Efficient biocatalytic synthesis of docosahexaenoic acid-enriched phosphatidylethanolamine (DHA-PE) by an engineered Streptomyces sp. Phospholipase D (PLD) with expanded active pocket.

Docosahexaenoic acid-enriched phosphatidylethanolamine (DHA-PE) represents a premium functional lipid with exceptional oxidative stability and bioavailability. However, the enzymatic synthesis of DHA-PE via Phospholipase D (PLD) is severely hindered by the extreme structural bulkiness of the polyunsaturated DHA acyl chain, resulting in low transphosphatidylation efficiency. To overcome this critical steric barrier, we employed a computationally guided semi-rational design strategy focused on remodeling the substrate-binding pocket of Streptomyces sp. PLD Sp-256. Among the 30 designed variants, the optimal mutant A409C was identified, delivering a remarkable 5.1-fold enhancement in apparent transphosphatidylation activity alongside a dramatically improved catalytic efficiency (kcat/Km). Molecular dynamics (MD) simulations unveiled the underlying structural basis: the A409C substitution effectively expands the active pocket volume and optimizes the substrate-binding free energy, thereby relieving the steric hindrance against the bulky DHA-phosphatidylcholine (DHA-PC). Through systematic process optimization (substrate molar ratio of 10:1, 40 μg enzyme loading, 40 °C, and a 1:1 v/v organic/aqueous biphasic system), the A409C biocatalyst achieved an unprecedented 88.6% conversion yield-the highest recorded for PLD-mediated DHA-PE synthesis to date. This study not only elucidates the structure-function relationship of the PLD pocket architecture but also establishes a robust and highly efficient biocatalytic platform for the industrial production of high-value polyunsaturated phospholipids.