Combining Screening and Metabolic Engineering to Construct an Improved Rhamnolipid-Producing Pseudomonas aeruginosa Strain

Rhamnolipids (RLs), a class of glycolipid biosurfactants, hold significant promise for applications in industry, pharmaceuticals, and agriculture. However, their commercialization remains constrained by the technical challenges of large-scale production. This study focused on the construction of a high-producing RLs producer in Pseudomonas aeruginosa through an integrated strategy combining screening and metabolic engineering. Mutant M1 was isolated from the random mutant libraries using the cetyltrimethylammonium bromide-methylene blue agar assay screening method, achieving a 1.2-fold increase in RLs production (3.48 g/L) relative to the parental strain. To further enhance RLs production, rhlA and rhlB were overexpressed under a strong promoter, while the algC gene and rmlBDAC operon were co-overexpressed to boost dTDP-L-rhamnose precursor supply and maximize the metabolic flux toward RLs biosynthesis. Additionally, a 1:1 mixture of glucose and glycerol was employed as a carbon source to balance strain growth and RLs accumulation. Through these combinatorial strategies, the RLs production in a 5 L fermenter reached 18.2 g/L at 72 h, representing a substantial enhancement in production performance.