title: EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.
abstract: Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Vratislav
authors.initials: V
authors.lastname: Stovicek
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Gheorghe M
authors.initials: GM
authors.lastname: Borja
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Jochen
authors.initials: J
authors.lastname: Forster
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. irbo@biosustain.dtu.dk.
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics; D003434:Crossing Over, Genetic; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D010427:Pentose Phosphate Pathway / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
keywords: Heterologous gene expression; Industrial yeast; Integrative vectors; Metabolic engineering; Xylose utilization