Extensive usage of plant-based oils, especially palm oil, has led to environmental and social issues, such as deforestation and loss of biodiversity, thus sustainable alternatives are required. Microbial oils, especially from Yarrowia lipolytica, offer a promising solution because of their similar composition to palm oil, low carbon footprint and ability to utilise low-cost substrates. In this study, we employed the Design-Build-Test-Learn (DBTL) approach to enhance lipid production in Y. lipolytica. We systematically evaluated predictions from the genome-scale metabolic model to identify and overcome bottlenecks in lipid biosynthesis. We tested the effect of predicted medium supplements (glutamate, leucine, methionine and threonine) and genetic intervention targets, including the overexpression of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS), diacylglycerol acyltransferase(DGA1), the deletion of citrate exporter gene (CEX1) and disruption of β-oxidation pathway (MFE1). This work revealed the critical roles of ACC, ACL, TS and DGA1 and the interaction of these genes with elevated intracellular citrate availability in lipid biosynthesis. Combining TS and DGA1 overexpression in the Δmfe_Δcex background achieved a remarkable 200% increase in lipid content (56% w/w) and a 230% increase in lipid yield on glycerol. These findings underscore the potential of Y. lipolytica as an efficient microbial cell factory for fatty acid production. Our study advances the understanding of lipid metabolism in Y. lipolytica and demonstrates a viable approach for developing sustainable and economically feasible alternatives to palm oil.