title: Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.
abstract: 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 putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-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 harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering 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 proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.
authors.affiliation: State Key Laboratory of Genetic Engineering, Morgan-Tan International Center for Life Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Ruxian
authors.initials: R
authors.lastname: Ding
authors.forename: Yourong
authors.initials: Y
authors.lastname: Chai
authors.forename: Mercedes
authors.initials: M
authors.lastname: Bonfill
authors.forename: Elisabet
authors.initials: E
authors.lastname: Moyano
authors.forename: Kirsi-Marja
authors.initials: KM
authors.lastname: Oksman-Caldentey
authors.forename: Tiefeng
authors.initials: T
authors.lastname: Xu
authors.forename: Yan
authors.initials: Y
authors.lastname: Pi
authors.forename: Zinan
authors.initials: Z
authors.lastname: Wang
authors.forename: Hanming
authors.initials: H
authors.lastname: Zhang
authors.forename: Guoyin
authors.initials: G
authors.lastname: Kai
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Liao
authors.forename: Xiaofen
authors.initials: X
authors.lastname: Sun
authors.forename: Kexuan
authors.initials: K
authors.lastname: Tang
mesh_terms: D000470:Alkaloids / Q000378:metabolism; D046508:Culture Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D032304:Hyoscyamus / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism*; D010957:Plasmids; D014326:Tropanes / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D014326:Tropanes