Altering the Substrate Specificity of RhII by Directed Evolution
Altering the Substrate Specificity of RhII by Directed Evolution
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DOI：
10.1002/cbic.200800636
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发表时间：
2009-02-13
期刊：
影响因子：
3.2
通讯作者：
Sun, Lianhong
中科院分区：
文献类型：
--
作者：
Kambam, Pavan Kumar Reddy;Eriksen, Dawn T.;Sun, Lianhong
群体感应调节人类条件致病菌铜绿假单胞菌生物膜形成和毒力因子产生。我们使用定向进化工程师RhII，在铜绿假单胞菌的RhII-RhIR群体感应系统中的酶，以改变其底物特异性，并深入了解群体感应的分子机制。通过使用遗传筛选，我们确定了一个突变体，提高生产RhII的两个信号分子，N-丁酰基和N-己酰基高丝氨酸内酯（BHL和HHL）。特别是，BHL的产量提高了两倍以上，HHL的合成从检测不到的水平提高到与BHL相似的水平;这种变化表明底物特异性发生了显著变化。未观察到基因表达水平的显著变化。序列比对表明，突变是最有可能促进酶和两个酰化ACP底物之间的相互作用。这项工作还表明，遗传筛选/选择应该是有用的工程额外的群体感应组件。
Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhII, an enzyme in the RhII-RhIR 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 RhII'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.