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...

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Autores principales:	Worarat Kruasuwan, Aekkachai Puseenam, Chitwadee Phithakrotchanakoon, Sutipa Tanapongpipat, Niran Roongsawang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2f0d6faccf15452a96ffe9e6c7669445
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spelling	oai:doaj.org-article:2f0d6faccf15452a96ffe9e6c76694452021-12-02T20:17:27ZModulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.1932-620310.1371/journal.pone.0258005https://doaj.org/article/2f0d6faccf15452a96ffe9e6c76694452021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258005https://doaj.org/toc/1932-6203The 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.Worarat KruasuwanAekkachai PuseenamChitwadee PhithakrotchanakoonSutipa TanapongpipatNiran RoongsawangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 9, p e0258005 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Worarat Kruasuwan Aekkachai Puseenam Chitwadee Phithakrotchanakoon Sutipa Tanapongpipat Niran Roongsawang Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
description	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.
format	article
author	Worarat Kruasuwan Aekkachai Puseenam Chitwadee Phithakrotchanakoon Sutipa Tanapongpipat Niran Roongsawang
author_facet	Worarat Kruasuwan Aekkachai Puseenam Chitwadee Phithakrotchanakoon Sutipa Tanapongpipat Niran Roongsawang
author_sort	Worarat Kruasuwan
title	Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
title_short	Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
title_full	Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
title_fullStr	Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
title_full_unstemmed	Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.
title_sort	modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast ogataea thermomethanolica tbrc 656 by crispr-cas9 system.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/2f0d6faccf15452a96ffe9e6c7669445
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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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