ABSTRACTIn order to enhance the growth performance of S. cerevisiae under harsh environmental conditions, mutant XCG001, which tolerates up to 1.5M NaCl, was isolated via adaptive laboratory evolution (ALE). Comparisons made via transcriptome data of XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. Overexpression of ELO2 increased the contents of inositol phosphorylceramide (IPC, t18:0/26:0), mannosylinositol phosphorylceramide (MIPC, t18:0/22:0(2OH)), MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide (M(IP)2C, d20:0/26:0), M(IP)2C (t18:0/26:0(2OH)) and M(IP)2C (d20:0/26:0(2OH)) by 88.3-, 166.9-, 63.3-, 23.9-, 27.9-, 113.8- and 208.1-fold at 1.0 M NaCl, respectively, compared those of strain XCG002. As a result, membrane integrity, cell growth and cell survival of the ELO2 overexpression strain (XCG010) increased by 24.4%, 29% and 22.1% at 1.0 M NaCl, respectively, compared those of strain XCG002. The findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance.IMPORTANCEThis study demonstrated a novel strategy for manipulation membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Osmotic tolerance was related to sphingolipid acyl chain elongase, Elo2, via transcriptome analysis of the wild-type strain and an osmotic tolerant strain generated from ALE. Overexpression of ELO2 increased complex sphingolipid with longer acyl chain, thus improved membrane integrity and osmotic tolerance.