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.

Subplenones A-J: Dimeric Xanthones with Antibacterial Activity from the Endophytic Fungus Subplenodomus sp. CPCC 401465.

Subplenones A-J (1-10), 10 new xanthone dimers, have been isolated and characterized from the endophytic fungus Subplenodomus sp. CPCC 401465, which resides within the Chinese medicinal plant Gentiana straminea. The isolation process was guided by antibacterial assays and molecular-networking-based analyses. The chemical structures of these compounds were elucidated through the interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Furthermore, the relative configuration of the compounds was determined using NMR and single-crystal X-ray diffraction analyses, and the absolute configuration was established using electronic circular dichroism calculations. All of the isolated compounds exhibited significant inhibitory activity against Gram-positive bacteria. Notably, compounds 1, 5, and 7 displayed remarkable inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 700698, with a minimum inhibitory concentration (MIC) of 0.25 µg/mL, and against vancomycin-resistant Enterococcus faecium (VRE) ATCC 700221, with MIC values ranging from 0.5 to 1.0 µg/mL.

Brochoscopic Airway Clearance Therapy vs. Conventional Sputum Aspiration: The Future of Flexible Brochoscopes in Intensive Care Units?

(1) Background: The aim of our study is to investigate the effectiveness of bronchoscopic airway clearance therapy (B-ACT) on severe pneumonia (SP) patients with invasive mechanical ventilation (IMV) in the intensive care unit (ICU). (2) Methods: Our study retrospectively enrolled 49 patients with sputum aspiration and 99 patients with B-ACT, and the latter were divided into the ≤once every 3 days group (n = 50) and >once every 3 days group (n = 49). (3) Results: We found most laboratory blood results were significantly improved in the B-ACT group as compared with those in sputum aspiration group (p < 0.05). Patients in the B-ACT group and those in ≤once every 3 days group also had significantly better survival to hospital discharge than those in their counterpart groups (Logrank p < 0.001). In patients with cardiopulmonary diseases or positive cultures for bacteria, the B-ACT group and those in the ≤once every 3 days group had significantly better survival outcomes to discharge than those in their counterpart groups (Logrank p < 0.001). B-ACT and the average frequency of ≤once every 3 days had significantly better impact on survival outcomes than their counterpart groups (HR: 0.444, 95% CI: 0.238-0.829, p = 0.011; HR: 0.285, 95% CI: 0163-0.498, p < 0.001). (4) Conclusions: In the future, flexible bronchoscopes may paly an important role in ACT for SP patients with IMV.

Interleukin-6 and pulmonary hypertension: from physiopathology to therapy.

Pulmonary hypertension (PH) is a progressive, pulmonary vascular disease with high morbidity and mortality. Unfortunately, the pathogenesis of PH is complex and remains unclear. Existing studies have suggested that inflammatory factors are key factors in PH. Interleukin-6 (IL-6) is a multifunctional cytokine that plays a crucial role in the regulation of the immune system. Current studies reveal that IL-6 is elevated in the serum of patients with PH and it is negatively correlated with lung function in those patients. Since IL-6 is one of the most important mediators in the pathogenesis of inflammation in PH, signaling mechanisms targeting IL-6 may become therapeutic targets for this disease. In this review, we detailed the potential role of IL-6 in accelerating PH process and the specific mechanisms and signaling pathways. We also summarized the current drugs targeting these inflammatory pathways to treat PH. We hope that this study will provide a more theoretical basis for targeted treatment in patients with PH in the future.

Verbalide A~F: new phthalide derivatives from the endophytic fungus Preussia sp. CPCC 400972.

There are six new phthalide derivatives Verbalide A ~ F (1-6) together with another known derivative (7) isolated from the endophytic fungus Preussia sp. CPCC 400972. Their structures were established by comprehensive spectroscopic analyses, including NMR and HRESIMS. In addition, compounds 1-7 exhibited excellent inhibitory effect against influenza A virus.

Genomic and Transcriptomic Approaches Provide a Predictive Framework for Sesquiterpenes Biosynthesis in Desarmillaria tabescens CPCC 401429.

Terpenoids constitute a structurally diverse class of secondary metabolites with wide applications in the pharmaceutical, fragrance and flavor industries. Desarmillaria tabescens CPCC 401429 is a basidiomycetous mushroom that could produce anti-tumor melleolides. To date, no studies have been conducted to thoroughly investigate the sesquiterpenes biosynthetic potential in Desarmillaria or related genus. This study aims to unravel the phylogeny, terpenome, and functional characterization of unique sesquiterpene biosynthetic genes of the strain CPCC 401429. Herein, we report the genome of the fungus containing 15,145 protein-encoding genes. MLST-based phylogeny and comparative genomic analyses shed light on the precise reclassification of D. tabescens suggesting that it belongs to the genus Desarmillaria. Gene ontology enrichment and pathway analyses uncover the hidden capacity for producing polyketides and terpenoids. Genome mining directed predictive framework reveals a diverse network of sesquiterpene synthases (STSs). Among twelve putative STSs encoded in the genome, six ones are belonging to the novel minor group: diverse Clade IV. In addition, RNA-sequencing based transcriptomic profiling revealed differentially expressed genes (DEGs) of the fungus CPCC 401429 in three different fermentation conditions, that of which enable us to identify noteworthy genes exemplified as STSs coding genes. Among the ten sesquiterpene biosynthetic DEGs, two genes including DtSTS9 and DtSTS10 were selected for functional characterization. Yeast cells expressing DtSTS9 and DtSTS10 could produce diverse sesquiterpene compounds, reinforced that STSs in the group Clade IV might be highly promiscuous producers. This highlights the potential of Desarmillaria in generating novel terpenoids. To summarize, our analyses will facilitate our understanding of phylogeny, STSs diversity and functional significance of Desarmillaria species. These results will encourage the scientific community for further research on uncharacterized STSs of Basidiomycota phylum, biological functions, and potential application of this vast source of secondary metabolites.

Metabolites from the plant endophytic fungus Penicillium sp. CPCC 401423 and their cytotoxic activity against MIA PaCa-2 cells.

Twenty-two metabolites were isolated from Penicillium sp. CPCC 401423 cultured on rice. The structures of all compounds were elucidated mainly by MS and NMR analysis as well as the necessary CD experimental evidence, of which penicillidione A (1), penicillidione B (2), (E)-4-[(4-acetoxy-3-methyl-2-butenyl)oxy]phenylacetic acid (3), (S)-2-hydroxy-2-{4-[(3-methyl-2-butenyl)oxy]phenyl} (4), (S)-4-(2,3-dihydroxy-3-methyl-butoxy)phenylacetic acid (5), (E)-4-[(3-carboxy-2-butenyl)oxy]benzoic acid (6), (Z)-4-[(4-hydroxy-3-methyl-2-butenyl)oxy]benzoic acid (7), open-cycled N-demethylmelearoride A (12), and penostatin M (16) were identified as new compounds. The cytotoxic activity against human pancreatic carcinoma cell line MIA PaCa-2a was detected. Among them, compounds 13-15 and 22 displayed significant cytotoxicity against MIA-PaCa-2 cells with IC50 values of 8.9, 36.5, 31.8, and 22.3 µM, respectively (positive control gemcitabine IC50 65.0 µM).

Mining and characterization of the PKS-NRPS hybrid for epicoccamide A: a mannosylated tetramate derivative from Epicoccum sp. CPCC 400996.

BACKGROUND: Genomic analysis indicated that the genomes of ascomycetes might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained enigmatic. The ascomycete genus Epicoccum, belonging to the family Didymellaceae, is ubiquitous that colonizes different types of substrates and is associated with phyllosphere or decaying vegetation. Species of this genus are prolific producers of bioactive substances. The epicoccamides, as biosynthetically distinct mannosylated tetramate, were first isolated in 2003 from Epicoccum sp. In this study, using a combination of genome mining, chemical identification, genetic deletion, and bioinformatic analysis, we identified the required BGC epi responsible for epicoccamide A biosynthesis in Epicoccum sp. CPCC 400996. RESULTS: The unconventional biosynthetic gene cluster epi was obtained from an endophyte Epicoccum sp. CPCC 400996 through AntiSMASH-based genome mining. The cluster epi includes six putative open reading frames (epiA-epiF) altogether, in which the epiA encodes a tetramate-forming polyketide synthase and nonribosomal peptide synthetases (PKS-NRPS hybrid). Sequence alignments and bioinformatic analysis to other metabolic pathways of fungal tetramates, we proposed that the gene cluster epi could be involved in generating epicoccamides. Genetic knockout of epiA completely abolished the biosynthesis of epicoccamide A (1), thereby establishing the correlation between the BGC epi and biosynthesis of epicoccamide A. Bioinformatic adenylation domain signature analysis of EpiA and other fungal PKS-NRPSs (NRPs) indicated that the EpiA is L-alanine incorporating tetramates megasynthase. Furthermore, based on the molecular structures of epicoccamide A and deduced gene functions of the cluster epi, a hypothetic metabolic pathway for biosynthesizing compound 1 was proposed. The corresponding tetramates releasing during epicoccamide A biosynthesis was catalyzed through Dieckmann-type cyclization, in which the reductive (R) domain residing in terminal module of EpiA accomplished the conversion. These results unveiled the underlying mechanism of epicoccamides biosynthesis and these findings might provide opportunities for derivatization of epicoccamides or generation of new chemical entities. CONCLUSION: Genome mining and genetic inactivation experiments unveiled a previously uncharacterized PKS - NRPS hybrid-based BGC epi responsible for the generation of epicoccamide A (1) in endophyte Epicoccum sp. CPCC 400996. In addition, based on the gene cluster data, a hypothetical biosynthetic pathway of epicoccamide A was proposed.

Discovery and Activation of the Cryptic Cluster from Aspergillus sp. CPCC 400735 for Asperphenalenone Biosynthesis.

Postgenomic analysis manifested that filamentous fungi contain numerous natural product biosynthetic gene clusters in their genome, yet most clusters remain cryptic or down-regulated. Herein, we report the successful manipulation of strain Aspergillus sp. CPCC 400735 that enables its genetic engineering via targeted overexpression of pathway-specific transcriptional regulator AspE. The down-regulated metabolic pathway encoded by the biosynthetic gene cluster asp was successfully up-activated. Analyses of mutant Ai-OE::aspE extracts led to isolation and characterization of 13 asperphenalenone derivatives, of which 11 of them are new compounds. All of the asperphenalenones exhibited conspicuous anti-influenza A virus effects with IC50 values of 0.45-2.22 µM. Additionally, their identification provided insight into biosynthesis of asperphenalenones and might benefit studies of downstream combinatorial biosynthesis. Our study further demonstrates the effective application of targeted overexpressing pathway-specific activator and novel metabolite discovery in microorganisms. These will accelerate the exploitation of the untapped resources and biosynthetic capability in filamentous fungi.

Antibacterial polyene-polyol macrolides and cyclic peptides from the marine-derived Streptomyces sp. MS110128.

Marine microbes provide an important resource to discover new chemical compounds with biological activities beneficial to drug discovery. In our study, two new polyene macrolides, pyranpolyenolides A (1) and B (2), and one new natural cyclic peptide (9), together with two known polyenes (7 and 8) and three known cyclic peptides (10-12), were isolated from a culture of the marine Streptomyces sp. MS110128. In addition, four new polyene macrolides, pyranpolyenolides C-F (3-6), were identified as olefin geometric isomers that were most likely produced by photochemical conversion during the cultivation or isolation procedures. The pyranpolyenolides are 32-membered macrolides endowed with a conjugated tetraene and several pairs of 1,3-dihydroxyl groups. Pyranpolyenolides that contain a hydropyran group have not been previously reported. Four cyclic peptides (9-12) showed significant activities against Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant S. aureus with supporting MIC values ranging from 0.025 to 1.25 µg/mL. These cyclic peptides containing piperazic moieties showed moderate activities with MIC values of 12.5 µg/mL against Bacille Calmette Guerin (BCG), an attenuated form of the bovine. Additionally, cyclic peptide 12 showed moderate antifungal activity against Candida albicans with an MIC value of 12.5 µg/mL. KEY POINTS: • Discovery of new polyenes and cyclic peptides from a marine-derived Actinomycete. • Cyclic peptides containing piperazic moieties exhibited good antibacterial activity.

New butyrolactone derivatives from the endophytic Fungus Talaromyces sp. CPCC 400783 of Reynoutria japonica Houtt.

Six new butyrolactone derivatives (1, 2a/2b, 3a/3b and 4), together with another two known derivatives (5 and 6) were isolated from the endophytic fungus Talaromyces sp. CPCC 400783. Their structures were established by a combination of spectroscopic analysis, including NMR and HRESIMS. The absolute configurations were elucidated by ECD experiments. Subsequently, compound 1, 3b, 4 and 5 exhibited good inhibitory effect against influenza A/WSN/33 (H1N1) virus with IC50 values of 21.93 ± 1.51, 21.54 ± 3.75, 18.36 ± 2.15 and 23.80 ± 3.05 µM respectively.

Ornithinimicrobium cerasi sp. nov., isolated from the fruit of Cerasus pseudocerasus and emended description of the genus Ornithinimicrobium.

Strain CPCC 203383T, isolated from the surface-sterilized fruit of Cerasus pseudocerasus (Lindl.) G. Don, was taxonomically characterized based on a polyphasic investigation. It had the highest 16S rRNA gene sequence similarities with Ornithinimicrobium pekingense DSM 21552 (97.2 %) and O. kibberense DSM 17687T (97.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a distinct phyletic branch within the genus Ornithinimicrobium and the whole genome sequence data analyses supported that strain CPCC 203383T was phylogenetically related to the Ornithinimicrobium species. The isolate shared a range of phenotypic patterns reported for members of the genus Ornithinimicrobium, but also had a range of cultural, physiological and biochemical characteristics that separated it from related Ornithinimicrobium species. The menaquinone was MK-8(H4). The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI) and unidentified lipids (ULs). The major fatty acids (>5 %) were iso-C15 : 0, anteiso-C15 : 0, iso-C16:0, 9-methyl C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. The cell wall peptidoglycan contains l-ornithine as diagnostic diamino acid and an interpeptide bridge consisting of L-Orn←L-Ala←Gly←D-Asp. The combined genotypic and phenotypic data indicated that the isolate represents a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cerasi sp. nov. is proposed, with CPCC 203383T(=NBRC 113522T=KCTC 49200T) as the type strain. The DNA G+C composition is 72.3 mol%. The availability of new data allows for an emended description of the genus Ornithinimicrobium.

New cryptotanshinone derivatives with anti-influenza A virus activities obtained via biotransformation by Mucor rouxii.

This paper provides an efficient platform to diversify the structure and pharmaceutical potentials of known natural products. Seven metabolites were obtained via the biotransformation of cryptotanshinone by the fungus Mucor rouxii AS 3.3447, and assigned as 13R-14R-hydroxy-anhydride of 16R-cryptotanshinone (1), 1S-hydroxy-anhydride of 16R-cryptotanshinone (2), 1R-hydroxy-anhydride of 16R-cryptotanshinone (3), 3S-hydroxy-epicryptoacetalide (4), 3S-hydroxy-cryptoacetalide (5), epicryptoacetalide (6), and cryptoacetalide (7). Among these compounds, 1-5 are novel. The ortho-naphthoquinone chromophore of cryptotanshinone was degraded and rearranged by M. rouxii. 1 and 3 showed good anti-influenza A virus activities with the reduced cytotoxic activities compared to the parent substrate cryptotanshinone (8). The structures of all the new compounds were determined on the basis of HRESIMS (high-resolution electrospray ionization mass spectroscopy) spectrometry, NMR (nuclear magnetic resonance) spectroscopy, ECD (electronic circular dichroism) calculations, and the CD (circular dichroism) of "in situ" method with [Rh2(OCOCF3)4].

Fungal biotransformation of tanshinone results in [4+2] cycloaddition with sorbicillinol: evidence for enzyme catalysis and increased antibacterial activity.

The biotransformation of tanshinone IIA to a new antibacterial agent tanshisorbicin (1) by the fungus Hypocrea sp. (AS 3.17108) is described. The structure of tanshisorbicin is a hybrid of tanshinone IIA (2) and sorbicillinol (3). The latter is a metabolite produced by Hypocrea sp. The structure of tanshisorbicin was determined using mass spectrometry, NMR spectroscopy, and ECD calculations. The anti-MRSA activity of 1 was found to be significantly higher than that of the parent substrate Tan IIA. Preliminary experiments indicate that tanshisorbicin is formed via a [4+2] cycloaddition reaction that is likely catalyzed by microbial enzyme.

Discovery of tanshinone derivatives with anti-MRSA activity via targeted bio-transformation.

Two potent anti-MRSA tanshinone glycosides (1 and 2) were discovered by targeted microbial biotransformation, along with rapid identification via MS/MS networking. Serial reactions including dehydrogenation, demethylations, reduction, glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447. In addition, tanshinosides B (2) showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 µg/mL. This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.

Bioassay-guided identification of bioactive molecules from traditional Chinese medicines.

Traditional Chinese medicines (TCMs) serve as a major source of a variety of drug lead compounds. In the process of natural products development, bioassay-guided isolation is a rapid and validated method for isolation of compounds with bioactivities. This chapter describes bioassay-guided separation and purification of compounds from the crude extracts of TCMs. Two approaches including size-exclusion chromatography (SEC) and high performance liquid chromatography (HPLC) are described in detail.

Biotransformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis.

Microbial transformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis CICC 3043 yielded seven metabolites. Their structures were elucidated on the basis of extensive spectroscopic analyses. R. chinensis could catalyze hydroxylation and further dehydrogenation at C-24 of 20(R)-panaxadiol, as well as hydroxylation at C-7, C-15, C-16, and C-29. Three of these compounds at 10µM could moderately inhibit growth of HepG2 human hepatocellular carcinoma cells with an inhibition rate of about 40%. Three compounds (also at 10µM) showed approximately 30% inhibition on NF-κB transcriptional activity in SW480 human colon carcinoma cells stably transfected with NF-κB luciferase reporter and induced by LPS.

Endophytic Streptomyces sp. Y3111 from traditional Chinese medicine produced antitubercular pluramycins.

As part of a search for antitubercular substances from natural sources, we screened a library of endophytic microbes (50 strains and 300 crude extracts in total) isolated from traditional Chinese medicines (TCMs) for growth inhibitory activity against Bacillus Calmette-Guérin (BCG). The crude extract of Streptomyces sp. strain Y3111, which was associated with the stems of Heracleum souliei, showed good anti-BCG activity with an MIC value of 12.5 µg/mL. Bioassay-guided isolation led to four new pluramycin-type compounds, heraclemycins A-D (1-4). Their structures were determined by different spectroscopic techniques including HRMSESI, 1D NMR, and 2D NMR. This is the first report of pluramycin analogues produced by TCM endophytic microbes as well as the first example of BCG-selective pluramycins. Heraclemycin C (3) showed selective antitubercular activity against BCG with a MIC value of 6.25 µg/mL and a potential new mode of action.

Caesanines A-D, new cassane diterpenes with unprecedented N bridge from Caesalpinia sappan.

Serial antibacterial furanoditerpenes caesanines A-D (1-4), possessing a cassane-type diterpenoid skeleton with an unusual N bridge between C-19/C-20, were identified from a Chinese herb Caesalpinia sappan Linn. In addition, caesanine D (4) showed the first class of dicassane diterpenoid ethers. Their structures were determined by different spectroscopic methods and ECD calculation. Caesanines A and B exhibited strong activities against MRSA suggesting a promising entry point for the development of anti-infective drugs.