Cell periphery-related proteins as major genomic targets behind the adaptive evolution of an industrial Saccharomyces cerevisiae strain to combined heat and hydrolysate stress.

BACKGROUND: Laboratory evolution is an important tool for developing robust yeast strains for bioethanol production since the biological basis behind combined tolerance requires complex alterations whose proper regulation is difficult to achieve by rational metabolic engineering. Previously, we reported on the evolved industrial Saccharomyces cerevisiae strain ISO12 that had acquired improved tolerance to grow and ferment in the presence of lignocellulose-derived inhibitors at high temperature (39 °C). In the current study, we used comparative genomics to uncover the extent of the genomic alterations that occurred during the evolution process and investigated possible associations between the mutations and the phenotypic traits in ISO12. RESULTS: Through whole-genome sequencing and variant calling we identified a high number of strain-unique SNPs and INDELs in both ISO12 and the parental strain Ethanol Red. The variants were predicted to have 760 non-synonymous effects in both strains combined and were significantly enriched in Gene Ontology terms related to cell periphery, membranes and cell wall. Eleven genes, including MTL1, FLO9/FLO11, and CYC3 were found to be under positive selection in ISO12. Additionally, the FLO genes exhibited changes in copy number, and the alterations to this gene family were correlated with experimental results of multicellularity and invasive growth in the adapted strain. An independent lipidomic analysis revealed further differences between the strains in the content of nine lipid species. Finally, ISO12 displayed improved viability in undiluted spruce hydrolysate that was unrelated to reduction of inhibitors and changes in cell wall integrity, as shown by HPLC and lyticase assays. CONCLUSIONS: Together, the results of the sequence comparison and the physiological characterisations indicate that cell-periphery proteins (e.g. extracellular sensors such as MTL1) and peripheral lipids/membranes are important evolutionary targets in the process of adaptation to the combined stresses. The capacity of ISO12 to develop complex colony formation also revealed multicellularity as a possible evolutionary strategy to improve competitiveness and tolerance to environmental stresses (also reflected by the FLO genes). Although a panel of altered genes with high relevance to the novel phenotype was detected, this study also demonstrates that the observed long-term molecular effects of thermal and inhibitor stress have polygenetic basis.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

BACKGROUND: A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. RESULTS: In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. CONCLUSIONS: We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

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

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.

Adaptive evolution of an industrial strain of Saccharomyces cerevisiae for combined tolerance to inhibitors and temperature.

INTRODUCTION: Development of industrial yeast strains with high tolerance towards the inhibitors released during biomass pretreatment is critical for bioethanol production. Combining this trait with increased thermotolerance would result in a more efficient production via Simultaneous Saccharification and Fermentation (SSF) as well as reduced cooling costs. The aim of the present work was to develop a yeast strain combining these traits. RESULTS: Using a long-term adaptation strategy a stable Saccharomyces cerevisiae isolate (ISO12) was evolved from the industrial strain Ethanol Red (ER). ISO12, contrary to the parental strain, is capable of growing and fermenting the liquid fraction of non-detoxified spruce hydrolysate at 39°C with an ethanol yield of 0.38 g ethanol . g hexoses-1. In contrast with previous studies, the superior phenotype of ISO12 does not rely on higher reductase activities for furaldehyde inhibitor conversion, but rather on a higher thermotolerance. ISO12 shows a higher capacity to ferment hydrolysate at 39°C and higher viability during heat-shock at 52°C than ER. In the absence of inhibitors, however, both ER and ISO12 displayed similar growth phenotype at 39°C. CONCLUSIONS: The evolved isolate ISO12 shows a superior phenotype than the parental strain ER when both stresses, temperature and inhibition by hydrolysate-derived compounds, are applied together. The results suggest that the presence of inhibitors depress the maximum temperature permissible for growth to a value below 39°C. As a result of the adaptation process and acquired improved thermotolerance, ISO12 is able to overcome this synergistic effect. Robust strains, such as ISO12, are interesting candidates for second generation ethanol production by SSF, as well as in tropical countries where fermentations at higher temperature can positively impact the production costs.