In silico characterization of Rhodotorula toruloides ELO-like elongases and production of very-long-chain fatty acids by expressing Rtelo2, RtKCR, RtHCD, and RtECR through IRES-mediated bicistrons.

Rhodotorula toruloides, an oleaginous yeast known for its high lipid productivity, produces lipids with low very-long-chain fatty acid (VLCFA) content. Meanwhile, the roles of enzymes, particularly the condensing enzymes, involved in VLCFA biosynthesis in R. toruloides remained unclear. In this study, two elongases, RtELO1 and RtELO2, were identified from R. toruloides U13N3 and their tertiary structure and catalytic mechanism were investigated using molecular dynamic methods. Both enzymes exhibited typical ELO-like characteristics, with active sites located within cavities formed by seven transmembrane helixes. RtELO2 displayed higher binding affinity to acyl-CoAs compared to RtELO1, and at least seven amino acid residues, including two crucial histidines in the "HXXHH" box, were identified as important for the condensation reaction. To enhance VLCFA production, an internal ribosome entry site (IRES)-mediated bicistronic strategy was developed to integrate multiple genes into the R. toruloides genome. The efficiency of IRES-mediated translation initiation reached 85.4% of cap-dependent upstream translation, based on EGFP fluorescent intensity. Using this strategy, four genes encoding enzymes involved in the VLCFA biosynthesis cycle (Rtelo2, RtKCR, RtHCD, and RtECR) were introduced into the U13N3 genome in various combinations. The results indicated that the expression of a single elongase had a modest effect on VLCFA production, but the simultaneous expression of multiple genes resulted in cumulative effects. Notably, the transformant harboring four genes exhibited a remarkable 436.8% increase in C22 and C24 VLCFA yield compared to the original strain.