Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain

Abstract Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene target...

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Autores principales:	Yumiko Nambu-Nishida, Keiji Nishida, Tomohisa Hasunuma, Akihiko Kondo
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a640bae8ea944b59b39961a085ac05ee
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spelling	oai:doaj.org-article:a640bae8ea944b59b39961a085ac05ee2021-12-02T11:52:55ZDevelopment of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain10.1038/s41598-017-08356-52045-2322https://doaj.org/article/a640bae8ea944b59b39961a085ac05ee2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08356-5https://doaj.org/toc/2045-2322Abstract Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (~50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.Yumiko Nambu-NishidaKeiji NishidaTomohisa HasunumaAkihiko KondoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yumiko Nambu-Nishida Keiji Nishida Tomohisa Hasunuma Akihiko Kondo Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
description	Abstract Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (~50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.
format	article
author	Yumiko Nambu-Nishida Keiji Nishida Tomohisa Hasunuma Akihiko Kondo
author_facet	Yumiko Nambu-Nishida Keiji Nishida Tomohisa Hasunuma Akihiko Kondo
author_sort	Yumiko Nambu-Nishida
title	Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
title_short	Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
title_full	Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
title_fullStr	Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
title_full_unstemmed	Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain
title_sort	development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant kluyveromyces marxianus yeast strain
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/a640bae8ea944b59b39961a085ac05ee
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Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain

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