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 lacks stereoselectivity and requires the use of toxic or expensive 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 RebHEvo4, 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 RebHEvo4, we devised an aminoacyl tRNA synthetase based halogenase biosensor and conducted over 500 hours of phage-assisted continuous evolution (PACE). Use of RebH _Evo4 in a bioreactor resulted in the production of 2.7 g/L of halogenated tryptophan. When coupled with a downstream enzyme, RebH _Evo4 allowed 36-fold increased yields of halogenated tryptamines compared to the wild-type enzyme. Additionally, RebH _Evo4 enabled efficient production of genetically encoded antimicrobial halogenated peptides. The efficient, site-specific halogenation by our evolved halogenase will accelerate sustainable biomanufacturing of halogenated drugs.