生物
异源的
清脆的
分泌蛋白
分泌物
内质网
突变体
未折叠蛋白反应
细胞生物学
分泌途径
Cas9
异源表达
基因
生物化学
高尔基体
重组DNA
作者
Worarat Kruasuwan,Aekkachai Puseenam,Chitwadee Phithakrotchanakoon,Sutipa Tanapongpipat,Niran Roongsawang
出处
期刊:PLOS ONE
[Public Library of Science]
日期:2021-09-28
卷期号:16 (9): e0258005-e0258005
被引量:3
标识
DOI:10.1371/journal.pone.0258005
摘要
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress ( sod1 Δ) and vacuolar and protein sorting ( vps1 Δ and ypt7 Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12 Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O . thermomethanolica and used it to activate SOD1 , VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O . thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.
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