Unraveling diterpenoid alkaloid biosynthesis in aconitum Lana Mutabdzija and Tomáš Pluskal Institute of Organic Chemistry and Biochemistry of the CAS, Czech Republic Diterpenoid alkaloids (DA) are specialized metabolites found mainly within the Aconitum plant genus which has a long history of use in Chinese traditional medicine. They are highly toxic upon ingestion and even topical applications, leading to heart failure or in the worst cases — death. The infamous reputation is owed to the presence of C 19 diterpenoid ester alkaloids, such as aconitine, a well-known DA representative. In addition, various medicinal roles (e.g. anticancer, antiarrhythmic, antimicrobial, anti-inflammatory) and structural complexity of DAs have long captivated scientists 1 . Out of over 1,000 reported DAs, only a handful can be produced by total synthesis, which limits their usage in the pharmaceutical industry. Furthermore, although some of the initial enzymes involved in their biosynthesis have been characterized, the complete biosynthetic network remains to be elucidated 2 . Overall, we hypothesize the involvement of over 20 enzymes in the transformation of diterpenoid precursor geranylgeranyl diphosphate to aconitine, belonging to the classes of terpene synthases, monooxygenases, lyases, methyltransferases, and BAHD acyltransferases. In particular, enzymes that create the aconitine-type skeleton through a Mannich-like reaction and Wagner-Meerwein-like rearrangement, remain elusive. In order to bridge this knowledge gap, we analyzed the chemical space of five Aconitum species by means of LC- MS/MS, and based on the uniqueness of their DA profiles, we selected two species ( Aconitum lycoctonum and Aconitum plicatum ) for de novo transcriptome assembly. Metabolomic analysis of A. plicatum and A. lycoctonum tissues (root, leaf, stem, flower, fruit) showed the highest presence of unique DAs in the roots, with all the tissues showing distinct metabolic fingerprints in relation to the amount of DAs. Following the biosynthetic pathway hypothesis, while combining the transcriptomic and metabolomic data 3 , we aim to identify the enzymes involved in the aconitine biosynthetic pathway. Upon completion, the obtained results will open an avenue for the biosynthetic production of medicinally valuable DAs using metabolic engineering. References 1. Shen, Y.; Liang, W.-J.; Shi, Y.-N.; Kennelly, E. J.; Zhao, D.-K. Structural Diversity, Bioactivities, and Biosynthesis of Natural Diterpenoid Alkaloids. Nat. Prod. Rep. 2020 , 37 (6), 763–796. 2. Mao, L.; Jin, B.; Chen, L.; Tian, M.; Ma, R.; Yin, B.; Zhang, H.; Guo, J.; Tang, J.; Chen, T.; Lai, C.; Cui, G.; Huang, L. Functional Identification of the Terpene Synthase Family Involved in Diterpenoid Alkaloids Biosynthesis in Aconitum Carmichaelii. Yao Xue Xue Bao 2021 . https://doi.org/10.1016/j.apsb.2021.04.008. 3. Torrens-Spence, M. P.; Fallon, T. R.; Weng, J. K. Chapter Four - A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms. In Methods in Enzymology; O’Connor, S. E., Ed.; Academic Press, 2016 ; Vol. 576, pp 69–97.
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