385706 Discovery of 16-Methoxytabersonine Hydratase in Vindoline Biosynthetic Pathway in Catharanthus Roseus

Thursday, November 20, 2014: 2:31 PM
204 (Hilton Atlanta)
Le Zhao1, Fuyuan Jing2, Zengyi Shao1 and Jacqueline V. Shanks1, (1)Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, (2)Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA

Catharanthus roseus is the unique natural source of the well-known high-value anticancer drugs, vinblastine and vincristine, which are derived from the coupling of two monomers, catharanthine and vindoline. At present, the two anticancer drugs are produced commercially via extraction and purification from plant. The uneconomic and laborious process leads to the high cost of the pharmaceutical compounds.  Chemical synthesis is one possible solution, but current synthetic routes are not economically feasible for large scale production. It’s a promising strategy to produce these valuable compounds from genetically engineered plant cells, plant tissues, or microbial cultures. However, genetic engineering approach is retarded by the uncharacterized genes in the biosynthetic pathway.

This study describes two strategies for discovering 16-methoxytabersonine hydratase (16-MOTH) in vindoline biosynthetic pathway: one is to screen candidate genes from cDNA library of C. roseus leaves expressed in microbial system, and another one is to separate and purify target proteins from total proteins of C. roseus leaves. To quickly screen the cDNA library and characterize the separated proteins, an enzymatic assay based high throughput approach is being developed for detecting the product of 16-MOTH, replacing the time-consuming and expensive liquid chromatography-mass spectrometry (LC-MS) detection technology. This approach couples two enzymatic reactions to produce a yellow color compound which can be easily detected by spectrophotometry. Since 16-MOTH is the last unknown enzyme in the vindoline branch of the diverse alkaloid pathways in C. roseus, the discovery of this enzyme will enable future metabolic engineering of the pathway in either native or heterologous hosts, providing a potential commercial production route for vinblastine and vincristine.

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