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

## TL;DR

This study engineered the yeast Yarrowia lipolytica to produce nervonic acid, a compound important for nerve repair, using metabolic engineering techniques.

## Contribution

The study introduces an integrated strategy combining organelle compartmentalization and dynamic promoter coordination to enhance nervonic acid production.

## Key findings

- The engineered strain achieved 451.80 mg/L of nervonic acid in shake-flask culture.
- Fed-batch fermentation in a 5-L bioreactor yielded 6.55 g/L, with nervonic acid comprising 29.81% of total fatty acids.
- Optimized β-ketoacyl-CoA synthases and precursor availability improved nervonic acid accumulation.

## Abstract

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 Yarrowia lipolytica 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.