Continuous evolution of a halogenase enzyme with improved solubility and activity for sustainable bioproduction

Halogenation enhances the stability and function of pharmaceuticals, biomaterials, and industrial compounds. However, chemical halogenation of molecules and peptides can lack stereoselectivity and require the use of toxic chemicals. Although enzymatic halogenation can improve selectivity and reduce environmental impact, current halogenases are inefficient and insoluble, leading to low yields that limit their applications. Here, we develop RebH_Evo4, a soluble and highly active tryptophan halogenase, containing 12 mutations that confer 37-fold and 44-fold increases in 7-chloro- and 7-bromotryptophan production respectively, in vivo. To create RebH_Evo4, we devise an aminoacyl-tRNA synthetase-based halogenase biosensor and conduct over 500 hours of phage-assisted continuous evolution (PACE). Use of RebH_Evo4 in a bioreactor results in the production of 2.7 g/L of halogenated tryptophan. When coupled with a downstream enzyme, RebH_Evo4 allows 36-fold increased yields of halogenated tryptamines compared to the wild-type enzyme. Additionally, RebH_Evo4 enables efficient production of genetically encoded antimicrobial halogenated peptides. The efficient, site-specific halogenation enabled by our evolved halogenase will accelerate sustainable biomanufacturing of halogenated drugs.