A modular approach for dCas9-mediated enzyme cascading via orthogonal bioconjugation
A modular approach for dCas9-mediated enzyme cascading via orthogonal bioconjugation
复制标题
DOI：
10.1039/d0cc04196c
复制
发表时间：
2020-09-29
影响因子：
4.9
通讯作者：
Chen, Wilfred
中科院分区：
文献类型：
--
作者：
Berckman, Emily A.;Chen, Wilfred
我们报告了一种新的模块化策略，通过采用正交翻译后化学来组装 dCas9 引导的酶级联。两个正交的 SpyCatcher 和 SnoopCatcher 对用于一锅酶生物缀合到两种不同的 dCas9 蛋白上，以使其能够引导组装到 DNA 支架上。所得的双组分纤维素体的还原糖产量比未组装的酶高 2.8 倍。该平台保留了 dCas9 蛋白提供的高结合亲和力，可轻松控制酶组装，同时为多种酶级联的体内和体外组装提供灵活性，只需最少的优化。
We report a new modular strategy to assemble dCas9-guided enzyme cascades by employing orthogonal post-translation chemistry. Two orthogonal SpyCatcher and SnoopCatcher pairs were used for the one-pot enzyme bioconjugation onto two different dCas9 proteins to enable their guided assembly onto a DNA scaffold. The resulting two-component cellulosomes exhibited 2.8-fold higher reducing sugar production over unassembled enzymes. This platform retains the high binding affinity afforded by dCas9 proteins for easy control over enzyme assembly while offering the flexibility for bothin vivoandin vitroassembly of a wide array of enzyme cascades with minimal optimization.