Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst

<p>Abstract</p> <p>Background</p> <p>Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6β-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-β hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from <it>Brugmansia candida</it>.</p> <p>Results</p> <p>The <it>h6h</it>cDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in <it>Saccharomyces cerevisiae </it>in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the <it>h6h</it>cDNA.</p> <p>Conclusion</p> <p>The strains obtained in this work are promising and potentially applicable in biocatalytic processes.</p

Analytical considerations for the successful evaluation of hyoscyamine biotransformation into 6β-hydroxyhyoscyamine and scopolamine

Hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine are anticholinergic agents that belong to the tropane alkaloids, a pharmacological important group of secondary metabolites. Hyoscyamine and scopolamine were historically used in medicine. Additionally, potential medical applications for 6β- hydroxyhyoscyamine were described in the last years. Previous works carried out in our lab allowed us the construction of a Saccharomyces cerevisiae strain harboring the Hyoscyamine-6β-hydroxylase (H6H) enzyme which is responsible of the conversion of hyoscyamine into 6β-hydroxyhyoscyamine and scopolamine. Several factors influenced and complicated the optimization of the hyoscyamine bioconversion process. The aim of this work was to evaluate the analytical factors that critically affect the performance of the alkaloid extraction and the detection and quantification method of the alkaloids implied in the biocatalytical process. The mechanical breakdown of yeast cells by continuous agitation at 4 °C in 2 mL tubes was the method of choice for an efficient recovery of the functional H6H enzyme. In addition, the different pH assayed for the alkaloid extraction caused significant variations in the recovery of the alkaloids, specifically impacting on scopolamine recovery which decreased a 35 % after the increase of the pH of the extraction. The development of robust and sensitive analytical methods was requisite for the correct monitoring and quantification of the alkaloids produced in order to evaluate the technological and economic feasibility of this process.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)

Brugmansia candida (Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-\u3b2 hydroxylase (H6H, EC 1.14.11.11) at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97% identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1)

Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)

Brugmansia candida ( Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-β hydroxylase (H6H, EC 1.14.11.11) at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97% identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1)

Western blot performed with cell lysates of producing the recombinant H6H enzyme from after different post-induction times: 0, 4, 8, 12, 16, 24, 27, 29 h

<p><b>Copyright information:</b></p><p>Taken from "Expression of Hyoscyamine 6beta-Hydroxylase gene in and its potential use as biocatalyst"</p><p>http://www.microbialcellfactories.com/content/7/1/17</p><p>Microbial Cell Factories 2008;7():17-17.</p><p>Published online 27 May 2008</p><p>PMCID:PMC2430943.</p><p></p