A novel synthetic pathway for tropolone ring formation via the olefin monoterpene intermediate terpinolene in cultured Cupressus lusitanica cells.
β-Thujaplicin is a wood monoterpene and tropolone compound with a unique conjugated 7-membered ring. Because of its strong antifungal and antitumor activities, β-thujaplicin is used in several fields. The biosynthesis pathway of β-thujaplicin has not yet been elucidated. Using Cupressus lusitanica cell cultures in a radioisotope feeding experiment, our group previously demonstrated that geranyl pyrophosphate (GPP) is the starting material of β-thujaplicin biosynthesis. The results of our previous terpene synthase assay suggested that terpinolene is the first olefin terpenoid intermediate from GPP to β-thujaplicin, although there was no experimental evidence of this at that time. In the present study, we fed deuterium-labeled terpinolene to cultured C. lusitanica cells to determine whether terpinolene is an intermediate metabolite of β-thujaplicin biosynthesis. A gas chromatography-mass spectroscopy analysis of the cell extracts from labeled terpinolene cultures revealed a peak of labeled β-thujaplicin that was not observed after treatment with non-labeled terpinolene. The identification of labeled β-thujaplicin was also performed by mass spectrum assignment. The outcome indicated that terpinolene is indeed an intermediate metabolite of β-thujaplicin biosynthesis. To the best of our knowledge, there has been no prior report that tropolone compounds are biosynthesized via a terpene biosynthesis system, and our results thus suggest the existence of a novel biosynthetic pathway that produces the conjugated 7-membered ring.
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: koki-fujita@agr.kyushu-u.ac.jp.
Koki
K
Fujita
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.
Yasufumi
Y
Bunyu
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.
Ken'ich
K
Kuroda
Department of Environment, Faculty of Agriculture, Yamagata University, Wakaba 1-23, Tsuruoka 997 0037, Japan.
Tatsuya
T
Ashitani
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.
Jun
J
Shigeto
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.
Yuji
Y
Tsutsumi
D002457:Cell Extracts; D002478:Cells, Cultured; D029781:Cupressus / Q000201:enzymology / Q000378:metabolism*; D000081005:Cyclohexane Monoterpenes; D003903:Deuterium / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D039821:Monoterpenes / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*; D014334:Tropolone / Q000031:analogs & derivatives* / Q000378:metabolism*
D002457:Cell Extracts; D000081005:Cyclohexane Monoterpenes; D039821:Monoterpenes; D013729:Terpenes; D014334:Tropolone; D003903:Deuterium; C027009:terpinolene; C009479:beta-thujaplicin
Biosynthesis; Cupressus lusitanica; Terpene; Tropolone; β-Thujaplicin