congresos y reuniones científicas
Recombinant yeast as an alternative biocatalysis system
Simposio; 14 th European Congress on Biotechnology; 2009
Hyoscyamine and scopolamine are tropane alkaloids traditionally applied in medicine due to their anticholinergic activity. Hyoscyamine is converted by hyoscyamine 6_-hydroxylase (H6H) enzyme into anisodamine and scopolamine. Recently, potential medical applications were also described for anisodamine. Nowadays, tropane alkaloids 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. Saccharamyces cerevisiae is frequently considered the start point in the development of industrial biocatalysts due to the robustness, availability of large scale process technology and regulatory advantages related to the wide use of this microorganism as a host for the expression of therapeutic proteins approved by the FDA. The aim of this work was the development and analysis of S. cerevisiae strains carrying the H6H enzyme as a potential biocatalyst for the anisodamine and scopolamine production by biotransformation. The alginate immobilization of the recombinant biocatalyst was also assayed. The h6h gene was amplified from total RNA preparations obtained from immature anthers of the South American tropane alkaloid producer plant, Brugmansia candida. The h6h cDNA obtained was cloned into the pYES2.1® and the pYES2.1/V5- His-TOPO® vectors to produce an untagged and a tagged enzyme, respectively. The constructions were introduced by chemical transformation in S. cerevisiae CEN PK2. Crude protein extracts of the induced yeast strains were assayed for the enzyme activity at 30◦C for 15 hours. The analysis of the alkaloids was carried out by HPLC with UV detection. The mobile phase used was octanesulfonic acid 0.01M pH 3/methanol (65:35), flow rate 1 ml/min. The results showed that the tagged and untagged enzymes were able to transform hyoscyamine, showing a functional expression of the h6hcDNA. The untagged enzyme presented a higher rate of hyoscyamine conversion comparing to the tagged enzyme. It is important to point out that the untagged H6H was able to produce two products (83.3% of anisodamine and 7.6% of scopolamine) and the tagged one produced only a 35.7% of anisodamine. The conversion of hyoscyamine by the free protein extract was more efficient than the conversion of the alkaloid by the immobilized protein extract and the free and immobilized transformed S. cerevisiae cells. According to the results obtained in this work it can be concluded that the recombinant S. cerevisiae strains obtained are promissory for technological applications in the production of scopolamine and anisodamine by biotransformation. doi:10.1016/j.nbt.2009.06.443