Affiliation:
1. Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, Zernike Institute for Advanced Materials, Kluyver Centre for Genomics of Industrial Fermentation, Groningen, the Netherlands
2. DSM Biotechnology Center, Delft, the Netherlands
Abstract
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 (
V
max
) 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.
Funder
Dutch Ministry of Economical Affairs
Be-basic
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
31 articles.
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