Rhamnolipids have the potential to play the important role in enhanced oil recovery (EOR) as the substitutes of chemical surfactants,and mainly produced from Pseudomonas aeruginosa that is pathogenic strain and limited or not suitable for future industrial development.Here,Escherichia coli strains were designed to produce either mono-rhamnolipids or di-rhamnolipids by introducing different gene combinations related to rhamnolipid biosynthesis from P.aeruginosa.Rhamnolipids from engineered strains showed different performance in EOR due to the different chemical structures.The directed evolution and subsequent site-directed mutagenesis of rhamnosyltransferase 1 resulted in the production of new types of rhamnolipids with the increased ratio of C10-C10 congener and different performance in EOR.Computational modeling of mutations elucidates the function and specificity for the production of rhamnolipids.Creation of these mutations fundamentally provides evidence for new biocatalyst design and the corresponding production of rhamnolipids,by which expanded the possibility to improve oil recovery.