Pichia pastoris X-33 ΔGT2缓解甘油对AOX1的阻遏并用于外源蛋白的高效表达

Pichia pastoris X-33 ΔGT2 Release the Glycerol Repression on AOX1 and Ef-ficiently Express Heterologous Proteins

巴斯德毕赤酵母是甲醇酵母,作为应用最广泛的真核表达系统之一,在以甲醇为唯一碳源时可以利用醇氧化酶启动子PAOX1进行外源蛋白的表达,但是这一过程会被甘油阻遏。近几年有研究表明,甘油转运体不仅有运输甘油的功能,还与甘油、甲醇的代谢有一定的联系。目的:构建了甘油转运体GT2(PAS_chr3_1076)缺失菌株P.pastoris X-33ΔGT2,研究该菌株的甘油去阻遏效应和在不同碳源培养基中诱导PAOX1启动子驱动外源蛋白的表达水平。方法:构建以甲醇诱导型启动子PAOX1调控外源基因EGFP的表达载体PAOX1-EGFP,经酶线性化后电转野生型菌株P.pastoris X-33获得重组菌株x-EGFP;通过同源重组的方法敲除GT2基因,获得ΔGT2-EGFP敲除菌株;以ΔGT2-EGFP和X-EGFP为出发菌株,在甘油、甲醇,以及甘油甲醇混合为碳源诱导醇氧化酶AOX1及绿色荧光蛋白EGFP的表达和生长情况,并检测在以甘油为唯一碳源时,胞外的甘油含量。结果:在以甘油甲醇混合碳源培养时,突变体ΔGT2-EGFP菌株中AOX1单位酶活比野生型菌株高出近35%,单位荧光强度要高出近70%;在以甘油为唯一碳源时,X-EGFP最终收获时的生物量比ΔGT2-EGFP多,且发酵液中甘油含量相对较少;以混合碳源培养时ΔGT2总外源蛋白表达水平最高。结论:实验表明,GT2参与甘油的吸收与代谢,ΔGT2突变株可在一定程度上解除甘油对甲醇的代谢抑制,暗示甘油转运体与PAOX1相关,且基于此研究结果有望优化出更高效的酵母表达系统。

Pichia pastoris is one of the most widely used eukaryotic expression systems. P. pastoris can express heterologous proteins with methanol as the sole carbon source. However,the expression can be repressed by glycerol. As reported recently,the glycerol transporter played a part not only in transporting glycerol,but also in the regulation between glycerol and methanol metabolism. Objective： A mutant P. pastoris X-33 ΔGT2（ PASchr3 1076） was constructed,and the glycerol de-repression effects was found. Methods： The X-EGFP andΔGT2-EGFP cells were constructed respectively based X-33 wild-type strain（ WT） and ΔGT2 cells,in which the EGFP was driven by PAOX1. The biomass and expression levels of AOX1 and EGFP were tested in different carbon resources（ glycerol,methanol,glycerol plus methanol） mediums. The extracellular glycerol contents were tested. Results： The results showed that,for each OD strain,the enzyme activity of AOX1 of ΔGT2-EGFP was35% higher than that of X-EGFP,and the fluorescence of ΔGT2-EGFP was 70% higher than that of x-EGFP.The x-EGFP harvested more biomass than ΔGT2-EGFP when glycerol as the sole carbon source resulting in less glycerol contents in the culture supernatant. Conclusion： GT2 involved in uptaking and metabolism of the glycerol,and the absence of GT2 could release the repression of glycerol on AOX1,which indicated that the glycerol transporter could be related to the transcription of PAOX1. The more efficient expression system of yeast is expected to be constructed based on these results.