Construction and characterization of centromeric, episomal and GFP-containing vectors for Saccharomyces cerevisiae prototrophic strains

• Published in: Journal of biotechnology Vol. 143; no. 4; pp. 247 - 254
• Main Authors: Baruffini, Enrico, Serafini, Fausta, Lodi, Tiziana
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.09.2009; [New York, NY]: Elsevier; Elsevier
• Subjects: antibiotic resistance; Antifungal Agents; Antifungal Agents - pharmacology; Biological and medical sciences; Biotechnology; Blotting, Northern; centromeres; Cloning, Molecular; Cloning, Molecular - methods; Dominant selectable markers; drug effects; Drug Resistance; Drug Resistance - genetics; Fundamental and applied biological sciences. Psychology; Gene Dosage; Genes, Reporter; Genes, Reporter - genetics; genetic transformation; Genetic Vectors; Genetic Vectors - genetics; Genetic Vectors - metabolism; geneticin; genetics; Genomic Instability; GFP; green fluorescent protein; Green Fluorescent Proteins; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; growth & development; hygromycin B; industrial strains; Intracellular Space; Intracellular Space - metabolism; metabolism; methods; molecular genetics; pharmacology; plasmid vectors; plasmids; Plasmids - genetics; Plasmids - metabolism; Prototrophic strains; reporter genes; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Selection, Genetic; Transfection; Transfection - methods; Vectors; Vitaceae; Wine; wine yeasts; yeasts; GFP; Dominant selectable markers; Vectors; Prototrophic strains; Saccharomyces cerevisiae; Ascomycota; Fungi; Characterization; Yeast; Green fluorescent protein; Prototrophy; Vector
• ISSN: 0168-1656; 1873-4863; 1873-4863
• DOI: 10.1016/j.jbiotec.2009.08.007

A set of shuttle yeast vectors containing the dominant selectable markers KanMX4 or HphMX4 cassettes, conferring resistance to geneticin and hygromycin B, respectively, was constructed. Dominant selectable markers are useful for genetic manipulation of natural, wine and industrial strains which do not contain any auxotrophic markers as well as of strains which cannot grow on synthetic mineral medium. Vectors were characterized by (i) copy number, (ii) mitotic stability both in selective and non-selective conditions, (iii) the efficiency and frequency of transformations, (iv) optimal adaptation times in non-selective media, (v) optimum conditions for transformation of various laboratory, commercial and wine strains, and (vi) expression level of an inserted gene. Furthermore we produced GFP-containing vectors that can be used for protein subcellular localization in prototrophic strains.