Enzymes are increasingly employed for chemical synthesis due to their high catalytic efficiency, high regio- and stereoselectivity, and extremely mild operating conditions. Perhaps the most attractive feature of these catalysts however, is their ability to be systematically optimized for a particular application using directed evolution (Fig. 1A). Thus, while the activity of a given enzyme may or may not be particularly general (with respect to substrate scope for example), this activity is highly generalizable such that activity toward a desired substrate can be rapidly improved using successive rounds of mutagenesis and screening. We are exploiting this property to engineer various halogenases for use in organic synthesis due to the importance of halogenated compounds as both building blocks and active pharmaceutical ingredients (Fig. 1B/C). We are using structure-guided and directed evolution schemes to expand the substrate scope and improve the practicality of these valuable catalysts. We then explore structure function relationships in improved enzymes to help rationalize mechanisms for the observed improvements and to inform subsequent engineering efforts.
Fig. 1. A) General scheme for directed enzyme evolution.
B) Work-flow for structure-guided and directed evolution of halogenases.
C) Representative substrate scope of the halogenase RebH.
We are also pursuing several other enzymatic solutions for new C-H bond hetero-functionalization reactions. These efforts include genome mining for new halogenases and exploring non-native atom transfer reactions of various metalloenzymes. As part of these efforts, we also develop new evolution and mutagenesis methodologies, including continuous evolution, in collaboration with the Dickinson lab. Ultimately, we hope to port these enzymatic transformations into living organisms to facilitate chemical production in vivo.