Browsing by Author "Pasquali, Giancarlo"
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- An assay for secologanin in plant tissues based on enzymatic conversion into strictosidinePublication . Hallard, Didier; Heijden, Robert Van Der; Contin, Adriana; Tomaz Jiméréz, Emília M.; Snoeijer, Wim; Verpoorte, Robert; Jensen, Soren R.; Cardoso, M. Inês Lopes; Pasquali, Giancarlo; Memelink, Johan; Hoge, J. Harry C.The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and in cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding strictosidine, in a reaction catalysed by the enzyme strictosidine synthase (STR; E.C. 4.3.3.2). Subsequently, the formation of strictosidine is quantified by high performance liquid chromatography (HPLC). STR was isolated from transgenic Nicotiana tabacum cells expressing a cDNA-derived gene coding for STR from Catharanthus roseus. The high specificity of STR for secologanin, in combination with a sensitive and selective HPLC system, allows a simple extraction of secologanin from plant tissue. The detection limit of this method is 15 ng secologanin. Using this assay, secologanin contents were determined in tissues of various plant species; Lonicera xylosteum hairy roots were found to contain 1% of secologanin on a dry weight basis. # 1998 John Wiley & Sons, Ltd.
- Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseusPublication . Canel, Camilo; Cardoso, Inês Lopes; Whitmer, Serap; Fits, Leslie Van Der; Pasquali, Giancarlo; Heijden, Robert van der; Hoge, J. Harry C.; Verpoorte, RobertCells 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 Agrobacteriummediated transformation showed wide phenotypic diversity, re¯ecting 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 á 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 in¯uenced 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 su cient, 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.