Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate

AMB Express, Jul 2014

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.

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Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate

AMB Express 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)


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Valeria Wallace-Salinas, Lorenzo Signori, Ying-Ying Li, Magnus Ask, Maurizio Bettiga, Danilo Porro, Johan M Thevelein, Paola Branduardi, María R Foulquié-Moreno, Marie Gorwa-Grauslund. Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate, AMB Express, 2014, pp. 56, Volume 4, Issue 1, DOI: 10.1186/s13568-014-0056-5