Engineering the Carbon Metabolism of <i>Yarrowia lipolytica</i> to Efficiently Synthesize 2′-Fucosyllactose via a De Novo Pathway

2'-Fucosyllactose (2'-FL), the most abundant oligosaccharide in human milk, exhibits diverse bioactivities and is added to infant formula to simulate breastfeeding. Herein, <i>Yarrowia lipolytica</i> was employed as a microbial chassis for the de novo biosynthesis of 2'-FL. A complete biosynthetic pathway was established via genomic integration, resulting in an initial titer of 2.16 g/L. Systematic metabolic analysis identified the fucosyltransferase (FutC)-catalyzed reaction as the key rate-limiting step, alleviated by promoter engineering upregulating FutC, enhancing 2'-FL production. Additional strategies, including enhancing GDP-fucose supply, knocking out the <i>PFK</i> gene to redirect carbon flux, and strengthening the pentose phosphate pathway (PPP), further boosted production. Fed-batch fermentation with a mixed carbon source led to a titer of 105.88 g/L and a productivity of 0.98 g/L/h in strain YL16-6, representing the highest reported 2'-FL production in a plasmid- and antibiotic-free microbial system to date. This work demonstrates the potential of <i>Y. lipolytica</i> as a robust, scalable cell factory for the sustainable and antibiotic-free industrial production of 2'-FL.