New strategies for the production of medicinal compounds: Tropane alkaloids.

CARDILLO AB; RODRÍGUEZ TALOU J; GIULIETTI AM

Giardini Naxos

Congreso; Biotrans 2011; 2011

Plants are able to produce a large diversity of natural products of vast interest for pharmaceutical purposes. Tropane alkaloids, such as hyoscyamine and scopolamine, are secondary metabolites traditionally applied in medicine according to their anticholinergic activity [1]. Hyoscyamine is converted by Hyoscyamine 6b-hydroxylase (H6H) into anisodamine and scopolamine [2]. Recently, potential medical applications were also described for anisodamine [3]. Nowadays, these compounds are obtained from natural producer plants due to the cost and complexity of the chemical synthesis of them. For this reason, tropane alkaloids production by biotransformation processes is an attractive strategy for the pharmaceutical industry [4]. In the present work we explored the development of an alternative strategy for the production of the most valuable alkaloids, anisodamine and scopolamine, using the H6H as biocatalyst. For this purpose the h6h gene was amplified from total RNA preparations obtained from immature anthers of the South American tropane alkaloid producer plant, Brugmansia candida and cloned into different vectors in order to produce tagged and untagged enzymes. The H6H enzyme was expressed fused at its C-terminus to a V5 epitope and a His-tag (H6H-V5-6His) [4]. On the other hand, it was fused at its N-terminus to a cellulose binding domain (CBD-H6H) in order to combine the purification step with the enzyme immobilization on cellulose, a low cost matrix [5]. The constructions were introduced by chemical transformation in S. cerevisiae CEN PK2. In order to explore the different strategies, crude protein extracts of the induced yeast strains were assayed for the enzyme activity at 30ºC for 15hs. The analysis of the alkaloids was carried out by HPLC with UV detection. The mobile phase used was octanesulfonic acid 0.01M pH3/methanol (65:35), flow rate 1ml/min [4]. The results showed that the tagged and untagged enzymes were able to transform hyoscymine, showing a functional expression of the h6hcDNA. However, the products obtained were different when the reaction was performed by the different enzyme constructions. The untagged H6H and the CBD-H6H were able to convert hyoscyamine into anisodamine and scopolamine while H6H-V5-6His only produced anisodamine. Surprisingly, the cellulose binding domain did not negatively affect the catalytic activity of the H6H as the V5 epitope and the Histidine tag affected it. These facts are encouraging for the development of a biocatalytic process using immobilized enzymes