Overexpression of a tryptophan decarboxylase cDNA inCatharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

Abstract

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus

“…Overexpression of CrWRKY1 results in an up to 3-fold increase of serpentine in C. roseus hairy roots (Fig. 8); however, overexpression of TDC does not result in similar levels of serpentine production (Goddijn et al, 1995;Hughes et al, 2004). Therefore, up-regulation of TDC alone is evidently not sufficient to explain the large increase of serpentine induced by CrWRKY1.…”

“…Attempts to increase TIA production by ectopic expression of genes encoding several rate-limiting enzymes have met with less than satisfactory results. For instance, despite the observation that overexpression of the Tryptophan Decarboxylase (TDC) gene has led to a moderate increase of alkaloid accumulation (Hong et al, 2006), it is generally believed that TDC activity does not correlate positively with TIA production (Goddijn et al, 1995;El-Sayed and Verpoorte, 2007). A more promising approach is the ectopic expression of transcription factors (TFs) that regulate the TIA pathway in order to enhance alkaloid production (Memelink and Gantet, 2007).…”

Metabolic Engineering of Plants for Alkaloid Production

“…The early availability of clones for STR and TDC led to a few metabolic engineering studies. Constitutive expression of STR in cell cultures showed a surprising positive correlation to alkaloid levels , but TDC expression resulted in elevated tryptamine levels only in transgenic cell cultures and crown gall calluses (Goddijn et al, 1995). Although elevated levels of TDC did not result in improved TIA accumulation, an antisense clone showed that TIAs were absent when TDC activity was ablated (Goddijn et al, 1995).…”

Assessing competence for adventitious shoot formation in hypocotyl explant cultures fromCatharanthus roseus cultivars

“…Hairy root cultures can be produced using Agrobacterium (Brillanceau et. al., 1989;Goddijn et al, 1995) and transgenic cell lines of C. roseus (van der Fits and Memelink, 1997;Hilliou et al, 1999). Although transgenic plants of C. roseus (via Agrobacterium) have been reported, no evidence of genetic transformation at the molecular level is available (Zarate et al, 1999).…”