High-titer retinal production in Yarrowia lipolytica via redox engineering coupled with intra- and extracellular sequestration.

Retinal is an industrially valuable retinoid widely used in cosmetic and pharmaceutical applications, but its microbial production remains limited due to its chemical instability and potential susceptibility to intracellular redox metabolism. Here, Yarrowia lipolytica was engineered for high-level retinal biosynthesis by integrating targeted alcohol dehydrogenase deletion, precursor enhancement, and lipid-associated sequestration. Deletion of endogenous alcohol dehydrogenases increased retinal accumulation, while expression of ERG20(F87S) improved precursor flux toward carotenoid biosynthesis. Notably, coupling enhanced biosynthetic flux with DGA1 overexpression produced a greater improvement in retinal accumulation than either strategy alone. To further protect the oxidation-sensitive product, a two-phase system using isopropyl myristate (IPM) supplemented with an antioxidant was implemented, enabling efficient in-situ extraction and stabilization during fermentation. As a result, a total retinal titer of 2253 mg/L was achieved, with the product highly concentrated in the IPM phase at 45.1 g/L. Given that IPM is a cosmetic-grade solvent, this approach provides a formulation-compatible product phase, highlighting the potential to integrate microbial production with downstream processing. Overall, this study demonstrates that coordinated metabolic engineering and phase-partitioning strategies are essential for the efficient production of oxidation-sensitive terpenoid aldehydes.