Disulfides with Anti-inflammatory Activity from the Brown Alga Dictyopteris membranacea

Six new (1, 2, and 4-7) and two previously reported (3 and 8) disulfides, along with 4-butyl-2,6-cycloheptadienone, γ-tocopherol, and δ-tocopherol, were isolated from an organic extract of the brown alga Dictyopteris membranacea, collected at Gerolimenas Bay, Greece. The structure elucidation of the isolated natural products was based on analysis of their spectroscopic data. Compounds 1, 3-6, and 8 were evaluated for their antibacterial and anti-inflammatory activities. None of the compounds displayed antibacterial activity against two resistant strains of Staphylococcus aureus and one strain of Escherichia coli. In contrast, metabolite 5 was able to cause strong inhibition of NO production with an IC50 value of 3.8 μM using an LPS stimulation assay. © 2016 The American Chemical Society and American Society of Pharmacognosy

Thuwalallenes A-E and Thuwalenynes A-C: New C15 Acetogenins with Anti-Inflammatory Activity from a Saudi Arabian Red Sea Laurencia sp.

Thuwalallenes A-E (1-3, 5 and 8) and thuwalenynes A-C (4, 6, 7), new C15 acetogenins featuring uncommon ring systems, along with cis-maneonene D (9), thyrsiferol (10) and 23-acetyl-thyrsiferol (11) were isolated from the organic extract of a population of the red alga Laurencia sp., collected at Rose Reef off the village of Thuwal in the Red Sea waters of the Kingdom of SaudiArabia. The structure elucidation of the isolated natural productswas based on extensive analysis of their spectroscopic data. Compounds 1-6, 8, 10 and 11 were evaluated for their anti-inflammatory activity by quantifying nitric oxide (NO) release in response to TLR4 stimulation in macrophages. Besides compound 4 that did not exhibit any activity, all other tested metabolites inhibited NO production from activated macrophages. Among them, thyrsiferol (10) and 23-acetylthyrsiferol (11) displayed activity with IC50 values in the low nM scale without cytotoxicity. © 2019 by the authors

Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast

Terpenes are a large class of natural products, many of which are used in cosmetics, pharmaceuticals, or biofuels. However, terpene[U+05F3]s industrial application is frequently hindered by limited availability of natural sources or low yields of chemical synthesis. In this report, we developed a modular platform based on standardized and exchangeable parts to reproduce and potentially expand the diversity of terpene structures in Saccharomyces cerevisiae. By combining different module-specific parts, we exploited the substrate promiscuity of class I diterpene synthases to produce an array of labdane-type scaffolds. These were subsequently modified by a scaffold decoration module consisting of a mutant library of a promiscuous cytochrome P450 to afford a range of hydroxylated diterpenes. Further P450 protein engineering yielded dedicated and efficient catalysts for specific products. Terpenes produced include precursors of pharmacologically important compounds, molecules that are difficult to obtain from natural sources, or new natural products. The approach described here provides a platform on which additional gene mining, combinatorial biosynthesis, and protein engineering efforts can be integrated to sustainably explore the terpene chemical space. © 2014 International Metabolic Engineering Society

Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities

Background: Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast. Results: Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L-1. Conclusions: In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids. © 2016 Ignea et al

Neorogioltriol and related diterpenes from the red alga Laurencia inhibit inflammatory bowel disease in mice by suppressing M1 and promoting M2-like macrophage responses

Macrophages are central mediators of inflammation, orchestrating the inflammatory response through the production of cytokines and nitric oxide. Macrophages obtain pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, which can be modulated by soluble factors, including natural products. Despite the crucial protective role of inflammation, chronic or deregulated inflammation can lead to pathological states, such as autoimmune diseases, metabolic disorders, cardiovascular diseases, and cancer. In this case, we studied the anti-inflammatory activity of neorogioltriol (1) in depth and identified two structurally related diterpenes, neorogioldiol (2), and O11,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3), with equally potent activity. We investigated the mechanism of action of metabolites 1-3 and found that all three suppressed macrophage activation and promoted an M2-like anti-inflammatory phenotype by inducing expression of Arginase1, MRC1, IRAK-M, the transcription factor C/EBPβ, and the miRNA miR-146a. In addition, they suppressed iNOS induction and nitric oxide production. Importantly, treatment of mice with 2 or 3 suppressed DSS-induced colitis by reducing tissue damage and pro-inflammatory cytokine production. Thus, all these three diterpenes are promising lead molecules for the development of anti-inflammatory agents targeting macrophage polarization mechanisms. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms. © 2014 International Metabolic Engineering Society

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes

Background: Salvia 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. Results: In 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. Conclusions: Our 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. © 2015 Trikka et al

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Funtional Characterization of the Three First Steps of the Pathway in Slavia Fruicosa and Rosmarinus officinalis

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 SfCPSand 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