Halogenation, once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways1. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants2. Given the impact that halogenation can have on the biological activity of natural products1, we rationalized that introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group– a halide– is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore appear to be a viable platform for synthetic biology efforts.

Many natural products contain the hexahydropyrrolo[2, 3-b]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C3-aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon–carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C3-aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli, we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.