RESUMEN
Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological and whole-genome analyses were conducted to obtain insights on molecular responses which potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated by adaptive laboratory evolution (ALE) experiments. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H+, iron, and glycerol transport (i.e., PMA1, FRE1/2, JEN1, VMA2, VCX1, KHA1, AQY3, and ATO2); transcriptional regulation of stress responses to drugs, reactive oxygen species and heat-shock (i.e., HSF1, SKN7, BAS1, HFI1, and WAR1); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1, GPA1/2, RAS2, IRA2, and REG1). At 30 °C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results revealed that evolved strains adjust their intracellular pH by H+ and acetic acid transport, modify their metabolism and stress responses via glucose signaling pathways, control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides, and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Moreover, the motifs analysis in mutated transcription factors suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. KEY POINTS: ⢠All the evolved strains overexpressed the plasma membrane H+ -ATPase PMA1 at optimal conditions ⢠Tolerant strain TAT12 mutated genes encoding weak acid and heat response TFs HSF1, SKN7, and WAR1 ⢠TFs HSF1 and SKN7 likely controlled the transcription of metabolic genes associated to heat and acid tolerance.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , ATPasas de Translocación de Protón Vacuolares , Saccharomyces cerevisiae/metabolismo , Temperatura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Acético/metabolismo , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/metabolismo , Proteína Fosfatasa 1/metabolismo , Transactivadores/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Saccharomyces cerevisiae scarcely grows on minimal media with acetic acid, acidic pH, and high temperatures. In this study, the adaptive laboratory evolution (ALE), whole-genome analysis, and reverse engineering approaches were used to generate strains tolerant to these conditions. The thermotolerant strain TTY23 and its parental S288C were evolved through 1 year, in increasing concentrations of acetic acid up to 12 g/L, keeping the pH ≤ 4. Of the 18 isolated strains, 9 from each ancestor, we selected the thermo-acid tolerant TAT12, derived from TTY23, and the acid tolerant AT22, derived from S288C. Both grew in minimal media with 12 g/L of acetic acid, pH 4, and 30 °C, and produced ethanol up to 29.25 ± 6 mmol/gDCW/h-neither of the ancestors thrived in these conditions. Furthermore, only the TAT12 grew on 2 g/L of acetic acid, pH 3, and 37 °C, and accumulated 16.5 ± 0.5 mmol/gDCW/h of ethanol. Whole-genome sequencing and transcriptomic analysis of this strain showed changes in the genetic sequence and transcription of key genes involved in the RAS-cAMP-PKA signaling pathway (RAS2, GPA2, and IRA2), the heat shock transcription factor (HSF1), and the positive regulator of replication initiation (SUM1), among others. By reverse engineering, the relevance of the combined mutations in the genes RAS2, HSF1, and SUM1 to the tolerance for acetic acid, low pH, and high temperature was confirmed. Alone, the RAS2 mutation yielded acid tolerance and HSF1 nutation thermotolerance. Increasing the thermo-acidic niche and acetic acid tolerance of S. cerevisiae can contribute to improve economic ethanol production. KEY POINTS: ⢠Thermo-acid tolerant (TAT) yeast strains were generated by adaptive laboratory evolution. ⢠The strain TAT12 thrived on non-native, thermo-acidic harmful conditions. ⢠Mutations in RAS2, HSF1, and SUM1 genes rendered yeast thermo and acid tolerant.