Alkaloid production in Duboisia hybrid hairy roots and plants overexpressing the h6h gene

In the biosynthetic pathway leading to the tropane alkaloid scopolamine, which is the 6,7β-epoxide of hyoscyamine, the enzyme hyoscyamine 6-hydroxylase (H6H; EC 1.14.11.11) catalyses two consecutive steps, hydroxylation of hyoscyamine followed by epoxide formation, to produce scopolamine. The h6h gene encoding H6H from Hyoscyamus niger, placed under the regulation of the CaMV 35S promoter, was introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the Agrobacterium rhizogenes strain LBA9402. The presence and overexpression of the transgene in the resulting kanamycin-resistant hairy roots and the regenerated plants obtained from them were confirmed by polymerase chain reaction and Northern blot analysis, respectively. The scopolamine levels in the resulting engineered hairy root lines increased up to three times compared to wild-type hairy roots, but there was no clear increase in the engineered regenerated plants. The best engineered line (H6H-5) produced 74.50 mg/l scopolamine, which to our knowledge is the highest content ever reported for hairy roots of tropane-alkaloid-producing plants containing the 35S-h6h transgene.