Acetate co-feeding increases ethylene glycol assimilation and glycolic acid production in Yarrowia lipolytica

• Y. lipolytica cannot grow on EG as the sole carbon source in minimal medium. • Co-feeding with acetate increases EG uptake and glycolic acid production. • EG oxidation supplies NADPH, enhances acetate use, and stabilizes pH. • 13 C-MFA shows EG carbon enters biomass via glyoxylate cycle and glycine synthesis. • Optimized fed-batch strategy yields 48.4 g/L GA with 73 % molar yield. Upcycling ethylene glycol (EG) and terephthalic acid (TPA), key monomers derived from the depolymerisation of polyethylene terephthalate (PET), is a promising strategy for advancing the circular economy of plastics. This study investigates the metabolic fate of EG and its conversion into glycolic acid (GA) by the non-conventional yeast Yarrowia lipolytica . While EG alone does not support growth in a minimal medium, the addition of acetate (Ac) significantly enhances EG assimilation and GA production. 13 C labeling experiments and metabolic flux analysis revealed that a small but measurable fraction of EG-derived carbon is incorporated into biomass via glycine and serine biosynthesis and the glyoxylate cycle when acetate is supplied as a carbon source. Ac-EG co-feeding resulted in the production of 48.4 ± 1.4 g/L of GA after 66 h, corresponding to a molar yield of 73 % and a productivity of 0.73 g/(L·h). These results highlight the potential of Y. lipolytica as a microbial platform for EG valorisation and contribute to sustainable strategies for the bioconversion of plastic waste.