Precision Molecular Editing: Predicting Substrate Scope and Regiochemistry for CHEESY1, a Flavin Dependent Halogenase

The ability to carry out C-H activation at any site on any heteroaromatic scaffold is a holy grail, offering the potential to revolutionize molecule making. Precision editing, activating, and replacing a C-H bond with a carbon halogen bond opens the way to almost any diversification imaginable. Here, through genome mining and in silico analysis, we present a previously undescribed halogenase tool for precision C-H activation and halogenation. While many halogenated metabolites have been found in the marine environment, indicating the operation of a vast array of halogenases, the presence of such halogenases in other salty environments is less well-known. Here, we describe the first discovery and utilization of a halogenase from a microbe associated with salty and fermented food: specifically brined cheese. Most halogenases explored so far have been identified through their association with a biosynthetic gene cluster of a known natural product. Based on their role in the biosynthesis of that natural product, their native substrate is predicted. Many exciting and unexplored halogenases exist discretely of identified biosynthetic clusters. We describe an approach of carrying out halogenase discovery (unrelated to and unguided by known biosynthetic pathways and their encoded natural products) and predicting non-native substrates that the enzyme can and cannot process as well as the regiochemistry of each biotransformation. Following carrying out discovery in silico, we demonstrate the validation of the discovery results in the laboratory. CHEESY1 (Chemistry Helper Enzyme Enabling SelectivitY1) is shown to regioselectively halogenate a broad structural range of medicinally relevant heterocycles, including quinolines, isoquinoline, phenylpyrazole, and flavonoids. Being able to predict from gene to substrate to product for this powerful class of enzymes, which together afford the opportunity for providing a general solution for precision molecule editing, ahead of carrying out wet experimentation opens up exciting opportunities for the future of chemical catalysis and synthesis.