多形汉逊酵母启动子的挖掘

Evaluation of promoters from Ogataea polymorpha

多形汉逊酵母可以利用甲醇作为唯一的碳源和能量来源,是构建微生物细胞工厂的潜在宿主,在代谢工程改造和重组蛋白生产中引起了广泛关注。在合成生物学研究和代谢工程改造过程中,通常需要改变相关基因的转录水平来调节代谢通量,而这一过程需要借助不同种类、不同表达强度的启动子来实现。因此,对汉逊酵母糖酵解途径和活性氧(ROS)防御途径相关基因启动子进行挖掘,将其与GFPuv融合,通过测定荧光值的大小,分析汉逊酵母中相关启动子对不同碳源的响应情况及其表达强度。结果发现:在汉逊酵母中,含有多种不同强度的甲醇诱导型和组成型启动子,包括:强甲醇诱导型启动子P_(SOD1),强组成型启动子P_(ADH21)和P_(GAP);中等强度甲醇诱导型启动子P_(PGM2)、P_(SOD3)和P_(SOD2),中等强度组成型启动子P_(PFK2)、P_(GPI)和P_(ADH22);弱甲醇诱导型启动子P_(GSH)、P_(MSR)和弱组成型启动子P_(PGM1)、P_(SOD4)和P_(GPX)。这一研究结果将促进多形汉逊酵母在代谢工程改造和相关基础研究中的应用,为实现汉逊酵母细胞工厂提供理论依据。

Ogataea polymorpha represents for a promising host for cell factory construction as its native assimilation of methanol as the sole carbon and energy source,and attracts numerous attentions in metabolic engineering and recombination protein production.As for synthetic biology and metabolic engineering,regulation of pathway flux is achieved at the transcriptional level via multiple promoters with different strengths.Hence,in this study,related promoters in glycolysis and reactive oxygen(ROS)defense pathway from O.polymorpha was evaluated,and responses to various carbon sources and strengths of these promoters are all analyzed by detecting fluorescence intensity while fusing with GFPuv.Results discovered numerous induced and constitutive promoters with different strengths in O.polymorpha,including strong induced promoter P_(SOD1),strong constitutive promoter P_(ADH2⁃1) and P_(GAP),medium induced promoter P_(PGM2),P_(SOD3) and P_(SOD2),medium constitutive promoter P_(PFK2),P_(GPI) and P_(ADH2⁃2),weak induced promoter P_(GSH),P_(MSR) and weak constitutive promoter P_(PGM1),P_(SOD4) and P_(GPX).All these findings contributed to promoting applications of O.polymorpha in metabolic engineering and related fundamental research,and provided theoretical basis for cell factory construction in O.polymorpha.