(375f) Directed Evolution of Rhli for Introducing a Novel Activity

LuxI and its homologs catalyze the synthesis of a variety of signaling molecules essential for a cell-cell communication mechanism, quorum sensing. These signaling molecules vary in the length of the side chain and the oxidation states, and these differences can be distinguished by corresponding cognate transcription factors. Such specificity allows the presence of multiple quorum sensing systems simultaneously without interfering with each other's functions. LuxI homologs exhibit high sequence similarity, and highly conserved amino acid residues across the family have been identified. In spite of the intensive biochemical characterizations of the LuxI enzymes, the molecular mechanisms of the LuxI enzyme substrate specificity, amino acid residues important for the substrate specificity in particular, remain unknown. Because quorum sensing is involved in a variety of infectious diseases, illuminating the mechanisms will facilitate the development of novel antibiotics specifically targeting quorum sensing to eliminate bacterial infections, a method regarded as a superior strategy to common antibiotics with the potential of not introducing bacterial drug resistance.

We believe engineering the substrate specificity of LuxI homologs will identify amino acids important for the substrate specificity. As a homolog of LuxI, RhlI catalyzes the synthesis of C4HSL (N-butanoyl homoserine lactone) and C6HSL (N-hexanoyl homoserine lactone) but not OHHL (3-oxo-hexanoyl homoserine lactone), which is synthesized by LuxI. Using a genetic selection we established for the engineering of LuxI, we performed directed evolution on RhlI to introduce a novel OHHL synthesis activity. After three rounds of directed evolution experiments, a mutant with a significant OHHL activity was identified, and the OHHL synthesized by the mutant was detected from the cell culture. In addition to the novel OHHL synthesis activity, this mutant shows a more than 10-fold increase in the C6HSL synthesis activity, an activity which is comparable to the C4HSL synthesis activity. To further increase OHHL synthesis activity, we have developed a strategy allowing for selecting such an activity. In this presentation, we will discuss our efforts in using both directed evolution and rational design in introducing and improving the OHHL synthesis activity.