A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes Odanir Garcia Guerra a,b , Ileana G.S. Rubio c , Claudionor Gomes da Silva Filho a , Regiane Aparecida Bertoni a , Rute Cardoso dos Santos Govea a , Elisabete José Vicente a, ⁎ a Department of Microbiology, Biomedical Sciences Institute, University of São Paulo-USP, Av. Prof. Lineu Prestes 1374, Cidade Universitária, São Paulo – SP, 05508-900, Brazil b Native Sciences Department, Campus Três Lagoas, Mato Grosso do Sul Federal University-UFMS, Brazil c Thyroid Study Unit (LIM-25), Division of Endocrinology, University of São Paulo Medical School, 05403-900- São Paulo, SP., Brazil Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Glucoamylase; Retrotransposon Ty1; S. cerevisiae transformation; Yeast linear transformation vector; Yeast transformation vector without selective marker; δ integration 1. Introduction The study of fungi was very important for the development of genetics. Fungi include yeasts, which are unicellular eukaryotic microorganisms of extremely simple handling. Particularly, Saccharomyces cerevisiae is a very useful organism due to the huge amount of genetic, molecular and cellular data currently available (Goffeau et al., 1996, 1997; Blandin et al., 2000; Cliften et al., 2001; Dunham et al., 2002; Brachat et al., 2003; Dietrich et al., 2004; Garfinkel, 2005). These micro- organisms have been classifieds as GRAS (Generally Journal of Microbiological Methods 67 (2006) 437 – 445 ⁎ Corresponding author. Tel.: +55 11 3091 7275; fax: +55 11 3091 7420. E-mail address: bevicent@usp.br (E.J. Vicente).