Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa

Abstract We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura−) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUM...

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Autores principales:	Yuki Kasai, Takuya Tsukahara, Fukiko Ikeda, Yoko Ide, Shigeaki Harayama
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8ba167cf36e2418999530200e8cb775f
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spelling	oai:doaj.org-article:8ba167cf36e2418999530200e8cb775f2021-12-02T15:07:46ZMetabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa10.1038/s41598-018-30254-72045-2322https://doaj.org/article/8ba167cf36e2418999530200e8cb775f2018-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-30254-7https://doaj.org/toc/2045-2322Abstract We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura−) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura− strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker–gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.Yuki KasaiTakuya TsukaharaFukiko IkedaYoko IdeShigeaki HarayamaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-11 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yuki Kasai Takuya Tsukahara Fukiko Ikeda Yoko Ide Shigeaki Harayama Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
description	Abstract We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura−) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura− strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker–gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.
format	article
author	Yuki Kasai Takuya Tsukahara Fukiko Ikeda Yoko Ide Shigeaki Harayama
author_facet	Yuki Kasai Takuya Tsukahara Fukiko Ikeda Yoko Ide Shigeaki Harayama
author_sort	Yuki Kasai
title	Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
title_short	Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
title_full	Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
title_fullStr	Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
title_full_unstemmed	Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa
title_sort	metabolic engineering using iterative self-cloning to improve lipid productivity in coccomyxa
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/8ba167cf36e2418999530200e8cb775f
work_keys_str_mv	AT yukikasai metabolicengineeringusingiterativeselfcloningtoimprovelipidproductivityincoccomyxa AT takuyatsukahara metabolicengineeringusingiterativeselfcloningtoimprovelipidproductivityincoccomyxa AT fukikoikeda metabolicengineeringusingiterativeselfcloningtoimprovelipidproductivityincoccomyxa AT yokoide metabolicengineeringusingiterativeselfcloningtoimprovelipidproductivityincoccomyxa AT shigeakiharayama metabolicengineeringusingiterativeselfcloningtoimprovelipidproductivityincoccomyxa
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Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa

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