A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes

• Published in: Journal of microbiological methods 2006-12, Vol.67 (3), p.437-445
• Main Authors: Guerra, Odanir Garcia, Rubio, Ileana G.S., da Silva Filho, Claudionor Gomes, Bertoni, Regiane Aparecida, dos Santos Govea, Rute Cardoso, Vicente, Elisabete José
• Format: Article
• Language: English
• Subjects: Aspergillus - enzymology; Aspergillus - genetics; Aspergillus awamori; Biological and medical sciences; Chromosomes, Fungal - genetics; DNA, Complementary; DNA, Fungal - genetics; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Genetic Engineering - methods; Genetic Vectors; Glucan 1,4-alpha-Glucosidase - genetics; Glucoamylase; Microbiology; Mycological methods and techniques used in mycology; Mycology; Recombinant Proteins - genetics; Recombination, Genetic; Retrotransposon Ty1; S. cerevisiae transformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Selection, Genetic; Transformation, Genetic; Yeast linear transformation vector; Yeast transformation vector without selective marker; δ integration
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2006.04.014

Increasing industrial competitiveness and productivity demand that recombinant yeast strains, used in many different processes, be constantly adapted and/or genetically improved to suit changing requirements. Among yeasts, Saccharomyces cerevisiae is the best-studied organism, and the most frequently employed yeast in industrial processes. In the present study, laboratory strains and industrial S. cerevisiae strains were stably transformed with a novel vector containing the glucoamylase cDNA of Aspergillus awamori flanked by δ-sequences (δGlucoδ), and lacking a positive selection marker. Co-transformation with known plasmids allowed selection by auxotrophic complementation of the leu2 mutation and/or geneticin resistance (G418 R). In all cases, several copies of the δGlucoδ vector were inserted into the genome of the yeast cell without selective pressure, showing 100% stability after 80 generations. Transformation frequency of the new vector was similar for S. cerevisiae laboratory strains and industrial wild-type S. cerevisiae strains. This novel genetic transformation system is versatile and suitable to introduce several stable copies of a desired expression cassette into the genome of different S. cerevisiae yeast strains.