Study Of Tropane Alkaloids Biosynthesis Related Genes From Hairy Roots Of Anisodus Tanguticus

Anisodus tanguticus (Maxim.) Pascher (Solanaceae) is one of the Chinese ethnological medicines used in Tibet and Qinghai Province in West China. It is an important medicinal plant species producing anticholinergic alkaloids such as hyoscyamine, anisodamine and scopolamine, but its cultivation is difficult.Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11) is a key enzyme in the biosynthesis of scopolamine. It belongs to the 2-oxoglutarate dependent dioxygenase family and catalyzes two consecutive oxidation reactions: the hydroxylation of hyoscyamine to anisodamine and the epoxidation of anisodamine to scopolamine.Hairy roots of A. tanguticus were the original materials for the cDNA preparation. RT-PCR was performed with primers that were designed on the basis of the known H6H sequences. Based on the sequence obtained by RT-PCR, several gene-specific primers were designed to obtain the sequences of 3'-end and 5'-end by RACE. After a PCR with primers based on the sequences of RACE, the full-length sequence of AtH6H cDNA was obtained. The full-length cDNA of AtH6H was 1,305 bp in size containing 5'UTR, 3'UTR, poly (A)~+ tail, and an ORF of 1,035 nucleotides encoding a deduced protein of 344 amino acid residues (GenBank Accession No.AY356396).The ORF fragment of AtH6H was sub-cloned into the expression vector pET-32a (+) and expressed in E. coli strain BL21trxB (DE3) cells. The recombinant AtH6H was purified by HiTrap affinity chromatography and DEAE Sepharose Fast Flow chromatography.The products catalyzed by AtH6H were analyzed by TLC, HPLC and NMR. It showed that AtH6H is a bifunctional enzyme, which could convert hyoscyamine to scopolamine via anisodamine.The biotransformation of hyoscyamine and anisodamine by E. coli cells harboring pETAtH6H plasmid was studied. It showed that the recombinant E. coli cells could convert L-hyoscyamine to L-scopolamine via L-anisodamine in vivo.