Abstract

This study investigated rhamnolipid synthesis in Pseudomonas aeruginosa ATCC 27853. Two constitutive promoters, PrpsJ and PoprL, were isolated and cloned upstream of the rhlABRI and rmlBDAC gene clusters to evaluate their impact on rhamnolipid titers. The overexpression of rhlB, driven by the PrpsJ promoter, significantly enhanced rhamnolipid production. Subsequent glycine-scanning mutagenesis of RhlB identified an optimal variant (RhlBM328G), which increased the titer 1.82-fold (to 24.6 g·L−1) compared to the wild type, achieving a product yield of 0.39 g·g−1. Characterization of the extracted rhamnolipids revealed a critical micelle concentration of 1 mg/L, a corresponding surface tension of 53.9 mN/m, and a hydrophilic–lipophilic balance (HLB) value of 14. This HLB value indicated that the synthesized rhamnolipids possess superior hydrophilicity, robust oil-in-water emulsifying capabilities, and excellent solubilization and dispersion properties. Furthermore, molecular docking and molecular dynamics simulations demonstrated that in the RhlBM328G mutant, the nucleophilic attack distances between the substrates and the catalytic moiety are optimized for catalysis, thereby boosting rhamnolipid production.

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