Implementing a Design-Build-Test pipeline for the Discovery of a Silyl Hydrolase

海洋海绵在其无机骨架中使用硅（以二氧化硅的形式），利用酶将硅酸（H4SiO4）聚合成二氧化硅。这些酶，被称为硅蛋白，是非常有趣的，因为它们催化硅- o键的形成。至关重要的是，我们实验室的工作表明，这些酶接受一系列有机硅化合物，并且可以通过操纵反应平衡来反向驱动（即Si-O键水解）。这些结果暗示了利用酶回收有机硅聚合物的有趣可能性。然而，由于活性低，这些硅蛋白目前在实际应用中表现不佳。该项目旨在开发高效和可推广的工艺，使这种酶的工程应用于化学加工。该项目将采用工程“设计-建造-测试”的方法，结合高度自动化和高通量筛选。因此，该项目将开发自动化流程（“管道”），以：(i)生成和筛选突变硅蛋白的虚拟库，以便与聚二甲基硅氧烷（PDMS，最常见的硅酮）有效结合；（ii）异源产生（在大肠杆菌中）聚焦的突变酶文库；（iii）实验筛选酶库的降解PDMS的能力，以验证虚拟筛选。

Marine sponges that use silicon (in the form of silica) in their inorganic skeleton utilise enzymesto polymerise silicic acid (H4SiO4) into silica. These enzymes, termed the silicateins, are uniquelyinteresting as they catalyse the formation of Si-O bonds. Crucially, work in our laboratory hasshown that these enzymes accept a range of organosilicon compounds, and can be driven in thereverse (i.e. Si-O bond hydrolysis) by manipulation of the reaction equilibrium. These results alludeto the intriguing possibility of utilising enzymes for the recycling of silicone polymers. However,these silicateins are currently poor candidates for practical application due to their low activity.The project will aim to develop efficient and generalizable processes that will enable theengineering of this enzyme towards its application in chemical processing. This project will adoptan engineering "design-build-test" approach, incorporating a high degree of automation and high-throughput screening. Thus, the project will develop automatable processes ("pipelines") to: (i)generate and screen virtual libraries of mutant silicateins for efficient binding withpolydimethylsiloxane (PDMS, the most common silicone); (ii) heterologously produce (in E. coli) afocused library of mutant enzymes; (iii) experimentally screen the library of enzymes for theirability to degrade PDMS, to validate the virtual screening.