Terpenoids are classified based on the number of isoprene units into major subclasses, which include monoterpenes, sesquiterpenes, diterpenes, triterpenes, and polyterpenes. Their biosynthesis is primarily governed by two fundamental metabolic routes: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. The MVA pathway occurs in the cytoplasm, where acetyl-CoA serves as the initial substrate for producing the essential C5 precursors—isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In contrast, the MEP pathway takes place in plastids, utilizing glyceraldehyde-3-phosphate and pyruvate to generate IPP and DMAPP. This compartmentalization is critical for regulating the temporal and spatial production of diverse terpenoid compounds (Nabavi et al., 2020; Kant et al., 2024).

Flavonoids possess a characteristic C6–C3–C6 skeleton, consisting of two aromatic benzene rings (A and B) linked by a three-carbon bridge. In plants, their biosynthesis derives from carbon units supplied by both the acetyl-CoA and shikimate–phenylpropanoid pathways. Chalcone synthase (CHS) catalyzes the condensation of malonyl-CoA with p-coumaroyl-CoA to form the core chalcone structure. This intermediate is subsequently isomerized by chalcone isomerase (CHI) into flavanone, which can be further modified into various flavonoid subclasses, such as isoflavones and anthocyanins, by multiple downstream enzymes (Liu et al., 2021).

Alkaloid biosynthesis typically begins with specific amino acid derivatives. For example, tyramine, a derivative of tyrosine metabolism, reacts with 3,4-dihydroxybenzaldehyde to form norbelladine. This key intermediate then undergoes sequential methylation and oxidative modifications to yield bioactive alkaloids such as galanthamine and colchicine (Tariq et al., 2023).

The biosynthetic pathways of terpenoids, flavonoids, and alkaloids are core components of the plant secondary metabolic network. CYPs pivotally catalyze key oxidative and modification reactions in these pathways, significantly enhancing the structural diversity of plant metabolites. Understanding these enzymatic functions enhances our knowledge of plant metabolism and provides a theoretical foundation and technical platform for developing bioactive compounds from medicinal plants and advancing metabolic engineering strategies. Further investigation into the precise catalytic and regulatory mechanisms of CYPs will facilitate the sustainable and scalable production of valuable plant-derived natural products.

Terpenoids are one of the most structurally diverse and chemically complex classes of primary and secondary metabolites in nature, playing crucial roles in plant biology (Bergman et al., 2024). This structural diversity underlies their significant research value and broad potential for applications, particularly in pharmacology and biotechnology, where they exhibit a range of bioactive properties such as antioxidant, anticancer, and immunomodulatory effects. Additionally, terpenoids are essential for plant growth, defense, and environmental adaptation (Nagegowda and Gupta, 2020; Zhang et al., 2023; Zhang et al., 2025).

To date, multiple cytochrome P450 subfamilies have been identified as key catalysts in the biosynthesis of plant terpenoids (Figure 3). These CYP enzymes mediate critical oxidative, hydroxylative, and cyclization reactions that modify the terpenoid scaffold, thereby greatly expanding structural diversity. For example, members of the CYP76 and CYP71 subfamilies contribute to the biosynthesis of diterpenoids such as gibberellins and paclitaxel precursors. The functional specificity of these enzymes is largely determined by the spatial configuration and chemical microenvironments of their active sites, which dictate substrate recognition and catalytic efficiency (Bathe and Tissier, 2019).

Flavonoids are natural polyphenolic compounds that serve as the primary pigments in plants. This group includes anthocyanins (imparting red, orange, blue, and purple hues), along with chalcones and aurones (yellow pigments), and flavanols and flavones (white to pale yellow pigments). These compounds exhibit a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antibacterial, and antihypertensive effects. As secondary metabolites, flavonoids possess both nutritional and therapeutic value. The cytochrome P450 family functions as a central molecular determinant of flavonoid chemical diversity across the plant kingdom. This functional diversity is achieved through mechanisms such as tissue-specific expression, developmental regulation, narrow substrate specificity, and site-selective catalysis (Figure 4).

Alkaloids are a prominent class of nitrogen-containing natural products, with structures ranging from simple monocyclic to highly complex polycyclic frameworks. Indole, steroidal, terpenoid, isoquinoline, and dibenzylisoquinoline alkaloids exhibit significant anti-inflammatory and antitumor activities in both human and animal models, underscoring their broad therapeutic potential (Oladeji et al., 2024). The cytochrome P450 family, with its catalytic versatility and organ-specific expression, serves as a dual regulatory system governing both the structural diversification and spatial organization of alkaloid biosynthetic pathways in plants.

Phenylpropanoids, defined by a C6–C3 skeleton, are widespread secondary metabolites with multiple physiological functions in plants. They contribute to cell wall reinforcement, UV protection, defense against herbivores and pathogens, and floral pigmentation for pollinator attraction. Additionally, phenylpropanoids exhibit diverse bioactive properties beneficial to human health, underscoring their potential for natural product development (Deng and Lu, 2017; Zhu et al., 2024).

Lignin monomers, key end-products of the phenylpropanoid pathway, are synthesized through a rate-limiting 3-hydroxylation step catalyzed by the cytochrome P450 subfamily CYP98. Beyond lignin biosynthesis, CYP98 enzymes also orchestrate carbohydrate and fatty acid metabolism, hormone biosynthesis, and signaling in primary metabolism, thereby serving as central regulatory hubs in the plant metabolic network (Xin et al., 2023). Lignin, a complex polymer, is primarily formed by the polymerization of phenylpropanoid monomers. For example, in Arabidopsis thaliana, CYP98A3 specifically catalyzes the 3-hydroxylation of 4-coumaroyl-shikimate to form caffeoyl-shikimate, thereby bridging the upstream phenylalanine metabolism and downstream lignin synthesis pathways at the molecular level. The homologous enzyme CYP98A27 in Populus trichocarpa performs the same 3-hydroxylation function, participating in lignin biosynthesis (Alber et al., 2019). CYP98A20 and its close homologs (e.g., CYP98A35) exhibit strict substrate specificity by hydroxylating the p-coumaroyl and tyrosol moieties of osmanthuside B to yield verbascoside. Furthermore, CYP98 enzymes convert p-coumaroyl shikimate or quinate into caffeoyl derivatives, representing key steps that channel carbon flux through the phenylpropanoid network (Yang et al., 2023).

The Baeyer-Villiger oxidation is a classic method for directly converting aliphatic or cyclic ketones into the corresponding esters or lactones, and the reaction occurs under acidic conditions provided by peracids or hydrogen peroxide. The Baeyer-Villiger oxidation involves the electrophilic attack of a peracid on the carbonyl group of a ketone, forming the key Criegee intermediate; subsequently, this intermediate undergoes a rearrangement in which an alkyl group or hydrogen migrates to the oxygen atom, thereby inserting an oxygen atom between the carbonyl carbon and the adjacent carbon atom, ultimately forming an ester or lactone (Fatima et al., 2024). Takahito Nomura and colleagues analyzed tomato CYP85A1 loss-of-function mutants, and, combined with heterologous expression in yeast and double mutant experiments in Arabidopsis, they for the first time elucidated that the cytochrome P450 enzymes CYP85A3 and CYP85A2 not only catalyze the C-6 hydroxylation reaction in brassinosteroid biosynthesis, but can further convert ketone intermediates into highly active final products with a seven-membered lactone ring, brassinolides, through Baeyer-Villiger oxidation. This reveals a novel enzymatic function of the plant P450 family in catalyzing lactone ring formation and its specific regulatory role in organ development (Nomura et al., 2005).

Rina Saito found that the CYP94 family of monooxygenases (especially SICYP94B18 and SICYP94B19) are mainly responsible for the catabolism of jasmonoyl-isoleucine in tomato leaves. They sequentially convert jasmonoyl-isoleucine into 12-hydroxy-jasmonoyl-isoleucine and 12-carboxy-jasmonoyl-isoleucine through a two-step oxidation reaction, thereby weakening the jasmonate signal and preventing its prolonged activation from harming the plant. In addition, these two enzymes can also catalyze the oxidative metabolism of other jasmonate-amino acid conjugates, indicating their broad role in jasmonate signal regulation. The metabolites they produce are oxidative products of jasmonate plant hormones, specifically hydroxylated and carboxylated derivatives generated via the ω-hydroxylation pathway (Saito et al., 2024).

Mining and studying specialized multifunctional CYPs is of vital importance for a profound understanding and efficient exploitation of plant secondary metabolic networks. These enzymes are not only key links in elucidating the complete biosynthetic pathways of high-value natural products, but also fundamental drivers promoting innovation in natural product science, synthetic biology, and agricultural biotechnology from the very origin.

CYPs are capable of performing a wide variety of oxidation reactions, based on a highly conserved protein structural framework and catalytic core. As mentioned in the introduction, motifs in the heme-binding domain, such as FxxGxRxCxG and ExxR, are retained in the vast majority of plant CYPs, ensuring the fundamental ability to correctly bind the heme cofactor, activate oxygen, and position substrates. This structural conservation allows different CYP family members, even when involved in different metabolic pathways, to follow similar catalytic cycles. For example, members of the CYP71 family can oxidize diterpene precursors to produce defense metabolites in maize, as well as oxidize indole substrates to participate in camalexin synthesis in Arabidopsis; fundamentally, both processes rely on using a high-valent iron-oxo reactive intermediate to selectively attack inert C-H bonds. Similarly, subfamilies such as CYP76 and CYP716 have been reported to primarily catalyze hydroxylation reactions in terpene, flavonoid, and even alkaloid pathways.

The evolutionary expansion and functional innovation of the cytochrome P450 gene family are key drivers of the diversification of plant secondary metabolites and lineage-specific adaptive strategies. Comparing CYP-mediated metabolic networks in monocots and dicots can reveal both their conserved catalytic logic and the uniquely differentiated defense niches. In terpene biosynthesis, monocots such as Zea mays L. rely on the CYP71Z subfamily to catalyze the production of zealexin terpenoids, which exhibit specific antifungal activity in grasses. In contrast, dicots utilize a more diverse array of CYP families, such as CYP71AV, CYP76AH, and CYP716, to construct structurally complex medicinal terpene backbones, such as artemisinin CYP71AV1 in Artemisia annua and ginsenoside CYP716A in Panax, whose functions extend from direct defense to interactions with mammalian systems. Flavonoid metabolic differences are also notable. Members of the CYP75 family in monocots primarily participate in B-ring hydroxylation of basic flavonoids to respond to abiotic stress. In dicots like Camellia sinensis, the CYP75A/B subfamily has further diverged, finely regulating the lineage-specific production of anthocyanins and catechins, directly affecting flower color, flavor, and antioxidant capacity, demonstrating co-evolution with insect pollination and seed dispersal strategies.

As a ubiquitous enzyme family, plant cytochrome P450s (CYPs) participate in nearly all major metabolic pathways (Fang et al., 2024). The catalytic versatility of the CYP98 family and other CYP450s illuminates the organization and regulation of complex metabolic networks. These insights provide a theoretical foundation for the targeted discovery of bioactive compounds and offer practical strategies for enhancing crop resilience through metabolic engineering.

Environmental stresses profoundly affect the activity of key enzymes in secondary metabolite biosynthesis (Guo et al., 2025). Under stress conditions, the expression of cytochrome P450 genes is precisely modulated to reprogram secondary metabolic pathways, thereby enhancing plant adaptability and stress tolerance.

The cytochrome P450 family plays a central role in the biosynthesis of plant natural products. However, limited understanding of substrate selectivity, structural dynamics, and enzyme–enzyme interactions continues to hinder the development of efficient heterologous production systems. In a study focusing on CYP76AH1—a key enzyme involved in tanshinone biosynthesis—Shi et al. employed directed evolution to modify residues within the substrate-binding pocket, significantly enhancing substrate affinity. Further optimization of the oxygen-binding motif, guided by naturally occurring sequence variations, led to improved catalytic efficiency (Shi et al., 2021). This work presents a clear strategy for the rational engineering of CYP enzymes.

Despite these advances, the majority of plants CYPs remain functionally uncharacterized, and the mechanisms underlying substrate recognition and conformational changes are still poorly understood. Combining high-resolution structural techniques such as cryo-electron microscopy and X-ray crystallography with machine learning models capable of predicting substrate specificity and turnover rates is expected to provide a robust foundation for targeted CYP engineering.

Equally critical is the need to understand how CYP enzymes function within complex and dynamic metabolic networks that mediate plant responses to environmental stress. In high-value biosynthetic pathways such as that of artemisinin, optimizing CYP expression to enhance metabolic flux and compound accumulation remains a major research objective. Future investigations should integrate multi-omics approaches—including transcriptomics and metabolomics—to characterize the tissue-specific expression profiles and co-expression networks of CYP genes under various stress conditions. Such integrative analyses will not only clarify the functional roles of CYPs in plant–environment interactions but also establish a theoretical basis for improving metabolite yields through genetic or environmental modulation.

Plant secondary metabolites are extensively utilized in medicine, agriculture, and the food industry; however, their naturally low abundance often fails to meet commercial demands. Therefore, developing scalable bioproduction strategies is critically important.

Bioreactors represent the core hardware of modern biotechnology. These systems maintain plant, animal, or microbial cells in vitro under precisely controlled conditions to facilitate biochemical reactions and fermentation processes, earning them the designation as the “heart” of biotechnological production. Designing bioreactor systems around the cytochrome P450 gene family presents a promising approach for the sustainable production of plant-derived natural products. CYP mono-oxygenases catalyze the hydroxylation of C–H bonds, thereby enabling subsequent modifications—such as acylations, methylations, and glycosylations—that enhance structural diversity and bioactivity. CYP monooxygenases have been successfully applied in the industrial biosynthesis of several high-value natural products. For example, by expressing CYP76AH3 and CYP76AK1 from Salvia miltiorrhiza in engineered yeast and combining this with fermentation optimization, efficient synthesis of tanshinone compounds can be achieved, with the target product accounting for more than 92% of the total tanshinones (Guo et al., 2016). Beyond native enzymes, rationally engineered P450 BM3 variants have demonstrated exceptional catalytic selectivity. The triple mutant A82F/F87V/L188Q hydroxylated sesquiterpenic acid at the C-7 position with 94% regioselectivity, while a quadruple mutant (A82F/F87V/T268A/L437Q) achieved 90% regioselectivity and complete stereoselectivity at the C-9 position, providing powerful tools for chiral drug synthesis (Urlacher and Girhard, 2019).

NADPH-cytochrome P450 reductase (CPR) is a structurally conserved diflavin protein, with its N-terminal and C-terminal containing domains derived from bacterial flavodoxin and ferredoxin-NADP⁺reductase, respectively, and is localized to the endoplasmic reticulum through a membrane-anchoring region. Its core function is to sequentially transfer electrons from NADPH via FAD and FMN to the heme center of cytochrome P450, driving catalytic oxidation reactions such as hydroxylation, thus playing a key role in the biosynthesis of plant secondary metabolites, defense responses, and environmental adaptation. Its highly conserved interaction interface allows CPRs from different species to partially complement each other, making it an ideal module for constructing novel biocatalytic systems in synthetic biology (Jensen and Møller, 2010; Wei et al., 2025). Regarding the biosynthesis of the core medicinal saponin aglycone Quillaic acid, this study systematically screened 110 CYP/CPR pairings through a combinatorial optimization strategy and successfully constructed an efficient oxidation cascade network composed of CYP716A12, CYP714E19, CYP72A567 (2 copies), and CYP716A262. This network enabled six-step selective oxidation of the substrate β-amyrin at the C-16, C-23, and C-28 positions. By engineering the endoplasmic reticulum (overexpressing ice2) and using the membrane sterol-binding protein MSBP1 to bring CYP enzymes closer together, QA production in 1L fed-batch fermentation was dramatically increased from the previously reported 314 mg/L to 2.23 g/L (approximately a 7.1-fold increase), with a 32-fold increase in specific productivity. Additionally, the purity of QA among oxidized triterpene products rose from 54% to 86%. This achievement highlights the core value of rationally designing and combinatorially optimizing the CYP gene family in achieving efficient, high-purity, and scalable biomanufacturing of complex natural products (Yang et al., 2023). In Artemisia annua, six key enzyme genes in the artemisinin biosynthesis pathway—IDI, FPS, ADS, CYP71AV1, AACPR, and DBR2—were co-introduced into the plants, and two core optimization techniques were employed: First, the cytochrome P450 monooxygenase (CYP71AV1) was co-expressed with its specific electron donor, cytochrome P450 reductase (AACPR/CPR), to provide an efficient redox catalytic cycle, addressing the common issue of insufficient reducing power when P450 enzymes are heterologously expressed; second, key enzymes were precisely modified with signal peptides to achieve subcellular compartmentalization. The ADS enzyme was targeted to mitochondria rich in the precursor FPP using the mitochondrial signal peptide cox4, while CYP71AV1, AACPR, and DBR2 were directed to the chloroplasts with chloroplast transit peptides. This non-glycosylated environment not only stabilizes enzyme activity but also prevents futile cycling of metabolic intermediates and potential toxicity in the cytoplasm. This combined strategy synergistically optimized precursor supply, catalytic efficiency, and the spatial environment for product accumulation, ultimately resulting in transgenic A. annua plants with artemisinin content reaching 2.69% of dry weight in the T2 generation—an increase of up to 232% compared to the wild type (0.81%)—and confirmed that this high-yield trait can be stably inherited according to Mendelian genetics in the next generation (Qamar et al., 2024).

Plant CYPs are key drivers of secondary metabolite diversification, catalyzing hydroxylation and epoxidation reactions that enhance the structural complexity and bioactivity of terpenoids, alkaloids, and flavonoids. Suspension culture complements bioreactor-based strategies by propagating callus or somatic embryos in liquid medium on orbital shakers until stable, homogeneous cell lines are established. Supplementing the culture with CYP-inducing elicitors further promotes metabolite accumulation (Bhatia and Bera, 2015). Coupling these cultures with an enhanced NADPH supply—supported by the pentose phosphate pathway and ATP-dependent electron transfer—facilitates the scaling from laboratory to industrial production (Figure 1). Integrating advanced bioreactor systems with CYP-centered metabolic engineering effectively circumvents the natural scarcity of plant secondary metabolites. This combined strategy significantly boosts product yield and scalability, improves economic viability across healthcare, agriculture, and green industries, and pushes the field of plant biotechnology to new frontiers.

The cytochrome P450 enzyme system is a core catalytic component in synthetic biology for constructing biosynthetic pathways of medicinal compounds, playing an irreplaceable role in the synthesis of key products such as artemisinin, taxol precursors, and ginsenosides. However, its engineered application still faces numerous bottlenecks: strict substrate specificity limits the expansion of catalytic scope; electron transfer efficiency dependent on CPR is low; protein folding and localization issues often occur in heterologous expression; and during large-scale fermentation, enzyme activity is unstable and by-product accumulation is common.

Salicylic acid (SA) is a key phytohormone regulating pathogen defense responses in plants. Substantial evidence shows that cytochrome P450 mono-oxygenase inhibitors suppress SA biosynthesis across a wide range of plant species (Wang et al., 2025). Molecular breeding strategies now employ targeted pathway engineering—through transcriptional or enzymatic modulation of CYP genes—to enhance the accumulation of high-value bioactive compounds in medicinal plants and fungi, while concurrently improving agronomic traits such as stress tolerance. Environmental stress acts as a potent inducer of secondary metabolism, enhancing plant resilience and metabolite biosynthesis both, thereby creating novel opportunities for trait improvement via metabolic engineering. CRISPR/Cas9 technology has significantly advanced these efforts. Shang et al. developed the first functional CRISPR-Cas9 system in the medicinal fungus Ganoderma lucidum. By delivering a Cas9–sgRNA ribonucleoprotein complex via PEG-mediated transformation of spore-derived protoplasts, they successfully disrupted cyp512a3, a key gene in triterpenoid ganoderic acid biosynthesis. The edited strains exhibited substantially reduced ganoderic acid levels, confirming the central regulatory role of cyp512a3. Furthermore, this study identified previously unknown CYPs in the type-II ganoderic acid (GA) biosynthetic pathway originally proposed by Yuan et al., and elucidated their catalytic sequences. This work provides direct evidence that CYP-targeted genome editing can effectively generate high-yield Ganoderma strains (Wang et al., 2020; Khanal et al., 2025).

In addition, artificial intelligence technology provides a new approach for the rational design of CYPs. Machine learning-based sequence-to-function predictions can guide the directed evolution of key residues; deep learning-assisted protein structure modeling can accurately elucidate substrate binding patterns; and integrated metabolic networks and computational models can virtually optimize CYP catalytic performance and metabolic flux distribution in synthetic pathways (Huang et al., 2019; Yadav et al., 2024).

A mechanistic understanding of individual CYP enzymes within secondary metabolic networks is therefore essential for redirecting metabolic flux and achieving sustained production of target compounds. This precision-regulation strategy not only ensures stable yields of medicinal resources but also supports the sustainable and scalable long-term development of plant biotechnology.

Future research on the cytochrome P450 family should prioritize interdisciplinary collaboration, integrating bioinformatics, chemical engineering, molecular biology, and synthetic biology. The integration of genomic, proteomic, metabolomic, and environmental datasets will enable a more comprehensive understanding of the functional diversity and complexity of CYP regulatory networks. When combined with artificial intelligence and advanced big-data analytics, these multidisciplinary approaches will allow for the systematic characterization of CYP functions in plant metabolism. This will provide a robust theoretical foundation and precise tools for the engineered production of valuable secondary metabolites.

Of course, it also raises potential ethical and ecological issues, such as unintended effects on non-target organisms and the possibility of gene flow to wild relatives. We can wait to use methods such as employing tissue-specific promoters to restrict the accumulation of metabolites in the edible parts to address the above issues.

In summary, a systematic analysis of CYP genes and their regulatory networks in medicinal plants and fungi is essential for discovering novel therapeutic agents and natural products, as well as for elucidating the roles of secondary metabolites in disease treatment. As our understanding of these regulatory mechanisms deepens, continued scientific and technological advances are poised to accelerate innovations in medicine, ultimately contributing to improved human health.