(532e) Decoupling Carbon and Energy Metabolism in Yarrowia Lipolytica for High-Yield Product Synthesis from C2 Substrates

Food security, land and water usage, and deforestation mandate biomanufacturing to seek alternative feedstocks other than biomass. Substrates directly generated from CO2 via electrocatalysis or biological fixation of CO2, encompassing various one-carbon (C1) and two-carbon (C2) compounds have become promising feedstock for sustainable biomanufacturing. Such substrates can be derived from direct CO2 fixation by acetogens or processes capitalizing on recent advances in electrochemistry combined with rapid deployment of affordable, carbon-free electricity. A model oleaginous yeast, Yarrowia lipolytica, has gained attention as a biomanufacturing chassis due to its ability to utilize various carbon feedstock and produce lipids at unprecedented titer and productivity. Notably, it has been shown to produce lipids at more than 100 g/L titer using acetate, a C2 substrate, as sole carbon and energy source. However, acetate has lower energy content than glucose, which leads to lower overall yield (carbon efficiency) in producing high energy products like lipids. To address this, we investigated two different approaches: 1) an alternative C2 substrate, ethanol, which contains more energy and can still be generated by electroreduction or microbial conversion of CO2 and energy; 2) co-feeding additional electron sources that can be efficiently generated from CO2 reduction, such as formate and methanol. To streamline metabolic carbon flux from C2 substrates to products, we leverage yeast organelle engineering to decouple carbon and energy metabolism, with the aim of achieving near-complete carbon efficiency of C2-to-lipid biosynthesis. Specifically, we investigated various energy metabolism pathways for the efficient conversion of electrons derived from reduced carbon sources to NADPH and ATP, essential cofactors required for lipid biosynthesis.