Our long-term goal is to understand the mechanisms by which Camptotheca acuminata regulates the biosynthesis, transport, and degradation of camptothecin (CPT), an antitumor and antiviral alkaloid. This work should provide insight into the coordinate regulation of other complex secondary product pathways and may suggest biotechnology strategies for increasing the production of CPT and other alkaloids. Our first specific aim is to investigate regulation at the molecular genetic level by identifying and characterizing genes that encode specific enzymes of the CPT pathway, including two previously isolated genes encoding tryptophan decarboxylase and 3-hydroxy-3-methylglutary CoA reductase, as well as two new genes encoding geraniol-10- hydroxylase from the terpenoid side of the pathway and CPT 10 -hydroxylase, an enzyme that occurs very late in the pathway. Characterization of the developmental and environmental conditions required for the expression of these genes will elucidate the overall regulation of the pathway. Our second specific aim is to investigate the regulation of this pathway at the intracellular and intercellular levels. We discovered that CPT accumulates to high levels in very young leaves, but it is not made there. Because CFT is insoluble in aqueous Solutions, it is likely that a water.soluble precursor is transported to young leaves where the final steps in the pathway are carried out. We will identify the initial sites of Synthesis by asking which tissues can incorporate radiolabeled precursors into intermediates in the CPT pathway. Transported intermediates will be identified by feeding labeled precursors to the sites of synthesis and examining labeled products in the vascular system of the plant The metabolic fate of CPT, including its likely conversion into 10-hydroxy-CPT, will be examined also. Our third specific aim is to develop procedures for the transformation and regeneration of Camptotheca. We have expressed foreign genes in transgenic callus and suspension culture cells, and we have regenerated multiple shoots from stem explants. We will develop methods to combine these two capabilities so that transgenic trees can be produced to allow facile analysis of alkaloid genes and over-expression of the CPT pathway.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA057592-04
Application #
2008106
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1992-08-01
Project End
1999-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Biology
Type
Schools of Earth Sciences/Natur
DUNS #
047006379
City
College Station
State
TX
Country
United States
Zip Code
77845
Lu, Hua; Gorman, Elizabeth; McKnight, Thomas D (2005) Molecular characterization of two anthranilate synthase alpha subunit genes in Camptotheca acuminata. Planta 221:352-60
Lorence, Argelia; Nessler, Craig L (2004) Camptothecin, over four decades of surprising findings. Phytochemistry 65:2735-49
Lorence, A; Medina-Bolivar, F; Nessler, C L (2004) Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata hairy roots. Plant Cell Rep 22:437-41
Vincent, R M; Lopez-Meyer, M; McKnight, T D et al. (1997) Sustained harvest of camptothecin from the leaves of Camptotheca acuminata. J Nat Prod 60:618-9
Lopez-Meyer, M; Nessler, C L (1997) Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress. Plant J 11:1167-75
Maldonado-Mendoza, I E; Nessler, C L (1997) Molecular characterization of the AP19 gene family in Arabidopsis thaliana: components of the Golgi AP-1 clathrin assembly protein complex. Plant Mol Biol 35:865-72
Maldonado-Mendoza, I E; Vincent, R M; Nessler, C L (1997) Molecular characterization of three differentially expressed members of the Camptotheca acuminata 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene family. Plant Mol Biol 34:781-90
Maldonado-Mendoza, I E; Lopez-Meyer, M; Galef, J R et al. (1996) Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family. Plant Physiol 110:43-9
Lopez-Meyer, M; Nessler, C L; McKnight, T D (1994) Sites of accumulation of the antitumor alkaloid camptothecin in Camptotheca acuminata. Planta Med 60:558-60
Nessler, C L (1994) Metabolic engineering of plant secondary products. Transgenic Res 3:109-15

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