Cloning and yeast expression of Brugmansia candida Hyoscyamine 6-hydroxylase.

CARDILLO, A B; RODRÍGUEZ TALOU, J AND GIULIETTI, A M; CARDILLO, A B; RODRÍGUEZ TALOU, J AND GIULIETTI, A M

Rosario, Argentina

Congreso; XLII Congreso de SAIB; 2006

Sociedad Argentina de Investigaciones Bioquimicas

Hyoscyamine and scopolamine are tropane alkaloids widely used in the pharmaceutical industry. Scopolamine is more useful for medicinal purposes. The conversion of hyoscyamine into scopolamine is carried out by Hyoscyamine 6b-hydroxylase (H6H, EC 1.14.11.11). In this work we report the cloning and expression of B. candida h6h gene in Saccharomyces cerevisiae as the first step in the development of a biological catalyst for the production of scopolamine. The h6h cDNA was cloned into the yeast pYES2.1-TOPO TA expression vector. The enzyme expression was induced by changing the carbon source and it was shown that the protein was expressed after 4 hour-induction. The recombinant and wild type S. cerevisiae strains were assayed for their tolerance to both alkaloids by growth inhibition assays. Both strains tolerated the maximum scopolamine concentration tested (150 mM) but only the recombinant strain tolerated 150 mM of hyoscyamine. In order to perform the in vitro enzyme activity assay, different cell disruption methods were tested using recombinant yeast harbouring the lacz gene instead of the h6h gene. Of them, the mechanical lysis gave the best results compared to the liquid nitrogen lysis, permeabilization with toluene and sonication. Processes of conversion of hyoscyamine into scopolamine by the recombinant strain carrying the h6h gene are currently under study.