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.