Sustainable microbial production of retinol in Yarrowia lipolytica: From strain engineering to formulation.

Retinol (vitamin A) is traditionally produced by chemical synthesis, but growing demand for sustainable alternatives has increased interest in microbial production. However, the intrinsic instability of retinol complicates both biosynthesis and downstream processing, requiring integrated strategies for product recovery and stabilization. In this study, we report the first purification and formulation of microbially produced retinol. We previously engineered Yarrowia lipolytica for retinol production and improved retinol recovery by supplementing the fermentation with Tween 80 and the antioxidant butylated hydroxytoluene (BHT). Here, retinol production was further enhanced 3.2-fold in shake flasks by increasing acetyl-CoA supply and pathway flux. To address purification challenges, we developed an extraction strategy for retinol encapsulated in micelles using n-hexane and ethanol, followed by two additional ethanol extractions. Replacing Tween 80 with the more hydrophilic Tween 20 improved purification efficiency. The engineered strain produced 7.16 g/L retinol in a 5-L fermenter supplemented with Tween 20 and BHT, and retinol was purified to 48.6% purity, demonstrating the importance of co-designing fermentation conditions with downstream processing. A prototype formulation containing 10% retinol was developed and validated for cytotoxicity and stability. Life cycle assessment quantified the carbon footprint and identified key environmental contributors. Together, this work establishes an integrated approach combining metabolic engineering, fermentation design, and downstream processing to enable sustainable microbial production of unstable high-value compounds such as retinol.