Acremopeptaibols A-F, 16-Residue Peptaibols from the Sponge-Derived Acremonium sp. IMB18-086 Cultivated with Heat-Killed Pseudomonas aeruginosa.

Six new 16-residue peptaibols, acremopeptaibols A-F (1-6), along with five known compounds, were isolated from the cultures of the sponge-associated fungus Acremonium sp. IMB18-086 grown in the presence of the autoclaved bacterium Pseudomonas aeruginosa on solid rice medium. The peptaibol sequences were established based on comprehensive analysis of 1D and 2D NMR spectroscopic data in conjunction with HRESIMS/MS experiments. The configurations of the amino acid residues were determined by advanced Marfey's analysis. Compounds 1-6 feature the lack of the highly conserved Thr6 and Hyp10 residues in comparison with other members of the SF3 subfamily peptaibols. A plausible biosynthetic pathway of compounds 1-6 was proposed on the basis of genomic analysis. Compounds 1, 5, 7, and 10 exhibited significant antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans. Compounds 7-10 showed potent cytotoxicities against the A549 and/or HepG2 cancer cell lines.

Antitumor effects of 3-bromoascochlorin on small cell lung cancer via inhibiting MAPK pathway.

Small cell lung cancer (SCLC) was defined as a recalcitrant cancer, and novel therapies are urgently needed. Marine natural products (MNPs) may bring continuing hope for treatment of SCLC. In this study, 3-bromoascochlorin (BAS), an MNP isolated from the coral-derived fungus Acremonium sclerotigenum GXIMD 02501, was primarily screened out with antiproliferative activity towards SCLC cell lines. Then western blot analysis (WB) and flow cytometry were conducted, and we found BAS could induce the apoptosis of H446 and H69AR cells. Besides, BAS could suppress the invasion and migration of H446. In an SCLC xenograft mice model, BAS inhibited the growth of tumor without affecting the body weight of mice. Finally, the underlying mechanisms were preliminarily explored. According to the results of RNA-seq, reverse transcription-quantitative polymerase chain reaction, and WB, our results revealed that BAS exerted antitumor activity via inhibiting mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) pathway. Collectively, these results indicated that BAS can be used as a promising compound for the treatment of human SCLC.

Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.

The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum.

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Acremotins A-D, peptaibiotics produced by the soil-derived fungus Acremonium persicinum SC0105.

Four new peptaibiotics, acremotins A-D (1-4) featuring three α,α-dialkylated amino acid-imino acid motifs and an unreduced C-terminal residue, along with the known peptaibiotic XR586 (5) were isolated from the solid cultures of the soil-derived fungus Acremonium persicinum SC0105. Their primary structures were characterized by detailed analysis of the HRESIMS/MS fragmentation pattern combined with comprehensive interpretation of the 1D and 2D NMR spectroscopic data. The absolute configurations of amino acid residues were determined by the advanced Marfey's method. Sequence alignment result shows that 1-4 are closely related to zervamicin IIB and emerimicin IIA, thus belong to peptaibiotic subfamily-3 (SF3). The three-dimensional (3D) structure of 4 was established by theoretical conformational analysis using the ab initio density functional theory (DFT) method, which, together with the CD spectrum, indicated an amphiphilic and helical structure for 4. 1-5 actively inhibited the growth of gram-positive bacterial pathogens, and amongst them 4 was the most potent compound showing MIC of 12.5 and 6.25 µg/ml against S. aureu and MRSA strains, respectively. 1-5 were also cytotoxic against three human cancer cell lines with IC50 ranging from 1.2 to 21.6 µM.

Deactivation of the autotrophic sulfate assimilation pathway substantially reduces high-level ß-lactam antibiotic biosynthesis and arthrospore formation in a production strain from Acremonium chrysogenum.

The filamentous ascomycete Acremonium chrysogenum is the only industrial producer of the ß-lactam antibiotic cephalosporin C. Synthesis of all ß-lactam antibiotics starts with the three amino acids l-α-aminoadipic acid, l-cysteine and l-valine condensing to form the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine tripeptide. The availability of building blocks is essential in every biosynthetic process and is therefore one of the most important parameters required for optimal biosynthetic production. Synthesis of l-cysteine is feasible by various biosynthetic pathways in all euascomycetes, and sequencing of the Acr. chrysogenum genome has shown that a full set of sulfur-metabolizing genes is present. In principle, two pathways are effective: an autotrophic one, where the sulfur atom is taken from assimilated sulfide to synthesize either l-cysteine or l-homocysteine, and a reverse transsulfuration pathway, where l-methionine is the sulfur donor. Previous research with production strains has focused on reverse transsulfuration, and concluded that both l-methionine and reverse transsulfuration are essential for high-level cephalosporin C synthesis. Here, we conducted molecular genetic analysis with A3/2, another production strain, to investigate the autotrophic pathway. Strains lacking either cysteine synthase or homocysteine synthase, enzymes of the autotrophic pathway, are still autotrophic for sulfur. However, deletion of both genes results in sulfur amino acid auxotrophic mutants exhibiting delayed biomass production and drastically reduced cephalosporin C synthesis. Furthermore, both single- and double-deletion strains are more sensitive to oxidative stress and form fewer arthrospores. Our findings provide evidence that autotrophic sulfur assimilation is essential for growth and cephalosporin C biosynthesis in production strain A3/2 from Acr. chrysogenum.

Secondary Metabolites from Acremonium Fungi: Diverse Structures and Bioactivities.

BACKGROUND: Acremonium fungi have been isolated from various sources, such as soil, plants, and marine organisms. METHOD: The species in Acremonium have been proved to be rich sources of novel and bioactive secondary metabolites. Up to now, 356 metabolites belonging to steroids (6 compounds), terpenoids (86), meroterpenoids (66), polyketides (89), alkaloids (28), peptides (75), and miscellaneous types (6) have been isolated from Acremonium fungi. These metabolites displayed a wide range of biological activities including antimicrobial, cytotoxic, antitumor, immunosuppressive, antioxidant, antiinflammatory, antimalarial, phytotoxic, tremorgenic, antiviral, neuritogenic, insecticidal and enzymesinhibiting activities. CONCLUSION: This review highlights the structures and bioactivities of the secondary metabolites from Acremonium fungi reported until July 2016.

Isolation and identification of two new compounds from marine-derived fungus Acremonium fusidioides RZ01.

Two new compounds, (22E)-25-carboxy-8ß,14ß-epoxy-4α,5α-dihydroxyergosta-2,22-dien-7-one (1) and fusidione (3), along with two known compounds, 5α,8α-epidioxy ergosta-6,22-diene-3ß-ol (2) and microperfuranone (4), were isolated from the fermentation products of the marine-sourced fungus Acremonium fusidioides RZ01. The structures of compounds 1 and 3 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data. Both new compounds showed inhibitory activity against HL-60 cells with IC50 values being16.6 and 44.9 µmol·L-1, respectively.

Bioaugmentation of soil contaminated with high-level crude oil through inoculation with mixed cultures including Acremonium sp.

Heavy contamination of soil with crude oil has caused significant negative environmental impacts and presents substantial hazards to human health. To explore a highly efficient bioaugmentation strategy for these contaminations, experiments were conducted over 180 days in soil heavily contaminated with crude oil (50,000 mg kg(-1)), with four treatments comprised of Bacillus subtilis inoculation with no further inoculation (I), or reinoculation after 100 days with either B. subtilis (II), Acremonium sp.(III), or a mixture of both organisms (IV). The removal values of total petroleum hydrocarbons were 60.1 ± 2.0, 60.05 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % for treatment (I-IV), respectively. Treatments (III-IV) significantly enhanced the soil bioremediation compared with treatments (I-II) (p < 0.05). Furthermore, significantly (p < 0.05) greater rates of degradation for petroleum hydrocarbon fractions were observed in treatments (III-IV) compared to treatments (I-II), and this was especially the case with the degradative rates for polycyclic aromatic hydrocarbons and crude oil heavy fractions. Dehydrogenase activity in treatment (III-IV) containing Acremonium sp. showed a constant increase until the end of experiments. Therefore reinoculation with pure fungus or fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation for soil heavily contaminated with crude oil.

Acremoxanthone E, a novel member of heterodimeric polyketides with a bicyclo[3.2.2]nonene ring, produced by Acremonium camptosporum W. GAMS (Clavicipitaceae) endophytic fungus.

Bioactivity-directed fractionation of the organic mycelium extract of the endophytic fungus Acremonium camptosporum W. Gams (Clavicipitaceae), isolated from the leaves of Bursera simaruba (Burseraceae), led to the isolation of six major heterodimeric polyketides, including one not previously characterized acremoxanthone derivative. In addition, the already known acremoxanthone C, acremonidins A and B, and acremoxanthones A and B were obtained. The structure of the new compound was established by extensive NMR studies, including DEPT, COSY, NOESY, HSQC, and HMBC methods. The trivial name proposed for this compound is acremoxanthone E. In addition, the structure of acremoxanthone C was unequivocally established for the first time, through X-ray crystal-structure analysis. The anti-oomycete activities of the pure compounds were tested against four economically important phytopathogenic oomycetes. Inhibitory concentration for 50% diameter growth reduction, IC50 , values for the four phytopathogens ranged from 6 to 38 µM. Also, in parallel, the cytotoxic activities against six cancer cell lines were evaluated showing IC50 values similar to those of cisplatin. To the best of our knowledge, this is the first report on three different groups of heterodimeric polyketides, linked by a bicyclo[3.2.2]nonene, such as xanthoquinodins, acremonidins, and acremoxanthones, which are isolated from an endophytic fungus. In addition, a common biosynthetic origin could be proposed.

Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes.

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.

The first promoter for conditional gene expression in Acremonium chrysogenum: iron starvation-inducible mir1(P).

The filamentous fungus Acremonium chrysogenum is of enormous biotechnological importance as it represents the natural producer of the beta-lactam antibiotic cephalosporin C. However, a limitation in genetic tools, e.g. promoters for conditional gene expression, impedes genetic engineering of this fungus. Here we demonstrate that in A. chrysogenum iron starvation induces the production of the extracellular siderophores dimerumic acid, coprogen B, 2-N-methylcoprogen B and dimethylcoprogen as well as expression of the putative siderophore transporter gene, mir1. Moreover, we show that the promoter of mir1, mir1(P), is suitable for conditional expression of target genes in A. chrysogenum as shown by mir1(P)-driven and iron starvation-induced expression of genes encoding green fluorescence protein and phleomycin resistance. The obtained iron-starvation dependent phleomycin resistance indicates the potential use of this promoter for selection marker recycling. Together with easy scorable siderophore production, the co-regulation of mir1 expression and siderophore production facilitates the optimization of the inducing conditions of this expression system.

[Microbial models in screening of inhibitors of sterol biosynthesis].

On the base of previously developed microbial models high effective scheme for screening of inhibitors of sterol biosynthesis (ISB) is proposed. It is based on cultivation of halophilic bacteria Halobacterium salinarum (former Halobacterium halobium), possessing mevalonate pathway of sterol biosynthesis, and cultivation of fungus Acremonium fusidioides (former Fusidium coccineum), that is producer of steroid antibiotic fusidin (fusidic acid), which biosynthesis has great similarity (with coincidence of its initial steps till squalene formation) to cholesterol biosynthesis in human organism. In H. salinarum model ISB are revealed as compounds that inhibit test-culture growth, whereas in A. fusidioides test-system they are revealed as compounds that strongly reduce fusidin production without any visible influence on producer's growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis remove inhibition induced by many microbial metabolites that is the evidence of their action at early stages of sterol biosynthetic pathway, including HMG-CoA reductase step. Both test-systems are developed as micromethod and could be easily mechanized due to miniaturization of microbiological procedures, cultivation in sterile 96-well plates and usage of automatic micropipettes and dispensers. Effectiveness of both test-systems, as well as their sensitiveness, laboriousness and ability to give false-positive or false-negative results in ISB screening work is compared. The proposed scheme of screening of ISB includes microbial models at early steps of screening procedures and Hep G2 test-system at the late step. The preliminary screening of microbial metabolites possessing antifungal activity at initial step is compulsory. Miniaturization and mechanization of microbial processes and purification of producers' culture broth with micro- and ultrafiltration are under consideration as well.

Comparative analysis of intracellular metabolites of Cephalosporium acremonium in pilot and industrial fermentation processes.

To get a better understanding of the characteristics of Cephalosporium acremonium with higher productivity, C. acremonium cells in pilot and industrial fermentation processes were analyzed using the profiles of metabolites. The different metabolic features of cells in pilot and industrial processes were caused by the different fermentation environments. The hierarchical cluster analysis of the data of metabolic profiling revealed that the concentrations of most of the metabolites were higher in the industrial process than in the pilot one, especially at the cephalosporin C accumulation stage. The analysis of important metabolites of primary metabolism indicated that the ability of the cephalosporin C biosynthesis was higher in the industrial process than that in the pilot one in C. acremonium. The analysis of the variations of cephalosporin C precursors and amino acids that were related to these precursors suggested that the metabolic flux changes of α-aminoadipic acid and cysteine between the primary metabolism and cephalosporin biosynthetic pathway in the industrial process. Furthermore, metabolites of C. acremonium, such as proline, spermine, inositol phosphate, and glycerol, were shown to respond to the fermentation environmental stress. These findings provide insights into the intracellular metabolite characteristics and feasible regulation scheme to improve the titer of cephalosporin C in the industrial process.

Production of cephalosporin C using crude glycerol in fed-batch culture of Acremonium chrysogenum M35.

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.

Characterization of an autoinducer of penicillin biosynthesis in Penicillium chrysogenum.

Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of ß-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.

Synthesis of a natural gamma-butyrolactone from nerylacetone by Acremonium roseum and Fusarium oxysporum cultures.

Natural gamma-butyrolactone - (4R, 5R)-5-(4'-methyl-3'pentenyl)-4-hydroxy-5-methyl-dihydrofuran-2-one (2) was isolated as the product of microbial transformation of nerylacetone (1) by fungal strains. This product was obtained as the enantiomer (+) in high yields 24% and 61% with ee=94% and 82% by the biotransformation in the cultures of Acremonium roseum AM336 and Fusarium oxysporum AM13 respectively.