Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system.
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.
Hughes
Stephen R
SR
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
Sterner
David E
DE
Bischoff
Kenneth M
KM
Hector
Ronald E
RE
Dowd
Patrick F
PF
Qureshi
Nasib
N
Bang
Sookie S
SS
Grynaviski
Nicole
N
Chakrabarty
Tania
T
Johnson
Eric T
ET
Dien
Bruce S
BS
Mertens
Jeffrey A
JA
Caughey
Robert J
RJ
Liu
Siqing
S
Butt
Tauseef R
TR
LaBaer
Joshua
J
Cotta
Michael A
MA
Rich
Joseph O
JO
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*
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