Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form "biosynthetic gene clusters" (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%-60% of the crude organic extracts.

Berkeleylactones and a Citreohybriddione Analogue from Penicillium turbatum.

In 2017 we reported the isolation and characterization of berkeleylactones A-H and A26771B from a coculture of two extremophilic Penicillium sp. isolated from an acid mine waste lake. Berkeleylactone A exhibited potent activity against several strains of multi-drug-resistant Staphylococcus aureus and Bacillus anthracis. A26771B, which is related to the berkeleylactones, also exhibited antibiotic activity. Although the berkeleylactones were novel compounds, A26771B was originally isolated by scientists at Eli Lilly Company from P. turbatum and reported in1977. We recently obtained P. turbatum and grew it in axenic culture. We isolated five new berkeleylactones (2 and 4-7), two berkeley-γ-lactones (8 and 9), and citreohybriddional (10), as well as the known compounds A26771B (1), berkeleylactone E (3), and gliovictin. The structures of the novel compounds were deduced from analysis of spectral data. Compounds 2 and 4 -7 are 16-membered macrolides, while 8 and 9 are γ-lactones that share the hexadecanoic acid skeleton. A26771B (1) and berkeleylactone I (2) were active against several strains of Staphylococcus aureus, including four multi-drug-resistant strains. Berkeleylactone N (8) was active only against Streptococcus pyogenes.

Cryptic Biosynthesis of the Berkeleypenostatins from Coculture of Extremophilic Penicillium sp.

Coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded berkeleypenostatins A-G (1-7) as well as the previously reported berkeleylactones A-H, the known macrolide A26771B, citrinin, and patulin. As was true with the berkeleylactones, there was no evidence of the berkeleypenostatins in either axenic culture. The structures were deduced from analyses of spectral data, and the absolute configuration of berkeleypenostatin A (1) was determined by single-crystal X-ray crystallography. Berkeleypenostatins A (1) and E (5) inhibited migration of human pancreatic carcinoma cells (HPAF-II). Both compounds were tested by the NCI Developmental Therapeutics Program. In the NCI 60 cell five-dose screen, berkeleypenostatin E (5) was the more active of the two, with 1-10 µM total growth inhibition (TGI) of all leukemia cell lines, as well as the majority of colon, CNS, melanoma, ovarian, prostate, renal, and breast cancer cell lines.

The Berkeleylactones, Antibiotic Macrolides from Fungal Coculture.

A carefully timed coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded eight new 16-membered-ring macrolides, berkeleylactones A-H (1, 4, 6-9, 12, 13), as well as the known antibiotic macrolide A26771B (5), patulin, and citrinin. There was no evidence of the production of the berkeleylactones or A26771B (5) by either fungus when grown as axenic cultures. The structures were deduced from analyses of spectral data, and the absolute configurations of compounds 1 and 9 were determined by single-crystal X-ray crystallography. Berkeleylactone A (1) exhibited the most potent antimicrobial activity of the macrolide series, with low micromolar activity (MIC = 1-2 µg/mL) against four MRSA strains, as well as Bacillus anthracis, Streptococcus pyogenes, Candida albicans, and Candida glabrata. Mode of action studies have shown that, unlike other macrolide antibiotics, berkeleylactone A (1) does not inhibit protein synthesis nor target the ribosome, which suggests a novel mode of action for its antibiotic activity.

The Ayurvedic plant Bacopa monnieri inhibits inflammatory pathways in the brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacopa monnieri (L) Wettst (common name, bacopa) is a medicinal plant used in Ayurveda, the traditional system of medicine of India, as a nootropic. It is considered to be a "medhya rasayana", an herb that sharpens the mind and the intellect. Bacopa is an important ingredient in many Ayurvedic herbal formulations designed to treat conditions such as memory loss, anxiety, poor cognition and loss of concentration. It has also been used in Ayurveda to treat inflammatory conditions such as arthritis. In modern biomedical studies, bacopa has been shown in animal models to inhibit the release of the pro-inflammatory cytokines TNF-α and IL-6. However, less is known regarding the anti-inflammatory activity of Bacopa in the brain. AIM OF THE STUDY: The current study examines the ability of Bacopa to inhibit the release of pro-inflammatory cytokines from microglial cells, the immune cells of the brain that participate in inflammation in the CNS. The effect of Bacopa on signaling enzymes associated with CNS inflammatory pathways was also studied. MATERIALS AND METHODS: Various extracts of Bacopa were prepared and examined in the N9 microglial cell line in order to determine if they inhibited the release of the proinflammatory cytokines TNF-α and IL-6. Extracts were also tested in cell free assays as inhibitors of caspase-1 and matrix metalloproteinase-3 (enzymes associated with inflammation) and caspase-3, which has been shown to cleave protein Tau, an early event in the development of Alzheimer's disease. RESULTS: The tea, infusion, and alkaloid extracts of bacopa, as well as Bacoside A significantly inhibited the release of TNF-α and IL-6 from activated N9 microglial cells in vitro. In addition, the tea, infusion, and alkaloid extracts of Bacopa effectively inhibited caspase 1 and 3, and matrix metalloproteinase-3 in the cell free assay. CONCLUSIONS: Bacopa inhibits the release of inflammatory cytokines from microglial cells and inhibits enzymes associated with inflammation in the brain. Thus, Bacopa can limit inflammation in the CNS, and offers a promising source of novel therapeutics for the treatment of many CNS disorders.

Bioactive Secondary Metabolites Produced by the Fungal Endophytes of Conifers.

This is a review of bioactive secondary metabolites isolated from conifer-associated endophytic fungi from 1990-2014. This includes compounds with antimicrobial, anti-inflammatory, anti-proliferative and cytotoxic activity towards human cancer cell lines, and activity against either plant pathogens or plant insect pests. Compounds that were originally reported without associated activity were included if other studies ascribed activity to these compounds. Compounds were not included if they were exclusively phytotoxic or if they were isolated from active extracts but were not determined to be the active component of that extract.

Azaphilones from an Acid Mine Extremophile Strain of a Pleurostomophora sp.

An extremophilic fungus identified as a Pleurostomophora sp. was isolated from the Berkeley Pit, an acid mine waste lake. When grown in liquid culture, the fungus produced berkchaetoazaphilones A-C (1, 2, and 5), the red pigment berkchaetorubramine (6), and the known compound 4-(hydroxymethyl)quinoline. These compounds were evaluated as inhibitors of matrix metalloproteinase-3, caspase-1, and proinflammatory cytokine production in induced THP-1 cells. Berkchaetoazaphilone B (2) inhibited IL-1ß, TNFα, and IL-6 production in the induced inflammasome assay and was cytotoxic toward human retinoblastoma cell line Y79 (IC50 = 1.1 µM), leukemia cell lines CCRF-CEM and SR, and the melanoma cell line LOX IMVI (IC50 = 10 µM).

Bioactive secondary metabolites from acid mine waste extremophiles.

The extremophilic microbes of the Berkeley Pit Lake are a valuable source of new and interesting secondary metabolites. It is of particular interest that these acidophilic microbes produce small molecule inhibitors of pathways associated with low pH and high Eh. These same small molecules also inhibit molecular pathways induced by reactive oxygen species (ROS) and inflammation in mammalian cells. Low pH is a hallmark of inflammation and high Eh is one of ROS, so the suitability of this collection as a source of bioactive metabolites is actually quite biorational. Compound isolation was guided by inhibition of caspase-1 and matrix metalloproteinase-3, and active compounds were sent to the National Cancer Institute-Developmental Therapeutics Program and Memorial Sloan Kettering Cancer center for evaluation as either antiproliferative or cytotoxic agents.

Phomopsolides and related compounds from the alga-associated fungus, Penicillium clavigerum.

For the past fifteen years we have studied the secondary metabolites of extremophilic fungi from the Berkeley Pit, an abandoned acid mine waste lake. Fungi associated with an acid-tolerant alga have also been harvested from the Pit. Penicillium clavigerum Demelius was isolated from the green alga Chlorella vulgaris Beyerinck [Beijerinck]. In culture it produced the known compounds phomfuranone (1), patulin (2), dimethylphthalides (3) and (4), phomopsolide A (5), phomopsolide C (6), phomopsolide B (7), phomopsolide E (8), phomopsolide F (9), and phompyrone (10) and the new compound berkbenzofuran thioester (11). Compounds 5 and 6 were potent inhibitors (IC50 < 10 microM) of specific and established human cancer cell lines.

Caspase-1 inhibitors from an extremophilic fungus that target specific leukemia cell lines.

Berkeley Pit Lake, Butte, Montana, is a 540 m deep abandoned open-pit copper mine filled with over 140 billion liters of acidic, metal-sulfate-contaminated water. This harsh environment has yielded several microorganisms that produce interesting biologically active compounds. Several polyketide metabolites including the new berkazaphilones A (1) and B (2) and octadienoic acid derivatives berkedienoic acid (13) and berkedienolactone (15), as well as previously reported azaphilone 4, vermistatin (6), dihydrovermistatin (7), penisimplicissin (8), aldehyde 9, and methylparaconic acid (11), were isolated from a culture broth of Penicillium rubrum taken from a depth of 270 m. The structures of these compounds were deduced by interpretation of spectroscopic data. The compounds were isolated either for their inhibition of the signal transduction enzyme caspase-1 or because of their structural similarity to these inhibitors. Selected compounds were further evaluated for their ability to inhibit interleukin-1ß production by inflammasomes in induced THP-1 cells. Berkazaphilones B (2) and C (4) and vermistatin analogue penisimplicissin (8) exhibited selective activity against leukemia cancer cell lines in the National Cancer Institute 60 human cell line assay.

Caspase-1 and -3 inhibiting drimane sesquiterpenoids from the extremophilic fungus Penicillium solitum.

Two new drimane sesquiterpene lactones and one new tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic fungus Penicillium solitum. The structures of these compounds were deduced by spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction enzymes caspase-1 and caspase-3 and mitigated the production of interleukin 1-ß in the induced THP-1 (pro-monocytic leukemia cell line) assay.

Berkeleyones and related meroterpenes from a deep water acid mine waste fungus that inhibit the production of interleukin 1-ß from induced inflammasomes.

The Berkeley Pit, an acid mine waste lake, is a source of extremophilic microorganisms that produce interesting bioactive compounds. We have previously reported the isolation of berkeleydione (1), berkeleytrione (2), the berkeleyacetals, and the berkeleyamides from the Pit Lake fungus Penicillium rubrum. In this paper we report the isolation and characterization of berkeleyones A-C (4, 5, and 7) as well as previously described preaustinoid A (3) and A1(6) from this same fungus. These compounds were evaluated as inhibitors of the signaling enzyme caspase-1 and as potential inhibitors of interleukin 1-ß production by inflammasomes in induced THP-1 cell line assays.

The berkeleyamides, amides from the acid lake fungus Penicillum rubrum.

We previously reported several novel bioactive hybrid polyketide-terpenoid metabolites from a deep water Penicillium rubrum isolated from Berkeley Pit Lake, Butte, Montana. In this paper we report the structures of four new amides, berkeleyamides A-D (1, 4, 5, 7), isolated from extracts of this fungus. The structures of these compounds were deduced by analysis of NMR data, chemical derivatization, and comparison of their spectroscopic data to those of known compounds.

The berkeleyacetals, three meroterpenes from a deep water acid mine waste Penicillium.

Berkeley Pit Lake is a 1500 ft deep abandoned open-pit copper mine filled with over 1140 billion liters of acidic, metal-sulfate-contaminated water. This harsh environment is proving to be a source of unusual microorganisms that produce novel bioactive compounds. We recently reported the structures of berkeleydione (1) and berkeleytrione (2), two novel hybrid polyketide-terpenoid metabolites isolated from a deep water Penicillium sp. growing in Berkeley Pit Lake. In this paper we report the structures of three new compounds, berkeleyacetals A-C ( 3-5) isolated from extracts of this fungus. The structures of these compounds were deduced by comparison of mass spectral and NMR data to that of berkeleydione (1).

Berkelic acid, a novel spiroketal with selective anticancer activity from an acid mine waste fungal extremophile.

Berkeley Pit Lake is an abandoned open-pit copper mine filled with 30 billion gallons of acidic, metal-contaminated water. This harsh environment is proving to be a source of unusual microorganisms that produce novel bioactive metabolites. Bioassay-guided fractionation using signal transduction enzyme assays led to the isolation of the novel spiroketal, berkelic acid 1, and of the known gamma-pyrone, spiciferone A 4. Berkelic acid has shown selective, nanomolar activity against OVCAR-3, an ovarian cancer cell line in the National Cancer Institute cell line screen. The isolation and characterization of these compounds are reported here.

Novel sesquiterpenoid matrix metalloproteinase-3 inhibitors from an acid mine waste extremophile.

Berkeley Pit Lake is a 1500 ft deep abandoned open-pit copper mine filled with 30 billion gallons of acidic, metal-contaminated water. This harsh environment is proving to be a source of unusual, biologically active microorganisms. Bioassay-guided fractionation using signal transduction enzyme assays led to the isolation of three novel bisabolane sesquiterpenes and a novel coumarin. The isolation and characterization of these compounds are reported here.

Berkeleydione and berkeleytrione, new bioactive metabolites from an acid mine organism.

[structure: see text] Two novel hybrid polyketide-terpenoid metabolites were isolated from a Penicillium sp. growing in the Berkeley Pit Lake of Butte, Montana. Their structures were deduced by spectroscopic analysis and confirmed by single-crystal X-ray analysis on berkeleydione (1). Both compounds inhibited matrix metalloproteinase-3 and caspase-1, and berkeleydione showed activity toward non-small-cell lung cancer in NCI's human cell line antitumor screen.

A novel 5-HT receptor ligand and related cytotoxic compounds from an acid mine waste extremophile.

Berkeley Pit Lake in Butte, Montana, is an acid mine waste reservoir rich in toxic metals. A Pithomycessp. isolated from the Pit Lake yielded three tyrosine derivatives (1-3), one of which acts as a 5-HT((2a)) receptor ligand. This type of activity has been associated with migraine preventative and antihypertensive drugs. The isolation and characterization of compounds 1-3 and three sesquiterpenes (5-7) that have been isolated previously from higher plants are reported here.

Sequoiamonascins a-d: novel anticancer metabolites isolated from a redwood endophyte.

Aspergillus parasiticus, a fungal isolate from the bark of a redwood tree (Sequoia sempervirens), has been shown to produce the antitumor metabolites sequoiatones A and B and more recently the sequoiatones C-F. We have also isolated another series of compounds with a new carbon skeleton, the sequoiamonascins. The structures of sequoiamonascins A-D were deduced by interpretation of their spectral data and that of some reaction products. The sequoiamonascins were isolated by brine shrimp lethality-guided fractionation and were submitted to the NCI for anticancer evaluation.