Discovery of New Antimicrobial Metabolites in the Coculture of Medicinal Mushrooms.

Bioactivity screening revealed that the antifungal activities of EtOAc extracts from coculture broths of Trametes versicolor SY630 with either Vanderbylia robiniophila SY341 or Ganoderma gibbosum SY1001 were significantly improved compared to that of monocultures. Activity-guided isolation led to the discovery of five aromatic compounds (1-5) from the coculture broth of T. versicolor SY630 and V. robiniophila SY341 and two sphingolipids (6 and 7) from the coculture broth of T. versicolor SY630 and G. gibbosum SY1001. Tramevandins A-C (1-3) and 17-ene-1-deoxyPS (6) are new compounds, while 1-deoxyPS (7) is a new natural product. Notably, compound 2 represents a novel scaffold, wherein the highly modified p-terphenyl bears a benzyl substituent. The absolute configurations of those new compounds were elucidated by X-ray diffraction, ECD calculations, and analysis of physicochemical constants. Compounds 1, 2, and 5-7 exhibited different degrees of antimicrobial activity, and the antifungal activities of compounds 6 and 7 against Candida albicans and Cryptococcus neoformans are comparable to those of fluconazole, nystatin, and sphingosine, respectively. Transcriptome analysis, propidium iodide staining, ergosterol quantification, and feeding assays showed that the isolated sphingolipids can extensively downregulate the late biosynthetic pathway of ergosterol in C. albicans, representing a promising mechanism to combat antibiotic-resistant fungi.

Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1.

Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C30 backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-ß-glucopyranosyl]-4,4'-diapolycopenedioc acid (1), a glycosylated C30 carotenoid. Furthermore, the genes related to the C30 carotenoid synthesis were investigated. Squalene, the precursor of the C30 carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1. By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4'-diapolycopene-4,4'-dioic acid to carotenoid 1. In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield.

Discovery of Novel Terpenoids from the Basidiomycete Pleurotus ostreatus through Genome Mining and Coculture Optimization.

In our previous work, postredienes A-C, three unusual linear sesterterpenes with high antifungal activities, were isolated from Pleurotus ostreatus SY10 when cocultured with Trametes robiniophila SY636. However, their titers were low, and exploration of newly biosynthesized trace analogues is required. Herein, genome mining analysis predicted that 17 gene clusters are involved in terpenoid biosynthesis in P. ostreatus. Thus, coculture conditions for strains SY10 and SY636 were optimized using a single-factor test and Box-Behnken design. As a result, the titers of postredienes A-C were increased by over 2.5-fold, reaching 1.28 to 8.40 mg/L. Moreover, five new terpenoids, named postredienes D-H (1-5), were successfully isolated. Compound 1 exhibited activities against the human pathogenic fungi Candida albicans and Cryptococcus neoformans comparable to those of amphotericin B. Compound 2 represents a novel sesterterpene with a five-membered ring at C-7. The absolute configurations of 1-5 were elucidated by making the methoxyphenylacetic acid esters and acetonide derivatives, combined with ECD and NMR calculation. Two potential gene clusters and relevant biosynthetic pathways for 1-5 were subsequently proposed based on real-time reverse transcription-quantitative PCR (RT-qPCR) analysis. The current study provides new insights into the research of terpenoid biosynthesis genes in P. ostreatus and other basidiomycetes.

Dothideomins A-D, Antibacterial Polycyclic Bisanthraquinones from the Endophytic Fungus Dothideomycetes sp. BMC-101.

Four new bisanthraquinones, dothideomins A-D (1-4), were identified from Dothideomycetes sp. BMC-101, an endophytic fungus isolated from Magnolia grandiflora L. leaves. Their chemical structures were established by NMR analysis, single-crystal X-ray crystallography, and ECD analysis. Dothideomins A-D (1-4) were characterized by an unusual 6/6/6/5/6/3/6/6 octocyclic scaffold (1 and 2) and a 6/6/6/5/6/6/6 heptacyclic scaffold (3 and 4), respectively. All compounds, especially 1 and 3, exhibited potent antibacterial activity with MIC values ranging from 0.4 to 0.8 µg/mL.

Secondary Metabolites Produced by Coculture of Pleurotus ostreatus SY10 and Pleurotus eryngii SY302.

A new isovaleryl-monoterpene, pleurotusin A (1), and a new cyclopentenone derivative, pleurotusin B (7), together with five related known terpenoids (2-6), were isolated from the coculture broth of two edible fungi, Pleurotus ostreatus SY10 and Pleurotus eryngii SY302. The absolute configurations of 1 and 7 were elucidated by comprehensively using the density functional theory calculation of NMR and ECD data, DP4+ probability analysis, Mo2 (OAc)4 -based CD experiment and optical rotation. The antimicrobial activities of these compounds except for the unstable compound 7, were evaluated against two types of human-pathogenic fungi, Candida albicans and Cryptococcus neoformans, and three types of human-pathogenic bacteria, Staphylococcus aureus, Escherichia coli, and Shigella sp. Compound 1 displayed moderate activity against S. aureus with an MIC50 value of 90.3 µM. In addition, the antioxidant activities of high-yielding 2-6 were tested using DPPH, and compound 4 showed moderate activity with an EC50 value of 573 µM.

Linear polyketides produced by co-culture of Penicillium crustosum and Penicillium fellutanum.

Two new polyketides, penifellutins A (1) and B (2), possessing a 22 carbon linear skeleton, were isolated from a co-culture of the deep-sea-derived fungi Penicillium crustosum PRB-2 and Penicillium fellutanum HDN14-323. Meanwhile, two esterification products of 1, penifellutins C (3) and D (4), were obtained because compound 1 could be esterified spontaneously when stored in methanol. Their configurations were difficult to determine because of chiral central crowdedness, structural flexibility and instability. As such, we solved this issue by comprehensively using Mo2(OAc)4-based CD experiments, density functional theory calculation of 13C NMR, DP4 + probability analysis and many chemical reactions, including making acetonide derivative, Mosher's method, PGME method, etc. Compounds 1 and 2 show obvious inhibitory activity on the liver hyperplasia of zebrafish larvae at a concentration of 10 µmol/L, while 3 and 4 show no activity, indicating that two carboxyls in the structure are important active sites. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-021-00125-8.

Coculture, An Efficient Biotechnology for Mining the Biosynthesis Potential of Macrofungi via Interspecies Interactions.

Macrofungi, which are also known as mushrooms, can produce various bioactive constituents and have become promising resources as lead drugs and foods rich in nutritional value. However, the production of these bioactive constituents under standard laboratory conditions is inefficiency due to the silent expression of their relevant genes. Coculture, as an important activation strategy that simulates the natural living conditions of macrofungi, can activate silent genes or clusters through interspecific interactions. Coculturing not only can trigger the biosynthesis of diverse secondary metabolites and enzymes of macrofungi, but is also useful for uncovering the mechanisms of fungal interspecific interactions and novel gene functions. In this paper, coculturing among macrofungi or between macrofungi and other microorganisms, the triggering and upregulation of secondary metabolites and enzymes, the potential medicinal applications, and the fungal-fungal interaction mechanisms are reviewed. Finally, future challenges and perspectives in further advancing coculture systems are discussed.

Identification of a novel non-ATP-competitive protein kinase inhibitor of PGK1 from marine nature products.

Phosphoglycerate kinase 1 (PGK1) acts as both a glycolytic enzyme and a protein kinase playing critical roles in cancer progression, thereby being regarded as an attractive therapeutic target for cancer treatment. However, no effective inhibitor of PGK1 has been reported. Here, we demonstrate that GQQ-792, a thiodiketopiperazine derivative from marine nature products, is a non-ATP-competitive inhibitor of PGK1 with the disulfide group within the structure of GQQ-792 as a key pharmacophore. The disulfide group of GQQ-792 binds to Cys379 and Cys380 of PGK1, resulting in occlusion of ATP from binding to PGK1. GQQ-792 treatment blocks hypoxic condition- and EGF stimulation-enhanced protein kinase activity of PGK1 that phosphorylates PDHK1 at T338 in glioblastoma cells; this treatment leads to decreased lactate production and glucose uptake, and subsequent apoptosis of glioblastoma cells. Animal studies reveal that GQQ-792 significantly inhibits the growth of tumor derived from glioblastoma cells. These findings underscore the potential of GQQ-792 as a promising anticancer agent and pave an avenue to further optimize the structure of GQQ-792 basing on its target molecule and pharmacophore in future.

Bupleuri Radix Prevents the Recurrences of Resected Colonic Polyps by Affecting Angiogenin-2-Induced Protein Kinase B/Akt Signaling.

AIM: We aimed to explore the effects of Bupleuri Radix (BR) on the recurrence of resected colonic polyp (CP) by measuring angiogenin-2-induced protein kinase B (Ang PKB)/Akt signaling. METHOD: The main ingredients of BR were extracted by using ethanol and measured by HPLC. One hundred twenty patients with CP >10 mm who underwent resected surgery were randomly allocated to an aspirin (AG) or a BR medicine (BG) group. The allocation ratio was 1 : 1 and the intervention duration was one year. The recurrence rate of resected CP was investigated and the plasma levels of Ang PKB/Akt and inflammatory cytokines were measured using ELISA kits. After one-year surgery, side effects were recorded. The relationship between the serum levels of the main compounds of BR and plasma levels of Ang PKB/Akt was analyzed. RESULTS: The main ingredients of CP are paeoniflorin, baicalin, saikosaponin A, and bupleurum saponin B2. Recurrence of resected CP was found in 17 patients from the AG group and eight patients from the BG group after one-year follow-up (p < 0.05). The levels of angiogenin-2 II and PKB/Akt in the AG group were higher than those in the BG group (p < 0.05). Meanwhile, BR treatment reduced the plasma levels of TNF-α, IL-1ß, and IL-6, and increased the level of IL-10(p < 0.05). Inflammatory cytokines are important factors that affect the recurrence of resected CP. Serum paeoniflorin, baicalin, saikosaponin A, and bupleurum saponin B2 in BR had a strong negative relationship with the plasma levels of Ang PKB/Akt. CONCLUSION: BR significantly reduces the recurrence risk of resected CP by affecting Ang PKB/Akt signaling.

Chemoreactive-Inspired Discovery of Influenza A Virus Dual Inhibitor to Block Hemagglutinin-Mediated Adsorption and Membrane Fusion.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, we reveal the discovery of an anti-IAV agent as a dual inhibitor to block hemagglutinin-mediated adsorption and membrane fusion using a chemoreactive ortho-quinone methide (o-QM) equivalent. Based on the o-QM equivalent nonenzymatically multipotent behavior, we created a series of clavatol-derived pseudo-natural products and found that penindolone (PND), a new diclavatol indole adduct, exhibited potent and broad-spectrum anti-IAV activities with low risk of inducing drug resistance. Distinct from current anti-IAV drugs, PND possesses a novel scaffold and is the first IAV inhibitor targeting both HA1 and HA2 subunits of virus hemagglutinin to dually block the IAV adsorption and membrane fusion process. More importantly, intranasal and oral administration of PND can protect mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. Thus, the use of chemoreactive intermediates could expand our understanding of chemical diversity and aid in the development of anti-IAV drugs with novel targets.

Secondary Metabolites Produced by Combined Culture of Penicillium crustosum and a Xylaria sp.

Four new alkyl aromatics, penixylarins A-D (1-4), along with the known biogenetically related 1,3-dihydroxy-5-(12-hydroxyheptadecyl)benzene (5) and 1,3-dihydroxy-5-(12-sulfoxyheptadecyl)benzene (6), were isolated from a mixed culture of the Antarctic deep-sea-derived fungus Penicillium crustosum PRB-2 and the mangrove-derived fungus Xylaria sp. HDN13-249. UPLC-MS data and an analysis of structural features showed that compounds 1 and 2 were produced by collaboration of the two fungi, while compounds 3-6 could be produced by Xylaria sp. HDN13-249 alone, but in noticeably increased quantities by cocultivation. Compounds 2, 3, 5, and 6 showed antibacterial activity against a panel of strains, and compound 3 possessed potential antituberculosis effects (MIC = 6.25 µM against Mycobacterium phlei).

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.

[Mining and discovery for active components of "Coptidis Rhizome-Magnoliae Officinalis Cortex" based on network pharmacology].

To mine and discover the active components of " Coptidis Rhizome-Magnoliae Officinalis Cortex( C&M) " based on the network pharmacology,integrate and analyze the potential targets and mechanisms. The TCMSP database was used to screen active ingredients. TTD and Drug Bank databases were used to predict the potential targets by referring to relevant literature,and the pathway annotation technology was used to enrich and analyze the active ingredients and potential targets of " C&M". A total of 29 potential target active ingredients were screened from " C&M",including 12 alkaloids components such as( R)-canadine,berberine,coptisine,and palmatine; 3 lignans consisting of magnolol,honokiol and obovatol; 6 volatile oils consisting of α-eudesmol,ß-eudesmol,eucalyptol and so on,and flavonoids including quercetin and neohesperidin. Corresponding 199 predicted targets were screened out,mainly including PTGS2,PTGS1,NCOA2,Hsp90 AB1,and so on. 72 signaling pathways were involved,8 of which were related to cancer,such as prostate cancer,bladder cancer,and pancreatic cancer; 9 of which were related to endocrine,including oxytocin signaling pathway,insulin signaling pathway,thyroid hormone signaling pathway and so on,as well as inflammation-related pathway. This study has preliminarily mined and discovered the main active components and potential targets of " C&M",providing material source for the study on the preparation of structural components of traditional Chinese medicine.

Methylsulfonylated Polyketides Produced by Neosartorya udagawae HDN13-313 via Exogenous Addition of Small Molecules.

Two new polyketides modified with a rare methylsulfonyl group, 3-methoxy-6-methyl-5-(methylsulfonyl)benzene-1,2,4-triol (1) and neosartoryone A (2), along with a biogenetically related compound (3), were isolated from Neosartorya udagawae HDN13-313 cultivated with the DNA methyltransferase inhibitor 5-azacytidine. The methylsulfonyl group of 1 and 2 was proven to be derived from DMSO, which was used as the solvent to dissolve 5-azacytidine. This is the first report of a fungus that can achieve a sulfonylation-like modification of natural products utilizing DMSO as a sulfur source. Compound 2 showed lipid-lowering activity in vitro comparable to simvastatin.

[Mechanism of flavonoid components in Astragali Radix in inhibiting tumor growth and immunoregulation in C57BL/6 tumor bearing mice based on "invigorating Qi for consolidation of exterior"].

Traditional Chinese medicine believes that the occurrence and development of tumors is related to the body's Qi deficiency. " Invigorating Qi for consolidation of exterior" has became an effective way to treat tumors by traditional Chinese medicine. This study is based on the " invigorating Qi for consolidation of exterior" to explore the effect of flavonoid components in Qi-invigorating herbs Astragali Radix( AR) on the growth and immune function of mouse Lewis lung cancer xenografts,and further explore its mechanism of action. In the present study,high performance liquid chromatography was performed to analyze the flavonoid components in AR.The Lewis lung cancer model of C57 BL/6 mice was constructed,and the tumor volume of mice was determined by Visual Sonics Vevo2100 high frequency color ultrasound. The levels of IL~(-1)7 and RORγt in serum and tumor tissues were detected by ELISA and immunohistochemistry. The expression of IRE~(-1)/XBP~(-1) pathway-related proteins in tumor tissues was detected by Western blot. The results revealed that treatment of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR significantly inhibited tumor growth of C57 BL/6 tumorbearing mice. The inhibition rates at the dose of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR were( 29. 5±4. 4) % and( 43. 4±5. 2) %,respectively. The expression of IL~(-1)7 and RORγt in serum and tumor tissues of Lewis lung cancer mice were decreased,and the spleen index and thymus index were significantly enhanced by the flavonoid components in AR. Flavonoid components in AR could decrease the expression of X-box binding protein 1( XBP1),inositol-requiring enzyme( IRE1) and glucose regulated protein 78 k D( GRP78),and increase the expression of C/EBP homologous protein( CHOP),and the high-dose group is better,suggesting that the anti-lung cancer effect of flavonoid components in AR is related to the regulation of XBP1 mediated ERs. This study provides new evidence that the flavonoid components in AR could inhibit the tumor growth of C57 BL/6 tumor-bearing mice by regulating the body's immune function through " invigorating Qi for consolidation of exterior".

Cytotoxic Tetrahydroxanthone Dimers from the Mangrove-Associated Fungus Aspergillus versicolor HDN1009.

Three new tetrahydroxanthone dimers, 5-epi-asperdichrome (1), versixanthones N (2), and O (3), were isolated from the mangrove-derived fungus Aspergillus versicolor HDN1009. Their structures, including the absolute configurations, were elucidated by NMR, HRMS, and circular dichroism (CD) experiments. Among them, compound 1 was the second example of tetrahydroxanthone dimers, which dimerized by a rare diaryl ether linkage and showed promising antibacterial activities against Vibrio parahemolyticus, Bacillus subtilis, Mycobacterium phlei, and Pseudomonas aeruginosa, with MIC values ranging from 100 µM to 200 µM; whilst compounds 2 and 3 exhibited extensive cytotoxicities against five cancer cell lines (HL-60, K562, H1975, MGC803, and HO-8910), with IC50 values ranging from 1.7 µM to 16.1 µM.

Structure-based discovery of cytotoxic dimeric tetrahydroxanthones as potential topoisomerase I inhibitors from a marine-derived fungus.

DNA topoisomerase I (Topo I) is an important anticancer drug target, and xanthone dimers are considered to be a new kind of Topo I inhibitor chemotypes. Based on the characteristics of dimeric xanthone structures, five new dimeric xanthones (1-5) and two known SAD isomers (6 and 7) were isolated from the mangrove-derived fungus Aspergillus vericolor. The absolute configurations of compounds 1-7, entailing both central and axial chirality elements, were established by a combination of ECD comparison, chemical conversions, and biogenetic considerations. Compounds 1-7 possessed high structural diversity and exhibited cytotoxicity at different levels. The selected new compounds 1, 2, and 5 showed Topo I inhibition properties and the most potent compound 1, an atropisomer of compound 2, was confirmed to inhibit Topo I-mediated DNA relaxation by targeting Topo I, thereby, arresting the cell cycle process and inducing necrosis in cancer cells. Molecular docking studies showed that compound 1 could bind DNA by π-π interaction and DNA Topo I by hydrogen bonds to form a ternary complex.

Lipid-Lowering Polyketides from the Fungus Penicillium Steckii HDN13-279.

Seven new polyketides, named tanzawaic acids R-X (1-6, 11), along with seven known analogues (7-10 and 12-14), were isolated from Penicillium steckii HDN13-279. Their structures, including the absolute configurations, were elucidated by NMR, MS, X-ray diffraction, circular dichroism (CD) analyses and chemical derivatization. Five compounds (2, 3, 6, 10 and 12) significantly decreased the oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells at the concentration of 10 µM, among which, four compounds (3, 6, 10 and 12) significantly decreased intracellular total cholesterol (TC) levels and three Compounds (3, 6, and 10) significantly decreased intracellular triglyceride (TG) levels. Moreover, the TG-lowering capacities of compounds 6 and 10 were comparable with those of simvastatin, with the TG levels being nearly equal to blank control. This is the first report on the lipid-lowering activity of tanzawaic acid derivatives.

[Effective components and signaling pathways of Epimedium brevicornumbased on network pharmacology].

The aim of this paper was to find out the active components of Epimedium brevicornum using network pharmacology, and find the potential targets and mechanisms. The TCMSP database was used to screen the active ingredients, and TTD and DrugBank databases were used to predict the potential targets with the literature mining. The pathway annotation was used to enrich and analyze the active ingredients and potential targets of E. brevicornum. The results showed that E. brevicornum had34 potential target active ingredients, including 21 flavones components, such as icariin, epimedin A, epimedin B, epimedin C, Yinyanghuo A, Yinyanghuo C and so on, 2 lignans involved in (+)-cycloolivil and olivil, 3 sterols consisting of sitosterol, 24-epicampesterol and poriferast-5-en-3beta-ol. The main predicted targets included Ptgs2, NCOA6, RANK, OPG, WNT9B, PTH1R, BMPs, SMAD4A and so on. There were 88 signaling pathways involved in 10 signaling pathways which was related to inflammation, such as NF-kappa B signaling pathway, T cell receptor signaling pathway, IL-17 signaling pathway and 10 pathways which was related to cancer included breast cancer, bladder cancer, pancreatic cancer and so on, and estrogen related signaling pathways included estrogen signaling pathway. This laid the foundation for the discovery of the active components of Epimedium and the study on its mechanism of action.