title: Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Engineering of the yeast Saccharomyces cerevisiae for improved utilization of pentose sugars is vital for cost-efficient cellulosic bioethanol production. Although endogenous hexose transporters (Hxt) can be engineered into specific pentose transporters, they remain subjected to glucose-regulated protein degradation. Therefore, in the absence of glucose or when the glucose is exhausted from the medium, some Hxt proteins with high xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely xylose.
  
  
  RESULTS
  The low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant), and Hxt5 are subjected to catabolite degradation as evidenced by a loss of GFP fused hexose transporters from the membrane upon glucose depletion. Catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. Therefore, N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination, were replaced for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely xylose concomitantly with markedly stimulated growth on xylose. The mutagenesis also improved the late stages of sugar fermentation when cells are grown on both glucose and xylose.
  
  
  CONCLUSIONS
  Substitution of N-terminal lysine residues in the endogenous hexose transporters Hxt1 and Hxt36 that are subjected to catabolite degradation results in improved retention at the cytoplasmic membrane in the absence of glucose and causes improved xylose fermentation upon the depletion of glucose and when cells are grown in d-xylose alone.
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Groningen, The Netherlands.
authors.forename: Jeroen G
authors.initials: JG
authors.lastname: Nijland
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Groningen, The Netherlands.
authors.forename: Erwin
authors.initials: E
authors.lastname: Vos
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Groningen, The Netherlands.
authors.forename: Hyun Yong
authors.initials: HY
authors.lastname: Shin
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
authors.forename: Paul P
authors.initials: PP
authors.lastname: de Waal
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
authors.forename: Paul
authors.initials: P
authors.lastname: Klaassen
authors.affiliation: Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials and Kluyver Centre for Genomics of Industrial Fermentation, Groningen, The Netherlands.
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
keywords: Sugar transporter; Ubiquitination; Xylose transport; Yeast