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EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expressio...

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Autores principales: Jensen, Niels B, Strucko, Tomas, Kildegaard, Kanchana R, David, Florian, Maury, Jérôme, Mortensen, Uffe H, Forster, Jochen, Nielsen, Jens, Borodina, Irina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BlackWell Publishing Ltd 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282123/
https://www.ncbi.nlm.nih.gov/pubmed/24151867
http://dx.doi.org/10.1111/1567-1364.12118
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author Jensen, Niels B
Strucko, Tomas
Kildegaard, Kanchana R
David, Florian
Maury, Jérôme
Mortensen, Uffe H
Forster, Jochen
Nielsen, Jens
Borodina, Irina
author_facet Jensen, Niels B
Strucko, Tomas
Kildegaard, Kanchana R
David, Florian
Maury, Jérôme
Mortensen, Uffe H
Forster, Jochen
Nielsen, Jens
Borodina, Irina
author_sort Jensen, Niels B
collection PubMed
description Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
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spelling pubmed-42821232015-01-15 EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae Jensen, Niels B Strucko, Tomas Kildegaard, Kanchana R David, Florian Maury, Jérôme Mortensen, Uffe H Forster, Jochen Nielsen, Jens Borodina, Irina FEMS Yeast Res Research Articles Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out. BlackWell Publishing Ltd 2014-03 2013-11-18 /pmc/articles/PMC4282123/ /pubmed/24151867 http://dx.doi.org/10.1111/1567-1364.12118 Text en © 2013 The Authors FEMS Yeast Research published by John Wiley & Sons on behalf of the European Federation of Microbiological Societies http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Jensen, Niels B
Strucko, Tomas
Kildegaard, Kanchana R
David, Florian
Maury, Jérôme
Mortensen, Uffe H
Forster, Jochen
Nielsen, Jens
Borodina, Irina
EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title_full EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title_fullStr EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title_full_unstemmed EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title_short EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae
title_sort easyclone: method for iterative chromosomal integration of multiple genes in saccharomyces cerevisiae
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282123/
https://www.ncbi.nlm.nih.gov/pubmed/24151867
http://dx.doi.org/10.1111/1567-1364.12118
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