Department of agricultural and natural resources biotechnology, University of Tehran, Karaj, Iran.
Protein Research Center, Shahid Beheshti University, G.C., Tehran, Iran.
Enzyme Microb Technol. 2020 Sep;139:109582. doi: 10.1016/j.enzmictec.2020.109582. Epub 2020 Apr 22.
AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.
AOX1 启动子（pAOX1）是一种强诱导启动子，非常适合在毕赤酵母（Pichia pastoris）中生产重组蛋白。然而，该启动子存在其他碳源抑制和甲醇诱导的显著缺点，甲醇是一种有火灾危险的化学物质，不适合工业生产。因此，非常需要新型强调控启动子。在本研究中，探索了甲醇氧化酶基因（pMOX）的启动子区域，用于蛋白酶缺陷型和野生型毕赤酵母菌株中外源蛋白的异源表达。分离了 MOX 的启动子区域，并将其替换为 pPINK-HC 质粒中的 pAOX1。使用内切葡聚糖酶 3（CMC3）和内切葡聚糖酶 II（EgII）作为报告蛋白，比较了 pMOX 和 pAOX1 的活性。对碳源对 pMOX 活性的影响进行评估，结果表明在存在木糖和山梨糖醇的情况下完全失活，而甘油、葡萄糖和甲醇进料则具有高活性。此外，结果表明，增加基因剂量和使用缺乏蛋白酶的特性，在 pMOX 控制下显著增加了 CMC3 和 EgII 的表达。总之，在本研究中，为酵母毕赤酵母（P. pastoris）中的重组蛋白生产引入了一种新的小型强甲醇自由启动子。
