Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.
Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
BackgroundSalvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems.ResultsIn the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera.ConclusionsOur work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2147-3) contains supplementary material, which is available to authorized users.
In this study, we investigated the antimicrobial activity of the young shoots of the smoke tree, Cotinus coggygria Scop., Anacardiaceae. The acetone extract and the derived ethyl acetate fraction effectively inhibited the growth of Gram-positive and Gram-negative bacteria (MIC 25-200 µg/ml), while the chloroform fraction showed pronounced activity against the yeast Candida albicans (MIC 3.12 µg/ml). The ethyl acetate fraction exhibited a significant ferric-reducing ability (10.7 mmol Fe(2+) /g extract), a very high DPPH radical scavenging activity (SC50 = 1.7 µg/ml) and inhibition of lipid peroxidation (IC50 = 41.8 µg/ml). High amounts of total phenolics (929.8 mg/g), tannins (833.8 mg/g) and flavonoids (35.5 mg/g) were determined in the ethyl acetate fraction, which also exerted significant anti-inflammatory (76.7%) and cytotoxic effects (IC50 = 15.6 µg/ml).
The chemical composition and antimicrobial activity of the essential oil of the Balkan endemic species Seseli rigidum Waldst. & Kit. (Apiaceae) was investigated. The monoterpene α-pinene was predominant in the volatile oil from aerial parts (57.4%) and fruit (23.3%). In the essential oil of the aerial parts limonene (6.7%), camphene (5.8%) and sabinene (5.5%) were also present in high amounts, and in the fruit oil, β-phellandrene (17.4%) and sabinene (12.9%). On the contrary, the root essential oil was composed almost entirely of the polyacetylene falcarinol (88.8%). The antimicrobial activity of the root essential oil was significant against Staphylococcus aureus, S. epidermidis, Micrococcus luteus and Enterococcus faecalis (MICs 6.25-25.00 μg/mL). Volatile constituents from the root strongly inhibited the growth of methicillin-resistant strains of S. aureus (MICs 6.25-50.00 μg/mL). Anti-staphylococcal activity can be attributed to the main volatile constituent of S. rigidum root, falcarinol.
Since the fruits of Lycium L. species (Fructus Lycii, goji berries) are promoted as a “superfood” with plenty of health benefits, there is extensive research interest in their nutritional and phytochemical composition. In the present study, the nutritional value, minerals, fatty acid composition, and bioactive compounds of L. barbarum L., red, yellow, and black goji berry (L. ruthenicum Murray.) cultivated in Serbia were investigated. Antioxidant and antimicrobial properties of their methanol extracts were assessed. Red goji berry had the highest content of fats, dietary fiber, iron, total carotenoids, and 2-O-β-d-glucopyranosyl-l-ascorbic acid (AA-2βG). The yellow goji berry extract showed the highest level of flavonoids and the most prominent antimicrobial (especially against Gram-negative bacteria) properties. The highest total phenolic content and the most potent antioxidant activity were observed for the extract of black goji berry. Therefore, all goji berries could be a valuable source of bioactive compounds in the food and pharmaceutical industry.
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