Engineering Flavin-Dependent Halogenases

In the two decades since the discovery of the first flavin-dependent halogenase (FDH), great strides have been made in elucidating the diversity of enzymes in this class as well as their structures and functions. More recently, efforts to engineer these enzymes for synthetic applications have yielded their first successes. FDH variants with improved stability, expanded substrate scope, and altered regioselectivity have been developed. Here, we review these efforts and provide representative protocols that have been employed for FDH engineering. Random and structure-guided mutagenesis approaches and screening methods, including HPLC, mass spectrometry, and spectrophotometric methods, are discussed. The protocols described herein should be generalizable to many FDHs and a wide variety of engineering goals.