Scientific research on elucidation of terpene-cyclic reactions in the biosynthesis of bioactive microbial terpenoids.

(1)从一株产淫羊藿碱的真菌中分离得到(+)-cyatha-3，12-diene，并对其立体结构进行了半合成研究。我们从一株大量生产褐霉素的真菌中分离得到了(+)-δ-花生油作为推测的双环烃中间体。还分离到(+)-Fusicocca-3(16)，10(14)-diene，其鉴定表明在生物合成途径中存在一个新的Fusicocenyl阳离子。(2)从杏仁拟南星GGDP合成酶基因中，通过染色体穿梭鉴定了两个与二萜生物合成相关的基因簇。这些基因簇使我们成功地分离和鉴定了两个编码二萜环酶的cDNA。(3)利用已报道的二萜环化酶基因序列，通过基于聚合酶链式反应的基因组步行技术，鉴定了蜂毒灵生物合成的基因簇。基于各种条件下的非酶环化反应和分子轨道计算，提出了APHIPDICONE骨架构筑的环化机理。(4)克隆了松油菌素生物合成所必需的两个二萜环化酶基因。通过重组酶的作用，发现一种酶能将GGDP转化为环化中间体(叔戊二烯醇二磷酸)，而另一种酶又能将其转化为萜三烯。(5)通过使用定点突变体和底物类似物，我们清楚地证明了活性中心残基的空间体积大小在多环化级联过程中决定角鲨烯底物折叠构象的重要作用。

(1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.