牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

Combinational expression of geranylgeranyl diphosphate synthase and taxadiene synthase in Coprinopsis cinerea

摘要:

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物，是一种重要的新型抗癌药物。然而，紫杉醇在植物中含量低且难提取，限制了高效应用。利用基因工程手段，借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径，构建含有牻牛儿基牻牛儿基焦磷酸 (Geranylgeranyl diphosphate, GGPP) 合酶和紫杉二烯合酶的融合基因表达载体pBgGGTS和独立表达盒表达载体pBgGGgTS，并分别转入灰盖鬼伞LT2菌株中，经过选择性筛选、PCR鉴定、Southern blotting杂交验证，分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株，分别提取菌丝体和发酵液分析。GC-MS分析表明，两种工程菌株与原出发菌株的菌丝提取物无明显差异峰，而与出发菌株的发酵液提取物相比，两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰，采用GC-MS特征质量离子分析方法判定为紫杉二烯，分别为44 ng/L (转化pBgGGgTS) 和30 ng/L (转化pBgGGTS)。结果表明，通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因，可以生物合成紫杉二烯。

Abstract:

Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.