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Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system
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Document Title
Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system
Author
Kruasuwan W., Puseenam A., Phithakrotchanakoon C., Tanapongpipat S., Roongsawang N.
Name from Authors Collection
Scopus Author ID
26427756600
Scopus Author ID
6602764100
Affiliations
Microbial Cell Factory Research Team, Microbial Biotechnology and Biochemicals Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Nueng, Khlong Luang, Pathum Thani, Thailand; Microbial Systems and Computational Biology Research Team, Thailand Bioresource Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Nueng, Khlong Luang, Pathum Thani, Thailand
Type
Article
Source Title
PLoS ONE
ISSN
19326203
Year
2021
Volume
16
Issue
44448
Open Access
All Open Access, Gold, Green
Publisher
Public Library of Science
DOI
10.1371/journal.pone.0258005
Format
Abstract
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 (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ 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. © 2021 Kruasuwan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
National Center for Genetic Engineering and Biotechnology; Thailand Science Research and Innovation
Funding Number
P-18-51484, RSA6180011
License
N/A
Rights
N/A
Publication Source
Scopus