Antibacterial Prenylated p-Hydroxybenzoic Acid Derivatives from Oberonia myosurus and Identification of Putative Prenyltransferases

Article abstract of DOI:10.1021/acs.jnatprod.0c01101

Twelve hitherto unknown tandem prenylated p-hydroxybenzoic acid derivatives, namely, oberoniamyosurusins A-L, together with five known derivatives, were isolated from an EtOH extract of the whole parts of the plant Oberonia myosurus. Compounds 10, 13, and 17 exhibited moderate inhibitory activity against Staphylococcus aureus subsp. aureus ATCC29213 with MIC50 values ranging from 7.6 to 23 μg/mL. To determine the biosynthetic pathway of this class of tandem prenyl-substituted compounds, the full-length transcriptome of O. myosurus was sequenced, yielding 19.09 Gb of clean data and 10 949 nonredundant sequences. Two isoforms of p-hydroxybenzoic acid prenyltransferases were annotated and functionally characterized as the enzymes that might be involved in the biosynthesis of nervogenic acid (13) in Pichia pastoris.

Full text of DOI:10.1021/acs.jnatprod.0c01101

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Article
Antibacterial Prenylated p‑Hydroxybenzoic Acid Derivatives from
Oberonia myosurus and Identification of Putative Prenyltransferases
Fu-Cai Ren, Li Liu, Yong-Feng Lv, Xue Bai, Qian-Jin Kang, Xiao-Jing Hu, Hong-Dan Zhuang, Liu Yang,
Jiang-Miao Hu,* and Jun Zhou*
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ABSTRACT: Twelve hitherto unknown tandem prenylated p-hydrox-
ybenzoic acid derivatives, namely, oberoniamyosurusins A−L, together
with five known derivatives, were isolated from an EtOH extract of the
whole parts of the plant Oberonia myosurus. Compounds 10, 13, and 17
exhibited moderate inhibitory activity against Staphylococcus aureus subsp.
aureus ATCC29213 with MIC50 values ranging from 7.6 to 23 μg/mL. To
determine the biosynthetic pathway of this class of tandem prenyl-
substituted compounds, the full-length transcriptome of O. myosurus was
sequenced, yielding 19.09 Gb of clean data and 10 949 nonredundant
sequences. Two isoforms of p-hydroxybenzoic acid prenyltransferases were
annotated and functionally characterized as the enzymes that might be
involved in the biosynthesis of nervogenic acid (13) in Pichia pastoris.
renylation is a major contributor to the diversity of natural
research on chemical constituents from natural sources with
Pproducts because of differences in prenylation position, pharmacological applications, phytochemical investigation of
whole plants of O. myosurus led to the isolation of 17
prenylated p-hydroxybenzoic acid derivatives, including 12 new
derivatives (1−12). All isolated compounds were evaluated for
their potential antibacterial activity against four bacterial
strains. ObPPT-1/ObPPT-2 were annotated from the full-
length transcriptome of O. myosurus and functionally
characterized as the enzymes that might be involved in the
biosynthesis of nervogenic acid (13) in Pichia pastoris. Details
of these efforts will be described below.
various lengths of prenyl chain, and further modifications of
the prenyl moiety.1,2 Prenylated aromatic compounds are
hybrid natural products containing isoprenoid moieties or
structures derived therefrom, and they display extensive
structural diversity with various biological activities. The
improvement of the lipophilicity and/or binding to the target
protein by prenylation might be one of the causes of enhanced
biological activities.3 Aromatic prenyltransferases catalyze the
transfer of prenyl moieties to aromatic acceptor molecules and
give rise to an astounding diversity of primary and secondary
metabolites.4 A group of prenyltransferases can catalyze the
transfer of allylic prenyl moieties to aromatic acceptor
molecules, forming C−C bonds between C-1 or C-3 of the
isoprenoid substrate and one of the aromatic carbons of the
acceptor substrate, and they were found to carry out aromatic
prenylations with different substrate, specificities, and
regiospecificities and to catalyze both regular and reverse
prenylations.4,5 In particular, prenylated p-hydroxybenzoic acid
derivatives are a class of aromatic compounds that exhibit
antimicrobial,6 anti-inflammatory,7 antiparasitic,8 and mollus-
cicidal6 activities.
RESULTS AND DISCUSSION
■
Compound 1 was isolated as a colorless oil. The IR spectrum
indicated the presence of hydroxy (3407 cm−1) and carbonyl
(1719 cm−1) groups and an aromatic ring (1591 and 771
cm−1). The 1H NMR spectrum (Table 1) displayed two meta-
coupled aromatic doublets at δH 7.51 (1H, d, J = 2.0 Hz) and
7.79 (1H, d, J = 2.0 Hz), a pair of vinyl protons at δH 6.69 (1H,
d, J = 16.2 Hz) and 6.98 (1H, d, J = 16.2 Hz), an exo-
methylene at δH 5.15 and 5.20 (each, 1H, s), a methyl singlet
at δH 2.00 (3H, s), and two methoxy groups at δH 3.84 and
The genus Oberonia includes approximately 300 species of
perennial herbs belonging to the plant family Orchidaceae.9
Oberonia myosurus Lindl. is used as a folk medicine by some
ethnic minorities in southwestern China for the treatment of
pneumonia, bronchitis, hepatitis, and urinary tract infections
and externally for rheumatic bone pain. Nevertheless, studies
on this plant are limited. In the course of our continual
Received: October 13, 2020
Published: January 25, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
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Chart 1
a
1
Table 1. H and 13C NMR Spectroscopic Data for Compounds 1−3
1
2
3
b
c
d
e
f
e
no.
δC, type
δH, m, J (Hz)
δC, type
δH, m, J (Hz)
δC, type
δH, m, J (Hz)
1
126.7, C
125.5, C
127.0, C
2
3
4
5
6
7
115.3, CH
148.9, C
148.3, C
130.7, C
119.9, CH
166.6, C
7.51, d (2.0)
115.1, CH
148.2, C
148.4, C
130.4, C
124.3, CH
166.8, C
7.52, d (2.0)
118.5, CH
149.3, C
150.0, C
127.9, C
121.1, CH
7.65, d (2.0)
7.79, d (2.0)
7.48, d (2.0)
7. 84, d (2.0)
1′
2′
3′
4′
121.7, CH
134.5, CH
141.9, C
6.69, d (16.2)
6.98, d (16.2)
123.6, CH
134.9, CH
141.5, C
6.43, d (12.3)
6.32, d (12.3)
136.4, CH
129.2, CH
198.2, C
7.73, d (16.5)
6.85, d (16.5)
118.8, CH2
5.15, s
5.20, s
2.00, s
3.90, s
119.0, CH2
5.02, s
5.02, s
1.66, s
3.88, s
3.88, s
28.0, CH3
2.41, s
5′
18.5, CH3
52.2, CH3
61.7, CH3
21.6, CH3
52.1, CH3
61.1, CH3
COOCH3
OCH3-4
52.4, CH3
62.4, CH3
3.91, s
3.88, s
3.84, s
a
b
c
d
e
Measured in CDCl3 (δH 7.26 ppm, δC 77.0 ppm). Recorded at 125 MHz. Recorded at 500 MHz. Recorded at 150 MHz. Recorded at 600
f
MHz. Recorded at 100 MHz.
3.90 (each, 3H, s). The 13C and DEPT spectra showed a total
of 14 carbon resonances including an aromatic ring [δC 126.7
(s, C-1), 115.3 (d, C-2), 148.9 (s, C-3), 148.3 (s, C-4), 130.7
(s, C-5), and 119.9 (d, C-6)], a carbonyl carbon (δC 166.3), an
isoprene chain including a double bond (δC 121.7, 134.5) and
an exo-methylene (δC 118.8), an ester methoxy group (δC
52.2), and an aromatic methoxy group (δC 61.7). Those
mentioned NMR data together with the HMBC correlations
(Figure S1) from the methoxy group at δH 3.84 (3H, s) to C-4,
from H-1′ to C-4, C-5, C-6, and C-3′, from H-2′ to C-5 and C-
4′, and from H-4′ to C-2′, C-3′, and C-5′ revealed that
compound 1 was a 3,4,5-trisubstituted benzoic acid methyl
ester derivative with a hydroxy group, a methoxy group, and a
“3-methyl-1,3-butadienyl” moiety located at C-3, C-4, and C-5,
respectively. The diagnostic coupling constant (J = 16.2 Hz)
between H-1′ and H-2′ suggested C-1′C-2′ was an E-form
olefin. Therefore, the structure of compound 1 was established
as (E)-3-hydroxy-4-methoxy-5-(3-methyl-1,3-butadienyl)-
benzoic acid methyl ester and named as oberoniamyosurusin
A.
The NMR data of compound 2 (Table 1) were very similar
1
to those of 1, except for the H NMR data of H-1′ and H-2′
418
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and the diagnostic coupling constant (J = 12.3 Hz) between
them which suggested a Z-form double bond of C-1′C-2′.
Then, the structure of compound 2 was established as (Z)-3-
hydroxy-4-methoxy-5-(3-methyl-1,3-butadienyl)benzoic acid
methyl ester and named as oberoniamyosurusin B.
The NMR spectra of compounds 5 and 6 showed a 3,4,5-
trisubstituted benzoic acid skeleton and a 3-hydroxy-3-
methylbutyl side chain at C-5 relating as in compound 4.
For compound 5, the remaining signals [δH 2.85 (1H, dd, J =
14.8, 2.3 Hz, H-1′), 2.98 (1H, dd, J = 14.8, 8.9 Hz, H-1′), 4.40
(1H, dd, J = 8.9, 2.3 Hz, H-2′), 4.88, 5.00 (each 1H, s, H-4′),
1.81 (3H, s, H-5′); δC 38.1, 77.8, 146.3, 111.3, 18.2] were due
to a 2-hydroxy-3-methylbut-3-en-1-yl unit, which was con-
firmed by the HMBC correlations from H-2′ to C-1′, C-3′, and
C-4′ and from H-5′ to C-2′, C-3′, and C-4′. This moiety was
assigned to C-3 according to the HMBC correlations from H-
1′ to C-2, C-3, and C-4 and from H-2′ to C-3. The small
rotation value and chiral-phase analysis suggested that
compound 5 is racemic. For compound 6, the remaining
protons at δH 5.64, 6.34 (each 1H, dd, J = 9.9 Hz) and 1.45
(6H, s) are typical for a fused 2,2-dimethylpyran ring being
attached to the C-3 and C-4 oxygen of the aromatic ring.
Hence, compounds 5 and 6 were established and named as
oberoniamyosurusins E and F, respectively.
Compound 3 was isolated as a colorless oil. The HRESIMS
data indicated the molecular formula C13H14O5. The 1D NMR
spectra of 3 displayed a pair of aromatic doublets, two methoxy
groups, a benzene ring, and a carboxylate, which indicated that
3 had a similar structure to oberoniamyosurusin A (1). The
main difference is only that 3 had a newly arising keto carbon
at δC 198.2 instead of the terminal olefin. Thus, compound 3
might be the oxidation derivative of 1. And this was confirmed
by the HMBC correlations from H-1′ (δH 7.73) to C-4, C-5,
C-6, and C-3′, from H-2′ (δH 6.85) to C-5 and C-4′, and from
H-4′ to C-2′ and C-3′. Hence, compound 3 was assigned as
(E)-3-hydroxy-4-methoxy-5-(3-oxo-1,3-butadienyl)benzoic
acid methyl ester and named as oberoniamyosurusin C.
1
Compound 7 was isolated as a colorless oil. The H NMR
1
spectrum revealed the presence of two meta-coupled aromatic
doublets at δH 7.90 and 8.15 (each, 1H, d, J = 2.2 Hz) and a
prenyl group similar to nervogenic acid, except for the
replacement of one prenyl group with a prenyl-derived unit.
The signals at δH 2.84 (2H, s), 1.40 (6H, s), and δC 191.9
indicated a 2,2-dimethyldihydropyran ring unit bearing a keto
group at C-1′, which was further confirmed from HMBC
correlations from H-2′ to C-1′ and C-3 and from H3-4′(5′) to
C-2′ and C-3′. This ring fused with the aromatic ring via the
C-3 and C-4 oxygen was determined by the HMBC
correlations from H-2 (δH 8.15) to C-1′ and from H-2′ (δH
2.84) to C-3 and C-3′. Therefore, compound 7 was established
and named as oberoniamyosurusin G.
The NMR data of compounds 8 and 9 were similar to those
of compound 7 except for the isoprenoid ring. For compound
8, a 2,2-dimethyl-3-hydroxydihydropyran ring was assigned
according to the typical signals at δH 1.15, 1.28 (each, 3H, s),
2.61 (1H, dd, J = 16.6, 7.9 Hz), 2.91 (1H, dd, J = 16.6, 5.2
Hz), and 3.64 (1H, dd, J = 7.9, 5.2 Hz). For compound 9, the
characteristic protons at δH 3.14 (2H, d, J = 8.7 Hz), 4.60 (1H,
t, J = 8.7 Hz), and 1.07, 1.12 (each 3H, s) due to a 2-(1-
hydroxy-1-methylethyl)-2,3-dihydrobenzofuran moiety were
obviously distinguished. The chiral-phase analysis as well as
the small rotation values implied that 8 and 9 were racemic.
Hence, the structures of compounds 8 and 9 were established
and named as oberoniamyosurusins H and I, respectively.
The NMR data of compound 10 (Table 4) showed it was a
nervogenic acid (13) ester. The esterification group was
established as a 2,3-dihydroxy-2-methylbutyryloxy unit accord-
ing to the HMBC correlations from H-4‴ (δH 1.36, d, J = 6.4
Hz) to C-3‴ (δC 74.5) and C-2‴ (δC 76.3) and from H-5‴ (δH
1.42, s) to C-1‴ (δC 178.7), C-2‴, and C-3‴. The chemical
shift of H-3‴ (δH 5.25) indicated that the hydroxy group at C-
3‴ was esterified, which was further confirmed by the HMBC
correlation from H-3‴ to C-7 (δC 165.8). The ECD
calculations suggested that 10b matched well with the
experimental data of 10 (Figure 1), revealing the absolute
configuration to be the R,R-form. Compound 10 was
established and named as oberoniamyosurusin J.
Table 2. H NMR Spectroscopic Data for Compounds 4−9
a
a
a
b
b
b
no.
2
4
5
6
7
8
9
7.75, s
7.68, d
(2.2)
7.61, d
(2.2)
8.15, d
(2.2)
7.48, s
7.56, s
6
7.75, s
7.78, d
(2.2)
7.77, d
(2.2)
7.90, d
(2.2)
7.47, s
7.50, s
1′ 3.38, d
2.85, dd
(14.8,
2.3)
6.34, d
(9.9)
2.61, dd
(16.6,
7.9)
3.14, d
(8.7)
(7.2)
2.98, dd,
(14.8,
8.9)
2.91, dd,
(16.6,
5.2)
2′ 5.33, t
4.40, dd
5.64, d
(9.9)
1.45, s
2.84, s
1.40, s
3.64, dd
4.60, t
(8.7)
1.12, s
(7.2)
(8.9, 2.3)
(7.9, 5.2)
4′ 1.77, s
4.88, s
5.00, s
1.81, s
2.75, m
1.28, s
5′ 1.76, s
1.45, s
2.68, m 3.29, d
(7.4)
1.40, s
1.15, s
3.18, d
(7.4)
1.07, s
3.20, d
(7.3)
1″ 2.75, t
(7.3)
2″ 1.80, t
1.79, t (7.6) 1.75, m 5.14, t
(7.4)
5.20, t (7.4) 5.23, t
(7.3)
(7.3)
4″ 1.31, s
5″ 1.31, s
1.29, s
1.29, s
1.31, s
1.31, s
1.69, s
1.69, s
1.67, s
1.67, s
1.66, s
1.65, s
a
b
Measured in CDCl3 (δH 7.26 ppm). Measured in DMSO-d6 (δH
2.49 ppm). Recorded at 600 MHz.
The 1H and 13C NMR spectra (Tables 2 and 3) of
compound 4, a light yellow oil, exhibited a set of signals at δH
7.75 (2H, s), δC 120.7, 130.5, 128.7, 157.6, 127.9, and 131.2,
characteristic of a 1,3,4,5-tetrasubstituted aromatic ring, a
carbonyl resonance at δC 172.0, and a prenyl group at δH 3.38
(2H, d, J = 7.2 Hz), 5.33 (1H, t, J = 7.2 Hz), 1.76 (3H, s), 1.77
(3H, s), δC 29.3, 121.6, 134.5, 25.8, and 17.9. The remaining
signals at δH 1.80 (2H, t, J = 7.3 Hz, H-2″), 2.75 (2H, t, J = 7.3
Hz, H-1″), 1.31 (6H, s, H-4″ and H-5″), δC 24.7, 42.7, 71.9,
29.4, and 29.4 were assigned as a 3-hydroxy-3-methylbutyl
moiety. The moiety was located at C-5 according to the
HMBC correlations from H-1″ to C-4, C-5, and C-6, from H-
2″ to C-5, and from H-4″ (H-5″) to C-2″ and C-3″. Then,
compound 4 was established as 4-hydroxy-3-prenyl-5-(3-
hydroxy-3-methylbutyl)benzoic acid, which biologically derives
from nervogenic acid (13),6 and was given the trivial name
oberoniamyosurusin D.
The NMR data of compound 11 showed the presence of a
nervogenic acid (13) moiety, and the remaining signals [δH
2.21, br dd, (J = 18.3, 6.1 Hz), 2.62, br. dd, (J = 18.3, 4.4 Hz)/
δC 27.1; δH 3.80, dd, (J = 6.1, 4.4 Hz)/δC 67.3; δH 5.16, dt, (J =
419
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Table 3. 13C NMR Spectroscopic Data for Compounds 4−9
a c
,
ad
,
ac
,
bd
,
b d
,
bd
,
no.
4
5
6
7
8
9
1
2
3
4
5
6
7
1′
2′
3′
4′
5′
1″
2″
3″
4″
5″
120.7, C
130.5, CH
128.7, C
157.6, C
127.9, C
130.3, CH
172.0, C
29.3, CH2
121.6, CH
134.5, C
25.8, CH3
17.9, CH3
24.7, CH2
42.7, CH2
71.9, C
120.6, C
131.8, CH
125.6, C
158.8, C
130.5, C
131.2, CH
171.1, C
38.1, CH2
77.8, CH
146.3, C
111.3, CH2
18.2, CH3
25.2, CH2
43.4, CH2
71.5, C
121.5, C
126.7, CH
120.4, C
155.3, C
129.9, C
131.9, CH
171.5, C
122.0, CH
130.6, CH
77.4, C
28.4, CH3
28.4, CH3
24.6, CH2
43.7, CH2
71.2, C
123.1, C
125.5, CH
119.2, C
160.3, C
131.1, C
135.7, CH
166.7, C
191.9, C
47.5, CH2
80.2, C
26.1, CH3
26.1, CH3
27.8, CH2
121.4, CH
132.6, C
25.6, CH3
17.8, CH3
124.7, C
129.2, CH
119.5, C
153.4, C
128.0, C
128.3, CH
168.4, C
31.1, CH2
67.8, CH
77.7, C
25.8, CH3
20.7, CH3
28.1, CH2
122.6, CH
131.3, C
25.6, CH3
17.7, CH3
122.7, C
124.2, CH
127.7, C
161.9, C
122.0, C
130.0, CH
167.7, C
29.7, CH2
90.0, CH
70.5, C
26.2, CH3
24.5, CH3
27.9, CH2
121.8, CH
132.5, C
25.8, CH3
17.9, CH3
29.4, CH3
29.4, CH3
29.2, CH3
29.2, CH3
29.1, CH3
29.1, CH3
a
b
c
d
Measured in CDCl3 (δC 77.0 ppm). Measured in DMSO-d6 (δC 39.5 ppm). Recorded at 100 MHz. Recorded at 150 MHz.
1
Table 4. H and 13C NMR Spectroscopic Data for Compounds 10−12
a
b
a
10
11
δH, m, J (Hz)
12
c
d
c
d
c
d
no.
δC, type
δH, m, J (Hz)
δC, type
δC, type
δH, m, J (Hz)
1
121.5, C
120.5, C
121.2, C
2
3
4
5
6
7
129.8, CH
127.0, C
157.4, C
127.0, C
129.8, CH
165.8, C
7.60, s
128.1, CH
128.5, C
157.1, C
128.5, C
128.1, CH
162.2, C
7.47, s
7.47, s
126.2, CH
120.4, C
155.2, C
129.3, C
131.4, CH
166.5, C
7.43, s
7.59, s
7.60, s
1′
2′
3′
4′
5′
1″
2″
3″
4″
5″
1‴
2‴
3‴
4‴
5‴
6‴
7‴
29.4, CH2
121.2, CH
134.9, C
25.7, CH3
17.8, CH3
29.4, CH2
121.2, CH
134.9, C
3.30, d (7.4)
5.25, overlapped
28.0, CH2
122.0, CH
132.4, C
25.5, CH3
17.7, CH3
28.0, CH2
122.0, CH
132.4, C
25.5, CH3
17.7, CH3
127.9, C
138.7, CH
65.6, CH
67.3, CH
70.2, CH
27.1, CH2
170.2, C
3.27, d (7.4)
5.24, t (7.4)
28.3, CH2
122.0, CH
132.3, C
25.7, CH3
17.9, CH3
122.1, CH
130.6, CH
77.2, C
28.2, CH3
28.2, CH3
129.6, C
137.8, CH
66.4, CH
69.1, CH
70.0, CH
27.8, CH2
3.21, d (6.8)
5.19, t (6.8)
1.76, s
1.74, s
3.30, d (7.4)
5.25, overlapped
1.70, s
1.64, s
3.27, d (7.4)
5.24, t (7.4)
1.67, s
1.68, s
6.27, d (9.8)
5.57, d (9.8)
25.7, CH3
17.8, CH3
178.7, C
1.76, s
1.74, s
1.70, s
1.64, s
1.38, s
1.38, s
76.3, C
6.68, br s
4.25, br s
3.80, dd, (6.1, 4.4)
5.16, dt, (6.1, 4.4)
6.92, br s
4.53, br s
4.03, br s
5.39, t (6.9)
74.5, CH
13.4, CH3
21.6, CH3
5.25, overlapped
1.36, d (6.4)
1.42, s
2.21, br dd (18.3, 6.1), 2.62, dd (18.3, 4.4)
2.40, br d (16.1) 2.87, br d, (16.1)
170.2, C
a
b
c
d
Measured in CDCl3 (δH 7.26 ppm, δC 77.0 ppm). Measured in DMSO-d6 (δH 2.49 ppm, δC 39.5 ppm). Recorded at 150 MHz. Recorded at
600 MHz.
6.1, 4.4 Hz)/δC 70.2; δH 6.68 (br s); δC 127.9, 170.2] could be
recognized as a shikimic acid ester.10 The esterification of the
C-5‴ hydroxy group and the C-7 carboxyl of nervogenic acid
(13) could be confirmed by the chemical shift of H-5‴ (δH
5.16) and the HMBC correlation from H-5‴ to C-7. The
NMR data of compound 12 indicated that 12 possesses a
similar structure to a shikimic acid ester moiety, except the 2,2-
dimethyl-8-prenylchromene-6-carboxylic acid (17) moiety
replaced the nervogenic acid (13) in compound 11. The
absolute configurations of compounds 11 and 12 were
postulated as R,R,R-form in light of a negative specific rotation
value (−89.6, −93.1, MeOH), consistent with that of (R,R,R)-
3-O-galloylshikimic acid (−108.3, MeOH).11 Hence, com-
pounds 11 and 12 were established and named as
oberoniamyosurusins K and L, respectively.
In addition, except for the new substances described above,
five known prenylated p-hydroxybenzoic acid derivatives were
also isolated from O. myosurus, which were identified as
nervogenic acid (13),6 (4-hydroxy-3-(2-hydroxy-3-methyl-3-
butenyl)-5-(3-methyl-2-butenyl)benzoic acid (14),8 1iparacids
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clean data and 207 191 circular consensus (CCS) reads,
including 165 339 full-length nonchimeric (FLNC) sequences.
By clustering FLNC sequences and performing polishing,
19 451 high-quality consensus sequences were obtained. Then,
10 949 nonredundant transcript sequences were captured, and
10 181 sequences were subjected to functional annotations
blasting with the NR, SwissProt, GO, COG, KOG, Pfam, and
KEGG databases. Subsequently, two unique isoforms (ObPPT-
1 and ObPPT-2) were annotated as encoding 4-hydroxyben-
zoate polyprenyltransferase enzymes.
Molecular phylogenetic analysis showed that the ObPPT-1
and ObPPT-2 formed a new sub-branch of prenyltransferases
in ubiquinone biosynthesis (Figure 2). Multiple sequence
alignments with plant aromatic prenyltransferases with known
functions showed that the protein sequence of ObPPT-1/
ObPPT-2 shared approximately 52% identity with AtPPT1,
which catalyzes isoprenylation with solanesyl diphosphate to
produce 3-solanesyl-4-hydroxybenzoate,16 and shared only
28.5% identity with XimB, which utilizes GPP as a substrate
(Figure S2).17 Sequence alignment showed that a putative
prenyl diphosphate binding site consisting of NDXXD and the
GXKSTAL motif was present in ObPPT-1 and ObPPT-2.14
The TMHMM 2.0 online analysis tool was used to predict the
transmembrane area of ObPPT-1/ObPPT-2 and clearly showed
that ObPPT-1/ObPPT-2 has six transmembrane areas (Figure
3). Because eukaryotic expression systems have many
advantages over prokaryotic expression systems for the
heterologous expression of membrane proteins,18 the Pichia
pastoris expression system with a substrate incubation method
was chosen to investigate the functions of ObPPT-1/ObPPT-2.
To analyze the functions of these two isoforms of putative 4-
hydroxybenzoate polyprenyltransferases, genetic approaches
were employed to create a DMAPP overproduction platform in
P. pastoris to efficiently identify the functions of ObPPT-1/
ObPPT-2. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) re-
ductase is generally considered as the rate-limiting enzyme in
the MVA pathway, and overexpression of a truncated HMG-
CoA reductase gene (tHMG1) has commonly been used to
increase the production of many terpenoids.19,20 The codon-
optimized ObPPT-1/ObPPT-2 gene (controlled by the AOX1
promoter) and the tHMG1 gene (controlled by the TEF1
promoter) were integrated into the expression vector pPICZB
(Invitrogen). The P. pastoris yeast strain SMD1168H was
transformed using electroporation.
Figure 1. ECD calculations for 10.
A and C (15, 16),12 and 2,2-dimethyl-8-prenylchromene-6-
carboxylic acid (17)13 by comparing their NMR data with
literature values.
In accordance with the background of folk medicine
application of O. myosurus, all isolated compounds were
evaluated for their potential antibacterial activity against the
four bacterial strains Escherichia coli ATCC25922, Staph-
ylococcus aureus subsp. aureus ATCC29213, Salmonella enterica
subsp. enterica ATCC14028, and Pseudomonas aeruginosa
ATCC27853. Ceftazidime and penicillin G sodium salt were
used as positive controls. Some isolates exhibited inhibitory
activity against Staphylococcus aureus subsp. aureus
ATCC29213. Compounds 10, 13, and 17 exhibited moderate
inhibitory activity with MIC50 values ranging from 7.6 to 23
μg/mL, whereas compounds 7, 12, 14, and 15 exhibited
modest inhibitory potency with MIC50 values ranging from 56
to 93 μg/mL (Table 5).
Table 5. MIC50 Values of Compounds 7, 10, 12, 13, 14, 15,
and 17 against Staphylococcus aureus subsp. aureus
ATCC29213
sample
MIC50 (μg/mL)
penicillin G sodium salt
compd 7
compd 10
compd 12
compd 13
0.53 0.03
93
23
56
2
1
1
7.6 1.0
SMD1168Hs harboring the ObPPT-1/ObPPT-2 and tHMG1
genes were grown in BMMY medium with PHB as the
supplemented precursor and induced with 0.5% MeOH for
120 h. As a result, two newly appearing ion peaks were
detected using GC-MS in the supernatant of the fermentation
broth compared with the control strain without the ObPPT-1/
ObPPT-2 gene inserted (Figures 4, S3). The fragment ion peak
with a retention time of 18.2 min occurring at m/z 162 led us
to speculate that this might be a decarboxylated fragment of 4-
hydroxy-3-prenylbenzoic acid, which was caused by heating.
To verify this inference and determine whether the ObPPT-1/
ObPPT-2 gene has the biological function of catalyzing the
second prenyl group, 4-hydroxy-3-prenylbenzoic acid was
chemically synthesized from 4-hydroxybenzoic acid with a
total yield of 43% through a three-step reaction (Scheme 2).21
Subsequently, the above deduction was confirmed in view of
the consistency between the supernatant of the fermentation
broth extract and the chemically synthesized standard
specimen using GC-MS (Figures 4, S3, S4). In addition, 4-
compd 14
compd 15
compd 17
89
81
15
1
5
1
All of the isolated compounds exhibited one or two prenyl
groups (cyclization and hydroxylation included) at C-3 or C-5,
and most of those could be obtained from nervogenic acid
(13) through enzymatic or nonenzymatic reactions (Scheme
1). Specifically, nervogenic acid (13) is a diprenyl-substituted
compound and could be directly biosynthesized from p-
hydroxybenzoic acid (PHB) and dimethylallyl diphosphate
(DMAPP).5,14 DMAPP is an important intermediate in the
biosynthesis of terpenes, whereas PHB can be obtained
through the shikimic acid pathway via chorismic acid as the
last intermediate.15
To gain insight into the biosynthetic pathway of this class of
tandem prenyl-substituted compounds, the full-length tran-
scriptome of O. myosurus was sequenced, yielding 19.09 Gb of
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Scheme 1. Proposed Routes of Formation of Compounds Isolated from Oberonia myosurus
hydroxy-3-prenylbenzoic acid was also used as a precursor
substrate for the fermentation. Subsequently, two significant
peaks both occurring at m/z 230 (decarboxylated fragments)
were detected using GC-MS in the supernatant of the
fermentation broth, and the peak at 25.0 min was recognized
as nervogenic acid (13) by comparison with the naturally
separated standard (Figures 4, S5). Regrettably, the structure
of the peak of 24.0 has not yet been determined, but it is clear
that it is not the product of an O-prenylated product
(compound 22 in the Supporting Information) by GC-MS.
Therefore, ObPPT-1/ObPPT-2 can produce low levels of the
prenylated PHB products and the enzymes might be involved
in the biosynthesis of nervogenic acid (13).
In summary, phytochemical investigation of whole plants of
O. myosurus supported the chemical basis of its traditional
medicine applications and provides a new idea for the
discovery of plant-derived antibiotics. With the help of genetic
engineering, we identified ObPPT-1 and ObPPT-2, two
isoforms of 4-hydroxybenzoate prenyltransferase that may be
involved in the prenylation of PHB from O. myosurus. Our
study provides an approach to the functional and mechanistic
characterization of prenyltransferases using the DMAPP
overproduction platform in P. pastoris, and this approach
might be further extended to other prenyltransferases of
various origins. This may speed up the process of discovering
new prenyltransferases and prenylated compounds with high
value added or various biological activities for drug discovery
or industrial applications.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured in MeOH with Horiba SEPA-300 and JASCO P-1020
polarimeters. UV spectra were recorded on a Shimadzu UV-2700
spectrophotometer. ECD were obtained on an Applied Photophysics
Chirascan apparatus. IR spectra (KBr) were obtained on a Bruker
Tensor-27 infrared spectrophotometer. NMR spectra were carried out
on Bruker Avance III 600, Bruker DRX-500, or Bruker Avance III 400
spectrometers with deuterated solvent signals used as internal
standards. ESIMS and HRESIMS were collected on an Agilent
G6230 time-of-flight or an Agilent 1290 UPLC/6540 Q-TOF mass
spectrometer. Column chromatography (CC) was performed on silica
gel (100−200 and 200−300 mesh, Qingdao Haiyang Chemical Co.,
Ltd.), Lichroprep RP-18 (43−63 μm, Merck), YMC*-GEL ODS-A
(12 nm, S-50 μm, YMC), MCI gel (CHP20P, 75−150 μm, Mitsubishi
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Figure 2. Phylogenetic analysis of ObPPT-1, ObPPT-2, and other aromatic prenyltransferases. The sequence information on aromatic
prenyltransferases is listed in Table S3. Branches are labeled with the percentage based on 1000 bootstrap replicates.
Extraction and Isolation. The whole herbs of O. myosurus (2.5
kg) were air-dried and powdered, then extracted three times with 95%
EtOH (15 L × 3) at room temperature, and then filtered. The extracts
were combined and concentrated to afford an organic extract (ca. 260
g), which was fractionated by silica gel CC and eluted successively
with a gradient of increasing acetone in petroleum ether (50:1, 15:1,
10:1, 5:1, 2:1, 1:1, 0:1; v/v) and then MeOH to afford fractions A−H,
respectively. Fraction B was separated by Sephadex LH-20 (CHCl3−
MeOH, 1:1) and preparative thin layer chromatography (TLC) to
yield compounds 1 (44 mg), 2 (17 mg), and 3 (8 mg). Fraction C
was further purified by passage over repeated silica gel (CHCl3−
MeOH, 100:0 → 20:1) and Sephadex LH-20 (CHCl3−MeOH, 1:1)
CC to yield compounds 10 (490 mg), 13 (910 mg), and 17 (112
mg). Fraction D was separated sequentially using silica gel (CHCl3−
MeOH, 100:0 → 10:1), MCI resin (MeOH−H2O, 100:0 → 4:1), and
Sephadex LH-20 (MeOH) and then preparative HPLC to afford
compounds 4 (97 mg), 6 (112 mg), 7 (12 mg), 8 (7 mg), 9 (9 mg),
14 (19 mg), and 15 (154 mg). Compounds 5 (136 mg) and 16 (257
mg) were isolated from fraction E using silica gel (CHCl3−MeOH,
100:1 → 10:1), Sephadex LH-20 (MeOH), and RP-18 CC (MeOH−
H2O, 70%), respectively. Fraction F was purified by passage over
repeated silica gel CC (CHCl3−MeOH gradient, 50:1 → 5:1),
followed by MCI resin (MeOH−H2O, 40%), to yield compounds 11
(240 mg) and 12 (174 mg).
Figure 3. Predictive analysis of the transmembrane section of ObPPT-
1 through the TMHMM 2.0 online analysis.
Chemical Corporation), and Sephadex LH-20 (Amersham Bio-
sciences AB). Semipreparative HPLC was carried out on an Agilent
1100 HPLC with a Zorbax SB-C18 (9.4 × 250 mm, Agilent) column.
Fractions were monitored by TLC plates (Si gel G and GF254
,
Qingdao Haiyang Chemical Co., Ltd.), and spots were visualized by
heating silica gel plates sprayed with 10% H2SO4 in EtOH.
Plant Material. The whole plants of Oberonia myosurus were
purchased from Wenshan Hengfeng farmers market, Wenshan
Zhuang and Miao Autonomous Prefecture, Yunnan Province, People’s
Republic of China, in September 2017. The plant was identified by
Dr. Ende Liu of Kunming Institute of Botany, Chinese Academy of
Sciences. A voucher specimen (KIBZJ-20170901) was deposited at
the State Key Laboratory of Phytochemistry and Plant Resources in
West China, Kunming Institute of Botany, Chinese Academy of
Sciences.
Oberoniamyosurusin A (1): colorless oil; UV (MeOH) λmax (log
ε) 212 (4.29), 260 (4.57), 293 (4.31) nm; IR (KBr) νmax 3407, 2949,
2931, 1719, 1591, 1435, 1324, 1248, 1103, 1003, 911, 893, 771 cm−1;
1H and 13C NMR data, Table 1; HRESIMS m/z 271.0945 [M + Na]+
(calcd for C14H16O4Na, 271.0941).
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and 3; HRESIMS m/z 331.1518 [M + Na]+ (calcd for C17H24O5Na,
331.1521).
Oberoniamyosurusin F (6): yellow oil; UV (MeOH) λmax (log ε)
195 (4.15), 240 (4.33), 275 (3.44) nm; IR (KBr) νmax 3424, 2969,
1
2930, 1682, 1643, 1603, 1378, 1233, 1126, 958, 906, 779 cm−1; H
and 13C NMR data, Tables 2 and 3; HRESIMS m/z 313.1413 [M +
Na]+ (calcd for C17H22O4Na, 313.1416).
Oberoniamyosurusin G (7): colorless oil; UV (MeOH) λmax (log
ε) 196 (4.34), 234 (4.41), 327 (3.41) nm; IR (KBr) νmax 3427, 2978,
2926, 1698, 1678, 1606, 1441, 1387, 1280, 1239, 1201, 1176, 925,
1
776 cm−1; H and 13C NMR data, Tables 2 and 3; HRESIMS m/z
311.1262 [M + Na]+ (calcd for C17H20O4Na, 311.1259).
Oberoniamyosurusin H (8): light yellow oil; [α]2D5 +4.2 (c 0.22,
MeOH); UV (MeOH) λmax (log ε) 212 (4.33), 262 (4.03) nm; IR
(KBr) νmax 3424, 2978, 2930, 1687, 1607, 1436, 1383, 1280, 1194,
1139, 1061, 950.9, 774 cm−1; 1H and 13C NMR data, Tables 2 and 3;
HRESIMS m/z 313.1410 [M + Na]+ (calcd for C17H22O4Na,
313.1410).
Oberoniamyosurusin I (9): light yellow oil; [α]2D5 −2.2 (c 0.21,
MeOH); UV (MeOH) λmax (log ε) 205 (4.20), 263 (3.92) nm; IR
(KBr) νmax 3424, 2977, 2931, 1687, 1383, 1282, 1188, 963, 909, 776
cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z
289.1445 [M − H]− (calcd for C17H21O4, 289.1440).
Oberoniamyosurusin J (10): light yellow oil; [α]2D5 +12.7 (c 0.23,
MeOH); UV (MeOH) λmax (log ε) 196 (4.47), 212 (4.41), 262
(4.09) nm; IR (KBr) νmax 3436, 2979, 2927, 1716, 1695, 1603, 1450,
1
1355, 1313, 1281, 1196, 1102, 1051, 771 cm−1; H and 13C NMR
data, Table 4; HRESIMS m/z 413.1932 [M + Na]+ (calcd for
C22H30O6Na, 413.1935).
Oberoniamyosurusin K (11): light yellow oil; [α]2D5 −89.6 (c 1.4,
MeOH); UV (MeOH) λmax (log ε) 195 (4.56), 209 (4.51), 264
(4.12) nm; IR (KBr) νmax 3420, 2974, 2916, 1698, 1604, 1437, 1229,
1104, 1207, 905, 770 cm−1; 1H and 13C NMR data, Table 4;
HRESIMS m/z 453.1884 [M + Na]+ (calcd for C24H30O7Na,
453.1884).
Figure 4. Total ion GC/MS chromatogram with PHB as the
substrate: (A) SMD1168H:: PEF1-tHMG1-TCYC1, (B) SMD1168H::
PAOX1-ObPPT-1-TAOX1-PTEF1-tHMG1-TCYC1, (C) SMD1168H:: PAOX1
-
ObPPT-2-TAOX1-PTEF1-tHMG1-TCYC1, (D) chemically synthesized
standard 4-hydroxy-3-prenylbenzoic acid and with 4-hydroxy-3-
prenylbenzoic acid as the substrate: (E) SMD1168H:: PEF1-tHMG1-
Oberoniamyosurusin L (12): light yellow oil; [α]2D5 −93.1 (c 0.41,
MeOH); UV (MeOH) λmax (log ε) 195 (4.55), 247 (4.49), 281
(3.74) nm; IR (KBr) νmax 3410, 2976, 2924, 1701, 1652, 1390, 1312,
1285, 1197, 1105, 1028, 907, 804 cm−1; 1H and 13C NMR data, Table
4; HRESIMS m/z 451.1728 [M + Na]+ (calcd for C24H28O7Na,
451.1727).
T
CYC1; (F) SMD1168H:: PAOX1-ObPPT-1-TAOX1-PTEF1-tHMG1-TCYC1;
(G) SMD1168H:: PAOX1-ObPPT-2-TAOX1-PTEF1-tHMG1-TCYC1; (H)
naturally separated standard nervogenic acid.
ECD Calculations. Conformational analysis was performed using
the MMFF94 molecular mechanics force field. The molecules of 10a,
10b, 10c, and 10d showed 15, 15, 20, and 20 stable conformers within
an energy window of 3.0 kcal/mol, respectively. All these conformers
were further optimized by the DFT calculation at the B3LYP/6-
311G(d,p) level by Gaussian 09. The ECD values were calculated
using time-dependent density functional theory (TDDFT) at the
B3LYP/6-311G(d,p) level in methanol. The ECD curves were drawn
using the Origin Pro 9 program (OriginLab Corporation, North-
ampton, MA, USA).
Scheme 2. Synthetic Route to 4-Hydroxy-3-prenylbenzoic
Acid
In Vitro Antimicrobial Assays. All of the compounds 1−17 were
tested for their potential antibacterial activity against the four bacterial
strains Escherichia coli ATCC25922, Staphylococcus aureus subsp.
aureus ATCC29213, Salmonella enterica subsp. enterica ATCC14028,
and Pseudomonas aeruginosa ATCC27853 (China General Micro-
biological Culture Collection Center) at the single concentration of
128 μg/mL. The tested bacteria strains were cultured in Mueller
Hinton broth (Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.)
at 37 °C overnight with shaking (200 rpm). Briefly, the assays were
performed in 96-well plates containing the mid log phase culture (50
μL) and the tested compound (150 μL). The final concentrations of
culture and compound were 5 × 105 CFU/mL and 128 μg/mL,
respectively. Plates were incubated for 24 h; then the absorbances at
625 nm were measured to determine the inhibition. All assays were
performed in triplicate. Ceftazidime (Shanghai Yuanye Bio-Technol-
ogy Co., Ltd.) and penicillin G sodium salt (Biosharp) were used as
the positive controls. Subsequently, MIC assays were carried out in
96-well plates with an initial bacterial inoculum of 5 × 105 CFU/mL.
The tested compounds 7, 10, 12, 13, 14, 15, and 17 were dissolved in
Oberoniamyosurusin B (2): colorless oil; UV (MeOH) λmax (log
ε) 213 (4.54), 254 (4.22), 307 (3.98) nm; IR (KBr) νmax 3426, 2953,
2927, 1718, 1604, 1436, 1383, 1330, 1222, 1169, 1014, 894, 771
1
cm−1; H and 13C NMR data, Table 1; HRESIMS m/z 247.0978 [M
− H]− (calcd for C14H15O4, 247.0976).
Oberoniamyosurusin C (3): colorless oil; UV (MeOH) λmax (log
ε) 209 (4.52), 247 (4.14), 307 (3.85) nm; IR (KBr) νmax 3420, 2954,
1721, 1639, 1586, 1434, 1356, 1269, 1248, 1214, 1013, 985, 972, 760
1
cm−1; H and 13C NMR data, Table 1; HRESIMS m/z 273.0736 [M
+ Na]+ (calcd for C13H14O5Na, 273.0733).
Oberoniamyosurusin D (4): light yellow oil; UV (MeOH) λmax
(log ε) 195 (4.54), 208 (4.49), 257 (4.05) nm; IR (KBr) νmax 3413,
1
2972, 2932, 1686, 1605, 1436, 1196, 906, 776 cm−1; H and 13C
NMR data, Tables 2 and 3; HRESIMS m/z 315.1570 [M + Na]+
(calcd for C17H24O4Na, 315.1572).
Oberoniamyosurusin E (5): light yellow oil; [α]2D7 −1.2 (c 0.29,
MeOH) (c 0.22, MeOH); UV (MeOH) λmax (log ε) 196 (4.46), 207
(4.45), 258 (4.00) nm; IR (KBr) νmax 3393, 2972, 1686, 1603, 1408,
1
1299, 1205, 1060, 904, 776 cm−1; H and 13C NMR data, Tables 2
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DMSO and further serial 2-fold diluted in medium, giving a final
different compound concentration of 8−128 μg/mL. Plates were
incubated for 24 h; then the absorbance at 625 nm was measured.
MIC value was calculated according to the Reed and Muench
method.22
Strains and Media. Escherichia coli strains were grown in low-salt
LB broth or agar at 37 °C. P. pastoris yeast strains were cultivated in
YPD medium, and for induced growth, in BMGY/BMMY medium.
The primers used in this study are summarized in Table S1, and
strains and plasmids are listed in Table S2.
Full-Length Transcriptome Sequences. RNA-seq and bio-
informatics analyses were conducted by Biomarker Technologies
Corporation. Total RNA was extracted from frozen fresh leaves
following the Biomarker protocol. After the samples are qualified,
total RNA was reverse transcribed into cDNA using a SMARTer PCR
cDNA synthesis kit; then the full-length cDNA was amplified by PCR.
Terminal repair was performed on the full length cDNA, which was
then connected to the SMRT dumbbell connector. The library was
obtained by digestion with exonuclease.
Raw reads were processed into error-corrected reads of inserts
(ROIs) using the Iso-seq pipeline with minFullPass = 0 and
minPredictedAccuracy = 0.90. Next, FLNC transcripts were
determined by searching for the polyA tail signal and the 5′ and 3′
cDNA primers in ROIs. ICE (iterative clustering for error correction)
was used to obtain consensus isoforms, and full-length consensus
sequences from ICE were polished using Quiver. High-quality full-
length transcripts were classified with the criteria postcorrection
accuracy above 99%. Then, Iso-Seq high-quality full-length transcripts
for removing redundancy using cd-hit (identity >0.99) were used.
The raw data were uploaded to the Sequence Read Archive (SRA)
(http://www.ncbi.nlm.nih.gov/) with accession SRR12052857. The
transcriptome assembly project was deposited at DDBJ/EMBL/
GenBank under the accession GISE00000000.
Expression of ObPPT-1 and ObPPT-2 in P. pastoris. P. pastoris
yeast strains SOB03−05 were cultivated in YPD media with shaking
in 15 mL culture tubes and then inoculated into 500 mL shake flasks
with 100 mL of BMGY medium at 30 °C to OD600 = 2−6. The cells
were collected by centrifugation and resuspended in BMMY medium.
Then, the resuspended cells were incubated at 30 °C for 120 h, with
the addition of 0.5% MeOH every 24 h.
Metabolite Extraction and GC-MS Analysis. The supernatant
of the fermentation was collected from 100 mL of culture by
centrifugation and extracted three times with 100 mL of EtOAc.
EtOAc extracts were combined and concentrated to afford an organic
extract. And then the exact was dissolved with 1.5 mL of CHCl3 and
analyzed using an Agilent 7890 GC-5975 MS instrument under
electronic impact at 70 eV. The oven temperature was initially set at
70 °C, raised from 70 to 130 °C (5 °C min−1), then elevated from
130 to 180 °C (10 °C min−1), and then elevated from 180 to 300 °C
(5 °C min−1).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01101.
■
sı
Complete description of methods, additional tables, and
figures, including structure elucidation and NMR spectra
for compounds 1−12 (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Jiang-Miao Hu − State Key Laboratory of Phytochemistry and
Plant Resources in West China and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
People’s Republic of China; orcid.org/0000-0002-9013-
8489; Email: hujiangmiao@mail.kib.ac.cn
Phylogenetic Analysis of Aromatic Prenyltransferases.
Multiple sequence alignment of aromatic prenyltransferases was
performed using clustalW. Evolutionary analyses were conducted in
MEGA X. The evolutionary history was inferred using the neighbor-
joining method.23 The evolutionary distances were computed using
the Poisson correction method and are in units of the number of
amino acid substitutions per site. This analysis involved 67 amino acid
sequences. All ambiguous positions were removed for each sequence
pair (pairwise deletion option). There were a total of 579 positions in
the final data set.
Jun Zhou − State Key Laboratory of Phytochemistry and Plant
Resources in West China and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, People’s
Republic of China; Email: jzhou@mail.kib.ac.cn
Construction of Plasmids and Mutants. The strain to express
sufficient precursor DMAPP in P. pastoris was constructed according
to the following procedure: ObPPT-1 and ObPPT-2 were made by
synthesis, with yeast-preferred codons incorporated into the sequence,
then introduced into the pUC57 vector (Genewiz). The plasmids
were double-digested with XhoI and NotI and ligated into the yeast
expression vector pPICZB to generate the plasmids pBOB01 and
pBOB02, respectively. The tHMG1 gene was PCR amplified from
Saccharomyces cerevisiae S288C chromosomal DNA, using the primer
pair P3/P4. The promoter PTEF1 and the TCYC1 terminator were
amplified from pPICZB using primer pairs P1/P2 and P5/P6,
respectively. Then, PTEF1, tHMG1, and TCYC1 were assembled using
splicing by overlap extension PCR (SOE-PCR) using the primer pair
P1/P6. The amplified products were cloned into the pBluescript
SK(+) vector and sequenced from both ends. Then, the plasmids
were digested with BamHI and inserted into same enzyme-digested
pPICZB, pBOB01, and pBOB02 for the construction of plasmid
pBOB03, pBOB04, and pBOB05, respectively. Plasmids pBOB03−05
was linearized with DraI and inserted into the AOX1 site of P. pastoris
yeast SMD1168H to generate the strains SOB03−05, respectively,
using electroporation according to the protocol described in the
EasySelect Pichia expression kit (Invitrogen).
Authors
Fu-Cai Ren − State Key Laboratory of Phytochemistry and
Plant Resources in West China and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
People’s Republic of China; University of Chinese Academy of
Sciences, Beijing 100049, People’s Republic of China
Li Liu − State Key Laboratory of Phytochemistry and Plant
Resources in West China and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, People’s
Republic of China; University of Chinese Academy of
Sciences, Beijing 100049, People’s Republic of China
Yong-Feng Lv − State Key Laboratory of Phytochemistry and
Plant Resources in West China and Yunnan Key Laboratory
of Natural Medicinal Chemistry, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201,
People’s Republic of China; University of Chinese Academy of
Sciences, Beijing 100049, People’s Republic of China
Xue Bai − State Key Laboratory of Phytochemistry and Plant
Resources in West China and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, People’s
Republic of China
Plasmid maps of pBOB03−05 were uploaded to Benchling and can
be obtained through the Web site freely: https://benchling.com/s/
seq-HpSu1N1hjpyS6Ycq0ChF, https://benchling.com/s/seq-
rW8bte6rTRsuCXbU3L4m, and https://benchling.com/s/seq-
Orjq9I7OVr1ttUA4Qhzg, respectively.
425
https://dx.doi.org/10.1021/acs.jnatprod.0c01101
J. Nat. Prod. 2021, 84, 417−426

Journal of Natural Products
pubs.acs.org/jnp
Article
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Qian-Jin Kang − State Key Laboratory of Microbial
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Metabolic & Developmental Sciences, and School of Life
Sciences & Biotechnology, Shanghai Jiao Tong University,
Shanghai 200240, People’s Republic of China
Xiao-Jing Hu − State Key Laboratory of Microbial
Metabolism, Joint International Research Laboratory of
Metabolic & Developmental Sciences, and School of Life
Sciences & Biotechnology, Shanghai Jiao Tong University,
Shanghai 200240, People’s Republic of China
Hong-Dan Zhuang − State Key Laboratory of Phytochemistry
and Plant Resources in West China and Yunnan Key
Laboratory of Natural Medicinal Chemistry, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming
650201, People’s Republic of China
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Liu Yang − State Key Laboratory of Phytochemistry and Plant
Resources in West China and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650201, People’s
Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01101
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Key
Research and Development Program of China
(2017YFD0201402) and Yunnan Provincial Science and
Technology Department (No. 2017ZF003-04).
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