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Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea
BACKGROUND: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489027/ https://www.ncbi.nlm.nih.gov/pubmed/26140053 http://dx.doi.org/10.1186/s13068-015-0277-0 |
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author | Kasai, Yuki Oshima, Kohei Ikeda, Fukiko Abe, Jun Yoshimitsu, Yuya Harayama, Shigeaki |
author_facet | Kasai, Yuki Oshima, Kohei Ikeda, Fukiko Abe, Jun Yoshimitsu, Yuya Harayama, Shigeaki |
author_sort | Kasai, Yuki |
collection | PubMed |
description | BACKGROUND: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. RESULTS: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. CONCLUSIONS: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0277-0) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4489027 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-44890272015-07-03 Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea Kasai, Yuki Oshima, Kohei Ikeda, Fukiko Abe, Jun Yoshimitsu, Yuya Harayama, Shigeaki Biotechnol Biofuels Research Article BACKGROUND: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. RESULTS: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. CONCLUSIONS: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0277-0) contains supplementary material, which is available to authorized users. BioMed Central 2015-06-30 /pmc/articles/PMC4489027/ /pubmed/26140053 http://dx.doi.org/10.1186/s13068-015-0277-0 Text en © Kasai et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Kasai, Yuki Oshima, Kohei Ikeda, Fukiko Abe, Jun Yoshimitsu, Yuya Harayama, Shigeaki Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title | Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title_full | Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title_fullStr | Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title_full_unstemmed | Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title_short | Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea |
title_sort | construction of a self-cloning system in the unicellular green alga pseudochoricystis ellipsoidea |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489027/ https://www.ncbi.nlm.nih.gov/pubmed/26140053 http://dx.doi.org/10.1186/s13068-015-0277-0 |
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