifuged at room temperature for 5 min at 8000 rpm. The supernatant
was separated and tested for production of 3-hydroxybutyryl-CoA using
HPLC.
Experimental Section
General methods, plasmids and strains: Methods for restriction enzyme
digestions, ligation, transformation, and other standard molecular biology
manipulations were based on methods described elsewhere[30] or as
suggested by the manufacturer. Protein was analyzed by polyacrylamide
gel electrophoresis (PAGE) under either denaturing condition using
sodium dodecyl sulfate (SDS) or native conditions on gels having 7.5-12%
poly-acrylamide. The gels were stained with Coomassie brilliant blue. All
oligonucleotides were purchased from Eurofins India. Table S1 in
Supporting Information summarizes the primers used in this study. One
Taq DNA polymerase, restriction enzymes and T4 DNA ligase were bought
from New England Biolabs. DNA purification, plasmid isolation and PCR
purification kits were purchased from Qiagen. Ni-NTA resin was bought
from Qiagen and PD10 desalting columns from GE-Healthcare. All other
reagents were of analytical grade. The pET-30a (+) plasmid and E. coli
BL21 (DE3)/DH5α strains were used as expression vector and host strains
and were procured from Invitrogen. The strains were propagated at 37 °C
in Luria-Bertani (LB) medium containing 50 µg mL-1 kanamycin for
selection with shaking at 220 rpm.
Cascade reaction with AdhE: The activities of FabH/ACP and AdhE were
coupled together in presence of NADH in a cascade reaction. The quantity
of AdhE and NADH to be added was optimized for the reaction. 100 μL of
the reaction mixture was used for the assay, to which 5 mM of NADH
solution and 25 μL of AdhE purified enzyme (200 μg) was added. The
reaction mixture was mixed thoroughly and incubated for 2.5 hours at
37 °C. The enzymatic mixture was centrifuged for 5 min at 8000 rpm. The
supernatant was analysed by HPLC and GC-MS. The production of 1,3-
BDO was confirmed using HPLC.
Analysis of 3-hydroxybutyryl-CoA, 1,3-BDO and 1,3-BD: 3-
Hydroxybutyryl-CoA and 1,3-BDO were analyzed on HPLC. The
conditions for the analysis of 3-hydroxybutyryl-CoA includes using a C-18
column and isocratic mobile phase consisting of 100 mM ammonium
acetate with 9% methanol and 0.1% formic acid at a flow rate of 1.0 mL
min-1. Column temperature was maintained at 25 °C. The retention time
for standard 3-hydroxybutyryl-CoA was 10.3 min.
Expression and purification of His-tagged proteins: For expression
tests, 10 mL of LB medium containing 50 µg mL-1 kanamycin was
inoculated with a freshly isolated colony of the host strain (E. coli
BL21(DE3)) carrying the recombinant plasmid of pET30a-FabH, pET30a-
ACP, and pET30a-Cbei_3832 respectively. The inoculated cultures were
incubated overnight at 37 ˚C and diluted 1:100 into 2 mL of fresh LB
medium containing antibiotics with shaking until OD600 = 0.6; induced with
IPTG at a final con-centration of 0.5 mM and were grown at 20 °C and
37 °C for 12 and 8 h respectively. 1 mL sample of induced cultures grown
under different conditions were centrifuged and resuspended in 1 mL of
lysis buffer. A portion was mixed with equal portion of 2X SDS loading
buffer and boiled for 10 min to prepare whole-cell lysate for expression
analysis on SDS-PAGE. The samples for FabH and AdhE was run on 12 %
SDS-PAGE gel and ACP was run on 16% Tricine gel; gels were visualized
by Coomassie Brilliant Blue R-250 staining. The target proteins were
detected by comparison with protein standard markers. Optimum
conditions for high expression were determined based on the observations
from the SDS-PAGE gel (data not shown).
The conditions for the analysis of 1,3-BDO includes using anion
exchange column and the mobile phase used was 5 mM sulfuric acid
solution at a flow rate of 0.6 mL min-1. Column temperature was maintained
at 50 °C. The retention time for standard 1,3-BDO was 18.0 min.
For analysis of 1,3-BD, headspace sample of the sealed vial was
withdrawn and injected on the GC system (DB-1 column, 100 x 0.5 mm,
ID: 0.25 mm). Parameters for GC analysis were: FID detector temperature:
160 °C, column temperature: 50 °C, gas flow rate: 25 mL min-1. The
retention time of standard 1,3-BD was 5.9 min.
1
Sample preparation for H-NMR: The head space aliquot was sampled
and bubbled into the CDCl3 solution in NMR tube which was kept at -10 °C.
The NMR tube was sealed and recorded instantly using Bruker NMR
instrument.
For purification of the protein, growth and expression were carried
out in 500 mL scale in optimal conditions identified by the expression tests.
The cells harvested from 500 mL of culture grown and induced under
optimum conditions were suspended in 10 mL of lysis buffer (50 mM
NaH2PO4, pH 8.0, 300 mM NaCl, and 5 mM imidazole) containing
lysozyme (1 mg mL-1) and cells were disrupted by sonication. Soluble and
insoluble cell fractions were separated by centrifugation at 15000 rpm for
10 min in cold. Supernatants carrying the soluble fractions were mixed with
1 mL Ni-NTA resin to purify target proteins according to manufacturer’s
manual. Bound His-tagged proteins were eluted in 1 mL of elution buffer
containing 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, at pH
Chemical catalysis reaction and conditions: All reactions were carried
out in oven dried glassware or in cylindrical reaction vials with rubber cork.
All solvents were purchased from local suppliers and were of laboratory
reagent grade. Dry acetone was prepared by standard protocol and was
stored under 4 Å molecular sieves. 1H-NMR (400 MHz) spectra was
recorded in CDCl3 and chemical shifts are given in part per million (ppm).
1H-NMR spectra are referenced to CDCl3 (δ =7.26 ppm). The multiplicity
5
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10.1002/cbic.202000565
ChemBioChem
FULL PAPER
is given as, s = singlet, d = doublet, m = multiple and coupling constants J
are reported in Hz.
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Chemical conversion of 1,3 BDO to 1,3 BD: To 1 g of 1,3-BDO, 85%
H3PO4 or zeolite NaY or a combination of both H3PO4 and zeolite NaY was
added and heated. The progress of the reaction was monitored using GC
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Acknowledgements
We would like to thank Reliance Industries Limited for providing
the financial support to carry out this work. Special thanks are also
due to Dr. Rakshvir Jasra for providing us with the zeolite NaY
and the fixed bed reactor facility.
Keywords: 1,3-Butadiene • Decarboxylative Claisen
condensation • Enzymes • Metabolism • Syngas
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6
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10.1002/cbic.202000565
ChemBioChem
Entry for the Table of Contents
Insert graphic for Table of Contents here.
A novel pathway to convert syngas to 1,3-butadiene using a combination of biological and chemical catalysis is proposed. In vitro
demonstration of Decarboxylative Claisen Condensation by FabH using unnatural substrates is the highlight of this study. A unique
combination of zeolites and inorganic acid facilitated the dehydration of 1,3-butanediol to 1,3-butadiene with 88 % yield in a fixed bed
reactor.
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