title: Combinatorial mutagenesis and selection to understand and improve yeast promoters.
abstract: Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6, 7491 Trondheim, Norway.
authors.forename: Laila
authors.initials: L
authors.lastname: Berg
authors.forename: Trine Aakvik
authors.initials: TA
authors.lastname: Strand
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics* / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D025141:Drug Resistance, Fungal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D016296:Mutagenesis; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
chemical_list: D001761:Bleomycin; C105427:Zeocin; D042931:Aldehyde Oxidase