The major objective of our work is to understand the regulation of indole alkaloid biosynthesis in Camptotheca acuminata, a chinese tree that produces the anti-cancer alkaloid camptothecin (CPT). In addition to its basic scientific interest, elucidation of the regulatory mechanisms of the indole alkaloid pathway may suggest biotechnology strategies to increase the amount or alter the spectrum of indole alkaloids produced by this plant and other species. Because complex pathways often are regulated at the early stages, our approach to studying the regulation of this pathway is to examine the effects of over-expression of key enzymes on other components of indole alkaloid biosynthesis. We have over-expressed the first two enzymes of the CPT pathway, tryptophan decarboxylase (TDC) and strictosidine synthase (SS), In transgenic tobacco plants as a model system. Both proteins are highly expressed in enzymatically-active forms. We have also devised a assays for two other steps in the CPT pathway, the formation of strictosamide from strictosidine and the hydroxylation of CPT at the C-10 position. These enzymes and their genes will be isolated so their effects on alkaloid metabolism can be analyzed as well. In addition to regulation by its own metabolism, the CPT pathway may be induced by external factors, such as biotic and abiotic stress, light regimes, or developmental cues. We will analyze expression of reporter genes from the transcriptional promoters of both TDC and SS under a variety of potentially inductive conditions. Factors that regulate both genes may be general inducers of the pathway.
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