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

Abstract

BackgroundLaboratory 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.ResultsThrough 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.ConclusionsTogether, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1737-4) contains supplementary material, which is available to authorized users.

A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains

“…The results observed here highlight the importance of intergenic polymorphisms and their potential effect on phenotype. Such mutations are commonly overlooked, for example in adaptive evolution studies (Wallace-Salinas et al 2015; Quarterman et al 2016; Krogerus et al 2018), and warrant more emphasis in future studies.…”

Adaptive laboratory evolution of β-caryophyllene producing Saccharomyces cerevisiae

“…The synonymous mutation in FLO9 changed the codon for threonine from ACT to the less preferred ACC, which may impact translation of the protein. Mutations and changes in copy number in the FLO genes have been found to be involved in tolerance to environmental stressors [33][34][35]. Mutations in all FLO9 and MDS3 were found in detectable frequencies in all populations sequenced, and the MTL1 mutation was present at ~ 49% frequency in the P11 population, suggesting that the increased oxidative stress tolerance conferred by these mutations likely contributed to their selection in the ALE experiment.…”

“…The MTL1 S257S mutation is also a synonymous mutation, with a serine codon change from TCA to a less preferred TCC, suggesting this mutation may lead to reduced translation of the protein. Mutations in FLO9 and MTL1 were found in an industrial yeast strain that was evolved for growth on hydrolysates inhibitors [33], which have been shown to induce oxidative stress in S. cerevisiae [36]. The increased hydrogen peroxide tolerance in YAG142 (STE6 T1025N) can be attributed to increased production, as without β-caryophyllene production there was no benefit to survival in oxidative stress challenge.…”

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications

“…For this purpose, rational metabolic engineering and evolutionary engineering strategies are commonly combined to further increase the robustness of metabolically engineered strains [98]. [113,114] Most of the evolutionary engineering studies to improve yeast for bioethanol production focus on increasing yeast growth rate and viability, decreasing by-product formation such as glycerol and biomass, improving utilization and transport of pentose sugars in lignocellulosic feedstocks for second-generation bioethanol production, and increasing tolerance to ethanol and lignocellulosic inhibitors. Examples of these evolutionary engineering studies that are discussed in Sections 4.1-4.4 are summarized in Table 2.…”