Utilization of Aspergillus niger for the fermentative production of azaphilone dye in YEPB medium.

The current research focuses on the production and optimization of a natural yellowish-brown Azaphilone dye using Aspergillus niger. A variety of culture media were tested to ascertain the best conditions for dye synthesis. The formation of the yellowish-brown dye was confirmed by a color shift in the reaction mixture, and UV-Vis spectroscopy detected the dye at 450 nm. Static conditions were found to be more favorable than shaking for higher dye yields, and fed-batch fermentation was more effective than batch fermentation. Maximum dye production was achieved after 28 days of incubation. Factors such as temperature, pH, and inoculum percentage were shown to influence dye synthesis, with the highest production (2.5 ml) occurring at 30 °C, pH 7, and a 3% spore suspension in yeast extract peptone broth (YEPB) medium under static conditions. Gas chromatography-mass spectrometry (GC-MS) analysis validated the presence of Azaphilone dye in the culture filtrate. The dye was successfully applied to a pretreated cotton cloth. These findings advance our understanding of optimizing fungal dye production for sustainable and eco-friendly textile coloration applications. This study appears to be the first of its kind to report azaphilone dye production by A. niger in the YEPB medium.

Pleiotropically activation of azaphilone biosynthesis by overexpressing a pathway-specific transcription factor in marine-derived Aspergillus terreus RA2905.

The genome sequencing of Aspergillus terreus reveals that the vast number of predicted biosynthetic gene clusters have not reflected by the metabolic profile observed under conventional culture conditions. In this study, a silent azaphilone biosynthetic gene cluster was activated by overexpressing a pathway-specific transcription factor gene2642 in marine-derived fungus A. terreus RA2905. Consequently, twenty azaphilone compounds were identified from the OE2642 mutant, including 11 new azaphilones and their precursors, azasperones C-J (1-5, 7-9) and preazasperones A-C (15-17). The structures of those new compounds were unambiguously determined on the basis of NMR and HRESIMS spectra analysis, and the absolute configurations were established depending on ECD calculations. Compounds 1 and 2 were the rarely reported naturally occurring azaphilones with 2-N coupled phenyl-derivative. The bioactivity assay revealed that compounds 18-20 exhibited significant anti-inflammatory activity. Based on the occurrence of diverse intermediates and the putative gene functions, a plausible biosynthetic pathway of these compounds was proposed. The above results demonstrated that overexpression of the pathway-specific transcription factor presents a promising approach for enriching fungal secondary metabolites and accelerating the targeted discovery of novel biosynthetic products.

Activation of a Silent Gene Cluster from the Endophytic Fungus Talaromyces sp. Unearths Cryptic Azaphilone Metabolites.

Fungal azaphilones have attracted widespread attention due to their significant potential as sources of food pigments and pharmaceuticals. Genome mining and gene cluster activation represent powerful tools and strategies for discovering novel natural products and bioactive molecules. Here, a putative azaphilone biosynthetic gene cluster lut from the endophytic fungus Talaromyces sp. was identified through genome mining. By overexpressing the pathway-specific transcription factor LutB, five new sclerotiorin-type azaphilones (1, 6, 8, and 10-11) together with seven known analogues (2-5, 7, 9, 12) were successfully produced. Compounds 8 and 9 exhibited antibacterial activity against Bacillus subtilis with MIC values of 64 and 16 µg/mL, respectively. Compound 11 showed cytotoxic activity against HCT116 and GES-1 with IC50 values of 10.9 and 4.9 µM, respectively, while 1, 4, 5, and 7-10 showed no obvious cytotoxic activity. Gene inactivation experiments confirmed the role of the lut cluster in the production of compounds 1-12. Subsequent feeding experiments unveiled the novel functional diversity of the dual megasynthase system. Furthermore, a LutC-LutD binary oxidoreductase system was discovered, and in combination with DFT calculations, the basic biosynthetic pathway of the sclerotiorin-type azaphilones was characterized. This study provided a good example for the discovery of new azaphilones and further uncovered the biosynthesis of these compounds.

Penicilazaphilone C alleviates allergic airway inflammation and improves the immune microenvironment by hindering the NLRP3 inflammasome.

AIMS: Penicilazaphilone C (PAC) is hypothesized to potentially serve as a therapeutic treatment for allergic airway inflammation by inhibiting the NLRP3 inflammasome and reducing oxidative stress. METHODS: An allergic asthma model was induced in female BALB/c mice of the OVA, OVA+PAC, OVA+PAC+LPS, and OVA+Dex groups by sensitizing and subsequently challenging them with OVA. The OVA+PAC and Normal+PAC groups were treated with PAC, while the OVA+PAC+LPS group also received LPS. The OVA+Dex group was given dexamethasone (Dex). Samples of serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected for histological and cytological analysis. RESULTS: Allergic mice treated with PAC or Dex showed inhibited inflammation and mucus production in the lungs. There was a decrease in the number of inflammatory cells in the BALF, lower levels of inflammatory cytokines in the serum and BALF, and a reduction in the protein expression of NLRP3, ASC, cleaved caspase-1, IL-1ß, activated gasdermin D, MPO, Ly6G, and ICAM-1. Additionally, oxidative stress was reduced, as shown by a decrease in MDA and DCF, but an increase in SOD and GSH. Treatment with PAC also resulted in a decrease in pulmonary memory CD4+ T cells and an increase in regulatory T cells. However, the positive effects seen in the PAC-treated mice were reversed when the NLRP3 inflammasome was activated by LPS, almost returning to the levels of the Sham-treated mice. SIGNIFICANCE: PAC acts in a similar way to anti-allergic inflammation as Dex, suggesting it may be a viable therapeutic option for managing allergic asthma inflammation.

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.

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

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.

Terminal carboxylation of branched carbon chain contributing to acidic stability of azaphilone pigments from a new isolate of Talaromyces amestolkiae.

Red Monascus pigments, a series of natural azaphilone alkaloids, have been utilized in China as a traditional food colorant for over 1000 years. However, instability under an acidic condition is its drawback. A new strain of Talaromyces amestolkiae was isolated in the present work, which produced the azaphilone talaromycorubrin and the corresponding azaphilone alkaloid (N-MSG-talaromycorubramine) exhibiting good stability even at pH below 3. The azaphilone alkaloid with acidic stability, an alternative of Chinese traditional red Monascus pigments, is potential for application as natural food colorant in acidic foods. The acidic stability of azaphilone alkaloid also benefits for direct fermentation of N-MSG-talaromycorubramine under a low pH condition. More importantly, correlation relationship between the terminal carboxylation of branched carbon chain of azaphilone and the stability of azaphilone alkaloids under an acidic condition is set up for the first time, which makes designing other acidic stable azaphilone alkaloids via genetic engineering become possible.

The screening for marine fungal strains with high potential in alkaloids production by in situ colony assay and LC-MS/MS based secondary metabolic profiling.

Background: Alkaloids are the second primary class of secondary metabolites (SMs) from marine organisms, most of which have antioxidant, antitumor, antibacterial, anti-inflammatory, and other activities. However, the SMs obtained by traditional isolation strategies have drawbacks such as highly reduplication and weak bioactivity. Therefore, it is significantly important to establish an efficient strategy for screening strains and mining novel compounds. Methods: In this study, we utilized in situ colony assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the strain with high potential in alkaloids production. The strain was identified by genetic marker genes and morphological analysis. The secondary metabolites from the strain were isolated by the combine use of vacuum liquid chromatography (VLC), ODS column chromatography, and Sephadex LH-20. Their structures were elucidated by 1D/2D NMR, HR-ESI-MS, and other spectroscopic technologies. Finally, these compounds bioactivity were assay, including anti-inflammatory and anti-ß aggregation. Results: Eighteen marine fungi were preliminarily screened for alkaloids production by in situ colony assay using Dragendorff reagent as dye, and nine of them turned orange, which indicated abundant alkaloids. By thin-layer chromatography (TLC), LC-MS/MS, and multiple approaches assisted Feature-Based Molecular Networking (FBMN) analysis of fermentation extracts, a strain ACD-5 (Penicillium mallochii with GenBank accession number OM368350) from sea cucumber gut was selected for its diverse alkaloids profiles especially azaphilones. In bioassays, the crude extracts of ACD-5 in Czapek-dox broth and brown rice medium showed moderate antioxidant, acetylcholinesterase inhibitory, anti-neuroinflammatory, and anti-ß aggregation activities. Three chlorinated azaphilone alkaloids, compounds 1-3 (sclerotioramine, isochromophilone VI, and isochromophilone IX, respectively), were isolated from the fermentation products of ACD-5 in brown rice medium guided by bioactivities and mass spectrometry analysis. Compound 1 had shown remarkable anti-neuroinflammatory activity in liposaccharide induced BV-2 cells. Conclusion: In summary, in situ colony screening together with LC-MS/MS, multi-approach assisted FBMN can act as an efficient screening method for strains with potential in alkaloids production.

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.

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.

Azaphilones produced by Penicillium maximae with their cell death-inducing activity on Adriamycin-treated cancer cell.

BACKGROUND: Heat shock proteins (Hsps) are overexpressed in several tumors and contribute to cell proliferation, metastasis, and anticancer drug resistance. Therefore, Hsp inhibitors have enhanced cytotoxicity as chemotherapeutic agents and may be effective with a reduced dosage for tumor therapy to avoid side effects. RESULTS: Four new azaphilones, maximazaphilones I-IV (1-4), and three known compounds (5-7) have been isolated from the airborne-derived fungus Penicillium maximae. Inhibitory effects of isolated compounds against induction of Hsp105 were evaluated by the luciferase assay system using Hsp105 promoter. In this assay, 2-4, 6, and 7 significantly inhibited hsp105 promoter activity without cytotoxicity. In addition, all isolated compounds except for 5 significantly induced the death of Adriamycin (ADR)-treated HeLa cells. Interestingly, 1-4, 6, and 7 didn't show anti-proliferative and cell death-inducing activity without ADR. CONCLUSION: This study revealed the chemical structures of maximazaphilones I-IV (1-4) and the potency of azaphilones may be useful for cancer treatment and reducing the dose of anticancer agents. In addition, one of the mechanisms of cell death-inducing activity for 2-4, 6, and 7 was suggested to be inhibitory effects of Hsp105 expression.

Absolute configuration of azaphilones from Monascus kaoliang KB9 and solvent effects on their keto and enol forms.

Monascus fermented rice, also known as red yeast rice, exhibits a broad spectrum of biological activities due to its chemical constituents, such as monacolins and azaphilone pigments. Here, we cultured Monascus kaoliang KB9 in a liquid malt medium instead of on rice as a carbon source. Eleven known compounds (1-11) containing azaphilones and their early intermediate were isolated and identified. However, this was the first time that angular tricyclic azaphilones, monasfluols A (4) and B (7), acetyl-monasfluol A (5) and monasfluore A (6), were isolated from this species. Interestingly, all isolated tricyclic azaphilones existed exclusively in enol form in CD3OD, as evidenced by NMR spectroscopy. The absolute configuration of compounds 4-7 was also first experimentally identified based on ECD spectroscopy combined with conformational analyses using computational techniques. The assigned stereochemistry of Monascus azaphilones in this work provides essential structural information that will benefit future biological and pharmaceutical investigations.

Induction of Secondary Metabolite Biosynthesis by Deleting the Histone Deacetylase HdaA in the Marine-Derived Fungus Aspergillus terreus RA2905.

Aspergillus terreus is well-known for its ability to biosynthesize valuable pharmaceuticals as well as structurally unique secondary metabolites. However, numerous promising cryptic secondary metabolites in this strain regulated by silent gene clusters remain unidentified. In this study, to further explore the secondary metabolite potential of A. terreus, the essential histone deacetylase hdaA gene was deleted in the marine-derived A. terreus RA2905. The results showed that HdaA plays a vital and negative regulatory role in both conidiation and secondary metabolism. Loss of HdaA in A. terreus RA2905 not only resulted in the improvement in butyrolactone production, but also activated the biosynthesis of new azaphilone derivatives. After scaled fermentation, two new azaphilones, asperterilones A and B (1 and 2), were isolated from ΔhdaA mutant. The planar structures of compounds 1 and 2 were undoubtedly characterized by NMR spectroscopy and mass spectrometry analysis. Their absolute configurations were assigned by circular dichroism spectra analysis and proposed biosynthesis pathway. Compounds 1 and 2 displayed moderate anti-Candida activities with the MIC values ranging from 18.0 to 47.9 µM, and compound 1 exhibited significant cytotoxic activity against human breast cancer cell line MDA-MB-231. This study provides novel evidence that hdaA plays essential and global roles in repressing secondary metabolite gene expression in fungi, and its deletion represents an efficient strategy to mine new compounds from A. terreus and other available marine-derived fungi.

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.

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.

Back to nature, microbial production of pigments and colorants for food use.

Pigments-producing microorganisms are quite common in Nature. However, there is a long journey from the Petri dish to the market place. Twenty-five years ago, scientists wondered if such productions would remain a scientific oddity or become an industrial reality. The answer is not straightforward as processes using fungi, bacteria or yeasts can now indeed provide carotenoids or phycocyanin at an industrial level. Another production factor to consider is peculiar as Monascus red colored food is consumed by more than one billion Asian people; however, still banned in many other countries. European and American consumers will follow as soon as "100%-guaranteed" toxin-free strains (molecular engineered strains, citrinin gene deleted strains) will be developed and commercialized at a world level. For other pigmented biomolecules, some laboratories and companies invested and continue to invest a lot of money as any combination of new source and/or new pigment requires a lot of experimental work, process optimization, toxicological studies, and regulatory approval. Time will tell whether investments in pigments such as azaphilones or anthraquinones were justified. Future trends involve combinatorial engineering, gene knock-out, and the production of niche pigments not found in plants such as C50 carotenoids or aryl carotenoids.

Inactivation of MrSir2 in Monascus ruber Influenced the Developmental Process and the Production of Monascus Azaphilone Pigments.

Monascus species are the producers of Monascus azaphilone pigments (MonAzPs) and lipid-lowering component Monacolin K, which have been widely used as food colorant and health products. In this study, silent information regulator 2 (Sir2) homolog (MrSir2) was characterized, and its impacts on the development and MonAzPs production of Monascus ruber were evaluated. Enzyme activity test in vitro showed that MrSir2 was an NAD+-dependent histone deacetylase. Compared to WT, Δmrsir2 strain accumulated more acetylated lysine residues of histone H3 subunit during its vegetative growth phase, and it exhibited accelerated mycelial aging, more spores, increased resistance to oxidative stress, and more MonAzPs production. RNA-Seq-based transcriptome analysis revealed that MrSir2 mainly regulated the gene expression in macromolecular metabolism such as carbohydrates, proteins, and nucleotides, as well as genes encoding cell wall synthesis and cell membrane component, indicating that MrSir2 probably facilitates the metabolic transition from the primary growth phase to the mycelial aging. Taken together, MrSir2 mainly targets H3 subunit at the vegetative growth phase and affects the development of M. ruber and MonAzPs production.

Formation of Azaphilone Pigments and Monasnicotinic Acid by the Fungus Aspergillus cavernicola.

Due to the ever-increasing demand for healthy and safe food, much attention has been gained by natural food colorants. This study showed the culture fluid extract of the fungus Aspergillus cavernicola VKM F-906 to contain red pigment and monasnicotinic acid (MNA) in predominant amounts. The structure of the pigment corresponded to cis-cavernamine (red pigment, RP). Two tautomers, NH and OH forms, in rapid equilibrium were present in a solution of RP. The critical factors for RP to form were the presence of NH4+ salt and pH 6.3-6.5. In vitro experiments showed that MNA was synthesized from RP as a result of chemical transformations without the participation of enzymes. In this case, the main influence on the reaction rate is exerted by the pH of the medium, which is associated with the keto-enol tautomerism of RP in solution. The culture broth extract and MNA exhibited antifungal activity against Fusarium fungi.