Department of Food Engineering, Erzincan Binali Yıldırım University, Erzincan, Türkiye.
Appl Microbiol Biotechnol. 2022 Nov;106(21):7073-7083. doi: 10.1007/s00253-022-12183-8. Epub 2022 Sep 27.
Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters (Kluyveromyces marxianus TPI, Hansenula polymorpha PMA, Candida tropicalis ICL, and Saccharomyces cerevisiae CUP) in Pichia pastoris. The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI, glucose for CUP, and ethanol for ICL. The DNA region showing the highest activity was determined as 1000 bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters. KEY POINTS: • Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host. • HpPMA has the highest promoter activity among the heterologous promoters tested. • Reporter gene expression with ScCUP is responsive to elevating Cuin P. pastoris.
启动子选择是重组蛋白生产的重要步骤，它直接决定了重组蛋白的表达方式是组成型还是诱导型，以及表达水平。本研究旨在探讨异源酵母启动子（克鲁维酵母 TPI、多形汉逊酵母 PMA、热带假丝酵母 ICL 和酿酒酵母 CUP）在毕赤酵母中的适用性。研究发现，CtICL 和 ScCUP 启动子在毕赤酵母中的调控模式为诱导型，而 KmTPI 和 HpPMA 启动子为组成型。通过启动子缺失分析，确定了各启动子在毕赤酵母中表现出最高活性的碳源分别为 PMA 和 TPI 的甘油、CUP 的葡萄糖和 ICL 的乙醇。研究结果表明，在毕赤酵母表达系统中，诱导型和组成型异源启动子具有表达不同条件下基因的潜力，为常用启动子提供了替代选择。
异源启动子在毕赤酵母中的表达模式与其天然宿主相似。
在测试的异源启动子中，HpPMA 的启动子活性最高。
ScCUP 表达报告基因对毕赤酵母中 Cu 的升高有响应。
