title: amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae.
abstract: Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Solis-Escalante
authors.forename: Niels G A
authors.initials: NG
authors.lastname: Kuijpers
authors.forename: Nadine
authors.initials: N
authors.lastname: Bongaerts
authors.forename: Irina
authors.initials: I
authors.lastname: Bolat
authors.forename: Lizanne
authors.initials: L
authors.lastname: Bosman
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
authors.forename: Pascale
authors.initials: P
authors.lastname: Daran-Lapujade
mesh_terms: D000081:Acetamides / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics*; D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D005463:Fluoroacetates / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D005799:Genes, Dominant; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D004718:Saccharomycetales / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D000081:Acetamides; D005463:Fluoroacetates; D005656:Fungal Proteins; D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; C030686:acetamide; C007741:fluoroacetamide; D000581:Amidohydrolases; C021445:acetamidase