ArticleFEMS yeast research2014
EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae.
Article in FEMS yeast research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 137 papers.
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137 citing papers in PubMed.
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- Reprogramming yeast metabolism for customized starch-rich micro-grain through low-carbon microbial manufacturing.Nature communications · 2025Article
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- BAHD acyltransferase from dragon fruit enables production of phyllocactin in engineered yeast.FEMS yeast research · 2025Article
- Exploring the interplay between yeast cell membrane lipid adaptation and physiological response to acetic acid stress.Applied and environmental microbiology · 2024Article
- Two ubiquitous aldo-keto reductases in the genus Papaver support a patchwork model for morphine pathway evolution.Communications biology · 2024Article
77 more citing papers are in PubMed but not listed here.
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9 authors.
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Abstract
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 Log10 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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