Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

Stephen R. Hughes, David E. Sterner, Kenneth M. Bischoff, Ronald E. Hector, Patrick F. Dowd, Nasib Qureshi, Sookie S. Bang, Nicole Grynaviski, Tania Chakrabarty, Eric T. Johnson, Bruce S. Dien, Jeffrey A. Mertens, Robert J. Caughey, Siqing Liu, Tauseef R. Butt, Joshua LaBaer, Michael A. Cotta, Joseph O. Rich

Research output: Contribution to journalArticle

22 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)22-38
Number of pages17
JournalPlasmid
Volume61
Issue number1
DOIs
StatePublished - Jan 1 2009

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Keywords

  • Automated plasmid-based robotic workcell
  • Pentose-utilizing yeast
  • SUMOduo plasmid set

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Hughes, S. R., Sterner, D. E., Bischoff, K. M., Hector, R. E., Dowd, P. F., Qureshi, N., Bang, S. S., Grynaviski, N., Chakrabarty, T., Johnson, E. T., Dien, B. S., Mertens, J. A., Caughey, R. J., Liu, S., Butt, T. R., LaBaer, J., Cotta, M. A., & Rich, J. O. (2009). Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system. Plasmid, 61(1), 22-38. https://doi.org/10.1016/j.plasmid.2008.09.001