Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

Abstract

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

“…We have observed spontaneous dedifferentiation of transformed roots to callus on phytohormone free basal media which is similar to reports available from several species of Solanaceae (Jaziri et al 1994;Moyano et al 1999Moyano et al , 2003Batra et al 2004). Bandyopadhyay et al (2007) have reported spontaneous dedifferentiation in LBA9402-transformed hairy roots of Withania somnifera.…”

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…Transcriptomics, coupled with silencing, metabolite, and biochemical analysis, identified a specialized role for ArAT4 in diverting L-Phe, via phenyllactate, into hyoscyamine and scopolamine in A. belladonna, establishing the identity of the first enzyme in this branch of the pathway. There have been many attempts to engineer increased production of hyoscyamine and scopolamine, particularly through overexpression of PMT and H6H (Moyano et al, 2003;Rothe et al, 2003;Zhang et al, 2004), which participate in early and late steps of the pathway, respectively (Figure 1). The identification of Ab-ArAT4 as the first step in the pathway that provides the phenyllactate moiety for the formation of littorine (Figure 1) provides a tool to potentially manipulate tropane alkaloid levels in the Solanaceae.…”

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

“…Moyano et al (2002) inserted the tobacco (N. tabacum L.) pmt gene into the hairy roots of a hybrid of Duboisia and the N-methylputrescine levels of the resulting engineered hairy roots increased (two-to fourfold) compared with wild-type roots, but there was no significant increase in either tropane or pyridinetype alkaloids. Moyano et al (2003) also introduced the transferred-DNA (T-DNA) of the root inducing plasmid (Ri plasmid) together with the tobacco pmt gene into the genome of Datura metel L. and Hyoscyamus muticus Linn. in order to influence tropane alkaloid production.…”