Azaphilone pigments from the marine-derived Penicillium sclerotium UJNMF 0503 and their neuroprotective potential against H2O2-induced cell apoptosis through modulating PI3K/Akt pathway.

Azaphilones represent a particular group of fascinating pigments from fungal source, with easier industrialization and lower cost than the traditional plant-derived pigments, and they also display a wide range of pharmacological activities. Herein, 28 azaphilone analogs, including 12 new ones, were obtained from the fermentation culture of a marine fungus Penicillium sclerotium UJNMF 0503. Their structures were elucidated by MS, NMR and ECD analyses, together with NMR and ECD calculations and biogenetic considerations. Among them, compounds 1 and 2 feature an unusual natural benzo[d][1,3]dioxepine ring embedded with an orthoformate unit, while 3 and 4 represent the first azaphilone examples incorporating a novel rearranged 5/6 bicyclic core and a tetrahydropyran ring on the side chain, respectively. Our bioassays revealed that half of the isolates exhibited neuroprotective potential against H2O2-induced injury on RSC96 cells, while compound 13 displayed the best rescuing capacity toward the cell viability by blocking cellular apoptosis, which was likely achieved by upregulating the PI3K/Akt signaling pathway.

Isolation and Characterization of Antimicrobial Metabolites from the Sophora tonkinensis-Associated Fungus Penicillium sp. GDGJ-N37.

Chemical investigation of Penicillium sp. GDGJ-N37, a Sophora tonkinensis-associated fungus, yielded two new azaphilone derivatives, N-isoamylsclerotiorinamine (1) and 7-methoxyl-N-isoamylsclerotiorinamine (2), and four known azaphilones (3-6), together with two new chromone derivatives, penithochromones X and Y (7 and 8). Their structures were elucidated based on spectroscopic data, CD spectrum, and semi-synthesis. Sclerotioramine (3) showed significant antibacterial activities against B. subtilis and S. dysentery, and it also showed most potent anti-plant pathogenic fungi activities against P. theae, C. miyabeanus, and E. turcicum.

New Hybrid Phenalenone Dimer, Highly Conjugated Dihydroxylated C28 Steroid and Azaphilone from the Culture Extract of a Marine Sponge-Associated Fungus, Talaromyces pinophilus KUFA 1767.

An undescribed hybrid phenalenone dimer, talaropinophilone (3), an unreported azaphilone, 7-epi-pinazaphilone B (4), an unreported phthalide dimer, talaropinophilide (6), and an undescribed 9R,15S-dihydroxy-ergosta-4,6,8 (14)-tetraen-3-one (7) were isolated together with the previously reported bacillisporins A (1) and B (2), an azaphilone derivative, Sch 1385568 (5), 1-deoxyrubralactone (8), acetylquestinol (9), piniterpenoid D (10) and 3,5-dihydroxy-4-methylphthalaldehydic acid (11) from the ethyl acetate extract of the culture of a marine sponge-derived fungus, Talaromyces pinophilus KUFA 1767. The structures of the undescribed compounds were elucidated by 1D and 2D NMR as well as high-resolution mass spectral analyses. The absolute configuration of C-9' of 1 and 2 was revised to be 9'S using the coupling constant value between C-8' and C-9' and was confirmed by ROESY correlations in the case of 2. The absolute configurations of the stereogenic carbons in 7 and 8 were established by X-ray crystallographic analysis. Compounds 1,2, 4-8, 10 and 11 were tested for antibacterial activity against four reference strains, viz. two Gram-positive (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212) and two Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), as well as three multidrug-resistant strains, viz. an extended-spectrum ß-lactamase (ESBL)-producing E. coli, a methicillin-resistant S. aureus (MRSA) and a vancomycin-resistant E. faecalis (VRE). However, only 1 and 2 exhibited significant antibacterial activity against both S. aureus ATCC 29213 and MRSA. Moreover, 1 and 2 also significantly inhibited biofilm formation in S. aureus ATCC 29213 at both MIC and 2xMIC concentrations.

Two New Species of Talaromyces Sect. Trachyspermi Discovered in China.

Two new species of sect. Trachyspermi isolated from soil are proposed, namely, T. albidus (ex-type AS3.26143T) and T. rubidus (ex-type AS3.26142T), based on the integrated taxonomic methods. Morphologically, T. albidus is characterized by slow growth, white gymnothecia, singly-borne asci and ellipsoidal echinulate ascospores. Talaromyces rubidus is distinguished by restricted growth, moderate to abundant red soluble pigment on CYA and YES, biverticillate penicilli, and  commonly ovoid to globose echinulate conidia. The two proposed novelties are further confirmed by the phylogenetic analyses of the concatenated BenA-CaM-Rpb2-ITS sequence matrix and the individual BenA, CaM, Rpb2 and ITS sequence matrices. Talaromyces albidus is closely related to T. assiutensis and T. trachyspermus, while T. rubidus is in the clade containing T. albobiverticillius, T. rubrifaciens, T. catalonicus, T. heiheensis, T. erythromellis, T. halophytorum, T. pernambucoensis, T. solicola and T. aerius.

Penazaphilones J-L, Three New Hydrophilic Azaphilone Pigments from Penicillium sclerotiorum cib-411 and Their Anti-Inflammatory Activity.

Penazaphilones J-L (1-3), three new hydrophilic azaphilone pigments, as well as six known compounds, were discovered from the filamentous fungus Penicillium sclerotiorum cib-411. Compounds 1-3 were structurally elucidated by the detailed interpretation of their 1D and 2D NMR spectroscopic data. Compound 1 is an unprecedented hybrid of an azaphilone and a glycerophosphate choline. Compounds 2 and 3 each contain an intact amino acid moiety. The bioassay showed that compound 3 exhibited significant anti-inflammatory activity. Concretely, compound 3 significantly suppressed the NO production, the expression levels of COX-2, IL-6, IL-1ß, and iNOS mRNA in LPS-stimulated RAW264.7 cells. Moreover, treatment of compound 3 prevented the translocation of NF-κB through inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3ß. Thus, the inhibition of compound 3 against LPS-induced inflammation should rely on its inactivation on NF-κB.

Characterization of a silent azaphilone biosynthesis gene cluster in Aspergillus terreus NIH 2624.

Filamentous fungal secondary metabolites are an important source of bioactive components. Genome sequencing ofAspergillus terreusrevealed many silent secondary metabolite biosynthetic gene clusters presumed to be involved in producing secondary metabolites. Activation of silent gene clusters through overexpressing a pathway-specific regulator is an effective avenue for discovering novel fungal secondary metabolites. Replacement of the native promoter of the pathway-specific activator with the inducible Tet-on system to activate thetazpathway led to the discovery of a series of azaphilone secondary metabolites, among which azaterrilone A (1) was purified and identified for the first time. Genetic deletion of core PKS genes and transcriptional analysis further characterized thetazgene cluster to consist of 16 genes with the NR-PKS and the HR-PKS collaborating in a convergent mode. Based on the putative gene functions and the characterized compounds structural information, a biosynthetic pathway of azaterrilone A (1) was proposed.

Azaphilone alkaloids: prospective source of natural food pigments.

Azaphilone, biosynthesized by polyketide synthase, is a class of fungal metabolites. In this review, after brief introduction of the natural azaphilone diversity, we in detail discussed azaphilic addition reaction involving conversion of natural azaphilone into the corresponding azaphilone alkaloid. Then, setting red Monascus pigments (a traditional food colorant in China) as example, we presented a new strategy, i.e., interfacing azaphilic addition reaction with living microbial metabolism in a one-pot process, to produce azaphilone alkaloid with a specified amine residue (red Monascus pigments) during submerged culture. Benefit from the red Monascus pigments with a specified amine residue, the influence of primary amine on characteristics of the food colorant was highlighted. Finally, the progress for screening of alternative azaphilone alkaloids (production from interfacing azaphilic addition reaction with submerged culture of Talaromyces sp. or Penicillium sp.) as natural food colorant was reviewed. KEY POINTS: • Azaphilic addition reaction of natural azaphilone is biocompatible • Red Monascus pigment is a classic example of azaphilone alkaloids • Azaphilone alkaloids are alterative natural food colorant.

Dynamic regulation of Monascus azaphilones biosynthesis by the binary MrPigE-MrPigF oxidoreductase system.

Monascus azaphilones (MAs) have been extensively applied as natural food coloring agents. MAs are classified into three categories: yellow MAs (YMAs), orange MAs (OMAs), and red MAs with various biological activities. However, the exact biosynthetic mechanism of OMAs and YMAs are not thoroughly elucidated. Firstly, we identified four DNA-binding residues of transcription factor MrPigB and constructed a multi-site saturation mutagenesis library of MrPigB. Then, comparative metabolite and gene expression of the mutants revealed that two oxidoreductases MrPigE and MrPigF were responsible for the formation of YMAs and OMAs. Finally, the in vitro and in vivo assays demonstrated the opposite roles of MrPigE and MrPigF in conversion of OMAs to YMAs. To our knowledge, this is the first report of a binary oxidoreductase system for dynamic regulation of fungal secondary metabolite biosynthesis. Broadly, our work also demonstrates the transcription factor engineering strategy for elucidating the biosynthetic pathway of secondary metabolite. KEY POINTS: • MrPigE converts orange Monascus azaphilones to yellow Monascus azaphilones • MrPigF oxidizes intermediates to afford orange Monascus azaphilones • MrPigE and MrPigF constitute a binary system in Monascus azaphilones biosynthesis.

Azaphilones from the Endophyte Diaporthe sp. and Their Toxicity.

Chemotherapy and targeted therapies are increasingly used as conventional means to control tumor growth and prolong survival. Patient treated with anti-neoplastic agents experience severe side effects, especially those cytotoxic chemotherapies. Exploring chemo agents with less side effects is the hot spot of anticancer research. In this study, three azaphilone derivatives (chaetoviridin A (1), chaetoviridin E (2) and chaetomugilin D (3)) were isolated from the endophyte of the plant Anoectochilus roxburghii (Wall.) Lindl, their structures were elucidated by NMR. The toxicity of these compounds was evaluated by zebrafish model. The results show that these compounds had no toxicity against zebrafish. These compounds may act as safe anticancer drug leads according to this result. These three azaphilone derivatives were first time reported isolated from Diaporthe species which mainly used to isolate from Chaetomium species.

Core Steps to the Azaphilone Family of Fungal Natural Products.

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.

Cytochalasans and azaphilones: suitable chemotaxonomic markers for the Chaetomium species.

The accurate taxonomic concept of the fungal Chaetomium species has been a hard work due to morphological similarity. Chemotaxonomy based on secondary metabolites is a powerful tool for taxonomical purposes, which could be used as an auxiliary reference to solve the problems encountered in the classification of Chaetomium. Among secondary metabolites produced by Chaetomium, cytochalasans and azaphilones exhibited a pattern of distribution and frequency of occurrence that establish them as chemotaxonomic markers for the Chaetomium species. This review attempted to elucidate the composition of the Chaetomium species and its relationship with classical taxonomy by summarizing the pattern of cytochalasans and azaphilones distribution and biosynthesis in the Chaetomium species. KEY POINTS: • Secondary metabolites from the genus Chaetomium are summarized. • Cytochalasans and azaphilones could be characteristic metabolites of the Chaetomium species. • Cytochalasans and azaphilones could be used to analyze for taxonomical purposes.

Anti-Inflammatory Azaphilones from the Edible Alga-Derived Fungus Penicillium sclerotiorum.

To discover the new medical entity from edible marine algae, our continuously natural product investigation focused on endophytes from marine macroalgae Grateloupia sp. Two new azaphilones, 8a-epi-hypocrellone A (1), 8a-epi-eupenicilazaphilone C (2), together with five known azaphilones, hypocrellone A (3), eupenicilazaphilone C (4), ((1E,3E)-3,5-dimethylhepta-1,3-dien-1-yl)-2,4-dihydroxy-3-methylbenzaldehyde (5), sclerotiorin (6), and isochromophilone IV (7) were isolated from the alga-derived fungus Penicillium sclerotiorum. The structures of isolated azaphilones (1-7) were elucidated by spectrometric identification, especially HRESIMS, CD, and NMR data analyses. Concerning bioactivity, cytotoxic, anti-inflammatory, and anti-fibrosis activities of those isolates were evaluated. As a result, compound 1 showed selective toxicity toward neuroblastoma cell line SH-SY5Y among seven cancer and one fibroblast cell lines. 20 µM of compounds 1, 3, and 7 inhibited the TNF-α-induced NFκB phosphorylation but did not change the NFκB activity. Compounds 2 and 6 respectively promoted and inhibited SMAD-mediated transcriptional activities stimulated by TGF-ß.

Azaphilones and Meroterpenoids from the Soft Coral-Derived Fungus Penicillium glabrum glmu003.

Two new azaphilone compounds, daldinins G (1) and H (2), together with nine known compounds daldinin D (3), sargassopenilline B (4), austalide V (5), austalide K (6), austalide P (7), austalide P acid (8), austalide H (9), 13-O-deacetyaustalide I (10), and 17-O-demethylaustalide B (11), were isolated from the soft coral-derived fungus Penicillium glabrum glmu003. The new structures of 1 and 2 were elucidated on the basis of 1D and 2D NMR, mass spectra, and electronic circular dichroism (ECD) data analysis. Compound 5 showed weak inhibitory activity against pancreatic lipase (PL) with IC50 value of 23.9 µg/mL.

Two new azaphilone pigments from Talaromyces albobiverticillius and their anti-inflammatory activity.

Two new azaphilone pigments, talaralbols A and B (3 and 7), along with five known azaphilone metabolites (1, 2, and 4-6), were isolated from the culture of Talaromyces albobiverticillius associated with the isopod Armadillidium vulgare. Their structures were elucidated by a combination of 1 D and 2 D NMR data, ECD calculations, chemical transformations, and NMR data analogy with model compounds. Talaralbol A (3) showed a moderate inhibition on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells with the inhibitory rate being 31.0% at the concentration of 10 µM.[Formula: see text].

Enhancement of Monascus yellow pigments production by activating the cAMP signalling pathway in Monascus purpureus HJ11.

BACKGROUND: Monascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life. Moreover, MonAzPs recently have received more attention because of their diverse physiological activities. However, the high microbial production of MonAzPs is still not always guaranteed. Herein, the aim of this study was to develop an efficient biotechnological process for MonAzPs production. RESULTS: In this study, exogenous cyclic adenosine monophosphate (cAMP) treatment not only induced MonAzPs production, but also stimulated the expression of a cAMP phosphodiesterase gene, named as mrPDE, in M. purpureus HJ11. Subsequently, MrPDE was identified as a cAMP phosphodiesterase by in vitro enzymatic reaction with purified enzyme. Further, a gene knockout mutant of mrPDE was constructed to verify the activation of cAMP signalling pathway. Deletion of mrPDE in M. purpureus HJ11 improved cAMP concentration by 378% and enhanced PKA kinase activity 1.5-fold, indicating that activation of cAMP signalling pathway was achieved. The ΔmrPDE strain produced MonAzPs at 8563 U/g, with a 2.3-fold increase compared with the WT strain. Moreover, the NAPDH/NADP+ ratio of the ΔmrPDE strain was obviously higher than that of the wild type strain, which led to a higher proportion of yellow MonAzPs. With fed-batch fermentation of the ΔmrPDE strain, the production and yield of MonAzPs achieved 332.1 U/mL and 8739 U/g. CONCLUSIONS: A engineered M. purpureus strain for high MonAzPs production was successfully developed by activating the cAMP signalling pathway. This study not only describes a novel strategy for development of MonAzPs-producing strain, but also provides a roadmap for engineering efforts towards the production of secondary metabolism in other filamentous fungi.

Atrorosins: a new subgroup of Monascus pigments from Talaromyces atroroseus.

A new series of azaphilone pigments named atrorosins have been isolated from the filamentous fungus Talaromyces atroroseus. Atrorosins have a similar azaphilone scaffold as the orange Monascus pigment PP-O, with a carboxylic acid group at C-1, but are unique by their incorporation of amino acids into the isochromene system. Despite that the atrorosin precursor PP-O, during fermentation, was initially produced as two isomers (3:2, cis:trans ratio), the atrorosins were surprisingly almost exclusively (99.5%) produced as the cis-form, possibly due to steric interactions with the incorporated amino acid. When grown on complex media, a whole range of atrorosins is produced, whereas individual atrorosins can be produced selectively during fermentation by supplementing with the desired primary amine-containing compound.

Unique processes yielding pure azaphilones in Talaromyces atroroseus.

Azaphilones are a class of fungal pigments, reported mostly in association with Monascus species. In Asian countries, they are used as food colourants under the name of "red yeast rice" and their production process is well described. One major limitation of current production techniques of azaphilones is that they always occur in a mixture of yellow, orange and red pigments. These mixtures are difficult to control and to quantify. This study has established a controlled and reproducible cultivation protocol to selectively tailor production of individual pigments during a submerged fermentation using another fungal species capable of producing azaphilone pigments, Talaromyces atroroseus, using single amino acids as the sole nitrogen source. The produced azaphilone pigments are called atrorosins and are amino acid derivatives of the known azaphilone pigment Penicillium purpurogenum-orange (PP-O), with the amino acid used as nitrogen source incorporated into the core skeleton of the azaphilone. This strategy was successfully demonstrated using 18 proteinogenic amino acids and the non-proteinogenic amino acid ornithine. Two cultivation methods for production of the pure serine derivative (atrorosin S) have been further developed, with yields of 0.9 g/L being obtained. Yielding pure atrorosins through switching from KNO3 to single amino acids as nitrogen source allows for considerably easier downstream processing and thus further enhances the commercial relevance of azaphilone producing fungal cell factories.

Identification of a Unique Azaphilone Produced by Chaetomium globosum Isolated from Polygonatum sibiricum.

A new azaphilone, chaephilone E, eight azaphilone derivatives, and three chaetoglobosins were isolated from endophytic fungi Chaetomium globosum. The structures of the compounds were elucidated by 1D and 2D NMR as well as HR-ESI-MS data, and the absolute configuration of chaephilone E was established on the basis of electronic circular dichroism and NOESY spectrum. The activity of chaephilone E was evaluated via the cytotoxic assay (human hepatoma cell lines HepG-2) and brine shrimp (Artemia salina) bioassay.

New Glutamine-Containing Azaphilone Alkaloids from Deep-Sea-Derived Fungus Chaetomium globosum HDN151398.

Three new azaphilone alkaloids containing glutamine residues, namely N-glutarylchaetoviridins A-C (1-3), together with two related compounds (4 and 5) were isolated from the extract of Chaetomium globosum HDN151398, a fungus isolated from a deep-sea sediment sample collected in South China Sea. Their structures were elucidated on the basis of extensive 1D and 2D NMR as well as HRESIMS spectroscopic data and chemical analysis. N-glutarylchaetoviridins A-C (1-3) represent the first class of chaetoviridins characterized by embedded glutamate residues. Amino acids incubation experiments produced five azaphilone laden different amino acids residues (6-10) which indicated that this method can enhanced the structural diversity of this strain by culturing with amino acids. Cytotoxicity of the isolated compounds were evaluated against a panel of human cancer cell lines.

New Azaphilones from Nigrospora oryzae Co-Cultured with Beauveria bassiana.

In this study, the co-culture of Nigrospora oryzae and Beauveria bassiana, the endophytes in the seeds of Dendrobium officinale, were examined for metabolite diversity. Five new azaphilones were isolated, and their structures were determined by spectral analysis. In terms of azaphilones, compound 2 had an unprecedented skeleton, with a bicyclic oxygen bridge. The antifungal selectivities of the metabolite produced by N. oryzae against its co-culture fungus, B. bassiana, and common pathogens exhibited competitive interaction in this mix-culture. Compounds 1 and 2 showed obvious nitric oxide (NO) inhibitory activity with ratios of 37%, and 39%, respectively, at a concentration of 50 µM.