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Biotechnology

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Jeroen G. Nijland, Hyun Yong Shin, Leonie G. M. Boender, Paul P. de Waal, Paul Klaassen, Arnold J. M. Driessen
Robert M. Kelly, Editor
Jeroen G. Nijland
aMolecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands
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Hyun Yong Shin
aMolecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands
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Leonie G. M. Boender
bDSM Biotechnology Center, Delft, the Netherlands
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Paul P. de Waal
bDSM Biotechnology Center, Delft, the Netherlands
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Paul Klaassen
bDSM Biotechnology Center, Delft, the Netherlands
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Arnold J. M. Driessen
aMolecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands
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Robert M. Kelly
North Carolina State University
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DOI: 10.1128/AEM.00095-17
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ABSTRACT

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16. Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.

IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

FOOTNOTES

    • Received 11 January 2017.
    • Accepted 24 March 2017.
    • Accepted manuscript posted online 31 March 2017.
  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00095-17 .

  • Copyright © 2017 American Society for Microbiology.

All Rights Reserved .

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Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae
Jeroen G. Nijland, Hyun Yong Shin, Leonie G. M. Boender, Paul P. de Waal, Paul Klaassen, Arnold J. M. Driessen
Applied and Environmental Microbiology May 2017, 83 (11) e00095-17; DOI: 10.1128/AEM.00095-17

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Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae
Jeroen G. Nijland, Hyun Yong Shin, Leonie G. M. Boender, Paul P. de Waal, Paul Klaassen, Arnold J. M. Driessen
Applied and Environmental Microbiology May 2017, 83 (11) e00095-17; DOI: 10.1128/AEM.00095-17
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KEYWORDS

Mutation, Missense
Repressor Proteins
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
xylose
sugar transporter
xylose transport
evolutionary engineering
transcriptome
yeast

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