Dataset Information

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

ABSTRACT: BACKGROUND: 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 (6beta-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-beta hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from Brugmansia candida. RESULTS: The h6hcDNA 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 h6hcDNA. CONCLUSION: The strains obtained in this work are promising and potentially applicable in biocatalytic processes.

SUBMITTER: Cardillo AB

PROVIDER: S-EPMC2430943 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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

Cardillo Alejandra B AB Talou Julián Rodríguez JR Giulietti Ana M AM

Microbial cell factories 20080527

<h4>Background</h4>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 (6beta-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 ...[more]

PMID: 18505565

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Project description:Purpose: In order to identify genes for steroid 14α-hydroxylase P450lun and its associated redox partner CPRlun in filamentous fungus Cochliobolus lunatus ATCC#12017 Methods: Strand-specific RNA-seq libraries were constructed for two samples, including (I) Mycelia of C. lunatus treated with mock N, N-dimethylformamide (DMF); (II) Mycelia of C. lunatus treated with 170 mg/L 11-deoxycortisol 21-acetate (RSA) dissolved in N,N-dimethylformamide. For preparation of RNA samples, germinated young mycelia of C. lunatus after 22-24 pre-culture in liquid potato dextrose broth medium were harvested and transferred into a new 30 ml potassium phosphate buffer supplement with either 170 mg/L RSA dissolved in DMF, or an equal volume of DMF as mock. Additional two-hours cultivation was performed to induction of the P-450lun gene expression. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The two 150-nt strand-specific paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). The clean reads were used to create a de novo transcriptome assembly using the Trinity pipeline (v2.4.0). The abundance for each of the resulted transcripts was calculated by RSEM (v1.2.0) using the Fragments Per Kilobase per Million (FPKM) method. Results: A total of 7.1 and 7.9 million 150-bp paired-end clean reads were separately generated from the DMF mock and RSA induced samples,and 23957 transcripts with length more than 600nt were assembled from the pooled reads of both samples using Trinity program. The translated sequences of these transcripts were predicted using TransDecoder, and then used as queries to search against the SWISS-PROT and the Pfam databases for functional annotation. Transcripts DN8493 and DN2353 were separately identified as C.lunatus P-450lun and CPRlun genes. Conclusions: The 14β-hydroxylase system of C.lunatus was deciphered by transcriptome analysis.

Dataset Information

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

Publications

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

Similar Datasets

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