Rhamnolipids are a class of biosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in . Four of the 5 proteins except RhlC were successfully over-expressed in and three of them (RhlA, RhlB and RhlI) were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins to favor the construction of super strains for rhamnolipids production.