Altering the Substrate Specificity of RhII by Directed Evolution

Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhII, an enzyme in the RhII-RhIR quorum-sensing system of P. aeruginosa, to alter its substrate specificity and gain insight into the molecular mechanisms of quorum sensing. By using a genetic screen, we identified a mutant with improved production of RhII's two signaling molecules, N-butanoyl- and N-hexanoyl-homoserine lactone (BHL and HHL). In particular, production of BHL has been enhanced by more than two-fold, and the synthesis of HHL has been improved from an undetectable level to a level similar to BHL; this change indicates a significant change in substrate specificity. No significant change in the gene expression level was observed. Sequence alignments suggest that the mutations are most likely to facilitate interactions between the enzyme and the two acylated ACP substrates. This work also demonstrates that the genetic screen/selection should be useful in engineering additional quorum-sensing components.