ORIGINAL RESEARCH

published: 10 August 2021

doi: 10.3389/fmicb.2021.714335

Construction of an Artificial
Biosynthetic Pathway for the
Styrylpyrone Compound 11-Methoxy-
Bisnoryangonin Produced in
Engineered Escherichia coli
Kyung Taek Heo1,2, Byeongsan Lee1, Jae-Hyuk Jang1,2, Jung-Oh Ahn 2,3 and
Young-Soo Hong1,2*
1
Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si, South Korea,
2
Department of Bio-Molecular Science, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon,
South Korea, 3Biotechnology Process Engineering Center, KRIBB, Cheongju-si, South Korea
Edited by:
Eung-Soo Kim,
Inha University, South Korea A cDNA clone (named pnpks), which shows high homology to the known chalcone
Reviewed by: synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The
Yeo Joon Yoon, PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene
Seoul National University,
South Korea formation. The new product was characterized as a styrylpyrone, 11-methoxy-
Kenji Arakawa, bisnoryangonin, which is the lactonization compound of a linear triketide formed as the
Hiroshima University, Japan
reaction product of PnPKS protein with ferulic acid. These results show that pnpks
*Correspondence:
encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with
Young-Soo Hong
hongsoo@kribb.re.kr feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are
promising for use in human healthcare, they are mainly obtained by extraction from raw
Specialty section:
This article was submitted to
plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the
Microbial Physiology and Metabolism, heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic
a section of the journal pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene.
Frontiers in Microbiology
The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic
Received: 25 May 2021
Accepted: 15 July 2021 genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h
Published: 10 August 2021 in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-
Citation: bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately
Heo KT, Lee B, Jang J-H, Ahn J-O
and Hong Y-S (2021) Construction of
8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-
an Artificial Biosynthetic Pathway for bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-
the Styrylpyrone Compound bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and
11-Methoxy-Bisnoryangonin
Produced in Engineered Escherichia its production titer was also increased using engineered strains. This study provides a
coli. useful reference for establishing the biological manufacture of styrylpyrone compounds.
Front. Microbiol. 12:714335.
doi: 10.3389/fmicb.2021.714335 Keywords: styrylpyrone, 11-methoxy-bisnoryangonin, styrylpyrone synthase, de novo biosynthesis, type III PKS

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

INTRODUCTION L-tyrosine. The pET-opT5M vector, containing the optal, sam5, and
com genes, is responsible for the production of ferulic acid (Kang
Styrylpyrones are among the most abundant metabolites found et al., 2015), while the pET22-4P vector, which contains the 4cl2nt
in Piper methysticum, a crop of the Pacific Islands, suggesting and pnpks genes, converts ferulic acid to 11-methoxy-bisnoryangonin
that their underlying biosynthetic mechanism likely emerged (Figure 1). For the efficient production of 11-methoxy-bisnoryangonin
after the diversification of the Piper genus (Whitton et al., 2003). in E. coli, the engineered L-tyrosine overproducing E. coli ∆COS1
In many island regions of the Pacific Ocean, the root extract strain (Kang et al., 2015), which harbors the artificial biosynthetic
of the plant has traditionally been used as a drink with anesthetic gene cluster (pET-opT5M and pET22-4P), was cultured for 24 h
and sedative properties. Recently, there have been reports of in a minimal glucose medium containing kanamycin and ampicillin.
styrylpyrone kavain, the major compound of the root extract As a result, 11-methoxy-bisnoryangonin production as high as
of P. methysticum, which directly interacts with γ-aminobutyric 52.8 mg/L was achieved after 24 h of culture, this level is approximately
acid type A receptors (Chua et al., 2016; Kautu et al., 2017). 8.5-fold higher than that in the parental E. coli strain harboring
Thus, the anxiolytic effect of P. methysticum is likely attributable five genes in two plasmids.
to the pharmacological effects of α-pyrones on the nervous
system (Bilia et al., 2002). Various mushrooms have also been
reported to produce different styrylpyrones, which have shown
MATERIALS AND METHODS
important biological effects, such as anti-diabetic, anti-oxidative,
anti-platelet, anti-inflammatory, anti-cancer, and antiviral activities Bacterial Strains, Plasmids, and Chemicals
(Lee and Yun, 2011). Thus, styrylpyrones hold great promise The bacterial strains and plasmids used in this study are listed
as natural compounds for food and medical industries to replace in Table 1. Antibiotics were added to the medium at the
synthetic single compounds and extracts of plants that have following concentrations: kanamycin, 50 mg/L; ampicillin,
been implicated in medical conditions. However, plants usually 100 mg/L. Ferulic acid, cinnamic acid, 4-coumaric acid, and
contain complex styrylpyrone mixtures, which are difficult to caffeic acid were purchased from Sigma-Aldrich (United States)
separate into larger amounts of desired individual compounds. and used as substrates for in vitro and/or in vivo reactions
In contrast, the functional combination of plant-specific and standard for product identification by high-performance
biosynthetic pathways into microorganisms allows for the liquid chromatography (HPLC). Coenzyme A (CoA), adenosine
production of individual styrylpyrones as a single compound, triphosphate (ATP), and malonyl-CoA were purchased from
which can be synthesized in large amounts and isolated more Sigma-Aldrich for enzyme activity assays. The restriction enzymes
easily (Facchini et al., 2012; Wang et al., 2016; Noda and Kondo, (NEB, United States; Takara, Japan), AccuPower Ligation kit
2017; Palmer and Alper, 2019). (Bioneer, Korea), and KOD-plus-DNA polymerase (TOYOBO,
Styrylpyrone synthase (SPS), a chalcone synthase (CHS)-like Japan) were used according to the manufacturer’s instructions.
plant-specific type III polyketide synthase (PKS), normally
accepts a 4-coumarate-CoA ligase (4CL)-generated
4-coumaroyl-CoA thioester as a starter substrate and catalyzes Isolation of the pnpks Gene
the iterative decarboxylative condensation of two-carbon ketone The pnpks gene was identified through a CHS homology search
units derived from its extender substrate, malonyl-CoA (Austin on the black pepper (P. nigrum) fruit transcriptome data as
and Noel, 2003; Abe and Morita, 2010; Yu et al., 2012). In described previously (Jin et al., 2018, 2020). Gene-specific primer
most cases, the linear triketide intermediate cyclizes via Claisen pairs were designed from the candidate genes to clone the
condensation to form the aromatic chalcone backbone. However, respective open reading frames (ORFs) from the cDNA library.
SPS yields a triketide intermediate that lactonizes to produce The deduced amino acid sequence of the pnpks gene showed
the pyrone backbone (Pluskal et al., 2019). On behalf of typical the same amino acid sequences as the CHS (MK058495) originating
CHS, functionally different CHS-like PKSs have been found from P. methysticum already deposited in GenBank (Pluskal
in plants. Their amino acid sequences are highly homologous et al., 2019). PCR products were purified using an IncloneTM
and likely belong to a family of type III PKSs, which show Gel & PCR purification kit (Inclone Biotech, Korea) and finally
similarities in catalytic domains. However, studies are still cloned into pMD19-T vector (pMD-pnPKS; Takara, Japan).
ongoing to identify the amino acid residues that affect the
specificity of the initial substrate and/or the final product Expression and Purification of His-Tagged
(Jez et al., 2000; Morita et al., 2011; Stewart et al., 2017). PnPKS and 4CL Enzyme
Here, we identified one CHS-like gene, pnpks, in the black The black pepper CHS (pnpks) gene was cloned into the expression
pepper (Piper nigrum) transcriptome. We found that pnpks encodes plasmid, pET28a(+), at the NdeI and HindIII double digestion
a protein with SPS enzymatic activity that condenses two malonyl-CoA sites with a 6 × His tag at the N-terminus. The pnpks genes
with feruloyl CoA-linked starter substrate and further cyclizes to were amplified with primers pnPKS_F (5'-CATATGTCGAA
pyrone ring, resulting in the formation of 11-methoxy-bisnoryangonin. GACGGTAGAGGAGATTC-3', with an NdeI site shown by
In addition, we constructed a microbial de novo biosynthetic pathway underlining and the start codon of pnpks shown in boldface letters)
to produce 11-methoxy-bisnoryangonin using this pnpks gene. and pnPKS_R (5'-AAGCTTAGTTGGCCTCGGCG-3', with a
11-methoxy-bisnoryangonin was synthesized by five enzymes HindIII site shown by underlining) and cloned into pET-28a(+),
expressed in plasmids (pET-opT5M and pET22-4P) from intracellular which resulted in pET-PnPKS. The 4cl2nt gene (4-coumarate, CoA

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

FIGURE 1 | Engineered biosynthetic pathways for styrylpyrone compound, 11-methoxy-bisnoryangonin, starting from L-tyrosine in E. coli and the proposed
kavalactone biosynthetic pathway. Tyrosine ammonia-lyase (TAL) gene (optal), C3H (4-coumarate 3-hydroxylase) gene (sam5), COMT (caffeic acid
O-methyltransferase) gene (com), CCL gene (4cl2nt), and SPS gene (pnPKS). Bold arrows indicate the major synthetic pathway used in this study.

ligase gene from Nicotiana tabacum; GenBank # AAB18638) was Enzymatic Assay of Pnpks With Phenolic
cloned into pET-28a(+), resulting in pET-4Cl2nt, as described Acids
previously (Kang et al., 2012). E. coli C41 (DE3) cells transformed Reaction mixtures (100 μl) containing Tris-HCl (50 mM, pH
with pET-PnPKS or pET-4Cl2nt were inoculated in 100 ml of 8.0), CoA (0.1 mM), malonyl-CoA (0.2 mM), ATP (5 mM),
Luria-Bertani (LB) medium supplemented with kanamycin and MgCl2 (1 mM), and all phenolic acids (cinnamic acid, 4-coumaric
cultured at 37°C. When the optical density of the culture broth acid, caffeic acid, and ferulic acid; 0.1 mM) with both His-tagged
at 600 nm (OD600) was 0.6, isopropyl-D-thiogalactopyranoside 4CL2nt (1 μM) and PnPKS (1 μM) were incubated at 30°C
(IPTG, 1 mM) was added to induce protein expression. After for 4 h. The reaction mixture was extracted with an equal
culturing for 16 h at 26°C, the cells were harvested by centrifugation volume of ethyl acetate. The organic layer was then evaporated
(2,000 × g, 20 min, 4°C). The cells were resuspended in 4 ml to dryness. The residual material was dissolved in 100 μl of
of lysis buffer [50 mM Tris-HCl (pH 7.4), imidazole 10 mM, methanol. Twenty microliters of the extract was applied to an
and NaCl 100 mM] with a protease inhibitor cocktail (Sigma- HPLC and liquid chromatography/mass spectrometry (LC/MS)
Aldrich, United States). The harvested cells were centrifuged at system, as described below.
15,000 × g for 10 min at 4°C after sonic treatment. The supernatant
was mixed with His-Hyper agarose resin (Lugen Sci. Co., Korea)
in a Poly-Prep chromatography column (Bio-Rad, United States) Construction of 11-Methoxy-
for 1 h. The mixed resins were washed with 50 ml of wash Bisnoryangonin Expression Vector
buffer containing 50 mM imidazole, and the His-tagged enzymes Using pET-PnPKS as a template, the pnpks gene was amplified
were eluted using 2 ml of an elution buffer containing 250 mM by PCR using the primers Nspe (5'-ACTAGTAGGTTGAGGCC
imidazole. The purified His-tagged enzymes were dialyzed against GTTGAGCACCGCC-3', which is located upstream of the T7
10 mM Tris-HCl (pH 8.0) containing 0.1 mM EDTA, 0.1 mM promoter region of the pET-28a(+) vector and contains the
DTT, and 10% glycerol. The purified enzyme concentrations were designed SpeI site shown by underlining) and pnPKS_R
estimated by the Bradford method, using the Coomassie Plus (5'-AAGCTTAGTTGGCCTCGGCG-3', with a HindIII site shown
Protein Assay kit (Bio-Rad, United States). The purified His-tagged by underlining). Using pET-4CL2nt as a template, the 4cl2nt
PnPKS (45.2 kDa) and 4CL2nt (61.6 kDa) proteins were analyzed gene was amplified with primers 4cl2nt_F (5'-CATATGGAGAAA
using 12% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide GACACGAAGCAAGTTGACATC-3', with an NdeI site shown
gel electrophoresis (PAGE) (Figure 2). by underlining and the start codon of 4cl2nt shown in boldface

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

TABLE 1 | Plasmids and strains used in this study.

A B
Plasmids Relevant characteristics References

pBR322 origin, f1 origin, T7

pET-28a(+) Novagen
promoter, KanR
pBR322 origin, f1 origin, T7
pET-22b(+) Novagen
promoter, AmpR
pMD19-T ColE1 origin, lacZα, AmpR Takara
pMD19-T carrying PKS gene
pMD-PnPKS (pnpks) from Piper nigrum cDNA This study
library
pET-28a(+) carrying PKS gene
pET-PnPKS This study
(pnpks) from Piper nigrum
pET-22b(+) carrying codon-
pET-4CL2nt optimized 4CL gene (4cl2nt) from Kang et al. (2012)
Nicotiana tabacum
pET-28a(+) carrying codon-
optimized TAL gene (optal),
codon-optimized C3H gene
pET-opT5M Kang et al. (2015)
(sam5) from Saccharothrix
espanaensis, and COMT gene
(com) from Arabidopsis thaliana FIGURE 2 | SDS-PAGE analysis of purified His-tagged PnPKS (A) and
pET-22b(+) carrying 4cl2nt and 4CL2nt (B). Purified His-tagged PnPKS (Lane A; 45.2 kDa) and 4CL2nt
pET22-4P This study (Lane B; 61.6 kDa) protein after affinity purification. M, protein size marker.
pnpks
Strains
E. coli DH5α For cloning Invitrogen
Derivative strain of E. coli supplemented with kanamycin and ampicillin. The culture was
E. coli C41(DE3) Miroux and Walker (1996)
BL21(DE3) grown at 37°C to an OD600 of 0.6, and IPTG was added to
Derivative strain of E. coli a final concentration of 1 mM, followed by incubation at 26°C
∆COS1 Kang et al. (2015)
C41(DE3); ΔtyrR::tyrAfbr, aroGfbr for 6 h. The cultured cells were harvested and resuspended
P1
E. coli C41(DE3) harboring
This study
in 30 ml minimal glucose medium (SM; 7.3 g/L K2HPO4,
pET22-4P 3 g/L KH2PO4, 8.4 g/L MOPS, 0.5 g/L NaCl, 2 g/L NH4Cl,
P2
E. coli C41(DE3) harboring pET-
This study 5 g/L MgSO4 7H2O, 5 g/L (NH4)2SO4, 0.1 ml/L trace elements,
opT5M and pET22-4P
15 g/L glucose, 1 mM IPTG, and appropriate antibiotics) in
∆COS1 harboring pET-opT5M 250-ml flasks. The resuspended cells were incubated at 26°C
P3 This study
and pET22-4P
for 48 h. For the bioconversion experiments, the recombinant
COS6-T5M E. coli C41(DE3); ΔtyrR::tyrAfbr,
Kang et al. (2018) E. coli strains that harbored the pET22-4P plasmid were cultured
aroGfbr; ΔbioC::optal, sam5, com
in the SM media supplemented with 0.1 mM each of cinnamic
P4 COS6-T5M harboring pET22-4P This study
acid, 4-coumaric acid, caffeic acid, and ferulic acid. The samples
AmpR, ampicillin; KmR, kanamycin. were collected after 24 h. The cultured samples were collected
over time and analyzed by HPLC and LC/MS.
letters) and Cspe (5'-ACTAGTTCCTCCTTTCAGCAAAAA
ACCCCTC-3', the sequence is located downstream of the
terminator region of the pET-28a(+) vector and contains the Isolation of 11-Methoxy-Bisnoryangonin
SpeI site shown by underlining). Each of the amplified fragments The recombinant E. coli strains harboring the pET22-4P plasmid
was digested with corresponding sites and cloned between the were cultured using the same method as described earlier,
NdeI- and HindIII-digested pET-22b(+), resulting in pET22-4P. and the culture volume and time were increased to 2 L for
The pET-opT5M vector for ferulic acid production has been 60 h. After ferulic acid (15 mg/L) supplementation, the ethyl
previously reported (Kang et al., 2012). acetate soluble material was further separated using semi-
preparative HPLC (Waters Atlantis T3 C18 column:
10 × 250 mm, 5 μm) with a gradient solvent system (35%
Culture Conditions for 11-Methoxy- CH3CN–H2O [0.05% trifluoroacetic acid (TFA)] to 45% CH3CN–
Bisnoryangonin Production and H2O (0.05% TFA) over 15 min, UV 254 nm detection, flow
Bioconversion rate: 3 ml/min). Finally, pure 11-methoxy-bisnoryangonin
Engineered E. coli strains were grown at 37°C in LB medium (5.2 mg) was eluted at 13.2 min. The structure of 11-methoxy-
containing appropriate antibiotics (100 μg/ml ampicillin for bisnoryangonin was determined based on 1H and 13C NMR
pET22-4P and 50 μg/ml kanamycin for pET-opT5M). The data with values reported in the literature
overnight culture was inoculated into a fresh LB medium (Supplementary Table S1; Zan et al., 2011).

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

Quantification of 11-Methoxy- of these conserved functional motifs is thought to facilitate

Bisnoryangonin repeated decarboxylation reactions and help orient substrates
The cultures were extracted using an equal volume of ethyl and intermediates during the condensation reactions. However,
acetate. Ethyl acetate extracts were then dried and resuspended other members of the CHS family with very similar amino
in methanol. Twenty microliters of the extract was injected acid sequences, such as SPS, 2-PS, STS, and BAS, result in
into a J’sphere ODS-H80 column (4.6 × 150 mm i.d., 5 μm; the formation of different polyketides using various starting
YMC, Japan) using an UltiMate U3000 system (CH3CN–H2O substrates, condensation reactions, and cycling processes. It is
(0.05% TFA) 20–100% acetonitrile (CH3CN) for 20 min and of interest to understand how these similar but distinct enzymes
100% CH3CN for 5 min, at a flow rate of 1 ml/min; Thermo function to produce specific final products. To date, very little
Fisher Scientific, Unites States) equipped with a photodiode is known about the amino acid residues responsible for their
array detector. The quantification of the compounds was based mode of cyclization and starter specificity. Therefore, it is
on the peak areas of absorbance at 320 nm. The quantification difficult to determine the end product of the newly acquired
data shown in this study were generated from independent PnPKS using its amino acid sequence alone.
experiments performed in triplicate. For LC/MS analysis, samples
were dissolved in methanol and analyzed by electrospray Characterization of the SPS Function With
ionization (ESI) using an UltiMate U3000-LTQ XL linear ion
Ferulic Acid of the PnPKS
trap mass spectrometer system (Thermo Fisher Scientific,
To identify the function of the pnpks gene originating from
United States). A 2-μl volume of the sample was applied to
P. nigrum, we assessed the enzymatic activity using purified
an ACQUITY UPLC HSS T3 C18 column (2.1 × 150 mm;
His-tagged PnPKS with 4-coumarate-CoA ligase (4CL, Figure 2).
2.5 μm particle size; Waters, United States), and the mobile
Based on the previous literature, the 4CL from Nicotiana
phase used for the linear gradient condition (5–100% CH3CN–
tabacum (4CL2nt) has already been identified as having a broad
H2O containing 0.1% formic acid for 15 min, 100% CH3CN
substrate specificity for cinnamic acid, 4-coumaric acid, caffeic
for 5 min, at a flow rate 0.3 ml/min). The mass spectrometry
acid, and ferulic acid. In addition, the relative specificities of
experiments were controlled and analyzed using menu-driven
4CL2 toward cinnamic acid, caffeic acid, and ferulic acid are
software provided with the Xcalibur system (version 2.2 SP1.48;
29, 25, and 62%, respectively, compared to the value of 4-coumaric
Thermo Fisher Scientific).
acid (Lee and Douglas, 1996). The reaction of ferulic acid
with the 4CL2nt enzyme in the presence of PnPKS led to the
formation of a clear new peak at 7.9 min, which was detected
RESULTS using HPLC [Figure 4A(c)]. The molecular weight of the new
peak was determined using LC/MS. The peak at 7.9 min
Identification of CHS-Like Gene (pnpks) exhibited parent mass ion peaks at m/z 261 [M+H]+ and m/z
From Piper nigrum 259 [M–H]− (Figure 4B), which corresponded to a molecular
Piper nigrum (black pepper) produces an alkaloid compound, weight of 260 Da, and significant amounts of ferulic acid that
piperine, which is derived from the polyketide pathway (Geisler was used as substrate were also detected at 6.6 min. The PnPKS
and Gross, 1990; Srinivasan, 2007). The corresponding PKS protein with ferulic acid did not catalyze the formation of
that provides the piperine polyketide chain has not been chalcone (proposed MW 302 Da) or stilbene (proposed MW
identified, but it is estimated that the CHS-like type III PKS 258 Da) compounds. In addition, we constructed a bioconversion
can produce it (Jin et al., 2020; Schnabel et al., 2020). In our system utilizing the pnpks gene and the 4cl2nt gene. The pnpks
investigation of piperine biosynthesis, we identified one CHS-like and 4cl2nt genes were cloned into the expression vector pET-22b
gene, pnpks, in the P. nigrum transcriptome data. The deduced using a previously described method, resulting in pET22-4P
length of the enzymes encoded by the pnpks gene was 389 (Table 1). Ferulic acid was added to the cultured recombinant
amino acids. Phylogenetic analyses revealed that PnPKS was E. coli C41 (DE3) strain (P1) harboring pnpks and 4cl2nt genes
clustered together with other plant CHS-superfamily type III (pET22-4P). The P1 culture broth and bacterial cells were
PKSs (Figure 3). The PnPKS was closer to that of typical type collected after 24 h of culture and were subjected to HPLC
III PKSs [CHS and STS (stilbene synthase)] from Arabidopsis analysis [Figure 4A(d)]. As a result of the enzymatic reaction,
thaliana and Vitis vinifera, with identities of 79.7 and 73.5%, the 7.9 min peak was also identified and the MW was the
respectively. In addition, the deduced amino acid sequence same at 260 Da.
showed more than 66% identity with those of other type III In addition, the functional study of this pnPKS enzyme
PKSs of plant origin: 81.5% identity with P. methysticum SPS, against other phenylpropanoic acids, cinnamic acid, 4-coumaric
68.4% identity with Gerbera hybrida 2-pyrone synthase (2-PS), acid, and caffeic acid was also conducted under the same
and 66.1% identity with Rheum palmatum benzalacetone synthase bioconversion conditions as those used for ferulic acid (Figure 5).
(BAS). PnPKS maintains a CoA-binding site similar to that As a result, cinnamic acid, 4-coumaric acid, and caffeic acid
of the catalytic triad of Cys169, His309, and Asn342 [AtCHS formed new peaks (at RT 12.7 min, 9.8 min, and 8.5 min,
(CHS from A. thaliana) numbering; Ferrer et al., 1999; Figure 3]. respectively) corresponding to the molecular weights of chalcone
The “gate-keeper” Phe220 is also located at the junction between compounds (Supplementary Figure S1), which are denoted
the CoA-binding tunnel and the active-site cavity. The presence by asterisks on Figure 5. In the case of cinnamic acid and

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

A B

FIGURE 3 | Alignment (A) and phylogenetic analysis (B) of PnPKS against known CHS-superfamily type III PKS. (A) The red triangle indicates the three amino
acids forming a Cys-His-Asn catalytic triad that is essential for starter substrate loading and malonyl-CoA condensation. The blue triangle indicates a “gate-keeper”
that is known to adjust the chain elongation. (B) Arrow indicates PnPKS in this study. The sources and GenBank accession numbers are AtCHS from Arabidopsis
thaliana (P13114), VvSTS from Vitis vinifera (ABE68894), RpBAS from Rheum palmatum (AAK82824), PmSPS1 from Piper methysticum (QCX36371), Gh2PS from
Gerbera hybrida (P48391), CHS from Medigo sativa (AAB41559), Glycine max (P19168.1), and Gerbera hybrid (P48390.1), CTAS from Hydrangea macrophylla
(BAA32733.1), OKS from Aloe arborescens (Q3L7F5.1), PCS from Aloe araborescens (Q58V97.1), VPS from Psilotum nudum (Q9SLX9.1), STS from Pinus strobus
(P480407.1), BPS from Hypericum androsaemum (Q8SAS8.1), and DCS from Curcuma longa (C0SVZ5.1). The nucleotide sequence of the pnpks gene from Piper
nigrum is provided in the Supplementary data section. Amino acid sequences were aligned using the ClustalW method and the phylogenetic tree constructed by
using the MegAlign program of the DNASTAR Lasergene software (DNASTAR Inc.).

A B

FIGURE 4 | Functional analysis of PnPKS using in vitro enzymatic reaction and bioconversion experiment with ferulic acid. (A) HPLC profile of the standard ferulic
acid (a), purified 11-methoxy-bisnoryangonin (b), in vitro reaction (c), and ferulic acid supplemented E. coli harboring pET22-4P (P1; d). The absorbance was
monitored at 320 nm. (B) Selected mass ion chromatogram of peak at 7.9 min (trace c) exhibited m/z 261 [M+H]+ and m/z 259 [M−H]−,which corresponded to
11-methoxy-bisnoryangonin. Peak at 10.9 min (trace d) exhibited m/z 371 [M+H]+, which corresponded to dimerized ferulic acid.

caffeic acid, the bioconversion clearly produced chalcone compound at RT 9.8 min (m/z 273 [M+H]+) and the other
compounds (m/z 257 [M+H]+ and m/z 289 [M+H]+, peak at RT 11.0 min (m/z 310 [M+H]+) which probable
respectively), but the 4-coumaric acid converted to chalcone corresponded to dehydrodimer (Supplementary Figure S1).

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

FIGURE 5 | HPLC profile of bioconversion experiments with cinnamic acid (A), 4-coumaric acid (B), and caffeic acid (C). Lower panels represent standard
phenylpropanoic acids and upper panels represent reaction results. The absorbance was monitored at 280 nm. Cinnamic acid, 4-coumaric acid, and caffeic acid
formed new peaks (12.7 min, 9.8 min, and 8.5 min, respectively) corresponding to the molecular weights of different chalcone compounds, which are denoted by
asterisks. The mass spectra of the asterisked peaks are shown in Supplementary Figure S1.

Although chalcone compounds were major products in It is estimated that oxidative coupling via the action of peroxidases
bioconversion experiment using cinnamic acid, 4-coumaric acid, in E. coli cells produces dehydrodimers. These dimer compounds
and caffeic acid, but in vitro enzyme reactions produced unknown have often been identified in bioconversion experiments with
peaks along with the different chalcone compounds (denoted phenylpropanoic acids and are thought to be shunt products.
by asterisk), respectively (Supplementary Figure S2). Taken together, the newly formed 7.9 min peak in in vitro
Interestingly, only ferulic acid does not produce chalcone enzymatic reaction and bioconversion experiment was identified
compound as major peak in these reactions. as 11-methoxy-bisnoryangonin. Thus, the PnPKS protein has
To obtain the required amount for structural analysis of SPS enzymatic activity that catalyzes the sequential condensation
the 260 Da product estimated to be a pyrone ring, 30 mg of of one feruloyl-CoA and two malonyl-CoA molecules to form
ferulic acid was added to a 2 L fermentation broth of the P1 a triketide intermediate, followed by ring closure of the triketide
strain. The structure of the purified 7.9 min peak was identified to form a styrylpyrone (Figure 1).
as 11-methoxy-bisnoryangonin through spectral data
interpretation and compared with the values reported in the
literature (Supplementary Table S1; Zan et al., 2011). In Construction of de novo Artificial
addition, the other peak at 10.9 min exhibited m/z 371 [M+H]+, Biosynthetic Pathways for 11-Methoxy-
which corresponded to ferulate dehydrodimer [Figure 4A(d)]. Bisnoryangonin
The results of the tandem MS analysis suggested that the Although styrylpyrone compounds are promising for use in
compound formed an ester bond between the carboxyl residue human healthcare, they are mainly obtained by extraction from
of one ferulic acid molecule and the hydroxyl group located plants, such as P. methysticum or some mushrooms. Therefore,
in the aromatic ring of another ferulic acid these compounds are important targets for production through
(Supplementary Figure S3). Generally, dimerization of ferulic the use of engineered microbial strains. Although the
acid provides a pathway for cross-linking polysaccharide chains aforementioned strains are capable of producing significant
in some plants (Ralph et al., 1994). A reactive radical can levels of 11-methoxy-bisnoryangonin, they require the exogenous
abstract a hydrogen atom from the carboxyl group of ferulic addition of the pathway precursor ferulic acid. We attempted
acid to form a phenoxy radical that is highly resonance-stabilized. a de novo synthesis to produce an 11-methoxy-bisnoryangonin

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

compound in E. coli by engineering an artificial biosynthetic of 11-methoxy-bisnoryangonin in P2 and P3 strain reached

pathway using the newly identified PnPKS. To this end, three 6.2 ± 1.0 mg/L (23.8 μM) and 52.8 ± 3.5 mg/L (203.1 μM)
enzyme activities were needed from tyrosine, an essential at 24 h (Figure 7), respectively. In addition, the slightly reduced
aromatic amino acid, to engineer the production of ferulic productivity of 11-methoxy-bisnoryangonin in 48 h (Figure 7)
acid in E. coli. First, we used a ferulic acid biosynthetic gene was assumed to be due to the instability of the compound.
cluster (pET-opT5M), consisting of the tyrosine ammonia-lyase The instability may be attributed to the hydrolysis of esters
(TAL; optal) gene, 4-coumarate 3-hydroxylase (C3H; sam5) due to pH changes in the fermentation process. This result
gene, and caffeic acid O-methyltransferase gene (COMT; com; means that a de novo artificial 11-methoxy-bisnoryangonin
Table 1; Kang et al., 2012, 2015). The pnpks and 4cl2nt genes biosynthetic pathway was engineered in E. coli containing the
(pET22-4P) were used as the next step to produce 11-methoxy- two-vector system, although previous bioconversion methods
bisnoryangonin from ferulic acid. Each biosynthetic gene was include precursor feeding.
configured to be regulated by the T7 promoter. In addition, to facilitate cell growth and fermentation in
Two plasmids, pET-opT5M and pET22-4P, were the production of 11-methoxy-bisnoryangonin, we used an
simultaneously transformed into E. coli C41 (DE3) (P2) and engineered E. coli strain (COS6-T5M), in which one copy of
the engineered L-tyrosine overproducing strain (P3). The the ferulic acid production module was inserted into the
L-tyrosine overproducing E. coli strain (∆COS1) was engineered chromosome of the L-tyrosine overproducing strain (∆COS1)
to overexpress the chorismate mutase-prephenate dehydrogenase (Kang et al., 2018). Using the RedET recombination system,
gene (tyrA) and D-erythrose-4-phosphate-lyase gene (aroG) in the ferulic acid production module containing the optal, sam5,
a ΔtyrR strain background (Kang et al., 2015). A detailed and com genes was inserted into the bioC gene region of the
description of the method is provided in the Supporting E. coli ∆COS1 chromosome, which encodes a malonyl-acyl
Information (Supplementary Figure S4). The recombinant carrier protein methyltransferase (Supplementary Figure S4).
strains (P2 and P3) that harbored the artificial biosynthetic The elimination of this bioC gene inhibits the consumption
gene clusters (pET-opT5M and pET22-4P) were cultured in of malonyl-CoA for fatty acid biosynthesis (Lin and Cronan,
minimal glucose medium (SM) containing ampicillin and 2012). The COS6-T5M strain produced ferulic acid under the
kanamycin in shake flasks at 26°C. The 11-methoxy- same culture conditions without the antibiotics [Figure 6(d)].
bisnoryangonin peaks were detected in the culture broth of A total of three peaks, including a peak with the same retention
the P2 and P3 strains by HPLC analysis (Figure 6). The amount time (6.6 min) as the ferulic acid standard, were detected.

FIGURE 6 | De novo biosynthesis of 11-methoxy-bisnoryangonin by the P2, P3, and P4 strains. Comparison of HPLC profiles of purified 11-methoxy-
bisnoryangonin (a); the culture broth of P2 (E. coli C41 strain harboring pET-opT5M and pET22-4P), (b); P3 (∆COS1 strain harboring pET-opT5M and pET22-4P), (c);
COS6-T5M (d); and P4 (COS6-T5M strain harboring pET22-4P), (e). The peaks correspond to caffeic acid (5.1 min), 4-coumaric acid (6.2 min), ferulic acid (6.6 min),
and 11-methoxy-bisnoryangonin (7.9 min).

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

overproducing strain (P3) was 8.5-fold higher than that of the

parental E. coli (P2) harboring five genes in two plasmids.
The titers of 11-methoxy-bisnoryangonin reached 52.8 mg/L
in flask culture in a minimal glucose medium. However, strain
P4, whose plasmid pET22-4P was transformed into ferulic
acid-producing E. coli COS6-T5M, produced a smaller amount
of 11-methoxy-bisnoryangonin than the parental E. coli C41
(DE) harboring five genes in two plasmids (Figure 6). It can
be seen that one copy of the modular genes for ferulic acid
production shows significantly lower amounts of 11-methoxy-
bisnoryangonin when compared to high copy numbers. In
general, the plasmid copy number is known to affect the ability
of cells to grow and influence cellular metabolism (Karim et al.,
2013). Although it is difficult to explain this phenomenon
clearly, it can be estimated that an appropriate proportion of
enzyme concentrations is required in the heterologous host
FIGURE 7 | Comparisons of the 11-methoxy-bisnoryangonin productivity. cell for the optimal rate of biosynthetic metabolic flux (Farasat
The data were obtained after 24–48 h culture of P2, P3, and P4 strains in a
et al., 2014; Kang et al., 2018). Therefore, this artificial biosynthetic
minimal glucose medium. Error bars show one standard deviation from
triplicate experiments. pathway of 11-methoxy-bisnoryangonin is a multi-enzyme
reaction that requires a balanced expression of several enzymes.
In the future, it will be necessary to improve the expression
The other two peaks were identified as caffeic acid (5.1 min) of these enzymes or culture conditions.
and 4-coumaric acid (6.2 min). These products can be produced Styrylpyrones hold great promise as natural compounds for
by the reactions of the three enzymes in the artificial ferulic food and medical industries to replace synthetic compounds
acid pathway. Plasmid pET22-4P was transformed into ferulic and plant extracts (Lee and Yun, 2011). As a potential
acid-producing E. coli COS6-T5M (P4) and cultured for 48 h styrylpyrone, 11-methoxy-bisnoryangonin was successfully
under the same flask culture conditions. However, the strain produced in E. coli from a glucose medium, and its production
P4 produced only a small amount of 11-methoxy-bisnoryangonin titer was also increased using a strain that had an engineered
(2.3 ± 0.2 mg/L at 24 h). This amount is less than that produced metabolic pathway for tyrosine. The production of plant-
by the parental P2 strain (6.2 ± 1.0 mg/L). However, the originated styrylpyrone compounds, such as 11-methoxy-
accumulation of 4-coumaric acid, not ferulic acid, was also bisnoryangonin by engineering microbes, suggests the potential
identified in the P4 strain [Figure 6(e)], while 4-coumaric for industrialization through further exploration of advanced
acid, caffeic acid, and ferulic acid were produced in the synthetic biology and metabolic engineering techniques.
COS6-T5M strain [Figure 6(d)]. Taken together, the final levels
of 11-methoxy-bisnoryangonin produced under two-vector
conditions from an engineered tyrosine-producing strain (P3) DATA AVAILABILITY STATEMENT
were determined to be 8.5-fold and 22.9-fold over the parental
E. coli P2 strain and the engineered E. coli P4 strain, respectively. The datasets presented in this study can be found in
The titers of the 11-methoxy-bisnoryangonin of the P2 strain online repositories. The names of the repository/repositories
reached 52.8 mg/L after 24 h in flask culture. and accession number(s) can be found in the
article/Supplementary Material.

DISCUSSION
AUTHOR CONTRIBUTIONS
In this study, we successfully demonstrated the de novo synthesis
of a styrylpyrone compound, 11-methoxy-bisnoryangonin, which KH, BL, J-HJ, and J-OA performed the experimental work,
is an abundant metabolite responsible for the anxiolytic effects whereas KH, BL, and Y-SH drafted the manuscript. All authors
of P. methysticum extracts. The system was developed using were involved in designing, discussing, and interpreting the
the PnPKS protein as an SPS that catalyzes pyrone formation results of the experiments.
with specificity to feruloyl-CoA. PnPKS catalyzes the formation
of 11-methoxy-bisnoryangonin from malonyl-CoA and ferulic
acid precursors when combined with 4CL enzyme. Furthermore, FUNDING
we designed an artificial biosynthetic pathway using the newly
identified pnpks gene with the other four genes, including those This work was supported by the Basic Science Research Program
involved in feruloyl-CoA production, to complete the de novo (NRF2020R1I1A2068713) of the Ministry of Education and
synthesis of 11-methoxy-bisnoryangonin in E. coli. The production the KRIBB Research Initiative Program (KGM5292113) funded
of 11-methoxy-bisnoryangonin from an engineered tyrosine by the Ministry of Science ICT (MSIT) of the Republic of Korea.

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Heo et al. Artificial Biosynthetic Pathway for the Styrylpyrone

ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL

We thank Professor Soo-Un Kim (Department of Agricultural The Supplementary Material for this article can be found online
Biotechnology, Seoul National University) for CHS cDNA at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.714335/
samples from black pepper (Piper nigrum) cDNA library. full#supplementary-material

inheritance and expression, and properties of recombinant proteins. Plant

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neuromuscular transmission. J. Exp. Neurosci. 11:1179069517705384. doi: authors and do not necessarily represent those of their affiliated organizations,
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