Construction of high efficient bio-production systems using filamentous fungi as host organism

研究了以黑曲霉为宿主菌的生物生产系统的构建。新克隆的番茄早疫病菌（Alternaria solani）聚酮合酶（PKS）基因pksN在α-淀粉酶启动子下表达。助理该化合物在十酮碳骨架上有8个甲硫氨酸衍生的甲基。为了分析萘并吡喃酮合酶WA的功能，表达了WA PKS的修饰衍生物。结果，发现迄今为止被认为是硫酯酶基序的C-末端区域催化涉及聚酮亚甲基链的环化/芳构化的Claisen型环化。通过烟曲霉alb 1和ayg 1基因的共表达实验，发现alb 1基因编码萘并吡喃酮YWA 1的PKS，ayg 1基因编码将YWA 1转化为1，3，6，8-四羟基萘的酶Ayglp。Ayglp的过表达，纯化和表征。为了研究植物生物合成酶的表达，构建了氧化角鲨烯环化酶的表达质粒，并将其导入A。- 是的然而，未检测到氧化鲨烯环化酶的表达。

Construction of biological production system using Aspergillus oryzae as a host was investigated. Newly cloned polyketide synthase (PKS) gene, pksN, from Alternaria solani, was expressed under a-amylase promoter. The A. oryzae transformant produced new polyketide compound named alternapyrone, which has 8 methyl groups derived from methionine on the decaketide carbon backbone. To analyze the function of naphthopyrone synthase WA, modified derivatives of WA PKS were expressed. As a result, the C-terminus region which was so far considered to be thioesterase motif was found to catalyze Claisen-type cyclization involved in cyclization/aromatization of polyketomethylene chain. By the co-expression experiment of alb1 and ayg1 genes from Aspergillus fumigatus, it was found that alb1 gene encodes PKS for naphthopyrone YWA1 and ayg1 encodes for the enzyme Ayglp which converts YWA1 to 1, 3, 6, 8-tetrahydroxynaphthalene. Ayglp was overexpressed, purified, and characterized. To examine the expression of plant biosynthetic enzyme, expression plasmid for oxidosqualene cyclase was constructed and introduced into A. oryzae. However, expression of oxidosqalene cyclase was not detected.