Potential role of two cytochrome P450s obtained from Lithospermum erythrorhizon in catalyzing the oxidation of geranylhydroquinone during Shikonin biosynthesis

Article abstract of DOI:10.1016/j.phytochem.2020.112375

Shikonin is a natural naphthoquinone derivative that specifically occurs in boraginaceous plants, and the major active ingredient of the medicinal plant Lithospermum erythrorhizon. Previously, a cytochrome P450 oxygenase (CYP) CYP76B74 catalyzing 3″-hydroxylation of geranylhydroquinone (GHQ) — a key intermediate of shikonin biosynthesis, was identified from cultured cells of Arnebia euchroma. However, the enzymes catalyzing oxidation of the geranyl side-chain of GHQ from L. erythrorhizon remain unknown. In this study, we performed transcriptome analysis of different tissues (red roots and green leaves/stems) from L. erythrorhizon using RNA sequencing technology. Highly expressed CYP genes found in the roots were then heterologously expressed in Saccharomyces cerevisiae and functionally screened with GHQ as the substrate. As the result, two CYPs of CYP76B subfamily catalyzing the oxidation of GHQ were characterized. CYP76B100 catalyzed the hydroxylation of the geranyl side-chain of GHQ at the C-3″ position to form 3″-hydroxyl geranylhydroquinone (GHQ-3″-OH). The enzyme CYP76B101 carried out oxidation reaction of GHQ at the C-3″ position to produce a 3″-carboxylic acid derivative of GHQ (GHQ-3″-COOH) as well as GHQ-3″-OH. This enzyme-catalyzed oxidation reaction with GHQ as the substrate is reported for the first time. This study implicates CYP76B100 and CYP76B101 as having a potential role in shikonin biosynthesis in L. erythrorhizon.

Full text of DOI:10.1016/j.phytochem.2020.112375

Phytochemistry175(2020)112375
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Potential role of two cytochrome P450s obtained from Lithospermum
erythrorhizon in catalyzing the oxidation of geranylhydroquinone during
Shikonin biosynthesis
Wan Songa,b,c, Yibin Zhuanga,b, Tao Liua,b,∗
a Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
b Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
c University of Chinese Academy of Sciences, Beijing, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lithospermum erythrorhizon
Boraginaceae
Shikonin
Cytochrome P450 monooxygenase
Geranylhydroquinone
Shikonin is a natural naphthoquinone derivative that specifically occurs in boraginaceous plants, and the major
active ingredient of the medicinal plant Lithospermum erythrorhizon. Previously, a cytochrome P450 oxygenase
(CYP) CYP76B74 catalyzing 3″-hydroxylation of geranylhydroquinone (GHQ) — a key intermediate of shikonin
biosynthesis, was identified from cultured cells of Arnebia euchroma. However, the enzymes catalyzing oxidation
of the geranyl side-chain of GHQ from L. erythrorhizon remain unknown. In this study, we performed tran-
scriptome analysis of different tissues (red roots and green leaves/stems) from L. erythrorhizon using RNA se-
quencing technology. Highly expressed CYP genes found in the roots were then heterologously expressed in
Saccharomyces cerevisiae and functionally screened with GHQ as the substrate. As the result, two CYPs of CYP76B
subfamily catalyzing the oxidation of GHQ were characterized. CYP76B100 catalyzed the hydroxylation of the
geranyl side-chain of GHQ at the C-3″ position to form 3″-hydroxyl geranylhydroquinone (GHQ-3″-OH). The
enzyme CYP76B101 carried out oxidation reaction of GHQ at the C-3″ position to produce a 3″-carboxylic acid
derivative of GHQ (GHQ-3″-COOH) as well as GHQ-3″-OH. This enzyme-catalyzed oxidation reaction with GHQ
as the substrate is reported for the first time. This study implicates CYP76B100 and CYP76B101 as having a
potential role in shikonin biosynthesis in L. erythrorhizon.
1. Introduction
(GBA) in yeast, an important intermediate in shikonin biosynthesis
(Wang et al., 2017).
Shikonin and its derivatives are the natural naphthoquinone deri-
vatives that have been considered as the primary active components of
root tissues from Lithospermum erythrorhizon. Shikonins exhibit anti-
microbial, wound-healing, anti-inflammatory, and anti-adenoviral
properties (Andújar et al., 2013), and are currently used in food, cos-
metics and pharmaceutical industry (Rai et al., 2018). Shikonins are
only detected in boraginaceous plants belonging to the genera Lithos-
permum, Arnebia, Anchusa, Echium, Onosma and Alkanna, and have been
used as natural dyes and herbal medicines in both Europe and the
Orient for many centuries (Malik et al., 2016; Yazaki, 2017). The in-
creasing demands for shikonins have made L. erythrorhizon at real risk
of extinction in recent years (Yazaki, 2017). This heightened risk has
inspired extensive efforts to focus on cell suspension cultures (Tabata
and Fujita, 1985), biosynthetic pathways (Okamoto et al., 1995) and
metabolic engineering production of 3-geranyl-4-hydroxybenzoic acid
Shikonin is derived from p-hydroxybenzoic acid and geranyl di-
phosphate, originating from the shikimate pathway (Inouye et al.,
1979) and mevalonate pathway (Li et al., 1998), respectively (Fig. 1).
The coupling of two key precursors is catalyzed by geranyl diphosphate:
4-hydroxybenzoate 3-geranyltransferase (PGT) to yield the important
intermediate GBA (Yazaki et al., 2002), which is subsequently con-
verted to geranylhydroquinone (GHQ) by unknown enzymes. A cyto-
chrome P450 monooxygenase (CYP) was identified from L. ery-
throrhizon suspension cultures catalyzing the C-3″ hydroxylation of
GHQ to form a key intermediate 3″-hydroxy-geranylhydroquinone
(GHQ-3″-OH) (Yamamoto et al., 2000). Recently, the first mono-
oxygenase CYP76B74 of shikonin biosynthesis, which catalyzed the key
3″-hydroxylation step was cloned and identified from Arnebia euchroma
(Wang et al., 2019). However, the enzymes involved in 3″-hydroxyla-
tion reaction of GHQ in L. erythrorhizon and the intriguing steps for
∗ Corresponding author. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
E-mail address: liu_t@tib.cas.cn (T. Liu).
https://doi.org/10.1016/j.phytochem.2020.112375
Received 25 September 2019; Received in revised form 5 April 2020; Accepted 6 April 2020
0031-9422/©2020ElsevierLtd.Allrightsreserved.

W. Song, et al.
Phytochemistry175(2020)112375
Fig. 1. The proposed biosynthetic pathway of shikonin in L. erythrorhizon. Single arrows represent one step reaction, while double arrows represent multiple step
reactions. Dashed arrows signify undefined steps or the enzymes have not been verified yet. The steps with red-colored enzymes indicate the oxidation reactions of
converting GHQ to GHQ-3″-OH or GHQ-3″-COOH by CYP76B100 and CYP76B101. PGT, geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase; GBA, 3-
geranyl-4-hydroxybenzoic acid; GHQ, geranylhydroquinone; GHQ-3″-OH, 3″-hydroxy-geranylhydroquinone; GHQ-3″-CHO, 3″-aldehyde-geranylhydroquinone; GHQ-
3″-COOH, 3″-carboxyl-geranylhydroquinone.
naphthoquinone skeleton formation from GHQ-3″-OH remain un-
known. A plausible mechanism for ring closure has been proposed via
intermediate 3″-aldehyde-geranylhydroquinone (GHQ-3″-CHO), which
could form the C–C bond with the aromatic nucleus by an electrophilic
reaction (Yamamoto et al., 2000) (Fig. 1).
the oxidation of GHQ. CYP76B100 catalyzed the 3″-hydroxylation of
GHQ to form GHQ-3″-OH. Interestingly, CYP76B101 catalyzed the
oxidation of GHQ at the C-3″ position to synthesize 3″-carboxyl-ger-
anylhydroquinone (GHQ-3″-COOH) besides one-step oxidation product
GHQ-3″-OH. The results might provide new insights into the bio-
synthesis of shikonin in L. erythrorhizon (Fig. 1).
RNA sequencing technology (RNA-Seq) is a powerful tool for ac-
celerating the discovery of novel genes through transcription pattern
and functional analysis of genes thought to be involved in medicinal
plant specialized metabolism. RNA-Seq thus plays important roles in
elucidating pathways of natural products such as the seco-iridoid
pathway in Catharanthus roseus (Krithika et al., 2015; Miettinen et al.,
2014) and the tanshinones biosynthesis pathway in Salvia miltiorrhiza
(Guo et al., 2012; Gao et al., 2014). The average lengths of the contigs
generated by next-generation sequencing (NGS) based on RNA-Seq
are < 500 bp, while long reads (average 4–8 kb) can be obtained by
single-molecule real-time (SMRT) sequencing that is carried out in a
third-generation sequencing platform (Xu et al., 2015). SMRT sequen-
cing offers access to more complete transcriptome data, fully revealing
the key advantage of obtaining full-length transcripts, and the asso-
ciated problem of a higher error rate (up to 15%) can be solved by
correction with NGS reads (Au et al., 2012). Thus, a hybrid sequencing
approach combining NGS short reads and SMRT long reads will yield a
more precise and extensive transcriptional database (Xu et al., 2015).
In plants, most of the reported terpene oxidations are carried out by
CYPs to give rise to alcohols, aldehydes or carboxylic acids (Bathe and
Tissier, 2019). In this work, we aimed to clone and functionally char-
acterize CYP enzymes catalyzing the oxidation of GHQ at the C-3″ po-
sition from L. erythrorhizon. The candidate genes were obtained by the
transcriptome sequencing and differential gene expression analysis of
green leaves/stems samples and red roots. Functional screening of the
highly expressed CYP genes in yeasts combined with in vitro enzymatic
activity assays resulted in identification of two CYP enzymes catalyzing
2. Results
2.1. Discovery of CYP candidate genes
According to previous reports, shikonin is detected only in the red
roots rather than the green leaves/stems of L. erythrorhizon and the
genes related to shikonin biosynthesis are highly expressed in all red
roots of three Lithospermeae plants, such as hydroxymethylglutaryl-
CoA reductase gene (HMGR), phenylalanine ammonia lyase gene (PAL),
PGT (Wu et al., 2017). Thus, the expression of the major genes that are
related to shikonin downstream biosynthesis may be up-regulated in
the roots. CYPs are frequently involved in the oxidation of terpenes, and
GHQ hydroxylation at the C-3″ position is catalyzed by CYPs
(Yamamoto et al., 2000; Wang et al., 2019).
Combined the analysis of differentially expressed unigenes and the
integrity of the open reading frames, a total of 40 CYP genes tran-
scriptionally up-regulated in the red roots (Supplementary Table S1)
were selected as the candidates for next step investigation. Cytochrome
P450 reductase from Arabidopsis thaliana (AtCPR1), which is re-
sponsible for the electron transfer from NADPH to CYPs, was cloned
into the yeast expression vector pCf302 under the control of the TEF1
promoter. Using the cDNA from red roots as the amplification template,
the full-length sequences of 40 CYP candidates were obtained with the
primers shown in Supplementary Table S2, and successfully cloned into
pCf302-AtCPR1 to yield CYP gene-carrying plasmids. These plasmids
2

W. Song, et al.
Phytochemistry175(2020)112375
were transformed into Saccharomyces cerevisiae BY4742 to yield en-
gineered strains for further functional characterization using GHQ as
the substrate.
H]+ = 277.1453, [M + Na]+ = 299.1271), indicating that product 2
was further oxidized by CYP76B101 into product 3 with a carboxyl
group (Fig. 3D).
To isolate sufficient quantities of the oxidized products of
CYP76B100 and CYP76B101 for chemical structure characterization,
the S. cerevisiae BY4742 strains harboring pCf302-AtCPR1-CYP76B100
or pCf302-AtCPR1-CYP76B101 were cultured and incubated with GHQ
for large-scale fermentation. The new products were purified and sub-
mitted for NMR analysis. Consequently, 1H-NMR and 13C-NMR spectra
data of the purified products 1 and 2 (Table 1) were found to perfectly
match with those of GHQ-3″-OH as previously reported (Wang et al.,
2019). 1H-NMR and 13C-NMR spectra combined with the data of the
two-dimensional experiment HMBC demonstrated that purified product
3 was GHQ-3″-COOH (Table 1 and Figs. S2, S3, S4). Full assignment of
the 1H and 13C chemical shifts were achieved by evaluating the two-
dimensional NMR spectra of product 3. Especially, the carbonyl carbon
at δ 171.5 was assigned C-3″ on the basis of its 3J-HMBC correlation
with the olefinic proton H-2′ (δ 5.92) and the methylene protons H2-4′
(δ 2.32). Thus, product 3 was identified as GHQ-3″-COOH, which was
the three-step oxidation product of GHQ at the C-3″ position.
2.2. Functional screening of the CYP candidates in recombinant yeast
To screen the encoded enzymes from the acquired candidate CYP
genes to oxidize GHQ, we exploited the simplicity of heterologous ex-
pression of these cDNAs in engineered strains of S. cerevisiae harboring
pCf302-AtCPR1-CYP. A control yeast strain expressed pCf302-AtCPR1
without carrying exogenous CYP. GHQ is not available commercially,
and was synthesized according to the method reported in the literature
(Baeza et al., 2012). The engineered strains were incubated with GHQ
for 48 h, and then the ethyl acetate extracts of the yeast culture medium
were analyzed by HPLC. As a result, two engineered strains harboring
CYPs produced new products as compared with the strain harboring an
empty plasmid. The two CYPs were respectively designated as
CYP76B100 and CYP76B101 by the Cytochrome P450 Nomenclature
Committee, and the gene sequence information of CYP76B100 and
CYP76B101 from L. erythrorhizon were deposited into GenBank under
accession numbers MN056183 and MN056184, respectively. A new
peak (product 1) with a retention time (RT) of 21.1 min was observed
on the chromatograph by analyzing metabolites of yeast harboring
CYP76B100 (Fig. 2A). By co-expressing CYP76B101 and AtCPR1, two
new products (2 and 3) with the corresponding retention time (RT) of
21.1 min and 21.6 min, were detected in the chromatogram (Fig. 3A).
Meanwhile, the starting material GHQ completely disappeared, which
meant total conversion into new products by CYP76B100 and
CYP76B101 (Figs. 2A and 3A).
2.4. In vitro enzyme activity assays
To verify the GHQ hydroxylation activity of CYP76B100 and
CYP76B101, microsomal proteins were isolated from the engineered
strains. With GHQ as the substrate, enzymatic assays of CYP76B100 or
CYP76B101-containing microsomes were conducted. Compared with
negative control assays, the enzymatically converted products showed
an identical HPLC retention time to those of the GHQ-3″-OH and GHQ-
3″-COOH produced in feeding experiments (Fig. 4). These results con-
vincingly demonstrated that CYP76B100 catalyzed 3″-hydroxylation of
GHQ to form GHQ-3″-OH. The other enzyme CYP76B101 catalyzed
three-step oxidation of GHQ to form GHQ-3″-COOH.
2.3. Products identification
To determine the molecular weight, the extracted metabolites were
further subjected to LC-MS analyses. As shown in Fig. 2, product 1
exhibited the molecular ions of 263.1639 ([M + H]+) and 285.1460
([M + Na]+), which corresponded to a hydroxylated GHQ derivative.
The mass spectrum of product 2 that was formed by the strain carrying
CYP76B101 was identical to that of product 1 (Fig. 3C). LC-MS analysis
demonstrated that product 3 had the molecular ions of ([M +
2.5. Phylogenetic analysis of CYP76B100 and CYP76B101
To illustrate the evolutionary relationship among the CYP76B100,
CYP76B101 and CYP76 family, phylogenetic analysis was constructed
with the full-length amino acid sequences (Fig. S5 and Table S3). As
Fig. 2. LC-MS analysis of the biosynthetic products from GHQ catalyzed by CYP76B100 in feeding experiments. (A) The HPLC chromatogram of authentic standard
GHQ (black line), the extracts from a control yeast strain expressing AtCPR1 alone (green line), and the extracts from the engineered yeast strain co-expressing
CYP76B100 and AtCPR1 (red line). The labelled peaks correspond to GHQ-3″-OH (1), and authentic substrate GHQ. (B) Oxidation at the C-3″ position of substrate
GHQ to GHQ-3″-OH (1) catalyzed by CYP76B100. (C) The mass spectrum for the product 1 eluting at 21.1 min.
3

W. Song, et al.
Phytochemistry175(2020)112375
Fig. 3. LC-MS analysis of the biosynthetic products from GHQ catalyzed by CYP76B101 in feeding experiments. (A) The HPLC chromatogram of authentic standard
GHQ (black line), the extracts from a control yeast strain expressing AtCPR1 alone (green line), and the extracts from the engineered yeast strain co-expressing
CYP76B101 and AtCPR1 (blue line). The labelled peaks correspond to GHQ-3″-OH (2), GHQ-3″-COOH (3), and authentic substrate GHQ. (B) Oxidation at the C-3″
position of substrate GHQ to GHQ-3″-OH (2), to possible intermediate GHQ-3″-CHO (undetectable) and finally to GHQ-3″-COOH (3), all catalyzed by CYP76B101.
(C) The mass spectra for the product 2 eluting at 21.1 min. (D) The mass spectra for the product 3 eluting at 21.6 min.
Table 1
1H and13
C
NMR spectroscopic data for products
2
and 3 (CD3OD, 400,
100 MHz, δ ppm).
position products 2
Products 3
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
147.5, C
128.2, C
115.9, CH
149.8, C
148.0, C
126.6, C
116.4, CH
149.7, C
6.54, d (3.0)
6.61, d (3.0)
5
6
112.8, CH
115.2, CH
27.8, CH2
126.1, CH
138.4, C
26.5, CH2
34.9, CH2
123.9, CH
130.9, C
6.46, dd (8.5, 3.0) 113.4, CH
6.53, dd (8.5, 3.0)
6.64, d (8.5)
3.67, d (8.0)
6.58, d (8.5)
3.33, d (7.6)
5.42, t (7.6)
115.5, CH
30.6, CH2
137.8, CH
132.7, C
1′
2′
3′
4′
5′
6′
7′
8′
9′
3″
5.92, t (7.8)
2.14, br s
2.15, br s
5.11, br s
34.6, CH2
27.2, CH2
123.2, CH
131.6, C
2.32, t (7.5)
2.17, t (7.5)
5.12, t (7.2)
24.4, CH3
16.3, CH3
58.8, CH2OH 4.20, s
1.64, s
1.58, s
24.4, CH3
16.3, CH3
1.66, s
1.60, s
Fig. 4. HPLC analysis of CYP76B100 and CYP76B101 microsomal enzyme as-
says in vitro incubated with GHQ. Microsomal preparation containing AtCPR1
alone was used as a negative control (black line). Microsomal proteins were
respectively prepared from the engineered yeast strain co-expressing
CYP76B100 and AtCPR1 (green line), and the engineered yeast strain co-ex-
pressing CYP76B101 and AtCPR1 (blue line).
171.5, COOH
expected, the neighbor-joining tree confirmed that both CYP76B100
and CYP76B101 were members of the CYP76B subfamily, which in-
cludes CYP76B74 from A. euchroma and the enzyme geraniol 10-hy-
droxylase (G10H) hydroxylating geraniol to form 10-hydroxygeraniol.
The three CYPs from Boraginaceae, CYP76B100, CYP76B101 and
4

W. Song, et al.
Phytochemistry175(2020)112375
Fig. 5. Multiple amino acid sequence alignment of geranylhydroquinone 3″-oxygenases. Alignments of cytochrome P450s CYP76B100 and CYP76B101 from L.
erythrorhizon and CYP76B74 from A. euchroma are illustrated. Red boxes indicate identical amino acid residues; Red fonts with no box indicate identical residues for
at least two enzyme sequences. The highly conserved I-helix motif AGT(V)DTT, the K-helix motif KEA(T)L(V)R and the conserved haem binding domain PFGAGRRS
(I)CPG are highlighted in blue. CYP76B74 (GenBank accession number: AZU97066) was functionally characterized as geranylhydroquinone 3″-hydroxylase from A.
euchroma.
CYP76B74, exhibited typical cytochrome P450 characteristics, which
included the conserved characteristic oxygen binding and activation I-
helix motif (AGxDT), the highly conserved haem binding domain
(PFGxGRRxCPG), the absolutely conserved cysteine at position 336 and
the K-helix (KExxR) (Fig. 5). Sequence alignment of the CYP76B100
and CYP76B101 amino acids with the reported plant CYP sequences of
the CYP76B subfamily showed significant identity scores. CYP76B100
shares 62.1% sequence identity with G10H from Ophiorrhiza pumila,
5

W. Song, et al.
Phytochemistry175(2020)112375
64.7% identity with enzyme CYP76B74 from A. euchroma and 62.9%
sequence identity with CYP76B101. In addition, CYP76B101 shares
93.6% identity with CYP76B74 and 60.5% sequence identity with
G10H from C. roseus.
amyrin into erythrodiol and oleanolic acid, and not oleanolic aldehyde
in S. cerevisiae (Andre et al., 2016). In addition, a variant redox partner
may modulate the catalytic activity of P450 enzymes by protein-protein
interactions (Zhang et al., 2014).
It was previously speculated that the naphthalene ring could be
formed by oxidation of GHQ-3″-OH to aldehyde, leading to C–C bond
formation between the oxidized prenyl side-chain and the aromatic
nucleus by an electrophilic reaction (Yamamoto et al., 2000).
CYP76B101 catalyzed successive oxidations of GHQ at the C-3″ position
to synthesize GHQ-3″-COOH. Numerous studies have described the
coupling of a carboxylic acid with an aromatic component by the
Friedel-Crafts acylation reaction, which permits the preparation of aryl
ketones using different chemical catalysts (Wilkinson, 2011; Babu et al.,
2007; Kangani and Day, 2008; Li and Fuchs, 2003). Thus, naphthalene
ring closure may be formed via GHQ-3″-COOH catalyzed by an un-
known enzyme (Fig. 1). Recently, Friedel-Crafts alkylation and acyla-
tion have also been reported by employing enzymes as catalysts, such as
prenyltransferase (Zhou et al., 2015) and acyltransferase (Schmidt
et al., 2017; Pavkov-Keller et al., 2019). Even though it is still possible
that the naphthoquinone skeleton can be formed directly from GHQ-3″-
CHO, our work might provide another possible mechanism for the ring
formation, which needs to be further confirmed by using multiple ap-
proaches including gene knockouts and characterization of downstream
enzymes catalyzing the naphthoquinone formation in the future.
Plentiful quantities of dihydroechinofuran derived from GHQ were
secreted and no naphthoquinone derivatives were detected in cell sus-
pension cultures of L. erythrorhizon in a growth medium (Fukui et al.,
1992). Since GHQ-3″-OH is the intermediate for the formation of both
shikonin and dihydroechinofuran (Yamamoto et al., 2000), it is likely
that the cyclization reaction that forms naphthoquinone skeleton or
dihydroechinofuran may be regulated by fine-tuning the expression of
CYP76B100 and CYP76B101 in cell suspension culture (Fig. 1). Further
studies aimed at disrupting the function of CYP76B100 or CYP76B101
in plant tissues, for example, RNA interference (RNAi) may provide
further insights into the physiological roles played by CYP76B100 and
CYP76B101.
3. Discussion
Shikonins are the red naphthoquinone pigments produced in the
root tissues of L. erythrorhizon, and display diverse medicinal properties.
The long-standing and extensive uses, together with special formation
of the naphthoquinone skeleton have aroused keen interest in shikonin
biosynthesis. Despite the commercial importance of shikonin, the bio-
synthesis of the compound remains poorly understood. To illuminate
shikonin biosynthesis, many whole-plants, suspension culture cell and
hairy root culture-based transcriptome studies that rely on NGS have
been previously reported (Takanashi et al., 2019; Wu et al., 2017; Rai
et al., 2018). However, these studies were usually limited by in-
complete transcriptomic analysis and inaccurate sequence information.
To obtain more accurate and complete transcriptome data available for
L. erythrorhizon, short-read NGS and long-read SMRT sequencing were
combined. This strategy established the foundation for the discovery of
genes involved in shikonin biosynthesis.
In this work, we focused on transcriptionally up-regulated candidate
CYP genes in the red roots from differentially expressed unigenes, since
shikonin accumulated in the red roots (Wu et al., 2017). Both hetero-
logous expression of CYPs in yeasts and in vitro enzymatic assays re-
vealed that CYP76B100 and CYP76B101 catalyzed the oxidation of
GHQ to the hydroxyl or carboxyl group at the C-3″ position. Milligram
quantities of the new products were isolated and chemical structures of
their oxidized products were identified by NMR spectroscopy.
It has been demonstrated that CYP76B74 catalyzes the C-3″ hy-
droxylation of GHQ and is required in shikonin biosynthesis of A. eu-
chroma (Wang et al., 2019). Phylogenetic analysis was carried out to
illustrate the evolutionary relationship of the identified CYPs from
CYP76 family. The neighbor-joining tree confirmed that CYP76B100
and CYP76B101 showed a proximal evolutionary relationship with
CYP76B74, and G10H hydroxylating geraniol to form 10-hydro-
xygeraniol. The overall high sequence identity of CYP76B100,
CYP76B101 and CYP76B74 indicates that these three enzymes may
arise from a common ancestor. In the phylogenetic tree, the CYP76B
subfamily is closest to the CYP76F subfamily, whose members involve
in santalol and bergamotol biosynthesis in Santalum album (Diaz-
Chavez et al., 2013). More distantly related CYPs of CYP76C subfamily
from Arabidopsis are shown to be involved in the oxidation of mono-
terpenes (Boachon et al., 2015; Höfer et al., 2014; Wang et al., 2019).
The members of CYP76 family play an important role in the synthesis of
terpenoids (Wang et al., 2019), and especially in diterpene metabolism,
more than half of all the identified angiosperm CYPs belong to the
CYP76 family. It was found that the identified subfamilies of CYP76s
underwent expansion in specific plant families that oxidized a group of
diterpenes (Bathe and Tissier, 2019), such as tanshinone and forskolin
(Fig. S5). Similarly, the CYP76Bs are expanded within Boraginaceae to
form a Boraginaceae-specific clade, which are likely relevant to the
biosynthesis of shikonin (Wang et al., 2019).
In summary, we report identification of two CYPs catalyzing oxi-
dation at C-3″ position of GHQ, which might provide more insights for
shikonin biosynthesis in L. erythrorhizon.
4. Methods
4.1. Chemicals and reagents
Chromatography-grade formic acid, isopropanol and methanol were
obtained from Sigma-Aldrich, USA. Lithium acetate, PEG 3350, ssDNA
and ampicillin were obtained from Solarbio, China. Geraniol, BF3⋅Et2O
and 1, 4-hydroquinone were purchased from Sigma-Aldrich. Restriction
enzymes, DNA polymerase and T4 ligase were purchased from Thermo
Fisher Scientific, USA. The authentic standard of geranylhydroquinone
(GHQ) was chemically synthesized in our lab as reported previously
(Baeza et al., 2012). All other chemicals were of commercial reagent
grade.
CYPs have been previously reported to catalyze multiple steps oxi-
dation to form the carboxyl group, such as enzymes that are involved in
the biosynthesis of artemisinic acid (CYP71AV1) (Teoh et al., 2006; Ro
et al., 2006), abietic acid (CYP720B1) (Ro et al., 2005), and carnosic
acid (C20Ox) (Scheler et al., 2016). In our current work, heterologous
expression of CYP76B101 in yeast revealed the accumulation of GHQ-
3″-OH and GHQ-3″-COOH, wherein the intermediate aldehyde GHQ-3″-
CHO was not detected. The results suggest that CYP76B101 is in-
effective at catalyzing the formation of the aldehyde GHQ-3″-CHO from
GHQ-3″-OH. The product profile of CYP76B101 may be affected by
heterologous expression systems, and it has been reported that
CYP716A80 and CYP716A81 from Barbarea vulgaris transformed β-
4.2. Plants materials and growth conditions
The mature seeds of Lithospermum erythrorhizon Sieb. et Zucc.
(Boraginaceae) were collected in the field of Chifeng city, Inner
Mongolia Autonomous Region, China (located at 42°15′23.50″ N;
118°52′58.14″ E) in October 2016. The seeds were germinated on moist
filter gauze as described in the reported methods (Fang et al., 2016).
The seedlings with two cotyledons were subsequently transferred into
1.5 L circular plastic pots (15 cm in diameter) and filled with peat
growth media. Plants were grown in a greenhouse at 23
16 h-light/8 h-dark photoperiod (Wu et al., 2017) and they were wa-
tered as needed. After 60 d, three plants at a height of 10 cm above
1 °C with a
6

W. Song, et al.
Phytochemistry175(2020)112375
ground were harvested and respectively divided into three green
leaves/stems samples and three red roots samples (each in triplicate).
All tissues were flash frozen by liquid nitrogen and stored at −80 °C till
used.
CYPs were cloned into pCf302-AtCPR1 to yield a series of CYPs carrying
plasmids using primer pairs as described in Supplementary Table S2. All
of the constructed CYPs expression plasmids were transformed into S.
cerevisiae BY4742 and the transformations were carried out with the
standard lithium acetate method (Gietz and Schiestl, 2007). The vector
pCf302-AtCPR1 was also transformed into S. cerevisiae BY4742 and
served as a control. The recombinant yeast cells were selected on a
uracil-minus plate (SD-Ura) at 30 °C for 3 d and verified by colony PCR.
4.3. Transcriptome sequencing and bioinformatics analysis
Six total RNA samples were extracted respectively from green
leaves/stems and red roots of L. erythrorhizon and used for subsequent
cDNA library construction for transcriptome sequencing in Genewiz,
China. Firstly, six mRNA samples from green leaves/stems and red roots
tissues were subjected to paired-end sequencing on the Illumina HiSeq
2000 platform; Meanwhile, the six mixed poly(A) RNA samples were
normalized and subjected to an SMRT sequencing using the Pacbio
platform to generate the full-length transcriptome; Eventually, all the
SMRT subreads were corrected using the NGS reads to resolve the high
error rates of the subreads. The FPKM (fragments per kilobase of
transcript per million mapped reads) value was used to quantify the
expression level of each unigene and the DEGSeq program (Wang et al.,
2009) was used to analyze the differentially expressed unigenes be-
tween green leaves/stems and red roots. With the set stringent
4.7. Feeding experiments
All the recombinant yeast strains carrying CYPs expression vectors
and the empty vector were cultured in 2 ml SD-Ura medium at 30 °C
and 220 rpm for 20 h, and three colonies were picked for each genotype
to reduce errors. Subsequently, GHQ that was synthesized in our la-
boratory was added to the cultures to a final concentration of 0.1 g l−1
and the cultures were further shaken at 30 °C and 220 rpm for 48 h. The
yeast cells were harvested by centrifugation at 12,000 rpm for 10 min,
and extracted twice with 1.5 ml methanol. Culture broth was extracted
twice with an equivalent volume of ethyl acetate. The methanol and
ethyl acetate extracts were mixed together, evaporated, and finally
redissolved in methanol for HPLC analysis.
To isolate sufficient quantities of the oxidation products for che-
mical structure characterization, the S. cerevisiae BY4742 strains that
respectively harbored pCf302-AtCPR1-CYP76B100 and pCf302-
AtCPR1-CYP76B101 were cultivated in 1.5 L SD-Ura medium for pur-
ification of GHQ-3″-OH and GHQ-3″-COOH. The positive colonies were
inoculated into a 250 ml flask containing 40 ml culture medium, and
grown at 30 °C and 220 rpm for 24 h. The resulting seed cultures were
transferred into 1.5 L fresh SD-Ura medium, and then cultivated them at
30 °C and 220 rpm for 24 h. Subsequently, the substrate GHQ (20 mg)
was added to the cultures, which were further shaken at 30 °C and
220 rpm for 48 h. A total of 1.5 L of the culture broth was harvested and
extracted twice with ethyl acetate as described above. The yeast cells
were collected by centrifugation at 4000 rpm for 10 min and extracted
twice with methanol. The methanol and ethyl acetate extracts were
mixed together, evaporated, redissolved in methanol and further pur-
ified by semi-preparative HPLC.
threshold (| Log2 (ratio)
| ≥ 1 and FDR < 0.001 and Max
(FPKM) ≥ 10), there are 52 CYP genes transcriptionally up-regulated in
red roots compared to green stem/leaf of L. erythrorhizon. Considering
the integrity of open reading frame, a total of 40 up-regulated CYPs
were selected as candidates for further functional screening
(Supplementary Table S1).
4.4. Strains, plasmid and growth conditions
S. cerevisiae BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), which is
a derivative of S288C, was used as the parent strain for all engineered
strains. Yeast strains were cultivated at 30 °C and 220 rpm in YPD
medium containing 10 g l−1 of yeast extract, 20 g l−1 of beef peptone,
and 20 g l−1of glucose or in SD-Ura medium (uracil-minus) containing
6.7 g l−1 of yeast nitrogen base without amino acids, 0.9 g l−1 of SD-
Ura, 20 g l−1 of glucose (Liu et al., 2018). Escherichia coli Trans-T1
(TransGen Biotech, China) was used for bacterial transformation and
recombinant vectors construction. The E. coli strains with recombinant
plasmids were grown at 37 °C and 200 rpm in Luria-Bertani medium
with 100 mg l−1 ampicillin.
4.8. Synthesis of the substrate geranylhydroquinone (GHQ)
4.5. cDNA cloning and P450 gene mining
A solution of 1, 4-hydroquinone (0.15 g, 1.25 mmol) and geraniol
(0.2 g, 1.25 mmol) was placed in a round bottom flask and dissolved in
freshly distilled 1, 4-dioxane (5 ml). Under nitrogen gas and with vig-
orous stirring, BF3⋅Et2O (0.057 g, 0.4 mmol) was added dropwise at
room temperature to the solution and the mixture was stirred at room
temperature under a nitrogen atmosphere (Baeza et al., 2012). After
24 h, the mixture was poured onto crushed ice (about 10 g) and the
organic layer extracted with ethyl acetate (3 × 10 ml). The organic
solutions that were obtained after the extractions were mixed and dried
over anhydrous sodium sulphate and filtered. The solvent was evapo-
rated under reduced pressure and the crude residue was redissolved in
methanol (2 ml). The resulting crude product was purified by semi-
preparative HPLC separation to obtain the substrate GHQ.
The total RNA from red roots was used as a template for first-strand
cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser
(TransGen Biotech) and the oligo(dT)15 primer following the manu-
facturer's recommended protocols. The deduced amino acid sequences
of CYP genes were analyzed by the NCBI's BLAST tool and the full-
length transcripts were cloned from the cDNA library of red roots. The
amplified DNA fragments were cloned into pEASY-Blunt (TransGen
Biotech) and the clones harboring the correct insert were confirmed by
sequencing. The expression vectors harboring the candidate CYP genes
were constructed for expression in yeasts.
4.6. Construction of plasmids and strains
Semi-preparative HPLC separation was performed using a Shimadzu
LC-6 AD with a SPD-20A detector and a Shim-pack PREP-ODS (H)
column (10 × 250 mm, 5 μm). The mobile phase contained 0.1%
formic acid in water (A) and 100% HPLC grade methanol (B). The
elution condition was adopted isocratically at 70% of B. Products were
detected and quantified by UV absorption at 294 nm. The solvent flow
rate was 4.0 ml min−1. The target fraction was collected manually,
dried, and resuspended in CD3OD for structural analysis. 1H NMR
spectra was obtained on a 400 MHz Bruker Avance III spectrometer.
The yeast expression vector pCf302 with the constitutive promoters
TEF1, PPGK1, PTDH3 was constructed in our lab (Jiang et al., 2018). For
P
the analysis of CYPs, the vector harboring the A. thaliana cytochrome
P450 reductase gene (AtCPR1) was constructed. The AtCPR1 gene with
the restriction sites XhoI and BamHI was synthesized by Generay Bio-
tech Co., Ltd (Shanghai, China) with codon optimization for improving
expression in S. cerevisiae. The AtCPR1 gene was cloned into the
plasmid pCf302 under the promoter PTDH3, resulting in CPR expression
vector pCf302-AtCPR1. The coding-region fragments for the candidate
7

W. Song, et al.
Phytochemistry175(2020)112375
4.9. HPLC, LC-MS and NMR analysis
1 mM EDTA, 600 mM Sorbitol, 1 mM PMSF) and disrupted on ice by
using a high-pressure homogenizer (JNBIO). The lysed cells were cen-
trifuged (11,000 g, 20 min) to remove the cell debris, mitochondria and
nuclei. The supernatant was transferred to ultracentrifugation tubes
and centrifuged at 150,000 g for 1.5 h. The supernatant was discarded
and the harvested microsomes were resuspended in 1 ml TEG buffer
(50 mM Tris-HCl pH 7.5, 1 mM EDTA, and 20% glycerol), aliquoted
(200 μl) and stored at −80 °C. All steps for microsomal preparation
were carried out at 4 °C.
The enzymatic assays were conducted in 200 μl reaction mixtures
containing 50 mM sodium phosphate buffer (pH 7.5), 1 mM NADPH,
50 μl microsomal proteins, and 100 μM substrate GHQ. After incubation
for 3 h at 30 °C, the reactions were terminated by adding 200 μl me-
thanol. The denatured proteins were removed by centrifugation at
12,000 rpm for 10 min and the supernatant was submitted to HPLC
analysis.
The samples for HPLC analysis were cleaned off impurities by cen-
trifugation (12,000 rpm) and by filtration using PTFE 0.2 μm syringe
filters (Axiva, Sigma Chemicals). In order to identify the function of the
two new CYPs, 30 μl methanol extracts from feeding experiments were
analyzed by a Shimadzu HPLC system with a UV detector set at 294 nm.
The analytical column used was an Agela Innoval C18 column
(4.6 × 250 mm, 5 μm). The mobile phase contained 0.1% formic acid
in water (A) and 100% HPLC grade methanol (B). The gradient con-
ditions were as follows: 0–3 min, 45% B; 3–22 min, a linear gradient of
45–100% B; 22–32 min, 100% B; 32–33 min, 100-45% B; 33–43 min,
45% B. The mobile phase flow was 1 ml min−1
.
To identify the oxidation products of CYP76B100 and CYP76B101,
the LC-MS analysis was carried out on an Agilent 1200 HPLC system
coupled with an Agilent Infinity UV detector and a Bruker-MicrOTOF-II
mass spectrometer that was equipped with an electrospray ionization
device. Data acquisition and processing were done with MicrOTOF
control version 3.0/Data Analysis Version 4.0 software. For HPLC
analysis, the Agela Innoval C18 column (4.6 × 250 mm, 5 μm) was
used. The mobile phase contained 0.1% formic acid in water (A) and
100% HPLC grade methanol (B). The gradient programs were as fol-
lows: 0–3 min, 45% B; 3–22 min, a linear gradient of 45–100% B;
22–32 min, 100% B; 32–33 min, 100-45% B; 33–43 min, 45% B. The
mobile phase flow was 1 ml min−1, and the column temperature was
set at 40 °C. The products were detected at 294 nm. Optimized MS
operating conditions were as follows: all spectra were obtained in po-
sitive mode over an m/z range of 50–1000 under a dry gas flow of
6.0 l min−1, a dry temperature of 180 °C, a nebulizer pressure of 1 bar
and a probe voltage of −4.5 kV.
4.11. Phylogenetic analysis
The phylogenetic tree was constructed using Mega 6.0 software
package (Tamura et al., 2013) and the neighbor-joining program based
on the Poisson model and a bootstrap of 1000 replicates. All the amino
acid sequences used for phylogenetic tree construction were listed in
Supplementary Table S3 and aligned by ClustalX. The scale bar in-
dicates 0.2 amino acid substitution per site. The numbers at the nodes
of each branch indicate the percentage of bootstrap values.
Funding information
This work was supported by the National Key Research and
Development Program (2019YFA0905700), the National Natural
Science Foundation of China (31400026, 31970065, U1902214), the
Sciences and Technology Planning Projects of Tianjin city
(18YFZCSY01350), and the Yunnan Key Research and Development
Program (2019ZF011-2).
To purify sufficient oxidation products from CYP76B100 and
CYP76B101, substances GHQ-3″-OH and GHQ-3″-COOH were sepa-
rated using semi-preparative HPLC operated in a Shimadzu LC-6 AD
with SPD-20A detector and
a Shim-pack PREP-ODS (H) column
(10 × 250 mm, 5 μm). The mobile phase contained 0.1% formic acid in
water (A) and 100% HPLC grade methanol (B). The column was equi-
librated with 45% B, and then 200 μl of the enriched sample were in-
jected, reaching 100% B after 18 min and running it for 6 min.
Subsequently, it was returned to 45% B to equilibrate the column for
6 min before the next injection. Products were detected and quantified
Notes
The authors declare the following competing financial interest(s):
Part of the work has been included in a patent application by the
Tianjin Institute of Industrial Biotechnology.
by UV absorption at 294 nm. The solvent flow rate was 4.0 ml min−1
.
The target fraction that was eluted between 15.6 and 16.3 min (GHQ-
3″-OH) or between 16.4 and 17.0 min (GHQ-3″-COOH) was repeatedly
collected, dried and resuspended in CD3OD for structural analysis. The
chemical structure of the newly acquired substance GHQ-3″-OH was
verified by 1H NMR and 13C NMR spectra. The chemical structure of
substance GHQ-3″-COOH was verified by 1H NMR, 13C NMR and het-
eronuclear multiple-bond correlation spectroscopy (HMBC) spectra.
The NMR spectra were collected on a 400 MHz Bruker Avance III
spectrometer.
Acknowledgements
We thank Dr. David R. Nelson (University of Tennessee) for naming
of the new CYPs.
Appendix B. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.phytochem.2020.112375.
4.10. Microsome isolation and in vitro enzyme assays
Declaration of interests
The strain S. cerevisiae BY4742 harboring pCf302-AtCPR1-
CYP76B100 or pCf302-AtCPR1-CYP76B101 was cultivated respectively
for microsome isolation. Microsomes extracted from the yeast carrying
the vector pCf302-AtCPR1 were assayed as a negative control. The
strains were inoculated into culture tubes containing 5 ml of SD-Ura
medium and grown at 30 °C and 220 rpm for 16 h. The seed cultures
were inoculated into 200 ml SD-Ura medium and further cultivated at
30 °C and 220 rpm for 36 h. The microsomal isolation was carried out
by differential centrifugation as described previously (Krithika et al.,
2015; Scheler et al., 2016) with the following modifications. The cells
were centrifuged (6000 g, 10 min) and washed twice with TEK buffer
(50 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM KCl) and centrifuged.
Collected cells were resuspended in TES buffer (50 mM Tris-HCl pH 7.5,
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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9