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

在其无机骨架中使用硅(以二氧化硅的形式)的海绵利用酶将硅酸(H4SiO4)聚合成二氧化硅。这些被称为硅酸盐的酶特别有趣，因为它们催化形成Si-O键。重要的是，我们实验室的工作表明，这些酶可以接受一系列有机硅化合物，并可以通过操纵反应平衡来驱动反之亦然(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，由于活性较低，这些硅酸盐目前不太适合实际应用。该项目的目标是开发高效和可推广的过程，使这种酶的工程能够在化学加工中应用。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过程(“管道”)，以：(1)生成和筛选突变硅酸盐的虚拟文库，以与聚二甲基硅氧烷(PDMS，最常见的有机硅)有效结合；(2)异源生产(在大肠杆菌中)突变酶的聚焦文库；(3)实验筛选酶文库，以了解其降解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.