To investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding beta-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.