Researchers from the Max-Planck Institute for Chemical Ecology have unraveled the complete biosynthetic pathway for strychnine, which is naturally produced by the poison nut tree (Strychnos nux-vomica). The researchers then reconstructed the pathway in tobacco plants, illustrating the possibility of harnessing this pathway to synthesize related compounds using metabolic engineering approaches.

For years, many chemists were excited by the architecture of the strychnine molecule and developed ways to produce this molecule artificially. In high doses, strychnine is very toxic to humans and other animals, and because of this, it became famous in films, novels, and crime reports.

Until now, however, no one succeeded in unraveling the entire chain of biosynthetic events that lead to the natural formation of strychnine in the poison nut tree. The new work, led by Benke Hong, PhD, and Sarah O’Connor, PhD, was published in a Nature article entitled “Biosynthesis of strychnine.”

“As a first step, we compared the expression of genes (transcriptome) from two species of the same genus (Strychnos),” explained Hong, the first author of the article. Only one of those species—the poison nut tree—produces strychnine.

They then selected candidate genes for the biosynthesis of strychnine based on the chemical structures and mechanisms in each step of the pathway.

“You could say that chemistry guided the discovery of the genes in our study,” said O’Connor, head of the Department of Natural Product Biosynthesis. “Our speculations about the biosynthetic enzyme families with catalytic functions were based on the chemical reaction of each step.”

As evidence that the identified genes were responsible for the proposed biosynthetic steps, the researchers modified tobacco plants (Nicotiana benthamiana) to temporarily produce the enzymes from the poison nut tree. They concluded that the reconstituted pathway in tobacco plants matched the physiologically relevant pathway in the poison nut tree.

In total, the researchers reported the discovery of nine enzymes involved in strychnine biosynthesis.

The researchers were not able to find a corresponding enzyme that catalyzed the last step of strychnine biosynthesis, the conversion of prestrychnine to strychnine. They realized instead that this conversion occurs spontaneously, without an enzyme.

“The spontaneous conversion of prestrychnine to strychnine was a chance discovery,” remarked Hong. “We initially thought that this process must be catalyzed by one or more enzymes. In fact, we studied many enzymes, but none of them was reactive. Surprisingly, one day I found that a prestrychnine sample stored at room temperature on the lab bench had slowly converted to strychnine over time.”

With the mystery of the last step solved, the researchers were thus able to elucidate the complete biosynthetic pathway of strychnine, as well as the related molecules brucine and diaboline. Although brucine is also produced by the poison nut, diaboline is produced by a related species of the genus Strychnos that does not produce either strychnine or brucine.

The current study opens up new possibilities for the production of natural plant products using metabolic engineering approaches.