Optimizing Systems for Robust Heterologous Production of Biosurfactants Rhamnolipid and Lyso-Ornithine Lipid in Pseudomonas Putida KT2440

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L−1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L−1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L−1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants.