Resorculins: hybrid polyketide macrolides from Streptomyces sp. MST-91080.

Fourteen-membered macrolides are a class of compounds with significant clinical value as antibacterial agents. As part of our ongoing investigation into the metabolites of Streptomyces sp. MST-91080, we report the discovery of resorculins A and B, unprecedented 3,5-dihydroxybenzoic acid (α-resorcylic acid)-containing 14-membered macrolides. We sequenced the genome of MST-91080 and identified the putative resorculin biosynthetic gene cluster (rsn BGC). The rsn BGC is hybrid of type I and type III polyketide synthases. Bioinformatic analysis revealed that the resorculins are relatives of known hybrid polyketides: kendomycin and venemycin. Resorculin A exhibited antibacterial activity against Bacillus subtilis (MIC 19.8 µg mL-1), while resorculin B showed cytotoxic activity against the NS-1 mouse myeloma cell line (IC50 3.6 µg mL-1).

Stereodivergent Hydroxylation of Berkeleylactones by Penicillium turbatum.

Penicillium turbatum has previously been reported to produce A26771B, a 16-membered macrocyclic polyketide with activity against Gram-positive bacteria, mycoplasma, and fungi, as well as the structurally related compounds berkeleylactone E and berkeleylactones I-O. In this work, large-scale cultivation of P. turbatum NRRL 5630 on rice yielded seven new berkeleylactone analogues, berkeleylactone E methyl ester, 14-epi-berkeleylactone F, berkeleylactones P-R, 12-epi-berkeleylactone Q, and 13-epi-berkeleylactone R, and six previously reported analogues, A26771B and berkeleylactones E-G and J-K. The structures of the berkeleylactones were elucidated by detailed analysis of spectroscopic data, molecular modeling, and comparison with literature values. Interestingly, six of the berkeleylactone analogues were isolated as pairs of hydroxy epimers, highlighting how Nature can exploit stereodivergence in biosynthetic pathways to increase chemical diversity. The genome of P. turbatum was sequenced, and a putative gene cluster (bekl) responsible for the biosynthesis of the berkeleylactones was identified. The new berkeleylactone analogues exhibited no significant biological activity against a panel of bacteria, fungi, the parasite Giardia duodenalis, or NS-1 murine myeloma cells, suggesting a hitherto undiscovered biological role.

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).

Synthesis, Characterization, and Bioactivity of the Lichen Pigments Pulvinamide, Rhizocarpic Acid, and Epanorin and Congeners.

The lichen natural products pulvinamide, rhizocarpic acid, and epanorin have been synthesized and characterized spectroscopically and by X-ray crystallography. The syntheses, by ring-opening of pulvinic acid dilactone (PAD), may well be biomimetic, given the well-known occurrence of PAD in lichen. The enantiomers, ent-rhizocarpic acid and ent-epanorin, and corresponding carboxylic acids, norrhizocarpic acid and norepanorin, were similarly prepared. All compounds were assessed for growth inhibitory activity against selected bacteria, fungi, a protist, a mammalian tumor cell line, and normal cells. Rhizocarpic acid is weakly antibacterial (Bacillus subtilis MIC = 50 µg/mL) and possesses modest but selective antitumor activity (NS-1 murine myeloma MIC = 3.1 µg/mL) with >10-fold potency relative to its enantiomer (MIC = 50 µg/mL).

Dendrillic Acids A and B: Nitrogenous, Rearranged Spongian Nor-Diterpenes from a Dendrilla sp. Marine Sponge.

Two nitrogenous rearranged spongian nor-diterpenoids, dendrillic acids A and B, were isolated from a marine sponge Dendrilla sp. (order: Dendroceratida; family: Darwinellidae). The structures of the metabolites were elucidated on the basis of spectroscopic analysis as well as density functional theory prediction of NMR chemical shifts and application of the DP4+ algorithm. The absolute configuration of the metabolites was established via comparison of experimental and time-dependent density functional theory predicted electronic circular dichroism data. An unusual epimerization reaction was observed leading to the interconversion of the metabolites upon storage in dimethyl sulfoxide solution, which is proposed to proceed via an anionic pathway as probed via isotopic incorporation experiments. Evaluation against a panel of micro-organisms and cell lines revealed that the compounds were devoid of any significant biological activity against all organisms tested, with the exception of mild antiprotozoal activity displayed by dendrillic acid B (2) against Giardia duodenalis.

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.

Type-I Hemins and Free Porphyrins from a Western Australian Sponge Isabela sp.

Two novel free porphyrins, isabellins A and B, as well as the known compounds corallistin D and deuteroporphyrin IX were isolated from a marine sponge Isabela sp. LC-MS analysis of the crude extract revealed that the natural products were present both as free porphyrins and iron(III) coordinated hemins, designated isabellihemin A, isabellihemin B, corallistihemin D and deuterohemin IX, respectively. Structures were determined via high-resolution mass spectrometry, UV-Vis spectroscopy and extensive NOESY NMR spectroscopic experiments. The type-I alkyl substitution pattern of isabellin A and isabellihemin A was assigned unambiguously by single crystal X-ray diffraction. Biological evaluation of the metabolites revealed potent cytotoxicity for isabellin A against the NS-1 murine myeloma cell line.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

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).

Semisynthesis and biological evaluation of a focused library of unguinol derivatives as next-generation antibiotics.

In this study, we report the semisynthesis and in vitro biological evaluation of thirty-four derivatives of the fungal depsidone antibiotic, unguinol. Initially, the semisynthetic modifications were focused on the two free hydroxy groups (3-OH and 8-OH), the three free aromatic positions (C-2, C-4 and C-7), the butenyl side chain and the depsidone ester linkage. Fifteen first-generation unguinol analogues were synthesised and screened against a panel of bacteria, fungi and mammalian cells to formulate a basic structure activity relationship (SAR) for the unguinol pharmacophore. Based on the SAR studies, we synthesised a further nineteen second-generation analogues, specifically aimed at improving the antibacterial potency of the pharmacophore. In vitro antibacterial activity testing of these compounds revealed that 3-O-(2-fluorobenzyl)unguinol and 3-O-(2,4-difluorobenzyl)unguinol showed potent activity against both methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MIC 0.25-1 µg mL-1) and are promising candidates for further development in vivo.

Chlorinated metabolites from Streptomyces sp. highlight the role of biosynthetic mosaics and superclusters in the evolution of chemical diversity.

LCMS-guided screening of a library of biosynthetically talented bacteria and fungi identified Streptomyces sp. MST- as a prolific producer of chlorinated metabolites. We isolated and characterised six new and nine reported compounds from MST-, belonging to three discrete classes - the depsipeptide svetamycins, the indolocarbazole borregomycins and the aromatic polyketide anthrabenzoxocinones. Following genome sequencing of MST-, we describe, for the first time, the svetamycin biosynthetic gene cluster (sve), its mosaic structure and its relationship to several distantly related gene clusters. Our analysis of the sve cluster suggested that the reported stereostructures of the svetamycins may be incorrect. This was confirmed by single-crystal X-ray diffraction analysis, allowing us to formally revise the absolute configurations of svetamycins A-G. We also show that the borregomycins and anthrabenzoxocinones are encoded by a single supercluster (bab) implicating superclusters as potential nucleation points for the evolution of biosynthetic gene clusters. These clusters highlight how individual enzymes and functional subclusters can be co-opted during the formation of biosynthetic gene clusters, providing a rare insight into the poorly understood mechanisms underpinning the evolution of chemical diversity.

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.

Biosynthesis of a New Benzazepine Alkaloid Nanangelenin A from Aspergillus nanangensis Involves an Unusual l-Kynurenine-Incorporating NRPS Catalyzing Regioselective Lactamization.

1-Benzazepine is a pharmaceutically important scaffold but is rare among natural products. Nanangelenin A (1), containing an unprecedented 3,4-dihydro-1-benzazepine-2,5-dione-N-prenyl-N-acetoxy-anthranilamide scaffold, was isolated from a novel species of Australian fungus, Aspergillus nanangensis. Genomic and retrobiosynthetic analyses identified a putative nonribosomal peptide synthetase (NRPS) gene cluster (nan). The detailed biosynthetic pathway to 1 was established by heterologous pathway reconstitution in A. nidulans, which led to biosynthesis of intermediates nanagelenin B-F (2-5 and 7). We demonstrated that the NRPS NanA incorporates anthranilic acid (Ant) and l-kynurenine (l-Kyn), which is supplied by a dedicated indoleamine-2,3-dioxygenase NanC encoded in the gene cluster. Using heterologous in vivo assays and mutagenesis, we demonstrated that the C-terminal condensation (CT) and thiolation (T3) domains of NanA are responsible for the regioselective cyclization of the tethered Ant-l-Kyn dipeptide to form the unusual benzazepine scaffold in 1. We also showed that NanA-CT catalyzes the regioselective cyclization of a surrogate synthetic substrate, Ant-l-Kyn-N-acetylcysteamine, to give the benzazepine scaffold, while spontaneous cyclization of the dipeptide yielded the alternative kinetically favored benzodiazepine scaffold. The discovery of 1 and the characterization of NanA have expanded the chemical and functional diversities of fungal NRPSs.

Comprehensive chemotaxonomic and genomic profiling of a biosynthetically talented Australian fungus, Aspergillus burnettii sp. nov.

Aspergillus burnettii is a new species belonging to the A. alliaceus clade in Aspergillus subgenus Circumdati section Flavi isolated from peanut-growing properties in southern Queensland, Australia. A. burnettii is a fast-growing, floccose fungus with distinctive brown conidia and is a talented producer of biomass-degrading enzymes and secondary metabolites. Chemical profiling of A. burnettii revealed the metabolites ochratoxin A, kotanins, isokotanins, asperlicin E, anominine and paspalinine, which are common to subgenus Circumdati, together with burnettiene A, burnettramic acids, burnettides, and high levels of 14α-hydroxypaspalinine and hirsutide. The genome of A. burnettii was sequenced and an annotated draft genome is presented. A. burnettii is rich in secondary metabolite biosynthetic gene clusters, containing 51 polyketide synthases, 28 non-ribosomal peptide synthetases and 19 genes related to terpene biosynthesis. Functional annotation of digestive enzymes of A. burnettii and A. alliaceus revealed overlapping carbon utilisation profiles, consistent with a close phylogenetic relationship.

The chemical gymnastics of enterocin: evidence for stereodivergence in Nature.

Stereodivergence in Nature encapsulates both enzymatic (biosynthetic) and non-enzymatic (chemical) diversification of natural product scaffolds arising from a single biosynthetic pathway. Herein, we report a fascinating example of stereodivergence for the bacterial polyketide enterocin, which we observed to undergo a series of facile skeletal rearrangements in solution, leading to four distinct isomeric structures. The final distribution of the four isomers was found to be highly sensitive to the conditions used, including solvent, temperature and pH. In this study, we have investigated the kinetics of these isomeric conversions, and using a combination of DFT and thermochemical calculations, were able to establish a mechanism detailing a concerted rearrangement and an unusual "gymnastic" sequence of pseudo-chair-boat conformational interconversions. In addition to these kinetic and mechanistic studies, we also performed a semisynthetic study aimed at stabilising the enterocin scaffold. In total, seven analogues of enterocin were synthesised and investigated for their stability and in vitro activity against a panel of bacteria, fungi, plants and mammalian cells.

Total Synthesis of the Antitumor-Antitubercular 2,6'-Bijuglone Natural Product Diospyrin and Its 3,6'-Isomer.

The 2,6'-bijuglone natural product diospyrin and its unnatural 3,6'-isomer idospyrin have been synthesized in seven steps each from N,N-diethylsenecioamide in overall yields of 12% and 13%, respectively. The syntheses diverge from ramentaceone (7-methyljuglone) and include a key Suzuki-Miyaura cross-coupling. Diospyrin, idospyrin, and several synthetic precursors exhibit potent and selective cytotoxicity to the murine myeloma NS-1 cell line over neonatal foreskin cells.

Talauxins: Hybrid Phenalenone Dimers from Talaromyces stipitatus.

Cultivation and extraction of the fungus Talaromyces stipitatus led to the isolation of five new oxyphenalenone-amino acid hybrids, which were named talauxins E, Q, V, L, and I based on the corresponding one-letter amino acid codes, along with their putative biosynthetic precursor, duclauxin. The rapid reaction of duclauxin with amino acids to produce talauxins was demonstrated in vitro and exploited to generate a small library of natural and unnatural talauxins. Talauxin V was shown to undergo spontaneous elimination of methyl acetate to yield the corresponding neoclauxin scaffold. This process was modeled using density functional theory calculations, revealing a dramatic change in conformation resulting from the syn elimination of methyl acetate.

Albanitriles A-G: Antiprotozoal Polyacetylene Nitriles from a Mycale Marine Sponge.

Seven new nitrile-bearing polyacetylenes, named albanitriles A-G, were isolated from a marine sponge of the Mycale genus (Order: Poecilosclerida, Family: Mycalidae) collected near Albany, Western Australia. Structural elucidation was achieved using a combination of high-resolution mass spectrometry and ultraviolet/visible, infrared, and nuclear magnetic resonance spectroscopy. The compounds were found to possess moderate activity against Giardia duodenalis when compared to a metronidazole positive control.