A biosynthetic paradox implicating dynamic zeaxanthin biosynthesis at the ER-LD interface

Traditional metabolic engineering often treats lipid droplets (LDs) as inert storage sinks and assumes shortening enzyme-substrate distance inherently enhances pathway efficiency. Harnessing Yarrowia lipolytica for zeaxanthin production, we uncovered a pivotal “spatial paradox” challenging this conventional dogma. A selective shielding strategy first identified the hydroxylase Pa CrtZ as the primary biosynthetic bottleneck. Subcellular localization trials revealed that directly targeting Pa CrtZ to the β-carotene-rich LD surface unexpectedly yielded the lowest zeaxanthin relative fraction (9.87%), significantly underperforming compared to cytosolic (27.15%) or peroxisomal targeting (43.16%). To investigate this counterintuitive phenomenon, multidimensional engineering strategies were implemented to boost zeaxanthin accumulation. Subsequently, in situ , label-free stimulated Raman scattering (SRS) microscopy unequivocally demonstrated the co-localization of β-carotene and zeaxanthin within LDs, confirming that substrate inaccessibility was not the limiting factor. Guided by principles of LD biogenesis, these findings led us to conceptualize a dynamic endoplasmic reticulum (ER)–LD interface functional microenvironment model. We propose that robust Pa CrtZ-catalyzed hydroxylation is sustained predominantly at transient ER–LD junctions, where hydrophobic β-carotene "skimming" is dynamically synchronized with efficient O 2 and NADPH cofactor exchange. This model redefines lipid organelles as active, organized metabolic hubs beyond mere storage, offering a novel transformative framework for future organelle-targeted metabolic interface design.