Biosynthesis of strychnine

Strychnine, a complex monoterpene indole alkaloid, has been a significant molecule in the history of organic chemistry, its isolation, structural elucidation, and synthesis driving advancements in the field. Isolated in 1818 from *Strychnos nux-vomica* seeds, it's currently used as a potent neurotoxic pesticide. Despite its importance, the biosynthetic pathway remained unknown until this study. The complex polycyclic architecture of strychnine presented a significant challenge, inspiring numerous total syntheses but leaving the natural biosynthesis a mystery. This research aimed to elucidate the complete biosynthetic pathway of strychnine, along with the related alkaloids brucine and diaboline, in order to understand how these complex molecules are produced in plants and to explore potential applications in metabolic engineering for the production of related compounds. The successful elucidation of this pathway opens avenues for the production of diverse strychnos alkaloids with potential pharmacological applications, making this a crucial step for synthetic biology.

Previous research provided a partial understanding of strychnine biosynthesis, suggesting that like all monoterpene indole alkaloids, it originates from tryptophan and geranyl pyrophosphate. Radioisotope-labeled substrate feeding studies in *S. nux-vomica* demonstrated the incorporation of these precursors into the final molecule. These studies indicated the involvement of geissoschizine and Wieland-Gumlich aldehyde as key intermediates. However, the steps converting these intermediates to strychnine remained elusive, particularly the incorporation of acetate to form the piperidone moiety and the subsequent hydroxylations and methylations leading to brucine. The existing literature offered a framework but lacked the detail necessary to fully understand the complex enzymatic machinery involved. This study builds upon this foundational knowledge to provide a complete picture of the pathway.

To identify the genes responsible for strychnine biosynthesis, the authors compared the transcriptomes of *S. nux-vomica* (a strychnine producer) and a non-producing *Strychnos* species. Metabolic analysis revealed the presence of various strychnos alkaloids in *S. nux-vomica*, including strychnine, isostrychnine, β-colubrine, and brucine, which were absent in the non-producing species. The researchers generated tissue-specific RNA sequencing data and identified candidate genes based on three criteria: high expression in *S. nux-vomica* roots, co-expression with putative upstream genes, and the potential for encoding proteins with catalytic functions consistent with the chemical logic of the proposed pathway. The geissoschizine pathway, already elucidated in *Catharanthus roseus*, served as a reference. Homologues of the *C. roseus* genes were identified in *S. nux-vomica*, indicating conservation of this initial part of the pathway. The authors then focused on identifying the genes involved in the transformation of geissoschizine to Wieland-Gumlich aldehyde and the subsequent steps to strychnine and brucine. This involved characterizing candidate genes through transient expression in *N. benthamiana* leaves, followed by metabolic analysis using liquid chromatography-mass spectrometry (LC-MS) to identify the products. Combinatorial expression of candidate genes allowed for the identification of the enzymes responsible for each step in the pathway. Furthermore, the authors investigated the biosynthesis of diaboline in the non-producing *Strychnos* species, leveraging co-expression analysis and functional characterization to identify the key enzymes involved. The study also included in vitro enzymatic assays, phylogenetic analysis, and homology modeling to further characterize the identified enzymes and provide mechanistic insights into their function. Hydroponic feeding experiments with deuterium-labeled Wieland-Gumlich aldehyde were conducted to investigate the rate-limiting steps in *S. nux-vomica*. Finally, the entire biosynthetic pathway was reconstituted in *N. benthamiana* to confirm the functionality and order of the identified enzymes.

This study successfully elucidated the complete biosynthetic pathway of strychnine, brucine, and diaboline. Nine key enzymes were identified and characterized. The pathway starts with geissoschizine, which undergoes a series of transformations, including oxidation, hydrolysis, decarboxylation, reduction, and acetylation/malonylation, to form Wieland-Gumlich aldehyde. The conversion of Wieland-Gumlich aldehyde to strychnine involves the formation of a new piperidone ring using malonyl-CoA as a precursor, resulting in an intermediate called prestrychnine, and this intermediate subsequently undergoes a spontaneous non-enzymatic cyclization to form strychnine. Brucine is synthesized from strychnine via hydroxylation and methylation steps. The authors found that a specific BAHD acyltransferase, SnvAT, in *S. nux-vomica* exhibits malonyltransferase activity, producing *N*-malonyl Wieland-Gumlich aldehyde, while the homologous enzyme in the non-producing *Strychnos* species shows acetyltransferase activity, leading to diaboline. The study also revealed that the conversion of prestrychnine to strychnine and isostrychnine is a slow, non-enzymatic process, potentially accelerated by heat or acidic conditions. All intermediates in the pathway (except 11-deMe brucine) were detected in *S. nux-vomica* roots, supporting the accuracy of the reconstituted pathway. The successful reconstitution of the entire pathway in *N. benthamiana*, producing strychnine, isostrychnine, β-colubrine, and brucine, further validates the findings.

This research provides a comprehensive understanding of strychnine biosynthesis, resolving a long-standing question in natural product chemistry. The identification of the nine key enzymes involved opens new possibilities for metabolic engineering approaches to produce various strychnos alkaloids. The discovery of the distinct activities of SnvAT (malonyltransferase) and SpAT (acetyltransferase), differing by a single amino acid, highlights the subtle evolutionary changes that can lead to significant alterations in metabolic output. The observation that the final step in strychnine biosynthesis is a slow, non-enzymatic process suggests potential for optimization through manipulation of environmental factors or the introduction of rate-enhancing enzymes. The findings have implications for understanding alkaloid biosynthesis in general, and provides a foundation for future studies aimed at modifying the pathway to produce novel compounds with potential therapeutic or industrial applications. The detailed characterization of the enzymes involved provides valuable insights for designing strategies for manipulating alkaloid production in plants.

This study successfully mapped the complete biosynthetic pathway of strychnine, brucine, and diaboline, identifying nine key enzymes. The findings highlight the power of combining chemical logic, -omics data, and enzymatic characterization in elucidating complex biosynthetic pathways. The successful reconstitution of the pathway in *N. benthamiana* opens exciting possibilities for metabolic engineering, allowing for the production of novel strychnos alkaloid derivatives. Further research could focus on optimizing the heterologous production system and exploring the potential of these engineered pathways for the production of valuable compounds.

The study primarily utilized transient expression in *N. benthamiana* for functional characterization of enzymes. While this approach is valuable, it may not fully reflect the complex regulatory mechanisms and interactions that occur in the native *S. nux-vomica* environment. Furthermore, the identification of the enzymes responsible for the final, slow, non-enzymatic steps in strychnine biosynthesis remains incomplete, although evidence supports a spontaneous conversion of an intermediate to the final product. Finally, the study focused on a limited number of *Strychnos* species, and the generalizability of the findings to other members of the genus requires further investigation.