期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 134, 期 -, 页码 -出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2019.109475
关键词
Directed evolution; High-throughput screening; Quorum sensing; Protein engineering
资金
- University Natural Science Research Project of Anhui Province [KJ2018A0399]
- Natural Science Foundation of Anhui Province [1908085QC125]
- Innovation Team of Scientific Research Platform of Anhui Province [KJ2015TD001]
Quorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhIL an enzyme in the RhlI-RhIR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HIE), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to identify RhII mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coll. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. FthlI.4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds.
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