Dendrobium species within the Orchidaceae family are predominantly of medicinal origin, containing various active products, including alkaloids, bibenzyls, polysaccharides, and flavonoids. Alkaloids are effective in cardiovascular, cataract, gastrointestinal, and respiratory diseases. Members of the cytochrome P450 monooxygenase (CYP450) gene family play important roles in alkaloid synthesis, as well as in plant growth and development by participating in the formation of alkaloid terpene skeletons and later modifications. Additionally, the CYP450 family has been studied in several other species. However, a systematic study of the CYP450 gene family in Dendrobium at a genome-wide level has not yet been conducted. This gap in research hinders our understanding of the alkaloid metabolic pathways in Dendrobium.

Dendrobium is an epiphytic herb with approximately 1500 species primarily found in tropical and subtropical regions spanning from Asia to Oceania. In China, 76 Dendrobium species are cultivated, primarily in provinces located south of the Qinling Mountains, particularly in southern Yunnan [1]. The Pharmacopoeia of the People's Republic of China lists several Dendrobium species with medicinal properties including D. chrysotoxum, D. nobile, D. catenatum, D. fimbriatum, and D. huoshanense [2]. Studies on the pharmacology of the Dendrobium genus have indicated that its primary medicinal components include alkaloids, bibenzyls, polysaccharides, and flavonoids [3,4]. Alkaloids were the first substances to be extracted and studied in 1932 when they were first obtained from the stems of D. nobile and named dendrobine [[4], [5], [6]]. Alkaloids are widely present in plants, animals, and microorganisms, with plants being the primary source [7]. Over 60 alkaloids have been obtained from 15 different types of Dendrobium plants and can be categorized into five primary groups: indolizidine alkaloids, amine alkaloids, terpene alkaloids, indole alkaloids, and other classes of alkaloids. Among them, as many as 24 terpene alkaloids containing sesquiterpene skeletons have been identified, and their content is the highest among the total alkaloids in D. nobile [8]. Alkaloids are the primary active components of Dendrobium and have demonstrated efficacy in the treatment of cardiovascular, cataract, gastrointestinal, and respiratory disorders [4]. Although Dendrobium alkaloids have been reported to be obtained by chemical synthesis, the process is complicated, energy-consuming, and polluting due to the complex structure of Dendrobium alkaloids [9,10]. Therefore, alkaloid resources are still being extracted from D. nobile. Massive market needs have led to the transitional development of D. nobile, which is currently in shortage of wild D. nobile resources. Most of the D. nobile available in the market is artificially cultivated, which is expensive and varies in quality. Microbial cell factories and cell-free systems represent two alternative methods for the chemical synthesis and natural extraction of biomolecules. These environmentally friendly and sustainable approaches have successfully met industrial standards for vitamins, amino acids, and certain natural products in terms of titers [11,12]. Additionally, studies have explored the integration of microbial cellular factories and cell-free systems to achieve excessive production of ergot alkaloids [13]. Understanding the alkaloid synthesis pathway in D. nobile will help biosynthesize alkaloids and increase alkaloid resources.

Terpenoid alkaloids are produced by the synthesis of isoprene structural units through two pathways, namely the mevalonate pathway (MVA) and the methylerythritol 4-phosphate pathway (MEP) [14,15]. Geranyl diphosphate with a C10 backbone, farnesyl diphosphate (FPP) with a C15 backbone, and geranylgeranyl diphosphate with a C20 backbone are synthesized by combining methylallyl diphosphate (DMAPP) from the MVA pathway with isopentenyl diphosphate (IPP) obtained from the MEP pathway [16]. The action of terpene synthase (TPS) produces terpene skeletons. Cytochrome CYP450 oxygenase usually carry out oxidation of the terpene skeleton. Oxidized terpenoids can be further functionalized by adding substituents to form terpenoid alkaloids and other terpenoid compounds. Bathe et al. summarized previous studies, which revealed that CYP450 genes involved in the oxidation of diterpenoid skeletons in plants mainly belong to the CYP71, CYP85, and CYP72 families. Specifically, CYP725A, a member of the CYP85 family, is involved in paclitaxel synthesis. Furthermore, all hydroxylation reactions on the paclitaxel ring of the paclitaxel skeleton are catalyzed by CYP450 oxygenase [17]. Simultaneously, these simple oxidations trigger numerous additional chemical modifications in the molecule, including methylation, acetylation, glycosylation, and structural rearrangements, through various pathways. These reactions significantly expand the structural diversity of terpenoids. They also provide anchoring points for further modifications by various transferases.

Current research has revealed that the synthesis of Dendrobium alkaloids encompasses three fundamental phases: the formation of IPP, creation of the sesquiterpene structure, and subsequent modification procedures. The formation of FPP occurs during the construction of the terpene skeleton via the MVA and MEP pathways. Subsequently, picrotoxane lactone is produced by TPS and cytochrome P450 monooxygenase. The final Dendrobium alkaloids are generated through the modification of picrotoxane-lactone by reductases, transaminases, methyltransferases, and cytochrome CYP450 monooxygenases [8,18] (Fig. S1). CYP72A1 functions as a strictosidine synthase (SCS) in the vinblastine and vincristine synthesis pathways in Catharanthus roseus [19]. In contrast, CYP76 exhibits both geraniol 10-hydroxylase (G10H) activity and flavonoid 30-hydroxylase activities, suggesting a dual role in the terpene synthesis pathway and flavonoid biosynthesis [20].

CYP are a superfamily of hemoglobin genes containing a class of monooxygenases with hemoglobin as a cofactor and are widely distributed across various biological domains. These include animals, plants, fungi, protozoa, bacteria, archaea, and viruses [21]. Cytochrome P450 monooxygenases facilitate oxidative metabolic reactions of diverse exogenous and endogenous compounds through the participation of oxygen and NADPH [[22], [23], [24]]. In 1958, Klingenberg was the first to discover CYP protein in rat liver microsomes [25]. This protein possesses the traits of a hemoglobin protein and was named cytochrome CYP450 protein because of its distinctive absorption peak at 450 nm [26]. In 1969, Fresh reported the first plant CYP gene in cotton [27]. In plants, membrane-bound CYP proteins are predominantly located in the endoplasmic reticulum, chloroplasts, mitochondria, and other secretory pathways [8]. These proteins have molecular weights ranging from 45 to 62 kDa, with an average of 55 kDa. They feature four crucial structural domains: a hemoglobin-binding domain, an I-helix, a K-helix, and a PERF/W structural domain [28]. Cysteine, a highly conserved residue, is part of the hemoglobin-binding signature motif containing 10 conserved residues. This motif enables the binding of oxygen and catalyzation of various reactions [29].

Advancements in sequencing technology have facilitated the identification and analysis of members of the CYP gene family across various species, including humans, mice, Arabidopsis, buckwheat, and grapes. Arabidopsis CYP genes are categorized into nine clans: 51, 72, 710, 711, 74, 85, 86, 97, and 71 [30]. Utilizing phylogenetic tree construction, CYP family members were categorized based on the classification of the CYP family in Arabidopsis. Transcriptomic data were used for the initial identification and analysis of members of the CYP gene family in D. huoshanense and D. catenatum. Nevertheless, the absence of comprehensive genomic identification of CYP450 genes in medicinal Dendrobium hinders the investigation of its alkaloid metabolic pathways. To address this issue, the genomes of four medicinal Dendrobium species, namely, D. chrysotoxum, D. nobile, D. catenatum, and D. huoshanense, were used to identify CYP450 genes.

In this study, we identified CYP450 genes in four medicinal Dendrobium species, analyzed their motif composition, gene replication events, and selection pressure. Additionally, we screened two genes involved in dendrobine synthesis in D. nobile. This study provides a preliminary analysis of the expression patterns of related CYP450 gene family members associated with alkaloid metabolic pathways in medicinal Dendrobium, and offers a molecular resource for future studies on alkaloid metabolic networks in medicinal Dendrobium.