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 terpenes of leaves, pollen, and nectar of thyme (Thymus vulgaris) inhibit growth of bee disease-associated microbes.

Honey bees are highly prone to infectious diseases, causing colony losses in the worst case. However, they combat diseases through a combination of their innate immune system and social defence behaviours like foraging for health-enhancing plant products (e.g. nectar, pollen and resin). Plant secondary metabolites are not only highly active against bacteria and fungi, they might even enhance selective foraging and feeding decisions in the colony. Here, we tested six major plant terpenes and their corresponding acetates, characterizing six natural Thymus vulgaris chemotypes, for their antimicrobial activity on bacteria associated with European foulbrood. Comparison of the inhibitory activity revealed the highest activity for carvacrol and thymol whereas the acetates mostly did not inhibit bacterial growth. All terpenes and acetates are present in the nectar and pollen of thyme, with pollen containing concentrations higher by several orders of magnitude. The physiological response was tested on forager and freshly emerged bees by means of antennal electroantennography. Both responded much stronger to geraniol and trans-sabinene hydrate compared to carvacrol and thymol. In conclusion, bee-forageable thyme product terpenes (mainly from pollen) yield effective antibiotic activity by reducing the growth of bee disease-associated bacteria and can be detected with different response levels by the honey bees' antennae. This is a further step forward in understanding the complex pathogen-pollinator-plant network.