title: CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast.
abstract: Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Tomas
authors.initials: T
authors.lastname: Strucko
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Line Due
authors.initials: LD
authors.lastname: Buron
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Zofia Dorota
authors.initials: ZD
authors.lastname: Jarczynska
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Christina Spuur
authors.initials: CS
authors.lastname: Nødvig
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Louise
authors.initials: L
authors.lastname: Mølgaard
authors.affiliation: DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Barbara Ann
authors.initials: BA
authors.lastname: Halkier
authors.affiliation: Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.
authors.forename: Uffe Hasbro
authors.initials: UH
authors.lastname: Mortensen
mesh_terms: D005784:Gene Amplification; D018628:Gene Dosage; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D005960:Glucosides / Q000096:biosynthesis; D021141:Nucleic Acid Amplification Techniques; D061065:Polyketides / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005960:Glucosides; D061065:Polyketides