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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 IC 50 ranging from 1.2 to 21.6 μM.

The novel antifungal agent ASP2397 (Vical's compound ID VL-2397) is produced by the fungal strain MF-347833 that was isolated from Malaysian leaf litter and is identified here as an Acremonium species based on its morphology, physiological properties and 28S ribosomal DNA sequence. Because of its potential importance for producing novel antifungal agents, we determined the taxonomic and biologic properties of MF-347833. We show here that ASP2397 is a cyclic hexapeptide that chelates aluminum ion and is therefore similar to ferrichrome, a hydroxamate siderophore. However, ASP2397 differs structurally from licensed antifungal agents such as amphotericin B, triazoles and echinocandins. To understand the relationship between chemical structure and biological function, we isolated certain ASP2397 derivatives from the culture broth, and we further chemically converted the metal-free form to other derivatives.

A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and -75 ° C with a half-life of 770 days.

The interaction between the receptor for advanced glycation end-product (RAGE) and amphoterin has an important role in tumor growth and metastasis. Because the abrogation of the interaction results in the inhibition of the tumor growth and metastasis, we designed a screening system for an inhibitor of the interaction between RAGE and amphoterin. In the course of our screening of the inhibitor, we isolated a novel natural compound NBRI17671 (1) from the fermentation broth of Acremonium sp. CR17671. We also modified 1 into a more active NBRI17671al (2). Although 1 at 50 g ml(-1) weakly inhibited binding of various cells to amphoterin, 2 at 50 g ml(-1) inhibited it by >50% of control. Compound 2 effectively inhibited the tumor growth of glioma and lung tumor xenografts in mice at 25 mg kg(-1). Furthermore, 2 was found to downregulate mitogen-activated protein kinase (MAPK) activity in the tumor cells.

Awajanoran (1), a new dihydrobenzofuran derivative, was isolated from an agar-culture of Acremonium sp. AWA16-1, which had been isolated from sea mud collected at Awajishima Island in Japan. The structure of 1 was elucidated on the basis of a spectroscopic analysis. This compound inhibited the growth of A549 cells, the human lung adenocarcinoma cell line, with an IC50 value of 17 microg/ml, and also showed antimicrobial activity.

FR235222, a novel immunosuppressant which possesses potent inhibitory effects on the activity of mammalian histone deacetylases (HDACs), has been isolated from the fermentation broth of a fungus, Acremonium sp. No. 27082. FR235222 exhibited marked immunosuppressive effects on mouse ex vivo splenic T-lymphocyte proliferation, mouse delayed type hypersensitivity (DTH) response, rat adjuvant-induced arthritis (AA) and rat heterotopic cardiac transplantation. These results showed potential clinical use of this compound as a new type immunosuppressant in the fields of autoimmune diseases and organ transplantations.

A cyclic tetrapeptide FR235222, a novel immunosuppressant, has been isolated from the fermentation broth of a fungus, Acremonium sp. No. 27082. FR235222 showed potent and selective inhibitory effects on both T cell proliferation and lymphokine production. Further study has revealed this compound exhibits potent inhibitory effects on the activity of mammalian histone deacetylases (HDACs).

Novel antimicroalgal substances halymecins A (1), B (2) and C (3) were isolated from the fermentation broth of a Fusarium sp. and halymecins D (4) and E (5) from an Acremonium sp. The structures of these halymecins, Fig. 1, were determined based on extensive 2D NMR studies as well as mass spectral data. These chemical structures are conjugates of di- and trihydroxydecanoic acid. Halymecin A showed antimicroalgal activity against Skeletonema costatum.

Xenovulene A, a novel inhibitor of benzodiazepine binding to the GABA-benzodiazepine receptor is produced by submerged fermentation of Acremonium strictum. It was isolated from the mycelium by solvent extraction and purified by chromatography on Sephadex LH-20 and octadecyl silica. The structure of xenovulene A was determined to be a novel oxygenated sesquiterpene containing a humulene moiety by interpretation of various spectroscopic data, especially from 2D NMR experiments. Xenovulene A inhibited binding of the benzodiazepine, flunitrazepam, with an IC50 of 40 nM in an in vitro assay using bovine synaptosome membrane preparations.

A novel compound, PS-990, which induces differentiation of neuroblastoma cells, was isolated from the culture broth of a fungus, Acremonium sp. KY12702. PS-990 inhibited brain calcium calmodulin-dependent cyclic nucleotide phosphodiesterase with an IC50 value of 3 micrograms/ml, and markedly induced neurite extension of mouse neuroblastoma, Neuro2A, at concentrations ranging from 10 to 30 micrograms ml.

A radioligand test to detect inhibitors of endothelin-1 binding to its receptors in bovine atrial and porcine cerebral membranes was used to screen fungal metabolites from stationary fermentations. Inhibitory activity, observed in culture extracts of two Acremonium species, led to the discovery of aselacins A, B and C. Aselacin A inhibits binding to both membrane fractions with IC50s of approximately 20 micrograms/ml.

Cephalosporin production by Cephalosporium acremonium strain C-10 was suppressed when the organic nitrogen source (1.2% L-asparagine) was replaced by 1.2% (NH4)2SO4. A higher level of (NH4)2SO4 (3.5%) led to even greater suppression. Ammonium repression was exerted on formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, together with that of expandase; a lesser effect by ammonium was observed on cyclase production. Inhibition of ACV synthetase activity byammonium was also observed (ca. 50% inhibition at 250 mM NH4+).

A variety of inorganic and organic nitrogen sources were added to fermentation media to determine their regulatory effects on the production of beta-lactam antibiotics by Cephalosporium acremonium. (NH4)2SO4 at concentrations higher than 100 mM (1.3%) strongly inhibited beta-lactam production. L-Asparagine and L-arginine proved to be the best nitrogen sources tested for beta-lactam production. The optimum concentration of asparagine was 1.2%. Higher concentrations led to NH3 accumulation, increase in pH, and lower growth rates. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] to the (NH4)2SO4-containing medium stimulated beta-lactam production markedly and ammonium repression of the ring-expansion enzyme was reversed. It appears that the ring-expansion step is a very sensitive part of beta-lactam biosynthesis in C. acremonium with respect to nitrogen source repression. Other enzymes may also be sensitive in view of the fact that nitrogen source derepression not only led to increases in cephalosporin C but, to a lesser extent, penicillin N and total beta-lactam titers.

The previously reported inability of methionine to stimulate cephalosporin C production in a cysteine auxotroph is due to cysteine interference with methionine uptake. In such a cse, "illicit transport" of alanylmethionine can be used to demonstrate the efficacy of methionine in such mutants blocked in the path from methionine to cysteine. This result supports the hypothesis that the stimulatory activity of methionine is not due to its ability to donate sulfur to the caphalosporin C molecule.

A number of lysine-requiring auxotrophs of Cephalosporium acremonium were investigated for incorporation of side-chain precursors and for accumulation of beta-lactam compounds. One of the auxotrophs, Acremonium chrysogenum ATCC 20389, producing cephalosporin C and penicillin N only if grown in media supplemented with DL-alpha-amino-adipic acid (DL-alpha-AAA), was found to use L-S-carboxymethylcysteine (L-CMC) as a side-chain precursor for the synthesis of a new penicillin (RIT 2214). No corresponding cephalosporin was detected. The penicillin present in the culture filtrate, was concentrated by adsorption on activated carbon and successive column chromatography on Amberlite IRA-68 and Amberlite XAD-4. Final purification was achieved by cellulose column chromatography. RIT 2214 was identified as 6-(D)-[(2-amino-2-carboxy)-ethylthio]-acetamido]-penicillanic acid by spectral analysis, bioactivity spectrum, elucidation of side-chain structure and finally by semisynthesis. Its biological properties were also evaluated.

A new metabolite was isolated from the culture filtrate of a deacetylcephalosporin C-producing mutant, derived from Cephalosporium acremonium ATCC 14553, by means of adsorption on activated carbon, column chromatography on DEAE-Sephadex A-25 and gel filtration through a Sephadex G-10 column. The compound was identified as D-5-amino-5-carboxyvaleramido-(5-formyl-4-carboxy-2H, 3H, 6H-tetrahydro-1, 3-thiazinyl) glycine by spectral analyses, elucidation of hydrolysis products of the compound, and comparison of characteristics of the compound with those of a synthetic authentic compound.

A mutant with enhanced potential to utilize sulfate for cephalosporin C production was isolated from a strain of Cephalosporium acremonium. The mutant displayed potency levels more than twofold that of the parent in the presence of sulfate but its productivity was severely inhibited by more than 0.5% of methionine which gave high cephalosporin C production with the parent. In a complex medium norleucine stimulated cephalosporin C production by the mutant in the presence of sulfate, whereas it showed no effect on the parent. In an incubation system with sulfur-starved cells of the mutant, L-methionine, but not the D-isomer, gave lower cephalosporin C production and a delayed production of penicillin N. However, it exhibited a stimulatory effect in the presence of valine or alpha-aminoadipic acid, the constituent amino acids of the antibiotic. Norleucine showed a similar effect to that of L-methionine in the presence of sulfate. On the basis of these results, characteristics of the mutant are discussed in connection with the effect of methionine.

The transformation of rifamycin S into rifamycins B and L was reinvestigated in order to establish more detailed pathways. Our results exclude rifamycin O as a common progenitor in the biosyntheses of rifamycins B and L. Rifamycins B and L are formed from rifamycin S (SV) by different pathways using different C3-precursors for the biosynthesis of their glycolic acid moieties. A thiamine-dependent enzyme (decarboxylase) seems to be involved in the transformation reaction.

The catalytic properties of the partially purified deacetoxycephalosporin C (DAOC)-synthetase and DAOC-hydroxylase from an industrial strain of Cephalosporium acremonium were studied. After mechanical breakage of the cells, purification was achieved by fractional (NH4)2SO4 precipitation, gel chromatography on Sephadex G-75, ion exchange chromatography on DEAE-Trisacryl M and two isoelectric focusing steps. The two enzyme activities could not be separated. Indirect evidence was obtained from SDS-polyacrylamide gel electrophoresis of the purest fractions obtained by isoelectric focusing that the two reactions are catalyzed by a single enzyme with a molecular weight of 33,000 +/- 2,000 and a pI of 4.6 +/- 0.1. Both reactions require alpha-ketoglutarate, FeSO4, ascorbate and O2, whereas additional ATP shows only a slight stimulation.

Microbonded propylamine silica with a solvent system containing acetic acid, methanol, acetonitrile and water (2:4:7.5:86.5) is suitable for an efficient separation of mixtures containing several closely related cephem derivatives. The same system with preparative columns was used for the isolation of cephalosporin C directly from the filtered broth of C. acremonium fermentation.

Characteristics of a mutant of Cephalosporium acremonium with enhanced potential to utilize sulfate for cephalosporin C production were investigated with sulfur-starved cells. DL-Norleucine showed an inhibitory effect on cephalosporin C and penicillin N production by the mutant in the presence of a sulfur source such as sulfate, sulfite, thiosulfate, and L-cystine, but it exhibited no effect when it was added after a certain period of incubation. On the contrary, antibiotic production by the parent was stimulated by norleucine regardless of the addition time. An increase in the intracellular cysteine pool was found when the cells were incubated with L-methionine or norleucine and sulfate. Enzymatic studies revealed that methionine and norleucine stimulated the cysteine desulfhydrase formation, and this effect was significant in the mutant. Finally the mutant was found to have an enhanced L-serine sulfhydrylase activity. The increase in this enzyme activity in the mutant seems responsible for the increase in the sulfate-utilizing ability and the methionine sensitivity by maintaining a high level of the cysteine pool. Accordingly, the effect of methionine and norleucine is assumed to be exerted through cysteine.

A new cepham metabolite has been isolated from the filtered broth of Cephalosporium acremonium by high performance liquid chromatography (HPLC) and identified as 7 beta-(5-D-amino-adipamido)-3 beta-hydroxy-3 alpha-methyl-cepham-4 alpha-carboxylic acid (I). Pure penicillin N was prepared using HPLC in the analytical mode. When I was added in place of penicillin N as substrate for the cell-free biosynthetic of cephalosporin, no formation of deacetoxycephalosporin C (II) was observed. A synthetic cepham derivative, 7 beta-(5-D-aminoadipamido)-3-exomethylene-cepham-4 alpha-carboxylic acid (III) was also tested in the cell-free system as a possible intermediate. The compound III was shown to be an inhibitor of the ring expansion enzyme that converts penicillin N to deacetoxycephalosporin C.

Incubation of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine (DLD-ACV) with a partially purified extract of Cephalosporium acremonium resulted in the production of deacetoxycephalosporin C. The extract contained isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) but no penicillin epimerase activity, and was incubated aerobically in the presence of the components of the cyclase and expandase reaction mixtures (Fe++, ascorbate, dithiothreitol, alpha-ketoglutarate and ATP). The reaction was sensitive to penicillinase, indicating penicillin N to be an intermediate. However, when ring expansion was prevented by omission of alpha-ketoglutarate and ATP, no penicillin N was detected.

A mutant of Cephalosporium acremonium producing high amounts of penicillin N was isolated. This antibiotic was purified and characterized. It was possible to convert this penicillin N to deacetoxycephalosporin C enzymatically. The reaction could be carried out with enzyme systems prepared from C. acremonium mutants producing either no beta-lactam antibiotics or excreting only penicillin N. It was surprising that a high level of transformation capacity was just found in a cephem negative mutant which overproduces penicillin N. For that reason the inability of the latter mutant to produce cephem compounds cannot be explained by a functional block of the ring expanding enzyme complex. The enzyme preparations used to carry out this reaction were made by ether-treatment or sonication of C. acraemonium cells, or by submitting them to osmotic shock. The ring expanding enzyme system is strongly dependent on ATP and behaves as a 2-oxoglutarate dependent dioxygenase.

The rate of microbiological ring-expansion of penicillin N to deacetoxycephalosporin C using protoplast lysates of the antibiotic-negative mutant Cephalosporium acremonium M-0198 has been increased some 70-fold over that of our earlier system. We confirmed the stimulatory effects of FeSO4 and ascorbate described by Hook et al. (Biochem. Biophys. Res. Commun. 87: 258, 1979); the optimum concentrations found were 0.04 mM FeSO4 and 0.67 mM ascorbate. Adenosine triphosphate concentration was lowered to 0.83 mM; phosphoenolpyruvate and pyruvate kinase were eliminated. The optimum pH and temperature for the reaction were 7.2 and 25 degrees C, respectively. Alpha-ketoglutarate and MnCl2 showed no marked effect on the reactions, MgSO4 and KCl were mildly stimulatory, and CuSO4 and ZnSO4 were very inhibitory. Penicillin N was optimal at a concentration of 0.07 mM. Specific ring-expansion activity reached its peak 13 hours after growth ceased and then disappeared rapidly.

Addition of a natural zeolite, a known ammonium ion-trapping agent, to a complex medium resulted in a several fold increase in cerulenin production by Cephalosporium caerulens. In the presence of zeolite, ammonium ion in the medium decreased, while mycelial growth increased to a small extent, and pH values remained constant. Small amounts of ammonium bicarbonate inhibited cerulenin production without affecting mycelial growth and pH values. It is thus assumed that zeolite stimulated cerulenin production by releasing the biosynthesis from the suppression by ammonium ions in C. caerulens.