Fungal Duel between Penicillium brasilianum and Aspergillus nomius Results in Dual Induction of Miktospiromide A and Kitrinomycin A.

Cocultivation of the fungi Penicillium brasilianum MST-FP1927 and Aspergillus nomius MST-FP2004 resulted in the reciprocal induction of two new compounds, miktospiromide A (1) from A. nomius and kitrinomycin A (2) from P. brasilianum. A third new compound, kitrinomycin B (3), was also identified from an axenic culture of P. brasilianum, along with the previously reported compounds austalide K (4), 17S-dihydroaustalide K (5), verruculogen (6), and fumitremorgin B (7). The structures of 1-3 were elucidated by detailed spectroscopic analysis and DFT calculations, while 4-7 were identified by comparison to authentic standards. The genome of A. nomius MST-FP2004 was sequenced, and a putative biosynthetic gene cluster for 1 was identified. Compound 2 showed activity against murine melanoma NS-1 cells (LD99 7.8 µM) and the bovine parasite Tritrichomonas foetus (LD99 4.8 µM).

Fuligopyrones from the Fruiting Bodies of Myxomycete Fuligo septica Offer Short-Term Protection from Abiotic Stress Induced by UV Radiation.

The myxomycete Fuligo septica, colloquially referred to as "dog vomit fungus", forms vibrant yellow fruiting bodies (aethalia) on wood chips during warm and humid conditions in spring. In 2018, ideal climatic conditions in Sydney, Australia, provided a rare opportunity to access abundant quantities of F. septica aethalia, which enabled the isolation, purification, structure elucidation, and biological screening of two avenalumamide pyrones, fuligopyrone (1) and fuligopyrone B (2). While 1 and 2 did not exhibit any appreciable biological activity, their significant UV absorption at 325 nm suggested they may be acting as transient sunscreens to help protect the fruiting mass from exposure to sunlight. In support of this hypothesis, exposing a solution of 2 to direct sunlight for 5 min resulted in rapid equilibration with a mixture of 2E,4Z-fuligopyrone B (10) and 2Z,4E-fuligopyrone B (11) photoisomers.

Turonicin A, an Antifungal Linear Polyene Polyketide from an Australian Streptomyces sp.

Turonicin A (1) was isolated from Streptomyces sp. MST-123921, which was recovered from soil collected on the banks of the Turon River in New South Wales, Australia. Turonicin A (1) is an amphoteric linear polyene polyketide featuring independent pentaene and tetraenone chromophores and is structurally related to linearmycins A-C (2-4). The structure of 1 was determined by detailed spectroscopic analysis and comparison to literature data. Bioinformatic analysis of the linearmycin biosynthetic gene cluster also allowed the previously unresolved absolute stereostructures of 2-4 to be elucidated. Turonicin A (1) exhibited very potent activity against the fungi Candida albicans (MIC 0.0031 µg/mL, 2.7 nM) and Saccharomyces cerevisiae (MIC 0.0008 µg/mL, 0.7 nM), moderate activity against the bacteria Bacillus subtilis (MIC 0.097 µg/mL, 85 nM) and Staphylococcus aureus (MIC 0.39 µg/mL, 340 nM), and no cytotoxicity against human fibroblasts, making it an attractive candidate for further development as a potential next-generation antibiotic scaffold.

A Pseudoalteromonas Clade with Remarkable Biosynthetic Potential.

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we report the biochemical and genomic analysis of Pseudoalteromonas sp. strain HM-SA03, isolated from the blue-ringed octopus, Hapalochlaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides, and four novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through the phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of 10 biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the underexplored potential of Pseudoalteromonas as a source of new natural products.IMPORTANCE This study demonstrates that the Pseudoalteromonas strain HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Characterisation and heterologous biosynthesis of burnettiene A, a new polyene-decalin polyketide from Aspergillus burnettii.

Chemical exploration of the recently described Australian fungus, Aspergillus burnettii, uncovered a new metabolite, burnettiene A. Here, we characterise the structure of burnettiene A as a polyene-decalin polyketide. Bioinformatic analysis of the genome of A. burnettii identified a putative biosynthetic gene cluster for burnettiene A (bue), consisting of eight genes and sharing similarity to the fusarielin gene cluster. Introduction of the reassembled bue gene cluster into Aspergillus nidulans for heterologous expression resulted in the production of burnettiene A under native promoters. Omission of bueE encoding a cytochrome P450 led to the production of preburnettiene A, confirming that BueE is responsible for catalysing the regiospecific multi-oxidation of terminal methyl groups to carboxylic acids. Similarly, bueF was shown to encode an ester-forming methyltransferase, with its omission resulting in the production of the tricarboxylic acid, preburnettiene B. Introduction of an additional copy of the transcription factor bueR under the regulation of the gpdA promoter significantly improved the heterologous production of the burnettienes. Burnettiene A displayed strong in vitro cytotoxicity against mouse myeloma NS-1 cells (MIC 0.8 µg mL-1).

Hancockiamides: phenylpropanoid piperazines from Aspergillus hancockii are biosynthesised by a versatile dual single-module NRPS pathway.

The hancockiamides are an unusual new family of N-cinnamoylated piperazines from the Australian soil fungus Aspergillus hancockii. Genomic analyses of A. hancockii identified a biosynthetic gene cluster (hkm) of 12 genes, including two single-module nonribosomal peptide synthetase (NRPS) genes. Heterologous expression of the hkm cluster in A. nidulans confirmed its role in the biosynthesis of the hancockiamides. We further demonstrated that a novel cytochrome P450, Hkm5, catalyses the methylenedioxy bridge formation, and that the PAL gene hkm12 is dispensable, but contributes to increased production of the cinnamoylated hancockiamides. In vitro enzymatic assays and substrate feeding studies demonstrated that NRPS Hkm11 activates and transfers trans-cinnamate to the piperazine scaffold and has flexibility to accept bioisosteric thienyl and furyl analogues. This is the first reported cinnamate-activating fungal NRPS. Expression of a truncated cluster lacking the acetyltransferase gene led to seven additional congeners, including an unexpected family of 2,5-dibenzylpiperazines. These pleiotropic effects highlight the plasticity of the pathway and the power of this approach for accessing novel natural product scaffolds.

Nanangenines: drimane sesquiterpenoids as the dominant metabolite cohort of a novel Australian fungus, Aspergillus nanangensis.

Chemical investigation of an undescribed Australian fungus, Aspergillus nanangensis, led to the identification of the nanangenines - a family of seven new and three previously reported drimane sesquiterpenoids. The structures of the nanangenines were elucidated by detailed spectroscopic analysis supported by single crystal X-ray diffraction studies. The compounds were assayed for in vitro activity against bacteria, fungi, mammalian cells and plants. Bioinformatics analysis, including comparative analysis with other acyl drimenol-producing Aspergilli, led to the identification of a putative nanangenine biosynthetic gene cluster that corresponds to the proposed biosynthetic pathway for nanangenines.

Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Geministatins: new depside antibiotics from the fungus Austroacremonium gemini.

Two new depside antibiotics, geministatins A (1) and B (2), were isolated from the fungus Austroacremonium gemini MST-FP2131 (Sordariomycetes, Ascomycota), which was recovered from rotting wood in the wet tropics of northern Australia. The structures of the geministatins were elucidated by detailed spectroscopic analysis, chemical degradation and comparison with literature values. Chemical degradation of 1 and 2 yielded three new analogues, geministatins C-E (3-5), as well as a previously reported compound dehydromerulinic acid A (6). Compounds 1, 2 and 6 exhibited antibacterial activity against the Gram-positive bacteria Bacillus subtilis (MIC 0.2-1.6 µg mL-1) and Staphylococcus aureus (MIC 0.78-6.3 µg mL-1), including methicillin-resistant S. aureus (MRSA), while 4 exhibited antifungal activity against the yeast Saccharomyces cerevisiae (MIC 13 µg mL-1).

Talcarpones A and B: bisnaphthazarin-derived metabolites from the Australian fungus Talaromyces johnpittii sp. nov. MST-FP2594.

Talcarpones A (1) and B (2) are rare bisnaphthazarin derivatives produced by Talaromyces johnpittii (ex-type strain MST-FP2594), a newly discovered Australian fungus, which is formally described and named herein. The talcarpones were isolated along with the previously reported monomeric naphthoquinone, aureoquinone (3), suggesting a biosynthetic link between these metabolites. Talcarpone A is a lower homologue of hybocarpone (4), which was first isolated from a mycobiont of the lichen Lecanora hybocarpa. The structures of 1 and 2 were elucidated by detailed spectroscopic analysis, molecular modelling and comparison with literature data. Talcarpones 1 and 2 exhibited moderate antifungal activity (MIC 0.78-3.1 µg ml-1) and weak activity against Gram-positive bacteria (MIC 13-25 µg ml-1). The talcarpones also demonstrated noteworthy chemical reactivities, with 2 converting rapidly to 1, which in turn converted slowly to the highly coloured 3. These post-biosynthetic reactions point to a potential ecological role for the talcarpones in providing ongoing (slow-release) physicochemical protection for T. johnpittii against solar irradiation.

Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals.

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.

Alternariol 9-O-methyl ether dimethyl sulfoxide monosolvate.

THE TITLE COMPOUND (SYSTEMATIC NAME: 3,7-dihy-droxy-9-meth-oxy-1-methyl-6H-benzo[c]chromen-6-one dimethyl sulfoxide monosolvate), C15H12O5·C2H6OS, was isolated from an unidentified endophytic fungus (belonging to class Ascomycetes) of Taxus sp. In the crystal, both the alternariol 9-O-methyl ether (AME) and the dimethyl sulfoxide (DMSO) mol-ecules exhibit crystallographic mirror symmetry. One of the hy-droxy groups makes bifurcated hydrogen bonds, viz. an intra-molecular bond with the carbonyl group and an inter-molecular bond with the carbonyl group in an inversion-related AME mol-ecule. In the crystal, the AME mol-ecules are organized into stacks parallel with the b axis by π-π inter-actions between centrosymmetrically related mol-ecules [the distance between the centroid of the central ring and the centroid of the meth-oxy-substituted benzene ring in the next mol-ecule of the stack is 3.6184 (5) Å]. Pairs of DMSO mol-ecules, linked via centrosymmetric C-H⋯O contacts, are inserted into the voids created by the AME mol-ecules, making O-H⋯O and C-H⋯O contacts with the hosts.

Unequivocal 13C NMR assignment of cyclohexadienyl rings in bromotyrosine-derived metabolites from marine sponges.

Bromotyrosine-derived compounds are commonly isolated from Verongida sponges and are a major class of marine natural products. Here we report on the unequivocal (13)C NMR assignment of the brominated carbons at positions C-2 and C-4 of the cyclohexadiene ring, two carbons whose resonances are often incorrectly assigned. Interpretation of HMBC data acquired for a series of known bromotyrosine analogues, which included ianthesine E(1), aerothionin (2), 11-hydroxyaerothionin (3), and 11,19-dideoxyfistularin-3 (4), allowed us to unequivocally assign the carbons in question, C-2 and C-4, through the observance of unique HMBC correlations from the C-1 hydroxyl proton. Here we present the complete 2D NMR data sets recorded in DMSO-d(6) for 2-4 that were used to confirm the assignment and establish the working model. Using this model, a survey of the literature revealed that many members of this structure class had been wrongly assigned. This paper serves to reassign those compounds whose (13)C NMR assignment at positions C-2 and C-4 of the cyclohexadiene ring should be reversed.

Bioactive natural products from Papua New Guinea marine sponges.

The discovery of novel natural products for drug development relies heavily upon a rich biodiversity, of which the marine environment is an obvious example. Marine natural product research has spawned several drugs and many other candidates, some of which are the focus of current clinical trials. The sponge megadiversity of Papua New Guinea is a rich but underexplored source of bioactive natural products. Here, we review some of the many natural products derived from PNG sponges with an emphasis on those with interesting biological activity and, therefore, drug potential. Many bioactive natural products discussed here appear to be derived from non-ribosomal peptide and polyketide biosynthesis pathways, strongly suggesting a microbial origin of these compounds. With this in mind, we also explore the notion of sponge-symbiont biosynthesis of these bioactive compounds and present examples to support the working hypothesis.

Discovery of brevijanazines from Aspergillus brevijanus reveals the molecular basis for p-nitrobenzoic acid in fungi.

The brevijanazines are novel p-nitrobenzoylated piperazines isolated from Aspergillus brevijanus. Their structures were elucidated by spectroscopic analysis, X-ray crystallography and total synthesis. Heterologous biosynthesis, precursor feeding and in vitro microsomal assays unveiled the biosynthetic pathway to the brevijanazines, featuring a cytochrome P450 oxygenase that converts p-aminobenzoic acid to p-nitrobenzoic acid.

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL.

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-ß-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.

Genome Mining of Aspergillus hancockii Unearths Cryptic Polyketide Hancockinone A Featuring a Prenylated 6/6/6/5 Carbocyclic Skeleton.

Activation of a cryptic polyketide synthase gene cluster hkn from Aspergillus hancockii via overexpression of the gene-cluster-specific transcription factor HknR led to the discovery of a novel polycyclic metabolite, which we named hancockinone A. The compound features an unprecedented prenylated 6/6/6/5 tetracarbocyclic skeleton and shows moderate antibacterial activity. Heterologous expression, substrate feeding, and in vitro assays confirmed the role of cytochrome P450 HknE in constructing the five-membered ring in hancockinone A from the precursor neosartoricin B.

Pestalactams A-C: novel caprolactams from the endophytic fungus Pestalotiopsis sp.

Chemical investigations of a fermentation culture from the endophytic fungus Pestalotiopsis sp. yielded three novel caprolactams, pestalactams A-C (). The structures of were determined by analysis of 1D and 2D-NMR, UV, IR, and MS data. The structure of pestalactam A was confirmed following single crystal X-ray diffraction analysis. Pestalactams A-C are the first C-7 alkylated caprolactam natural products to be reported. Pestalactams A () and B () were tested against two different strains of the malaria parasite Plasmodium falciparum (3D7 and Dd2), and the mammalian cell lines, MCF-7 and NFF, and showed modest in vitro activity in all assays.

Genome mining of a fungal endophyte of Taxus yunnanensis (Chinese yew) leads to the discovery of a novel azaphilone polyketide, lijiquinone.

Genome mining of Ascomycete sp. F53 (F53), a fungal endophyte of the traditional Chinese medicinal plant Taxus yunnanensis (Chinese yew), revealed 35 putative specialized metabolite biosynthesis gene clusters, one of which encodes a rarely seen tandem polyketide synthase pathway with close homology to azaphilone biosynthesis pathways. A novel compound, lijiquinone 1, was subsequently isolated from F53 and structurally and functionally characterized. The m/z 385 [M + H+ ]+ compound, comprised of a cyclohexenone side group attached to a core bicyclic ring, displayed cytotoxicity against human myeloma cells (IC50  = 129 µM), as well as antifungal activity against Candida albicans (IC50  = 79 µM) and Cryptococcus albidus (IC50  = 141 µM). Our results suggest that enzymes encoded on the lij gene cluster are responsible for the synthesis of 1 and that the medicinal properties of T. yunnanensis could be partially mediated by this novel azaphilone. This study highlights the utility of combining traditional knowledge with contemporary genomic approaches for the discovery of new bioactive compounds.

Production of novel pladienolide analogues through native expression of a pathway-specific activator.

Aberrant splicing of pre-mRNA is implicated in many human genetic disorders. Small molecules that target the spliceosome are important leads as therapeutics and research tools, and one compound of significant interest is the polyketide natural product pladienolide B. Here, we describe the reactivation of quiescent pladienolide B production in the domesticated lab strain Streptomyces platensis AS6200 by overexpression of the pathway-specific activator PldR. The resulting dysregulation of the biosynthetic genes led to the accumulation and isolation of five additional intermediate or shunt metabolites of pladienolide B biosynthesis, including three previously unreported congeners. These compounds likely comprise the entire pladienolide biosynthetic pathway and demonstrate the link between polyketide tailoring reactions and bioactivity, particularly the importance of the 18,19-epoxide. Each congener demonstrated specific inhibitory activity against mammalian cell lines, with successive modifications leading to increased activity (IC50: 8 mM to 5 µM).