Directed evolution of LuxI for enhanced OHHL production

Abstract

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.

Tunable synthetic phenotypic diversification on Waddington’s landscape through autonomous signaling

“…For autonomous signal production in our diversification, we placed a luxI variant, designed from known variants (20), downstream of the LacI-coding sequence on pHT toggle to obtain the diversity-generator plasmid, pHT_luxI1.5C (Fig. S1A).…”

“…The pHT_luxI1.5C plasmid was obtained by mutating A117T/A188G. LuxI1A and luxI2A contain the A101G and A101G/A117T/A188G mutations, respectively (20).…”

Design and Control of Synthetic Biological Systems

“…In traditional directed evolution, a gene encoding the desired protein activity or the desired transcription rate of a promoter is selected from a library of the randomly mutated gene, by the application of selective pressure. Directed evolution has been applied to increase the activities of various proteins, such as enzyme [18] or signal receptor [28], and has also been utilized to increase the protein synthesis rate [29]. Further advances in directed evolution strategies, such as library construction [30], will allow us to more easily tune the parameter values of genetic parts and result in the successful construction of more complex synthetic circuits.…”

“…The initial cell density was controlled by the inoculation volume of the cell culture. The LuxI activity was modified by mutations of the gene encoding LuxI, and the mutation sites were reported previously [18].…”

Altering the Substrate Specificity of RhlI by Directed Evolution

“…[11][12][13][14] We have also developed a genetic selection protocol and subsequently used the method for the directed evolution of LuxI to improve its activity; LuxI is an enzyme that is responsible for the synthesis of the signaling molecule 3-oxo-N-hexanoyl homoserine lactone (OHHL) of the LuxI-LuxR quorumsensing system in V. fischeri. [15,16] By using a similar method, we report the alteration of the substrate specificity of RhlI, which is an enzyme that is responsible for the synthesis of the signaling molecules of the RhlI-RhlR quorum-sensing system in P. aeruginosa.…”