Engineering Rhodotorula toruloides as a platform organism for de novo synthesis of fatty-acid esters

Fatty acid esters are building blocks in next-generation biofuels, eco-friendly detergents, crop-enhancing adjuvants, and high-value cosmetic emollients. However, their industrial production relies on two unsustainable approaches: petrochemical-derived chemical catalysis that poses sustainability challenges and botanical extraction processes that exacerbates land-use conflicts. Here we develop a de novo microbial biosynthesis platform using the non-conventional oleaginous yeast Rhodotorula toruloides as a chassis, and achieve high-level biosynthesis of structurally diversified esters without the addition of alcohol or lipid precursors. By screening specific pathway enzymes and conducting modular pathway engineering, we successfully reprogram the native lipogenic metabolism of R. toruloides for de novo synthesis of fatty acid ethyl esters (FAEEs) at 579 mg/L, fatty acid short-branched chain esters (FASBEs) at 169 mg/L, and wax esters (WEs) at 1.30 g/L in shake-flask fermentation. As a case study, we optimize the synthesis of WEs in a 5 L fermenter, and achieve a production of 13.04 g/L. These engineered strains potentially offer an efficient, economical and environmentally friendly platform for the industrial production of fatty acid esters and oleochemicals.