salicylates and olivetolic-acid

Investigations of antibacterial activities revealed that the incorporation of longer alkyl chains to the C-6 position in resorcylic acid conferred antibacterial properties against Staphylococcus aureus and Bacillus subtilis. The resultant olivetolic acid (OA) derivatives with n-undecyl and n-tridecyl side-chains, even those lacking the hydrophobic geranyl moiety from their C-3 positions, exhibited strong antibacterial activities against B. subtilis at a MIC value of 2.5 μM. Furthermore, the study demonstrated that the n-heptyl alkyl-chain modification at C-6 of cannabigerolic acid (CBGA) effectively enhanced the activity against B. subtilis, demonstrating the importance of the alkyl side-chain in modulating the bioactivity. Overall, the findings in this study provided insight into further evaluations of the antibacterial activities, as well as other various biological activities of OA and CBGA derivatives, especially with optimized hydrophobicities at both the alkyl and prenyl side-chain positions of the core skeleton for the discovery of novel drug seeds.

Topics: Anti-Bacterial Agents; Cannabinoids; Microbial Sensitivity Tests; Salicylates

NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in in vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis.

Topics: Cannabinoids; Dimethylallyltranstransferase; Salicylates; Substrate Specificity

The therapeutic effects of Δ

Topics: Salicylates

Engineering microbes to produce plant-derived natural products provides an alternate solution to obtain bioactive products. Here we report a systematic approach to sequentially identify the rate-limiting steps and improve the biosynthesis of the cannabinoid precursor olivetolic acid (OLA) in Yarrowia lipolytica. We find that Pseudomonas sp LvaE encoding a short-chain acyl-CoA synthetase can efficiently convert hexanoic acid to hexanoyl-CoA. The co-expression of the acetyl-CoA carboxylase, the pyruvate dehydrogenase bypass, the NADPH-generating malic enzyme, as well as the activation of peroxisomal β-oxidation pathway and ATP export pathway are effective strategies to redirect carbon flux toward OLA synthesis. Implementation of these strategies led to an 83-fold increase in OLA titer, reaching 9.18 mg/L of OLA in shake flask culture. This work may serve as a baseline for engineering cannabinoids biosynthesis in oleaginous yeast species.

Topics: Cannabinoids; Metabolic Engineering; Salicylates; Yarrowia

The microbial synthesis of cannabinoids and related molecules requires access to the intermediate olivetolic acid (OA). Whereas plant enzymes have been explored for

Topics: Aspergillus nidulans; Biosynthetic Pathways; Cannabinoids; Metabolic Engineering; Multigene Family; Polyketide Synthases; Salicylates

Phytocannabinoids are a group of plant-derived metabolites that display a wide range of psychoactive as well as health-promoting effects. The production of pharmaceutically relevant cannabinoids relies on extraction and purification from cannabis (

Topics: Cannabidiol; Cannabinoids; Cannabis; Dronabinol; Glucosides; Metabolic Engineering; Molecular Structure; Nicotiana; Plant Proteins; Saccharomyces cerevisiae; Salicylates; Synthetic Biology

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia

Topics: Acyl Coenzyme A; Alkyl and Aryl Transferases; Benzoates; Biosynthetic Pathways; Cannabinoids; Cannabis; Dronabinol; Fermentation; Galactose; Metabolic Engineering; Mevalonic Acid; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Salicylates

Type III polyketide synthases (PKS IIIs) contribute to the synthesis of many economically important natural products, most of which are currently produced by direct extraction from plants or through chemical synthesis. Olivetolic acid (OLA) is a plant secondary metabolite sourced from PKS III catalysis, which along with its prenylated derivatives has various pharmacological activities. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for OLA, here we utilize a synthetic approach to engineer Escherichia coli for the production of OLA. In vitro characterization of polyketide synthase and cyclase enzymes, OLA synthase and OLA cyclase, respectively, validated their requirement as enzymatic components of the OLA pathway and confirmed the ability for these eukaryotic enzymes to be functionally expressed in E. coli. This served as a platform for the combinatorial expression of these enzymes with auxiliary enzymes aimed at increasing the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units, respectively. Through combining OLA synthase and OLA cyclase expression with the required modules of a β-oxidation reversal for hexanoyl-CoA generation, we demonstrate the in vivo synthesis of olivetolic acid from a single carbon source. The integration of additional auxiliary enzymes to increase hexanoyl-CoA and malonyl-CoA, along with evaluation of varying fermentation conditions enabled the synthesis of 80 mg/L OLA. This is the first report of OLA production in E. coli, adding a new example to the repertoire of valuable compounds synthesized in this industrial workhorse.

Topics: Escherichia coli; Polyketide Synthases; Salicylates; Synthetic Biology

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future.. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively.

Topics: Amino Acid Sequence; Amino Acid Substitution; Cannabis; Catalytic Domain; Crystallography, X-Ray; Isomerases; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Plant Proteins; Polyketides; Protein Conformation; Recombinant Proteins; Salicylates; Sequence Homology, Amino Acid

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

Topics: Amino Acid Sequence; Cannabis; Crystallization; Intramolecular Transferases; Molecular Sequence Data; Molecular Weight; Plant Proteins; Salicylates

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Topics: Base Sequence; Cannabis; Dronabinol; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Intramolecular Transferases; Molecular Sequence Data; Plant Proteins; Polyketide Synthases; Polyketides; Salicylates

The defensive chemistry of two species of ants from Brunei in the genus Crematogaster (Physocrema group) has been investigated. Ants in this group release a white secretion from hypertrophied metapleural glands on their thorax when they are disturbed. Previously, one species in this group has been shown to produce alkylphenols and alkylresorcinols. In the present investigation, similar compounds along with salicylic acids and resorcylic acids that are anacardic acid and olivetolic acid homologs, respectively, are described from two species. The structures of these compounds were suggested by their spectroscopic data and confirmed by direct comparison with synthetic samples. Some of these compounds occur in lichens and have well documented physiological activities.

Topics: Anacardic Acids; Animals; Ants; Brunei; Exocrine Glands; Hydroxybenzoates; Lichens; Phenols; Salicylates; Species Specificity

A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.

Topics: Alkyl and Aryl Transferases; Benzoates; Cannabis; Dimethylallyltranstransferase; Diphosphates; Dronabinol; Plant Extracts; Protein Prenylation; Resorcinols; Salicylates