Supplementary materials for

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its

application in production of geraniol in Escherichia coli

Fei Chen 1,3 Wei Li 1,3 Liangzhen Jiang 1 Xiang Pu 1,3 Yun Yang 1,3 Guolin Zhang 1 Yinggang Luo 1,2

1 Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China

2 State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, People’s Republic of China

3 University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

*Corresponding Author: Tel: +86-28-82890813; Fax: +86-28-82890288; E-mail: yinggluo@cib.ac.cn

Contents

Bioinformatics properties and 3-D structure prediction of CaGES…………………………………………..S2

Quantitative real-time PCR analysis of CaGES………………………………………………………………S3

References…………………………………………………………………………………………………….S3

Fig. S1……………………………………………………………………………………………….………..S5

Fig. S2……………………………………………………………………………………………….………..S7

Fig. S3…………………………………………………………………………………………………….…..S8

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Bioinformatics properties and 3-D structure prediction of CaGES

The theoretical isoelectric point of the deduced CaGES was predicted to be 5.4 using the ExPASy online tools.

The molecular weight of CaGES is 67.73 kDa. The conserved DDXXD and NSE/DTE binding domains of the

diphosphate substrate are present in CaGES. The N-terminal transit peptide containing RRX8W motif is

present in many plant monoterpene synthases for its plastidic localization [1]. The directional transit peptide

will be cleaved after the protein is inserted into the organelle [1]. Although RRX 8W motif is absent in CaGES,

the CaGES was predicted to be a plastid target monoterpene synthases by TargetP software

(http://www.cbs.dtu.dk/services/TargetP/), owing to the hydrophobic characteristic of its amino acid residues.

Similarity search showed that CaGES shares 32.0 ~ 56.4 % amino acid residue identities with characterized

plant GESs. To gain insights into the relationship among GESs and terpene synthases from different plants,

the characterized plant GESs (highlighted in bold, Fig. S1a) and terpene synthases with same catalytic

mechanism were retrieved from NCBI database to align with CaGES. A phylogenetic tree was generated by

neighbor-joining method using MEGA 6.0 software (Fig. S1a). CaGES is phylogenetically close to CrGES,

which suggested CaGES’s involvement in the biosynthetic pathway of camptothecin, a member of MIAs.

To predict the 3-dimensional structure of a protein, a structurally closest protein with crystal structure

should be used as a basic model. However, there is no crystal structure of plant GESs can be found in the PDB

database (http://www.rcsb.org/pdb). When the amino acid residue sequence of CaGES was submitted to the

SWISS-MODEL online software (http://www.swissmodel.expasy.org) to perform multiple sequences

alignment, the limonene synthase from Mentha spicata (MsLS, PDB ID: 2ongA) showed 41 % amino acid

residue identities with CaGES and MsLS was suggested to be a model template. The MsLS, a metal ion-

dependent monoterpene cyclase, catalyzes the conversion of GPP into limonene via ionization, rearrangement,

and cyclization reactions [2]. The crystal structure of MsLS with ligands was reported at a 2.7-Å resolution

[2]. Using the template MsLS crystal structure, the 3-dimensional structure of CaGES was constructed and

refined by employing KoBaMIN web service (Fig. S1b). The stereochemical qualities of the energy refined

predicted model of CaGES was validated by the PROCHECK online service. Ramachandran plot analysis of

CaGES showed 83.4% residues in the most favorable region, 14.1% residues in the additional allowed region,

2.1% in the generously allowed region and 0.4% in the disallowed region. The results of the PROCHECK

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analysis presenting a relatively low percentage of amino acid residues in the disallowed regions suggested that

the 3-dimensional structure of CaGES was credible. The metal ion Mn 2+ (highlighted as green ball, Fig. S1b)

was predicted to be embraced by the conserved DDXXD (highlighted in yellow ribbon, Fig. S1b) and

NSE/DTE motifs (highlighted in pink ribbon, Fig. S1b) of CaGES.

Quantitative real-time PCR analysis of CaGES

To quantitate the relative expression level of CaGES in different plant tissues, the C. acuminata seedling

growth, total RNA extraction, cDNA synthesis, real-time PCR amplification and analysis were performed,

according to the reported procedures with minor modification [3]. A pair of specific primers, rtCaGES-F and

rtCaGES-R (Table 1), was designed for real-time PCR amplification of CaGES, according to the instructions

of real-time PCR system. The SYBR green PCR amplification was performed using the PIKOREAL24 real-

time PCR System (Thermo Scientific, USA), based on the manufacturer’s instructions. Briefly, 25 L of

SYBR green PCR amplification solution was prepared from 2 μL cDNA, 0.5 μL SYBR Green I (Zeesan

Biotech Co., Ltd., Xia’men, China), 0.5 μL of the primers (10 μM stock solution), 17.25 μL of dd H2O, 2.5 μL

of Buffer II, 1.5 μL of dNTP, and 0.25 μL of HiFi Taq DNA polymerase (TransGen Biotech). The real-time

PCR amplification was performed under the following cycling conditions: 1 cycle of 95 °C for 5 min; 40

cycles of 95 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s. The transcript levels of CaGES in different tissues

were calculated using the corresponding transcript levels of the reference gene encoding actin in the same

tissue as control. The mRNA amount of CaGES in stems was set as control to compare the expression levels

of CaGES in different tissues.

The real-time PCR amplification results showed that CaGES was expressed in all tissues of C. acuminata

seedlings. The highest transcript level of CaGES was found in roots and it is ~ 1.8-fold higher than that of the

stems. While the leaves showed a slight higher (~ 1.2-fold) transcript level than that of the stems.

References

1. Iijima Y, Gang DR, Fridman E, Lewinsohn E, Pichersky E (2004) Characterization of geraniol synthase

from the peltate glands of sweet basil. Plant Physiol 134: 370-379.

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2. Hyatt DC, Youn B, Zhao Y, Santhamma B, Coates RM, Croteau RB, Kang CH (2007) Structure of limonene

synthase, a simple model for terpenoid cyclase catalysis. Proc Natl Acad Sci USA 104: 5360-5365.

3. Qu X, Pu X, Chen F, Yang Y, Yang L, Zhang G, Luo Y (2015) Molecular cloning, heterologous expression,

and functional characterization of an NADPH-cytochrome P450 reductase gene from Camptotheca

acuminata, a camptothecin-producing plant. PLoS ONE 10, e0135397.

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Fig. S1 Phylogenetic tree (a) of plant GESs and terpene synthases and predicted 3-dimensional structure of

CaGES (b). The amino acid residue sequences were obtained using the National Center for Biotechnology

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Information search engine (http://www.ncbi.nim.bih.gov/). Phylogenetic analysis was performed using the

MEGA 6.0 software package and neighbor-joining program (http://www.megasoftware.net). The scale bar

indicates the phylogenetic distance calculated from the number of differences. The biochemically

characterized plant GESs were highlighted in bold type. The crystal structure of limonene synthase from

Mentha spicata (MsLS, PDB ID: 2ongA) was used as model template to construct the 3-dimensional structure

of CaGES. The conserved DDXXD motif, NSE/DTE motif, and Mn2+ were illustrated in yellow, pink, and

green, respectively

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Fig. S2 SDS-PAGE analyses of the purification of CaGES (I) and tCaGES (II). M, protein ruler; Lane 1 - 6,

the 1st – 6th tube (each 0.5 mL) of eluent from the nickel-nitrolotriacetic acid resin. The target band was

indicated with an arrow

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Fig. S3 Characterization of recombinant tCaGES. The effects of various pH (a), temperature (b), and metal

ions (c) on the catalytic activity of tCaGES. d, the steady-state kinetic constants of recombinant tCaGES

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