ABSTRACT
Labdane-related diterpenoids (LRDs) in fungi are a pharmaceutically important, but underexplored family of natural products. In the biosynthesis of fungal LRDs, bifunctional terpene cyclases (TCs) consisting of αßγ domains are generally used to synthesize the polycyclic skeletones of LRDs. Herein, we conducted genome mining of LRDs in our fungal genome database and identified a unique pair of TCs, AsPS and AsCPS, in the fungus Arthrinium sacchari. AsPS consists of catalytically active α and inactive ß domains, whereas AsCPS contains ßγ domains and a truncated α domain. Heterologous expression in Aspergillus oryzae and biochemical characterization of recombinant proteins demonstrated that AsCPS synthesized copalyl diphosphate and that AsPS then converted it to (-)-sandaracopimaradiene. Since AsPS and AsCPS have distinct domain organizations from those of known fungal TCs and are likely generated through fusion or loss of catalytic domains, our findings provide insight into the evolution of TCs in fungi.
ABSTRACT
By mining fungal genomic information, a noncanonical iterative type I PKS fused with an N-terminal adenylation-thiolation didomain, which catalyzes the formation of naringenin chalcone, was found. Structural prediction and molecular docking analysis indicated that a C-terminal thioesterase domain was involved in the Claisen-type cyclization. An enzyme responsible for formation of (2S)-flavanone in the biosynthesis of fungal flavonoids was also identified. Collectively, these findings demonstrate unprecedented fungal biosynthetic machinery leading to plant-like metabolites.
Subject(s)
Acyltransferases , Flavonoids , Molecular Docking Simulation , Flavonoids/chemistryABSTRACT
Diterpenoid pyrones are a type of mainly fungal meroterpenoid metabolite consisting of a diterpene connected to a pyrone, some of which show potent bioactivity. Through genome mining and heterologous expression, nine new diterpenoid pyrones, shearones A-I (1-9), were discovered from the fungus Eupenicillium shearii IFM 42152, and their biosynthetic enzyme activities were revealed. Some of these heterologously biosynthesized diterpenoid pyrones exhibited moderate antiaggregative ability against amyloid ß42 in vitro.
Subject(s)
Diterpenes , Pyrones , Diterpenes/metabolism , Diterpenes/pharmacology , Penicillium , Pyrones/pharmacology , Synthetic BiologyABSTRACT
Correction for 'Synthetic-biology-based discovery of a fungal macrolide from Macrophomina phaseolina' by Yohei Morishita et al., Org. Biomol. Chem., 2020, DOI: 10.1039/d0ob00519c.
ABSTRACT
A synthetic biology approach based on genome mining and heterologous biosynthesis is a powerful tool for discovering novel natural products from a tremendous gene resource. We carried out fungal genome mining guided by a polyketide synthase gene using a public database and found a putative macrolide biosynthetic gene cluster with a highly reducing polyketide synthase gene and a thioesterase gene in Macrophomina phaseolina. Reconstitution of the cluster in Aspergillus oryzae, a model heterologous host for fungal natural product biosynthesis, produced a new 12-membered macrolide, phaseolide A. The absolute stereochemistry was elucidated by vibrational circular dichroism spectroscopy and the crystalline sponge method.
Subject(s)
Ascomycota/chemistry , Macrolides/metabolism , Ascomycota/enzymology , Ascomycota/metabolism , Macrolides/chemistry , Molecular Structure , Polyketide Synthases/genetics , Polyketide Synthases/metabolismABSTRACT
Polyketide synthase (PKS) gene-guided genome mining in a cricket-associated fungus, Penicillium soppi, revealed a cryptic biosynthetic gene cluster that contained a highly reducing PKS (HR-PKS), a type III PKS, and a P450 gene. Heterologous expression of the cluster in Aspergillus oryzae led to the isolation of novel alkylresorcinols with a unique Z,E,Z-triene motif. This study displays an unusual biosynthetic mechanism of an HR-PKS and a new releasing mechanism via a type III PKS in fungi.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Penicillium/chemistry , Polyketide Synthases/antagonists & inhibitors , Resorcinols/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Molecular Structure , Polyketide Synthases/metabolism , Resorcinols/chemistry , Resorcinols/isolation & purificationABSTRACT
Plant hormones were evaluated for their ability to activate fungal secondary metabolite production. Three synthetic cytokinins, kinetin, 6-benzylaminopurine, and forchlorfenuron, showed remarkable enhancement of the production of aromatic polyketides derived from emodin in a fungus, Arthrinium sacchari, and allowed us to isolate a new polyketide. Furthermore, we firstly demonstrated the potential of plant hormones to activate a wide range of fungal secondary metabolite production processes.
Subject(s)
Ascomycota/metabolism , Plant Growth Regulators/metabolism , Polyketides/metabolism , Spiders/microbiology , Animals , Ascomycota/isolation & purification , Molecular Structure , Polyketides/chemistry , Polyketides/isolation & purificationABSTRACT
Pazopanib was administered to a 44-year-old man with local recurrence of retroperitoneal liposarcoma. Computed tomography showed an intestinal edema, which gradually progressed 15 months after pazopanib administration although he had no clinical symptoms. Upper gastrointestinal endoscopy implicated marked edematous hypertrophy of the Kerkling's fold. Pathological findings showed crystal deposition and fat accumulation, without a malignant component. All these abnormal findings resolved after pazopanib discontinuation.
Subject(s)
Antineoplastic Agents/adverse effects , Edema/chemically induced , Intestinal Diseases/chemically induced , Neoplasm Recurrence, Local/drug therapy , Pyrimidines/adverse effects , Sulfonamides/adverse effects , Adult , Edema/diagnostic imaging , Edema/pathology , Endoscopy, Gastrointestinal , Humans , Indazoles , Intestinal Diseases/diagnostic imaging , Intestinal Diseases/pathology , Intestinal Mucosa/diagnostic imaging , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Kidney Neoplasms/drug therapy , Liposarcoma/drug therapy , Male , Retroperitoneal Neoplasms/drug therapy , Tomography, X-Ray ComputedABSTRACT
Exposure of the fungus Chaetomium cancroideum to an NAD(+)-dependent HDAC inhibitor, nicotinamide, enhanced the production of aromatic and branched aliphatic polyketides, which allowed us to isolate new secondary metabolites, chaetophenol G and cancrolides A and B. Their structures were determined using spectroscopic analyses, and their absolute configuration was elucidated by electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and chemical transformations. Biosynthesis of the branched aliphatic polyketide skeletons in cancrolides A and B was evidenced by conducting a feeding experiment using compounds labeled with a (13)C stable isotope.
Subject(s)
Chaetomium/drug effects , Chaetomium/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , NAD/metabolism , Polyketides/metabolism , HCT116 Cells , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Humans , Molecular Conformation , NAD/pharmacology , Polyketides/chemistry , Structure-Activity RelationshipABSTRACT
Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in classâ I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including classâ I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.
ABSTRACT
The addition of a DNA methyltransferase inhibitor, 5-azacytidine, to Aspergillus sydowii fungus culture broth changed its secondary metabolites profile. Analysis of the culture broth extract led to the isolation of three new bisabolane-type sesquiterpenoids: (7S)-(+)-7-O-methylsydonol (1), (7S,11S)-(+)-12-hydroxysydonic acid (2) and 7-deoxy-7,14-didehydrosydonol (3), along with eight known compounds. The isolated compounds were evaluated for their anti-diabetic and anti-inflammatory activities. Among the isolates, (S)-(+)-sydonol (4) did not only potentiate insulin-stimulated glucose consumption but also prevented lipid accumulation in 3T3-L1 adipocytes. Additionally, (S)-(+)-sydonol (4) exhibited significant anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by fMLP/CB-induced human neutrophils. This is the first report on isolating a secondary metabolite with anti-diabetic and anti-inflammatory activities from microorganisms.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Aspergillus/metabolism , Azacitidine/metabolism , Hypoglycemic Agents/pharmacology , Industrial Microbiology , Sesquiterpenes/pharmacology , 3T3-L1 Cells , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/metabolism , Aspergillus/drug effects , Aspergillus/enzymology , DNA Modification Methylases/antagonists & inhibitors , Glucose/metabolism , Humans , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/isolation & purification , Hypoglycemic Agents/metabolism , Mice , Neutrophils/drug effects , Neutrophils/immunology , Sesquiterpenes/chemistry , Sesquiterpenes/isolation & purification , Sesquiterpenes/metabolism , Superoxides/antagonists & inhibitorsABSTRACT
The addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid to a culture of the filamentous fungus Beauveria felina significantly changed its secondary metabolite profile and led to the isolation of eight compounds, including three new cyclodepsipeptides, desmethylisaridin E (1), desmethylisaridin C2 (2), and isaridin F (3), along with five known cyclodepsipeptide compounds. Isaridin F (3) possesses a cyclodepsipeptide ring with N-methylbutyric acid, which is rare in natural peptides. Absolute configurations of the new cyclodepsipeptides were achieved by Marfey's method. The anti-inflammatory activity of the isolated compounds was investigated through evaluating their effect on superoxide anion production and elastase release by FMLP-induced human neutrophils. Among the tested compounds, desmethylisaridin E (1) inhibited superoxide anion production and desmethylisaridin C2 (2) inhibited elastase release, with IC50 values of 10.00 ± 0.80 and 10.01 ± 0.46 µM, respectively.
Subject(s)
Beauveria/chemistry , Depsipeptides/isolation & purification , Depsipeptides/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Anti-Inflammatory Agents/pharmacology , Depsipeptides/chemistry , Humans , Inhibitory Concentration 50 , Molecular Structure , Neutrophils/drug effects , Nuclear Magnetic Resonance, Biomolecular , Superoxides/metabolism , VorinostatABSTRACT
Discovery of natural products that possess novel chemical structures and pharmaceutical activities increases opportunities of drug development. Filamentous fungi have been recognized as an attractive source for pharmaceutically beneficial natural products. Genome sequencing innovation represented by Next-generation sequencer opened fungal genomes one after another, suggesting that one fungal strain has far more biosynthetic gene clusters than that are estimated from the number of previously isolated natural products. In addition, bioinformatics analyses have indicated that most biosynthetic gene clusters are silent under laboratory culture conditions and there are a huge number of natural products hidden in the fungal genome. Therefore, we focused on those silent biosynthetic gene clusters as a potential source for novel natural products and developed methods to activate silent biosynthetic gene clusters by using low molecular weight molecules. In this review, we describe on discovery of novel natural products through activating fungal silent biosynthesis by addition of epigenetic modifiers and plant hormones.
Subject(s)
Biological Products , Biological Products/chemistry , Epigenesis, Genetic , Fungi/chemistry , Fungi/genetics , Genes, Fungal , Multigene FamilyABSTRACT
Genome mining and bioinformatics analyses allowed us to rationally find a candidate biosynthetic gene cluster for a new cyclic depsipeptide of Chaetomium mollipilium. A heterologous reconstitution of the identified biosynthetic pathway predictably afforded a new cyclic depsipeptide composed of l-leucine, l-tryptophan, and a polyketide moiety. Interestingly, the 10-membered macrocycle structure generated equilibrium to an unprecedented cyclol structure. This study demonstrates the advantage of a synthetic biology method in achieving rational access to new natural products.
Subject(s)
Biological Products , Chaetomium , Depsipeptides , Polyketides , Biological Products/chemistry , Biosynthetic Pathways , Chaetomium/genetics , Depsipeptides/chemistry , Multigene Family , Polyketides/chemistryABSTRACT
Chemical investigation of the glandular trichome exudate from Geranium carolinianum L. (Geraniaceae) led to the characterization of unique disaccharide derivatives, n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1â2)-6-O-isobutyryl-ß-D-glucopyranoside (1), n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1â2)-6-O-(2-methylbutyryl)-ß-D-glucopyranoside (2) and n-octyl 4-O-(2-methylbutyryl)-α-L-rhamnopyranosyl-(1â2)-6-O-isobutyryl-ß-D-glucopyranoside (3), named caroliniasides A-C, respectively. These structures were determined by spectral means. n-Alkyl glycoside derivatives have been isolated from the glandular trichome exudates for the first time. This rare type of secondary metabolites could be applicable to chemotaxonomic perspective because they are found in glandular trichome exudates of plants belonging to the genus Geranium, according to our studies.
Subject(s)
Disaccharides/chemistry , Geranium/chemistry , Glycosides/chemistry , Disaccharides/isolation & purification , Glycosides/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Conformation , Plant Exudates/chemistryABSTRACT
Through genome mining for fungal macrolide natural products, we discovered a characteristic family of putative macrolide biosynthetic gene clusters that contain a glycosylphosphatidylinositol-ethanolamine phosphate transferase (GPI-EPT) homologue. Through the heterologous expression of two clusters from Aspergillus kawachii and Colletotrichum incanum, new macrolides, including those with phosphoethanolamine or phosphocholine moieties, were formed. This study is the first demonstration of the tailoring steps catalyzed by GPI-EPT homologues in natural product biosynthesis, and it uncovers a new gene resource for phospholipid-resembling fungal macrolides.
Subject(s)
Aspergillus/chemistry , Colletotrichum/chemistry , Ethanolamines/chemistry , Fungal Proteins/metabolism , Glycosylphosphatidylinositols/biosynthesis , Macrolides/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Glycosylphosphatidylinositols/chemistry , Macrolides/chemistry , Molecular Structure , Multigene Family , Protein BiosynthesisABSTRACT
A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.
Subject(s)
Diterpenes/pharmacology , Fungi/chemistry , Naphthalenes/pharmacology , Pyrones/pharmacology , Synthetic Biology , Amyloid beta-Peptides/metabolism , Animals , Anti-HIV Agents/pharmacology , Aspergillus/chemistry , Biosynthetic Pathways/drug effects , Biosynthetic Pathways/genetics , Cell Proliferation/drug effects , Diterpenes/chemistry , Drosophila/drug effects , Fungi/genetics , Genome, Fungal , HIV-1/drug effects , Humans , MCF-7 Cells , Naphthalenes/chemistry , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Protein Aggregates , Pyrones/chemistry , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , StereoisomerismABSTRACT
Heterologous expression of a unique biosynthetic gene cluster (BGC) comprising a highly reducing polyketide synthase and stand-alone thioesterase genes in Aspergillus oryzae enabled us to isolate a novel 34-membered polyene macrolide, phaeospelide A (1). This is the first isolation of a fungal polyene macrolide and the first demonstration of fungal aliphatic macrolide biosynthetic machinery. In addition, sequence similarity network analysis demonstrated the existence of a large number of BGCs for novel fungal macrolides.
Subject(s)
Macrolides/metabolism , Polyenes/metabolism , Polyketides/metabolism , Thiolester Hydrolases/metabolism , Trans-Activators/metabolism , Aspergillus oryzae/enzymology , Cyclization , Macrolides/chemistry , Macrolides/isolation & purification , Molecular Conformation , Polyenes/chemistry , Polyenes/isolation & purification , Polyketides/chemistry , Thiolester Hydrolases/genetics , Trans-Activators/chemistryABSTRACT
BACKGROUND AND AIMS: Recent studies have shown that small structures on plant surfaces serve ecological functions such as resistance against herbivores. The morphology, distribution, chemical composition and changes during shoot and leaf development of such small structures were examined on Paulownia tomentosa. METHODS: The morphology and distribution of the structures were studied under light microscopy, and their chemical composition was analysed using thin-layer chromatography and high-performance liquid chromatography. To further investigate the function of these structures, several simple field experiments and observations were also conducted. KEY RESULTS: Three types of small structures on P. tomentosa were investigated: bowl-shaped organs, glandular hairs and dendritic trichomes. The bowl-shaped organs were densely aggregated on the leaves near flower buds and were determined to be extrafloral nectarines (EFNs) that secrete sugar and attract ants. Nectar production of these organs was increased by artificial damage to the leaves, suggesting an anti-herbivore function through symbiosis with ants. Glandular hairs were found on the surfaces of young and/or reproductive organs. Glandular hairs on leaves, stems and flowers secreted mucilage containing glycerides and trapped small insects. Secretions from glandular hairs on flowers and immature fruits contained flavonoids, which may provide protection against some herbivores. Yellow dendritic trichomes on the adaxial side of leaves also contained flavonoids identical to those secreted by the glandular hairs on fruits and flowers. Three special types of leaves, which differed from the standard leaves in shape, size and identity of small structures, developed near young shoot tips or young flower buds. The density of small structures on these leaf types was higher than on standard leaves, suggesting that these leaf types may be specialized to protect young leaves or reproductive organs. Changes in the small structures during leaf development suggested that leaves of P. tomentosa are primarily protected by glandular hairs and dendritic trichomes at young stages and by the EFNs at mature stages. CONCLUSIONS: The results indicate that P. tomentosa protects young and/or reproductive organs from herbivores through the distribution and allocation of small structures, the nature of which depends on the developmental stage of leaves and shoots.