Improvement of tropane alkaloids production in hairy root cultures of Atropa belladonna by overexpressing pmt and h6h genes

Atropa belladonna L. is the commercial plant to produce tropane alkaloids including hyoscyamine and scopolamine, which are widely used as anticholinergic agents. In the present study, the genes encoding the key enzymes including putrescine N-methyltransferase (PMT) and hyoscyamine 6β-hydroxylase (H6H) were simultaneously overexpressed in transgenic hairy root cultures of A. belladonna. All of the nine transgenic hairy root lines ovexpressing both pmt and h6h produced tropane alkaloids at higher levels. The best line T3 produced 2.2 mg/g dry weight (DW) hyoscyamine, which was about 11 times more than that in non-transgenic hairy root cultures and 24 times more than that in the wild type (intact A. belladonna). Simultaneously, T3 produced 1.0 mg/g DW scopolamine, which was five times more than that in untransgenic hairy root cultures and four times more than that in the wild type. This is the first report of engineering the tropane alkaloids biosynthetic pathway via overexpressing both pmt and h6h in A. belladonna and the result demonstrated that the metabolic engineering strategy of BREAKING can facilitate accumulation of tropane alkaloids in A. belladonna. Further, it is also proved that transgenic hairy root cultures of A. belladonna can be used as bioreactor to produce pharmaceutical tropane alkaloids.