An artificial synthetic pathway for acetoin, 2,3-butanediol, and 2-butanol production from ethanol using cell free multi-enzyme catalysis

Article abstract of DOI:10.1039/c7gc02898a

Upgrading ethanol to higher order alcohols is desired but difficult using current biotechnological methods. In this study, we designed a completely artificial reaction pathway for upgrading ethanol to acetoin, 2,3-butanediol, and 2-butanol in a cell-free bio-system composed of ethanol dehydrogenase, formolase, 2,3-butanediol dehydrogenase, diol dehydratase, and NADH oxidase. Under optimized conditions, acetoin, 2,3-butanediol, and 2-butanol were produced at 88.78%, 88.28%, and 27.25% of the theoretical yield from 100 mM ethanol, respectively. These results demonstrate that this artificial synthetic pathway is an environmentally-friendly novel approach for upgrading bio-ethanol to acetoin, 2,3-butanediol, and 2-butanol.

Full text of DOI:10.1039/c7gc02898a

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Page 1 of 42
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DOI: 10.1039/C7GC02898A
Artificial synthetic pathway for acetoin, 2,3-butanediol, and
2-butanol production from ethanol using cell free multi-enzyme
catalysis
†
†
†
Liaoyuan Zhang*,a,b, Raushan Singh,b, Sivakumar D,b Zewang Guo,a Jiahuan Li,a
Fanbin Chen,a Yuanzhi He,a Xiong Guan,a Yun Chan Kang,*,c and JungꢀKul Lee*,b
aKey Laboratory of Biopesticide and Chemical Biology, Ministry of Education,
College of Life Sciences, Gutian Edible Fungi Research Institute, Fujian Agriculture
and Forestry University, Fuzhou, Fujian province, 350002, PR China
bDepartment of Chemical Engineering, Konkuk University, Seoul 05029, Republic of
Korea
cDepartment of Materials Science and Engineering, Korea University, Seoul 02841,
Republic of Korea
†: These authors contributed equally to this work.
*Corresponding author: Key Laboratory of Biopesticide and Chemical Biology,
Fujian Agriculture and Forestry University, Ministry of Education, FuZhou, Fujian
Province, 350002, PR China Tel.: +86ꢀ591ꢀ83789492; Fax: +86ꢀ591ꢀ83789121;
Eꢀmail: zliaoyuan@126.com
*Corresponding author: Department of Materials Science and Engineering, Korea
University, Seoul 143ꢀ701, Republic of Korea Tel.: +82ꢀ2ꢀ4503505; Fax:
+82ꢀ2ꢀ4583504;
Eꢀmail: yckang@korea.ac.kr
*Corresponding author: Department of Chemical Engineering, Konkuk University,
Seoul 05029, Republic of Korea Tel.: +82ꢀ2ꢀ4503505; Fax: +82ꢀ2ꢀ4583504;
Eꢀmail: jkrhee@konkuk.ac.kr
† Electronic supplementary information
1

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Abstract
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3
Upgrading ethanol to higher order alcohols is desired but difficult by using
current biotechnological methods. In this study, we designed a completely artificial
reaction pathway for upgrading ethanol to acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol in a
cellꢀfree bioꢀsystem composed of ethanol dehydrogenase, formolase, 2,3ꢀbutanediol
dehydrogenase, diol dehydratase, and NADH oxidase. Under optimized conditions,
acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol were produced at 88.78%, 88.28%, and 27.25%
of the theoretical yield from 100 mM ethanol, respectively. These results demonstrate
an environmentꢀfriendly novel approach for upgrading bioꢀethanol to acetoin,
2,3ꢀbutanediol, and 2ꢀbutanol.
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The production of biofuels from renewable biomass has been given increased
attention because of the finite supply of fossil fuels.1–3 It is estimated that the global
biofuel market consists of approximately 15% biodiesel and 85% bioethanol.4,5
Bioethanol, a direct product of biomass fermentation, is considered a renewable
energy source that can replace or blend with gasoline for transportation use.6,7
Gasoline blended with the appropriate bioethanol as a fuel additive may increase the
octane level and reduce toxic emissions of pollutants.8 However, several
disadvantages including the low energy density, high hygroscopicity, and corrosivity
to engine technology and fuel pipelines limit the broad implementation of bioethanol
in global transportation.7,9,10 This has shifted research interests to higherꢀorder
alcohols such as 2,3ꢀbutanediol and butanol.7,11,12
2,3ꢀButanediol is an important platform chemical and potential aviation fuel with
a heating value of 27.2 kJ gꢀ1 and can be used to produce butadiene (a monomer of
synthetic rubber), acetoin (a volatile compound used in foods, plant growth promoters,
and biological pest controls), diacetyl (a flavor enhancer), and methyl ethyl ketone
(2ꢀbutanone, an excellent organic solvent).13ꢀ15 Furthermore, 2ꢀbutanone can be
hydrogenated to produce 2ꢀbutanol, which shows the highest octane number and
lowest boiling point among the four stereoisomers of butanol.1,16 Compared to
bioethanol, butanol has a higher energy density and lower hygroscopicity and can be
directly blended with gasoline without the need for modifying current vehicle
system.17,18 In recent years, many studies have been conducted to engineer microbes
for 1ꢀbutanol and isobutanol production through genetic modifications.19,20 However,
bioꢀproduction of 2ꢀbutanol has not been widely explored. Few studies of 2ꢀbutanol
production have been conducted by extending the terminal product of 2,3ꢀbutanediol
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or acetolactate using metabolic engineering methods.1,16,21 The final 2ꢀbutanol
concentration was still quite low because of metabolic flux limitations and its
inevitable toxicity to microbial cells.
One of the potential solutions for overcoming these limitations is to construct a
simplified pathway in vitro using a limited number of enzymes, a process known as
cellꢀfree metabolic engineering (CFME). CFME is a cellꢀfree bioꢀsystem that uses in
vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates
of cells to produce target products.22,23 It can efficiently eliminate cellꢀassociated
process barriers, such as substrate or product toxicity, intracellular flux balance that
results in low target product yield and unwanted byꢀproducts, and product excretion
constraints by intracellular transport barriers.4,22,24 Compared with traditional
metabolic engineering, CFME has many unique advantages including no requirement
for cell growth, shorter synthetic pathway, faster reaction rate, higher theoretical yield
and productivity, and easier manipulation of reaction conditions, although some
challenges such as activity, stability, and cost of enzymes must be resolved.25,26
The costs of acetoin (10,000–30,000 $/ton), 2,3ꢀbutanediol (10,000–50,000
$/ton), and 2ꢀbutanol (10,000–78,000 $/ton) are over 10ꢀfold that of ethanol (800–950
$/ton), according to the global trade data (www.alibaba.com). To upgrade ethanol to
higherꢀorder alcohols, we designed a completely artificial CFMEꢀbased reaction
pathway composed of ethanol dehydrogenase, formolase, 2,3ꢀbutanediol
dehydrogenase, diol dehydratase, and NADH oxidase for the conversion of ethanol
into C4 alcohols. Furthermore, protein engineering was conducted to improve the
catalytic efficiencies of formolase and diol dehydratase to increase the conversion rate
of this artificial pathway. In addition, a novel NAD(P)H purge valve regulatory node
was developed to prevent NADH buildup in the artificial reaction. The results showed
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that C4 compounds including acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol can be efficiently
produced from ethanol using the CFMEꢀbased artificial reaction pathway.
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Enzymes and chemicals
Restriction enzymes, DNA Polymerase High Fidelity and T4 DNA ligase, were
purchased from TaKaRa Biotech (Shiga, Japan) and New England Biolabs (Ipswich,
MA, USA), respectively. DNA and protein markers were obtained from Tiangen
Biotech (Shanghai, China). IsopropylꢀbetaꢀDꢀthiogalactopyranoside (IPTG),
dithiothreitol (DTT), and dimethyl sulfoxide (DMSO) were purchased from
SigmaꢀAldrich (St. Louis, MO, USA) and Sinopharm (Shanghai, China), respectively.
The standards, including (3S/3R)ꢀacetoin, (2S,3S)ꢀ2,3ꢀbutanediol, (2R,3R)ꢀ2,3ꢀ
butanediol, mesoꢀ2,3ꢀbutanediol, butanone, and 2ꢀbutanol, were obtained from
SigmaꢀAldrich. All other chemicals, unless otherwise indicated, were of analytical
grade and commercially available.
Bacterial strains, plasmids, and bacterial growth conditions
The strains and plasmids used in this study are presented in Table 1. Escherichia
coli DH5α and BL21(DE3) as the cloning and expression hosts were cultured at 37°C.
The plasmid pET28a was used to construct the expression vector. LuriaꢀBertani (LB)
medium was used for strain cultivation and recombinant protein expression.
Kanamycin was added to the LB medium for cultivation of recombinant strains at a
final concentration of 50 ꢁg mLꢀ1.
Recombinant proteins expression and purification
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The genes for ethanol dehydrogenase (EtDH), formolase (FLS), 2,3ꢀbutanediol
dehydrogenase (BDH), and diol dehydratase (DDH), and NADH oxidase (NOX) from
Cupriavidus necator,27 Pseudomonas fluorescens,28 Clostridium autoethanogenum,29
Lactobacillus brevis,1,30 and Lactobacillus rhamnosus31 were synthesized by General
Biosystems, Inc. (Anhui, China) and cloned into the expression plasmid pET28a (The
details of enzyme sequences are presented in the ESI). The protein expression
plasmids were introduced into E. coli BL21(DE3). Recombinant E. coli BL21(DE3)
harboring pETꢀEtDH, pETꢀFLS, pETꢀBDH, pETꢀDDH, pETꢀdhaR, pETꢀDDHꢀdhaR,
and pETꢀNOX were cultured at 37°C in LB medium and induced by adding 0.5 mM
IPTG when the optical density was 0.6 at 600 nm. After induction for 24 h at 18°C,
the cells were harvested by centrifugation and disrupted by sonication in an ice bath.
The cell lysate was centrifuged at 8000 ×g for 10 min to remove the cell debris. For
the EtDH, FLS, BDH, dhaR, and NOX enzymes, the soluble fraction was subjected to
purification with a HisTrap HP column according to the purification protocol (GE
Healthcare, Little Chalfont, UK). DDH purification was carried out as described
previously.32 The purified enzymes were concentrated and desalted by ultrafiltration,
and then detected by SDSꢀPAGE.
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Development of the enzyme variants
The EtDH:D46G and BDH:S199A variants were generated by siteꢀdirected
mutagenesis, which was performed using the primers EtDH1/EtDH2 and
BDH1/BDH2 as shown in Table S1†. The recombinant plasmids pETꢀEtDH and
pETꢀBDH containing the wildꢀtype EtDH and BDH genes were used as DNA
templates for PCRꢀamplification, respectively. Recombinant plasmids harboring the
correct mutant genes were transformed into E. coli BL21(DE3) and colonies selected
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via kanamycin resistance were used for protein expression. After purification, the
activities and kinetic parameters of the EtDH and BDH variants were determined.
To improve the catalytic efficiency of the FLS enzyme, the HotSpot Wizard 2.0
server was used to analyze hot spots by inputting the FLS structure (PDB No.:
4QPZ).28,33 Six residuals as hot spots (T396, T446, M473, S477, L482, and L499)
were identified and subjected to siteꢀsaturated mutagenesis using the primers
FLS1–FLS12 (Table S1†). The recombinant plasmid pETꢀFLS containing the
wildꢀtype FLS gene was used as a DNA template. Recombinant plasmids containing
the mutant genes were transformed into E. coli BL21(DE3). FLS variants were
screened using the wholeꢀcell biocatalytic method with acetaldehyde as a substrate.
Briefly, the colonies were inoculated in LB medium and protein expression was
induced by adding 0.5 mM IPTG for 24 h at 18°C when the optical density at 600 nm
reached 0.6. The cells were harvested by centrifugation and used to carry out
wholeꢀcell biocatalysis in a reaction mixture containing 50 mM phosphate buffer (pH
8.0), 100 mM acetaldehyde, and 40 g Lꢀ1 wet cell weight (WCW) at 30°C for 6 h. The
product acetoin was quantified using the VogesꢀProskauer (VP) reaction (See the ESI).
Moreover, positive variants were verified by enzyme activity and kinetic parameter
assays after purification. Variants were also analyzed by commercial sequencing.
DDH variants including S302A, Q337A, F375I, S302A/Q337A, S302A/F375I,
Q337A/F375I, and S302A/Q337A/F375I were developed to assess their catalytic
efficiencies compared with the wildꢀtype DDH enzyme. Siteꢀdirected mutagenesis
was performed using the primers DDH1ꢀDDH6 as shown in Table S1†. The
recombinant plasmid pETꢀDDHꢀdhaR harboring the wildꢀtype DDH and its
reactivating factor dhaR genes were used as DNA templates for PCR amplification.
The PCR products were transformed into E. coli BL21(DE3) for protein expression,
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which was conducted in LB medium containing 0.5 mM IPTG at 18°C for 24 h. These
variants were used to evaluate the catalytic activities using wholeꢀcell biocatalysis
with mesoꢀ2,3ꢀbutanediol as a substrate. The reaction mixture consisted of 50 mM
HEPES buffer (pH 7.0), 50 mM mesoꢀ2,3ꢀbutanediol, 20 ꢁM coenzyme B12, and 40 g
Lꢀ1 wet cell weight, and wholeꢀcell biocatalysis was carried out at 30°C for 6 h in the
dark. The product butanone was analyzed and quantified by gas chromatography.
Enzyme activity assays
EtDH activity: The activities of EtDH and its variant were determined in a
reaction mixture containing 100 mM glycineꢀNaOH buffer (pH 9.5), 5 mM Mg2+, 3
mM NAD+/NADP+, and 10 mM ethanol at 25°C. The activity was defined by the
reduction rate of NAD+/NADP+ at 340 nm using a spectrophotometer (UVꢀ1800,
MAPADA, Shanghai, China). One unit of EtDH activity was defined as the amount of
enzyme required to reduce 1 ꢁmol of NAD+/NADP+ per minute.
FLS activity: The activities of FLS and its variants were assayed in a reaction
mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM Mg2+, 0.1 mM TPP, and
20 mM acetaldehyde. After the reaction was conducted at room temperature for 1 h,
acetoin concentration from acetaldehyde was determined by the VP reaction and
calculated from calibration curves of standard acetoin. One unit of FLS activity was
defined as the amount of enzyme that produced 1 ꢁmol acetoin from acetaldehyde per
minute.
BDH activity: The activities of BDH and its variant were measured in a reaction
mixture containing 50 mM TrisꢀHCl buffer (pH 7.5), 0.2 mM NADPH, 1 mM DTT,
and 20 mM acetoin or 5 mM butanone at room temperature. The activity was defined
by the oxidation rate of NADPH at 340 nm using a spectrophotometer (UVꢀ1800,
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MAPADA). One unit of BDH activity was defined as the amount of enzyme required
to oxidize 1 ꢁmol of NADPH per minute.
DDH activity: The activities of DDH and its variants with its reactivating factor
dhaR were determined in a reaction mixture containing 50 mM phosphate buffer (pH
7.0), 1 mM coenzyme B12, 100 mM ATP, 1 mM Mg2+, and 50 mM
mesoꢀ2,3ꢀbutanediol. After conversion at room temperature in the dark for 1 h, the
reaction was stopped by adding an equal volume of citrate buffer (100 mM, pH 3.6).
The product butanone was detected and quantified by gas chromatography. One unit
of DDH activity was defined as the amount of enzyme that produces 1 ꢁmol butanone
from mesoꢀ2,3ꢀbutanediol per minute.
NOX activity: The NOX activity was determined in a reaction mixture
containing 50 mM HEPESꢀNaOH buffer (pH 8.0) and 0.2 mM NADH at room
temperature. The activity was defined by the oxidation rate of NADH at 340 nm using
a spectrophotometer (UVꢀ1800, MAPADA). One unit of NOX activity was defined as
the amount of enzyme required to oxidize 1 ꢁmol of NADH per minute.
Determination of kinetic parameters
The kinetic parameters of EtDH and EtDH:D46G were determined in a reaction
mixture containing 100 mM glycineꢀNaOH buffer (pH 9.5), 5 mM Mg2+, 3 mM
NAD+/NADP+, and 0.5ꢀ100 mM ethanol at room temperature. The Km and kcat values
were obtained by nonlinear regression fitting of the MichaelisꢀMenten equation. All
assays were carried out in triplicate.
The kinetic parameters of FLS and its variants were assayed in a reaction mixture
containing 100 mM phosphate buffer (pH 8.0), 1 mM Mg2+, 0.1 mM TPP, and 0.5ꢀ20
mM acetaldehyde at room temperature. The Km and kcat values were obtained by
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nonlinear regression fitting of the MichaelisꢀMenten equation. All assays were carried
out in triplicate.
The kinetic parameters of BDH and BDH:S199A were measured in a reaction
mixture containing 50 mM TrisꢀHCl buffer (pH 7.5), 0.2 mM NADPH, 1 mM DTT,
and 0.5ꢀ100 mM acetoin or 0.5–10 mM butanone at room temperature. The Km and
kcat values were obtained by nonlinear regression fitting of the MichaelisꢀMenten
equation. All assays were carried out in triplicate.
Cell-free multi-enzyme catalysis system
The synthesis of acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol from ethanol by cellꢀfree
multiꢀenzyme catalysis was conducted in a 0.5ꢀmL reaction mixture containing
substrate, coenzymes, metal ions, and the corresponding enzymes. The reaction
conditions including temperature, pH, coenzyme, and metal ions were optimized to
improve the flux of the artificial reaction pathway (See the ESI). The optimal reaction
conditions for acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol from ethanol were as follows.
Acetoin production was carried out in a 0.5ꢀmL reaction mixture containing 50
mM HEPES buffer (pH 8.0), 1 mM NAD+, 0.1 mg mLꢀ1 EtDH, 0.2 mg mLꢀ1
FLS:L482S, 0.1 mg mLꢀ1 NOX, 0.1 mM TPP, 1 mM Mg2+, 1 mM DTT, 20 % DMSO,
and 100 mM ethanol. The reaction was conducted at 30 °C.
2,3ꢀButanediol was produced in a 0.5ꢀmL reaction mixture containing 50 mM
HEPES buffer (pH 8.0), 1 mM NAD+, 1 mM NADP+, 0.1 mg mLꢀ1 EtDH:D46G, 0.2
mg mLꢀ1 FLS:L482S, 0.1 mg mLꢀ1 NOX, 0.1 mg mLꢀ1 BDH:S199A, 0.1 mM TPP, 1
mM Mg2+, 1 mM DTT, 20 % DMSO, and 100 mM ethanol. The reaction was
conducted at 30 °C.
2ꢀButanol was produced in a 0.5ꢀmL reaction mixture containing 50 mM HEPES
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buffer (pH 8.0), 1 mM NAD+, 1 mM NADP+, 0.1 mg mLꢀ1 EtDH:D46G, 0.2 mg mLꢀ1
FLS:L482S, 0.1 mg mLꢀ1 NOX, 0.1 mg mLꢀ1 BDH:S199A, 0.2 mg mLꢀ1
DDH:Q337A/F375I, 0.2 mg mLꢀ1 dhaR, 0.1 mM TPP, 1 mM Mg2+, 1 mM DTT, 20 %
DMSO, 1 mM coenzyme B12, 100 mM ATP, and 100 mM ethanol. The reaction was
carried out at 30 °C.
All reactions were carried out for 6 h and the products were identified by
gas chromatography (GC). The product percentage yields were calculated using the
following equation: percentage yield (%) = product yield (mM)/theoretical yield
(mM). Theoretically, two moles of ethanol can generate one mole of acetoin or
2,3ꢀbutanediol or 2ꢀbutanol.
Recyclability of cascade reactions
Purified enzymes were mixed with activated silicon oxide nanoparticles and
incubated for 12 h at 4°C. Before immobilization, the silicon oxide nanoparticles
(4830HT; Nanostructured & Amorphous Materials, Houston, TX, USA) were
activated by treating the nanoparticles with glutaraldehyde (Sigma). Immobilization
yield (%) and immobilization efficiency (%) were calculated for the immobilized
enzymes as follows: immobilization efficiency = (αi/αf) ×100, immobilization yield =
[{Piꢀ(Pw + Ps)}/Pi] × 100, where αi is the total activity of the immobilized enzyme and
αf is the total activity of the free enzyme. Pi is the total protein content of the crude
enzyme preparation and Pw and Ps are the protein concentrations in the wash solution
and supernatant after immobilization, respectively.
For acetoin production, the reaction was performed in a 0.5ꢀmL reaction mixture
containing 50 mM HEPES buffer (pH 8.0), 1 mM NAD+, 1.06 U mLꢀ1 EtDH, 0.05 U
mLꢀ1 FLS:L482S, 0.98 U mLꢀ1 NOX, 0.1 mM TPP, 1 mM Mg2+, 1 mM DTT, 20%
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DMSO, and 100 mM ethanol. Similarly, 2,3ꢀbutanediol production was performed in
a 0.5ꢀmL reaction mixture containing 50 mM HEPES buffer (pH 8.0), 1 mM NAD+, 1
mM NADP+, 0.1 mg mLꢀ1 EtDH:D46G, 0.2 mg mLꢀ1 FLS:L482S, 0.1 mg mLꢀ1 NOX,
0.1 mg mLꢀ1 BDH:S199A, 0.1 mM TPP, 1 mM Mg2+, 1 mM DTT, 20% DMSO, and
100 mM ethanol. The reaction was conducted at 30°C for 6 h. The reusability of
immobilized enzymes was examined under the same reaction conditions. After each
reaction cycle, the immobilized enzyme was removed by centrifugation at 4000 ×g for
30 min. The immobilized enzyme was collected and washed with deionized water and
buffer. For the second reaction cycle, the immobilized enzyme was dissolved in fresh
buffer, added to the substrate, and processed as in the first cycle.
Analytical methods
Cell growth was determined by measuring the optical density at 600 nm with a
spectrophotometer (UVꢀ1800, MAPADA). Protein concentration was determined
using the Bradford method, and bovine serum albumin served as the standard protein.
The concentrations of ethanol, acetaldehyde, acetoin, 2,3ꢀbutanediol, butanone, and
2ꢀbutanol were determined with the addition of isoamylol as an internal standard and
quantified using a gas chromatograph system (Agilent GC9860, Santa Clara, CA,
USA) equipped with a chiral column (Supelco βꢀDEX™ 120, 30ꢀm length, 0.25ꢀmm
inner diameter). The operation conditions were as follows: N2 was used as the carrier
gas at flow rate of 1.2 mL minꢀ1; injector temperature and detector temperature were
215 and 245°C, respectively; and column temperature was maintained at 50°C for 1.5
min, and then increased to 180°C at a rate of 15°C minꢀ1.
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Construction of artificial pathway for C4 compound production from ethanol
We designed a novel artificial reaction pathway for the conversion of ethanol into
the C4 compounds acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol, which required cascade
enzymes (Fig. 1). Ethanol is first dehydrogenated by the NAD(P)Hꢀdependent EtDH
to afford acetaldehyde, which undergoes a condensation reaction to yield acetoin by
FLS, a computationally designed enzyme. Subsequently, acetoin is reduced by the
NADPHꢀdependent BDH to produce 2,3ꢀbutanediol. Finally, 2ꢀbutanol is obtained via
dehydration and hydrogenation reaction by DDH and BDH, respectively. NOX is used
to regenerate NAD+ throughout the reaction process. Considering that oxygen is used
as a substrate for NOX, aeration may be required for a largeꢀscale reaction. By
stoichiometry, 1 M ethanol is converted completely into acetoin or 2,3ꢀbutanediol and
generates 1 or 0.5 M NADH, respectively, which requires 0.5 M oxygen for acetoin
production or 0.25 M oxygen for 2,3ꢀbutanediol production (1 M NADH oxidized by
NOX requires 0.5 M oxygen as a substrate). Thus, 1 L of the reaction mixture
containing 1 M ethanol as a substrate requires 11.2 or 5.6 L oxygen for acetoin or
2,3ꢀbutanediol production throughout the reaction process. For 2ꢀbutanol production,
the oxygen demand in the reaction was lower than that of 2,3ꢀbutanediol production.
Compared to traditional fermentation, the aeration demand in the current reaction
system is much lower supporting the scaleꢀup potential of the artificial reaction
pathway. In this artificial reaction pathway, we chose EtDH from C. necator,27 BDH
from C. autoethanogenum,29 DDH from L. brevis1,30 with its reactivating factor dhaR,
and NOX enzymes from L. rhamnosus31 as candidate enzymes because of their
relatively high catalytic efficiencies for their corresponding substrates as
demonstrated in previous studies. However, the FLS enzyme was only reported to
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catalyze three molecules of formaldehyde into one molecule of dihydroxyacetone.28
To verify the catalytic ability of acetaldehyde conversion to acetoin by the FLS
enzyme, the catalytic reaction was carried out using wholeꢀcell biocatalyst
overꢀexpressing FLS with acetaldehyde as a substrate. The VP test and GC/GCꢀMS
analysis indicated that racemic acetoin ((3S)ꢀacetoin and (3R)ꢀacetoin) was produced
from acetaldehyde, demonstrating that the FLS enzyme can catalyze the conversion of
acetaldehyde into acetoin (Fig. S1†, Fig. S14† and Fig. S15†). These results show that
C4 compounds including acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol can be produced from
ethanol using the artificial reaction pathway.
Characterization of enzymes in the artificial pathway
Furthermore, to determine the catalytic efficiencies of these enzymes in the
artificial pathway, we systematically determined the kinetic parameters of EtDH, FLS,
BDH, and DDH after purification (The results of enzyme expression and purification
are presented in the ESI, Fig. S2†, and Fig. S3†). As shown in Table 2, the kcat/Km
values of EtDH for ethanol with NAD+, FLS for acetaldehyde with TPP, and BDH for
acetoin/butanone with NADPH were 17.09, 7.69 × 10ꢀ3, 18.50, and 19.31 sꢀ1 mMꢀ1,
respectively. However, purified DDH enzyme with its reactivating factor dhaR
showed low activity (0.02 U mgꢀ1) towards mesoꢀ2,3ꢀbutanediol as a substrate, partly
because of its low stability.32 A wholeꢀcell catalytic method was used to determine the
conversion of three 2,3ꢀbutanediol isomers (mesoꢀ2,3ꢀbutanediol, (2R,
3R)ꢀ2,3ꢀbutanediol, and (2S, 3S)ꢀ2,3ꢀbutanediol) to butanone by E. coli/
pETꢀDDHꢀdhaR cells. The results showed that 20.56 mM butanone was produced
from 50 mM mesoꢀ2,3ꢀbutanediol after 6 h (Fig. S4†), indicating that the DDH
enzyme with dhaR had high catalytic activity towards mesoꢀ2,3ꢀbutanediol in vivo. In
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contrast, no butanone was detected during wholeꢀcell catalysis in the presence of (2R,
3R)ꢀ2,3ꢀbutanediol and (2S, 3S)ꢀ2,3ꢀbutanediol as substrates (Fig. S4†). These results
are consistent with those of a previous study showing that only mesoꢀ2,3ꢀbutanediol
was dehydrated by DDH enzyme.34 The enzyme thermostability assay showed that the
three enzymes had relatively higher stabilities and retained 87.91% (EtDH), 70.43%
(FLS), and 91.30% (NOX) of their initial activities after incubation at 30°C for 6 h,
while all enzymes except NOX showed decreased stability at 37 and 45°C (Fig. S5†).
Acetoin production from ethanol via artificial pathway
Rate limiting step for acetoin production from ethanol. To test the viability of
the artificial pathway, we first investigated acetoin production from ethanol using
three enzymes, EtDH, FLS, and NOX, as presented in Fig. 1A. The initial reaction
was carried out in a 0.5ꢀmL reaction mixture containing 50 mM HEPES buffer (pH
7.0), 0.1 mg mLꢀ1 EtDH, 0.2 mg mLꢀ1 FLS, 0.1 mg mLꢀ1 NOX, 4 mM NAD+, 0.1 mM
TPP, 1 mM Mg2+, 1 mM DTT, 20% DMSO, and 100 mM ethanol as the starting
substrate. The reaction was conducted at 30°C for 6 h and 17.98 mM of acetoin at
35.96% of the theoretical yield was generated in the reaction solution after 6 h,
suggesting that the reaction pathway for upgrading ethanol to acetoin is feasible.
Further, the reaction conditions including temperature, pH, coenzyme (NAD+ and
TPP), and metal ions were optimized to improve the reaction flux from ethanol to
acetoin. The optimal reaction conditions including temperature (30°C), pH (8.0),
NAD+ (1 mM), TPP (0.1 mM), and Mg2+ (1 mM) were determined (Fig. S6†). Under
the optimized conditions, 22.75 mM of acetoin at 45.50% of the theoretical yield was
produced from ethanol (100 mM) after 6 h (Fig. S7†). To determine the rateꢀlimiting
step in the reaction, the reaction pathway was systematically reconstituted with 1/10
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the starting amount of each enzyme, while the other enzymes were kept constant. The
results are shown in Fig. 2. For EtDH and NOX, lowering the concentration to 10%
had minimal effects on the production of acetoin from ethanol. The exception was
FLS, indicating that FLS represents potential bottlenecks.
Engineering of FLS to improve its activity. To improve the catalytic
efficiency of the FLS enzyme, mutational hotspots in the FLS amino acid sequence
were analyzed using the HotSpot Wizard 2.0 server.33 Six hot spot residues (T396,
T446, M473, S477, L482, and L499) were identified and altered by siteꢀsaturated
mutagenesis (Fig. S8†). The results revealed that site 482 in FLS plays an important
role in enzyme activity. The FLS variants L482S, L482R, and L482E showed specific
activity increases of 59.03%, 36.89%, and 34.12%, respectively (Fig. 3 and Table
S2†). Furthermore, the kinetic parameters of the three FLS variants were assayed
using the substrate acetaldehyde with TPP as a coenzyme. The kcat/Km values of the
FLS variants L482S, L482R, and L482E for acetaldehyde were 1.33 × 10ꢀ2, 1.06 ×
10ꢀ2, and 9.66 × 10ꢀ3 with improvements of 72.95%, 37.84%, and 25.62%,
respectively, compared to wildꢀtype FLS (Table 2).
The mutant FLS:L482S showed higher activity than wildꢀtype FLS.
Computational studies for FLS variants at atomic resolution using molecular
dynamics simulation for 100 ns provided insight into the structural changes for the
mutation L482S and its correlation with enzymatic activity. Computational analysis
revealed that W480 in the mutant FLS:L482S made stronger contacts (2.1Å) with the
substrate acetaldehyde than the wildꢀtype (2.8Å) (Fig. S9AꢀB†). Siegel et al (2015)
reported W480 as an active site residue in FLS (4QPZ), supporting the higher activity
of FLS:L482S.28 The 100ꢀns molecular dynamics analysis also shows that the
FLS:L482S retained more hydrogen bond contacts than the wildꢀtype enzyme (Fig.
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S9CꢀD†).
Acetoin production from ethanol using mutant FLS:L482S The FLS:L482S
rather than the wildꢀtype enzyme was used to conduct the reaction from ethanol to
acetoin under optimized reaction conditions. As shown in Fig. 4A, a maximum
concentration of acetoin (44.39 mM) was obtained from ethanol (100 mM) at 4 h,
with an acetoin yield of 88.78% of the theoretical yield. In addition to acetoin, the
substrate ethanol and intermediate acetaldehyde were measured during the reaction.
The substrate ethanol was rapidly consumed and 6.25 mM residual ethanol remained
in the reaction solution at 4 h. Acetaldehyde accumulated up to 9.58 mM during the
first 2 h of the reaction, and then decreased to 5.65 mM at 6 h. No accumulation of
other undesired byꢀproducts was detected, indicating that the artificial synthetic
pathway was specific for the substrate. Subsequently, the effects of substrate
concentrations (200, 300, 400, and 500 mM) on acetoin production were investigated
using the optimized reaction mixture. As shown in Fig. 4B, the acetoin concentration
was increased by increasing the initial substrate concentration. A maximum acetoin
concentration of 117.5 mM was achieved at an ethanol concentration of 400 mM after
6 h. However, acetoin yield was only 58.71% of its theoretical yield. The intermediate
acetaldehyde showed significant accumulation when the initial ethanol concentration
was increased, suggesting that the FLS:L482S enzyme limits the productivity of this
CFMEꢀbased artificial pathway.
2,3-Butanediol production from ethanol via artificial pathway
NAD(P)H purge valve regulatory node to prevent NADH buildup.
Subsequently, we investigated 2,3ꢀbutanediol production from ethanol with the
enzyme BDH added to the CFME system. However, the artificial pathway from
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ethanol to 2,3ꢀbutanediol generates more reducing equivalents than is required to
produce 2,3ꢀbutanediol (1 NADH per ethanol, with 0.5 NADH required). Herein, we
designed a NAD(P)H purge valve regulatory node to prevent the buildup of NADH.
As shown in Fig. 1B, we developed the mutant enzyme EtDH (D46G) to
simultaneously utilize NAD+ and NADP+ as coenzymes in the conversion of ethanol
to acetaldehyde by siteꢀdirected mutagenesis according to the results of sequence
alignment (Table 2 and Fig. S10†). Additionally, NADPHꢀdependent BDH1,29 from C.
autoethanogenum was used to catalyze acetoin into 2,3ꢀbutanediol in the presence of
NADPH. During the reaction process, NADH produced was rapidly recycled back to
NAD+ via the H2Oꢀforming NOX, while NADPH generated was used to produce
2,3ꢀbutanediol from acetoin. When NADP+ levels were high (NADPH low), the
mutant enzyme EtDH:D46G regenerated NADPH via the oxidation of ethanol.
However, when NADPH levels were high (NADP+ low), BDH generated
2,3ꢀbutanediol from acetoin and regenerate NADP+ without the buildup of additional
reducing equivalents (NADH produced from ethanol to acetaldehyde immediately
recycled for purging the excess). To achieve high catalytic efficiency, the mutant BDH
enzyme (S199A) was developed via siteꢀspecific mutagenesis as reported
previously.35
Characterization of mutants EtDH and BDH in the artificial pathway. As
shown in Table 2 and Table S2†, the wildꢀtype EtDH enzyme only employed NAD+ as
a coenzyme for ethanol oxidation with 17.09 sꢀ1 mMꢀ1 of the kcat/Km value, whereas its
variant EtDH:D46G simultaneously used NAD+ and NADP+ as coenzymes and the
kcat/Km values for ethanol with NAD+ and NADP+ were 9.97 and 1.65 sꢀ1 mMꢀ1,
respectively. Compared with the wildꢀtype NADPHꢀdependent BDH enzyme, the
kcat/Km value of its variant BDH:S199A for acetoin with NADPH was up to 19.31 sꢀ1
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mMꢀ1 (Table 2 and Table S2†). In addition, the enzyme thermostability of
EtDH:D46G and BDH:S199A was assayed at 30, 37, and 45 °C. EtDH:D46G activity
levels of 86.53% and 86.67% were retained after incubation at 30°C for 6 h when
acetaldehyde was used as a substrate with the coenzymes NAD+ and NADP+,
respectively (Fig. S5†). For the BDH:S199A enzyme, 80.81% of the initial enzyme
activity for acetoin with the coenzyme NADPH was retained at 30 °C for 6 h (Fig.
S5†).
2,3-Butanediol production from ethanol via improved artificial pathway.
The four enzymes, EtDH:D46G, FLS:L482S, BDH:S199A, and NOX, were reacted
with ethanol in the presence of 1 mM NAD+ and/or 1 mM NADP+. As shown in Fig.
S11†, no 2,3ꢀbutanediol was detected when NAD+ was used as a coenzyme in the
reaction system. This was primarily because BDH is an NADPHꢀdependent enzyme
in the conversion of acetoin to 2,3ꢀbutanediol. In contrast, 18.20 mM 2,3ꢀbutanediol
was produced from 100 mM ethanol as a substrate in the presence of NADP+,
indicating that NADP+ is required for 2,3ꢀbutanediol production in the artificial
cascade reaction. When 1 mM NAD+ and 1 mM NADP+ were simultaneously used in
the reaction, a maximum 2,3ꢀbutanediol concentration of 44.14 mM at 88.28 % of the
theoretical yield was produced from 100 mM ethanol after 5 h (Fig. 5A). The result
showed that use of the NAD(P)H purge valve regulatory node for preventing the
buildup of NADH was feasible and efficient. Throughout the reaction process, low
levels of acetoin accumulated in the reaction solution, demonstrating high catalytic
efficiency from acetoin to 2,3ꢀbutanediol by BDH:S199A (Fig. 5A). Furthermore,
different substrate concentrations were used to investigate the effects on
2,3ꢀbutanediol production in the reaction pathway. The maximum 2,3ꢀbutanediol
concentration was 127.3 mM when the initial ethanol concentration in the reaction
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mixture was 500 mM (Fig. 5B). However, 2,3ꢀbutanediol yield decreased with
increasing initial ethanol concentration, although higher 2,3ꢀbutanediol concentration
was obtained. Analysis of the intermediates in the reaction solution revealed
accumulation of a large amount of acetaldehyde in the reaction system, indicating that
the conversion of acetaldehyde to acetoin by the FLS:L482S enzyme remained the
rateꢀlimiting step. Compared with the results for acetoin production from ethanol, the
concentration of acetaldehyde in the 2,3ꢀbutanediol production system was
significantly decreased (Fig. 4B and Fig. 5B), suggesting that FLS:L482S enzyme
activity is partially inhibited by the product acetoin. However, the relatively low
catalytic efficiency of the FLS:L482S enzyme remained the major cause of
acetaldehyde accumulation.
2-Butanol production from ethanol
Construction of an artificial pathway for 2-butanol production from ethanol.
Finally, we employed an additional B12ꢀdependent DDH to convert
mesoꢀ2,3ꢀbutanediol into butanone,30,32,36 which can further be transformed into
2ꢀbutanol by BDH:S199A from C. autoethanogenum as shown in Fig. 1C.35 Previous
studies showed that DDH enzyme with its reactivating factor dhaR from L. brevis was
the best candidate for catalyzing mesoꢀ2,3ꢀbutanediol into butanone in vivo.1 To
determine its reaction conditions in vitro, the effects of four factors including
coenzyme B12, ATP, Mg2+, and dhaR on the conversion of mesoꢀ2,3ꢀbutanediol to
butanone by DDH were investigated. The results suggested that the coenzyme B12
and ATP were required for the catalytic reaction, whereas dhaR and Mg2+ efficiently
enhanced butanone production from mesoꢀ2,3ꢀbutanediol (Table S3†). Therefore,
coenzyme B12, ATP, Mg2+, and dhaR were supplemented into the artificial reaction
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pathway for efficient butanone production from mesoꢀ2,3ꢀbutanediol. To convert
butanone to 2ꢀbutanol, the BDH:S199A enzyme was used because it can reduce
acetoin and butanone into 2,3ꢀbutanediol and 2ꢀbutanol, respectively.35 As shown in
Table 2 and Table S2†, the BDH:S199A enzyme showed high catalytic efficiency for
the substrate butanone with NADP+ (kcat/Km =37.76 sꢀ1 mMꢀ1), which was improved
by 145.0% compared to wildꢀtype BDH. Considering that 2ꢀbutanol exists in two
forms (Rꢀ2ꢀbutanol and Sꢀ2ꢀbutanol), we also analyzed the configuration of the
product from butanone by BDH:S199A. As shown in Fig. S16†, Rꢀ and Sꢀ2ꢀbutanol in
a ratio of 1.23:1 were produced from butanone by BDH:S199A at 30 °C after 6 h.
Theoretically, two moles of ethanol are oxidized to generate two moles of
reducing equivalents, which satisfies the need for the reduction of acetoin and
butanone into 2,3ꢀbutanediol and 2ꢀbutanol in the artificial pathway (Fig. 1C).
However, the DDH enzyme only catalyzed mesoꢀ2,3ꢀbutanediol into butanone,
whereas (2R,3R)ꢀbutanediol and (2S,3S)ꢀbutanediol are not substrates for DDH (Fig.
S4†). Thus, reducing equivalents would be excessive if the products from acetoin by
BDH were other forms of 2,3ꢀbutanediol except mesoꢀ2,3ꢀbutanediol. Therefore, the
configurations of acetoin and 2,3ꢀbutanediol were determined using a GC system
equipped with a chiral column. Chiral analysis indicated that 2,3ꢀbutanediol generated
from ethanol contained mesoꢀ2,3ꢀbutanediol (65%) and (2R,3R)ꢀbutanediol (35%)
from (3S)ꢀacetoin and (3R)ꢀacetoin, which were produced from acetaldehyde by
FLS:L482S (Fig. S14† and Fig. S15†). Considering that only mesoꢀ2,3ꢀbutanediol
was converted into butanone by DDH,34 we used the same strategy for 2ꢀbutanol
production as 2,3ꢀbutanediol production from ethanol by employing an NAD(P)H
purge valve regulatory node to prevent the buildup of NADH (Fig. 1C).
Rate limiting step for 2-butanol production from ethanol. The reaction for
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2ꢀbutanol production was carried out to determine the rateꢀlimiting step using
EtDH:D46G, FLS:L482S, BDH:S199A, DDH with its reactivating factor dhaR, and
NOX enzymes. The rateꢀlimiting experiment showed that only 1.84 mM 2ꢀbutanol
with a significant decrease of 79.16% was obtained from 100 mM ethanol when 1/10
concentration of the DDH enzyme was used, whereas lowering the concentration of
BDH:S199A to 10% had a little effect on 2ꢀbutanol production (Fig. 6A). This
demonstrates that DDH became a potential rateꢀlimiting factor in the artificial reaction
for 2ꢀbutanol production. Based on previous studies,1,30 S302A, Q337A, F375I, and
their combinatorial variants in the DDH enzyme were developed by siteꢀdirected
mutagenesis. The results of wholeꢀcell catalysis indicated that the mutant Q337A and
F375I DDH enzymes with dhaR produced 27.20 and 25.63 mM butanone from 50
mM mesoꢀ2,3ꢀbutanediol, while 19.76 mM butanone was obtained using the wildꢀtype
DDH enzyme with dhaR. Furthermore, a maximum butanone concentration of 30.35
mM, which was an increase of 53.60%, was observed by the combinatorial variant of
DDH:Q337A/F375I with its reactivating factor dhaR (Fig. 6B and Fig. S12†).
Computational analysis showed that proton donor residue E171 of the double
mutant DDH (Q337A/F375I) (1.9Å) contacted the substrate 2,3ꢀbutanediol more
strongly than the wildꢀtype (2.6Å) (Fig. S13AꢀB†). E171 showed more hydrogen
bond contacts with the substrate in the double mutant compared to the wildꢀtype.
Longꢀrange simulation (100 ns) results also showed that the substrate had stronger
interaction networks including hydrogen bonding and water bridges with E171 in the
double mutant compared to in the wildꢀtype (Fig. S13C–D†).
2-Butanol production from ethanol by improved artificial pathway.
DDH:Q337A/F375I with dhaR rather than wildꢀtype DDH was used to perform the
cascade reaction from ethanol to 2ꢀbutanol (Fig. 1C). As shown in Fig. 7, 13.62 mM
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2ꢀbutanol at 27.24% of the theoretical yield was produced from 100 mM ethanol after
6 h in the cellꢀfree bioꢀsystem. Furthermore, high levels of 2,3ꢀbutanediol (up to 32.55
mM) accumulated in the reaction solution, whereas butanone was not detected during
the reaction process. These results indicate that the conversion of 2,3ꢀbutanediol to
butanone by DDH:Q337A/F375I was a rateꢀlimiting step. Although the wholeꢀcell
biocatalyst coꢀexpressing the DDH:Q337A/F375I enzyme and its reactivating factor
dhaR exhibited higher catalytic efficiency for the conversion of mesoꢀ2,3ꢀbutanediol
(50 mM) to butanone (30.35 mM), the in vitro enzyme activity assay showed a low
specific activity of 0.05 U mgꢀ1 for mesoꢀ2,3ꢀbutanediol (Table S2† and Fig. 6B). The
thermal instability of the DDH:Q337A/F375I enzyme also limited the conversion of
2,3ꢀbutanediol to butanone. As shown in Fig. S5†, the DDH:Q337A/F375I enzyme
only retained 1.21% of its initial activity after incubation at 30°C for 6 h, and no
activity was observed when the enzyme was incubated at 37 and 45°C for 6 h. During
the reaction process, 2ꢀbutanol production rapidly increased during the first 4 h and
then stopped increasing after 5 h, likely because of inactivation (Fig. 7). Therefore,
further improving the enzyme activity and stability of DDH by protein engineering or
searching for a new enzyme with high catalytic efficiency to convert 2,3ꢀbutanediol to
butanone would improve 2ꢀbutanol yield in the artificial synthetic pathway.
Recyclability of cascade reactions
The recyclability of the catalytic system is an attractive parameter that dictates
the economic feasibility of using these enzymes in the cellꢀfree bioꢀsystem. To
evaluate the recyclability of the catalytic system, the enzymes for acetoin production
(EtDH, FLS:L482S, and NOX) and 2,3ꢀbutanediol production (EtDH:D46G,
FLS:L482S, BDH:S199A, and NOX) were immobilized on glutaraldehydeꢀ
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functionalized silicon oxide nanoparticles.37,38 Immobilized enzymes showed more
than 90% immobilization efficiency. Acetoin production by the immobilized enzymes
was evaluated and a recyclability test of the catalytic system was performed (Fig. 8).
Enzymes immobilized on silicon oxide nanoparticles exhibited 94% of their initial
product concentrations, even after 10 cycles of reuse to produce acetoin. This
indicates that there was no significant decrease in acetoin production using the
immobilized enzymes during repeated use. Additionally, the enzymes immobilized on
silicon oxide nanoparticles were stable because of the covalent linkages between the
enzymes and silicon oxide nanoparticles. Similarly, 2,3ꢀbutanediol production by
immobilized enzymes and recyclability of the catalytic system were evaluated (Fig. 8).
Here, the immobilized enzymes exhibited 73% of their initial 2,3ꢀbutanediol
production activity after 10 cycles of reuse. Upon immobilization, biocatalysts
catalyzed the production of acetoin and 2,3ꢀbutanediol for many cycles without
significant losses of activity. Hence, the immobilized catalytic system can be
efficiently recycled for industrial production of acetoin and 2,3ꢀbutanediol.
Unfortunately, we were not able to produce 2ꢀbutanol under the same experimental
conditions because of the low stability of DDH.
In recent years, acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol as platform chemicals
have gained more attention because of their increasing market demands. The
derivatives of 2,3ꢀbutanediol have a potential global market of approximately 32
million tons per annum.29 As a precursor of 2,3ꢀbutanediol, acetoin has been classified
as one of the 30 platform chemicals that are given priority for development and
utilization by the US Department of Energy, and its market demand has reached more
than 10 kilotons per annum,39 while the market capacity of bioꢀbutanol is
approximately 5 million tons per annum.40 Previous studies have shown that acetoin
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and 2,3ꢀbutanediol could be produced by microbial fermentation or chemical
synthesis.39,41 Compared to chemical synthesis, microbial fermentation for acetoin and
2,3ꢀbutanediol production has attracted greater attention due to its mild condition.
However, during the fermentation process, many metabolic byproducts are inevitably
produced, resulting in difficulty in product isolation and high purification cost. In
addition, the resulting microbial biomass causes environmental problems.
Commercially available 2ꢀbutanol is primarily obtained industrially by the hydration
of 1ꢀbutene using sulfuric acid as a catalyst. However, the harsh reaction condition
results in higher cost for 2ꢀbutanol production and potential environmental pollution.
Compared to chemical synthesis and microbial fermentation, the artificial
multiꢀenzyme pathway in the current work provides an environmentꢀfriendly, fewer
byproductꢀforming, and recyclable process with high conversion efficiency, thus
supporting the viability of producing acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol from
ethanol by CFME.
Conclusions
In conclusion, we developed an artificial multiꢀenzyme pathway capable of
upgrading ethanol to C4 compounds (Table 3). High activity (>88% conversion) was
obtained for acetoin or 2,3ꢀbutanediol production from ethanol. Additionally, the
artificial synthetic pathway showed potential for butanone and 2ꢀbutanol production,
although their yields were relatively low because of the low activity and stability of
DDH enzyme. This environmentally friendly novel approach can be used to upgrade
bioꢀethanol to acetoin, 2,3ꢀbutanediol, and 2ꢀbutanol. Ongoing efforts are focused on
developing modified FLS and DDH enzymes with high catalytic efficiency as well as
identifying new enzymes. Overall, this artificial CFME approach is a promising and
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highly selective strategy for ethanolꢀtoꢀC4 compounds conversion using our novel
cascade enzymes system.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(No. 81673542), New Century Excellent Talents Supporting Plan of the Provincial
Education Department of Fujian Province of China (No. K8015056A), the
Development Platform of Edible Fungi Industry in Fujian Province (No. K5114001A),
and Basic Science Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT & Future Planning
(2017R1A2B3011676, 2017R1A4A1014806, 2013M3A6A8073184).
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