Discovery and heterologous biosynthesis of glycosylated polyketide luteodienoside A reveals unprecedented glucinol-mediated product offloading by a fungal carnitine O-acyltransferase domain.

Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus Aspergillus luteorubrus MST-FP2246, consisting of an unusual 1-O-ß-d-glucopyranosyl-myo-inositol (glucinol) ester of 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid. Mining the genome of A. luteorubrus identified a putative gene cluster for luteodienoside A biosynthesis (ltb), harbouring a highly reducing polyketide synthase (HR-PKS, LtbA) fused at its C-terminus to a carnitine O-acyltransferase (cAT) domain. Heterologous pathway reconstitution in Aspergillus nidulans, substrate feeding assays and gene truncation confirmed the identity of the ltb cluster and demonstrated that the cAT domain is essential for offloading luteodienoside A from the upstream HR-PKS. Unlike previously characterised cAT domains, the LtbA cAT domain uses glucinol as an offloading substrate to release the product from the HR-PKS. Furthermore, the PKS methyltransferase (MT) domain is capable of catalysing gem-dimethylation of the 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid intermediate, without requiring reversible product release and recapture by the cAT domain. This study expands the repertoire of polyketide modifications known to be catalysed by cAT domains and highlights the potential of mining fungal genomes for this subclass of fungal PKSs to discover new structurally diverse secondary metabolites.

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.

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.

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.

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).

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.

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).

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.

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.

Suertides A-C: selective antibacterial cyclic hexapeptides from Amycolatopsis sp. MST-135876v3.

Amycolatopsis sp. MST-135876 was isolated from soil collected from the riverbank of El Pont de Suert, Catalonia, Spain. Cultivation of MST-135876 on a range of media led to the discovery of a previously unreported dichlorinated cyclic hexapeptide, suertide A (D-Ser, 5-Cl-D-Trp, 6-Cl-D-Trp, L-Ile, D-Val, D-Glu), featuring an unprecedented pair of adjacent 5/6-chlorotryptophan residues. Supplementing the growth medium with KBr resulted in production of the mono- and dibrominated analogues suertides B and C, respectively. Suertides A-C displayed selective activity against Bacillus subtilis (MIC 1.6 µg ml-1) and Staphylococcus aureus (MIC 3.1, 6.3, and 12.5 µg ml-1, respectively), while suertides A and B showed appreciable activity against methicillin-resistant S. aureus (MIC 1.6 and 6.3 µg ml-1, respectively).

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.

Yeppoonic acids A - D: 1,2,4-trisubstituted arene carboxylic acid co-metabolites of conglobatin from an Australian Streptomyces sp.

Streptomyces sp. MST-91080 was isolated from a soil sample collected in Queensland, Australia. From this strain, yeppoonic acids A - D were purified and spectroscopically characterised. The yeppoonic acids are a family of diene enecarboxylic acids on a 1,2,4-trisubstituted benzene scaffold, structurally related to other Streptomyces secondary metabolites MF-EA-705α/ß, NFAT-133 and the lorneic acids. Yeppoonic acids B and C show strong cytotoxicity against the NS-1 mouse myeloma cell line (IC50 2.3 µg ml-1 and 3.8 µg ml-1, respectively) and moderate activity against the DU 145 human prostate cancer cell line (IC50 32.8 µg ml-1 and 49.6 µg ml-1, respectively).

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.

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).

Testing the effectiveness of community-engaged citizen science to promote physical activity, foster healthier neighborhood environments, and advance health equity in vulnerable communities: The Steps for Change randomized controlled trial design and methods.

While low-income midlife and older adults are disproportionately affected by non-communicable diseases that can be alleviated by regular physical activity, few physical activity programs have been developed specifically with their needs in mind. Those programs that are available typically do not address the recognized local environmental factors that can impact physical activity. The specific aim of the Steps for Change cluster-randomized controlled trial is to compare systematically the initial (one-year) and sustained (two-year) multi-level impacts of an evidence-based person-level physical activity intervention (Active Living Every Day [ALED] and age-relevant health education information), versus the ALED program in combination with a novel neighborhood-level citizen science intervention called Our Voice. The study sample (N = 300) consists of insufficiently active adults ages 40 years and over living in or around affordable senior public housing settings. Major study assessments occur at baseline, 12, and 24 months. The primary outcome is 12-month change in walking, and secondary outcomes include other forms of physical activity, assessed via validated self-report measures supported by accelerometry, and physical function and well-being variables. Additional intervention impacts are assessed at 24 months. Potential mediators and moderators of intervention success will be explored to better determine which subgroups do best with which type of intervention. Here we present the study design and methods, including recruitment strategies and yields. TRIAL REGISTRATION: clinicaltrial.gov Identifier = NCT03041415.

Evaluation of Benzguinols as Next-Generation Antibiotics for the Treatment of Multidrug-Resistant Bacterial Infections.

Our recent focus on the "lost antibiotic" unguinol and related nidulin-family fungal natural products identified two semisynthetic derivatives, benzguinols A and B, with unexpected in vitro activity against Staphylococcus aureus isolates either susceptible or resistant to methicillin. Here, we show further activity of the benzguinols against methicillin-resistant isolates of the animal pathogen Staphylococcus pseudintermedius, with minimum inhibitory concentration (MIC) ranging 0.5-1 µg/mL. When combined with sub-inhibitory concentrations of colistin, the benzguinols demonstrated synergy against Gram-negative reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MICs of 1-2 µg/mL in the presence of colistin), whereas the benzguinols alone had no activity. Administration of three intraperitoneal (IP) doses of 20 mg/kg benzguinol A or B to mice did not result in any obvious adverse clinical or pathological evidence of acute toxicity. Importantly, mice that received three 20 mg/kg IP doses of benzguinol A or B at 4 h intervals exhibited significantly reduced bacterial loads and longer survival times than vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. We conclude that the benzguinols are potential candidates for further development for specific treatment of serious bacterial infections as both stand-alone antibiotics and in combination with existing antibiotic classes.

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.

TriTOX: A novel Trichomonas vaginalis assay platform for high-throughput screening of compound libraries.

Trichomonas vaginalis is a neglected urogenital parasitic protist that causes 170 million cases of trichomoniasis annually, making it the most prevalent non-viral, sexually transmitted disease. Trichomoniasis treatment relies on nitroheterocyclics, such as metronidazole. However, with increasing drug-resistance, there is an urgent need for novel anti-trichomonals. Little progress has been made to translate anti-trichomonal research into commercialised therapeutics, and the absence of a standardised compound-screening platform is the immediate stumbling block for drug-discovery. Herein, we describe a simple, cost-effective growth assay for T. vaginalis and the related Tritrichomonas foetus. Tracking changes in pH were a valid indicator of trichomonad growth (T. vaginalis and T. foetus), allowing development of a miniaturised, chromogenic growth assay based on the phenol red indicator in 96- and 384-well microtiter plate formats. The outputs of this assay can be quantitatively and qualitatively assessed, with consistent dynamic ranges based on Z' values of 0.741 and 0.870 across medium- and high-throughput formats, respectively. We applied this high-throughput format within the largest pure-compound microbial metabolite screen (812 compounds) for T. vaginalis and identified 43 hit compounds. We compared these identified compounds to mammalian cell lines, and highlighted extensive overlaps between anti-trichomonal and anti-tumour activity. Lastly, observing nanomolar inhibition of T. vaginalis by fumagillin, and noting this compound has reported activity in other protists, we performed in silico analyses of the interaction of fumagillin with its molecular target methionine aminopeptidase 2 for T. vaginalis, Giardia lamblia and Entamoeba histolytica, highlighting potential for fumagillin as a broad-spectrum anti-protistal against microaerophilic protists. Together, this new platform will accelerate drug-discovery efforts, underpin drug-resistance screening in trichomonads, and contributing to a growing body of evidence highlighting the potential of microbial natural products as novel anti-protistals.

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.

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.