GIULIETTI Ana Maria
congresos y reuniones científicas
Biotransformation of hyoscyamine by recombinant Escherichia coli whole cells
ALEJANDRA CARDILLO; MARIELA SARTUQUI; SABRINA CARDILLO; JULIÁN RODRÍGUEZ TALOU; ANA MARIA GIULIETTI
Congreso; Biotrans 2013; 2013
Tropane alkaloids, such as hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine, are secondary metabolites produced by Solanaceous plants. These compounds were traditionally applied in medicine according to their anticholinergic activity. Hyoscyamine is converted by Hyoscyamine-6-hydroxylase (H6H) into 6β-hydroxyhyoscyamine and scopolamine. Nowadays, these bioactive 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. In the present work we explored the development of an alternative strategy for the production of the most valuable alkaloids, 6β-hydroxyhyoscyamine and scopolamine, using Escherichia coli whole cells harboring the H6H enzyme as biocatalyst. For this purpose the h6h gene was amplified from total RNA preparations obtained from immature anthers of Brugmansia candida, a South American tropane alkaloid producer plant. The h6hcDNA was cloned into the pET32(a) vector (Novagen) to generate the pH6Htrx(2). Three E. coli strains (Origami, Origami BL21 and Origami pLys) were used as host for the H6H production. In order to analyze the recombinant whole cells as successful biocatalysts a preliminary biotransformation assay was carried out by the addition of 40 ppm of hyoscyamine to the culture medium in conjunction to 1 mM IPTG. These compounds were added at an OD600= 0.5. Samples were taken at 20h, 40h and 80h post-induction. 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. The results showed that Origami and Origami BL21 E. coli strains transformed with the pH6Htrx(2) were able to produce a 20% and 5% of 6β-hydroxyhyoscyamine, respectively. Although Origami pLys strain synthesizes the enzyme, no transformation products were found in the medium. The recombinant strains assayed were unable to produce scopolamine under the conditions described. In addition, E. coli strains transformed with the pET32 empty vector were unable to convert the hyoscyamine added to the culture medium. Further studies are needed in order to optimize and develop a biotransformation process of hyoscyamine using E. coli whole cells expressing the H6H enzyme as biocatalyst.