Heterologous production of rhamnolipids in Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 based on the endogenous production of N-acyl-homoserine lactones.

Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.