Molecular characterization of phenylethanol resistance in Saccharomyces cerevisiae

Abstract

The alcohol production industry faces the challenge of producing high-concentration alcohol at low cost. Yeast, the main organism used in alcohol production, has limited alcohol tolerance, with many industrial strains unable to efficiently ferment at concentrations above 13% ethanol. Increasing yeast's alcohol tolerance could improve overall efficiency and reduce distillation costs. However, the diverse detrimental effects of alcohol on cellular structures and pathways make it difficult to obtain resistant strains through rational design. Evolutionary engineering, an inverse metabolic engineering approach, can be used to overcome such challenges of obtaining alcohol-resistant strains. This strategy mimics natural evolution by diversifying the gene pool of an organism in vitro with mutagenic agents, and applying artificial stress to create selective pressure, resulting in adapted organisms with the desired phenotype. This approach has successfully produced various resistant yeast strains, including those resistant to different types of alcohols and heavy metals. Phenylethanol (2-PE), a common aromatic alcohol in many plant essential oils, is structurally similar to ethanol and is of interest for resistance development. Current 2- PE production relies on chemical synthesis, which uses raw materials toxic to human health. An alternative production method could reduce the use of harmful materials. However, research on the negative effects of 2-PE and its metabolic pathways is limited. Previously, a Saccharomyces cerevisiae, a yeast strain with a three-fold increase in 2- PE resistance was obtained using evolutionary engineering approach. The molecular mechanisms of 2-PE resistance in an evolved yeast strain were investigated through whole-genome transcriptomic analysis. However, the underlying genomic causes of this resistance remained unknown, necessitating further analysis at the genomic level. The aim of this study was to perform genomic analysis on the 2-PE-resistant strain and combine these data with transcriptomic and metabolic analyses to identify the molecular mechanisms underlying resistance. Another goal of this study was to generate a high-quality assembled and annotated reference genome of the 2-PE-resistant S. cerevisiae strain for use by academic and industrial yeast research communities. This will involve sequencing, de novo assembly and annotation of the resistant strain. Another purpose of this study was to perform a deeper characterization of the physiological properties of the 2-PE-resistant evolved strain through a series of experiments, including assessing cross-resistance to various stress conditions, analyzing growth and metabolite profiles under control and stress conditions, and comparing cell wall characteristics, ultimately to obtain a comprehensive knowledge of the resistance mechanisms and potential applications of the evolved strain. According to growth analysis, the 2-PE-resistant S. cerevisiae strain C9 mutant exhibited a superior growth rate in comparison to the reference strain for 2-PE stress, while the reference strain demonstrated a higher maximum specific growth rate in the absence of stress. The cross-resistance analysis showed that C9 showed increased sensitivity to NaCl at 48-hour incubation, particularly at 0.5M and 0.75M concentrations. There were no significant difference detected between the reference and evolved strains in 1M and 2M sorbitol media. ALD3 and ALD4 a genes encoding cytoplasmic and mitochondrial aldehyde dehydrogenase respectively, were upregulated in the evolved strain. The phenylacetaldehyde sensitivities of both strains did not differ. However, the evolved strain had significantly cross resistance to phenylacetate. The results may suggest a resistance mechanism that includes conversion of 2-phenylethanol to phenylacetate by those dehydrogenases. HPLC analysis of metabolite production revealed similarities between the C9 and the reference strains in glucose consumption and ethanol production under non-stress and 3 g/L 2-PE stress conditions. However, the C9 strain exhibited higher acetate and glycerol production, up to six-fold higher than the reference strain, under both conditions. The C9 strain contained three to five times more trehalose than the reference strain, while glycogen content remained largely unchanged. Lyticase sensitivity assays were conducted to assess cell wall remodeling in response to 2-PE adaptation. The C9 strain demonstrated significantly increased lyticase resistance compared to reference strain, suggesting cell wall remodeling occurred due to 2-PE adaptation. Whole-genome sequencing of the reference and stress-resistant evolved S. cerevisiae strains revealed 53 genomic changes in the C9 strain not present in the reference strain. Among these mutations, 32 were located in exonic regions, indicating that EMS induced mutations without causing lethal damage. Significantly upregulated MAPK signaling pathways harbored three of the 53 mutations. One such change, Hog1p.F318L, was located in the phosphorylation lip of the Hog1p enzyme, which is typically activated in reaction to hyperosmotic stress in order to facilitate glycerol biosynthesis and retention. Another mutation was identified in the CRH1 gene, which encodes a trans-glycosidase involved in the cell wall remodelling via formation of cross-links between chitin and β 1,6 glucan. Homology modeling was used to compared wild type and mutant protein structures for candidate genes, revealing significant conformational and structural changes in Crh1 and Hog1 proteins due to the mutations. The mutations in these proteins may contribute to 2-PE resistance in the mutant strain. Several mutated genes, including RPN4, HOG1, CRH1, SSK2, MCA1, and PDE2, were identified as potentially contributing to 2-PE-resistance. The roles of these genes in resistance mechanisms were explored, revealing various functional impacts such as transcriptional activation, signal transduction pathways, cell wall architecture, and osmotic stress response. A comprehensive genomic analysis of the 2-PE-resistant S. cerevisiae strain was conducted to detect the altered pathways in the evolved strain. Genes with significant expression differences between the evolved and the reference strains were used for pathway analysis. Pathways were extracted from the KEGG database and filtered based on adjusted p-values. The analysis results indicated that the evolved strain displayed an Environmental Stress Response (ESR) like expression profile, and one- sample t-tests were applied to identify the statistical significance of gene set tests. The presence of transcription factors correlated with the pathway enrichment analysis, which highlighted the MAPK signaling pathway, proteasome, autophagy, and ribosome biogenesis. Additionally, KEGG pathway analysis of the MAPK signaling pathway revealed that differentially expressed genes in the evolved strain were primarily associated with cell wall remodeling, osmolyte synthesis, filamentation, and mating. In summary, in this thesis, detailed molecular characterization of 2-PE-resistant S. cerevisiae was carried out to elucidate the molecular mechanisms of 2-PE-resistance. Transcriptomic analyses showed significant expression changes in MAPK and cell wall signaling pathways. The HOG1 mutation detected by genome sequencing may be associated with this expression profile. The metabolite analyses data were also consistent with increased HOG1 activity and an ESR-like transcriptomic profile. In addition, characterization studies performed on the cell wall and the mutation in CRH1 gene indicated that phenylethanol resistance may be related to the alteration in the molecular architecture of the cell wall. The cross-resistance of the evolved strain against phenylacetate and its increased expression of ALD3 and ALD4 genes also suggest that 2-PE-resistance may be associated with conversion of 2-PE to phenylacetate by the overexpressed aldehyde dehydrogeneases.