Journal Article

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

Strucko, Tomas, Maury, Jérôme, Mortensen, Uffe H., Nielsen, Jens, Borodina, Irina, David, Florian, Jensen, Niels B., Forster, Jochen, Kildegaard, Kanchana R.

2014

Overview

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. EasyClone genetical toolbox allows faster development of yeast strains for biotechnological applications.

• Publisher: Blackwell Publishing Ltd,Oxford University Press,BlackWell Publishing Ltd
• Subject: bakers yeast / CASSETTES / chromosomes / Chromosomes, Fungal / CLONING / Cloning vectors / Cloning, Molecular - methods / Cre recombinase / DELTA SEQUENCES / DNA / drugs / excision / EXPRESSION / Expression vectors / Flow Cytometry / fluorescence / fluorescent proteins / Gene Expression / Gene Order / genes / Genes, Reporter / Genetic engineering / genetic markers / genetic vectors / genetically engineered microorganisms / genome editing / Homologous Recombination / integrative vectors / Metabolic engineering / molecular cloning / Mutagenesis, Insertional / PCR / Plasmids / Plasmids - genetics / protein synthesis / Proteins / Recombination / Saccharomyces cerevisiae / Saccharomyces cerevisiae - genetics / SELECTION / SHUTTLE VECTORS / STABILITY / Uracil / USER cloning / YEAST