Directed evolution of LuxI for enhanced OHHL production
Directed evolution of LuxI for enhanced OHHL production
复制标题
DOI：
10.1002/bit.21901
复制
发表时间：
2008-10-01
影响因子：
3.8
通讯作者：
Sun, Lianhong
中科院分区：
文献类型：
--
作者：
Kambam, Pavan Kumar Reddy;Sayut, Daniel J.;Sun, Lianhong
群体感应是细菌用于协调群体行为的常见机制，并且涉及多种生物过程，例如生物发光、毒力因子合成、抗生素产生和生物膜形成。为了工程化LuxI-L，uxR群体感应系统的LuxI酶，我们开发了高通量遗传选择以鉴定在E.杆菌利用这种遗传选择，我们通过定向进化创建了具有改进的OHHL合成速率和产量的LuxI突变体，在两代后鉴定了三个LuxI突变体。允许验证遗传筛选的体内半定量方法和使用HPLC-MS/MS定量的OHHL产率，揭示了与野生型培养物相比，突变培养物中增加了80倍。除了OHHL之外，C6 HSL（己酰基高丝氨酸内酯）和C8 HSL（辛酰基高丝氨酸内酯）的产率也得到提高，并且观察到对C6 HSL生产的底物特异性的轻微变化。根据与EsaI（LuxI的同源物）的晶体结构的对齐，两个突变最有可能参与增强酶与底物之间的相互作用。高通量的遗传选择和半定量的方法可以方便地修改为LuxI同源基因的定向进化。这些LuxI突变体的鉴定在合成生物学中具有意义，它们可用于人工遗传电路的构建。此外，特异性靶向群体感应以减弱革兰氏阴性感染性细菌发病机制的药物的开发也可能受益于对氨基酸取代揭示的群体感应分子机制的见解。
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-L,uxR 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.