Molecular metabolic engineering of clinically-used anti-cancer plant products

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Camptothecin derivatives are clinically used anti-tumor alkaloids. Transformed plant cell cultures may be an alternative source of production of camptothecin and related compounds, although they are now producted by extraction from intact plants. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) incited by Agrobacteriun rhizogenes strain 15834. This hairy root grew well 16-fold times for 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amount of camptothecin. The camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20), to which camptothecin was absorbed. Camptothecin was easily recovered from the resin. Our established method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture. We have cloned and characterized cDNAs encoding strictosidine synthase (OpSTR ; EC4.3. 3.2) and tryptophan decarboxylase (OpTDC ; EC4.1.1. 28), two key enzymes in the biosynthesis of camptothecin from hairy roots of 0. Pumila. We also idolated the cDNA coding for NADPH : cytochrome P450 reductase (OpCPR ; EC1. 6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSRC and OpTDC were quite similar, unlike those of OpCPR The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid (SA) repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.