1. Plant materials
The seeds of T. cucumerina L., obtained from Prof. Dr. Weena Jiratchariyakul, Faculty of Pharmacy, Mahidol University, Thailand (BKF No. 70279), were used in this experiment. Seeds were cultivated in Sra Kaew provinces, Thailand, from 2017 to 2018, until bearing fruits. The fruits were harvested, and their fruit peels, fruit loofah, immature seeds (white seeds), and mature seeds (black seeds) were separated. Moreover, seeds were germinated to prepare seedlings, including seedling leaves, stems, and roots. The callus was prepared as previously described (Lertphadungkit, et al. 2020).
2. Full-length cDNA Preparation and Cloning candidate TcCYPs
Total RNA from T. cucumerina L. samples was extracted using RNeasy plant mini kit following the manufacture’s protocol treated with RNase free DNase to remove contaminant genomic DNA. The purity and concentration were observed by agarose gel electrophoresis and Nano Dot microspectrophotometer (Hercuvan, Malaysia). First-strand cDNA was synthesized from the total RNA using RevertAid First Strand cDNA Kit and oligo(dt) adapter primers (Takara, Japan) as described previously (Lertphadungkit, et al. 2021)
Using cDNA as the template, the open reading frame (ORF) of each gene was cloned with specific primers (Table S1) using TransStart FastPfu Fly DNA Polymerase (TransGen Biotech, China). The purified PCR product was individually inserted into pEASY Blunt simple cloning vector before Trans1-T1 transformation on LB agar plate with 50 µg/mL of kanamycin. Positive transformants were selected to perform colony PCR and sequencing.
3. Nomenclature and Accession Numbers
All CYPs were named according to their amino acid sequences identity by the cytochrome P450 nomenclature committee ([email protected]). Sequence data in this experiment have been submitted to GenBank databases under the following accession number: TcCYP82D364 (OR611132), TcCYP749A368 (OR611133), TcCYP712D39 (OR611134), TcCYP712D38 (OR611135), TcCYP81B203 (OR611136), TcCYP704A289 (OR611137), TcCYP72D41 (OR611138), and TcCYP78A448 (OR611139).
4. Phylogenetic analysis and multiple sequence alignment
The protein sequences of candidate TcCYPs and characterized CYPs responsible for triterpenoid oxidation were aligned using Mega X software. The phylogenetic tree was constructed based on the Neighbour-Joining method with default parameters. Confidence values for individual branches were measured with bootstrapping 1000 replicates. The tree was graphically generated using EvolView (https://www.evolgenius.info/evolview/). Multiple sequence alignments of CYPs were aligned using ClustalW (https://www.genome.jp/tools-bin/clustalw).
5. Quantitative real-time PCR (qRT-PCR)
The expression of candidate TcCYPs was analyzed by qRT-PCR with KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, USA). The primers used in this experiment were designed by Primer3 (Table S2). The PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 3 s, 55°C for 20 s, 72°C for 10 s, and dissociation at 95 °C for 1 min. TcActin was used as an endogenous control for normalization (Lertphadungkit, et al. 2021). The gene expression was calculated using the 2-DDCt method on three independent biological replicates.
6. Plasmid construction
The DNA fragments of candidates amplified by PCR were ligated into the vector pESC-Leu by pEASY Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China). Briefly, the pESC-Leu vector was linearized at the cloning site by BamHI restriction enzyme. The end of linearized plasmid, which contained a 25-bp homologous region, was identical to the end of the insert. The reaction was performed following the manufacture’s protocol, obtaining pESC-Leu-TcCYP712D39. The reaction mixture was directly transformed to Trans1-T1 competent cell and confirmed by colony PCR.
7. Expression of TcIMS and TcCYP712D39 in yeast system
S. cerevisiae WAT11 was used for functional characterization of TcCYP712D39, which was engineered with ATR gene, a cytochrome reductase from A. thaliana. The WAT11 yeast competent cells were prepared by Frozen-EZ yeast transformation II kit (Zymo Research, USA). The construct of pYES2-TcIMS was first introduced into WAT11 yeast for producing a substrate isomultiflorenol. The yeast with recombinant plasmid pYES2-TcIMS was spread on SD-Ura agar plate to select positive colony, which was then confirmed by colony PCR. The positive yeast was used to prepare yeast competent cells. The construct of pESC-Leu-TcCYP712D39 was introduced into the competent yeast containing pYES2-TcIMS. The transformant was selected on SD-Ura-Leu agar plate and confirmed by colony PCR.
The yeast contained pYES-TcIMS and pESC-Leu-TcCYP712D39 was cultured on 40 mL of SD-Ura-Leu (2% glucose) liquid medium for 3 days at 30°C and 200 rpm. The culture was then pelleted at 3000 rpm for 5 min and re-suspended with SD-Ura-Leu (2% galactose) The yeast culture was then harvested after 2 days.
8. Analysis of bryonolic acid production in engineering yeast
Yeast pellets were extracts with 10 mL ethyl acetate for 3 times by 30 min sonication. The evaporated extracts were derivatized by incubating with 50 µL of TMS-HT solution (70°C, 30 min). The solution was then mixed with 100 µL of MeOH and centrifuged to remove residues. The supernatant was analyzed by GC-MS (Agilent 7890A/5975C, DB-5, 30m, 0.25 mm, 0.25µm). A 1 µL of sample was injected into GC inlet. The injection temperature was 250°C. The GC oven was programmed as follows: 170°C for 2 min, 170°C to 300°C with 20°C/min and held for 11 min. The ion trap heating temperature was 250°C with 60eV electron ionization and mass spectra were recorded in the scan range of 40-700 m/z.
9. Homology modeling of TcCYP712D39 and Molecular Docking
The three-dimensional structure model of TcCYP712D39 was created by a homology modeling method using SWISS-MODEL. Due to no experimental crystal structure of CYP712 being available, CYP4B1 (PDB: 6c94.1.A) was used as the template with 26.21% sequence identity. The substrate recognition sites (SRSs) of TcCYP712D39 were predicted as previously described by Gotoh 1992. The heme group was added to the model before docking with bryonolic acid as a ligand in the web-based tool PCPLD (http://p450.biodesign.ac.cn/). The 3D structure of the ligand was generated and minimized with default parameters using MarvinSketch (Marvin 23.8), 2023 ChemAxon (http://www.chemaxon.com). The generated model was visualized using Pymol.