Generating an EMS Mutant Population and Rapid Mutant Screening by Thin-Layer Chromatography Enables the Studies of Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus Roseus

Mohammadamin Shahsavarani; Maisha Farzana; Vincenzo De Luca; Yang Qu

doi:10.1007/978-1-0716-2349-7_13

Generating an EMS Mutant Population and Rapid Mutant Screening by Thin-Layer Chromatography Enables the Studies of Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus Roseus

Methods Mol Biol. 2022:2505:181-190. doi: 10.1007/978-1-0716-2349-7_13.

Authors

Mohammadamin Shahsavarani¹, Maisha Farzana², Vincenzo De Luca³, Yang Qu^{4

5}

Affiliations

¹ Department of Chemical Engineering, University of New Brunswick, Fredericton, NB, Canada.
² Department of Chemistry, University of New Brunswick, Fredericton, NB, Canada.
³ Department of Biological Sciences, Brock University, St. Catharines, ON, Canada.
⁴ Department of Chemical Engineering, University of New Brunswick, Fredericton, NB, Canada. yang.qu@unb.ca.
⁵ Department of Chemistry, University of New Brunswick, Fredericton, NB, Canada. yang.qu@unb.ca.

PMID: 35732945
DOI: 10.1007/978-1-0716-2349-7_13

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.

Keywords: Catharanthus roseus; EMS mutagenesis; Monoterpenoid indole alkaloid (MIA) biosynthesis; Thin-layer chromatography (TLC).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Catharanthus* / genetics
Catharanthus* / metabolism
Chromatography, Thin Layer
Ethyl Methanesulfonate
Gene Expression Regulation, Plant
Plant Proteins / metabolism
Secologanin Tryptamine Alkaloids*
Vinblastine

Substances

Plant Proteins
Secologanin Tryptamine Alkaloids
Vinblastine
Ethyl Methanesulfonate