Characterization of a Sesquiterpene Synthase Catalyzing Formation of Cedrol and Two Diastereoisomers of Tricho-Acorenol from Euphorbia fischeriana

Article abstract of DOI:10.1021/acs.jnatprod.1c00126

A sesquiterpene synthase gene was identified from the transcriptome of Euphorbia fischeriana Steud, and the function of its product EfTPS12 was characterized by in vitro biochemical experiments and synthetic biology approaches. EfTPS12 catalyzed conversion of farnesyl diphosphate into three products, including cedrol (1) and eupho-Acorenols A (2) and B (3) (two diastereoisomers of tricho-Acorenol), thereby being named EfCAS herein. The structures of 2 and 3 were determined by spectroscopic methods and comparison of experimental and calculated electronic circular dichroism spectra. EfCAS is the first example of a plant-derived sesquiterpene synthase that is capable of synthesizing acorane-Type alcohols. This study also documents that synthetic biology approaches enable large-scale preparation of volatile terpenes and thereby substantially facilitate characterization of corresponding terpene synthases and elucidation of the structures of their products.

Full text of DOI:10.1021/acs.jnatprod.1c00126

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Article
Characterization of a Sesquiterpene Synthase Catalyzing Formation
of Cedrol and Two Diastereoisomers of Tricho-Acorenol from
Euphorbia fischeriana
Jianxun Zhu,# Lihong Liu,# Maobo Wu, Guiyang Xia, Pengcheng Lin, and Jiachen Zi*
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ABSTRACT: A sesquiterpene synthase gene was identified from
the transcriptome of Euphorbia fischeriana Steud, and the function
of its product EfTPS12 was characterized by in vitro biochemical
experiments and synthetic biology approaches. EfTPS12 catalyzed
conversion of farnesyl diphosphate into three products, including
cedrol (1) and eupho-acorenols A (2) and B (3) (two
diastereoisomers of tricho-acorenol), thereby being named
EfCAS herein. The structures of 2 and 3 were determined by
spectroscopic methods and comparison of experimental and
calculated electronic circular dichroism spectra. EfCAS is the
first example of a plant-derived sesquiterpene synthase that is
capable of synthesizing acorane-type alcohols. This study also
documents that synthetic biology approaches enable large-scale preparation of volatile terpenes and thereby substantially facilitate
characterization of corresponding terpene synthases and elucidation of the structures of their products.
Euphorbia fischeriana Steud is a plant belonging to the
lant volatile oils with sesquiterpenes as their major
Pcomponents have been widely used in the flavor, perfume, Euphorbiaceae family. Its dry roots, known as “langdu” in
cosmetic, and health care industries,1,2 such as sandalwood
traditional Chinese medicine, have been used to treat edema,
ascites, and tuberculosis.12 Although this herb is well known
for its diterpene constituents, such as the anti-HIV candidate
drug prostratin13 and the potent cytotoxic agent 17-
hydroxyjolkinolide B,14 its volatile oil (with sesquiterpenes as
the major components) has been reported to possess radical
scavenging activity.15 In the present study, we identified the
sesquiTPS EfTPS12 from E. fischeriana and obtained sufficient
amounts of its products for NMR and ECD analysis using an E.
coli-based synthetic biology approach. The results showed that
EfTPS12 catalyzed cyclization of FPP to yield cedrol (1) and
eupho-acorenols A (2) and B (3) (two diastereoisomers of
tricho-acorenol).
oils,3 patchouli oils,4 and turmeric oils.5 Volatile sesquiterpenes
also play a variety of critical roles in plant physiology and
ecological interactions including plant−plant, plant−herbivore,
plant−pathogen, and plant−mutualist interactions.2,6,7 Sesqui-
terpenes are synthesized from their common precursor farnesyl
diphosphate (FPP) under catalysis of sesquiterpene synthases
(sesquiTPSs).8 Most sesquiTPSs are catalytically promiscuous,
thereby producing more than one product with diverse planar
skeletons and configurations.8,9 This makes sesquiterpenes a
group of structurally diverse compounds. Due to great
complexities of volatile sesquiterpene patterns in plants, the
low content of each single sesquiterpene component, and their
close chemical properties (e.g., polarity), it is difficult to
completely separate these sesquiterpene components and
thereby elucidate the complete structures (particularly the
absolute configurations). In most cases, volatile sesquiterpenes
of plant extracts are simply identified and analyzed by GC-MS-
based methods and comparing mass spectra of target peaks
with standard spectra.10 We constructed microbial platforms
for the production of plant-derived sesquiterpenes in previous
studies,3,11 which not only laid a foundation for biological
production of the corresponding compounds on an industrial
scale, but also provided a promising approach to obtain
sufficient amounts of products of sesquiTPSs for unambiguous
elucidation of their structures.
RESULTS AND DISCUSSION
■
To identify sesquiTPS-encoding genes from E. fischeriana, the
total RNAs were isolated from a mixture of leaves, stems, and
roots and sequenced on an Illumina Novaseq platform. By
Received: February 5, 2021
Published: May 20, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
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GC-MS, compared with the control sample (the extract of the
reaction mixture containing FPP and the inactive EfTPS12,
which was heated at 80 °C for 30 min in advance) (Figure 1b−
f). The MS spectra of 1−3 showed the same molecular ion
peaks at m/z 222 (Figure 1c−e), indicating that they are all
sesquiterpene alcohols. Compound 1 was identified to be
cedrol, as its MS spectrum is identical with that of the reported
one (Figure 1c).19 This result was consistent with the
phylogenetic analysis (Figure S1) in which EfTPS12 was
closest to Lc-CedS, a cedrol synthase from Leucosceptrum
canum.19
To unambiguously determine the structures of these three
products, we used a synthetic biology approach to obtain
sufficient amounts for spectroscopic analysis. EfTPS12 was
expressed in an E. coli strain in which three lowly expressed
genes encoding 1-deoxy-D-xylulose-5-phosphate synthase
(DXS), 1-deoxy-D-xylulose-5-phosphate reductase (DXR),
and isopentenyl diphosphate isomerase (IDI) of the
methylerythritol 4-phosphate (MEP) pathway were overex-
pressed.20 After fermentation of the resulting E. coli strain, the
products 1−3, as well as two other compounds, 4 and 5, were
detected in the culture (Figure 2). These five compounds were
extracted and isolated, and their structures were elucidated.
Compound 1 is indeed cedrol, as its NMR data and specific
rotation value ([α]22D +11.6, c 0.3, CH2Cl2) are identical with
the reported data of cedrol.21,22 Compound 5 was determined
using a Trinity de novo assembler,16 57 million clean reads
including 8.55 G clean bases were assembled after removing
reads containing adapters, reads containing poly-N, and low-
quality reads, with a Q30 of 92.4%. Subsequently, we used the
previously characterized sesquiTPS genes17,18 (Table S1) as
the queries to perform a BLAST search, and 10 candidate
genes were identified, including five full-length and five
truncated genes. The full-length sequences of five truncated
genes were acquired by Rapid Amplification of cDNA ends.
After expressing all of the candidate genes in E. coli, soluble
expression of EfTPS12 was observed (Figure 1a). EfTPS12
was incubated with FPP, and then the reaction mixture was
extracted with n-hexane. From the resulting extract, three peaks
(1−3), as well as the peak of farnesol (FOH), were detected by
Figure 1. Functional characterization of EfTPS12 by in vitro biochemical assays. (a) SDS-page analysis of the recombinant EfTPS12; (b) GC-MS
chromatograms of the extracts of the reaction mixtures of FPP with either EfTPS12 (red) or boiled EfTPS12 (black); (c−f) Mass spectra of 1, 2, 3,
and farnesol (FOH), respectively.
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presence of a cyclohexene with a methyl group substituted at
C-4 and a hydroxy group at C-3, which is consistent with the
hydroxy group signal (3302 cm−1) in the IR spectrum. The
HMBC correlations from H3-14 to C-1, C-9, and C-10, from
H3-12 and H3-13 to C-7 and C-11, from H-11 to C-1 and C-8,
and from H2-8 to C-9 (Figure 3) suggested the presence of a
five-membered ring with an isopropyl group at C-7 and a
methyl group at C-10. Thus, the planar structure of 2 was
determined and is the same as tricho-acorenol.24 Because the
NMR data of 2 were not completely identical to those of
tricho-acorenol24,25 and (+)-(3R)-hydroxy-4-acorene (also
called as ent-epi-tricho-acorenol),25,26 2 is not one of these
compounds or their enantiomers.
The relative configuration of 2 was determined by the
NOESY correlations between H2-2 and H3-14, between H-3
and H-7, between H-5 and H3-13, between H2-6 and H3-12,
and between H2-6 and H3-13, as shown in Figure 3. The
absolute configuration of 2 was subsequently determined by
comparing the experimental and calculated ECD spectra
(Figure 4).27 Moreover, cedrol (1) is one of the co-products
of EfTPS12 and was synthesized from the intermediate v,
which is also the biosynthetic intermediate of 2 (Scheme 1).28
This further confirms the absolute configuration of 2, e.g., 1R,
3S, 7S, 10R, a diastereoisomer of tricho-acorenol.29 Compound
2 was obtained by a chemical synthesis approach before,30
while this study documents that it is also a natural product. We
named it eupho-acorenol A.
Compound 3 was isolated as a colorless oil, [α]22 +4.9 (c
D
0.3, CH2Cl2). Its molecular formula, C15H26O, was deduced
from the molecular ion peak [M]+ at m/z 222.1976 in the
1
HREIMS spectrum. The H and 13C NMR data of 2 and 3
were very similar (Table 1). A careful analysis of the HSQC,
COSY, and HMBC spectra of 3 indicated that it possesses the
same planar structure as 2 (Figure 3). Because the cross-peaks
in the NOESY spectrum of 3 acquired in CDCl3 are too
crowded to be clearly identified, additional NMR experiments
were performed in pyridine-d5 (Table 1). The NOESY
spectrum of 3 acquired in pyridine-d5 showed the correlations
of H-3/H3-14, H-2a (δH 2.23)/H3-14, and H-2b (δH 1.86)/H-
7 (Figure 3), suggesting the relative configuration of 3 as
shown in Figure 3. Then, its absolute configuration (1R, 3R,
7S, 10R) was also determined by comparing its experimental
and calculated ECD spectra (Figure 4). The absolute
configuration was consistent with the proposed biogenesis
(Scheme 1). Like compound 2, 3 was obtained in the same
synthesis effort.30 We named it eupho-acorenol B.
Figure 2. Functional characterization of EfTPS12 by synthetic biology
approaches. (a) GC-MS chromatogram of the extracts of the cultures
of the E. coli C41 strains containing either petDuet-1-ERG20-
EfTPS12 (red) or petDuet-1-ERG20 (black); (b and c) mass spectra
of 4 and 5.
as (E)-β-curcumen-12-ol by comparing its MS (Figure 2c) and
NMR data with the literature data,23 as well.
Compound 2 was obtained as a colorless oil, [α]22D +14.4 (c
0.3, CH2Cl2). Its HREIMS spectrum showed a molecular ion
[M]+ at m/z 222.1976, indicating that its molecular formula is
1
C15H26O, with three indices of hydrogen deficiency. The H
NMR spectrum showed the signals for four methyl groups (δH
1.77, 0.89, 0.86, and 0.81) (Table 1). Fifteen carbon signals
were preliminarily assigned by the 13C and DEPT NMR
spectra, including a trisubstituted double bond (δC 134.7 and
125.1), an oxygen-bearing methine group (δC 69.3), a
quarternary carbon (δC 45.7), three nonoxygenated methine
groups (δC 28.1, 45.1, and 56.2), four methylene groups (δC
45.5, 30.8, 25.6, and 22.6), and four methyl groups (δC 25.4,
19.3, 19.0, and 15.6), which are consistent with four methyl
proton signals in the 1H NMR spectrum (Table 1). After one-
bond linkage relationships between carbons and protons were
determined by the HSQC experiment, the planar structure of 2
was constructed by the COSY and HMBC experiments. The
correlations of H2-2/H-3 and H-5/H2-6 were observed in the
COSY spectrum of 2, and the long-range correlations from H3-
15 to C-3, C-4, and C-5, from H-3 to C-1, C-2, C-4, and C-5,
from H-2 to C-3, C-4, and C-6, and from H-6 to C-1 were
observed in the HMBC spectrum (Figure 3), indicating the
Compound 4 was obtained as a colorless oil. Its 1H and 13
C
NMR data showed the signals assigned to three methyl groups
(δH 1.54, δC 15.5; δH 1.58, δC 15.8; δH 1.63, δC 14.0), three
trisubstituted double bonds (δH 5.27, 5.20, and 4.89; δC 135.7,
133.4, 133.2, 125.8, 125.0, and 124.3), and two oxygen-bearing
methylene groups (δH 4.12 and 3.94; δC 77.7 and 69.3) (Table
1). The HSQC experiment was used to assign the one-bond
linkage of the protons and carbons (Table 1), and then the
structure of 4 was constructed by analysis of its COSY and
HMBC spectra. Three structural fragments C-3/C-4/C-5, C-
7/C-8/C-9, and C-11/C-12 were obtained by the COSY
experiment (Figure S2). In the HMBC spectrum, the
correlations from H3-15 to C-1, C-2, and C-3, from H3-14 to
C-5, C-6, and C-7, from H3-13 to C-9, C-10, and C-11, from
H-1 to C-12, and from H-12 to C-1 constructed a 13-
membered ether ring and indicated that three methyl groups
(C-15, C-14, and C-13) are located at C-2, C-6, and C-10,
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Table 1. NMR Data for Compounds 2, 3, and 4 (1H 400 MHz and 13C 100 MHz)
2
3
4
a
a
a
a
b
b
a
a
no.
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
45.7, C
45.5, CH2
46.7, C
45.9, CH2
46.64
46.61
77.7, CH2
133.2, C
3.94, s
c
1.85
1.88, dd (12.6, 9.9)
1.72
4.25, m
2.23, dd (12.8, 10.1)
1.86, ddd (12.8, 6.2, 1.7)
4.51, m
c
c
1.60
4.29, brt (6.9)
3
4
5
6
69.3, CH
134.7, C
125.1, CH
25.6, CH2
69.6, CH
135.2, C
125.1, CH
25.2, CH2
68.1
125.8, CH
24.1, CH2
38.7, CH2
133.4, C
5.20, t (6.6)
c
137.4
123.4
25.2
2.18
5.52, ddd (6.1, 3.0, 1.4)
5.52, m
2.00, dd (17.6, 5.3)
5.51, m
2.13, m
c
c
1.84
1.96
1.58
1.17
1.54
1.18
1.69
1.15
1.70
c
c
1.66
1.28
1.29
c
c
c
7
8
56.2, CH
22.6, CH2
1.51
58.0, CH
25.8, CH2
58.0
26.1
125.0, CH
24.7, CH2
4.89, t (6.9)
c
c
c
c
1.59
2.18
c
c
1.50
c
c
c
9
30.8, CH2
1.74
31.6, CH2
1.74
1.27
1.73
1.66
31.6
38.6, CH2
2.10, m
c
c
1.18, m
1.68, m
c
c
10
11
12
13
14
15
45.1, CH
28.1, CH
25.4, CH3
19.0, CH3
15.6, CH3
19.3, CH3
44.6, CH
28.3, CH
24.6, CH3
21.9, CH3
18.5, CH3
18.9, CH3
44.8
28.4
24.3
21.9
18.5
19.6
135.7, C
c
c
c
1.75
1.53
124.3, CH
69.3, CH2
15.5, CH3
15.8, CH3
14.0, CH3
5.27, t (6.3)
4.12, d (6.3)
1.54, s
1.58, s
1.63, s
0.89, d (6.9)
0.81, d (6.6)
0.86, d (6.8)
0.98, d (6.7)
0.87, d (6.7)
0.82, d (6.5)
1.74, brs
0.85, d (6.7)
0.77, d (6.6)
0.87, d (6.5)
1.99, brs
1.77, brs
a
b
c
The data were measured in CDCl3. The data were measured in pyridine-d5. Overlapped by other signals.
A cedrol synthase and an epi-cedrol synthase have been
identified from other plants,19,31 and the biosynthetic
mechanism of 1 has been reported (Scheme 1).28 In the
reaction catalyzed by EfTPS12 (Scheme 1), formation of 1, 2,
and 3 competes to consume intermediate v. In this process,
1,5-hydride shift (path B) may be more favorable than attack
of the double bond (path A), which could result in the
amounts of 2 and 3 being bigger than that of 1 (Figure 1b). In
addition, nucleophilic attack of a water molecule above the
plane of the cyclohexene ring could be hindered by CH3-14
and CH3-15 (Scheme 1 and Figure 3), due to which more 3
was produced than 2 in the in vitro reaction (Figure 1b). In
order to clarify the role of EfTPS12 in the formation of 4 and
5, a handful of experiments were conducted. We synthesized
tris(tetrabutylammonium) 12-hydroxyfarnesyl diphosphate
and used it as the substrate to perform an in vitro reaction,
but no product was detected (Figure S5), suggesting EfTPS12
cannot catalyze conversion of 12-hydroxyfarnesyl diphosphate
into 4 and 5. We mutated EfTPS12 by respectively replacing
Asp residues of its catalytic motif (DDTYD) with Ala to yield
three mutant enzymes, EfCTSD303A, EfCTSD304A, and
EfCTSD307A. Coexpression of three mutant enzymes with
ERG20 did not result in production of 1−5 (Figure S6),
indicating that EfTPS12 is indispensable for production of 4
Figure 3. Key COSY, HMBC, and NOESY correlations of 2 and 3.
respectively (Figure S2). In the NOESY spectrum, the
correlation between H2-1 and H-3 was observed, indicating
the configuration of the double bond between C-2 and C-3 is
E. Thus, the structure of 4 was determined.
It has been reported that E. fischeriana volatile oil contains
many sesquiterpenes including cedrol (1).15 We detected
sesquiterpenes in the roots, stems, and leaves of E. fischeriana
by GC-MS. 1 and an unknown compound (6) were identified
from the root extract (Figure S8). The mass spectrum of 6 is
very similar to that of acorenone (or ent-acorenone) (Figure
S8),25 indicating 6 is one of the stereoisomers of acorenone.
The results implied that 2 and 3 could be further modified in
E. fischeriana, which may explain the reason that 2 and 3 were
not detected in the plant tissues (Figure S8).
Figure 4. Comparison of the experimental and calculated ECD spectra of 2 and 3.
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Scheme 1. Plausible Biosynthetic Pathway for 1−3
MS analysis was respectively performed on an Agilent 7890 GC
instrument equipped with a Saturn 2100 ion trap mass spectrometer
and an Agilent 7250 GC/Q-TOF system in electron ionization (70
eV) mode, using an HP-5MS column. HPLC separation was
performed on an Ultimate 3000 instrument (Thermo Scientific),
using a YMC-Pack Pro C18 column (250 × 4.6 mm, 5 μm) or a
Phenomenex FMG-AC5-A01-NFC Chiral ND (2) column (250 × 4.6
mm, 5 μm, Guangzhou FLM Scientific Instrument Co. Ltd.). Silica gel
(200−300 mesh, Qingdao Marine Chemical Inc.) was used for
gravity-driven column chromatography.
Plant Material. The whole herbs of Euphorbia fischeriana were
collected in Shenyang, Liaoning Province, People’s Republic of China,
in May 2017, and identified by Prof. Yingni Pan of Shenyang
Pharmaceutical University (voucher specimen No. EFIYP-201705).
After being washed with tap water and cut into small pieces, the plant
tissue pieces were immediately frozen in liquid nitrogen and stored at
−80 °C.
Transcriptome Analysis. RNA isolation, sequencing, assembling,
annotation, and FPKM calculation were performed according to the
previous methods.11
RNA Isolation and Gene Cloning. Total RNAs were isolated
from a mixture of E. fischeriana leaves, stems, and roots and
transcribed to cDNAs using RNA Prep Pure Plant kit (TIANGEN)
and HiScript II Q Select RT SuperMix kit (Vazyme), respectively.
The full-length transcript of EfCAS was amplified using the gene-
specific primers by Phanta Max Super-Fidelity DNA polymerase
(Vazyme). The amplified fragments were first gel-purified and then
introduced into the pEASY-Blunt vector (TransGen Biotech) for
sequence analysis.
In Vitro Assays. The coding sequence of EfTPS12 was inserted
into the NdeI/XhoI site of the pCold TF vector and transferred into
E. coli BL21 (DE3) for heterologous expression. The transformants
were grown at 37 °C, shaking at 200 rpm. When the OD600 of the
cultures reached 0.4−0.6, 0.2 mM IPTG was used to induce protein
expression overnight at 16 °C. The cells were harvested by
centrifugation at 5000g for 10 min at 4 °C. Pellets were then
resuspended in Tris-HCl buffer (100 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 2 mM DTT). The cells were disrupted by sonication, and the
lysate was centrifuged at 13000g for 20 min to obtain the supernatant
crude protein, which was subjected to a Ni-NTA (Takara) affinity
column successively eluted with buffer A (50 mM Tris-HCl, 5 mM
MgCl2, 250 mM NaCl, 10% glycerol, pH 8.4), buffer B (50 mM Tris-
HCl, 5 mM MgCl2, 250 mM NaCl, 10% glycerol, 100 mM imidazole,
pH 8.4), and buffer C (50 mM Tris-HCl, 5mM MgCl2, 250 mM
NaCl, 10% glycerol, 300 mM imidazole, pH 8.4). After removal of
imidazole by dialysis, the resulting enzyme solution was concentrated
and 5. We then coexpressed EfTPS12 with ERG20 in E. coli
BL21, and production of 1−5 was observed (Figure S7), as
well. These results indicate that although 4 and 5 do not
appear to be the direct products of EfTPS12, they arise by
unknown routes that do involve the participation of EfTPS12.
Hence, only 1, 2, and 3 are the direct products of EfTPS12.
Herein, we named it EfCAS.
In the in vitro reaction, a large amount of FOH was
produced (Figure 1), but no evident FOH peak was detected
from the extracts of E. coli expressing EfTPS12 and ERG20
(Figure 2). The 6xHis tag may cause a slight change in the
structure of the recombinant EfTPS12 used in the in vitro
reaction, which could result in FOH production.
In conclusion, a new sesquiterpene synthase, EfCAS, was
characterized by in vitro biochemical experiments and synthetic
biological approaches. It can catalyze cyclization of FFP to
afford cedrol (1) and two tricho-acorenol stereoisomers,
eupho-acorenols A (2) and B (3). Acorenol isomers (α-
acorenol, β-acorenol, epi-α-acorenol, and epi-β-acorenol) are
the indispensable components of highly valuable plant volatile
oils,32−34 and tricho-acorenol is one of the major volatile
metabolites of Trichoderma fungi24,25 which can be used as
biocontrol and biostimulant agents.35,36 Microbial sesquiter-
pene synthases producing α-acorenol and tricho-acorenol were
reported,37−39 while for plant sesquiterpene synthases, TEAS
(a tobacco sesquiterpene synthase) was reported to only
produce acorane olefins (α-acoradiene, 3.9%; 4-epi-α-acor-
adiene, 1.3%) in small quantities.40 EfCAS is the first example
of a plant-derived sesquiterpene synthase that is capable of
synthesizing acorane-type alcohols. Additionally, this study
documents that synthetic biology approaches enable large-scale
preparation of volatile terpenoids, thereby substantially
facilitating characterization of corresponding terpene synthases
and elucidation of the complete structures of their products.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotations were
measured on a JASCO P-2000 polarimeter. UV spectra were obtained
on a JASCO V-550 spectrophotometer. ECD spectra were measured
using a Chirascan spectropolarimeter (Applied Photophysics). NMR
spectra were recorded on a Bruker Avance III-400 spectrometer, and
TMS was used as the internal standard. Low- and high-resolution GC-
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by centrifugation. Enzymatic reactions were performed in 500 μL of
buffer A containing 5 μM enzyme and 3 mM FPP purchased from
Sigma. The reaction mixtures were incubated at 30 °C for 3 h,
followed by extraction with n-hexane for GC-MS analysis.
calculations of ECD spectra of 2 and 3; GC-MS
chromatogram of the extract of the reaction mixture of
tris(tetrabutylammonium) 12-hydroxyfarnesyl diphos-
phate with EfTPS12; GC-MS chromatogram of the
extract of the culture of the E. coli strain coexpressing
ERG20 and each EfTPS12 mutant; GC-MS chromato-
gram of the extract of the culture of the E. coli BL21
strain containing petDuet-1-ERG20-EfTPS12; extraction
of E. fischeriana tissues; GC-MS chromatograms of the
extracts of roots, stems, and leaves of E. fischeriana;
HREIMS, IR, and NMR spectra of 2, 3, and 4;
sequences of the recombinant EfCAS and its encoding
gene (PDF)
Characterization of EfTPS12 by a Synthetic Biology
Approach. The EfTPS12 gene was introduced into the NdeI/XhoI
site (the first cloning site) of petDuet-1 vector by the cut-paste
method to generate the plasmid petDuet-1-EfTPS12. Then, ERG20
(accession no. 97 NC_001142.9) was inserted in the NcoI/NotI site
(the second cloning site) of petDuet-1-EfTPS12 to afford petDuet-1-
ERG20-EfTPS12. E. coli was transformed with petDuet-1-ERG20-
EfTPS12 and pIRS,20 and the resulting constructs were grown in TB
media (containing 12 g/L peptone, 24 g/L yeast extracts, 8 mL of
50% glycerol, 9.4 g of K2HPO4, 2.2 g of KH2PO4) supplied with 50
mg/L ampicillin in 250 mL flasks. At an OD600 of 0.4−0.6, expression
was induced by adding 1 mM IPTG, and the temperature was set to
16 °C. The cultures were fermented for an additional 72 h at 16 °C
and then extracted with 50 mL of n-hexanes.
AUTHOR INFORMATION
■
Compound Isolation and Structural Elucidation. A 30 L
culture of the recombinant E. coli strain containing petDuet-1-ERG20-
EfTPS12 and pIRS20 was grown in TB media. After a 72 h growth,
the culture was extracted by n-hexane. Then, the extracts were dried
under vacuum and the residue was subjected to a silica gel column
eluted with petroleum ether−EtOAc (50:1 to 10:1) to yield four
fractions, Fr1−Fr4. 1 (83 mg) in Fr2 and 2 (72 mg) and 3 (75 mg) in
Fr3 were further purified by normal phase HPLC, using a
Phenomenex FMG-AC5-A01-NFC chiral-phase column (the mobile
phase for 1: n-hexane−isopropanol = 200/1; the mobile phase for 2
and 3: n-hexane−isopropanol = 99/1). Compounds 4 (124 mg) and
5 (70 mg) were respectively purified from Fr1 and Fr4 by reverse
phase HPLC, using a YMC-Pack Pro C18 column and methanol−
water (80/20) as the mobile phase.
Corresponding Author
Jiachen Zi − College of Pharmacy, Jinan University,
Guangzhou 510632, People’s Republic of China;
orcid.org/0000-0002-0212-1282; Email: tzjc@
jnu.edu.cn
Authors
Jianxun Zhu − College of Pharmacy, Jinan University,
Guangzhou 510632, People’s Republic of China
Lihong Liu − College of Pharmacy, Jinan University,
Guangzhou 510632, People’s Republic of China
Maobo Wu − College of Pharmacy, Jinan University,
Guangzhou 510632, People’s Republic of China
Guiyang Xia − Key Laboratory of Chinese Internal Medicine of
Ministry of Education and Beijing, Dongzhimen Hospital,
Beijing University of Chinese Medicine, Beijing 100700,
People’s Republic of China
Eupho-acorenol A (2): colorless oil; [α]22D +14.4 (c 0.3, CH2Cl2);
ECD (4.5 × 10−3 M, MeOH) λmax (Δε) 207 (−2.4) nm; IR (KBr)
νmax 3302, 2950, 2919, 2867, 1461, 1378, 1274, 1052, 1020, 937, 814
1
cm−1; H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR
data, Table 1; HREIMS m/z 222.1976 [M]+ (calcd for C15H26O,
222.1984).
Pengcheng Lin − College of Pharmacy, Qinghai Nationalities
University, Xining 810007, People’s Republic of China
Eupho-acorenol B (3): colorless oil; [α]22 +4.9 (c 0.3, CH2Cl2);
D
ECD (4.5 × 10−3 M, MeOH) λmax (Δε) 207 (+2.7) nm; IR (KBr)
1
νmax 3318, 2952, 2922, 2868, 1456, 1377, 1052, 1017, 811 cm−1; H
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.1c00126
(400 MHz, CDCl3 or pyridine-d5) and 13C (100 MHz, CDCl3 or
pyridine-d5) NMR data, Table 1; HREIMS m/z 222.1976 [M]+
(calcd for C15H26O, 222.1984).
Author Contributions
#J. Zhu and L. Liu contributed equally.
Compound 4: colorless oil; IR (KBr) νmax 2906, 2846, 1438, 1380,
1
1251, 1087, 1065, 920, 816, 542 cm−1; H (400 MHz, CDCl3) and
13C (100 MHz, CDCl3) NMR data, Table 1; HREIMS m/z 220.1820
Notes
[M]+ (calcd for C15H24O, 220.1827).
The authors declare no competing financial interest.
Synthesis of Tris(tetrabutyl ammonium) 12-Hydroxyfarne-
syl Diphosphate. Following the previous method,41,42 tris(tetrabutyl
ammonium) 12-hydroxyfarnesyl diphosphate was synthesized. Briefly,
farnesol reacted with methanesulfonyl chloride to afford farnesyl
chloride,41 which was subsequently oxidized into 12-hydroxyfarnesyl
chloride by selenium dioxide and tert-butyl hydroperoxide,42 and 12-
hydroxyfarnesyl chloride was then converted into tris-
(tetrabutylammonium) 12-hydroxyfarnesyl diphosphate under treat-
ment of tris(tetrabutylammonium) hydrogen pyrophosphate.42
ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of China (Nos. 82073953 and 81673530),
Guangdong Provincial Key R&D Programme
(2020B1111120002), Natural Science Foundation of Qinghai
Province (No. 2018-ZJ-907), Qinghai Provincial Key Labo-
ratory of Phytochemistry for Tibetan Plateau (2017-ZJ-Y19),
and Local Innovative and Research Teams Project of
Guangdong Pearl River Talents Program (2017BT01Y036).
ASSOCIATED CONTENT
■
sı
* Supporting Information
REFERENCES
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