Engineering Yarrowia lipolytica for nervonic acid production by organelle compartmentalization and dynamic promoter coordination

strain capable of producing NA. The β-oxidation pathway was first disrupted and evaluated β-ketoacyl-CoA synthases (KCSs) with different substrate specificities. The substrate specificity of KCS was further optimized through semi-rational design. Oleic acid biosynthesis and the desaturation of tetracosanoic acid (C24:0) were enhanced to increase precursor availability. Furthermore, organelle compartmentalization and dynamic promoter coordination were implemented to synergistically improve NA accumulation. Finally, an orthogonal malonyl-CoA biosynthetic route was introduced together with an NADPH regeneration system to boost reducing power supply, leading to a substantial increase in production. The engineered strain achieved a titer of 451.80 mg/L in shake-flask culture, and reached 6.55 g/L during fed-batch fermentation in a 5-L bioreactor, where NA accounted for 29.81% of TFAs. This study presents an integrated metabolic engineering strategy providing novel insights into the microbial biosynthesis of NA.