Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

The thermotolerant methylotrophic yeastOgataea thermomethanolicaTBRC 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 (sod1Δ) and vacuolar and protein sorting (vps1Δ andypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy relatedatg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) inO.thermomethanolicaand used it to activateSOD1,VPS1andYPT7genes. 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 understandingO.thermomethanolicagenes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.