Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Total Synthesis of the Phenylnaphthacenoid Type II Polyketide Antibiotic Formicamycin H via Regioselective Ruthenium-Catalyzed Hydrogen Auto-Transfer [4 + 2] Cycloaddition.

The first total synthesis of the pentacyclic phenylnaphthacenoid type II polyketide antibiotic formicamycin H is described. A key feature of the synthesis involves the convergent, regioselective assembly of the tetracyclic core via ruthenium-catalyzed α-ketol-benzocyclobutenone [4 + 2] cycloaddition. Double dehydration of the diol-containing cycloadduct provides an achiral enone, which upon asymmetric nucleophilic epoxidation and further manipulations delivers the penultimate tetracyclic trichloride in enantiomerically enriched form. Subsequent chemo- and atroposelective Suzuki cross-coupling of the tetracyclic trichloride introduces the E-ring to complete the total synthesis. Single-crystal X-ray diffraction analyses of two model compounds suggest that the initially assigned stereochemistry of the axially chiral C6-C7 linkage may require revision.

New Bioactive Polyketides from the Mangrove-Derived Fungus Penicillium sp. SCSIO 41411.

Three new polyketides, including three ester derivatives (1, 3, and 5) and a new natural product, which was a benzoquinone derivative, embelin A (4), together with nine known ones (2 and 6-13), were isolated from the mangrove-derived fungus Penicillium sp. SCSIO 41411. Their structures were determined by detailed NMR and MS spectroscopic analyses. The X-ray single-crystal diffraction analysis of 4 was described for the first time. Compound 9 displayed obvious inhibition against PDE4 with an inhibitory ratio of 40.78% at 10 µM. Compound 12 showed DPPH radical scavenging activity, with an EC50 of 16.21 µg/mL, compared to the positive control (ascorbic acid, EC50, 11.22 µg/mL). Furthermore, compound 4 exhibited cytotoxicity against PC-3 and LNCaP with IC50 values of 18.69 and 31.62 µM, respectively.

The Cytochalasins and Polyketides from a Mangrove Endophytic Fungus Xylaria arbuscula QYF.

Twelve compounds, including four undescribed cytochalasins, xylariachalasins A-D (1-4), four undescribed polyketides (5-8), and four known cytochalasins (9-12), were isolated from the mangrove endophytic fungus Xylaria arbuscula QYF. Their structures and absolute configurations were established by extensive spectroscopic analyses (1D and 2D NMR, HRESIMS), electronic circular dichroism (ECD) calculations, 13C NMR calculation and DP4+ analysis, single-crystal X-ray diffraction, and the modified Mosher ester method. Compounds 1 and 2 are rare cytochalasin hydroperoxides. In bioactivity assays, Compound 2 exhibited moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 12.5 µM for both Compound 10 exhibited significant cytotoxic activity against MDA-MB-435 with an IC50 value of 3.61 ± 1.60 µM.

Reaping the Chemical Diversity of Morinagamyces vermicularis Using Feature-Based Molecular Networking.

Moringadepsin (6) and chaetone B (7) were isolated by us in the course of a conventional chemical screening of Morinagamyces vermicularis CBS 303.81, a fungus belonging to the relatively underexplored family Schizotheciaceae of the phylum Ascomycota. Since these metabolites did not account for the antifungal activity observed in a crude extract of this fungus, we utilized an MS/MS-based molecular networking approach to get a thorough insight into the secondary metabolites produced by this strain. Manual annotation of high-resolution fragmentation mass spectra by CANOPUS classified a major molecular family as putatively new thiodiketopiperazines. However, these results were opposite to the results of ChemWalker analysis based solely on MS/MS data, assigning these metabolites as various polyketides. Thus, targeted preparative HPLC isolation focusing on the most abundant features within this major molecular family resulted in the isolation of five secondary metabolites. Their structures were elucidated based on HRMS and NMR data as four new thiodiketopiperazine derivatives, botryosulfuranols D-G (1-4), alongside the known botryosulfuranol A (5). Compounds 1-3 and 5 exhibited moderate to weak antifungal activity against different test strains, accounting for the initial antifungal activity observed for its crude extract. Our study stressed the importance of full NMR-based structure elucidation for metabolomics research.

Penidaleodiolides A and B, Cage-Like Polyketides with Neurotransmission-Regulating Activity from the Soil Fungus Penicillium daleae L3SO.

Penicillium daleae L3SO is a fungus isolated from the rhizospheric soil of the chloroplast-deficient plant Monotropa uniflora. A chemical study on the rice fermentation of this fungus led to the isolation and identification of two cage-like polyketides, penidaleodiolide A (1) and its biosynthetic-related congener penidaleodiolide B (2). The structures of 1 and 2 were determined by a combination of extensive spectroscopic analysis, biosynthetic consideration, chemical derivatization, and computational methods. Compound 1 harbors an unusual tricyclo[4.3.04,9]nonane scaffold, unprecedented in polyketide natural products. The hypothetical biosynthetic pathways for 1 and 2 were postulated and were supported by CRISPR/Cas9 genome editing results. Penidaleodiolide A (1) showed a significant inhibitory effect on the action potentials of murine hippocampal basket neurons and decreased the frequency of spontaneous excitatory postsynaptic currents in a concentration-dependent manner (the inhibition ratios were 0.30 ± 0.02 for 1 µM, 0.37 ± 0.03 for 10 µM, and 0.50 ± 0.07 for 20 µM) while being devoid of cytotoxicity against the nerve cells.

Recent Advances in Discovery, Structure, Bioactivity, and Biosynthesis of trans-AT Polyketides.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex enzymes, which are responsible for generating a wide range of natural products, identified as trans-AT polyketides. These polyketides have received significant attention in drug development due to their structural diversity and potent bioactivities. With approximately 300 synthesized molecules discovered so far, trans-AT PKSs are found widespread in bacteria. Their biosynthesis pathways exhibit considerable genetic diversity, leading to the emergence of numerous enzymes with novel mechanisms, serving as a valuable resource for genetic engineering aimed at modifying small molecules' structures and creating new engineered enzymes. Despite the systematic discussions on trans-AT polyketides and their biosynthesis in earlier studies, the continuous advancements in tools, methods, compound identification, and biosynthetic pathways require a fresh update on accumulated knowledge. This review seeks to provide a comprehensive discussion for the 27 types of trans-AT polyketides discovered within the last seven years, detailing their sources, structures, biological activities, and biosynthetic pathways. By reviewing this new knowledge, a more profound understanding of the trans-AT polyketide family can be achieved.

From the forest floor to the lab: Insights into the diversity and complexity of mushroom polyketide synthases.

Mushroom-forming fungi exhibit a distinctive ecology, which is unsurprisingly also reflected in unique and divergent biosynthetic pathways. We review this phenomenon through the lens of the polyketide metabolism, where mushrooms often deviate from established principles and challenge conventional paradigms. This is evident not only by non-canonical enzyme architectures and functions but also by their propensity for multi-product synthases rather than single-product pathways. Nevertheless, mushrooms also feature many polyketides familiar from plants, bacteria, and fungi of their sister division Ascomycota, which, however, are the result of an independent evolution. In this regard, the captivating biosynthetic pathways of mushrooms might even help us understand the biological pressures that led to the simultaneous production of the same natural products (via convergent evolution, co-evolution, and/or metaevolution) and thus address the question of their raison d'être.

Induction of Three New Secondary Metabolites by the Co-Culture of Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 Isolated from the Chinese Mangrove Plant Rhizophora mangle.

Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1-6), two sterols (7, 8), and six polyketides (9-14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC50 values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.

Polyketide Derivatives from the Mangrove-Derived Fungus Penicillium sp. HDN15-312.

Four new polyketides, namely furantides A-B (1-2), talamin E (3) and arugosinacid A (4), and two known polyketides were obtained from the mangrove-derived fungus Penicillium sp. HDN15-312 using the One Strain Many Compounds (OSMAC) strategy. Their chemical structures, including configurations, were elucidated by detailed analysis of extensive NMR spectra, HRESIMS and ECD. The DPPH radicals scavenging activity of 3, with an IC50 value of 6.79 µM, was better than vitamin C.

Gatekeeping Activity of Collinear Ketosynthase Domains Limits Product Diversity for Engineered Type I Polyketide Synthases.

Engineered type I polyketide synthases (type I PKSs) can enable access to diverse polyketide pharmacophores and generate non-natural natural products. However, the promise of type I PKS engineering remains modestly realized at best. Here, we report that ketosynthase (KS) domains, the key carbon-carbon bond-forming catalysts, control which intermediates are allowed to progress along the PKS assembly lines and which intermediates are excluded. Using bimodular PKSs, we demonstrate that KSs can be exquisitely selective for the upstream polyketide substrate while retaining promiscuity for the extender unit that they incorporate. It is then the downstream KS that acts as a gatekeeper to ensure the fidelity of the extender unit incorporation by the upstream KS. We also demonstrate that these findings are not universally applicable; substrate-tolerant KSs do allow engineered polyketide intermediates to be extended. Our results demonstrate the utility for evaluating the KS-induced bottlenecks to gauge the feasibility of engineering PKS assembly lines.

Diversity of African fungi, chemical constituents and biological activities.

Besides plants and animals, the fungal kingdom consists of several species characterized by various forms and applications. Fungi are amazing producers of bioactive natural products with applications in medicine and agriculture. Though this kingdom has been extensively investigated worldwide, it remains relatively underexplored in Africa. To address the knowledge gaps, encourage research interest, and suggest opportunities for the discovery of more bioactive substances from African fungi, we considered it appropriate to extensively review the research work carried out on African fungi since 1988. This review summarizes the diversity and distribution of fungi throughout Africa, the secondary metabolites yet reported from studied fungi, their biological activities and, the countries where they were collected. The studied fungi originated from eleven African countries and were mainly endophytic fungi and higher fungi (macrofungi). Their investigation led to the isolation of five hundred and three (503) compounds with polyketides representing the main class of secondary metabolites. The compounds exhibited varied biological activities with antibacterial and antiproliferative properties being the most prominent.

Biosynthesis of Atypical Angucyclines Unveils New Ring Rearrangement Reactions Catalyzed by Flavoprotein Monooxygenases.

Six new angucycline structures, including spirocyclione A (1), which contains an unusual oxaspiro[5.5]undecane architecture, and its ring-A-cleaved product spirocyclione B (2), were discovered by heterologous expression of a type II polyketide biosynthetic gene cluster captured from a marine actinomycete strain Streptomyces sp. HDN155000. Three flavoprotein monooxygenases are confirmed to be responsible for the oxidative carbon skeleton rearrangements in the biosynthesis of compounds 1 and 2. The obtained compounds showed promising cytotoxicity against different types of cancer cells.

Recent progresses in the cyclization and oxidation of polyketide biosynthesis.

Polyketides represent an important class of natural products, renowned for their intricate structures and diverse biological activities. In contrast to common fatty acids, polyketides possess relatively more rigid carbon skeletons, more complex ring systems, and chiral centers. These structural features are primarily achieved through distinctive enzymatic cyclizations and oxidations as tailoring steps. In this opinion, we discuss the recent progress in deciphering the mechanisms of cyclization and oxidation within polyketide biosynthesis. By shedding light on these enzymatic processes, this article seeks to motivate the community to unravel the remaining mysteries surrounding cyclase and oxidase functionalities and to explore novel polyketide natural products through genome mining.

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. The recently updated module boundary has been successful for engineering short synthases, yet larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases to test every module combination of the pikromycin synthase. Anticipated products are detected from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. We determine ketosynthase gatekeeping and module-skipping are the principal impediments to obtaining functional synthases. The platform is also employed to construct active hybrid synthases by incorporating modules from the erythromycin, spinosyn, and rapamycin assembly lines. The relaxed gatekeeping of a ketosynthase in the rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

Cladoic Acid, an Anti-Trypanosoma cruzi Polyketide from Cladosporium sp. TP-F2020.

A new polyketide, cladoic acid, was isolated from a fungus of the genus Cladosporium. The structure of the highly oxygenated trans-decalin ring with an all-E triene side chain was elucidated by extensive spectroscopic analysis. The unique chair/twist-boat conformation of the trans-decalin core and the flexibility of the B-ring were demonstrated by computer-aided conformational analysis. Cladoic acid was active against Trypanosoma cruzi and inhibited the proliferation of amastigotes and epimastigotes with IC50 values of 27 and 46 µM, respectively, but it did not show any appreciable activity against P388 murine leukemia cells, bacteria, or fungi, indicating it is a potential candidate for drug development against Chagas disease.

MFS Transporter as the Molecular Switch Unlocking the Production of Cage-Like Acresorbicillinol C.

Sorbicillinoids are a class of fungal polyketides with diverse structures and distinguished bioactivities. Although remarkable progress has been achieved in their chemistry and biosynthesis, the efflux of sorbicillinoids is poorly understood. Here, we found MFS transporter AcsorT was responsible for the biosynthesis of sorbicillinoids in Acremonium chrysogenum. Combinatorial knockout and subcellular location demonstrated that the plasma membrane-associated AcsorT was responsible for the transportation of sorbicillinol and subsequent formation of oxosorbicillinol and acresorbicillinol C via the berberine bridge enzyme-like oxidase AcsorD in the periplasm. Homology modeling and site-directed mutation revealed that Tyr303 and Arg436 were the key residues of AcsorT, which was further explained by molecular dynamics simulation. Based on our study, it was suggested that AcsorT modulates sorbicillinoid production by coordinating its biosynthesis and export, and a transport model of sorbicillinoids was proposed in A. chrysogenum.

Herbidomicins, two pairs of polyketide tautomers produced by an actinomycete of the genus Herbidospora.

Herbidospora is one of the underexplored actinomycete genera from which only a limited number of secondary metabolites are reported. In our continuing investigation on less explored actinomycetes, a liquid culture of Herbidospora sp. RD 11066 was found to contain unknown metabolites that had no match in our in-house UV database. Chromatographic separation and following structural analysis using NMR and MS identified these metabolites to be chromanone and chromene derivatives, which were respectively composed of an inseparable mixture of two isomeric forms. The former polyketides, designated to be herbidomicins A1 (1) and A2 (2), are positional isomers in terms of a methyl substituent on an aromatic ring that mutually interconvert by acetal exchange by two phenolic hydroxy groups. The latter pair, herbidomicins B1 (3) and B2 (4), is Z/E-isomers regarding an enol ether double bond. Herbidomicins 1-4 were weakly antifungal against a dermatophytic fungus Trichophyton rubrum and were moderately cytotoxic against murine leukemia P388 cells.

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses.

The type 1 polyketide synthase (PKS) assembly line uses its modular structure to produce polyketide natural products that form the basis of many pharmaceuticals. Currently, several cryoelectron microscopy (cryo-EM) structures of a multidomain PKS module have been constructed, but much remains to be learned. Here we utilize ion-mobility mass spectrometry (IM-MS) to record size and shape information and detect different conformational states of a 207 kDa didomain dimer comprised of ketosynthase (KS) and acyl transferase (AT), excised from full-length module. Furthermore, gas-phase stability differences between these different conformations are captured by collision induced unfolding (CIU) technology. Additionally, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time.

New Zosteropenillines and Pallidopenillines from the Seagrass-Derived Fungus Penicillium yezoense KMM 4679.

Ten new decalin polyketides, zosteropenilline M (1), 11-epi-8-hydroxyzosteropenilline M (2), zosteropenilline N (3), 8-hydroxyzosteropenilline G (4), zosteropenilline O (5), zosteropenilline P (6), zosteropenilline Q (7), 13-dehydroxypallidopenilline A (8), zosteropenilline R (9) and zosteropenilline S (10), together with known zosteropenillines G (11) and J (12), pallidopenilline A (13) and 1-acetylpallidopenilline A (14), were isolated from the ethyl acetate extract of the fungus Penicillium yezoense KMM 4679 associated with the seagrass Zostera marina. The structures of isolated compounds were established based on spectroscopic methods. The absolute configurations of zosteropenilline Q (7) and zosteropenilline S (10) were determined using a combination of the modified Mosher's method and ROESY data. The absolute configurations of zosteropenilline M (1) and zosteropenilline N (3) were determined using time-dependent density functional theory (TD-DFT) calculations of the ECD spectra. A biogenetic pathway for compounds 1-14 is proposed. The antimicrobial, cytotoxic and cytoprotective activities of the isolated compounds were also studied. The significant cytoprotective effects of the new zosteropenilline M and zosteropenillines O and R were found in a cobalt chloride (II) mimic in in vitro hypoxia in HEK-293 cells. 1-Acetylpallidopenilline A (14) exhibited high inhibition of human breast cancer MCF-7 cell colony formation with IC50 of 0.66 µM and its anticancer effect was reduced when MCF-7 cells were pretreated with 4-hydroxitamoxifen. Thus, we propose 1-acetylpallidopenilline A as a new xenoestrogen with significant activity against breast cancer.