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Experimental Evolution of Ethanol Tolerance

 

Figure 1.tif

 

  • Ethanol shock evolution — S. cerevisiae PE-2_H4 populations evolved through 68–82 cycles of acute ethanol stress (19–30% v/v).                                 

  • Key mutations — Adaptive variants enriched in the cAMP/PKA signaling and trehalose degradation pathways.                                                                               

  • Trade-offs observed — Enhanced ethanol tolerance accompanied by reduced growth under non-stress conditions.                                                                    

  • Reverse engineering with EasyGuide CRISPR — ALE-derived mutations were reconstructed using the EasyGuide system to validate their functional contribution to ethanol tolerance.​

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Saccharomyces cerevisiae PE-2

PROJECT SUMMARY 

We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production.

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People involved: Dr. Jeferson Gross and Dr. Ana Paula Jacobus

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Genome Engineering and Evolution of Microorganisms

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