Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Department of Biotechnology, College of Science, University of Tehran, Iran.
Iran Biomed J. 2021 Jul 1;25(4):255-64. doi: 10.52547/ibj.25.4.255.
The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized.
A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains.
The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.
The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.
甲醇营养型酵母毕赤酵母是各种重组蛋白（包括生物制剂）的有吸引力的生产宿主。从这个意义上说，已经实施了各种基于遗传和非遗传的技术来提高这个表达平台的生产效率。缺失抑制（Los1）编码酿酒酵母中一种非必需的核 tRNA 输出蛋白，其缺失可延长复制寿命。在此，生成并表征了毕赤酵母的 los1 缺陷株。
制备基因敲除盒并转化到表达抗 CD22 的毕赤酵母菌株中。分离并确认了一个 δ los1 突变体。还评估了突变体的药物敏感性。比较了亲本和突变菌株之间的生长模式和抗 CD22 单链可变片段（scFv）表达水平。
发现 los1 同源物在毕赤酵母中是一个非必需基因。此外，los1 缺失株对蛋白质合成抑制剂的敏感性发生了改变。该菌株对外源表达的抗 CD22 scFv 的水平提高了约 1.85 倍（p < 0.05）。δ los1 和亲本菌株分泌的总蛋白的最大浓度分别为 125 mg/L 和 68 mg/L。
所提供的数据表明，毕赤酵母中 los1 同源物的靶向敲除可导致目标蛋白的表达水平提高。本研究的结果可能会改进用于优化重组毕赤酵母菌株生产力的现有策略。
