De novo biosynthesis of halogenated benzoxazinoids in Nicotiana benthamiana

Halogenation of natural products can improve their bioactivity, pharmacokinetics, and commercial value, yet sustainable production of halogenated small molecules remains challenging. Benzoxazinoids (BXDs) are indole-derived plant specialized metabolites whose halogenated derivatives possess promising pharmaceutical and agrochemical properties. Here, we engineered Nicotiana benthamiana to produce halogenated BXDs through both precursor-directed and de novo biosynthetic strategies. Screening 22 BXD biosynthetic enzymes from diverse plant species identified a subset of enzymes with substrate promiscuity toward halogenated indole analogs, enabling the formation of chlorinated and fluorinated BXDs in transiently transformed N. benthamiana plants supplied with halogenated precursors. To establish a fully plant-based production pathway, we co-expressed this enzyme set with a biosynthetic module that provides chlorinated indole analogs and includes bacterial tryptophan halogenases, a bacterial flavin reductase, and a bacterial tryptophanase. This strategy resulted in the de novo production of substantial amounts of chlorinated BXDs in planta. Furthermore, chlorinated BXDs could be converted into chlorinated benzoxazolinone analogs, compounds with pharmaceutical relevance. These findings demonstrate that N. benthamiana provides a versatile and sustainable platform for the biosynthesis of halogenated BXDs and related high-value halogenated natural products. • Halogenated benzoxazinoids have promising pharmaceutical and agrochemical properties • Engineering Nicotiana benthamiana to produce halogenated benzoxazinoids • Combinatorial biosynthetic approach using independently evolved enzymes • Precursor-directed and de novo biosynthesis of halogenated benzoxazinoids