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

ZHANG Zhen-yang;YANG Yan-kun;ZHAN Chun-jun;LI Xiang;LIU Xiu-xia;BAI Zhong-hu;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University;The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University;The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University;  
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( PAS_chr3_ 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.
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