Genetic characterization and construction of an auxotrophic strain of Saccharomyces cerevisiae JP1, a Brazilian industrial yeast strain for bioethanol production.
Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notably bioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanol production. JP1 is a dominant industrial S. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanol production in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its genetic manipulation. 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, an auxotrophic 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.
Centro de Biotecnologia Molecular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF 70910-900, Brazil.
Viviane Castelo Branco
VC
Reis
André Moraes
AM
Nicola
Osmar
O
de Souza Oliveira Neto
Vinícius Daniel Ferreira
VD
Batista
Lidia Maria Pepe
LM
de Moraes
Fernando Araripe Gonçalves
FA
Torres
D056804:Biofuels / Q000600:supply & distribution; D001709:Biotechnology; D001938:Brazil; D004171:Diploidy; D000431:Ethanol / Q000378:metabolism*; D005799:Genes, Dominant; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability; D012441:Saccharomyces cerevisiae / Q000145:classification* / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D031786:Saccharum; D013172:Spores, Fungal / Q000502:physiology; D014170:Transformation, Genetic
D056804:Biofuels; D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D000431:Ethanol