Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent.Here,we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme pu-trescine N-methyltransferase(PMT)and the downstream enzyme hyoscyamine6β-hydroxylase(H6H)of scopolamine biosynthesis in transgenic henbane(Hyoscyamus niger)hairy root cultures.Transgenic hairy root lines expressing both pmt and h6h produced significantly higher(P<0.05)levels of scopolamine compared with the wild-type and transgenic lines har-boring a single gene(pmt or h6h).The best line(T 3 )produced411mg/liter scopolamine,which was over nine times more than that in the wild type(43mg/liter)and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184mg/liter).To our knowledge,this is the highest scopolamine content achieved through genetic engi-neering of a plant.We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield,which was more efficient than plants harboring only one of the two genes.It seems that the pulling force of the downstream enzyme(the faucet enzyme)H6H plays a more important role in stimulating scopolamine accumulation in H.niger whereas the functioning of the upstream enzyme PMT is increased propor-tionally.This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.