Biosynthesis of fraxetin from three different substrates using engineered Escherichia coli

Abstract

Fraxetin, which is a simple coumarin, is a phytochemical present in medicinal plants, such as Fraxinus rhynchophylla, and Cortex Fraxini. In plants, it serves as a controller of iron homeostasis. The health-enhancing activities of fraxetin, such as anticancer, neuroprotective and antibacterial activities, are known. Scopoletin 8-hydroxylase (S8H) is a key enzyme involved in the synthesis of fraxetin from scopoletin. Scopoletin can be synthesized either from esculetin by O-methylation or from ferulic acid by feruloyl CoA 6′-hydroxylase (F6′H) and 4-coumaric acid CoA ligase (4CL). To enable fraxetin synthesis, the fraxetin biosynthesis pathway was introduced into Escherichia coli. Three distinct routes, from ferulic acid, esculetin, and scopoletin, were designed for the synthesis of fraxetin. In the first approach, E. coli strain harboring S8H was used and found to synthesize 84.8 μM fraxetin from 100 μM scopoletin. Two E. coli strains were used for the other two approaches because these approaches required at least two enzymatic reactions. Through this approach, 41.4 μM fraxetin was synthesized from 100 μM esculetin, while 33.3 μM fraxetin was synthesized from 100 μM ferulic acid.

Full text

Anetal. Appl Biol Chem (2020) 63:55
https://doi.org/10.1186/s13765-020-00543-9
NOTE
Biosynthesis offraxetin fromthree dierent
substrates using engineered Escherichia coli
Seung Hoon An, Gyu‑Sik Choi and Joong‑Hoon Ahn*
Abstract
Fraxetin, which is a simple coumarin, is a phytochemical present in medicinal plants, such as Fraxinus rhynchophylla,
and Cortex Fraxini. In plants, it serves as a controller of iron homeostasis. The health‑enhancing activities of fraxetin,
such as anticancer, neuroprotective and antibacterial activities, are known. Scopoletin 8‑hydroxylase (S8H) is a key
enzyme involved in the synthesis of fraxetin from scopoletin. Scopoletin can be synthesized either from esculetin
by O‑methylation or from ferulic acid by feruloyl CoA 6′‑hydroxylase (F6′H) and 4‑coumaric acid CoA ligase (4CL).
To enable fraxetin synthesis, the fraxetin biosynthesis pathway was introduced into Escherichia coli. Three distinct
routes, from ferulic acid, esculetin, and scopoletin, were designed for the synthesis of fraxetin. In the first approach,
E. coli strain harboring S8H was used and found to synthesize 84.8 μM fraxetin from 100 μM scopoletin. Two E. coli
strains were used for the other two approaches because these approaches required at least two enzymatic reactions.
Through this approach, 41.4 μM fraxetin was synthesized from 100 μM esculetin, while 33.3 μM fraxetin was synthe‑
sized from 100 μM ferulic acid.
Keywords: Coumarin, Fraxetin, Scopoletin 8‑hydroxylase
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Introduction
Coumarins (benzo-alpha-pyrones) were first isolated
from the tonka bean (Dipteryx odorata) in 1820. Since
then, their presence has been detected in various parts of
different plants, including the fruit (e.g., in Bael fruit or
Aegle marmelos), seed (e.g., in tonka beans or Calophyl-
lum inophyllum), root (e.g., in Ferulago campestris), and
leaf (e.g., Murraya paniculata) [1]. All coumarins have
a hydroxy or methoxy group at position 7. Scopoletin,
esculetin, umbelliferone, fraxetin, as well as their respec-
tive glycosides, are termed simple coumarins; they are
widespread in higher plants [2]. ese coumarins play
a pivotal role in protecting plants against pathogens [3];
furthermore, a simple coumarin, such as fraxetin, was
found to modulate vital physiological processes such as
iron homeostasis [4]. As naturally occurring phytochemi-
cals, coumarins possess health-enhancing properties,
including anticancer [1], neuroprotective [5], and anti-
bacterial properties [6].
Coumarins were synthesized from hydroxycinnamic
acids, such as p-coumaric acid, caffeic acid, and feru-
lic acid, in plants; p-coumaric acid, caffeic acid, and
ferulic acid resulted in the synthesis of umbelliferone,
esculetin, and scopoletin, respectively. e key enzyme
for coumarin biosynthesis was p-coumaroyl CoA
2′-hydroxylase (C2′H) or feruloyl CoA 6′-hydroxylase
(F6′H); the corresponding genes were cloned in Arabi-
dopsis thaliana [7], Ruta graveolens [8], Ipomoea bata-
tas [9], Manihot esculenta [10], Angelica decursiva [11],
and Peucedanum praeruptorum [12]. is enzyme is a
2-oxglutarate-dependent dioxygenase; the hydroxylation
of hydroxycinnamoyl-CoA resulted in the formation of a
pyrone ring.
Fraxetin belongs to the family of simple coumarins and
is synthesized from scopoletin by the hydroxylation of its
carbon at position 8. Fraxetin is involved in iron metab-
olism in plants [4, 13]. Similar to other phytochemicals,
fraxetin was found to exert beneficial effects in humans.
ese included antitumor [10, 14], neuroprotective [15],
Open Access
*Correspondence: jhahn@konkuk.ac.kr
Department of Bioscience and Biotechnology, Bio/Molecular Informatics
Center, Konkuk University, Seoul 05029, Republic of Korea

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Anetal. Appl Biol Chem (2020) 63:55
antihyperglycemic [16], and anti-inflammatory [17]
effects.
Since the metabolic pathway responsible for the syn-
thesis of simple coumarins is well established, these
compounds have been synthesized in E. coli. Scopole-
tin, esculetin, umbelliferone, skimming (umbelliferone
7-O-glucoside), and herniarin (7-O-methyl umbellifer-
one) were synthesized in E. coli [18, 19]. For the fraxetin
synthesis process in E. coli, two routes were postulated
(Fig.1). e first route involved the synthesis of escule-
tin from glucose, followed by the conversion of esculetin
into fraxetin by 7-O-methylation and 8-hydroxylation.
e second route started with the synthesis of scopoletin
from ferulic acid, followed by 8-hydroxylation. In the pre-
sent study, one coumarin, namely fraxetin, was synthe-
sized using E. coli via these two routes.
Materials andmethods
Plasmid construction
Reverse transcription polymerase chain reaction (RT-
PCR) was used to clone cDNA of scopoletin 8-hydroxy-
lase (S8H) from Arabidopsis thaliana (AtS8H; GenBank:
DQ446658.1). Two primers 5ʹ-aagaattcaATG GGT ATC
AAT TTC GAG GA-3ʹ and 5ʹ-aagcggccgcTCA CTC GGC
ACG TG-3ʹ were used (restriction sites for EcoRI and
NotI have been underlined). Additionally, the S8H
homologue from Oryza sativa (OsS8H; GenBank:
XM_026024461) was cloned by RT-PCR using two
primers: 5ʹ-aagaattcaATG CCG TCC GGC TAC GAC -3ʹ
and 5ʹ-aagcggccgcCTA ATC TAG ACT AGC GGC GG-3ʹ
(restriction sites for EcoRI and NotI have been under-
lined). AtS8H was digested using the EcoRI and NotI sites
and subcloned into pGEX 5X-3 (pG-AtS8H), pET-duet1
(pE-AtS8H), pRSF-duet1 (pR-AtS8H), and pCDF-duet1
(pC-AtS8H). OsS8H was subcloned into EcoRI/NotI sites
of pGEX 5X-3 (pG-OsS8H).
F6′H2 from Ipomoea batatas (IbF6′H2; GenBank :
AB636154) and 4CL (Os4CL; 4-coumarate: CoA ligase)
from O. sativa had been previously cloned using RT-PCR
[18]. F6′H2 was first cloned into pET-duet1 (EcoRI/NotI)
using PCR and, then, Os4CL was subcloned into pET-
duet1 containing F6′H2 to generate pE-IbF6′H2-Os4CL
(NdeI/XhoI). Subsequently, Os4CL was re-amplified
with a forward primer, adding a NotI site and riboso-
mal-binding site(RBS), and a reverse primer, containing
a XhoI site. ereafter, Os4CL was subcloned into the
NotI/XhoI sites of pET-duet1 containing IbF6′H2 to gen-
erate pE-IbF6′H-Os4CL controlled by a single promoter
(operon). e IbF6′H-Os4CL operon was subcloned into
pGEX 5X-3 (EcoRI/XhoI).
POMT7 (flavone 7-O-methyltransferase) [20] and
POMT9 from Populus deltoids [21] and ROMT9 (flavo-
noid 3′-O-methyltransferase) from O. sativa [22] have
also been cloned previously. ese genes were subcloned
into pGEX 5X-3 vector.
Production andanalysis ofmetabolites
For the synthesis of fraxetin from scopoletin, an over-
night culture of an E. coli transformant containing
pG-OsS8H, pG-AtS8H, pC-AtS8H, pE-AtS8H, or pR-
AtS8H was inoculated into fresh LB medium contain-
ing 50 μg/mL ampicillin and grown at 37 °C until the
OD600 reached 0.8; following this, isopropyl β-D-1-
thiogalactopyranoside (IPTG) was added to the medium
at a final concentration of 0.1mM or 1mM and incu-
bated at 18°C for 16h. Cells were harvested and the cell
concentration was adjusted to an OD600 of 3.0. e cells
were resuspended in M9 medium containing 2% glucose,
ampicillin (50μg/mL), and either 0.1mM or 1mM IPTG
POMT7AtS8H
Os4CLIbF6’H AtS8H
Esculetin
HO
HO
OO
Ferulic acid
OH
O
HO
OCH3
Feruloyl-CoA
OH
SCoA
HO
OCH
3
OH
SCoA
HO
OCH
3
OH
HO
H
3
CO
OO
Scopoletin
Fraxetin
HO
H3CO
OO
OH
Fraxetin
HO
H3CO
OO
OH
HO
H
3
CO
OO
Scopoletin
a
b
Fig. 1 Biosynthetic pathways of fraxetin from esculetin (a) and ferulic acid (b). POMT7 is an O‑methyltransferase, which converts esculetin into
scopoletin. AtS8H is a scopoletin 8‑hydroxylase. Os4CL catalyzes the attachment of CoA to ferulic acid. IbF6′H encodes feruloyl CoA 6′‑hydroxylase
(F6′H), which converts feruloyl CoA into scopoletin

Page 3 of 6
Anetal. Appl Biol Chem (2020) 63:55
in a test tube. A total concentration of 100μM of the
substrate (esculetin, isoscopoletin, scopoletin, or scopar-
one) was added, and the resulting culture was incubated
at 30°C for 24h. An E. coli transformant containing the
pG-AtS8H construct was employed to determine the
substrate (scopoletin) concentration. e cell concentra-
tion was adjusted to an OD600 of 3.0. e substrate was
added to the appropriate M9 medium at 0.1, 0.2, 0.3, or
0.5mM. e reaction culture was incubated at 30°C for
24h.
e E. coli transformant harboring ROMT9 was used to
methylate esculetin to scopoletin, isoscopoletin, and sco-
poletin. ree reaction products were purified using thin
layer chromatography (silica gel 60 F254, Millipore). A
mixture of benzene and ethyl acetate (3:1) was used as a
solvent. e E. coli transformant harboring POMT9 was
used to synthesize scopoletin from esculetin. e meth-
ylation reaction using E. coli was carried out as described
by Kim etal. [20].
Analysis of the reaction products was carried out using
ermo Ultimate 3000 HPLC [23]. Mass spectrometry
and proton nuclear magnetic resonance (NMR) were per-
formed as previously described [24, 25]. e 1H NMR of
fraxetin in acetone-d6 (in ppm) is δ 3.87 (3H, s, 6-OCH3),
6.15 (1H, d, J = 9.3Hz, H-3), 6.76 (1H, s, H-5), 7.91 (1H,
d, J = 9.3Hz, H-4) [26].
Results anddiscussion
Biotransformation ofscopoletin intofraxetin using E. coli
harboring scopoletin 8‑hydroxylase
Fraxetin is 8-hydroxy scopoletin. S8H from A. thali-
ana (AtS8H) and its homologue from rice (OsS8H) were
cloned as a glutathione S-transferase fusion protein and
expressed in E. coli. Scopoletin was tested, along with
other structurally-related coumarins, such as esculetin,
isoscopoletin, and scoparone. ese four compounds
have esculetin derivatives. ree methylated esculetins
(isoscopoletin, scopoletin, and scoparone) were synthe-
sized using E. coli harboring ROMT9, purified, and used
as substrates.
E. coli harboring AtS8H or OsS8H was tested for the
conversion of esculetin, isoscopoletin, scopoletin, and
scoparone by the administration of each compound. E.
coli harboring OsS8H did not convert any coumarins
used. However, for E. coli harboring AtS8H, scopole-
tin and isoscopoletin were converted into novel com-
pounds that had retention times different from those of
the corresponding substrates (Fig. 2). Other substrates
0.02.04.06.08.010.0 12.0 14.0 16.0 18.0 20.0
0
2500
2500
2500
250
400
100
140
200
350
340 nm
P1
P2
S1
S2
S3
a
b
c
d
e
f
min
mAU
Fig. 2 HPLC analysis of the reaction in E. coli harboring AtS8H. E. coli harboring AtS8H was administered scopoletin (a), isoscopoletin (b), and
esculetin (c); the reaction product was analyzed. d–f denote standard scopoletin, isoscopoletin, and esculetin, respectively. P1, reaction product
from scopoletin; P2, reaction product from isoscopoletin; S1, scopletin; S2, isoscopoletin; S3, esculetin

Page 4 of 6
Anetal. Appl Biol Chem (2020) 63:55
(esculetin and scoparone) did not generate any new prod-
uct. e molecular mass of the products from scopoletin
and isoscopoletin was 207.937Da, which is the molecular
mass obtained if hydroxylation occurs. S8H utilized one
methylated esculetins (scopoletin and isoscopoletin) as a
substrate and did not utilize dimethylated (scoparone) or
unmethylated esculetin. Scopoletin was a better substrate
than isoscopoletin; 84.8% of scopoletin was converted, as
opposed to the conversion of only 55% isoscopoletin. To
determine the structure of the biotransformation product
from scopoletin, the reaction product was purified, and
its structure was analyzed using proton NMR. e reac-
tion product was determined to be fraxetin (see Materials
and Methods). E. coli harboring different constructs (pG-
AtS8H, pE-AtS8H, pR-AtS8H, or pC-AtS8H) synthesized
the approximately same amount fraxetin from scopoletin.
To optimize the initial concentration of scopoletin and
the final yield of fraxetin, E. coli harboring AtS8H was
prepared at an OD600 of 3.0 after induction of AtS8H.
Subsequently, four different concentrations of scopole-
tin (100, 200, 300, and 500μM) were added. e highest
rate of conversion of scopoletin into fraxetin was seen
at 100μM scopoletin; 84.8μM fraxetin was synthesized
(84.8% conversion rate). However, fraxetin produc-
tion was highest at 200 μM scopoletin; approximately
139.5 μM fraxetin was synthesized (69.8% conversion
rate). Above 200μM scopoletin, the production level of
fraxetin registered a decline. e optimum initial cell
concentrations were also determined. Five initial cell
concentrations (OD600 = 1.0, 2.0, 3.0, 4.0, and 5.0) were
tested and 200μM scopoletin was administered. As the
initial cell concentration increased, the conversion of
scopoletin also registered a concomitant increase. At an
OD600 of 5.0, approximately 152.0μM of scopoletin was
converted into fraxetin.
Synthesis offraxetin fromesculetin andferulic acid
Fraxetin may also be synthesized from esculetin. Two
enzymatic reactions are required; the first is the conver-
sion of esculetin into scopoletin by an O-methyltrans-
ferase (OMT), and the second is the synthesis of fraxetin
from scopoletin by S8H. For the synthesis of scopoletin
from esculetin, three OMT genes (POMT7, POMT9,
and ROMT9) were evaluated. E. coli harboring POMT7
synthesized 56.8μM scopoletin from 100μM esculetin
(Fig.3a). However, E. coli harboring ROMT9 produced
three methylated esculetins (isoscopoletin, scopoletin,
and scoparone), with isoscopoletin as a major product.
e ratio of isoscopoletin, scopoletin, and scoparone was
83: 13: 3. POMT9 generated almost the same amounts of
isoscopoletin (38.1 μM) and scopoletin (37.4μM) from
100μM esculetin. erefore, E. coli harboring POMT7
was utilized to synthesize scopoletin from esculetin.
A two-step reaction was conducted using two E. coli
transformants to augment the final yield of fraxetin. e
first reaction was carried out using E. coli harboring
POMT7. Approximately 56.9μM scopoletin was synthe-
sized from 100 μM esculetin (Fig. 3a). Further incuba-
tion did not result in the conversion of more esculetin.
ereafter, the culture filtrate from the first reaction
was combined with E. coli harboring AtS8H. Approxi-
mately 41.4μM fraxetin was synthesized from 56.9μM
scopoletin (Fig. 3b), indicating that there was approxi-
mately 72.7% conversion from the synthesized fraxetin.
0.02.04.06.08.010.0 12.0 14.016.0 18.0 20.0
0
100
180
0
100
200
300
S1
P1
S1
P1
P2
a
b
min
340 nm
mAU
Fig. 3 Synthesis of fraxetin from esculetin using two E. coli transformants. a Conversion of esculetin into scopoletin using E. coli harboring POMT7.
E. coli harboring POMT7 was administered esculetin (S1), following which the reaction product was analyzed. P1 denotes the reaction product from
scopoletin. b Synthesis of fraxetin from scopoletin using E. coli harboring AtS8H. The culture filtrate from E. coli harboring POMT7 was administered
to the E. coli harboring AtS8H, and the reaction product was analyzed

Page 5 of 6
Anetal. Appl Biol Chem (2020) 63:55
Fraxetin was successfully synthesized from esculetin by
a two-step reaction. e final yield of fraxetin synthe-
sized from esculetin was lower than that from scopoletin;
moreover, the conversion rate of scopoletin into fraxetin
in the two-step reaction was lower than that seen for the
direct conversion. is could possibly be attributed to the
metabolite(s) in the first step inhibiting the second reac-
tion. It was attempted herein to synthesize fraxetin from
esculetin using an E. coli transformant harboring both
POMT7 and AtS8H. Only 3.4 μM fraxetin and 17.2μM
scopoletin were synthesized from 100μM esculetin.
Next, fraxetin was synthesized from ferulic acid. ree
enzymatic reactions are required for this. Previously,
scopoletin was successfully synthesized from ferulic
acid using E. coli harboring IbF6′H2 and Os4CL. It was
reasoned that introducing AtS8H into E. coli harbor-
ing IbF6′H2 and Os4CL could result in the synthesis of
fraxetin from ferulic acid. ree genes (IbF6′H, Os4CL,
and AtS8H) were introduced into E. coli, and the result-
ing transformant was administered ferulic acid. e E.
coli transformant synthesized fraxetin from ferulic acid.
To optimize fraxetin synthesis, several initial ferulic acid
concentrations (100, 200, 300, and 500μM) were tested.
e synthesis of fraxetin was optimal at 100μM of initial
ferulic acid, and approximately 33.3μM fraxetin was syn-
thesized (Fig. 4). Unreacted ferulic acid and scopoletin
were accumulated at the higher concentrations of ferulic
acid.
Fraxetin was synthesized from three different sub-
strates (scopoletin, esculetin, and ferulic acid). As shown
in Fig. 1, more enzymes are required when fraxetin is
synthesized from ferulic acid or esculetin than when it is
synthesized from scopoletin. Consequently, the final yield
of fraxetin was higher (84.8μM) when it was synthesized
from scopoletin (100μM). Its yield was decreased when
synthesis was carried out from esculetin (41.4μM) or fer-
ulic acid (33.3μM).
An attempt was made to synthesize fraxetin from
esculetin or ferulic acid. One E. coli transformant
harboring both POMT7 and AtS8H synthesized a lower
amount of fraxetin from esculetin than the other two E.
coli transformants, each of which conducted one reac-
tion. However, fraxetin was successfully synthesized
from ferulic acid using one E. coli transformant harbor-
ing three genes (Os4CL, IbF6′H, and AtS8H). Esculetin
may compete with scopoletin for AtS8H. In the E. coli
transformant harboring both POMT7 and AtS8H, escu-
letin served as a substrate for POMT7 and an inhibitor
of AtS8H. erefore, following the synthesis of sco-
poletin by POMT7, AtS8H could not utilize scopoletin
because it was inhibited by esculetin. Conversely, when
two independent E. coli transformants were used, more
scopoletin synthesized by the first E. coli transformant
harboring POMT7 was present in the medium and was
converted into fraxetin by the second E. coli transfor-
mant harboring AtS8H. When fraxetin was synthesized
from ferulic acid, only scopoletin was synthesized;
therefore, it was possible to synthesize fraxetin using E.
coli harboring Os4CL, IbF6′H, and AtS8H.
Acknowledgements
The present study was supported by grants from the Next‑Generation Bio‑
Green 21 Program (PJ01326001), Rural Development Administration, Republic
of Korea.
Authors’ contributions
SHA and JHA designed the experiments. SHA, GSC, and JHA performed the
experiments and analyzed the data. SHA, GSC, and JHA wrote the manuscript.
All authors read and approved the final manuscript.
Funding
Funding was received from the Next‑Generation BioGreen 21 Program, Rural
Development Administration (PJ01326001).
Availability of data and materials
All data generated or analyzed during the present study are included in this
published article.
Competing interests
The authors declare that they have no competing interests.
Received: 5 August 2020 Accepted: 9 September 2020
0.02.04.06.08.010.0 12.0 14.0 16.0 18.0 20.0
0
100
200
S1
P1
P2
min
340 nm
mAU
Fig. 4 Synthesis of fraxetin from ferulic acid using E. coli harboring Os4CL, IbF6′H, and AtS8H. S1, ferulic acid; P1, scopoletin; P2, fraxetin

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Anetal. Appl Biol Chem (2020) 63:55
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