title: Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system.
abstract: A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.
authors.affiliation: United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL 61604, USA. Stephen.Hughes@ars.usda.gov
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hughes
authors.forename: David E
authors.initials: DE
authors.lastname: Sterner
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Bischoff
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Hector
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Dowd
authors.forename: Nasib
authors.initials: N
authors.lastname: Qureshi
authors.forename: Sookie S
authors.initials: SS
authors.lastname: Bang
authors.forename: Nicole
authors.initials: N
authors.lastname: Grynaviski
authors.forename: Tania
authors.initials: T
authors.lastname: Chakrabarty
authors.forename: Eric T
authors.initials: ET
authors.lastname: Johnson
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Dien
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Mertens
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Caughey
authors.forename: Siqing
authors.initials: S
authors.lastname: Liu
authors.forename: Tauseef R
authors.initials: TR
authors.lastname: Butt
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cotta
authors.forename: Joseph O
authors.initials: JO
authors.lastname: Rich
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics* / Q000378:metabolism; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014994:Xylose / Q000378:metabolism*
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins; D013111:Spider Venoms; C110534:lycotoxin I; D014994:Xylose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase