Reaction pathway engineering converts a radical hydroxylase into a halogenase

• DOI: 10.1038/s41589-021-00944-x
• Publication Date: 2021-12-22T17:03:24Z
• Authors: Monica E. Neugebauer; Elijah N. Kissman; Jorge A. Marchand; Jeffrey G. Pelton; Nicholas A. Sambold; Douglas C. Millar; Michelle C. Y. Chang
• Keywords: Protein Engineering; Synthetic Biology; Metabolic Engineering

FeII/α-ketoglutarate (FeII/αKG)-dependent enzymes offer a promising biocatalytic platform for halogenation chemistry owing to their ability to functionalize unactivated C-H bonds. However, relatively few radical halogenases have been identified to date, limiting their synthetic utility. Here, we report a strategy to expand the palette of enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. This approach could allow us to tap the broader class of FeII/αKG-dependent hydroxylases as catalysts by their conversion to halogenases. Toward this goal, we discovered active halogenases from a DNA shuffle library generated from a halogenase-hydroxylase pair using a high-throughput in vivo fluorescent screen coupled to an alkyne-producing biosynthetic pathway. Insights from sequencing halogenation-active variants along with the crystal structure of the hydroxylase enabled engineering of a hydroxylase to perform halogenation with comparable activity and higher selectivity than the wild-type halogenase, showcasing the potential of harnessing hydroxylases for biocatalytic halogenation.