Abstract
Decades of research on the medicinal plant Catharanthus roseus have led to the complete elucidation of the 29-step pathway for the biosynthesis of the anticancer drug vinblastine from geraniol and tryptophan precursors. Several approaches have been used to identify the enzymes involved in this iconic and remarkably complex pathway. This chapter describes the use of the classic ethyl methanesulfonate (EMS) mutagenesis to create a selfed M2 mutant population, which can be rapidly screened to select mutants with altered monoterpenoid indole alkaloid (MIA) biosynthesis with a simple, high-throughput thin-layer chromatography (TLC)-based screening strategy. This TLC-based MIA screening has led to the discovery and characterization of three enzymes responsible for vinblastine biosynthesis.
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Acknowledgments
This work was supported by an NSERC Discovery Grant to YQ and an NSERC Discovery Grant to VDL. We thank Dr. Alan Blowers and colleagues from Ball Horticultural Company for preparing the M2 mutant seeds. YQ is supported by a Cannabis Health Research Chair (New Brunswick Health Research Foundation/Tetra Bio Pharma Inc.). VDL is supported by an NSERC Tier 1 Canada Research Chair in Plant Biotechnology.
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Shahsavarani, M., Farzana, M., De Luca, V., Qu, Y. (2022). Generating an EMS Mutant Population and Rapid Mutant Screening by Thin-Layer Chromatography Enables the Studies of Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus Roseus. In: Courdavault, V., Besseau, S. (eds) Catharanthus roseus. Methods in Molecular Biology, vol 2505. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2349-7_13
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DOI: https://doi.org/10.1007/978-1-0716-2349-7_13
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