A Genetic Circuit System Based on Quorum Sensing Signaling for Directed Evolution of Quorum‐Quenching Enzymes

Abstract

Quorum sensing is a cell-cell communication mechanism that is involved in the regulation of biological functions such as luminescence, virulence, and biofilm formation. Quorum-quenching enzymes, which interrupt quorum-sensing signaling through degradation of quorum-sensing molecules, have emerged as a new approach to controlling and preventing bacterial virulence and pathogenesis. In an effort to develop quorum-quenching enzymes with improved catalytic activities, a genetic circuit system based on acylhomoserine-lactone (AHL)-mediated quorum-sensing signaling was constructed. The genetic circuit system was composed of lux-R, lux-I promoter, beta-lactamase, and beta-lactamase inhibitor, and designed to confer antibiotic resistance on host cells expressing an AHL-degrading enzyme, thereby enabling rapid screening of quorum-quenching enzymes. To demonstrate the utility of the genetic circuit system, we attempted the directed evolution of the AHL hydrolase from Bacillus sp. The genetic circuit system was shown to be effective in screening of quorum-quenching enzymes with high catalytic efficiency. From these results it is expected that the genetic circuit system can be widely used for the isolation and directed evolution of quorum-quenching enzymes with greater potential.

Directed evolution: an evolving and enabling synthetic biology tool

“…Directed evolution has been applied to improve the catalytic efficiency of such enzymes from Bacillus sp. and Geobacillus kaustophilus HTA426 by 7-fold and 72-fold, respectively [43,44]. …”

Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

“…Another type of reporter system is based on the expression of a b-lactamase (i.e. a b-lactam antibiotic-degrading enzyme) and the expression of a b-lactamase inhibitor under regulation of a QS-sensitive promoter (45). When AHLs are present and not degraded by the QQ enzyme, the b-lactamase inhibitor is expressed, resulting in the death of the host strain grown in the presence of the b-lactam antibiotics.…”

“…When AHLs are present and not degraded by the QQ enzyme, the b-lactamase inhibitor is expressed, resulting in the death of the host strain grown in the presence of the b-lactam antibiotics. Upon selection of an effective QQ enzyme, AHLs are degraded, the b-lactamase inhibitor is not expressed, and b-lactamase can degrade the antibiotic, allowing the growth of the host cells (45). This biosensor strain was used to identify three improved variants of Bacillus sp.…”

“…This biosensor strain was used to identify three improved variants of Bacillus sp. AiiA, screening an estimated library of 5 3 10 5 clones (45).…”

“…Using the above mentioned b-lactamase-based assay, the single variant V69L, with a 3.7-fold increase in catalytic efficiency (k cat /K m ) for C6-HSL was obtained. This mutant was further improved, leading to the identification of double (V69L/I190F) and triple (V69L/ I190F/G207V) mutants, with 7-and 6.1-fold enhancements in k cat /K m values for C6-HSL, respectively (45).…”