Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants.

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 monooxygenases, was purified from a cell suspension culture of the higher plant Catharanthus roseus. Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identify of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animals species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.