Poisonous plants hold keys to potent medicines

The Science    

Metabolites are compounds produced through the chemical processes organisms use to stay alive. Plants make a wide range of specialized metabolites that help fend off diseases and pests, protect from external stresses, and signal to other organisms, such as pollinators or helpful microbes. Diterpenoids and alkaloids are two classes of metabolites used in both traditional and modern medicine. At the intersection of these classes are a group of extremely complex molecules called diterpenoid alkaloids (DAs). Found in plants including monkshood and larkspur, these metabolites include potent poisons as well as highly effective drugs. DAs tend to accumulate in the roots of slow-growing plants, and isolating pure products from lots of similar — and potentially toxic — metabolites makes them difficult to harvest from natural sources. And despite years of research, scientists have been unable to build these molecules in the lab. 

Michigan State University scientists with the Great Lakes Bioenergy Research Center used a combination of advanced genetic mapping techniques to identify six steps in the formation of one DA, atisinium, in two plant species. These steps include one unique and previously unidentified enzyme that adds nitrogen, a defining feature of the DA backbone. Through additional experiments using labeled molecules and computer analyses, researchers showed that nitrogen comes from an unexpected chemical source. The researchers were able to produce atisinium — a possible foundation of more complex products — in the lab by transplanting key genes into tobacco plants, which have simpler genetic codes and generate the product more quickly.  

The Impact

Diterpenoid alkaloids have a wide range of uses, from natural pesticides to treatments for cancer, malaria, inflammation, pain, and nausea. But their complex structures make them extremely difficult to create in the lab, meaning natural production, or biosynthesis, is the most promising alternative. These findings mark the first established pathway for a diterpenoid alkaloid, enabling biosynthetic production and serving as a basis for discoveries that could lead to production of more complex products.

Summary

Researchers performed transcriptome sequencing on Delphinium grandiflorum, Aconitum plicaturm, and Aconitum lycoctonum and incorporated public data from four other Aconitum species, all of which accumulate diterpenoid alkaloids. Comparative transcriptomics across tissue types and genera led to identification of six enzymes active in the DA pathway. Coexpression analysis of a public root tissue time course study of A. vilmorinianum resulted in the identification of a novel reductase activity in the pathway with little homology to previously characterized enzymes. This reductase enzyme catalyzes a key step in the pathway supporting formation of atisinium, a bioactive diterpenoid alkaloid and potential intermediate in the biosynthesis of more complex metabolites. 

Using isotope labeling in Aconitum callus cultures and a computational metabolomics approach, researchers showed that ethanolamine is the preferred substrate and primary nitrogen source for the majority of detected DA, despite the abundance of DAs with ethylamine groups attached to their central terpene scaffolds. Identification of these entry steps allowed for reconstruction of a minimal pathway sufficient for de novo biosynthesis of atisinium and will serve as a basis for further pathway discovery towards more complex diterpenoid alkaloid products and their biosynthetic production in heterologous hosts.