Substrate-dependent L-carnitine handling in Yarrowia lipolytica grown on biofuel waste

L-carnitine plays an important role in fatty acid metabolism associated with peroxisomal β-oxidation in Yarrowia lipolytica during growth on lipid-rich substrates. This study investigated substrate-dependent L-carnitine handling in two Y. lipolytica strains, A-101 and ATCC 9793, cultivated in lipid-rich industrial biofuel waste (SK medium) and carbohydrate-based YPG medium under various conditions. In YPG medium, free L-carnitine reached up to 22.85 mg/100 g wet biomass in ATCC 9793 and 7.09 mg/100 g wet biomass in A-101. In YPG-grown Y. lipolytica A-101, supplementation with trimethyllysine (TML), iron, and L-ascorbic acid, selected based on their proposed roles in L-carnitine biosynthesis, increased the free L-carnitine concentration by up to 2.5-fold relative to the unsupplemented control. In contrast, the biomass of both strains grown in SK medium contained less than 1 mg of L-carnitine per 100 g of wet biomass, indicating marked substrate-dependent differences in L-carnitine handling under lipid-rich (SK) growth conditions. The addition of TML, iron, and L-ascorbic acid to SK medium resulted in approximately 1.2–1.3-fold higher OD₅₅₀ values after 10 h of cultivation. To further characterize substrate-dependent L-carnitine handling, untargeted metabolomic profiling of post-culture supernatants of the A-101 strain and comparative in silico analyses, including sequence- and structure-based homology assessment, were performed. Despite the presence of putative homologues of L-carnitine biosynthesis-related proteins, the combined metabolomic and comparative in silico evidence supported substrate-dependent L-carnitine handling rather than predominant de novo biosynthesis under the tested conditions. These findings improve our understanding of the metabolic adaptation of Y. lipolytica to lipid-rich industrial substrates and highlight the importance of cultivation environment in determining L-carnitine availability and accumulation.