Regulatory Mechanisms of Terpene Biosynthesis in Plant Cultured Cells

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1. A Cyclization of farnesyl pyrophophate (FPP) to bisabolane class sesquiterpenes including alpha-cedrene by cell-free extract from Larix leptolepis callus was investigated. The cell-free extracts specifically converted the 2Z-isomers of FPP to form alpha- cedrene. The cell-free extract was fractionated by centrifugation. The activity to form alpha-cedrene was present in 2000 g supernatant, but absent in 46,200 g supernatant. The activity was solubilized by detergents, but not EDTA. The results suggested that the enzyme is amphipathic or hydrophobic membrane protein. The absolute configuration of alpha-cedrene which is accumulated in Larix leptolepis callus was determined as (+)-alpha- isomer by GC and GC/MS using a chiral capillary column. The occurrence of (+)-alpha-cedrene was confirmed for the first time in nature. These properties of enzymes and a stereochemical course to form (+)-alpha-cedrene were markedly different from those reported for the enzymes from intact plants.2. Lower terpenes produced by liverwort (Heteroscyphus planus) suspension cells were isolated. Structures of (-)-(1S)-7-Methoxy- 1,2-dihydrocadalene, (+)-(1S, 4R)-7-methoxycalamenene (a nobel compound), (+)-(1S,4R)-hydroxycalamenene (a nobel compound), and 7-methoxycadalene were determined by their spectral data and the chemical correlation. Feeding experiments of the specifically deuterated mevalonates indicated several hydride shifts during biosynthesis of cadalene derivatives. A cell-free extract from the suspension cells specifically converted the 6E-isomers of FPP to form sesquiterpenes of cadinane-type.3. The hydride shifts during the formation of bisabolane class compound in Perilla (Akachirimenshiso) callus were confirmed by the feeding experiment using the specifically deuterated mevalonates.