PhD Thesis Defense

Directed evolution of enzymes can reveal activities that do not occur in the natural world. While most examples of directed evolution of new-to-nature chemistry have been applied in a synthetic direction, enzymatic biodegradation typically relies on wild-type enzymes. Here, we show how directed evolution can generate enzymes capable of degrading non-biodegradable anthropogenic compounds, focusing on efforts to break silicon–carbon bonds. The volatile siloxane building blocks of silicones are produced at megaton scale and are non-biodegradable, leading to concerns over their potential for environmental persistence, long-range transport, and bioaccumulation. We discovered an engineered variant of bacterial cytochrome P450BM3 that can cleave siloxane Si–C bonds, an activity not previously demonstrated with an enzyme. To accomplish this transformation, the enzyme catalyzes two chemically distinct steps: C–H hydroxylation produces a carbinol species whose Si–C bond is subsequently broken via a [1,2]-Brook-like rearrangement followed by hydrolysis. We enhanced this promiscuous function on cyclic and linear siloxanes using directed evolution.