Alejandra B. Cardillo

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

BackgroundTropane 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 Brugmansia candida.ResultsThe h6h 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 Saccharomyces cerevisiae 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 h6h cDNA.ConclusionThe strains obtained in this work are promising and potentially applicable in biocatalytic processes.

Analysis of 6-methoxy podophyllotoxin and podophyllotoxin in hairy root cultures of Linum album Kotschy ex Boiss

Linum album is a herbaceous medicinal plant that contains some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, hairy root cultures of L. album were established by transformation with Agrobacterium rhizogenes strains LBA9402, A4, AR15834 and Agrobacterium tumefaciens strain C58C1 (pRiA4). The presence of PTOX and MPTOX in the hairy roots was verified by ESI/MS in positive ion mode. MPTOX was confirmed and its enantiomer determined by nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy, respectively. PTOX and MPTOX production was determined by HPLC, in different lines of hairy roots. The results showed that all obtained hairy root lines produced higher yield of lignans than mother plant roots. In addition, the lignan content in the roots derived from A. rhizogenes strain LBA9402 was higher than in those obtained from A. tumefaciens strain C58C1.

Anisodamine Production from Natural Sources: Seedlings and Hairy Root Cultures of Argentinean and Colombian Brugmansia candida Plants

The tropane alkaloid anisodamine ( 2) is obtained by 6 beta-hydroxylation of hyoscyamine ( 1). The application of this alkaloid in medicine is gaining attention due to the wide range of therapeutic applications described in addition to its anticholinergic activity. In this work, the production of anisodamine ( 2) by IN VITRO cultures of BRUGMANSIA CANDIDA (Argentinean and Colombian samples) was studied. This alkaloid was estimated in different organs of IN VITRO-germinated seedlings as well as in hairy roots obtained from seedlings from both sources. Colombian roots exhibited the highest content of tropane alkaloids, with anisodamine ( 2) being the main alkaloid measured. In the leaves, the main alkaloid was scopolamine ( 3) and no significant differences were observed between Argentinean and Colombian leaves. The tropane alkaloid content in Argentinean hairy roots was significantly higher than in Colombian ones. Also, in the Argentinean samples the main alkaloid detected was anisodamine ( 2). Argentinean and Colombian B. CANDIDA seedlings and hairy roots appear to be a promising system for the production of anisodamine ( 2).

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.

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).

Biosynthesis of Sesquiterpene Lactones in Plants and Metabolic Engineering for Their Biotechnological Production

In the present chapter, we review some aspects of the biosynthesis of sesquiterpene lactones and its regulation in different medicinal and aromatic plants used in the pharmaceutical industry. In this sense, we describe the mevalonate and the 2-C-methyl-D-erythritol 4-phosphate pathways, which generate the corresponding isoprenoid precursors (isopentenyl diphosphate and dimethylallyl diphosphate), as well as the late pathways that lead to sesquiterpene lactone biosynthesis. This chapter also analyses the role of the transcription factors involved in the regulation of sesquiterpene lactone biosynthesis and the different biotechnological approaches that have been developed for sesquiterpene lactone production. In vitro plant cell cultures (comprising micropropagation and plant cell suspension, shoot and root cultures) have emerged as a production platform for many plant secondary metabolites, since they allow their production under controlled conditions and shorter production cycles. The characterisation and isolation of genes involved in the regulation of sesquiterpene lactone biosynthetic pathways have allowed the design of metabolic engineering strategies to increase the production of these metabolites. Moreover, we discuss different strategies to increase sesquiterpene lactone production through genetic engineering. We also focus on the metabolic engineering of the artemisinin biosynthetic pathway in Artemisia annua. This metabolic pathway has become a model system not only for the biotechnological production of sesquiterpene lactones but also for the improvement of other plant secondary metabolic pathways. Finally, we analyse the successful expression of the complete artemisinin biosynthetic pathway in Escherichia coli and Saccharomyces cerevisiae, which has led to the efficient accumulation of artemisinic acid in these microorganisms.