Biosynthesis of Taxol Precursors in Transgenic Moss

Summary

Principal Investigator: Aldwin Anterola
Abstract: DESCRIPTION (provided by applicant): Taxol is a chemotherapeutic drug used to treat breast cancer. It was originally isolated from the bark of Pacific Yew in a process that kills the trees. These trees produce Taxol in such small amounts that it will take hundreds of trees to treat a patient. Chemical synthesis of the drug is not an economical alternative because of Taxol's very complex diterpenoid-based structure. Hence, Taxol and the related drug Taxotere (another anticancer agent) are now manufactured semi-synthetically from a Taxol precursor isolated from the leaves of the more common European Yew. Yew leaves, however, are not a reliable source of Taxol and Taxotere, because the yield is heavily dependent on environmental conditions and harvesting procedures. Thus, a new method of producing Taxol and other taxoids is required. The goal of this project is to provide an economical, reliable and renewable source of Taxol and other taxoids by overexpressing Taxol biosynthetic genes in the moss Physcomitrella patens. While there are other heterologous hosts available, P. patens is chosen because it is a prolific producer of diterpenoids, is amenable to genetic manipulation, grows well photoautotrophically in culture, is already being used in industrial bioreactors, and is a plant cell, which altogether suggest a reasonable hypothesis that this moss is by far the best heterologous host to overexpress Taxol biosynthetic genes. To test the latter hypothesis, this project aims to (1) compare the enzymatic activities of taxoid hydroxylases and taxoid acetyltransferases expressed in P. patens with those expressed in other heterologous hosts;and (2) identify the taxoid metabolites produced by multi-transgenic P. patens overexpressing the early part of the Taxol biosynthetic pathway. Transgenic mosses will be generated using custom-made plasmids that undergo homologous recombination targeted to a specific site in the P. patens genome. Overexpression of the genes will be achieved using constitutive and/or inducible promoters that have been demonstrated to work well in mosses. Enzymatic activities of each of the heterologously expressed proteins will be measured using protocols that have been used previously in yeasts and yews, while taxoid metabolites will be analyzed by both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The data from these experiments will guide future efforts to develop moss bioreactors for the production of Taxol and other anticancer drugs. PUBLIC HEALTH RELEVANCE: The manufacture of the anticancer drugs Taxol and Taxotere relies on a semi-synthetic procedure that uses Yew leaves as starting material. This process, however, is very expensive and do not provide a steady supply of the drug. The goal of this project is to develop an economical method of producing Taxol and Taxotere using moss bioreactors.
Funding Period: 2009-05-08 - 2012-04-30
more information: NIH RePORT