A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes.
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 delta-sequences (deltaGlucodelta), 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). In all cases, several copies of the deltaGlucodelta 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.
Guerra
Odanir Garcia
OG
Department of Microbiology, Biomedical Sciences Institute, University of São Paulo-USP, Av. Prof. Lineu Prestes 1374, Cidade Universitária, São Paulo, Brazil.
Rubio
Ileana G S
IG
da Silva Filho
Claudionor Gomes
CG
Bertoni
Regiane Aparecida
RA
Dos Santos Govea
Rute Cardoso
RC
Vicente
Elisabete José
EJ
D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D015825:Chromosomes, Fungal / Q000235:genetics*; D018076:DNA, Complementary; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic; D014170:Transformation, Genetic
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase