Regulatory Mechanisms of Terpene Biosynthesis in Plant Cultured Cells

1.研究了兴安落叶松愈伤组织的无细胞提取物催化焦磷酸法尼酯(FPP)环化成含α-柏木烯的倍半萜类化合物。无细胞提取物能特异性地将FPP的2Z-异构体转化为α-雪松烯。用离心法对无细胞提取物进行分级。在2000克上清液中有生成α-雪松烯的活性，而在46,200克上清液中没有。酶活可被洗涤剂增溶，但不能被EDTA增溶。结果表明，该酶为两亲性或疏水性的膜蛋白。采用GC和GC/MS联用技术，采用手性毛细管色谱柱，确定了细叶落叶松愈伤组织中积累的α-雪松烯的绝对构型为(+)-α-异构体。在自然界中首次证实了(+)-α-雪松的存在。酶的这些性质和形成(+)-α-柏木烯的立体化学过程与报道的完整植物中的酶明显不同。分离了地钱悬浮细胞产生的下萜类化合物。(-)-(1S)-7-甲氧基-1，2-二氢杜松烯、(+)-(1S，4R)-7-甲氧基龙骨烯(诺贝尔化合物)、(+)-(1S，4R)-羟基龙骨烯(诺贝尔化合物)和7-甲氧基龙骨烯(7-甲氧基-1，2-二氢杜松烯)的结构由光谱数据和化学关联确定。对特定的重氢甲戊酸酯的进料实验表明，在生物合成杜松烯衍生物的过程中发生了几次氢化物移动。从悬浮细胞中提取的无细胞提取物将FPP的6E-异构体特异性地转化为Cadinane类型的倍半萜。紫苏愈伤组织在形成双酚类化合物的过程中发生了氢化物漂移，这是通过特殊的重氢甲基戊酸盐类化合物的饲喂实验证实的。

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.