毕赤酵母组成型高效表达启动子的筛选鉴定

为获得高产量、高活性的葡萄糖氧化酶(GOD)酵母表达菌株,优化了GOD的T132S/T56V双突变编码序列,将之克隆到表达载体pGAP_k上得到表达载体pGAP_k-h₂-GOD,以pGAP启动GOD基因的表达。将pGAP_k-h₂-GOD上的pGAP启动子替换为pGCW14和pAOX1,并对pGCW14启动子进行改造,得到3种pGCW14的改造体,最终得到包括pGAP_k-h₂-GOD在内的6种不同启动子的重组表达载体。将这6种载体转化毕赤酵母GS115,建立了一种简便高效的筛选方法初步筛选出GOD重组菌株,再对筛选到的转化子进行发酵培养,测定发酵上清液的GOD酶活力,以此来比较各种启动子启动效率的强弱。结果显示:pGCW14的启动效率是pGAP的3~5倍,改造后的启动子pGCW14+G20A/C-467T(0.767)和pGCW14-UA(0.689)的启动效率较原始的pGCW14(0.574)都有明显的提高,尤其pGCW14+G20A/C-467T启动效率最高,比原始pGCW14提高了32.5%左右。与诱导型启动子pAOX1(1.187)相比,pGCW14+G20A/C-467T(1.109)的启动效率仅比其低6.6%左右。结论:改造后的启动子可望在毕赤酵母组成型高效表达的研究应用中具有一定的前景。

Screening and Identification of Constitutive Promoter for High-Level Expression in Pichia pastoris

In order to get a yeast strain which express glucose oxidase(GOD)with higher production and activity,the double mutanted GOD encoding sequence T132S/T56V was optimized and cloned into pGAPk,got pGAPk-h2-GOD which express GOD gene by pGAP promoter. The promoter pGAP of pGAPk-h2-GOD was replaced with pGCW14 and pAOX1,then pGCW14 promoter was modified into 3 variants,totally obtained 6 different vectors including pGAPk-h2-GOD. The vectors was transformed into Pichia pastoris GS115,and a simple and efficient approach was used for initial screening of recombinant GOD strains. The transformants was cultured to measure their GOD enzyme activity of their fermentation supernatant,the efficiency of their promoters was compared based on the GOD enzyme activity. The results showed that the efficiency of pGCW14 promoter was 3~5 times more than pGAP,both the efficiency of promoter pGCW14+G20A/C-467T(0.767)and pGCW14-UA(0.689)were better than that of the original pGCW14(0.574). Especially the efficiency of pGCW14+G20A/C-467T was the highest which increased by 32.5% than that of the original pGCW14.The efficiency of pGCW14 G20A/C-467T1.109)was only 6.6% lower than that of induced promoter pAOX1(1.187). Conclusion:the efficiency of the modified promoters are significantly improved,which is expected to have a certain prospect in the research and application of constitutive high efficient expression of Pichia pastoris.