Strychnine biosynthesis described

Researchers from Jena show how poisonous nut trees form strychnine

A research team at the Max Planck Institute for Chemical Ecology in Jena revealed a complete biosynthetic pathway for strychnine formation in the plant species Strychnos nux-vomica (poison pea). The researchers identified all the genes involved in the biosynthesis of strychnine and other metabolites and expressed them in the plant model Nicotiana benthamiana. This allowed them to demonstrate that this highly complex and pharmacologically important molecule could be synthesized using “metabolic engineering” methods.

Poisonous nut tree Strychnos nux-vomica

© Danny Kessler, Max Planck Institute for Chemical Ecology

Poisonous nut tree Strychnos nux-vomica

© Danny Kessler, Max Planck Institute for Chemical Ecology

Many of us know strychnine from crime reports, novels or movies. Agatha Christie has had several of her victims die from strychnine poisoning. He described what is perhaps the most famous fictional murder case involving a highly toxic alkaloid used as a rat poison in his first novel “The Mysterious Affair at Styles”. The final clue to solving the case is discovered by the famous detective character Hercule Poirot in his first literary appearance.

In science too, investigative instincts and detective work are sometimes needed. Researchers led by Benke Hong and Sarah O’Connor of the Department of Natural Products Biosynthesis must not only find one missing link, but also uncover the entire chain of biosynthetic events that lead to the formation of strychnine in the toxic nut tree. To stay in the language of crime literature, one could say: They solved the case!

The chemist and Nobel Prize winner Robert Robinson, who was one of the first to explain the structure of strychnine in the 1940s, once described this indole monoterpene alkaloid as the most complex chemical substance for its molecular size. Many chemists were interested in the molecular architecture of strychnine and developed ways to produce this molecule using chemical synthesis. Surprisingly, however, no one has yet succeeded in discovering how plants produce this natural product.

Comparison of gene activity

Benke Hong’s team has now tackled this huge task: “Our key question is how to find the gene responsible for strychnine biosynthesis in poison peas. As a first step, we compared gene expression (transcriptomes) of two species of the same genus (Strychnos), but only the poisonous pea tree produces strychnine. We selected candidate genes for each step based on the proposed chemical transformation, which we don’t know to be true or not,” explains Benke Hong.

The gene upstream of strychnine biosynthesis for the formation of an important intermediate (geissoschizine) has been fully described in the medicinal plant Catharanthus roseus (Madagascar periwinkle), which is also being studied in Sarah O’Connor’s department, and homologous genes have been identified in the poison nut tree.

Chemical logic

Further progress required detective talents to combine molecular and genetic clues, which scientists call “chemical logic.” “You could say that chemistry guided the discovery of genes in our study. Based on the chemical structure and mechanism, each step in the metabolic pathway results in the proposed chemical transformation. In turn, our speculation about a family of biosynthetic enzymes with catalytic functions is based on the chemical reactions of each step,” said Sarah O’Connor, head of the Department of Biosynthesis of Natural Products, explaining the research approach.

As evidence that the identified gene is responsible for the proposed biosynthetic steps, the researchers modified the tobacco plant (Nicotiana benthamiana) to temporarily produce an enzyme from Strychnos. After adding the appropriate feed ingredients, they then investigated whether the hypothesized product was produced by the altered tobacco plant. This method allows high-throughput testing of multiple genes simultaneously, which shortens the time needed to solve the puzzle.

Prestrychnine is converted to strychnine

The researchers were unable to find a suitable enzyme that catalyzes the final step of strychnine biosynthesis, the conversion of prestrychnine to strychnine. Instead, they realized that this conversion occurred spontaneously, without enzymes. As is often the case in detective work and science, chance came to the rescue: “The spontaneous conversion of prestrychnine to strychnine was a coincidental discovery. This requires several intermediate steps, and we initially thought that this process should be catalyzed by one or more enzymes. In fact, we have studied many enzymes, but none of them are reactive. Surprisingly, one day I discovered that a sample of prestrychnine stored at room temperature on a lab bench slowly turned into strychnine over time,” said Benke Hong.

With the mystery of the final step solved, the researchers were thus able to elucidate the complete biosynthetic pathway of strychnine, as well as the related molecules of brucine and diabolin. While brucine is also produced by poison peas, diabolin is produced by related species of the genus Strychnos, which do not produce either strychnine or brucine. Notably, the researchers also found that only one amino acid change in one of the biosynthetic enzymes was responsible for the differences in alkaloid accumulation in poison pea and other Strychnos species.

The elucidation of the biosynthesis of plant metabolites and the use of biotechnology from a genetic basis for the formation of medically important plant compounds in model plants are promising areas of research. The current study opens up new possibilities for the production of previously unknown plant natural products using a “metabolic engineering” approach.

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