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

Marine sponges that use silicon (in the form of silica) in their inorganic skeleton utilise enzymes
to polymerise silicic acid (H4SiO4) into silica. These enzymes, termed the silicateins, are uniquely
interesting as they catalyse the formation of Si-O bonds. Crucially, work in our laboratory has
shown that these enzymes accept a range of organosilicon compounds, and can be driven in the
reverse (i.e. Si-O bond hydrolysis) by manipulation of the reaction equilibrium. These results allude
to 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 the
engineering of this enzyme towards its application in chemical processing. This project will adopt
an 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 with
polydimethylsiloxane (PDMS, the most common silicone); (ii) heterologously produce (in E. coli) a
focused library of mutant enzymes; (iii) experimentally screen the library of enzymes for their
ability to degrade PDMS, to validate the virtual screening.