CARDILLO Alejandra Beatriz
congresos y reuniones científicas
Cloning and yeast expression of Hyoscyamine 6b-hydroxylase as a tool for the development of an industrial biocatalyst
CARDILLO, ALEJANDRA BEATRIZ; GIULIETTI, ANA MARÍA
Congreso; Biocat 2006 International Congress on Biocatalysis; 2006
Brugmansia candida is a South American native plant that produces tropane alkaloids, a pharmacological important group of secondary metabolites. Of these, hyoscyamine and scopolamine are the most important. Scopolamine is more useful for medicinal purposes and is more valuable than hyoscyamine. The conversion of hyoscyamine into scopolamine is carried out by Hyoscyamine 6b-hydroxylase (H6H, EC 18.104.22.168). In this work we report the isolation, cloning and expression of the B. candida h6h gene in S. cerevisiae as a first step of obtaining a biological catalyst for the production of scopolamine. The h6h mRNA was isolated from immature anthers of B. candida. The gene was cloned into the yeast pYES2.1-TOPO TA expression vector and the constructions obtained were confirmed by sequencing. The yeast clone YpAC3-15 (4) was isolated by cultures in YNBD medium without uracil and induced for the H6H expression by changing the dextrose carbon source for galactose. The protein is expressed after 4 hours induction. The recombinant and wild type S. cerevisiae strains were assayed for their tolerance to both alkaloids by growth inhibition assays. It was seeing that both strains tolerated the maximum scopolamine concentration tested (150 mM) but regarding to hyoscyamine, only the recombinant strain tolerated 150 mM of hyoscyamine. This could be attributed to the presence of the H6H enzyme in the induced recombinant yeast that allowed the cells to convert hyoscyamine into scopolamine that is better-tolerated improving yeast survival. Processes of conversion of hyoscyamine into scopolamine using the recombinant strain obtained are under study.