Hyperproduction of Rhamnolipid in <i>P. putida</i> by Protein and Metabolic Engineering

Rhamnolipids are eco-friendly biosurfactants, but their native producer Pseudomonas aeruginosa is an opportunistic pathogen. Herein, the biosafety strain Pseudomonas putida was engineered for efficient rhamnolipid production. The key enzyme RhlA was improved by rational and computer-aided design, which yields RhlA F43W/G130N with enhanced catalytic activity. Molecular dynamics simulations elucidated the mechanism behind its improved performance. Metabolic engineering was further employed to enhance the synthesis efficiency of rhamnolipids. Overexpression of rhlA F43W/G130N and rhlB driven by promoters Plac and Ptac, coupled with heterologous expression of the rmlBDAC operon in flagella deletion strain Δflag, enhanced the availability of two precursors, dTDP-L-rhamnose and β-hydroxy fatty acids, yielding the final strain E3. The strain E3 produced 5.1 g/L rhamnolipid in shake-flask and 28.6 g/L with a productivity of 0.3 g/L/h in a 5 L bioreactor, making the highest titer reported for engineered P. putida KT2440.