The biosynthesis of thymol,carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto–enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana. Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.

The phenolic monoterpenes of the Lamiaceae are widely used constituents of pharmaceuticals, cosmetics, and food products (1). Extracts of plants containing thymol or carvacrol are employed in medicine for their antibacterial, anti-spasmodic, antioxidant, and anti-cancer properties. Because of their pungent, warm, and aromatic odors, they also serve as additives to cosmetics and are used in aromatherapy. Thymol and carvacrol are best known as the aroma compounds of oregano and thyme, in which they provide the herbal, pizza-like tastes that are traditionally used in Mediterranean cuisine and food preservation (2). The occurrence of phenolic monoterpenes is restricted to a few genera in the Lamiaceae (Thymus, Origanum, Satureja, and Thymbra), Apiaceae (Trachyspermum), and Verbenaceae (Lippia). Of these, the essential oils of Thymus are the most important commercial source of phenolic monoterpenes (3). Thymus vulgaris L. and Origanum species also produce the structurally related monoterpenes thymohydroquinone and thymoquinone, which were first described in the essential oil of Nigella sativa L. black seed (4). Thymohydroquinone was shown to exhibit anti-cancer activity (5), and thymoquinone displays anti-inflammatory, hepatoprotective, antioxidant, cytotoxic, and anti-cancer activities (6).To date, only a few biosynthetic pathways to pharmaceutically valuable, oxidized terpenes have been completely elucidated, such as those leading to artemisinin, paclitaxel, and the phenolic, labdane-type diterpenes of sage and rosemary (7–9). For monoterpenes, a complex biosynthetic pathway has only been described for menthol and its derivatives in Mentha (10). However, the biosynthetic pathways for phenolic monoterpenes like thymol or carvacrol remain uncharacterized. Most monoterpenes are biosynthesized by fusion of the ubiquitous C₅ intermediates, isopentenyl diphosphate, and its isomer dimethylallyl diphosphate, resulting in the formation of a C₁₀ compound, geranyl diphosphate (GDP). This acyclic intermediate is the substrate for the large enzyme family of monoterpene synthases that form cyclic or acyclic products with an enormous variety of carbon skeletons (11, 12). In previous studies in thyme and oregano, the cyclic monoterpene olefin γ-terpinene was proposed as a precursor of thymol and carvacrol (Fig. 1A). Studies with ³H-labeled γ-terpinene showed that this compound was converted into thymol and carvacrol after incubation with young thyme leaves (13). Furthermore, the essential oils of plant species rich in either thymol or carvacrol have always been reported to contain substantial amounts of γ-terpinene (3). Terpene synthases forming γ-terpinene have been identified and characterized from various Lamiaceae species (14–19). In oregano, the expression of the γ-terpinene synthase OvTPS2 was found to correlate with thymol and carvacrol content in leaves (14). Beyond γ-terpinene, however, no further precursors of phenolic monoterpenes have been identified. The aromatic hydrocarbon p-cymene was suggested to be an intermediate in thymol and carvacrol formation from γ-terpinene (20), but its participation in the phenolic monoterpene pathway and the nature of the enzymes involved in formation of the aromatic ring still remain unknown. Moreover, there is little information about the conversion of the thymol and carvacrol to thymohydroquinone.Open in a separate windowFig. 1.T. vulgaris is composed of different monoterpene chemotypes. (A) Proposed biosynthetic pathway to the phenolic monoterpenes thymol and carvacrol as well as thymohydroquinone and thymoquinone. γ-terpinene and p-cymene were suggested as intermediates in the formation of thymol and carvacrol (17). (B) Essential oil composition of the T. vulgaris chemotypes dominated by carvacrol (C type), thymol (T type), and geraniol (G type). Terpenes were extracted with hexane and analyzed by GC-MS. The following terpenes were identified: 1, α-thujene; 2, α-pinene; 3, myrcene; 4, α-terpinene; 5, p-cymene; 6, γ-terpinene; 7, cis-sabinene hydrate; 8, linalool; 9, nerol; 10, neral; 11, thymoquinone; 12, geraniol; 13, geranial; 14, thymol; 15, carvacrol; 16, geranyl acetate; 17, (E)-β-caryophyllene; 18, thymohydroquinone; and 19, germacrene D. Nonyl acetate (10 µl/mL) was added as internal standard (IS) for quantification.In this study, we investigated the biosynthetic pathway leading to the formation of thymol, carvacrol, and thymohydroquinone in thyme and oregano. We isolated and characterized six cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily from thyme and oregano accessions producing high levels of thymol and carvacrol. When these CYP genes were heterologously expressed and combined with a short-chain dehydrogenase from thyme in vitro or coexpressed in vivo in Nicotiana benthamiana, thymol or carvacrol were formed. Based on the characteristics of the expressed enzymes and their reaction with other substrates, we constructed the biosynthetic pathway leading to thymol and inferred the nature of unstable intermediates. Furthermore, we identified and characterized two P450s of the CYP76S and CYP736A subfamilies that hydroxylate thymol and carvacrol to thymohydroquinone when expressed in vivo in yeast and in N. benthamiana.