SUMMARY PARAGRAPH: Few complete pathways are established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture. 1 A lead example is colchicine, an FDA-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species. 2 - 5 Here we use a combination of transcriptomics, metabolic logic, and pathway reconstitution to elucidate a near complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome, or genetic tools in the native host. We have uncovered eight genes from Gloriosa superba for the biosynthesis of N -formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine. 6 Notably, in doing so we have identified a non-canonical cytochrome P450 that catalyzes the remarkable ring expansion reaction required to produce the distinct carbon scaffold of colchicine. We further utilize the newly identified genes to engineer a biosynthetic pathway (16 enzymes total) to N -formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This work establishes a metabolic route to tropolone-containing colchicine alkaloids and provides new insights into the unique chemistry plants use to generate complex, bioactive metabolites from simple amino acids.

Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have independently arisen through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing/symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum, the symbiont of soybean, and Sinorhizobium fredii, a broad-host-nodulating species, contain a putative GA biosynthetic operon/gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer’s disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate–dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

The gibberellin (GA) phytohormones are produced not only by plants but also by fungi and bacteria. Previous characterization of a cytochrome P450 (CYP)-rich GA biosynthetic operon found in many symbiotic, nitrogen-fixing rhizobia led to the elucidation of bacterial GA biosynthesis and implicated GA9 as the final product. However, GA9 does not exhibit hormonal/biological activity and presumably requires further transformation to elicit an effect in the legume host plant. Some rhizobia that contain the GA operon also possess an additional CYP (CYP115), and here we show that this acts as a GA 3-oxidase to produce bioactive GA4 from GA9. This is the first GA 3-oxidase identified for rhizobia, and provides a more complete scheme for biosynthesis of bioactive GAs in bacteria. Furthermore, phylogenetic analyses suggest that rhizobia acquired CYP115 independently of the core GA operon, adding further complexity to the horizontal gene transfer of GA biosynthetic enzymes among bacteria.