TY - JOUR

T1 - The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase

AU - Krause, Sandra T.

AU - Liao, Pan

AU - Crocoll, Christoph

AU - Boachon, Benoit

AU - Foerster, Christiane

AU - Leidecker, Franziska

AU - Wiese, Natalie

AU - Zhao, Dongyan

AU - Wood, Joshua C.

AU - Buell, C. Robin

AU - Gershenzon, Jonathan

AU - Dudareva, Natalia

AU - Degenhardt, Joerg

PY - 2021

Y1 - 2021

N2 - 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 gamma-terpinene, followed by a series of oxidations via p-cymene. Here, we show that gamma-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 gamma-terpinene in in vitro assays or in vivo in Nicotiana ben-thamiana 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 gamma-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.

AB - 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 gamma-terpinene, followed by a series of oxidations via p-cymene. Here, we show that gamma-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 gamma-terpinene in in vitro assays or in vivo in Nicotiana ben-thamiana 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 gamma-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.

KW - aromatic monoterpenes

KW - Lamiaceae

KW - carvacrol

KW - thymol

KW - thymohydroquinone

KW - GAMMA-TERPINENE SYNTHASE

KW - FUNCTIONAL-CHARACTERIZATION

KW - MONOTERPENES

KW - METABOLISM

KW - EXPRESSION

KW - ENZYME

KW - THYMOQUINONE

KW - CLONING

KW - HYDROXYLATION

KW - (+)-SABINENE

U2 - 10.1073/pnas.2110092118

DO - 10.1073/pnas.2110092118

M3 - Journal article

C2 - 34930840

VL - 118

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 52

M1 - e2110092118

ER -