Astaxanthin is a high-value chemical with strong antioxidant, anti-inflammatory and coloration properties. Here, a set of novel genetic engineering and culturing strategies for efficient astaxanthin bioproduction in Yarrowia lipolytica is reported. Modular pathway engineering and the fusion of two key enzymes (geranylgeranyl pyrophosphate synthase and bifunctional lycopene cyclase/phytoene synthase) yielded a recombinant strain with enhanced β-carotene and astaxanthin production, accumulating astaxanthin up to 131.9 mg/L in small-scale cultures with sucrose and 587.3 mg/L in a 3-L bioreactor with glucose as carbon sources, respectively. Notably, formation of intracellular carotenoid crystals was observed. Addition of oil overlayer at different regimes in 3-L bioreactors promoted secretion of astaxanthin and increased astaxanthin titer up to 973.4 g/L. Lastly, fed-batch fermentation in a 100-L bioreactor with glycerol as the carbon source resulted in a final astaxanthin titer of 812.3 mg/L after 114 h, representing the highest astaxanthin titer reported so far in large-scale cultures.