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Engineering Tropane Biosynthetic Pathway in Hyoscyamus Niger Hairy Root Cultures

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Engineering Tropane Biosynthetic Pathway in Hyoscyamus Niger Hairy Root Cultures Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures Lei Zhang*†, Ruxian Ding†, Yourong Chai‡§, Mercedes Bonfill¶, Elisabet Moyanoʈ, Kirsi-Marja Oksman-Caldentey**, Tiefeng Xu§, Yan Pi*, Zinan Wang*, Hanming Zhang†, Guoyin Kai§, Zhihua Liao*, Xiaofen Sun*, and Kexuan Tang*†† *State Key Laboratory of Genetic Engineering, Morgan-Tan International Center for Life Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China; †School of Pharmacy, Second Military Medical University, Shanghai 200433, China; ¶Unidad de Fisiologı´aVegetal, Facultad de Farmacia, Universidad de Barcelona, Diagonal 643, E-08028 Barcelona, Spain; ʈDepartament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avenida Dr. Aiguader 80, E-08003 Barcelona, Spain; **VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT (Espoo), Finland; ‡School of Agronomy and Life Sciences, Southwest Agricultural University, Chongqing 400716, China; and §Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China Communicated by Jiazhen Tan, Fudan University, Shanghai, China, March 2, 2004 (received for review July 10, 2003) Scopolamine is a pharmaceutically important tropane alkaloid methylation of putrescine catalyzed by PMT is the first commit- extensively used as an anticholinergic agent. Here, we report the ted step in the biosynthesis of these alkaloids (7). Scopolamine, simultaneous introduction and overexpression of genes encoding which is the 6,7-␤-epoxide of hyoscyamine, is formed from the rate-limiting upstream enzyme putrescine N-methyltransferase hyoscyamine by means of 6␤-hydroxyhyoscyamine. Hyoscya- (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase mine 6␤-hydroxylase (H6H, EC 1.14.11.11), a 2-oxo-glutarate- (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscy- dependent dioxygenase, catalyzes the hydroxylation of hyoscy- amus niger) hairy root cultures. Transgenic hairy root lines express- amine to 6␤-hydroxyhyoscyamine, as well as the epoxidation of ing both pmt and h6h produced significantly higher (P < 0.05) 6␤-hydroxyhyoscyamine to scopolamine (1, 8, 9) (Fig. 1). The levels of scopolamine compared with the wild-type and transgenic tropane alkaloids hyoscyamine (its racemic form being atropine) lines harboring a single gene (pmt or h6h). The best line (T3) and scopolamine are structurally related and are derived from a produced 411 mg͞liter scopolamine, which was over nine times common intermediate, the N-methylpyrrolinium cation. They more than that in the wild type (43 mg͞liter) and more than twice are used medicinally as anticholinergic agents that act on the the amount in the highest scopolamine–producing h6h single-gene parasympathetic nerve system. Because they differ in their transgenic line H11 (184 mg͞liter). To our knowledge, this is the actions on the central nervous system, there is currently a 10-fold highest scopolamine content achieved through genetic engineer- higher commercial demand for scopolamine, in the N- ing of a plant. We conclude that transgenic plants harboring both butylbromide form, than for hyoscyamine and atropine com- pmt and h6h possessed an increased flux in the tropane alkaloid bined (9). Hyoscyamine and scopolamine are mostly synthesized biosynthetic pathway that enhanced scopolamine yield, which was in young root cells and translocated to the aerial parts of the more efficient than plants harboring only one of the two genes. It plant (10). Hence, cultured roots are capable of accumulating seems that the pulling force of the downstream enzyme (the faucet high concentrations of these metabolites. Small-scale jar fer- enzyme) H6H plays a more important role in stimulating scopol- menters for the hairy roots of several Solanaceous species have amine accumulation in H. niger whereas the functioning of the been developed as prospective in vitro systems for commercial upstream enzyme PMT is increased proportionally. This study production of tropane alkaloids, but the scopolamine levels in provides an effective approach for large-scale commercial pro- these systems are often much lower than those of hyoscyamine duction of scopolamine by using hairy root culture systems as (11). Researchers have also carried out genetic engineering of bioreactors. pharmaceutically important tropane alkaloids (12), in which the conversion of hyoscyamine to the much more valuable scopol- Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine amine is the major goal. Releasing the expression of key N-methyltransferase ͉ scopolamine ͉ transformation enzymatic activities from the strict regulation to which they are normally subjected is expected to increase product formation. A econdary metabolites are low-molecular-weight compounds rough correlation has been found between H6H activity and the Sproduced widely throughout the plant kingdom. Plant alka- ratio of scopolamine to hyoscyamine in scopolamine-producing loids constitute the largest groups of natural products, providing cultured roots (9). H6H, therefore, is a promising target enzyme many pharmacologically active compounds. The in-depth un- that, if overexpressed in hyoscyamine-accumulating tissues, derstanding of biosynthetic pathways, along with the increasing would result in increased scopolamine levels in the transfor- number of cloned genes involved in biosynthesis, enable the mants. Several hyoscyamine-rich but scopolamine-poor plants exploration of metabolic engineering as a potential effective that had been considered unattractive for commercial exploita- approach to increase the yield of specific metabolites by enhanc- tion may now become promising candidates for large-scale ing rate-limiting steps or by blocking competitive pathways. A scopolamine production by means of cultured roots. The hy- few genera of the plant family Solanaceae, including Hyoscyamus, droxylase gene from Hyoscyamus niger has been introduced into Duboisia, Atropa, and Scopolia, are able to produce biologically Atropa belladonna, a typical hyoscyamine-rich tropane alkaloid- active nicotine and tropane alkaloids simultaneously (1–3). Both producing plant species (13). Several transgenic root clones tropane and pyridine alkaloid biosynthetic pathways share a showed 5-fold higher concentrations of scopolamine than the common polyamine metabolism in their early steps. Putrescine wild-type hairy roots. By overexpressing h6h in Hyoscyamus is a common precursor of both polyamines, such as spermidine muticus hairy root cultures, the best transgenic clone had a and spermine, and tropane͞pyridine alkaloids (4–6). Putrescine N-methyltransferase (PMT; EC 2.1.1.53) is the enzyme involved Abbreviations: H6H, hyoscyamine 6 ␤-hydroxylase; PMT, putrescine N-methyltransferase; P, in the removal of putrescine from the polyamine pool because it transgenic hairy root lines generated from pmt single gene transformation; H, transgenic catalyses the N-methylation of this diamine to form N- hairy root lines generated from h6h single gene-transformation; T, transgenic hairy root methylputrescine (mP). Because both the tropane ring moiety of lines generated from pmt͞h6h double gene-transformation. the tropane alkaloids and the pyrrolidine ring of nicotine are ††To whom correspondence should be addressed. E-mail: [email protected]. derived from putrescine by way of mP synthesis, the N- © 2004 by The National Academy of Sciences of the USA 6786–6791 ͉ PNAS ͉ April 27, 2004 ͉ vol. 101 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401391101 Downloaded by guest on October 1, 2021 genes that control the expression of multiple pathway enzyme genes, or both. A good example is the bioengineering of the ␤-carotene biosynthetic pathway in the major staple food crop rice (Oryza sativa). By overexpressing the combination of genes encoding for three key enzymes (phytoene synthase, phytoene desaturase, and lycopene ␤-cyclase), a final ␤-carotene (previ- tamin A) concentration of 2 mg͞kg was detected in dry rice endosperm (18). So far, explants of H. niger have not been used for scopolamine bioengineering. Commercial cultivars of H. niger contain the natural amounts of scopolamine, which are not very high. Hence, there is a very strong need to increase scopolamine production rates for commercial production. Here, we report the introduc- tion of gene constructs containing cDNA clones of pmt and h6h, driven by the constitutive cauliflower mosaic virus 35S promoter, into H. niger, either one gene at a time, or both genes together. The morphology, growth rate, activities of alkaloid pathway enzymes, and alkaloid production capacities of these engineered hairy root lines were investigated. Materials and Methods Construction of pmt and h6h Binary Expression Vector. The mono- valent pmt expression plasmid pBMI was previously constructed (16). Disarmed Agrobacterium tumefaciens strain C58C1 harbor- ing both pBMI and Agrobacterium rhizogenes Ri plasmid pRiA4, containing a single pmt gene, were used for plant transformation (16, 19). 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). A positive clone, after confirmation by PCR and enzymatic digestion analysis for the presence of the h6h gene, was used for plant transformation. The pBMI and pLAL21 were used to construct a bivalent expression plasmid containing both pmt and h6h genes. The pBMI
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