Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate
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Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate
Valeria Wallace-Salinas 0 1
Lorenzo Signori 0 3
Ying-Ying Li 2 5
Magnus Ask 4
Maurizio Bettiga 4
Danilo Porro 3
Johan M Thevelein 2 5
Paola Branduardi 3
María R Foulquié-Moreno 2 5
Marie Gorwa-Grauslund 1
0 Equal contributors
1 Applied Microbiology, Department of Chemistry, Lund University , P.O. Box 124, SE-22100 Lund , Sweden
2 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology , Leuven, KU , Belgium
3 University of Milano Bicocca , Piazza della Scienza 2, 20126 Milan , Italy
4 Department of Chemical and Biological Engineering, Industrial Biotechnology, Chalmers University of Technology , SE-41296 Gothenburg , Sweden
5 Department of Molecular Microbiology , VIB, Kasteelpark Arenberg 31, Leuven, B-3001 Heverlee, Flanders , Belgium
Development of robust yeast strains that can efficiently ferment lignocellulose-based feedstocks is one of the requirements for achieving economically feasible bioethanol production processes. With this goal, several genes have been identified as promising candidates to confer improved tolerance to S. cerevisiae. In most of the cases, however, the evaluation of the genetic modification was performed only in laboratory strains, that is, in strains that are known to be quite sensitive to various types of stresses. In the present study, we evaluated the effects of overexpressing genes encoding the transcription factor (YAP1) and the mitochondrial NADH-cytochrome b5 reductase (MCR1), either alone or in combination, in an already robust and xylose-consuming industrial strain of S. cerevisiae and evaluated the effect during the fermentation of undiluted and undetoxified spruce hydrolysate. Overexpression of either gene resulted in faster hexose catabolism, but no cumulative effect was observed with the simultaneous overexpression. The improved phenotype of MCR1 overexpression appeared to be related, at least in part, to a faster furaldehyde reduction capacity, indicating that this reductase may have a wider substrate range than previously reported. Unexpectedly a decreased xylose fermentation rate was also observed in YAP1 overexpressing strains and possible reasons behind this phenotype are discussed.
Saccharomyces cerevisiae; Hydrolysate; Inhibitors; YAP1; MCR1; Ethanol
Introduction
Production of second-generation bioethanol from
lignocellulosic biomass requires robust Saccharomyces cerevisiae
strains with improved capacity to cope with the toxic
compounds formed during the biomass pre-treatment, among
which are 5-hydroxymethylfurfural (HMF), furfural, weak
organic acids and phenolic compounds
(Parawira and
Tekere 2011)
. This has led to extensive studies to decipher
mechanisms behind the compounds toxicity and the yeast
natural tolerance responses to them and, among others,
genes involved in detoxification and yeast tolerance to
individual inhibitors have been identified, such as ADH6,
HAA1 or PMA1
(Haitani et al. 2012; Mira et al. 2010;
Petersson et al. 2006)
; for a more exhaustive review, see
(Almeida et al. 2009a; Liu 2011)
. YAP1 is another
interesting candidate for industrial strain engineering because it
encodes a transcription factor (Yap1p) that simultaneously
controls a wide range of stress-related targets
(Toone and
Jones 1999)
. Notably, its overexpression has a beneficial
role in the response of laboratory S. cerevisiae towards
HMF, furfural, and different concentrations of hydrolysate
(Alriksson et al. 2010; Kim and Hahn 2013; Ma and Liu
2010; Sundström et al. 2010)
. Another interesting and
complementary candidate for gene overexpression is MCR1
that encodes the mitochondrial NADH-cytochrome b5
reductase
(Hahne et al. 1994; Meineke et al. 2008)
. Previous
experiments performed in our group revealed that
overexpression of MCR1 in S. cerevisiae resulted in a reduced lag
phase and faster growth rate when the yeast was grown
with high concentrations of acetic acid (Signori et al.,
personal communication). This weak acid is one of the
inhibitors directly affecting xylose metabolism in S.
cerevisiae
(Almeida et al. 2011; Bellissimi et al. 2009; Casey et al.
2010; Helle et al. 2003)
. Still, considering that many of the
studies about strain improvement towards
hydrolysate-derived inhibitors concern laboratory strains, it is difficult to
predict the real effect of these changes in an industrial,
and more robust, strain background.
The objective of the present study was to evaluate the
effect of overexpressing YAP1 and MCR1, either alone
or in combination, in process-like conditions, that is
using a robust industrial S. cerevisiae strain and
undetoxified lignocellulosic hydrolysate. For this, the strain GSE16
was chosen as background strain for engineering since it
combines a robust industrial background with the ability
to ferment xylose, using the xylose i (...truncated)