An　evolution　and　resequencing　strategy　was　used　to　research　the　genetic　basis　of　Saccharomyces　cerevisiae　BR20　(with　18　vol%　ethanol　tolerance)　and　the　evolved　strain　F23　(with　25　vol%　ethanol　tolerance).　Whole-genome　sequencing　and　RNA　sequencing　(RNA-seq)　indicated　that　the　enhanced　ethanol　tolerance　under　10　vol%　ethanol　could　be　attributed　to　amino　acid　metabolism,　whereas　18　vol%　ethanol　tolerance　was　due　to　fatty　acid　metabolism.　Ultrastructural　analysis　indicated　that　F23　exhibited　better　membrane　integrity　than　did　BR20　under　ethanol　stress.　At　low　concentrations　(＜5　vol%),　the　partition　of　ethanol　into　the　membrane　increased　the　membrane　fluidity,　which　had　little　effect　on　cell　growth.　However,　the　toxic　effects　of　medium　and　high　ethanol　concentrations　(5　to　20　vol%)　tended　to　decrease　the　membrane　fluidity.　Under　high　ethanol　stress　(＞10　vol%),　the　highly　tolerant　strain　was　able　to　maintain　a　relatively　constant　fluidity　by　increasing　the　content　of　unsaturated　fatty　acid　(UFA),　whereas　lesstolerant　strains　show　a　continuous　decrease　in　fluidity　and　UFA　content.　OLE1,　which　was　identified　as　the　only　gene　with　a　differential　single-nucleotide　polymorphism　(SNP)　mutation　site　related　to　fatty　acid　metabolism,　was　significantly　changed　in　response　to　ethanol.　The　role　of　OLE1　in　membrane　fluidity　was　positively　validated　in　its　overexpressed　transformants.　Therefore,　OLE1　lowered　the　rate　of　decline　in　membrane　fluidity　and　thus　enabled　the　yeast　to　better　fight　the　deleterious　effects　of　ethanol.　©　2019　American　Society　for　Microbiology.