The Evolution Of AiiA Quorum Sensing Inhibitor

aiiA gene cloned from Bt.4Q7 (B. thuringiensis 4Q7) encodes an enzyme which inactivating QS (quorum sensing) signal molecules AHLs (N-acylhomoserine lactones) involved in regulation of divers biological functions especially the expression of pathogenic gene in the Gram negative bacteria. Phenotypic characterization could be abolished or reduced further more when the AHLs inactivated by AiiA with higher enzyme activity which was desired in our molecule evolution. Library of aiiA gene was constructed by DNA shuffling including steps DNase I digestion, three round PCR without primers and one round PCR with primers. Reporter strain CV026(Chromobacterium violaceum 026) was used for the high throughput screening. Nine mutant strains with high enzyme activity were abtained and the evolved aiiA genes were cloned and sequenced. The evolution of AiiA quorum sensing inhabitor was achieved as the activity of AiiAS29/S36 was increased to 2.2 times as the original AiiA4Q7 by the relatively enzyme activity assay of AiiA samples.