Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

被引:35
|
作者
Whitmer, S
Canel, C
van der Heijden, R
Verpoorte, R
机构
[1] Leiden Univ, Gorlaeus Labs, Div Pharmacognosy, Leiden Amsterdam Ctr Drug Res, NL-2300 RA Leiden, Netherlands
[2] Corning Inc, Life Sci, Corning, NY 14831 USA
关键词
cell culture; genetic engineering; secondary metabolism; strictosidine synthase; terpenoid indole alkaloids; transgene expression; tryptophan decarboxylase;
D O I
10.1023/A:1023368309831
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the beta-glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.
引用
收藏
页码:73 / 80
页数:8
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