Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

Abstract

In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6b-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the ®rst time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more ecient gene-expression systems.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

“…These findings suggest that high H6H activity has a positive influence on the flow of metabolites through the pathway. This positive correlation between H6H activity and scopolamine production has also been found in tissues of A. belladoma (13), H. muticus (14), and N. tabacum (31). These data demonstrate the desirability of overexpressing h6h for elevated scopolamine production.…”

“…3B). Generally, a fairly high content of secondary metabolites in the tissues is associated with poor growth, and the actual total productivity of secondary metabolites therefore remains low (14). We found in this study, however, that root morphology had a considerable influence on secondary metabolite production whereas the growth rate in the high scopolamine-producing lines was not reduced as compared with those with low scopolamine production.…”

“…By overexpressing h6h in Hyoscyamus muticus hairy root cultures, the best transgenic clone had a 100-fold increase of scopolamine. Furthermore, this clone produced hyoscyamine as the major alkaloid in similar amounts compared with controls (14). Recent efforts have been aimed at increasing the flux through the biosynthetic pathways.…”

“…The monovalent h6h expression plasmid pLAL21, constructed by Jouhikainen et al (14), was used in the study. Plasmid LAL21 was isolated from Escherichia coli strain DH5␣ and transformed into A. rhizogenes LBA9402 by electroporation (20).…”

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

“…From a pharmaceutical point of view, the work of Hashimoto et al (1992) on the cloning of the h6h gene and the subsequent introduction of scopolamine in this plant is an excellent illustration of the great potential of applying metabolic engineering for trimming the plant cell factory. Subsequently, Jouhikainen et al (1999) reported that the 35S-h6h transgene, which coded for the enzyme H6H, was introduced into H. muticus strain Cairo (Egyptian henbane). The best clone produced 17 mg/L scopolamine, which was over 100-fold more than that produced by control clones.…”