Genome editing-based mutagenesis stably modifies composition of wax esters synthesized by Euglena gracilis under anaerobic conditions

[Display omitted] •Stably altered wax ester composition in Euglena gracilis using CRISPR/Cas9.•Knocked out beta-oxidation enzymes, enabling controlled wax ester synthesis.•Developed mutants with potential for diverse chemical and fuel applications.•Demonstrated additive effects in mutations that shorten wax ester chains.•Established a foundation for customized wax ester production in E. gracilis. Microalgal oil production represents a promising renewable biofuel source. Metabolic engineering can enhance its utility, transforming it into an improved biofuel and expanding its applications as a feedstock for commodity chemicals, thereby increasing their value in biorefineries. This study focused on anaerobic wax ester production by the microalga Euglena gracilis, aiming to develop stable mutant strains with altered wax ester profiles through genome editing. Two enzymes in the fatty acid beta-oxidation pathway involved in wax ester production were targeted—3-ketoacyl-CoA thiolase and acyl-CoA dehydrogenase—using clustered regularly interspaced short palindromic repeats/Cas9. The results revealed one genetic mutation that lengthened and three that shortened the distribution of wax ester compositions compared to the wild-type (WT). The triple-knockout mutant, combining mutations that shorten wax ester chains, produced wax esters with acyl chains two carbons shorter than WT. This study established a methodology to stably modify wax ester composition in E. gracilis.