A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.

A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L. Pornpatsorn Lertphadungkit, Paktaporn Mekloy, Somnuk Bunsupa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3896957/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bryonolic acid, a characteristic triterpenoid in Trichosanthes cucumerina L., is well-known due to its pharmacological activities. The biosynthetic pathway of this compound is similar to other triterpenoids, which are known to be initiated by oxidosqualene cyclase for squalene cyclization and cytochrome P450s for oxidation. However, the final step in this pathway has not been identified. This study presents a discovery of a novel enzyme, CYP712D39, with the remarkable ability to catalyze the crucial C-29 oxidation step in bryonolic acid production. We utilized previous transcriptome analysis with eight promising P450 candidates, exhibiting greater expression levels in callus tissue, which shows a high bryonolic acid production. Functional characterization experiment confirmed its capacity to convert isomultiflorenol into bryonolic acid in the WAT11 yeast system. Furthermore, we also predicted key amino acids, including F113, crucial for binding with C3-OH of isomultiflorenol for C29-oxidation. This discovery fills a critical knowledge gap and offers significant biotechnological potential for pharmaceutical and agricultural applications. Bryonolic acid C-29 oxidation isomultiflorenol Trichosanthes cucumerina L. triterpenoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message TcCYP712D39, discovered through transcriptome-guided selection, crucially catalyzes C-29 oxidation in Trichosanthes cucumerina L., unveiling a key biosynthetic step for bryonolic acid. Introduction The plant kingdom continues to astound with its ability to synthesize an array of biologically active natural products. Triterpenoids emerge as a diverse and fascinating class of compounds. Trichosanthes cucumerina L., renowned for its ethnomedicinal significance in the Cucurbitaceae family, has drawn attention due to its rich triterpenoid content, particularly bryonolic acid. This remarkable compound in pharmacological properties, including anti-allergic activity (Tanaka, et al. 1991 ), anti-inflammatory activity (Gatbonton-Schwager, et al. 2012 ), and cytotoxicity (Takeda, et al. 1994 ), has propelled it into the spotlight as a potential therapeutic agent with low toxicity against cells. Despite its promising potential, the downstream biosynthetic pathway of bryonolic acid remains undiscovered. Similar to other triterpenoids, bryonolic acid is biosynthesized by a series of enzymatic reactions that convert acyclic precursors to pentacyclic triterpenoids. The initial committed step involves the cyclization of 2,3-oxidosqualene to isomultiflorenol, catalyzed by isomultiflorenol synthase. This key oxidosqualene cyclase has been characterized in various cucurbitaceous plants such as Luffa cylindrica , Momordica charantia , and T. cucumerina where they play a crucial role in the production of bryonolic acid (Hayashi, et al. 2001 ; Lertphadungkit, et al. 2021 ; Takase, et al. 2019a ). In the following step, the generated triterpenoid scaffolds undergo for further modification through triterpene-modifying enzymes such as cytochrome P450, acyltransferase, and sugar transferase, leading to the formation of the diverse triterpenoid profiles observed in different plant species. (Thimmappa, et al. 2014 ). Cytochrome P450s (CYPs) are particularly important enzymes in the oxidative tailoring of these compounds through regio- and stereo-selective hydroxylation reactions by introducing hydroxy, oxo, carboxy, or epoxide moieties. Up to date, over 150 CYPs have been functionally characterized in various plants to be involved in the biosynthesis and modification of triterpenoids, with more than 30% belonging to the CYP716 family (Malhotra and Franke 2022 ). The increasing availability of technology and advances, including transcriptome and genome data, have greatly facilitated the exploration, identification, and functional characterization of these enzymes, even in non-model plants lacking reference databases. While advancements have been made in elucidating triterpenoid biosynthetic pathways across plant species, the specific enzymatic machinery governing unique structural modifications such as the C29 oxidation on isomultiflorenol remains poorly understood. In our previous study, we reported several putative enzymes annotated as CYPs and displayed elevated expression levels in T. cucumerina L. callus based on transcriptome analysis (Lertphadungkit, et al. 2021 ). Herein, we undertake a comprehensive exploration of this knowledge gap by dissecting the elusive pathway leading to bryonolic acid in T. cucumerina L. This research represents an important contribution as it provides novel insight into a previously unknown pathway for triterpenoid biosynthesis in a non-model plant species, highlighting the diversity and complexity of triterpenoid biosynthetic pathways. Materials and Methods 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 - DD Ct 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 Bam HI 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 Tc IMS and Tc CYP712D39 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- Tc IMS 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 Tc CYP712D39 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 Tc CYP712D39 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. Results 1. Transcriptome-Guided P450 Candidate Selection The investigation into the biosynthesis of bryonolic acid from T. cucumerina L. has illuminated a crucial knowledge gap of the pathway remains unexplored. We utilized the previous information of transcriptomic data derived from three tissues of T. cucumerina L., including callus, leaf, and fruit peels, to uncover candidates involved in the C-29 oxidation on isomultiflorenol, a critical step in bryonolic acid biosynthesis. This comprehensive approach led to the identification of eight full-length P450 candidates exhibiting higher expression in callus compared to leaf and fruit peel tissues (Lertphadungkit, et al. 2021 ). Subsequent cloning of these potential P450s from T. cucumerina L. cDNA allowed for further functional characterization. Upon successful cloning, the full-length sequences of these candidates revealed open reading frames (ORFs) ranging from 1515 to 1608 bp, encoding proteins consisting of 504 to 535 amino acids. Following the P450 nomenclature guidelines by Dr. David Nelson (Nelson 2006 ), the candidates were designated as Tc CYP82D364 (DN2096-1), Tc CYP749A368 (DN2218-4), Tc CYP712D39 (DN2280-7), Tc CYP712D38 (DN2506-9), Tc CYP81B203 (DN7422-10), Tc CYP704A289 (DN7647-3), Tc CYP72D41 (DN9219-3), and Tc CYP78A448 (DN20692-6). 2. Tissue-Specific Expression and Phylogenetic analysis As callus tissue is intricately linked to bryonolic acid biosynthesis, we evaluated tissue-specific expression levels of the candidate P450s. Among them, TcCYP712D38 , TcCYP712D39 , TcCYP82D364 , and TcCYP704A289 displayed higher expression in callus compared to other tissues (Fig. 1 ). Furthermore, a comparative analysis of the amino acid sequences of these P450 candidates with those of functionally characterized plant P450s involved in triterpenoid oxidation revealed intriguing patterns (Table S3). Notably, through phylogenetic analysis, Tc CYP712D38 and Tc CYP712D39 clustered closely with the CYP712K groups from Tripterygium wiifordii and Maytenus ilicifolia , recognized for their C-29 oxidation activity converting friedelin to maytenonic acid (Fig. 2 ). Despite diverse expression patterns, TcCYP712D38 and TcCYP712D39 displayed pronounced expression in callus and immature seeds, with TcCYP712D39 exhibiting tenfold higher expression compared to TcCYP712D38 . Thus, Tc CYP712D39 emerges as a strong candidate for bryonolic acid biosynthesis. Notably, TcCYP712D39 exhibited significantly higher expression in immature seeds, challenging the typical localization of bryonolic acid in roots (Kongtun, et al. 2009 ; Lertphadungkit, et al. 2020 ). Our earlier investigation of isomultiflorenol synthase, a gene encoding an intermediate for bryonolic acid, revealed high expression across multiple tissues, particularly in immature seeds and roots, prompting the question of why higher bryonolic acid levels are predominantly found in roots. Additionally, our tissue-specific expression study also provides interesting information on the cucurbitacin B biosynthesis. TcCYP81B203 exhibited the highest expression in immature seeds of T. cucumerina L. and grouped into the groups of CYP81Q58, CYP81Q59, and CYP81AQ19 (Shang, et al. 2014 ; Takase, et al. 2019b ) from other cucurbitaceous plants, which are related in the cucurbitacin biosynthesis. This finding supported our earlier hypothesis based on cucurbitadienol expression, which also showed the highest expression in immature seeds, although a large amount of cucurbitacin B was detected in fruit and fruit juice (Lertphadungkit, et al. 2022 ). The collective findings from our study provide compelling evidence that the production of two characteristic triterpenoids, bryonolic acid and cucurbitacin B, is primarily produced in immature seeds of T. cucumerina L. during seed development. In summary, our findings provide evidence that the production of two characteristic triterpenoids, bryonolic acid and cucurbitacin B, are primarily produced in immature seeds of T. cucumerina L. This highlights the need for further exploration into the intricate regulatory mechanisms governing the spatial distribution of bryonolic acid and cucurbitacin B in T. cucumerina L. 3. Functional Domain and Conservation analysis CYPs are typically categorized based on the similarity of amino acid sequences. The structures of CYPs normally contain α helices (A – L helices) and β sheets (β1–5), with a heme prosthetic group located in the catalytic center of the enzymes (Sirim, et al. 2010 ). According to the multiple sequence alignment, we observed that amino acid sequences in the specific domains, such as proline-rich region, oxygen-binding and activation site, EXXR, PXRX, and heme-binding motif, were highly conserved within CYP712 subfamily (Fig. 3 ). The core regions of Tc CYP712D39 were predicted as follows: proline-rich domain (44–48); oxygen activation domain (315–320); ExxR (372–375); PxRx (426–429); heme-binding domain (456–465). Most importantly, the EXXR and heme-binding sites of Tc CYP712D39 exhibited almost complete conservation compared to the CYP712K groups. Typically, these two specific motifs are characteristic in individual CYP families for maintaining the CYP tertiary structure and hold the heme during oxygenation (Syed and Mashele 2014 ). Moreover, it also presented a conserved residue in oxygen activating region as AGT(S/D)TS. This region positioned near the heme group to produce the formation of active Fe-O hydroxylating species, which plays an important role in proton transfer during catalysis (Guengerich 2018 ). Thus, the role of CYP712Ks in C29-modification on friedelin could be utilize to predict the function of Tc CYP712D39 whether this enzyme could demonstrate the ability to catalyze C29-oxidation in bryonolic acid biosynthesis. 4. Functional characterization of TcCYP712D39 To investigate the function of selected P450s in the oxidation of isomultiflorenol, TcCYP712D39 was co-expressed with TcIMS to provide isomultiflorenol as a substrate in the yeast system. The ethyl acetate extracts from the yeast cultures were subsequently analyzed by GC-MS. The yeast strain WAT11, harboring Arabidopsis cytochrome reductase for serving an electron donor, was used in this experiment. In the co-expression system of pYES2- TcIMS and pESC-Leu- TcCYP712D39 , isomultiflorenol and bryonolic acid were detected at retention times of 13.3 and 15.7 min, respectively, while the yeast with empty vector showed no observable peaks (Fig. 4 ). This clear evidence indicates that Tc CYP712D39 possesses the capability to oxidize isomultiflorenol at C29, resulting in the production of bryonolic acid. 5. Key Amino Acid Candidates Governing C29-Oxidation in Pentacyclic Triterpenoids The enzymatic functions of CYPs are intricately linked to their substrate recognition sites (SRSs), which play a pivotal role in substrate specificity and the formation of substrate-binding pocket (Sugimoto and Shiro 2012 ). In this study, we predicted the SRSs of Tc CYP712D39 based on definitions provided by Gotoh ( 1992 ) and Zawaira, et al. ( 2011 ). Briefly, SRS1 corresponds to the region situated between B and C helices, SRS2 is in the C-terminal end of the F helix, SRS3 extends from the F-G loop to N-terminal end of the G helix, SRS4 is the N-terminal segment of the I helix, SRS5 spans the region between the K helix and β-sheet 1, and SRS6 is the turn in β-sheet 4 (Fig. 5 ). To predict the key amino acids involved in C-29 modification process during bryonolic acid biosynthesis, we conducted homology modeling of Tc CYP712D39, and subsequently employed the PCPLD web-based platform for molecular docking. Figure 6 displays the docking model of Tc CYP712D39 and bryonolic acid for C-29 oxidation. Bryonolic acid lies directly above the heme group with the distance of 3.5 angstroms from C-29. We identified 13 amino acid residues located within 3 angstroms from the heme-binding ligand of Tc CYP712D39 and bryonolic acid. Remarkably, 11 of these residues were predicted to be situated within conserved regions commonly observed in CYPs. Among of them, 3 residues were located in SRS1 (R110, F113, and F127), 3 residues were located in SRS2 (M221, G224, and A228), 3 residues were in SRS4 (I314, G315, and T319), and 2 residues were in SRS5 (A379, and V380). Most of them are non-polar amino acids, which can form a central hydrophobic cavity to stabilize the protein’s tertiary structure and allow the hydrophobic interactions with methyl group on bryonolic acid (Guengerich 2018 ; Peterson and Graham 1998 ). These amino acids are noteworthy due to their proximity to the ligand-binding site and their potential role in shaping the substrate-binding environment. This predictive analysis suggests that these key amino acid candidates may play important roles in determining substrate specificity and catalytic activity in the biosynthesis of pentacyclic triterpenoids, further experimental validation will be essential to confirm their functional significance. Significantly, isomultiflorenol and friedelin, both classified in oleanane-type triterpenoids, are known as substrates for C-29 oxidation by CYP712 family. Typically, SRS1 to SRS3 are responsible for conferring substrate specificity, while the latter three SRSs are strategically positioned close to the heme catalytic center, where they directly interact with the substrate during oxygenation (Jóźwik, et al. 2016 ; Sugimoto and Shiro 2012 ). Additionally, the oxygen activating domain is conventionally located within the I-helix, positioned above the heme group in SRS4. We then combined the docking result with CYP712 sequence alignments. Interestingly, F113 (SRS1) is positioned in close proximity to the C3-OH of bryonolic acid with the distance of 3.1 angstroms, while serine (S) is conserved in other CYP712Ks. This observation suggests that F113 forms a cation-π interaction with C3-OH of isomultiflorenol, whereas CYP712Ks form hydrogen bonds with C3-oxo of friedelin, which allow them to be placed above the heme-iron for C-29 modification for bryonolic acid and maytenonic acid, respectively. In summary, our analysis highlights the putative significance of these predicted key amino acids in governing substrate specificity in pentacyclic triterpenoid biosynthesis. Conclusion This study represents a comprehensive exploration of the triterpenoid biosynthesis. Our transcriptome-guided candidate selection led to the discovery of a novel enzyme, Tc CYP712D39, with the remarkable ability to catalyze the C-29 oxidation of bryonolic acid in Trichosanthes cucumerina L. Our investigation also revealed key amino acid residues within Tc CYP712D39 in bryonolic acid production, enhancing our understanding of substrate specificity in pentacyclic triterpenoid production. This research bridges a critical knowledge gap, offering promising prospects for bioengineering valuable natural products. Our finding contributes to the understanding of triterpenoid biosynthesis in non-model plants and hold potential for applications in pharmaceuticals. Further experimental validation of the identified candidates is warranted to confirm their functional significance and potential biotechnological applications. Declarations Conflict of interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Funding This work was supported by grants from Mahidol Medical Scholars Program (MSP) and the New Discovery and Frontier Research Grant (NDFR 50/2564), Mahidol University, Thailand. Author contributions Conceptualization: PL, and SB; Methodology: PL, and PM; Analysis: PL, and PM; Resources: SB; Writing - original draft preparation: PL; Writing - review and editing: SB; Funding acquisition: SB; Supervision: SB. Acknowledgments The authors would like to thank Dr. David Nelson for the P450 nomenclature. We also would like to thank Prof. Dr. Weena Jiratchariyakul for providing bryonolic acid standard. We express our deep appreciation to Prof. Dr. Min Ye and Prof. Dr. Xue Qiao for their generosity in providing essential resources for our experiments. Their contributions, including the plasmids pYES2 and pESC-Leu, along with the yeast strain WAT11, have been instrumental to the success of our work. References Gatbonton-Schwager TN, Letterio JJ, Tochtrop GP (2012) Bryonolic acid transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo. J Nat Prod 75:591–598 Gotoh O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90 Guengerich FP (2018) Mechanisms of cytochrome P450-Catalyzed Oxidations. 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Planta Med 57:527–530 Thimmappa R, Geisler K, Louveau T, O'Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257 Zawaira A, Ching LY, Coulson L, Blackburn J, Wei YC (2011) An expanded, unified substrate recognition site map for mammalian cytochrome P450s: analysis of molecular interactions between 15 mammalian CYP450 isoforms and 868 substrates. Curr Drug Metab 12:684–700 Supplementary Files 20240202supplement.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3896957","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273822255,"identity":"7a090527-fc8c-48d7-ac07-8e8a57717d4e","order_by":0,"name":"Pornpatsorn Lertphadungkit","email":"data:image/png;base64,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","orcid":"","institution":"Mahidol University Faculty of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Pornpatsorn","middleName":"","lastName":"Lertphadungkit","suffix":""},{"id":273822256,"identity":"9f415068-223a-438c-a47a-9e7d2066c30d","order_by":1,"name":"Paktaporn Mekloy","email":"","orcid":"","institution":"Mahidol University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Paktaporn","middleName":"","lastName":"Mekloy","suffix":""},{"id":273822257,"identity":"a0589b84-82bd-4d32-b88b-d6f0cba2dac8","order_by":2,"name":"Somnuk Bunsupa","email":"","orcid":"https://orcid.org/0000-0002-0945-9737","institution":"Mahidol University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Somnuk","middleName":"","lastName":"Bunsupa","suffix":""}],"badges":[],"createdAt":"2024-01-25 11:20:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3896957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3896957/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51500052,"identity":"3febe9cd-ee0f-463d-8aaf-55fb356aecbd","added_by":"auto","created_at":"2024-02-22 16:52:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127883,"visible":true,"origin":"","legend":"\u003cp\u003eRT-qPCR analysis of \u003cem\u003eT. cucumerina\u003c/em\u003e L. cytochrome P450s, including TcCYP82D364, TcCYP749A368, TcCYP712D39, TcCYP712D38, TcCYP81B203, TcCYP704A289, TcCYP72D41, and TcCYP78A448. FP: fruit peels; FL: fruit loofahs; MS: mature seeds; IS: immature seeds; CA: calli; SR: seedling roots; SS: seedling stems; SL: seedling leaves. TcActin was used as a reference. Data represent the mean and the error bars show standard deviation from three independent samples.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/aeb7a8be0b0930e22b8425e2.jpg"},{"id":51500054,"identity":"39942160-8d06-4a63-b92e-979b3651e0b9","added_by":"auto","created_at":"2024-02-22 16:52:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":464414,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis constructed form amino acid residues evaluated using the Bootstrap method replicated 1000 times using Mega X software. Multiple amino acid sequence alignments of candidate CYPs from \u003cem\u003eT. cucumerina \u003c/em\u003eL (black stars) were compared with characterized CYPs responsible for triterpenoid oxidations. GenBank accession numbers are shown on the tree.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/aea14881a1e7519647d9c4b1.jpg"},{"id":51500475,"identity":"6f20b45a-7678-4f9c-a0f7-c63afbbb15f3","added_by":"auto","created_at":"2024-02-22 17:00:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":265970,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequence alignment of \u003cem\u003eTc\u003c/em\u003eCYP712D39 compared with CYP712K1-3 from \u003cem\u003eT. wiifordii\u003c/em\u003e. Multiple sequence alignment was carried out following CLUSTALW. Amino acid residues were predicted to be in the conserved CYP domains and substrate recognition sites (SRS) related to functional analysis. Predicted specific domains were highlighted in blue: proline-rich region; oxygen binding region; ExxR region; PxRx region; heme-binding region. Predicted SRSs were highlighted in pink: SRS1 – 6.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/8ba9f2281f0af56fa0f39902.jpg"},{"id":51500053,"identity":"77a6e762-bd06-46e4-848a-39256ff95886","added_by":"auto","created_at":"2024-02-22 16:52:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112909,"visible":true,"origin":"","legend":"\u003cp\u003e(a) GC-MS chromatogram of ethyl acetate extracts from yeasts co-expressing pYES2-\u003cem\u003eTc\u003c/em\u003eIMS and pESC-Leu-\u003cem\u003eTc\u003c/em\u003eCYP712D39 compared with bryonolic acid standard. The label shows a produced compound (retention time of 15.7 min) proposed as bryonolic acid. (b) The mass spectrum of bryonolic acid produced from pESC-Leu-\u003cem\u003eTc\u003c/em\u003eCYP712D39 compared with authentic bryonolic acid standard.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/ed9330c74aba385c6835d6d5.jpg"},{"id":51500055,"identity":"e01ca253-5013-481d-90ec-4231cf88ff89","added_by":"auto","created_at":"2024-02-22 16:52:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":283204,"visible":true,"origin":"","legend":"\u003cp\u003eHomology modeling of \u003cem\u003eTc\u003c/em\u003eCYP712D39 with predicted substrate recognition sites (SRS) and CYP conserved domains. The SRS regions were highlighted in pink: SRS1 (108 – 128); SRS2 (218 – 233); SRS3 (237 – 254); SRS4 (310 – 323); SRS5 (377 – 387); SRS6 (484 – 495). The CYP specific regions were highlighted in blue: proline-rich (44 – 48); oxygen-binding (315 – 320); ExxR (372 -375); PxRx (426 – 429); heme-binding (456 – 465).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/732d8d114b66679a72862852.jpg"},{"id":51500057,"identity":"da8e1e15-5d65-4a78-ba95-d69e6293b95f","added_by":"auto","created_at":"2024-02-22 16:52:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":242861,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Molecular docking of \u003cem\u003eT. cucumerina\u003c/em\u003e L. CYP712D39 and bryonolic acid (BA). The residues in green show amino acids within the SRS regions (3 angstroms) from ligands. The circle marks the predicted amino acid responsible for C3-OH interaction of BA (F113 of \u003cem\u003eTc\u003c/em\u003eCYP712D39) and C3-oxo interaction of MA (S110 of CYP712K1). (b) Amino acid sequence alignment of CYP712s in the SRS regions. The highlighted amino acids, in pink and blue, indicate the residues in 3 angstroms. (c) The chemical structure of bryonolic acid (BA) and maytenonic acid (MA).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/882233a9cf37862d6b4b3bd3.jpg"},{"id":52615696,"identity":"129fd4e1-e42f-4d24-93f3-cb92503da9e6","added_by":"auto","created_at":"2024-03-13 15:57:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1295794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/d9653503-3e63-4307-98f1-4f93f007ae6c.pdf"},{"id":51500059,"identity":"ae758e52-9f62-466e-854b-7b275bdfa4ac","added_by":"auto","created_at":"2024-02-22 16:52:20","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":42307,"visible":true,"origin":"","legend":"","description":"","filename":"20240202supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-3896957/v1/2b69aec970f7238bcb2105e1.docx"}],"financialInterests":"","formattedTitle":"A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.","fulltext":[{"header":"Key Message","content":"\u003cp\u003eTcCYP712D39, discovered through transcriptome-guided selection, crucially catalyzes C-29 oxidation in \u003cem\u003eTrichosanthes cucumerina\u003c/em\u003e L., unveiling a key biosynthetic step for bryonolic acid.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe plant kingdom continues to astound with its ability to synthesize an array of biologically active natural products. Triterpenoids emerge as a diverse and fascinating class of compounds. \u003cem\u003eTrichosanthes cucumerina\u003c/em\u003e L., renowned for its ethnomedicinal significance in the Cucurbitaceae family, has drawn attention due to its rich triterpenoid content, particularly bryonolic acid. This remarkable compound in pharmacological properties, including anti-allergic activity (Tanaka, et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), anti-inflammatory activity (Gatbonton-Schwager, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and cytotoxicity (Takeda, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), has propelled it into the spotlight as a potential therapeutic agent with low toxicity against cells. Despite its promising potential, the downstream biosynthetic pathway of bryonolic acid remains undiscovered.\u003c/p\u003e \u003cp\u003eSimilar to other triterpenoids, bryonolic acid is biosynthesized by a series of enzymatic reactions that convert acyclic precursors to pentacyclic triterpenoids. The initial committed step involves the cyclization of 2,3-oxidosqualene to isomultiflorenol, catalyzed by isomultiflorenol synthase. This key oxidosqualene cyclase has been characterized in various cucurbitaceous plants such as \u003cem\u003eLuffa cylindrica\u003c/em\u003e, \u003cem\u003eMomordica charantia\u003c/em\u003e, and \u003cem\u003eT. cucumerina\u003c/em\u003e where they play a crucial role in the production of bryonolic acid (Hayashi, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lertphadungkit, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Takase, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). In the following step, the generated triterpenoid scaffolds undergo for further modification through triterpene-modifying enzymes such as cytochrome P450, acyltransferase, and sugar transferase, leading to the formation of the diverse triterpenoid profiles observed in different plant species. (Thimmappa, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Cytochrome P450s (CYPs) are particularly important enzymes in the oxidative tailoring of these compounds through regio- and stereo-selective hydroxylation reactions by introducing hydroxy, oxo, carboxy, or epoxide moieties. Up to date, over 150 CYPs have been functionally characterized in various plants to be involved in the biosynthesis and modification of triterpenoids, with more than 30% belonging to the CYP716 family (Malhotra and Franke \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The increasing availability of technology and advances, including transcriptome and genome data, have greatly facilitated the exploration, identification, and functional characterization of these enzymes, even in non-model plants lacking reference databases.\u003c/p\u003e \u003cp\u003eWhile advancements have been made in elucidating triterpenoid biosynthetic pathways across plant species, the specific enzymatic machinery governing unique structural modifications such as the C29 oxidation on isomultiflorenol remains poorly understood. In our previous study, we reported several putative enzymes annotated as CYPs and displayed elevated expression levels in \u003cem\u003eT. cucumerina\u003c/em\u003e L. callus based on transcriptome analysis (Lertphadungkit, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Herein, we undertake a comprehensive exploration of this knowledge gap by dissecting the elusive pathway leading to bryonolic acid in \u003cem\u003eT. cucumerina\u003c/em\u003e L. This research represents an important contribution as it provides novel insight into a previously unknown pathway for triterpenoid biosynthesis in a non-model plant species, highlighting the diversity and complexity of triterpenoid biosynthetic pathways.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Plant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds of \u003cem\u003eT. cucumerina\u003c/em\u003e 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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Full-length cDNA Preparation and Cloning candidate \u003cem\u003eTcCYPs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from \u003cem\u003eT. cucumerina\u0026nbsp;\u003c/em\u003eL. samples was extracted using RNeasy plant mini kit following the manufacture\u0026rsquo;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)\u003c/p\u003e\n\u003cp\u003eUsing 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 \u0026micro;g/mL of kanamycin. Positive transformants were selected to perform colony PCR and sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Nomenclature and Accession Numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Phylogenetic analysis and multiple sequence alignment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of candidate \u003cem\u003eTcCYPs\u003c/em\u003e 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\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 3 s, 55\u0026deg;C for 20 s, 72\u0026deg;C for 10 s, and dissociation at 95 \u0026deg;C for 1 min. \u003cem\u003eTcActin\u003c/em\u003e was used as an endogenous control for normalization (Lertphadungkit, et al. 2021). The gene expression was calculated using the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eDD\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method on three independent biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Plasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u003cem\u003eBam\u003c/em\u003eHI 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\u0026rsquo;s protocol, obtaining pESC-Leu-\u003cem\u003eTcCYP712D39\u003c/em\u003e. The reaction mixture was directly transformed to Trans1-T1 competent cell and confirmed by colony PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Expression of \u003cem\u003eTc\u003c/em\u003eIMS and \u003cem\u003eTc\u003c/em\u003eCYP712D39 in yeast system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e WAT11 was used for functional characterization of \u003cem\u003eTcCYP712D39\u003c/em\u003e, which was engineered with ATR gene, a cytochrome reductase from \u003cem\u003eA. thaliana\u003c/em\u003e. The WAT11 yeast competent cells were prepared by Frozen-EZ yeast transformation II kit (Zymo Research, USA). The construct of pYES2-\u003cem\u003eTcIMS\u003c/em\u003e was first introduced into WAT11 yeast for producing a substrate isomultiflorenol. The yeast with recombinant plasmid pYES2-\u003cem\u003eTcIMS\u003c/em\u003e 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-\u003cem\u003eTcCYP712D39\u0026nbsp;\u003c/em\u003ewas introduced into the competent yeast containing pYES2-\u003cem\u003eTcIMS\u003c/em\u003e. The transformant was selected on SD-Ura-Leu agar plate and confirmed by colony PCR.\u003c/p\u003e\n\u003cp\u003eThe yeast contained pYES-\u003cem\u003eTc\u003c/em\u003eIMS and pESC-Leu-\u003cem\u003eTcCYP712D39\u003c/em\u003e was cultured on 40 mL of SD-Ura-Leu (2% glucose) liquid medium for 3 days at 30\u0026deg;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Analysis of bryonolic acid production in engineering yeast\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast pellets were extracts with 10 mL ethyl acetate for 3 times by 30 min sonication. \u0026nbsp;The evaporated extracts were derivatized by incubating with 50 \u0026micro;L of TMS-HT solution (70\u0026deg;C, 30 min). The solution was then mixed with 100 \u0026micro;L of MeOH and centrifuged to remove residues. The supernatant was analyzed by GC-MS (Agilent 7890A/5975C, DB-5, 30m, 0.25 mm, 0.25\u0026micro;m). A 1 \u0026micro;L of sample was injected into GC inlet. The injection temperature was 250\u0026deg;C. The GC oven was programmed as follows: 170\u0026deg;C for 2 min, 170\u0026deg;C to 300\u0026deg;C with 20\u0026deg;C/min and held for 11 min. The ion trap heating temperature was 250\u0026deg;C with 60eV electron ionization and mass spectra were recorded in the scan range of 40-700 m/z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Homology modeling of TcCYP712D39 and Molecular Docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe three-dimensional structure model of \u003cem\u003eTc\u003c/em\u003eCYP712D39 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 \u003cem\u003eTc\u003c/em\u003eCYP712D39 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1. Transcriptome-Guided P450 Candidate Selection\u003c/h2\u003e \u003cp\u003eThe investigation into the biosynthesis of bryonolic acid from \u003cem\u003eT. cucumerina\u003c/em\u003e L. has illuminated a crucial knowledge gap of the pathway remains unexplored. We utilized the previous information of transcriptomic data derived from three tissues of \u003cem\u003eT. cucumerina\u003c/em\u003e L., including callus, leaf, and fruit peels, to uncover candidates involved in the C-29 oxidation on isomultiflorenol, a critical step in bryonolic acid biosynthesis. This comprehensive approach led to the identification of eight full-length P450 candidates exhibiting higher expression in callus compared to leaf and fruit peel tissues (Lertphadungkit, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Subsequent cloning of these potential P450s from \u003cem\u003eT. cucumerina\u003c/em\u003e L. cDNA allowed for further functional characterization. Upon successful cloning, the full-length sequences of these candidates revealed open reading frames (ORFs) ranging from 1515 to 1608 bp, encoding proteins consisting of 504 to 535 amino acids. Following the P450 nomenclature guidelines by Dr. David Nelson (Nelson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), the candidates were designated as \u003cem\u003eTc\u003c/em\u003eCYP82D364 (DN2096-1), \u003cem\u003eTc\u003c/em\u003eCYP749A368 (DN2218-4), \u003cem\u003eTc\u003c/em\u003eCYP712D39 (DN2280-7), \u003cem\u003eTc\u003c/em\u003eCYP712D38 (DN2506-9), \u003cem\u003eTc\u003c/em\u003eCYP81B203 (DN7422-10), \u003cem\u003eTc\u003c/em\u003eCYP704A289 (DN7647-3), \u003cem\u003eTc\u003c/em\u003eCYP72D41 (DN9219-3), and \u003cem\u003eTc\u003c/em\u003eCYP78A448 (DN20692-6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2. Tissue-Specific Expression and Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eAs callus tissue is intricately linked to bryonolic acid biosynthesis, we evaluated tissue-specific expression levels of the candidate P450s. Among them, \u003cem\u003eTcCYP712D38\u003c/em\u003e, \u003cem\u003eTcCYP712D39\u003c/em\u003e, \u003cem\u003eTcCYP82D364\u003c/em\u003e, and \u003cem\u003eTcCYP704A289\u003c/em\u003e displayed higher expression in callus compared to other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, a comparative analysis of the amino acid sequences of these P450 candidates with those of functionally characterized plant P450s involved in triterpenoid oxidation revealed intriguing patterns (Table S3). Notably, through phylogenetic analysis, \u003cem\u003eTc\u003c/em\u003eCYP712D38 and \u003cem\u003eTc\u003c/em\u003eCYP712D39 clustered closely with the CYP712K groups from \u003cem\u003eTripterygium wiifordii\u003c/em\u003e and \u003cem\u003eMaytenus ilicifolia\u003c/em\u003e, recognized for their C-29 oxidation activity converting friedelin to maytenonic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Despite diverse expression patterns, \u003cem\u003eTcCYP712D38\u003c/em\u003e and \u003cem\u003eTcCYP712D39\u003c/em\u003e displayed pronounced expression in callus and immature seeds, with \u003cem\u003eTcCYP712D39\u003c/em\u003e exhibiting tenfold higher expression compared to \u003cem\u003eTcCYP712D38\u003c/em\u003e. Thus, \u003cem\u003eTc\u003c/em\u003eCYP712D39 emerges as a strong candidate for bryonolic acid biosynthesis. Notably, \u003cem\u003eTcCYP712D39\u003c/em\u003e exhibited significantly higher expression in immature seeds, challenging the typical localization of bryonolic acid in roots (Kongtun, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lertphadungkit, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our earlier investigation of isomultiflorenol synthase, a gene encoding an intermediate for bryonolic acid, revealed high expression across multiple tissues, particularly in immature seeds and roots, prompting the question of why higher bryonolic acid levels are predominantly found in roots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, our tissue-specific expression study also provides interesting information on the cucurbitacin B biosynthesis. \u003cem\u003eTcCYP81B203\u003c/em\u003e exhibited the highest expression in immature seeds of \u003cem\u003eT. cucumerina\u003c/em\u003e L. and grouped into the groups of CYP81Q58, CYP81Q59, and CYP81AQ19 (Shang, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Takase, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e) from other cucurbitaceous plants, which are related in the cucurbitacin biosynthesis. This finding supported our earlier hypothesis based on cucurbitadienol expression, which also showed the highest expression in immature seeds, although a large amount of cucurbitacin B was detected in fruit and fruit juice (Lertphadungkit, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The collective findings from our study provide compelling evidence that the production of two characteristic triterpenoids, bryonolic acid and cucurbitacin B, is primarily produced in immature seeds of \u003cem\u003eT. cucumerina\u003c/em\u003e L. during seed development. In summary, our findings provide evidence that the production of two characteristic triterpenoids, bryonolic acid and cucurbitacin B, are primarily produced in immature seeds of \u003cem\u003eT. cucumerina\u003c/em\u003e L. This highlights the need for further exploration into the intricate regulatory mechanisms governing the spatial distribution of bryonolic acid and cucurbitacin B in \u003cem\u003eT. cucumerina\u003c/em\u003e L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3. Functional Domain and Conservation analysis\u003c/h2\u003e \u003cp\u003eCYPs are typically categorized based on the similarity of amino acid sequences. The structures of CYPs normally contain α helices (A \u0026ndash; L helices) and β sheets (β1\u0026ndash;5), with a heme prosthetic group located in the catalytic center of the enzymes (Sirim, et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). According to the multiple sequence alignment, we observed that amino acid sequences in the specific domains, such as proline-rich region, oxygen-binding and activation site, EXXR, PXRX, and heme-binding motif, were highly conserved within CYP712 subfamily (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The core regions of \u003cem\u003eTc\u003c/em\u003eCYP712D39 were predicted as follows: proline-rich domain (44\u0026ndash;48); oxygen activation domain (315\u0026ndash;320); ExxR (372\u0026ndash;375); PxRx (426\u0026ndash;429); heme-binding domain (456\u0026ndash;465). Most importantly, the EXXR and heme-binding sites of \u003cem\u003eTc\u003c/em\u003eCYP712D39 exhibited almost complete conservation compared to the CYP712K groups. Typically, these two specific motifs are characteristic in individual CYP families for maintaining the CYP tertiary structure and hold the heme during oxygenation (Syed and Mashele \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, it also presented a conserved residue in oxygen activating region as AGT(S/D)TS. This region positioned near the heme group to produce the formation of active Fe-O hydroxylating species, which plays an important role in proton transfer during catalysis (Guengerich \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, the role of CYP712Ks in C29-modification on friedelin could be utilize to predict the function of \u003cem\u003eTc\u003c/em\u003eCYP712D39 whether this enzyme could demonstrate the ability to catalyze C29-oxidation in bryonolic acid biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4. Functional characterization of TcCYP712D39\u003c/h2\u003e \u003cp\u003eTo investigate the function of selected P450s in the oxidation of isomultiflorenol, \u003cem\u003eTcCYP712D39\u003c/em\u003e was co-expressed with \u003cem\u003eTcIMS\u003c/em\u003e to provide isomultiflorenol as a substrate in the yeast system. The ethyl acetate extracts from the yeast cultures were subsequently analyzed by GC-MS. The yeast strain WAT11, harboring Arabidopsis cytochrome reductase for serving an electron donor, was used in this experiment. In the co-expression system of pYES2-\u003cem\u003eTcIMS\u003c/em\u003e and pESC-Leu-\u003cem\u003eTcCYP712D39\u003c/em\u003e, isomultiflorenol and bryonolic acid were detected at retention times of 13.3 and 15.7 min, respectively, while the yeast with empty vector showed no observable peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This clear evidence indicates that \u003cem\u003eTc\u003c/em\u003eCYP712D39 possesses the capability to oxidize isomultiflorenol at C29, resulting in the production of bryonolic acid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e5. Key Amino Acid Candidates Governing C29-Oxidation in Pentacyclic Triterpenoids\u003c/h2\u003e \u003cp\u003eThe enzymatic functions of CYPs are intricately linked to their substrate recognition sites (SRSs), which play a pivotal role in substrate specificity and the formation of substrate-binding pocket (Sugimoto and Shiro \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, we predicted the SRSs of \u003cem\u003eTc\u003c/em\u003eCYP712D39 based on definitions provided by Gotoh (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and Zawaira, et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Briefly, SRS1 corresponds to the region situated between B and C helices, SRS2 is in the C-terminal end of the F helix, SRS3 extends from the F-G loop to N-terminal end of the G helix, SRS4 is the N-terminal segment of the I helix, SRS5 spans the region between the K helix and β-sheet 1, and SRS6 is the turn in β-sheet 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo predict the key amino acids involved in C-29 modification process during bryonolic acid biosynthesis, we conducted homology modeling of \u003cem\u003eTc\u003c/em\u003eCYP712D39, and subsequently employed the PCPLD web-based platform for molecular docking. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e displays the docking model of \u003cem\u003eTc\u003c/em\u003eCYP712D39 and bryonolic acid for C-29 oxidation. Bryonolic acid lies directly above the heme group with the distance of 3.5 angstroms from C-29. We identified 13 amino acid residues located within 3 angstroms from the heme-binding ligand of \u003cem\u003eTc\u003c/em\u003eCYP712D39 and bryonolic acid. Remarkably, 11 of these residues were predicted to be situated within conserved regions commonly observed in CYPs. Among of them, 3 residues were located in SRS1 (R110, F113, and F127), 3 residues were located in SRS2 (M221, G224, and A228), 3 residues were in SRS4 (I314, G315, and T319), and 2 residues were in SRS5 (A379, and V380). Most of them are non-polar amino acids, which can form a central hydrophobic cavity to stabilize the protein\u0026rsquo;s tertiary structure and allow the hydrophobic interactions with methyl group on bryonolic acid (Guengerich \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Peterson and Graham \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). These amino acids are noteworthy due to their proximity to the ligand-binding site and their potential role in shaping the substrate-binding environment. This predictive analysis suggests that these key amino acid candidates may play important roles in determining substrate specificity and catalytic activity in the biosynthesis of pentacyclic triterpenoids, further experimental validation will be essential to confirm their functional significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSignificantly, isomultiflorenol and friedelin, both classified in oleanane-type triterpenoids, are known as substrates for C-29 oxidation by CYP712 family. Typically, SRS1 to SRS3 are responsible for conferring substrate specificity, while the latter three SRSs are strategically positioned close to the heme catalytic center, where they directly interact with the substrate during oxygenation (J\u0026oacute;źwik, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sugimoto and Shiro \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, the oxygen activating domain is conventionally located within the I-helix, positioned above the heme group in SRS4. We then combined the docking result with CYP712 sequence alignments. Interestingly, F113 (SRS1) is positioned in close proximity to the C3-OH of bryonolic acid with the distance of 3.1 angstroms, while serine (S) is conserved in other CYP712Ks. This observation suggests that F113 forms a cation-π interaction with C3-OH of isomultiflorenol, whereas CYP712Ks form hydrogen bonds with C3-oxo of friedelin, which allow them to be placed above the heme-iron for C-29 modification for bryonolic acid and maytenonic acid, respectively.\u003c/p\u003e \u003cp\u003eIn summary, our analysis highlights the putative significance of these predicted key amino acids in governing substrate specificity in pentacyclic triterpenoid biosynthesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study represents a comprehensive exploration of the triterpenoid biosynthesis. Our transcriptome-guided candidate selection led to the discovery of a novel enzyme, \u003cem\u003eTc\u003c/em\u003eCYP712D39, with the remarkable ability to catalyze the C-29 oxidation of bryonolic acid in \u003cem\u003eTrichosanthes cucumerina\u003c/em\u003e L. Our investigation also revealed key amino acid residues within \u003cem\u003eTc\u003c/em\u003eCYP712D39 in bryonolic acid production, enhancing our understanding of substrate specificity in pentacyclic triterpenoid production. This research bridges a critical knowledge gap, offering promising prospects for bioengineering valuable natural products. Our finding contributes to the understanding of triterpenoid biosynthesis in non-model plants and hold potential for applications in pharmaceuticals. Further experimental validation of the identified candidates is warranted to confirm their functional significance and potential biotechnological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from Mahidol Medical Scholars Program (MSP) and the New Discovery and Frontier Research Grant (NDFR 50/2564), Mahidol University, Thailand.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization: PL, and SB; Methodology: PL, and PM; Analysis: PL, and PM; Resources: SB; Writing - original draft preparation: PL; Writing - review and editing: SB; Funding acquisition: SB; Supervision: SB.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Dr. David Nelson for the P450 nomenclature. We also would like to thank Prof. Dr. Weena Jiratchariyakul for providing bryonolic acid standard. We express our deep appreciation to Prof. Dr. Min Ye and Prof. Dr. Xue Qiao for their generosity in providing essential resources for our experiments. Their contributions, including the plasmids pYES2 and pESC-Leu, along with the yeast strain WAT11, have been instrumental to the success of our work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGatbonton-Schwager TN, Letterio JJ, Tochtrop GP (2012) Bryonolic acid transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo. J Nat Prod 75:591\u0026ndash;598\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGotoh O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. 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Biosci Biotechnol Biochem 83:251\u0026ndash;261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakase S, Kera K, Nagashima Y, Mannen K, Hosouchi T, Shinpo S, Kawashima M, Kotake Y, Yamada H, Saga Y, Otaka J, Araya H, Kotera M, Suzuki H, Kushiro T (2019b) Allylic hydroxylation of triterpenoids by a plant cytochrome P450 triggers key chemical transformations that produce a variety of bitter compounds. J Biol Chem 294:18662\u0026ndash;18673\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeda T, Kondo T, Mizukami H, Ogihara Y (1994) Bryonolic acid production in hairy roots of \u003cem\u003eTrichosanthes kirilowii\u003c/em\u003e Max. var \u003cem\u003eJaponica\u003c/em\u003e Kitam. transformed with \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e and its cytotoxic activity. Chem Pharm Bull (Tokyo) 42:730\u0026ndash;732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka S, Uno C, Akimoto M, Tabata M, Honda C, Kamisako W (1991) Anti-allergic effect of bryonolic acid from \u003cem\u003eLuffa cylindrica\u003c/em\u003e cell suspension cultures. Planta Med 57:527\u0026ndash;530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThimmappa R, Geisler K, Louveau T, O'Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225\u0026ndash;257\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZawaira A, Ching LY, Coulson L, Blackburn J, Wei YC (2011) An expanded, unified substrate recognition site map for mammalian cytochrome P450s: analysis of molecular interactions between 15 mammalian CYP450 isoforms and 868 substrates. Curr Drug Metab 12:684\u0026ndash;700\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bryonolic acid, C-29 oxidation, isomultiflorenol, Trichosanthes cucumerina L., triterpenoids","lastPublishedDoi":"10.21203/rs.3.rs-3896957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3896957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBryonolic acid, a characteristic triterpenoid in \u003cem\u003eTrichosanthes cucumerina\u003c/em\u003e L., is well-known due to its pharmacological activities. The biosynthetic pathway of this compound is similar to other triterpenoids, which are known to be initiated by oxidosqualene cyclase for squalene cyclization and cytochrome P450s for oxidation. However, the final step in this pathway has not been identified. This study presents a discovery of a novel enzyme, CYP712D39, with the remarkable ability to catalyze the crucial C-29 oxidation step in bryonolic acid production. We utilized previous transcriptome analysis with eight promising P450 candidates, exhibiting greater expression levels in callus tissue, which shows a high bryonolic acid production. Functional characterization experiment confirmed its capacity to convert isomultiflorenol into bryonolic acid in the WAT11 yeast system. Furthermore, we also predicted key amino acids, including F113, crucial for binding with C3-OH of isomultiflorenol for C29-oxidation. This discovery fills a critical knowledge gap and offers significant biotechnological potential for pharmaceutical and agricultural applications.\u003c/p\u003e","manuscriptTitle":"A novel C-29 oxidase, CYP712D39, catalyzes the formation of bryonolic acid from isomultiflorenol in Trichosanthes cucumerina L.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-22 16:52:15","doi":"10.21203/rs.3.rs-3896957/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"98ba3220-0c79-43e5-ad9b-82a48e897b3f","owner":[],"postedDate":"February 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T15:49:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-22 16:52:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3896957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3896957","identity":"rs-3896957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}