Citation

Genetic characterization and construction of an auxotrophic strain of Saccharomyces cerevisiae JP1, a Brazilian industrial yeast strain for bioethanol production

Reis VCB, Nicola AM, de Souza Oliveira Neto O, Batista VDF, de Moraes LMP, Torres FAG

J Ind Microbiol Biotechnol. Springer-Verlag; 2012 Nov 1;39(11):1673–83

Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notablybioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanolproduction. JP1 is a dominant industrial S. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanolproduction in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its geneticmanipulation. Using flow cytometry, mating type, and sporulation analysis, we verified that JP1 is diploid and homothallic. Vectors with dominant selective markers for G418, hygromycin B, zeocin, and ρ-fluoro-DL-phenylalanine were used to successfully transform JP1 cells. Also, anauxotrophic ura3 mutant strain of JP1 was created by gene disruption using integration cassettes with dominant markers flanked by loxP sites. Marker excision was accomplished by the Cre/loxP system. The resulting auxotrophic strain was successfully transformed with an episomal vector that allowed green fluorescent protein expression.

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