Waste-to-SAF Precursors from Mixed Volatile Fatty Acids: Stepwise Metabolic Engineering of Yarrowia lipolytica

Volatile fatty acids (VFAs) are key intermediates produced during anaerobic processing of organic wastes and represent a recyclable carbon stream for resource recovery. Converting mixed VFA streams into microbial lipids offers a potential route to generate lipid feedstocks that can serve as precursors for sustainable aviation fuel (SAF) production. Here, we evaluated stepwise metabolic engineering strategies in the oleaginous yeast Yarrowia lipolytica to improve lipid accumulation from a mixed VFA substrate designed to mimic waste-derived streams (acetic, butyric, and hexanoic acids). While Y. lipolytica efficiently converts acetate into lipids, utilization of mixed-VFA substrates can impose physiological constraints that limit conversion performance. To address this challenge, we first overexpressed DGA1, a key enzyme for triacylglycerol (TAG) synthesis, resulting in lipid production of 0.54 g/L (1.83-fold increase). In contrast, deletion of PEX10 (peroxisomal biogenesis factor 10), a commonly applied strategy to enhance lipid accumulation in Y. lipolytica, led to reduced lipid production due to impaired utilization of butyric and hexanoic acids as substrates, as the ΔPEX10 strain showed significantly lower consumption rates of butyric and hexanoic acids compared to the control, accompanied by reduced lipid accumulation, suggesting that disruption of peroxisomal biogenesis and β-oxidation impairs the utilization of C4–C6 fatty acids as carbon sources under VFA-based cultivation. To further improve lipid biosynthesis, a heterologous acetyl-CoA synthetase from Salmonella enterica(seACS) was overexpressed to enhance acetyl-CoA supply, achieving lipid titer of 0.82 g/L. Overall, these stepwise engineering efforts resulted in a 2.77-fold increase in lipid production from mixed VFAs relative to the parental strain, demonstrating that targeted metabolic engineering can significantly improve the VFA-to-lipid bioconversion. Taken together, our findings highlight the feasibility of upgrading complex, waste-derived VFAs mixtures into microbial lipid feedstocks, providing a foundation for future waste-to-SAF and circular bioresource platforms.