RECOMBINANT ESCHERICHIA COLI WHOLE CELLS AS A TOOL FOR THE PRODUCTION OF BIOACTIVE COMPOUNDS

CARDILLO, ALEJANDRA BEATRIZ; PERASSOLO, MARÍA; MINOIA, JUAN; RODRIGUEZ TALOU, JULIAN; GIULIETTI, ANA MARIA

Budapest

Congreso; Biotrans 2017 - 13th International Symposium on Biocatalysis and Biotransformations; 2017

Tropane alkaloids, such as hyoscyamine, 6B-hydroxyhyoscyamine and scopolamine, are secondary metabolites that were traditionally applied in medicine due to their anticholinergic activity[1]. Hyoscyamine is converted into 6B-hydoxyhyoscyamine and scopolamine by two sequential reactions catalyzed by Hyoscyamine 6B-hydroxylase (H6H, EC 1.14.11.11) (Fig. 1). Nowadays, these bioactive compounds are obtained from natural producer plants due to the cost and complexity of their chemical synthesis[2]. For these reasons, the production of the more valuable alkaloids by biocatalytic processes using genetically modified organisms appears to be a promising and attractive strategy for the pharmaceutical industry. Figure 1. Hyoscyamine 6B-hydroxylase scheme reaction, including the co-factors and co-substrates implicated in the transformation. (1)Hyoscyamine, (2) 6B-hydroxyhyoscyamine and (3) Scopolamine.The aim of this work was 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 biocatalysts. In addition, the protein extracts of the induced bacteria were assayed for the transformation of hyoscyamine into the more valuable alkaloids. For this purpose the h6hcDNA, previously amplified from Brugmansia candida total RNA preparations, was inserted into the pET32a(+) vector. E. coli Origami strains were used as host for the expression. The strategy allowed us to produce enough quantities of a soluble and functional enzyme. Protein extracts and whole cells of the induced bacteria were able to transform hyoscyamine into the valuable products. In addition, we found that except from 2-oxoglutarate, no supplementation of the reaction mixture with the cofactors and co-substrate was needed. The process developed in this work is attractive since it could become an alternative to the traditional isolation of 6B-hydroxyhyoscyamine and scopolamine.[1] Dehghan, E.; Hakkinen, S.T.; Oksman-Caldentey, K.M.; Shahriari Ahmadi, F. Plant Cell, Tissue and Organ Culture (PCTOC) 2012, 110, 35-44.[2] Palazon, J.; Navarro-Ocaña, A.; Hernandez-Vazquez, L.; Mirjalili, M.H. Molecules 2008, 13, 1722-42.