在Pichia pastoris中建立和应用RNAi系统
Shupeng Ruan1,2, Chenfeng He1,2, Aoxue Wang1,2
1Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Frontiers in bioengineering and biotechnology
|March 20, 2025
概括
对于代谢工程,RNA干扰 (RNAi) 能够在Pichia pastoris中更快地抑制基因. 这种方法增强异质蛋白质分泌和3-基酸生产,优化酵母作为微生物细胞工厂.
科学领域:
- 微生物代谢工程微生物代谢工程
- 合成生物学 合成生物学
- 分子生物学分子生物学
背景情况:
- 减少内源基因表达对于微生物代谢工程至关重要.
- 传统的基因淘汰和抑制方法耗时且复杂.
- RNA干扰 (RNAi) 提供了一个快速的基因调节方法,使用头RNA等离子体.
研究的目的:
- 在Pichia pastoris中建立和评估RNA干扰 (RNAi) 系统,以有效抑制基因.
- 研究单基因和双基因抑制对异质蛋白质分泌的影响.
- 通过抑制脂肪酸合成,评估RNAi在增强3-基酸 (3-HP) 生产中的效用.
主要方法:
- 在Pichia pastoris中开发了一个RNAi系统,使用编码毛RNA的等离子体,针对特定的内源基因.
- 使用表达细胞表面EGFP的记者菌株量化蛋白质分泌水平.
- 包括YAP1,YPS1,PRB1和PEP4在内的关键基因是目标,以及参与脂肪酸合成的基因.
主要成果:
- 抑制YAP1和YPS1显著降低了EGFP的表现 (分别为83%和48.8%).
- 抑制PRB1和PEP4增加了EGFP的表现 (分别为33.8%和26.5%),表明蛋白质分泌的调节.
- 通过RNAi介导的脂肪酸合成抑制成功地改善了3-HP的生产.
结论:
- 该研究成功地在Pichia pastoris中建立了功能性RNAi系统,以有效调节基因.
- RNAi为优化异质蛋白分泌和代谢途径提供了快速有效的工具.
- 这种RNAi系统具有很大的潜力,可以在工业应用中推进Pichia pastoris作为微生物细胞工厂.
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