# Comparative Expression of Diacylglycerol Acyltransferases for Enhanced Accumulation of Punicic Acid-Enriched Triacylglycerols in Yarrowia lipolytica

## TL;DR

Researchers engineered the yeast Yarrowia lipolytica to produce punicic acid, a rare fatty acid, using waste glycerol as a low-cost carbon source.

## Contribution

The study identifies PgDGAT1 as the optimal enzyme for punicic acid accumulation in Yarrowia lipolytica using crude glycerol.

## Key findings

- PgDGAT1 showed the best balance between lipid content and punicic acid accumulation.
- Crude glycerol supported punicic acid production of up to 147.8 mg/L in scale-up experiments.
- Yarrowia lipolytica is a robust host for sustainable production of punicic acid from waste substrates.

## Abstract

Punicic acid is an uncommon ω-5 conjugated fatty acid with significant biological activity, mainly found in pomegranate seed oil. Due to limited natural availability, heterologous production of punicic acid in oleaginous yeasts offers a sustainable alternative. In this study, Yarrowia lipolytica was engineered for punicic acid biosynthesis by expressing the PgFADX gene from Punica granatum and subsequently modified to evaluate the influence of distinct diacylglycerol acyltransferases on punicic acid accumulation. The effects of seven acyltransferases, originating from P. granatum or Y. lipolytica, were compared under various cultivation conditions. The PgDGAT1 enzyme demonstrated the most favorable balance between total lipid content and punicic acid accumulation. Medium containing crude glycerol as a low-cost carbon source was initially tested in flask experiments with punicic acid accumulation in yeast cells of 129 mg/L. Further optimization of crude glycerol medium and subsequent scale-up experiments confirmed the potential of crude glycerol as an effective substrate, yielding up to 147.8 mg/L of punicic acid. Overall, this work identifies key enzymatic determinants for efficient punicic acid biosynthesis and supports Y. lipolytica as a robust host for the sustainable production of conjugated fatty acids from waste substrates.