Altering the Substrate Specificity of RhlI by Directed Evolution

Abstract

REDUCING VIRULENCE: RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (BHL), with a minor product N-hexanoyl homoserine lactone (HHL). By using directed evolution and a genetic screen, RhlI has been engineered for enhanced production of both BHL and HHL at a similar level. Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhlI, an enzyme in the RhlI-RhlR 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 RhlI'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.

Precision metabolic engineering: The design of responsive, selective, and controllable metabolic systems

“…Transcription factors involved in quorum sensing have been the target of extensive protein engineering efforts to accomplish such goals (Collins et al, 2005; Hawkins et al, 2007; Kambam et al, 2008, 2009). For example, by screening a large library of LuxR mutants, a library of transcription factors with up to a 100 fold increase in sensitivity to specific molecules was developed (Collins et al, 2005).…”

The enzymes of bacterial census and censorship

“…The roles played by individual amino acid residues in the AHL synthase reaction are unclear, although mutagenesis studies of conserved and random residues have identified critical residues in RhlI [14, 15]. The pH-dependence of kinetic parameters in the RhlI reaction show that the ionization states of two residues are important for catalysis and binding [9].…”

Directed evolution: new parts and optimized function

“…In addition, these authors demonstrated how artificial positive feedback loops can increase the effective sensitivity of LuxR-based circuits. The signal synthase component of QS systems has also been engineered: the substrate specificity of the RhlI synthase was modified to increase production of the non-native signal hexanoyl-homoserine lactone [14]. …”

“…A similar approach was used recently to generate a genetic AND gate using LuxR and LacI [14]. Ribosome binding sites (RBS) are good targets for tuning mismatches in component activities.…”