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

研究了以米曲霉为寄主的生物生产体系的构建。在a-淀粉酶启动子下，对新克隆的茄孢霉PKS基因pksN进行了表达。在A. oryzae转化过程中产生了新的聚酮化合物alternapyrone，该化合物在十羟基碳主链上由蛋氨酸衍生出8个甲基。为了分析萘吡喃合成酶WA的功能，我们表达了WA PKS的修饰衍生物。结果发现，目前认为是硫酯酶基序的c端区催化clisen型环化，参与聚酮乙烯链的环化/芳构化。通过烟曲霉alb1和ayg1基因的共表达实验，发现alb1基因编码萘吡喃YWA1的PKS， ayg1编码将YWA1转化为1,3,6,8 -四羟基萘的Ayglp酶。Ayglp过表达、纯化和表征。为了检测植物生物合成酶的表达，构建了氧化角鲨烯环化酶表达质粒，并将其引入米芽孢杆菌中。氧化二烯环化酶未见表达。

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.