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

Rhamnolipids are eco-friendly biosurfactants, but their native producer <i>Pseudomonas aeruginosa</i> is an opportunistic pathogen. Herein, the biosafety strain <i>Pseudomonas putida</i> 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 <i>rhlA</i>^F43W/G130N and <i>rhlB</i> driven by promoters Plac and Ptac, coupled with heterologous expression of <i>the rmlBDAC</i> 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 <i>P. putida</i> KT2440.