Further Insights on the <i>Datura innoxia</i>Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

Cloning of H6H Gene from Atropa belladonna and Construction of the Efficient Plant Expression Vector

[Objective] The aim was to clone H6H gene from Atropa belladonna and construct an efficient plant expression vector.[Method] The coding sequence of H6H（Hyoscyamine 6β-hydroxylase）was cloned from Atropa belladonna with RT-PCR.Then,the sequence was subcloned into the reconstructed plant binary expression vector p2301 to construct the recombinant vector p2301-H6H,which was then introduced into Agrobacterium tumefaciens strain LBA4404 and Agrobacterium rhizogenes strain C58C1,respectively.[Result] The engineering bacteria p2301-H6H-LBA4404 and p2301-H6H-C58C1 which could be directly used in genetic improvement were obtained.[Conclusion] The present research provided basis for the increasing of alkaloid content of Atropa belladonna by plant genetic engineering technology.

Alkaloids Production from Callus of Hyoscyamus niger L, in Vitro

Callus cultures of Hyoscyamus niger L. were initiated from leaf segments cultured on Murashige and Skoog （MS） medium supplemented with 0.5 mg/L Benzyl Adenine （BA） and 0, 1, 2 and 3 mg/L Naphthalene acetic acid （ NAA ）. Half of cultures were incubated under light of 16 hr/day, while the other half was incubated under complete darkness. The incubation temperature was 25 ± 1 ℃in both incubation conditions. The fresh and dry weight of the produced callus was obtained after six weeks of incubation. Callus produced were recultured on medium that gave the highest production of callus. Constant weight （300 mg） of callus was cultured in each of these medium supplemented with abiotic elicitor of 50 g/L sucrose, 200 mg/L NaCI, 50 or 100 mg/L proline and 2 mg/L BA each one added separately and incubated under complete darkness. The fresh and dry weights of callus were measured after six weeks. HPLC was used to determine the tropane alkaloids （Hyoscyamine and Scopolamine）. The results showed that the significant highest average of fresh and dry weight of callus （112 and 89.6 mg） achieved using the medium contained 0.5 mg/L BA and 2 mg/L NAA under dark condition. The amount of fresh and dry weight of callus produced under dark condition was significantly higher than that produced under light condition, with increase in percentage of 51.3 and 37.62% respectively. The addition of abiotic elicitors caused reduction in both fresh and dry weight of callus, therefore the highest fresh weight average was 1,727 mg using 100 mg/L proline. The results indicated that addition of 50 or 100 mg/L proline led to increase in Hyoscyamine concentration of 58.03 and 21.37% respectively compared with the control. While other abiotic elicitors were found to cause reduction in Hyoscyamine concentration. Percentage of Scopolamine concentration were increased to 129.03, 166.94, 205.51 and 149.20% after addition of sucrose （50 g/L）, NaC1 （200 mg/L） and proline （50 or 100 mg/L） respectively compared with the control.

A chromosome-level genome assembly of anesthetic drug–producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids

Tropane alkaloids(TAs),which are anticholinergic agents,are an essential class of natural compounds,and there is a growing demand for TAs with anesthetic,analgesic,and spasmolytic effects.Anisodus acutan-gulus(Solanaceae)is a TA-producing plant that was used as an anesthetic in ancient China.In this study,we assembled a high-quality,chromosome-scale genome of A.acutangulus with a contig N50 of 7.4 Mb.A recent whole-genome duplication occurred in A.acutangulus after its divergence from other Solanaceae species,which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis.The catalytic activities of H6H enzymes were determined for three Solanaceae plants.On the basis of evolution and co-expressed genes,AaWRKY11 was selected for further analyses,which revealed that its encoded tran-scription factor promotes TA biosynthesis by activating AaH6H1 expression.Thesefindings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Abstract: Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.