S. hexandrum, a perennial herb belonging to the Berberidaceae family, is a traditional Chinese medicinal material 1. It is primarily distributed in Southeast Asia, thriving in alpine grasslands and forest edges at altitudes ranging from 1300 to 4500 meters 2. This herb has long been used in traditional Chinese medicine for various therapeutic purposes, making it a valuable addition to the pantheon of Chinese medicinal plants 3. Podophyllotoxin, a primary medicinal component of S. hexandrum, exhibits a diverse array of biological activities including cancer cell growth inhibition, antiviral activity, and insecticidal properties 4–7. After rigorous clinical research using podophyllotoxin and its derivatives on various cancers such as leukemia, prostate cancer, cervical cancer, thymoma, and glioblastoma multiforme, it was found that podophyllotoxin exhibits broad-spectrum and highly efficient anticancer activity 6,8,9. Subsequently, podophyllotoxin-based podophyllin antitumor drugs were continuously developed 10. The first batch of podophyllin antitumor drugs, such as etopophos, etoposide (VP-16) and teniposide (VM-26), were approved for marketing in Switzerland and the United States in the 1980s11. Due to the further development of novel epipodophyllotoxin antineoplastic drugs in the fields of medicine, biology, chemistry, and beyond, the demand for podophyllotoxin, its precursor substance podophyllotoxin, in the international market has been increasing 12,13. Currently, the natural source of podophyllotoxin is primarily derived from S. hexandrum and some other Epipodophyllaceae plants 3,14.S. hexandrum The growth of S. hexandrum is closely associated with various ecological factors such as altitude, temperature, and rainfall15,16. It is typically found in remote and high-altitude regions that are characterized by cold temperatures, including Tibet, Yunnan, Sichuan, and Qinghai provinces in China17. In recent years, environmental damage has caused a reduction in the suitable habitat range of S. hexandrum, while market demand and economic interests have driven humans to overexploit this species through excessive harvesting3,18. This has led to a growing scarcity and rarity of S. hexandrum’s wild resources. Extracting podophyllotoxin from plants results in low extraction rates and causes significant damage to wild plant resources, making it unsustainable19. Chemical synthesis methods, due to their cumbersome procedures, high costs, and slow production rates, are unsuitable for large-scale production of podophyllotoxin and its derivatives20. In contrast, the emergence of Biosynthesis methods has provided a novel and reliable approach for the efficient synthesis of significant amounts of podophyllotoxin, thus fulfilling the commercial demand21,22.
Methyltransferases (MTs) are widely present in nature and participate in various important biological processes, including signal transduction, transcriptional regulation, and biosynthesis of metabolic products23. Based on the type of methyl acceptor, MTs can be classified as oxygen-, nitrogen-, carbon-, sulfur-, and other methyltransferases24. Among them, oxygen methyltransferases (OMTs) account for 54% of MTs and usually use S-adenosylmethionine as the methyl donor25. In animals, O-methylation plays a significant role not only in the detoxification of reactive hydroxyl groups but also in the biosynthesis of hormone melatonin and catecholamine neurotransmitters26. OMTs in plants typically act on compounds containing phenolic hydroxyl groups, such as phenylpropanoids, flavonoids, and alkaloids, to methylate their oxygen atoms27,28. They are a crucial regulatory enzyme in the phenylpropanoid metabolic pathway, which is derived from the shikimate pathway29. Pinane, the common precursor of podophyllotoxin, is one of the important natural secondary metabolites in the phenylpropanoid metabolic pathway of plants30. Previous research has demonstrated that caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoMT), two types of OMTs, play a critical regulatory role in the biosynthesis pathway of pinane31,32. In previous studies, our team conducted transcriptome analysis of S. hexandrum from three different provenances in Gansu and Shaanxi. The results indicated significant differences in the expression of the Cluster-2923.2731 gene. Upon alignment, it was found that the Cluster-2923.2731 gene had a consistent sequence with the KT390157.1 and MW531736.1 genes in the NCBI database that encode OMT3 protein in S. hexandrum. In this study, ShOMT3 was identified as an important regulatory enzyme gene involved in the downstream synthesis of podophyllotoxin from pluviatolide to deoxypodophyllotoxin in S. hexandrum. The regulatory role of this enzyme gene in the podophyllotoxin biosynthesis pathway has been characterized33,34. Lau and Sattely 35 conducted a transcriptome analysis of P. hexandrum in the database and identified several candidate enzymes involved in the downstream biosynthesis of podophyllotoxin: O-methyltransferases (OMT1, OMT2, OMT3, OMT4), CYPs, and a 2-oxopentanedioic acid/Fe(II)-dependent dioxygenase (2-ODD). These enzymes were co-expressed with CYP719A23 in tobacco leaves, and it was found that only ShOMT3 could catalyze the C-4′ hydroxylation of (−)-pluviatolide to produce (−)-5′-desmethoxy-yatein34,36.
Currently, the biosynthetic pathways for important intermediates, namely deoxypodophyllotoxin and 4’-desmethyl-epipodophyllotoxin, have been well characterized37. However, the key enzymes involved in the downstream process of podophyllotoxin biosynthesis have not been definitively identified. Domestic and international scholars have conducted extensive research on the pharmacological effects, derivative synthesis, and biosynthetic pathway of podophyllotoxin6,38. However, the understanding of the critical regulatory genes involved in its biosynthesis is not yet fully developed39. Moreover, most of the existing references on plant oOMTs have focused on the study of COMT, which regulates lignin biosynthesis27,40. However, the research on the ShOMT3 gene in the podophyllotoxin biosynthetic pathway of S. hexandrum is relatively limited. Therefore, in this study, the ShOMT3 gene from samples S1 and S2 was cloned and comprehensively bioinformatics analyzed. The research findings contribute to the understanding of the molecular mechanism of podophyllotoxin biosynthesis in S. hexandrum, and have important theoretical and practical implications for the industrial production of podophyllotoxin and the molecular breeding of S. hexandrum.