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

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 enginee...

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2021-09, Vol.16 (9), p.e0258005-e0258005
Main Authors: Kruasuwan, Worarat, Puseenam, Aekkachai, Phithakrotchanakoon, Chitwadee, Tanapongpipat, Sutipa, Roongsawang, Niran
Format: Article
Language:English
Subjects:
Antibiotics
Ascomycota
Autophagy
Bacteria, Thermophilic
Biology and Life Sciences
Biotechnology
CRISPR
Endoplasmic reticulum
Fungi
Gene expression
Genes
Genetic aspects
Genetic engineering
Genomes
Mutagenesis
Mutants
Oxidative stress
Phagocytosis
Physiological aspects
Phytase
Plasmids
Protein biosynthesis
Protein folding
Protein research
Protein transport
Proteins
Research and Analysis Methods
Secretion
Superoxide dismutase
Transcription activation
Xylanase
Yeast
Yeasts
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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[DELTA]) and vacuolar and protein sorting (vps1[DELTA] and ypt7[DELTA]) genes. However, xylanase secretion was unaffected in an autophagy related atg12[DELTA] 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0258005