title: Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae.
abstract: The development of efficient tools for genetic modification of industrial yeast strains is one of the challenges that face the use of recombinant cells in industrial processes. In this study, we examine how the construction of two complementary integrative vectors can fulfill the major requirements of industrial recombinant yeast strains: the use of lactose assimilation genes as a food-grade yeast selection marker, and a system of integration that does not leave hazardous genes in the host genome and involves minimal interference in the yeast physiology. The pFB plasmid set was constructed to co-integrate both LAC4-based and LAC12-based cassettes into the ribosomal DNA (rDNA) locus to allow yeast cells to be selected in lactose medium. This phenotype can also be used to trace the recombinant cells in the environment by simply being plated on X-gal medium. The excisable trait of the LAC12 marker allows the introduction of many different heterologous genes, and makes it possible to introduce a complete heterologous metabolic pathway. The cloned heterologous genes can be highly expressed under the strong and constitutive TPI1 gene promoter, which can be exchanged for easy digestion of enzymes if necessary. This platform was introduced into Saccharomyces cerevisiae JP1 industrial strain where a recombinant with high stability of markers was produced without any change in the yeast physiology. Thus, it proved to be an efficient tool for the genetic modification of industrial strains.
authors.affiliation: Interdepartmental Metabolic Engineering Research Group, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Fernanda Cristina Bezerra
authors.initials: FC
authors.lastname: Leite
authors.forename: Rute Salgues Gueiros
authors.initials: RS
authors.lastname: Dos Anjos
authors.forename: Anna Carla Moreira
authors.initials: AC
authors.lastname: Basilio
authors.forename: Guilherme Felipe Carvalho
authors.initials: GF
authors.lastname: Leal
authors.forename: Diogo Ardaillon
authors.initials: DA
authors.lastname: Simões
authors.forename: Marcos A
authors.initials: MA
authors.lastname: de Morais
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D007785:Lactose / Q000378:metabolism; D030781:Organisms, Genetically Modified / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D015415:Biomarkers; D003470:Culture Media; D004275:DNA, Ribosomal; D007785:Lactose