title: 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.
authors.affiliation: Department of Chemical Engineering, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Pavan Kumar Reddy
authors.initials: PK
authors.lastname: Kambam
authors.forename: Dawn T
authors.initials: DT
authors.lastname: Eriksen
authors.forename: Jason
authors.initials: J
authors.lastname: Lajoie
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sayut
authors.forename: Lianhong
authors.initials: L
authors.lastname: Sun
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D015394:Molecular Structure; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D053038:Quorum Sensing; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C088386:homoserine lactone; D015107:4-Butyrolactone