Elucidation of Enzymatic Routes Underlying Odd‐Chain Fatty Acid Synthesis and Propionate Assimilation in <scp> <i>Euglena gracilis</i> </scp>

Wax esters (WEs) produced by Euglena gracilis under anaerobic conditions contain significant proportions of odd-chain fatty acids and fatty alcohols (30%-40%). Propionyl-CoA, the primer for odd-chain fatty acid synthesis, is generally thought to be generated via the methylmalonyl-CoA pathway; however, the enzymatic basis of this pathway in E. gracilis remains unclear. Here, we generated knockout (KO) mutants of candidate enzymes in the methylmalonyl-CoA pathway-succinyl-CoA synthetase (SCS), methylmalonyl-CoA mutase (MCM), methylmalonyl-CoA epimerase (MCE), and propionyl-CoA carboxylase (PCC)-using the CRISPR/Cas9 system and examined their roles in odd-chain fatty acid synthesis and propionate assimilation. The methylmalonyl-CoA pathway exhibited direction-dependent functions. KO of MCE and PCC did not affect the proportion of odd-chain WEs, indicating that these enzymes are not required for odd-chain WE synthesis and suggesting an alternative route converting methylmalonyl-CoA to propionyl-CoA. In contrast, both enzymes were required for propionate assimilation. Functional differentiation was observed between SCSβ isozymes: SCSβ1 contributed to odd-chain WE synthesis, whereas SCSβ2 primarily functioned in propionate assimilation. Partial impairment of propionate assimilation in SCSα or SCSβ2 KO strains suggests involvement of additional routes. These findings improve our understanding of direction-dependent roles in the methylmalonyl-CoA pathway in E. gracilis and support further studies of odd-chain lipid biosynthesis.