Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen

Stephen R. Hughes, Joseph O. Rich, Kenneth M. Bischoff, Ronald E. Hector, Nasib Qureshi, Badal C. Saha, Bruce S. Dien, Siqing Liu, John S. Jackson, David E. Sterner, Tauseef R. Butt, Joshua LaBaer, Michael A. Cotta

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to use pentose sugars. Because S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI), which catalyzes conversion of xylose to xylulose. In this study, an automated two-hybrid interaction protocol was used to find yeast genes encoding proteins that bind XI to identify potential targets for improving xylose utilization by S. cerevisiae. A pDEST32 vector re-engineered for TRP selection and containing the Gal4 binding domain fused with the Piromyces sp. E2 XI open reading frame (ORF) was used as bait with a library of LEU-selectable pOAD vectors containing the Gal4 activation domain in fusion with members of the S. cerevisiae genome ORF collection. Binding of a yeast ORF protein to XI activates two chromosomally located reporter genes in a PJ69-4 yeast strain to give selective growth. Five genes, including ADH1, were identified in the two-hybrid screen, suggesting the proteins encoded by these genes bind to XI. The effect of ADH1 overexpression was examined using the pSUMOduoHisADH1 vector in an automated protocol to transform eight previously identified yeast strains that showed anaerobic growth on xylose. One transformant consumed all available glucose, xylose, and arabinose during growth on wheat straw hydrolysate.

Original languageEnglish (US)
Pages (from-to)200-212
Number of pages13
JournalJALA - Journal of the Association for Laboratory Automation
Volume14
Issue number4
DOIs
StatePublished - Aug 2009
Externally publishedYes

Fingerprint

xylose isomerase
Cellulosic ethanol
Xylose
Robotics
Protein Binding
Yeast
Open Reading Frames
Saccharomyces cerevisiae
Ethanol
Yeasts
Engineers
Xylulose
Piromyces
Genes
Growth
Pentoses
Arabinose
Proteins
Reporter Genes
Glucose

Keywords

  • automated yeast transformation protocol
  • cellulosic fuel ethanol
  • proteins binding xylose isomerase
  • Saccharomyces cerevisiae genome ORFs
  • work cell-based yeast two-hybrid high-throughput screen

ASJC Scopus subject areas

  • Medical Laboratory Technology
  • Computer Science Applications

Cite this

Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen. / Hughes, Stephen R.; Rich, Joseph O.; Bischoff, Kenneth M.; Hector, Ronald E.; Qureshi, Nasib; Saha, Badal C.; Dien, Bruce S.; Liu, Siqing; Jackson, John S.; Sterner, David E.; Butt, Tauseef R.; LaBaer, Joshua; Cotta, Michael A.

In: JALA - Journal of the Association for Laboratory Automation, Vol. 14, No. 4, 08.2009, p. 200-212.

Research output: Contribution to journalArticle

Hughes, Stephen R. ; Rich, Joseph O. ; Bischoff, Kenneth M. ; Hector, Ronald E. ; Qureshi, Nasib ; Saha, Badal C. ; Dien, Bruce S. ; Liu, Siqing ; Jackson, John S. ; Sterner, David E. ; Butt, Tauseef R. ; LaBaer, Joshua ; Cotta, Michael A. / Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen. In: JALA - Journal of the Association for Laboratory Automation. 2009 ; Vol. 14, No. 4. pp. 200-212.
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AU - Bischoff, Kenneth M.

AU - Hector, Ronald E.

AU - Qureshi, Nasib

AU - Saha, Badal C.

AU - Dien, Bruce S.

AU - Liu, Siqing

AU - Jackson, John S.

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