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

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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.