Characterization of a sesquiterpene cyclase from the glandular trichomes of Leucosceptrum canum for sole production of cedrol in Escherichia coli and Nicotiana benthamiana

Article abstract of DOI:10.1016/j.phytochem.2019.03.009

Cedrol is an extremely versatile sesquiterpene alcohol that was approved by the Food and Drug Administration of the United States as a flavoring agent or adjuvant and has been commonly used as a flavoring ingredient in cosmetics, foods and medicine. Furthermore, cedrol possesses a wide range of pharmacological properties including sedative, anti-inflammatory and cytotoxic activities. Commercial production of cedrol relies on fractional distillation of cedar wood oils, followed by recrystallization, and little has been reported about its biosynthesis and aspects of synthetic biology. Here, we report the cloning and functional characterization of a cedrol synthase gene (Lc-CedS) from the transcriptome of the glandular trichomes of a woody Lamiaceae plant Leucosceptrum canum. The recombinant Lc-CedS protein catalyzed the in vitro conversion of farnesyl diphosphate into the single product cedrol, suggesting that Lc-CedS is a high-fidelity terpene synthase. Co-expression of Lc-CedS, a farnesyl diphosphate synthase gene and seven genes of the mevalonate (MVA) pathway responsible for converting acetyl-CoA into farnesyl diphosphate in Escherichia coli afforded 363 μg/L cedrol as the sole product under shaking flask conditions. Transient expression of Lc-CedS in Nicotiana benthamiana also resulted in a single product cedrol with a production level of 3.6 μg/g fresh weight. The sole production of cedrol by introducing of Lc-CedS in engineered E. coli and N. benthamiana suggests now alternative production systems using synthetic biology approaches that would better address sufficient supply of cedrol.

Full text of DOI:10.1016/j.phytochem.2019.03.009

Phytochemistry 162 (2019) 121–128
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Characterization of a sesquiterpene cyclase from the glandular trichomes of
Leucosceptrum canum for sole production of cedrol in Escherichia coli and
Nicotiana benthamiana
Fei Luoa,b,c, Yi Linga,b, De-Sen Lia,b, Ting Tanga,b, Yan-Chun Liua,b,c, Yan Liua,b,∗∗
Sheng-Hong Li
,
a,b,∗
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China
Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming, 650201, PR China
University of Chinese Academy of Sciences, Beijing, 100049, PR China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Leucosceptrum canum
Lamiaceae
Cedrol is an extremely versatile sesquiterpene alcohol that was approved by the Food and Drug Administration
of the United States as a flavoring agent or adjuvant and has been commonly used as a flavoring ingredient in
cosmetics, foods and medicine. Furthermore, cedrol possesses a wide range of pharmacological properties in-
cluding sedative, anti-inflammatory and cytotoxic activities. Commercial production of cedrol relies on frac-
tional distillation of cedar wood oils, followed by recrystallization, and little has been reported about its bio-
synthesis and aspects of synthetic biology. Here, we report the cloning and functional characterization of a
cedrol synthase gene (Lc-CedS) from the transcriptome of the glandular trichomes of a woody Lamiaceae plant
Leucosceptrum canum. The recombinant Lc-CedS protein catalyzed the in vitro conversion of farnesyl diphosphate
into the single product cedrol, suggesting that Lc-CedS is a high-fidelity terpene synthase. Co-expression of Lc-
CedS, a farnesyl diphosphate synthase gene and seven genes of the mevalonate (MVA) pathway responsible for
converting acetyl-CoA into farnesyl diphosphate in Escherichia coli afforded 363 μg/L cedrol as the sole product
under shaking flask conditions. Transient expression of Lc-CedS in Nicotiana benthamiana also resulted in a single
product cedrol with a production level of 3.6 μg/g fresh weight. The sole production of cedrol by introducing of
Lc-CedS in engineered E. coli and N. benthamiana suggests now alternative production systems using synthetic
biology approaches that would better address sufficient supply of cedrol.
Cedrol
Sesquiterpene cylase
Cedrol synthase
Heterologous production
Synthetic biology
1. Introduction
a peroxy-containing sesquiterpene lactone extracted from Artemisia
annua, is a prominent antimalarial drug, which has saved countless
Terpenoids comprise a large group of structurally diverse natural
lives (Klayman, 1985). In addition, the desirable aromatic and com-
bustible properties of sesquiterpenoids also inspired their applications
as flavors, fragrances and biofuel, as exemplified by the well-known
sesquiterpenoids bisabolene, bisabolol, β-farnesene and nootkatone
(Peralta-Yahya et al., 2011; Schempp et al., 2018).
On the basis of their valuable properties, sesquiterpenoids have
found a broad spectrum of medical and commercial applications.
Nevertheless, many sesquiterpenoids are difficult to be chemically
synthesized in an environmentally friendly and economic way, and
most sesquiterpenoids are still only available through extraction and
purification from natural sources, which is constrained by their low
products with a wide range of biological functions, covering both
ecological roles and pharmacological activities (Gershenzon and
Dudareva, 2007). Among them, sesquiterpenoids (C15) represent an
enormously diversified sub-class of terpenoids, and are widespread in
higher plants and microorganisms (Fraga, 2013). The chemical di-
versity of sesquiterpenoids is reflected by their important ecological
roles in antagonistic or mutualistic interactions among organisms
Gershenzon and Dudareva, 2007), as well as their extensive bioactiv-
ities including antimalarial, antitumor, anti-inflammatory and anti-
microbial properties (Chadwick et al., 2013). For example, artemisinin,
(
∗
Corresponding author. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, PR China
∗∗
Corresponding author. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, PR China
E-mail addresses: liuyan@mail.kib.ac.cn (Y. Liu), shli@mail.kib.ac.cn (S.-H. Li).
https://doi.org/10.1016/j.phytochem.2019.03.009
Received 30 January 2019; Received in revised form 8 March 2019; Accepted 9 March 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

F. Luo, et al.
Phytochemistry 162 (2019) 121–128
concentrations in raw materials and limited resources. Recently, the
development of synthetic biology has provided a potential to manu-
facture sesquiterpenoids, taking advantage of the pronounced regio-
and stereo-selectivity of enzymes involving in the relevant biosynthetic
pathway. It is well known that the diverse sesquiterpenoids are derived
from the common precursor farnesyl diphosphate (FDP), which is then
cyclized and/or rearranged by different sesquiterpene synthases
far little has been done about its biosynthesis and synthetic biology,
except for a patent about four sesquiterpene synthases from J. vir-
giniana, which could produce cedrol and thujopsene (Schalk et al.,
2016). Leucosceptrum canum Sm. (Lamiaceae) is a large woody plant
producing an unusual dark brown flower nectar that is caused by a
unique proline-benzoquinone pigment (Luo et al., 2012). A large
number of terpenoids including sesquiterpenoids, diterpenoids and
sesterterpenoids have been isolated from the glandular trichomes,
flowers and leaves of L. canum (Guo et al., 2019; Luo et al., 2010, 2013),
making L. canum an excellent system for studying terpenoid diversity
and biosynthesis. We have cloned and functionally characterized a
geranylfarnesyl diphosphate synthase for biosynthesis of the C25 prenyl
diphosphate precursor of sesterterpenoids from the glandular trichomes
of L. canum (Liu et al., 2016). Further investigation on the terpenoid
biosynthesis in L. canum led to the cloning and identification of a cedrol
synthase (Lc-CedS). Herein, we reported on the functional character-
ization of Lc-CedS, as well as the sole production of cedrol using en-
gineered E. coli and N. benthamiana.
(
Durairaj et al., 2019). Sesquiterpene synthases are encoded by genes
which are members of a supergene family widely existing in plants and
microorganisms, and a large number of sesquiterpene synthases have
been identified in recent years, as exemplified by amorpha-4,11-diene
synthase (Chang et al., 2000; Mercke et al., 2000), bisabolene synthase
(
2
Peralta-Yahya et al., 2011), valencene synthase (Beekwilder et al.,
014) and germacrene A synthase (Hu et al., 2017), and so on. By in-
troducing the above-mentioned sesquiterpene synthases, the en-
gineered microbes were obtained for producing amorphadiene, bisa-
bolene, valencene or germacrene A (Keasling, 2012; Peralta-Yahya
et al., 2011; Waltz, 2015; Hu et al., 2017), and the engineered yeasts
(
Saccharomyces cerevisiae) produced valencene has already been on the
market (Waltz, 2015). Most notably, an engineered yeast strain with the
yield of artemisinic acid as high as 25 g/L was reconstructed through
introduction of an amorpha-4,11-diene synthase along with three oxi-
dases and modification of the endogenous metabolic network (Paddon
et al., 2013). In addition, plant-based production systems such as Ni-
cotiana benthamiana have become increasingly attractive as a hetero-
logous expression platform for producing plant sesquiterpenoids in-
cluding costunolide, parthenolide, valencene and artemisinic acid
2. Results and discussion
2.1. Gene cloning and heterologous expression of candidate cedrol synthase
in Escherichia coli
To investigate cedrol synthase with high fidelity and activity, the
transcriptome of L. canum glandular trichome (Liu et al., 2016) was
searched with plant terpene synthase sequences. A unigene, sharing
38% identity with cedrol and thujopsene synthases from J. virginiana
(Schalk et al., 2016), was selected as a candidate cedrol synthase.
However, both the 5′ sequence and 3′ sequences of the candidate gene
were missing. Therefore, 5′ and 3’ rapid amplification of cDNA ends
(RACE) were performed using mRNA isolated from L. canum leaves as
template, and a full-length cDNA of 1873 bp was ultimately obtained.
Further analysis indicated that the open reading frame (ORF) of the
candidate was 1650 bp, which was predicted to encode a protein of 549
amino acids with a calculated molecular weight of 63 kDa. The se-
quence of Lc-CedS has been submitted to the GenBank database under
accession number of MK431785.
The candidate protein contained the typical “DDXXD” and “NSE/
DTE” motifs (Fig. 1), which were shown involved in the binding of
divalent metal ions (Christianson, 2017). The amino acid sequence of
the candidate protein showed 27%–29% identity with those of J. vir-
giniana cedrol and thujopsene synthases, but exhibited high similarity
to that of γ-cadinene synthase from Pogostemon cablin with identity of
67% (Deguerry et al., 2006). Phylogenetic analysis was performed
using the neighbor joining method with amino acid sequences of 37
additional plant terpene synthases obtained from the NCBI, and the
candidate protein was shown to belong to the TPS-a superfamily, which
mainly consists of angiosperm sesquiterpene synthases (Bohlmann
et al., 1998) (Fig. 2). However, J. virginiana cedrol and thujopsene
synthase JvCP1206-6 clustered in the TPS-b clade, which consists of
angiosperm monoterpene synthases (Bohlmann et al., 1998). Moreover,
the candidate cedrol synthase was predicted to be a protein without
transit peptide by Target P software (http://www.cbs.dtu.dk/services/
TargetP/) with reliability class of 2 (5 reliability class from 1 to 5; the
lower the value of reliability class, the safer the prediction).
(
Reed and Osbourn, 2018). However, the synthetic biology of a ma-
jority of valuable sesquiterpenoids still remains unachieved, probably
due to the poor selectivity and low activity of the present sesquiterpene
synthases. Therefore, mining of highly active and selective sesqui-
terpene synthases are still worthwhile, which should help create novel
systems for sesquiterpenoid production in the future.
Cedrol, a sesquiterpene alcohol with weak aroma, was found par-
ticularly in the wood part of several conifers, including Cedrus atlantica,
Cupressus sempervirens, and Juniperus virginiana (Sells, 2006). Cedrol
was approved by the Food and Drug Administration of the United States
as a flavoring agent or adjuvant (https://www.accessdata.fda.gov/
scripts/fdcc/index.cfm?set=FoodSubstances&id=CEDROL) and has
been commonly used as a flavor enhancer in cosmetics, foods, medi-
cine, fine fragrances, and so on. (Bhatia et al., 2008). In addition, cedrol
has also been reported to possess extensive pharmacological effects.
Cedrol showed potent platelet-activating factor (PAF) receptor-binding
antagonistic activity with IC50 value of 13 μM (Yang et al., 1995), and
was widely used as a positive control in relevant bioassays (Singh et al.,
2013). Moreover, cedrol also served as a sedative agent on autonomic
nervous system (Daiji et al., 2003; Dayawansa et al., 2003; Yada et al.,
2
2
007), an antileishmanial (Kar et al., 2017) and cytotoxic (Loizzo et al.,
008) agent, an enhancer of extracellular matrix production in dermal
fibroblasts in a MAPK-dependent manner (Jin et al., 2012), an inhibitor
of cytochrome P450 enzymes (Jeong et al., 2014), and a promoter of
hair growth (Zhang et al., 2016). Besides, cedrol also exhibited a broad
range of biological functions including antimicrobial (Su et al., 2012)
and antitermitic (Chang et al., 2001) effects, as well as contact toxicity
against the black-legged tick (Eller et al., 2014). The significant biolo-
gical functions and the rare 5/5/6 tricyclic carbon skeleton of cedrol
made it highly attractive for total chemical synthesis (Stork and Clarke,
1
955, 1961), biotransformation (Collins and Reese, 2001) and drug
For functional characterization, full-length cDNA of the candidate
was subcloned to a pCold TF expression vector (Takara), which contains
an N-terminal His tag, and then transferred into E. coli strain Rosetta
(DE3). SDS-PAGE analysis revealed that the E. coli transformant showed
high expression of recombinant protein with the expected molecular
mass in the soluble fraction after induction with IPTG and low tem-
perature. The recombinant protein was subsequently purified with a Ni-
NTA agarose column.
delivery system (Kar et al., 2017).
Cedrol is prepared from cedar wood oils by fractional distillation
followed by recrystallization (Sells, 2006). Although its chemical
synthesis has been reported long time ago, this is complicated in view of
its complicated structure and stereochemistry. It is thus quite attractive
to clone genes that code for enzymes being involved in corresponding
biosynthesis pathways, and to get them eventually heterologously ex-
pressed in systems like E. coli and S. cerevisiae for high productivity. So
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Phytochemistry 162 (2019) 121–128
Fig. 1. Alignment of the deduced amino sequences of Lc-CedS with plant sesquiterpene synthases. The DDXXD and NSE/DTE motifs are underlined. Aa-eCedS:
Artemisia annua epi-cedrol synthase; Hi-MuS: Handroanthus impetiginosus (Z)-muuroladiene synthase; JvCP1206-6: Juniperus virginiana (+)-cedrol and (−)-thujopsene
synthase; Lc-CedS: Leucosceptrum canum cedrol synthase; Si-γCDS: Sesamum indicum γ-cadinene synthase.
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Phytochemistry 162 (2019) 121–128
Fig. 2. Phylogenetic analysis of Lc-CedS and other plant terpene synthases. The sequence information of terpene synthases is listed in Supplementary Table S1.
2
.2. Functional characterization of candidate cedrol synthase in vitro
GGDP was used as a substrate, the hydrolyzed reaction mixture accu-
mulated a new peak, which was tentatively identified as linalool and
geranyl linalool, respectively, based on their mass spectra (Figure S2).
However, no specific hydrolyzed product was detected in the reaction
mixture with FDP as a substrate. By contrast, a new peak, tentatively
identified as nerolidol based on its mass spectrum, was found in the
hydrolyzed control reaction mixture that contained the same reaction
buffer and FDP, but not the recombinant protein. Linalool, nerolidol,
and geranyllinalool are likely to be the hydrolyzed products of GDP,
FDP, GGDP, further confirming that the candidate cedrol synthase
could utilize FDP as a prenyl diphosphate substrate and give cedrol as
its sole product. Therefore, the candidate was renamed as L. canum
cedrol synthase (Lc-CedS).
Lc-CedS is a high-fidelity terpene synthase, as it utilized FDP as a
substrate to generate a single product cedrol. In contrast, most plant
terpene synthases, including cedrol and thujopsene synthases from J.
virginiana (Schalk et al., 2016) and epi-cedrol synthase from Artemisia
annua (Hua and Matsuda, 1999; Mercke et al., 1999), gave multiple
products from prenyl diphosphate substrates, which might be due to the
To verify the enzymatic activity of recombinant protein, in vitro
enzyme activity reaction was carried out with geranyl diphosphate
GDP), FDP or geranylgeranyl diphosphate (GGDP) as a substrate. After
(
incubating at 30 °C for 3 h, the reaction mixture was extracted with n-
hexane and analyzed using GC-MS. Clearly, the recombinant candidate
cedrol synthase did utilize FDP to produce a very specific product peak
with retention time of 22.54 min in the total ion chromatogram. Its
characteristic fragmental ions were present at m/z 222, 150 and 95 in
the mass spectrum, which was very similar to that of cedrol in the NIST
library (version 2.0) with a high match value of 974 (out of 1000).
Through comparing its retention time and mass spectrum with those of
a commercial cedrol (Aladdin), the enzymatic product was further
confirmed as cedrol (Fig. 4). However, when GDP or GGDP was used as
a substrate in the enzyme assay, no product was detected (Figure S1).
The above-described reaction mixtures were further hydrolyzed
with HCl and analyzed using GC-MS to explore whether any product
containing a phosphate moiety was formed by Lc-CedS. When GDP or
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Phytochemistry 162 (2019) 121–128
Fig. 3. Proposed mechanism for the conversion of FDP into cedrol catalyzed by Lc-CedS. OPP indicates the diphosphate moiety.
electrophilic reaction mechanism in which several unstable carbocation
intermediates were formed. The cyclization mechanism of cedrol by A.
annua epi-cedrol synthase that could produce 4% cedrol and 96% epi-
cedrol was proposed (Mercke et al., 1999). The 3(R)-nerolidol dipho-
sphate is a catalytic intermediate of sesquiterpene synthase, which was
well studied by isotopic labelling experiments (Christianson, 2017). It
was hypothesized that a C1-C6 cyclization takes place, resulting in a
monocyclic bisabolyl cation, because A. annua epi-cedrol synthase also
produced (E)-α-bisabolene as its minor product, which might be gen-
erated via deprotonation of the bisabolyl cation (Mercke et al., 1999).
Next, a 1,2-hydride transfer occurs and the resulting 7(R)-homo-
bisabolyl cation undergoes two additional C6-C10 and C2-C11 cycli-
zations to yield a tricyclic cedryl cation, which is then quenched by
addition of a water molecule followed by proton loss (Fig. 3). The syn-
periplanar addition generates cedrol, while anti-periplanar addition
gives epi-cedrol. However, syn-periplanar addition should be much
preferred during the enzyme reaction of Lc-CedS, which is different
from that of A. annua epi-cedrol synthase. Nevertheless, the highly se-
lective mechanism of Lc-CedS deserves further investigation.
neither cedrol nor epi-cedrol was detected. The cloning and identifica-
tion of a gene encoding a cedrol synthase from L. canum implied that
cedrol might have been made in the plant, but was rapidly metabolized
to other downstream products.
2.3. Production of cedrol in engineered E. coli
Considering the high economical and pharmacological value of ce-
drol, the heterologous synthesis of cedrol was studied. An engineered E.
coli strain was constructed, in which Lc-CedS, a FDP synthase gene from
E. coli and seven genes of the MVA pathway including an acetoacetyl-
CoA thiolase (AtoB) gene from E. coli, and a HMG-CoA synthase
(
HMGS), a truncated HMG-CoA reductase (tHMGR), a mevalonate ki-
nase (ERG12), a phosphomevalonate kinase (ERG8) and a mevalonate
pyrophosphate decarboxylase (MVD1) genes from S. cerevisiae with
codon-optimization, along with an IPP isomerase (IDI) gene from E.
coli, were included (Peralta-Yahya et al., 2011). GC-MS analysis of the
extracts of the engineered E. coli strain showed a major compound with
characteristic fragmental ions at m/z 222, 150 and 95 in its mass
spectrum, which was consistent with those of commercial cedrol
The acetone extracts prepared from the leaves, stems, flowers and
roots of L. canum were then examined using GC-MS. Unfortunately,
(
Fig. 5). The yield of cedrol was then quantified by GC-MS using
Fig. 4. GC-MS analysis of the Lc-CedS enzyme assay product with FDP as a substrate. (A) Total ion chromatogram (TIC) of the Lc-CedS enzyme assay product. (B)
Mass spectrum of the Lc-CedS enzyme assay product. (C) TIC of the standard cedrol and its chemical structure. (D) Mass spectrum of the standard cedrol.
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Phytochemistry 162 (2019) 121–128
Fig. 6. GC–MS analysis of the extracts of N. benthamiana leaves expressing Lc-
CedS. (A and B) Total ion chromatogram (TIC) of the extracts of N. benthamiana
leaves co-infiltrated with A. tumefaciens harboring the empty vector pEAQ-HT
as a control (A) and the recombinant plasmid pEAQ-HT/Lc-CedS (B). (C) TIC of
the standard cedrol. The specific cedrol product formed by N. benthamiana
expressing Lc-CedS is shaded in (B).
the acetone extracts of N. benthamiana harboring pEAQ-HT/Lc-CedS
(
Figure S4), suggesting that either cedrol was not further transformed in
N. benthamiana, or the transformed cedrol derivatives were not thor-
oughly extracted and under detection limit, which warrants further
investigation.
Fig. 5. GC–MS analysis of the products formed by the engineered E. coli ex-
pressing Lc-CedS. (A) Total ion chromatogram (TIC) of the product of en-
gineered E. coli harboring the recombinant plasmid pCold TF/Lc-CedS. The
shaded part represents the specific cedrol product. (B) TIC of the product of
control E. coli harboring the empty vector pCold TF. (C) TIC of the standard
cedrol.
3. Conclusions
In this study, a cedrol synthase gene (Lc-CedS) with high-fidelity
was cloned from the transcriptome of L. canum glandular trichomes and
then functionally characterized through in vitro enzyme assay.
Recombinant Lc-CedS accepted FDP as a prenyl diphosphate substrate
to generate a single product cedrol. The identification of a high-fidelity
enzyme for cedrol biosynthesis, which resulted in the yield of 363 μg/L
of cedrol in engineered E. coli and 3.6 μg/g fresh weight in N. ben-
thamiana, will allow for further synthetic biology studies. To the best of
our knowledge, this is the first report on the characterization of a cedrol
synthase for heterologous production of cedrol, thereby providing an
alternative strategy for manufacturing the high-value cedrol using
synthetic biology approaches.
commercial cedrol as an external standard. It was found that 363 μg/L
cedrol was produced under shaking flask culture conditions for 72 h.
The results demonstrated that the transformed E. coli strain could in-
deed produce cedrol, which lays the foundation for synthetic biology of
cedrol in microbes.
2.4. Production of cedrol through transient expression of Lc-CedS in
Nicotiana benthamiana
Because Lc-CedS was cloned from a higher plant, we further at-
tempted to express Lc-CedS in N. benthamiana. The full-length cDNA of
Lc-CedS was inserted into a polylinker of CPMV-HT expression cassette
in the pEAQ-HT vector (Sainsbury et al., 2009) (Figure S3). After con-
firming by PCR and DNA sequencing, the recombinant pEAQ-HT
plasmid harboring Lc-CedS (pEAQ-HT/Lc-CedS) and empty vector
pEAQ-HT (control) was transformed into Agrobacterium tumefaciens,
respectively. A. tumefaciens strains containing different plasmid were
then used for agro-infiltration of N. benthamian leaves. After 5 days
post-infiltration, leaves of N. benthamian expressing Lc-CedS and control
were harvested and extracted with acetone, followed by condensation
and phase partition with petroleum ether, respectively. After con-
centration in vacuo, the extracts were dissolved in tetrahydrofuran and
analyzed using GC-MS. Consequently, expression of Lc-CedS in N. ben-
thamiana resulted in the occurrence of a specific peak, which was
identified as cedrol through comparison of its retention time and mass
spectrum with those of commercial cedrol (Fig. 6). The finding con-
firmed the enzymatic activity of Lc-CedS in planta. Meanwhile, the yield
of cedrol attained in N. benthamiana was 3.6 μg/g fresh weight.
4. Experimental
4.1. Plant, microbe and vector materials
Leucosceptrum canum Sm. (Lamiaceae) growing in Kunming
Botanical Garden was used in this experiment. Seeds of N. benthamiana
were kindly provided by Prof. Jinsong Wu at Kunming Institute of
Botany, Chinese Academy of Sciences (CAS). Seedlings of N. ben-
thamiana were grown in a constant greenhouse at 23 °C with a 16-h-
light/8-h-dark cycle for 4–6 weeks. Agrobacterium tumefaciens LBA4404
was kindly provided by Prof. Jianqiang Wu at Kunming Institute of
Botany, CAS. Plasmid pBbA5c-MevT-MBIS was kindly provided by Prof.
Tao Liu at Tianjin Institute of Industrial Biotechnology, CAS. Vector
pEAQ-HT was kindly provided by Dr. Qing Zhao at Shanghai Chenshan
Plant Science Research Center, CAS.
4.2. Cloning, expression, and purification of cedrol synthase
Considering the complex metabolic system of N. benthamiana, the
acetone extracts of infiltrated leaves were also analyzed using HPLC to
detect whether more polar products such as oxygenated or glycosylated
compounds were generated. However, no specific peak was detected in
The total RNA was extracted from the young leaves of L. canum
using a Trizol Plus RNA Purification Kit (Invitrogen) according to
manufacturer's instruction. The full-length sequence was cloned using
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Phytochemistry 162 (2019) 121–128
the SMART RACE cDNA amplication kit (Clontech), and the primers
used were listed in Supplementary Table S2. Lc-CedS cDNA was then
amplified with gene-specific primers (Table S2) using high-fidelity
PrimeSTAR Max DNA Polymerase (Takara) from the first strand cDNA
synthesized by a HiFiScript cDNA Synthesis Kit (CWBIO). The resultant
fragment was subcloned into a pCold TF vector (Takara), which was
digested with KpnI and HindIII (New England Biolabs), and then
transferred into E. coli (DH5α) using a heat shock method. After con-
firmation through sequencing five independent clones, an exact positive
plasmid named pCold TF/Lc-CedS was selected and transferred into the
E. coli strain Rosetta (DE3) (Novagen) for heterologous expression,
whereas the empty pCold TF vector was transferred as a negative
control.
The transformants were grown at 37 °C to the optimal optical den-
sity (OD600) of 0.5–0.6, and then induced with 0.3 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) overnight at 16 °C. The cells were har-
vested by centrifugation (3900 rpm, 4 °C, 5 min), resuspended in ex-
traction buffer (pH 8.0, 0.5 M NaCl, 10 mM imidazole and 5 mM DTT),
and sonicated. The lysate was obtained by centrifugation (12,000 rpm,
500 mL LB liquid medium containing 2% glucose and corresponding
antibiotics at 37 °C, 200 rpm/min to an OD600 of 0.5–0.6, and then
induced by 0.3 mM IPTG at 16 °C overnight, followed by continuing
cultivation at 25 °C for 72 h. Subsequently, the culture media were
extracted with n-hexane, and the obtained organic phase was con-
densed to 500 μL for the GC-MS analysis as described above.
4.5. Expression of Lc-CedS in N. benthamiana and product analysis
Lc-CedS was amplified from plasmid pCold TF/Lc-CedS and sub-
cloned into the Cowpea mosaic virus-based (CPMV-HT) plant expres-
sion vector, pEAQ-HT (Sainsbury et al., 2009). The PCR-amplified
products of Lc-CedS and the fragments of vector pEAQ-HT digested with
AgeI and XhoI were purified by the FastPure Gel DNA Extraction Mini
Kit (Vazyme) according to the manufacturer's instructions. The digested
vector pEAQ-HT and PCR-amplified products of Lc-CedS was combined
®
in molar ratio 1:2 in ClonExpress II One Step Cloning Kit (Vazyme) to
obtain the plant expression vector pEAQ-HT/Lc-CedS.
After verification by PCR and DNA sequencing, PEAQ-HT/Lc-CedS
and the empty PEAQ-HT (control) plasmids were transferred into the A.
tumefaciens strain LBA4404 using a freeze-thaw method, respectively.
The positive clones were cultured at 28 °C in Luria-Bertani liquid media
containing 25 μg/mL rifampicin, 50 μg/mL streptomycin and 50 μg/mL
kanamycin to OD600 of 1.2–1.8. The A. tumefaciens cells were collected
by centrifugation at 4000 g for 10 min and then gently resuspended in
MMA buffer (10 mM 2-[N-morpholino]ethanesulfonic acid (MES) pH
4
°C, 10 min), and the solubility of recombinant protein was analyzed
by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). A Ni-NTA agarose column (Qiagen) was used to purify re-
combinant protein according to manufacturer's instructions and an
elution buffer (pH 8.0, 0.5 M NaCl, 250 mM imidazole and 5 mM DTT)
was applied to elute the target protein. Both the crude lysate and the
purified protein were analyzed by SDS-PAGE.
5
2
.6, 10 mM MgCl and 100 μM acetosyringone) to make a solution with
4.3. Enzyme assays and product identification in vitro
final OD600 of 0.4. The suspensions were incubated at room tempera-
ture for about 2 h and aspirated into a sterile needle-free syringe (1 mL)
to infiltrate the leaves of 4- to 6-week-old N. benthamiana.
To determine the in vitro enzyme activity, 40 μg purified re-
combinant protein and 10 μg prenyl diphosphate substrate (GDP, FDP
or GGDP purchased from Sigma) were mixed with the reaction buffer
(
After growing in a glasshouse for five days, 3.2 g fresh leaves of
infiltrated plants were harvested and immediately frozen with liquid
nitrogen and ground into power. 50 mL acetone was used to extract
infiltrated leaves in an ultrasonic bath for 30 min, and the extraction
was repeated thrice. Subsequently, the extracts were combined, fil-
trated, and concentrated using a rotary evaporator, followed by ex-
traction with petroleum ether. After evaporating to dryness under ni-
trogen gas, the extracts were dissolved in tetrahydrofuran, which was
fixed the volume to 1 mL for the above-mentioned GC-MS and the fol-
lowing HPLC-DAD analyses.
An Agilent 1260 system was adopted for the HPLC-DAD analysis.
10 μL of each sample was injected into an Agilent Poroshell 120 EC-C18
analytical column (4 μm, 4.6 × 150 mm), and HPLC conditions were set
as follows: column temperature, 35 °C; UV detection wavelength,
254 nm; flow rate, 1 mL/min; mobile phase, mixture of acetonitrile
(solvent A) and water (solvent B) (0–25.00 min, linear gradient of
5%–95% of A; 25.01–40.00 min, isocratic 95% of A).
100 mM Tris–HCl pH 7.4, 5% glycerol, 5 mM MgCl
2
, 100 mM KCl,
2
mM DTT) with total volume of 200 μL. The protein purified from E.
coli cells containing the empty pCold TF vector was used as a control.
The reaction mixture was covered with 200 μL n-hexane and incubated
at 30 °C for 3 h, followed by extraction with n-hexane. The organic
phase was collected by centrifugation, and concentrated under nitrogen
gas before the GC–MS analysis.
After extraction with n-hexane, 20 μL HCl (3 M) was added into the
above- described reaction mixture. After hydrolysis at 30 °C for 30 min,
the reaction mixture was extracted with an equal volume of n-hexane,
and the organic phase was collected by centrifugation followed by
concentration under nitrogen gas before the GC–MS analysis.
The GC-MS analysis was performed on an Agilent GC (7890A)-MSD
5975C) instrument equipped with a HP-5MS quartz capillary column
30 mm × 250 μm × 0.25 μm) and conducted under electron-impact
EI) mode with ionization energy of 70 eV. Helium was used as carrier
(
(
(
gas at a constant flow rate of 3 mL/min. The temperature program was
set as follows: initial temperature 60 °C, holding for 2 min; ramp at rate
4.6. Quantitative analysis of cedrol in engineered E. coli and N.
benthamiana
5
°C/min to 240 °C, holding for 2 min; ramp at rate 10 °C/min to 280 °C,
holding for 2 min. 5 μL of each sample was injected in splitless mode.
The product of Lc-CedS recombinant protein was identified through
comparison of the mass spectrum and retention time with those of the
standard cedrol (CAS: 77-53-2) purchased form Aladdin.
A calibration curve of cedrol was generated by using GC-MS with
the method as described above. Seven concentration gradients of
standard sample (500, 100, 50, 25, 12.5, 6.25, 3.125 μg/mL) were
prepared. Each concentration was injected for three replicates, and the
linearity of standard curve was made by plotting the peak area versus
concentration. The equation and correlation coefficient obtained from
the linearity study was y = 8 × 10 x + 9 × 10 (R = 0.9918). The
yields of cedrol in E. coli and N. benthamiana were then calculated ac-
cording to the peak area and calibration curve of cedrol.
4.4. Introduction of Lc-CedS into E. coli cell and product analysis
6
7
2
The recombinant plasmid pCold TF/Lc-CedS was co-transferred into
E. coli strain BL21 with pBbA5c-MevT-MBIS, which integrated the en-
tire MVA pathway genes responsible for converting acetyl-CoA into
farnesyl diphosphate and a FDP synthase gene (Peralta-Yahya et al.,
4.7. Phylogenetic analysis
2011). The transformants were selected on the LB agarose plate con-
taining 34 μg/mL chloramphenicol and 100 μg/mL ampicillin. An E. coli
strain containing empty vector pCold-TF and pBbA5c-MevT-MBIS was
used as a negative control. The confirmed clones were inoculated in
The amino acid sequences were obtained from the NCBI protein
database (Supplementary Table S1). The phylogenetic tree was con-
structed using the MEGA7 software based on the Neighbor-Joining
127

F. Luo, et al.
Phytochemistry 162 (2019) 121–128
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Acknowledgements
We thank Prof. Tao Liu (Tianjin Institute of Industrial
Biotechnology, CAS), Dr Qing Zhao (Shanghai Chenshan Plant Science
Research Center, CAS), and Prof. Jinsong Wu and Prof. Jianqiang Wu
(
Kunming Institute of Botany, CAS) for kindly providing plasmid
2
28, 1049–1055.
pBbA5c-MevT-MBIS, vector pEAQ-HT, A. tumefaciens LBA4404 and N.
benthamiana seeds, respectively. This research was financially sup-
ported by National Science Fund for Distinguished Young Scholars
Liu, Y., Luo, S.H., Schmidt, A., Wang, G.D., Sun, G.L., Grant, M., Kuang, C., Yang, M.J.,
Jing, S.X., Li, C.H., Schneider, B., Gershenzon, J., Li, S.H., 2016. A geranylfarnesyl
diphosphate synthase provides the precursor for sesterterpenoid (C25) formation in
the glandular trichomes of the mint species Leucosceptrum canum. Plant Cell 28,
(
31525005), National Natural Science Foundation of China (31770390
8
04–822.
and 31770340), Yunnan Innovative Research Team for Discovery and
Biosynthesis of Bioactive Natural Products (2018HC012), Science
Foundation of Yunnan (2018FA017), and Youth Innovation Promotion
Association and “Western Light” Program of CAS (awarded to Yan Liu).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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Luo, S.H., Luo, Q., Niu, X.M., Xie, M.J., Zhao, X., Schneider, B., Gershenzon, J., Li, S.H.,
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