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

Nervonic acid (NA) is of great significance in repairing damaged nerve fibers and promoting the regeneration of neural cells. This study aimed to construct a <i>Yarrowia lipolytica</i> 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.