Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae

Article abstract of DOI:10.1080/09168451.2017.1386085

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α]_D, and gas chromatography–mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata “Udo” and a Tps gene (named AeTps1) in Aralia elata “Taranoki” were studied resulting in identifying functionalities of these cryptic Tps genes.

Full text of DOI:10.1080/09168451.2017.1386085

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Purification and structural analysis of volatile
sesquiterpenes produced by Escherichia coli
carrying unidentified terpene synthase genes from
edible plants of the family Araliaceae
Kazutoshi Shindo, Jun-ichiro Hattan, Mariko Kato, Miho Sato, Tomoko Ito,
Yurika Shibuya, Arisa Watanabe, Maki Sugiyama, Yuri Nakamura & Norihiko
Misawa
To cite this article: Kazutoshi Shindo, Jun-ichiro Hattan, Mariko Kato, Miho Sato, Tomoko Ito,
Yurika Shibuya, Arisa Watanabe, Maki Sugiyama, Yuri Nakamura & Norihiko Misawa (2017):
Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying
unidentified terpene synthase genes from edible plants of the family Araliaceae, Bioscience,
Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2017.1386085
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Date: 02 January 2018, At: 04:46

Bioscience, Biotechnology, and Biochemistry, 2017
https://doi.org/10.1080/09168451.2017.1386085
Purification and structural analysis of volatile sesquiterpenes produced by
Escherichia coli carrying unidentified terpene synthase genes from edible
plants of the family Araliaceae
Kazutoshi Shindoa, Jun-ichiro Hattanb, Mariko Katoa, Miho Satoa, Tomoko Itoa, Yurika Shibuyaa,
Arisa Watanabea, Maki Sugiyamaa, Yuri Nakamuraa and Norihiko Misawab
adepartment of Food and nutrition, Japan Women’s University, tokyo, Japan; bresearch institute for Bioresources and Biotechnology,
ishikawa Prefectural University, ishikawa, Japan
ARTICLE HISTORY
ABSTRACT
received 21 august 2017
accepted 17 september 2017
A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was
developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2
(ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of
volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were
isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant
cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium),
and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition,
silica gel column chromatography, and reversed-phase preparative high-performance liquid
chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic
resonance, [α]D, and gas chromatography–mass spectrometry analyses. Herein, the products
of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata “Udo”
and a Tps gene (named AeTps1) in Aralia elata “Taranoki” were studied resulting in identifying
functionalities of these cryptic Tps genes.
KEYWORDS
Tps genes; Araliaceae;
recombinant Escherichia
coli; volatile sesquiterpene;
purification and structural
determination
Functional identification of cryptic terpene synthase
(Tps) genes have ofen been performed using recombi-
nant micro-organisms containing Escherichia coli that
can functionally express the corresponding Tps genes
and generate the sesquiterpene synthase (TPS) prod-
ucts [1–4]. In many cases, the generated sesquiterpenes
(the TPS products) have been analyzed by gas chroma-
tography–mass spectrometry (GC–MS) using the MS
library or co-chromatography with the authentic sam-
ples [2,3,5–8]. However, such methods are sometimes
insufficient for the unambiguous determination of their
chemical structures and consequent identification of the
Tps genes.
To recover the generated volatile sesquiterpenes with-
out losses, recombinant micro-organisms that express
the Tps genes, such as E. coli and Saccharomyces cerevi-
siae, have ofen been cultured with two phases [n-do-
decane (C12 alkane)/H2O (medium), e.g. 20% (v/v)] to
recover the products in n-dodecane [5,6,8,9]. e two-
phase partition is adequate when the researchers iden-
tify the products using the GC–MS, whereas, because
of its high boiling point, n-dodecane is an inconvenient
solvent when the researchers purify the products. e
present report shows a simple method to purify vola-
tile sesquiterpenes from recombinant E. coli followed
by functional identification of Aralia-derived cryptic
Tps genes and structural identification of the generated
products.
Material and methods
Tps genes and mevalonate pathway-engineered
E. coli
e ZzZss2 (ZSS2) and ZoTps1 genes were described
previously [5,8]. Entire Tps gene sequences were isolated
by similar methods described [2]. Firstly, total RNA was
extracted from young leaves of Araria cordata “Udo”
or Aralia elata “Taranoki” using RNeasy Plant Mini kit
(Qiagen). Next, the cDNA pool was synthesized from
the total RNA using Clontech SMARTer RACE cDNA
amplification kit (Takara Bio) and used as the template
of following polymerase chain reactions (PCRs). Full
length sequence of Tps gene was cloned using degenerate
PCR and subsequent RACE method, isolating two Tps
gene (named AcTps1 and AcTps2) in A. cordata and a Tps
gene (named AeTps1) in A. elata. e cloned Tps gene
was transferred into a mevalonate (MVA) pathway-en-
gineered E. coli [10] using the E. coli expression vector
pETDuet-1 (Merck). e MVA pathway-engineered
CONTACT Kazutoshi shindo
kshindo@fc.jwu.ac.jp
supplemental data for this article can be accessed at https://doi.org/10.1080/09168451.2017.1386085.
© 2017 Japan society for Bioscience, Biotechnology, and agrochemistry

2
K. SHINDO ET AL.
E. coli was prepared by introducing plasmid pAC-MeV/
Scidi/Aacl into E. coli Bl21 (DE3). e plasmid contains
the MVA-pathway gene cluster from Streptomyces sp.
strain CL190, the Saccharomyces cerevisiae TPP isomer-
ase gene (Scidi), and acetoacetate-CoA ligase gene (Aacl)
for the utilization of lithium acetoacetate (LAA) as the
substrate [2,10].
was collected and washed with a equal amount of alka-
line 50% MeOH (0.5 M KOH in 50% MeOH) twice.
e washed alkane layer was concentrated to a small
volume of 5 mL in vacuo. is was applied on silica gel
(Silica Gel 60, Kanto Chemical) column chromatogra-
phy (20 i.d. × 200 mm) filled with n-pentane and devel-
oped with n-pentane (200 mL) (fraction (fr.) 1–30) →
n-pentane-EtOAc (5:1) (200 mL) (fr. 31 -60), stepwisely.
Eluates including sesquiterpenes in subsequent fractions
were detected as blue spots on silica gel TLC (fr.s 1–30
Preparation of LAA
Ethylacetoacetate (Tokyo Chemical Industry) (5.2 g),
H2O (10 mL), and 4 M LiOH (10 mL) were added into
capped 200 mL glass container and the container was
kept at 40 °C in water bath for 4 h for saponification
reaction. e solution was concentrated to dryness in
vacuo, and a quantity of 45 mL anhydrous MeOH was
then added into the flask and sonicated for 5 min. e
solution was filtered, and the filtrate was recovered in a
500 mL Erlenmeyer flask. Furthermore, 255 mL of dieth-
ylether was added in the recovery flask; it was shaken
well and stored overnight at 5 °C. e precipitation was
then recovered by filtration to obtain pure LAA (2.23 g).
were developed by n-pentane and fr.s 31–60 were devel
-
oped by n-pentane–EtOAc (5:1)) by heating TLC plates
afer spraying molybdophophoric acid solution (12 g
disodium molybdate (VI) dihydrate, 1.5 mL H3PO4,
25 mL H2SO4 in 500 mL distilled water).
Purification from E coli (pET-ZSS2 + pAC-MeV/Scidi/
Aacl) (ZzZss2 (ZSS2) in Zingiber zerumbet) [5] (10
flasks)
Fraction 38–42, which contained one silica gel TLC
(Merck) spot (Rf 0.40, solvent: n-pentane-EtOAc (5:1)),
was combined and concentrated to a small volume of
3 mL in vacuo and further purified through prepara-
tive high-performance liquid chromatography (HPLC)
(column: C30-UG-5 (10 i.d. × 250 mm, Developsil), sol-
vent: 80% CH3CN, flow rate: 3.0 mL/min), detection:
DAD (200–300 nm)); the peak that eluted at 9.6 min
was collected.
e eluate (30 mL) were mixed with a equal amount
of water, and partitioned with n-pentane (60 mL) in a
separatory funnel. e n-pentane layer was dried over
Na2SO4 for 30 min and concentrated to dryness in vacuo
(100 mm Hg) to procure pure β-eudesmol (1) (6.2 mg).
Production of volatile sesquiterpenes by
fermentation of recombinant E. coli
e recombinant E. coli was inoculated into the
100 mL TB medium in a 500 mL Erlenmeyer flask con-
taining 1 mM ampicillin (Ap) and chloramphenicol
(Cm) and was cultured at 25 °C with 150 rpm until the
OD (600 nm) of the culture reached 1.0. Furthermore,
2 mL of the culture was inoculated into a 500 mL
Sakaguchi flask containing 100 mL TB medium that
included 1 mM Ap, 1 mM Cm, 1 mM isopropylthio-
galactoside, and 100 mM LAA. In addition, 20 mL of
n-octane was added to each flask, and two-layer-cultured
at 20 °C with 150 rpm for 48 h. e culture without the
n-octane addition was also carried out, as needed.
Crude extracts from the recombinant E. coli cells cul-
tured without the n-octane addition [2], which included
all produced sesquiterpnes, were subjected to a 7890A
gas chromatography system coupled to a 5975C mass
spectrometer detector (GC–MS; Agilent Technologies,
CA, USA) to determine the production of volatile ses-
quiterpenes. A DB-WAX column (60 m, 0.25 mm ID,
0.25 μm Df) and N2 (carrier gas 1.12 mL/min) was uti-
lized. e GC oven was programmed to start at 40 °C
for 10 min, then ramped at 5 °C/min to 230 °C, and
held for 12 min.
Purification from E coli (pET-ZoTps1＋pAC-MeV/
Scidi/Aacl) (ZoTps1 in Zingiber officinale) [8] (10
flasks)
Fraction 8–10, which contained one silica gel TLC
spot (Rf 0.61, solvent: n-pentane), was combined and
concentrated to a small volume of 3 mL in vacuo and
further purified through preparative HPLC (column:
C30-UG-5 (10 i.d. × 250 mm), solvent: 95% CH3CN,
flow rate: 3.0 mL/min, detection: DAD (200–300 nm)),
and the peak that eluted at 18.0 min was collected. e
eluate (40 mL) was further treated as described above
to procure β-bisabolene [11] (2) (20.1 mg).
Purification from E. coli (pET‐AcTps1＋pAC‐MeV/
Purification of volatile sesquiterpenes from two-
layer cultures with recombinant E. coli cells
Scidi/Aacl) (AcTps1 in Araria cordata) (20 flasks)
Fraction 10–14, which contained one silica gel TLC spot
(Rf 0.61, solvent: n-pentane), was combined and con-
centrated to dryness in vacuo (100 mm Hg) to procure
pure germacrene A [11] (3) (10.1 mg).
e 10 or 20 flasks of each culture were mixed with 200 or
400 mL of n-hexane, and poured into a separatory funnel
and shaken well. e alkane layer (n-octane + n-hexane)

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
3
Research Analytical), and GC–MS as described above.
NMR spectra were measured in CDCl3 using the resid-
ual solvent peak as an internal standard (δC 77.0, δH
7.26 ppm); [α]D was measured in CHCl3.
Purification from E. coli (pET‐AcTps2＋pAC‐MeV/
Scidi/Aacl) (AcTps2 in Araria cordata) (20 flasks)
Fraction 8–12 (Rf 0.81, solvent: n-pentane) and fr. 13–17
(Rf 0.75, solvent: n-pentane), containing one silica gel
TLC spot, was combined and concentrated to dryness in
vacuo (100 mm Hg) to procure pure β-panasinsene [12]
(4) (fr. 8–12 5.6 mg) and β-caryophyllene [12] (5) (fr.
13–17 4.9 mg), respectively. Fr. 22–24, which contained
two silica gel spots (Rf 0.58 and 0.52, solvent: n-pen-
tane), was further purified through preparative HPLC
(column: ADME (10 i.d. × 250 mm, Shiseido), solvent:
80% CH3CN, flow rate: 3.0 mL/min, detection: DAD
(200–300 nm)), and the peaks that eluted at 21.5 and
22.5 min were collected separately. Subsequent eluates
(20 mL (tR 21.5 min) and 25 mL (tR 22.5 min)) were
further treated as described above to procure pure ger-
macrene A (3) (tR 21.5 min, 0.8 mg) and α-humulene
(6) (tR 22.5 min, 0.6 mg), respectively.
Physico-chemical properties of β-eudesmol (1)
GC–MS tR 48.1 min, m/z 222 (M+). [α]D20 32.9 (c 0.52).
1H NMR (Figure S1) δ: 0.69 (s, 3H, H-15), 1.20 (s,
6H, H-13 and H-14), 1.20–1.22 (2H, H-1b and H-9b),
1.35(m, 1H, H-7), 1.45–1.52 (2H, H-1a and H-9a), 1.55–
1.65 (6H, H-2a, H-2b, H-6a, H-6b, H-8a, and H-8b),
1.76 (m, 1H, H-5), 1.95 (m, 1H, H-3b), 2.30 (m, 1H,
H-3a), 4.44 (s, 1H, H-11b), and 4.70 (s, 1H, H-11a). 13C
NMR (Figure S2) δ: 16.3 (C-15), 22.4 (C-6), 23.5 (C-2),
25.0 (C-8), 27.1 (C-13), 27.2 (C-14), 35.9 (C-10), 36.9
(C-3), 41.1 (C-9), 41.8 (C-1), 49.4 (C-7), 49.8 (C-5), 72.9
(C-12), 105.3 (C-11), and 151.1 (C-4).
Physico-chemical properties of β -bisabolene (2)
Purification from E. coli (pET-AeTps1＋pAC-MeV/
GC–MS tR 38.4 min, m/z 204 (M+). [α]D24-69.3 (c 2.0).1H
NMR (Figure S3) δ: 1.45 (m, 1H, H-4b), 1.61 (s, 3H,
H-15), 1.65 (s, 3H, H-13), 1.69 (s, 3H, H-12), 1.81 (m,
1H, H-4a), 1.90 (m, 1H, H-3b), 2.05–2.15 (6H, H-6b,
H-6a, H-8b, H-8a, H-9b, and H-9a), 2.09 (m, 1H, H-5),
2.10 (m, 1H, H-3a), 4.74 (s, 1H, H-14b), 4.76 (s, 1H,
H-14a), 5.12 (m, 1H, H-10), 5.41 (m, 1H, H-1). 13C NMR
(Figure S4) δ: 17.7 (C-15), 23.5 (C-13), 25.7 (C-12), 26.8
(C-9), 28.3 (C-4), 30.8 (C-3), 31.4 (C-6), 34.9 (C-8), 39.8
(C-5), 107.1 (C-14), 120.8 (C-1), 124.3 (C-10), 131.5
(C-11), 133.8 (C-2), 154.3 (C-7).
Scidi/Aacl) (AeTps1 in A. elata) (20 flasks)
e fr.s that contained one silica gel TLC spot (fr. 8–9 (Rf
0.75, solvent: n-pentane) and fr. 17–25 (Rf 0.51, solvent:
n-pentane) were combined and concentrated to dryness
in vacuo (100 mm Hg), respectively, to procure pure
α-copaene [13] (7) (fr. 8–9, 5.0 mg) and δ-cadinene [14]
(8) (fr. 17–25, 4.2 mg).
Fraction 11–12 that contained two silica gel TLC
spots (Rf 0.65 and 0.61, solvent:n-pentane) were com-
bined and concentrated to dryness in vacuo (100 mm
Hg), and further purified through preparative HPLC
(column: C30-UG-5 (10 i.d. × 250 mm), solvent: 90%
CH3CN, flow rate: 3.0 mL/min, detection: DAD (200–
300 nm)), and the peaks that eluted at 27.0 min and
33.0 min were collected separately. Each eluate (30 mL
(tR 27.0 min) and 40 mL (tR 33.0 min)) was further
treated as described above to procure pure β-cubebene
[15] (9) (tR 27.0 min, 6.3 mg) and germacrene D [16]
(10) (tR 33.0 min, 1.8 mg), respectively.
Fraction 13–16, which contained several spots (Rf
0.58 (main spot), solvent: n-pentane), was combined
and concentrated to small volume (3 mL) in vacuo and
further purified through preparative HPLC (column:
C30-UG-5 (10 i.d. × 250 mm), solvent: 90% CH3CN,
flow rate: 3.0 mL/min, detection: DAD (200–300 nm)),
and the main peak eluted at 27.0 min was collected. e
eluate (30 mL) was further treated as described above to
procure pure δ-cadinene (8) (4.1 mg).
Physico-chemical properties of germacrene A (3)
24
GC–MS tR 34.4 min (β-elemene), m/z 204 (M+). [α]D
1
−1.90 (c 0.10). e signals in H NMR spectrum of
gemacrene A (3) were very broad because of its confor-
mational equilibrium in the solution, and we could not
assign them. e 1H spectrum of 3 was shown in Figure
S5 as supporting material.
Physico-chemical properties of β-panasinsene (4)
GC–MS tR 33.4 min, m/z 204 (M+). [α]D24-37.3 (c
0.29).1H NMR (Figure S6) δ: 0.75 (s, 3H, H-14), 0.86
(s, 3H, H-12), 1.07 (s, 3H, H-13), 1.09 (m, 1H, H-5b),
1.24 (m, 1H, H-5a), 1.38 (2H, H-3b and H-6b), 1.60 (m,
1H, H-6a), 1.69 (m, 1H, H-3a), 1.70 (m, 1H, H-2b), 1.95
(m, 1H, H-2a), 1.97 (m, 1H, H-7b), 2.10 (m, 1H, H-1),
2.20 (m, 1H, H-7a), 4.80 (s, 1H, H-15b), 4.90 (s, 1H,
H-15a). 13C NMR (Figure S7) δ: 18.2 (C-14), 23.1 (C-6),
24.7 (C-2), 24.8 (C-12), 30.5 (C-10), 30.6 (C-13), 33.8
(C-7), 35.7 (C-11), 36.0 (C-5), 41.1 (C-3), 45.6 (C-4),
52.6 (C-1), 52.8 (C-9), 108.3 (C-15), 152.3 (C-8).
Structural analyses of the isolated volatile
sesquiterpenes
Each isolated sesquiterpene was analyzed by NMR
(Bruker, AMX400), [α]D (AUTOPOL IV, Rudolph

4
K. SHINDO ET AL.
(m, 1H, H-8b), 0.89 (d J = 6.8 Hz, 3H, H-14), 0.92 (d
J = 6.8 Hz, 3H, H-15), 0.98 (d J = 6.8 Hz, 3H, H-12), 1.10
(m, 1H, H-9), 1.38 (m, 1H, H-1), 1.40 (m, 1H, H-8a),
1.54 (m, 1H, H-4b), 1.60 (2H, H-7a and H-13), 1.78 (m,
1H, H-6), 1.97 (m, 1H, H-4a), 2.02 (m, 1H, H-3b), 2.11
(m, 1H, H-3a), 4.57 (s, 1H, H-11b), 4.76 (s, 1H, H-11a).
13C NMR (Figure S15) δ: 18.8 (C-12), 19.4 (C-14), 20.1
(C-15), 26.3 (C-8), 28.5 (C-10), 29.0 (C-3), 30.8 (C-4),
31.6 (C-7), 33.4 (C-13), 35.8 (C-1), 38.1 (C-5), 44.1
(C-9), 100.8 (C-11), 155.3 (C-2).
Physico-chemical properties of β-caryophyllene (5)
GC–MS tR 37.2 min, m/z 204 (M+). [α]D24 -16 (c 0.16).1H
NMR (Figure S8) δ: 0.90 (s, 3H, H-12), 1.19 (s, 3H,
H-13), 1.35–1.55 (2H, H-2a and H-2b), 1.53 (s, 3H,
H-14), 1.55 (m, 1H, H-11b), 1.72 (m, 1H, H-11a), 1.74
(m, 1H, H-7b), 1.88 (ddd J = 2.5, 12.0, 12.0 Hz, 1H,
H-3b), 2.00 (m, 1H, H-6b), 2.08 (m, 1H, H-3a), 2.12 (m,
1H, H-1), 2.33 (m, 1H, H-6a), 2.48 (dd J = 7.0, 12.5 Hz,
1H, H-7a), 2.90 (ddd J = 8.3, 8.3, 11.8 Hz, 1H, H-9), 4.79
(s, 1H, H-15b), 4.86 (s, 1H, H-15a), 5.11 (dd J = 3.2,
11.6 Hz, 1H, H-5). 13C NMR (Figure S9) δ: 18.6 (C-14),
25.1 (C-2), 25.3 (C-12), 27.3 (C-6), 29.8 (C-13), 32.9
(C-10), 38.1 (C-11), 39.5 (C-7), 41.6 (C-3), 42.9 (C-9),
53.7 (C-1), 112.7 (C-15), 121.8 (C-5), 136.5 (C-4), 149.7
(C-8).
Physico-chemical properties of germacrene D (10)
GC–MS tR 38.0 min, m/z 204 (M+). [α]D24 -209 (c 0.17).
1H NMR (Figure S16) δ: 0.80 (d J = 6.8 Hz, 3H, H-13),
0.86 (d J = 6.8 Hz, 3H, H-14), 1.40 - 1.52 (2H, H-8b,
and H-8a), 1.42 (m, 1H, H-12), 1.52 (s, 3H, H-15), 1.95
(m, 1H, H-2b), 2.02 (m, 1H, H-7), 2.08 (m, 1H, H-3b),
2.10 - 2.30 (2H, H-9b, and H-9a), 2.40 (2H, H-2a, and
H-3a), 4.74 (s, 1H, H-11b), 4.79 (s, 1H, H-11a), 5.13
(m, 1H, H-1), 5.25 (dd J = 10.0, 15.8 Hz, 1H, H-6), 5.80
(d J = 15.8 Hz, 1H, H-5). 13C NMR (Figure S17) δ: 15.9
(C-15), 19.3 (C-13), 20.7 (C-14), 26.5 (C-8), 29.2 (C-2),
32.7 (C-12), 34.5 (C-3), 40.7 (C-9), 52.9 (C-7), 109.0
(C-11), 129.6 (C-1), 133.5 (C-6), 134.0 (C-10), 135.5
(C-5), 148.9 (C-4).
Physico-chemical properties of α-humulene (6)
1
GC–MS tR 37.0 min, m/z 204 (M+). H NMR (Figure
S10) δ: 1.06 (s, 6H, H-14 and H-15), 1.43 (s, 3H,
H-13), 1.64 (s, 3H, H-12), 1.91 (d J = 7.4 Hz, 2H, H-7),
2.05–2.15 (4H, H-3b, H-3a, H-4b, and H-4a), 2.51 (d
J = 7.3 Hz, 2H, H-11), 4.87 (t J = 7.4 Hz, 1H, H-6), 4.93
(m, 1H, H-2), 5.16 (d J = 15.4 Hz, 1H, H-9), 5.59 (dt
J = 7.3, 15.9 Hz, 1H, H-10). 13C NMR (Figure S11) δ:
15.1 (C-13), 17.9 (C-12), 23.3 (C-3), 27.1 (C-14 and
C-15), 37.4 (C-8), 39.7 (C-4), 40.4 (C-11), 42.0 (C-7),
125.0 (C-6), 125.8 (C-2), 127.7 (C-10), 133.2 (C-5), 139.2
(C-1), 141.0 (C-9).
Accession numbers
Accession numbers of the new genes deposited are
LC307165 (AcTps1), LC307166 (AcTps2), and LC307167
(AeTps1).
Physico-chemical properties of α-copaene (7)
GC–MS tR 31.9 min, m/z 204 (M+). e assignment of
α-copaene was performed by co-GC–MS analysis with
the authentic sample procured from Taiyo Corporation.
e physico-chemical properties of 7 will be described
in another report (Higuchi et al. in preparation).
Results
In the present study, we isolated three novel Tps genes
(AcTps1 and AcTps2 from A. cordata, and AeTps1 from
A. elata). Amino acid sequences deduced from these
genes were phylogenetically analyzed (Figure 1). e
AcTps1 and AcTps2 gene products were closely related.
Although they fall into the same clade, the AeTps1 gene
product was found to be categorized into another single
clade.
To recover volatile sesquiterpenes – TPS protein prod-
ucts that are produced by E. coli carrying the correspond-
ing Tps genes—the recombinant E. coli had ofen been
cultured with two phases (n-dodecane (C12 alkane)/
H2O (medium); 20% (v/v)) to recover the products in
n-dodecane [5,6,8]. e use of n-dodecane was adequate
when the researchers would identify the products (vola-
tile sesquiterpenes) using GC–MS, whereas n-dodecane
was an inconvenient solvent when the researchers would
purify the products because of its high boiling point
(n-dodecane is not removable using diaphragm pump).
us, we screened more suitable alkane for two-phase
culture to purify the products using E. coli that harbored
plasmid pET-ZSS2 carrying the Z. zerumbet Tps gene
Physico-chemical properties of δ-cadinene (8)
GC–MS tR 39.1 min, m/z 204 (M+). [α]D24 -57.6 (c 0.12,
CHCl3). Electron ionization-MS m/z 204 (M+). 1H NMR
(Figure S12) δ: 0.78 (d J = 7.0 Hz, 3H, H-14), 0.96 (d
J = 7.0 Hz, 3H, H-15), 1.05 (m, 1H, H-9), 1.58 - 1.64 (2H,
H-8b and H-8a), 1.65 (s, 3H, H-12), 1.67 (s, 3H, H-11),
1.90 - 2.00 (4H, H-3b, H-3a, H-7b, and H-7a), 2.05 (m,
1H, H-13), 2.06 (m, 1H, H-4b), 2.50 (m, 1H, H-10), 2.71
(m, 1H, H-4a), 5.44 (m, 1H, H-1). 13C NMR (Figure S13)
δ: 15.6 (C-14), 18.5 (C-12), 21.2 (C-8), 21.7 (C-15), 23.6
(C-11), 26.6 (C-4), 26.7 (C-13), 32.0 (C-3), 32.3 (C-7),
39.4 (C-10), 45.4 (C-9), 124.5 (C-6), 124.7 (C-1), 129.9
(C-5), 134.2 (C-2).
Physico-chemical properties of β-cubebene (9)
1
GC–MS tR 33.7 min, m/z 204 (M+). H NMR (Figure
S14) δ: 0.57 (m, 1H, H-7b), 0.85 (m, 1H, H-10), 0.88

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
5
Figure 1. Phylogenetic tree of plant tPss.
notes: ZzZss2, ZotPs1, actPs1, actPs2, aetPs1 (underlined) and known tPss were phylogenetically analyzed. tPs accession numbers are as follows:
ZmtPs4 (aas88571), ZmtPs5 (aas88574), ZmtPs6 (aas88576), ZmtPs10 (aaX99146), ZmtPs11 (acF58240), obZis (aaV63788), agag4 (aac05727),
agag5 (aac05728), VvaBs (nP_001267972), VvBcas (adr74193), VvBcus (adr74200), Vvgds (Q6Q3h3), adgds (aaX16121), ZotPs1 (Bai67934), Zogds
(aaX40665), ZzZss1 (Bag12020), ZzZss2 (Bag12021), PttPs5 (KF776503), PttPs7 (KF776505), PttPs8 (KF776506), PttPs9 (KF776507), PttPs11 (KF776509),
PttPs14 (KF776512), sasesquitPs (acF24768), sausesquitPs (hQ343281), smsesquitPs (JF746810), sspisesquitPs (hQ343282). Circles indicate tPss in the
same genus or the same species.
(ZzZss2; ZSS2) [5] and E. coli harbored plasmid pET-
ZoTps1 carrying the Z. officinale Tps gene (ZoTps1) [8].
We tested n-hexane (C-6), n-octane (C-8), and n-decane
(C-10), and found that n-hexane was toxic to recombi-
nant E. coli; it did not grow because of high solubility of
n-hexane in water, and n-octane and n-decane could be
used as alternative phases, which do not have any effect
on the growth of E. coli. Because n-octane was removable
using diaphram pump, we selected n-octane as the new
material for the two-phase culture. Consequently, pure
β-eudesmol (1) (6.2 mg) and β-bisabolene (2) (20.1 mg)
were obtained from total 1 L culture of E. coli carrying
plasmid pET-ZSS2 and E. coli carrying plasmid pET-
ZoTps1, respectively.
Using the purification method described in the
Material and Methods section, we successfully isolated
the volatile sesquiterpenes that were produced not only
by the above-mentioned recombinant E. coli but also
by the recombinant E. coli carrying the unidentified
Araliaceae Tps genes. e extracts from the recombinant
E. coli cells were analyzed by GC–MS to investigate the
product property of the enzyme. e results are shown in
Supplementary Figures S18 and Table S1–S4 (the result
of ZzZSS2 (ZSS2) is not shown, since it was described
[5]) . Each purified volatile sesquiterpene was subjected
to spectroscopic analysis along with GC–MS analysis to
identify its chemical structure.
e molecular formula for 1 was determined as
C15H26O by the observation of M+ ion peaks in GC–MS
1
analyses (m/z 222). e analyses of H NMR (Figure
S-1), 13C NMR (Figure S-2), and HSQC spectrum of
1 proved the presence of three singlet methyl carbons
[δC 17.0 (C-15), δC 27.8 (C-13), and δC 27.9 (C-14)],
six sp2 methylene carbons [δC 23.1 (C-6), δC 24.4 (C-2),
δC 25.8 (C-8), δC 37.7 (C-3), δC 42.0 (C-9), and δC 42.7
(C-1)], two sp3 methine carbons [δC 50.1 (C-7) and δC
50.5 (C-5)], two sp3 quaternary carbons [δC 36.5 (C-10)
and δC 72.4 (C-12)], one sp2 (exo methylene) carbon [δC
106.3 (C-11)], and one quaternary sp2 carbon [δC 151.6
(C-4)] in 1. e attachment of oxygen at C-12 was also
confirmed according to its 13C chemical shif.
1
e analyses of H-1H DQF COSY and HSQC-
TOCSY spectra of 1 proved the presence of 1H-1H vicinal
spin networks C-1 ∼ C-3 and C-5 ∼ C-9 in 1 (Figure 2).
e linkages of C-1, C-5, and C-9 through C-10 were
proved by the observation of 1H-13C long-range coupling

6
K. SHINDO ET AL.
Figure 2. nmr spectral analyses for β-eudesmol (1), β-bisabolene (2), and δ-cadinene (8).
Figure 3. the structures of purified volatile sesquiterpenes.
from H-15 (δH 0.78) to C-1, C-5, and C-9 in the HMBC
spectrum of 1, and the linkages of C-3 - C-4 - C-5 and
C-7 – C-12 – C-13 (C-14) were also proved by the 1H-13C
long-range coupling from H-11 (δH 4.65 and δH 4.89) to
C-3, C-4, and C-5 and from H-13 and H-14 (δH 1.07)
to C-7 and C-12, respectively (Figure 2). From these
observations, the planar structure of 1 was established.
e key NOEs observed among H-5, H-7, and H-9β
(1,3-diaxials) and between H-9α and H-15) (Figure 3)
in NOESY spectrum of 1 clarified the relative configura-
tions of 1, and the absolute configuration of 1 was proved
as shown in Figure 2 by the [α]D value (32.9 (c 0.52) [16].
From these observations, 1 was proved to identical to
β-eudesmol. is ZzZSS2 product was already identified

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
7
by Yu et al. [5] while its NMR and [α]D analysis was first
shown here.
and the ZoTps1 gene by GC–MS analyses (identification
using GC–MS library and the authentic standards) to be
β-eudesmol [5] (1) and (S)- β-bisabolene [8] (2), respec-
tively. Recently, we were further required to purify 1 and
2 to analyze their structures in detail. For these purposes,
we established an isolation method to purify the volatile
sesquiterpenes using the E. coli cultures of pET-ZSS2
(carrying ZzZss2) and pET-ZoTps1 (carrying ZoTps1).
n-Octane was first added to the culture to trap gener-
ated terpenes, followed by two-phase partition, silica gel
column chromatography, and preparative ODS-HPLC,
which is generally available in the modern chemical lab-
oratories. We also successfully clarified the structures of
1 and 2 by NMR, [α]D analyses, and GC–MS analyses.
Furthermore, we applied the above isolation method
to the volatile sesquiterpenes, produced by Araria TPSs
[pET-AcTps1 and pET-AcTps2 (AcTps1 and AcTps2 in
A. cordata), and pET-AeTps1 (AeTps1 in A. elata)] that
were not reported previously; we successfully identi-
fied the volatile sesquiterpenes. is is the first study to
report on the volatile sesquiterpenes of these Tsp genes
and their physico-chemical data (1H and 13C NMR, and
[α]D), resulting in achievement of the functional identi-
fication of these cryptic Tps genes.
e molecular formula for 2 was determined as
C15H24 by the observation of M+ ion peaks in GC–MS
1
analyses (m/z 204). e analyses of H NMR (Figure
S-3), 13C NMR (Figure S-4), and HSQC spectrum of 2
proved the presence of three singlet methyl carbons [δC
17.7 (C-15), δC 23.5 (C-13), and δC 25.7 (C-12)], five sp2
methylene carbons [δC 26.8 (C-9), δC 28.3 (C-4), δC 30.8
(C-3), δC 31.4 (C-6), δC 34.9 (C-8)], one sp3 methine
carbons [δC 39.8 (C-5)], one sp2 (exo methylene) carbon
[δC 107.1 (C-14)], two sp2 methine carbons [δC 120.8
(C-1) and δC 124.3 (C-10)], and three sp2 quaternary
sp2 carbons [δC 131.5 (C-11), δC 133.8 (C-2), and δC
154.3 (C-7)] in 2.
1
e analyses of H-1H DQF COSY and HSQC-
TOCSY spectra of 2 proved the presences of 1H-1H vic-
inal spin networks C-3 ∼ C-6 – C-1, and C-8 ∼ C-10 in
2 (Figure 2). e linkages of C-1–C-2–C-3 was proved
by the observation of 1H-13C long-range coupling from
H-13 (δH 1.65) to C-1, C-2, and C-3 in the HMBC spec-
trum of 2, and the linkages of C-5 - C-7 - C-8 and the
linkage of C-10, C-12, and C-15 through C-11 were also
1
proved by the H-13C long-range coupling from H-14
(δH 4.74 and δH 4.76) to C-5, C-7, and C-8 and from
H-12 (δH 1.69) and H-15 (δH 1.61) to C-11 and C-10,
respectively (Figure 2). From these observations, 2 was
proved to be identical to β-bisabolene. e absolute con-
figuration of 2 was shown to be 5S by chiral GC–MS
analysis in the previous study [8], and it was also proved
by the [α]D value (−69.3 (c 2.0)) [17] (Figure 2). is
ZoTPS1 product was already identified by Fujisawa et al.
[8] while its NMR and [α]D analysis was first shown here.
e planar structures of 4–10 produced by E. coli car-
rying the Araliaceae Tps genes were also determined by
NMR analyses, whereas germacrene A (3) was identified
by the observation of β-elemene in the GC–MS analy-
Author contributions
Kazutoshi Shindo (KS), Jun-ichiro Hattan, Mariko
Kato, Miho Sato, Tomoko Ito, Yurika Shibuya, Arisa
Watanabe, Maki Sugiyama, Yuri Nakamura performed
experiments. Norihiko Misawa supervised the present
study, and wrote the manuscript along with KS.
Ethical statement
is article does not contain any studies with human
participants or animals performed by any of the authors.
1
sis because of its broadened (unassignable) H signals
(Figure S-5). e key NOEs observed in NOESY spectra
to clarify the relative configurations of 8 are shown in
Figure 2, whereas the relative stereochemistry of 4–7,
9, and 10 were not confirmed by NOESY analyses. e
absolute configurations of 3–10 were certified by match-
ing with the GC–MS library (the presence of all the
compounds (1–10) was certified by the GC–MS library
(Supplementary Figure S18 and Table S1-S4) and [α]D
values. e structures of the isolated volatile sesquiter-
penes from each TPS are listed in Figure 3.
Acknowledgments
e authors thank to Miyuki Tsuneda and Erina Karaya
for their experiments involved in cloning and functional
analysis of the Aralia Tps genes with GC–MS as a bachelor
study. is work was in part supported by the commission
for Development of Artificial Gene Synthesis Technology
for Creating Innovative Biomaterial from the Ministry of
Economy, Trade and Industry (METI), Japan.
Disclosure statement
No potential conflict of interest was reported by the authors.
Discussion
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