Expression of Brugmansia candida Hyoscyamine 6â-Hydroxylase in Saccharomyces cerevisiae and its potential use as biocatalyst

ALEJANDRA B. CARDILLO; JULIÁN RODRIGUEZ TALOU; ANA M. GIULIETTI

MICROBIAL CELL FACTORIES

BIOMED CENTRAL LTD

Lugar: Barcelona; Año: 2008 vol. 7 p. 17 - 24

1475-2859

Background: Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity.Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6B-hydroxyhyoscyamine) is the intermediate in the conversion ofhyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable inmedicine. The gene that codifies for Hyoscyamine 6-Bhydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from Brugmansia candida. Results: The h6hcDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in Saccharomyces cerevisiae in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the h6hcDNA. Conclusion: The strains obtained in this work are promising and potentially applicable in biocatalytic processes.