期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：52
DOI：10.1073/pnas.2110092118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
The monoterpene alcohols thymol, carvacrol, and thymohydroquinone are characteristic flavor compounds of thyme, oregano, and other Lamiaceae. These specialized metabolites are also valuable for their antibacterial, anti-spasmolytic, and antitumor activities. We elucidated the complete biosynthetic pathway of these compounds, which starts with the formation of γ-terpinene from geranyl diphosphate. The aromatic backbone of thymol and carvacrol is formed by P450 monooxygenases in combination with a dehydrogenase via an unstable intermediate. Additional P450s hydroxylate thymol and carvacrol to form thymohydroquinone. Our findings demonstrate a mechanism for the formation of phenolic monoterpenes that differs from previous predictions and provides targets for metabolic engineering of high-value terpenes in plants.
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.
