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PubMed1992

The cefG gene of Cephalosporium acremonium is linked to the cefEF gene and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase.

The gene (cefG) encoding the acetyl coenzyme A:deacetylcephalosporin C acetyltransferase of Cephalosporium acremonium (synonym Acremonium chrysogenum) C10 has been cloned. It contains two introns and encodes a protein of 444 amino acids with an M(r) of 49,269 that correlates well with the M(r) deduced by gel filtration. The cefG gene is linked to the cefEF gene (encoding the bifunctional deacetoxycephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the cefEF gene. Two transcripts of 1.2 and 1.4 kb were found in C. acremonium that correspond to the cefEF and cefG genes, respectively; the degree of expression of the cefG gene was clearly lower than that of the cefEF gene in 48-h cultures. The cloned cefG complemented the deficiency of deacetylcephalosporin acetyltransferase in the nonproducer mutant C. acremonium ATCC 20371 and restored cephalosporin biosynthesis in this strain. Heterologous expression of the cefG genes took place in Penicillium chrysogenum. The deacetylcephalosporin acetyltransferase showed a much higher degree of homology with the O-acetylhomoserine acetyltransferases of Saccharomyces cerevisiae and Ascobolus immersus than with other O-acetyltransferases. The cefEF-cefG cluster of genes encodes the enzymes that carry out the three late steps of the cephalosporin biosynthetic pathway and is not linked to the pcbAB-pcbC gene cluster that encodes the first two steps of the pathway.

Acetyltransferases↗
PubMed1975

Differentition of mutants of Cephalosporium acremonium in complex medium: the formation of unicellular arthrospores and their germination.

Differentiation of swollen hyphal fragments to unicellular arthrospores accompanied the synthesis of cephalosporin C by a series of Cephalosporium acremonium mutants during propagation in a complex medium. The complex medium supported significantly higher synthesis than the defined medium used in previous studies of differentiation in C. acremonium. The mutants differed in their ability to form unicellular arthrospores and to synthesize cephalosporin C, but a one-to-one correspondence between the two properties was not observed. An inverse relation was observed between the growth rates of the mutants and their ability to synthesize cephalosporin C: each mutant produced more antibiotic but grew more slowly than its parent strain. Germination of the unicellular arthrospores occurred in complex medium but differed significantly from the germination of conidia in seed medium. The unicellular arthrospores were examined by electron microscopy and compared with swollen hyphal fragments and slender hyphal filaments. The unicellular arthrospores had a thicker cell wall, rougher cell surface, and had one or more small identations in their surface. The internal structure of the unicellular arthrospore resembled those of the swollen hyphal fragment and slender hyphal filament. Filaments had lower concentrations of lipid-containing vacuoles which were prevalent in both the swollen hyphal fragments and the unicellular arthrospores.

Acremonium↗
PubMed1979

Relationship between carbon source and susceptibility of Cephalosporium acremonium to selected amino acid analogues.

The susceptibility of Cephalosporium acremonium to selected amino acid analogues was markedly influenced by the carbon source used in the test media. Lysine hydroxamate, beta-hydroxy norvaline, and hexafluorovaline were toxic when tested with ribose, ribose or fructose, and ribose or galactose, respectively. In contrast, thialysine and thiaisoleucine inhibited C. acremonium with glucose, fructose, galactose, sucrose, mannitol, sorbitol, and soluble starch. Neither of these analogues was toxic at levels tested when glycerol was used as a carbon source. The minimal inhibitory concentrations (MIC) of thialysine, homoserine, and alpha-methylserine were greater than 1000, greater than 1000, and 250 microgram/mL, respectively, with glycerol. In contrast, the MIC values for the same three analogues were 31, 62, and 125 microgram/mL, respectively, with mannitol. The matching of the carbon sources with the specific amino acid analogues expands the number of analogues useful for selecting derepressed mutants. Thialysine-resistant mutants (tlysR) of C. acremonium which excrete lysine were isolated on a medium containing mannitol.

Acremonium↗
PubMed1983

Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium.

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive. In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.

Acremonium↗
PubMed2001

Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37 degrees C. Soluble expression was restored at a reduced temperature of 25 degrees C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.

Acremonium↗
PubMed2005

Characterization of graphislactone A as the antioxidant and free radical-scavenging substance from the culture of Cephalosporium sp. IFB-E001, an endophytic fungus in Trachelospermum jasminoides.

The extracts derived from cultures of 1626 endophytic strains harbored in Trachelospermum jasminoides were assayed for more potent antioxidant and/or free radical-scavenging agents. The free radical-scavenging assessment was carried out using l,l-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical assays, and the antioxidant actions on linoleic acid and human low-density lipoprotein (LDL) models. After extensive spectroscopic analyses, graphislactone A was characterized as the most bioactive secondary metabolite of endophytic Cephalosporium sp. IFB-E001 with its free radical-scavenging (in a dose-dependent manner) and antioxidant activities ascertained in vitro to be stronger than those of butylated hydroxytoluene (BHT) and ascorbic acid, the two positive references coassayed in the study. From the demonstrated efficacy of graphislactone A in preventing and protecting against oxidative injury, it can be predicted that this metabolite could be a potential agent in the management of oxidative damage-initiated diseases.

Acremonium↗
PubMed1993

Substrate specificity of alkaline proteases from Cephalosporium sp. KM388.

Serine alkaline proteases from Cephalosporium sp. KM388 were specific against esters of aromatic and hydrophobic amino acids. Against oxidized insulin B-chain, the enzymes initially cleaved the site of Leu-Tyr(15-16). The cleavage specificity of KM388 protease D was broader than those of other alkaline proteases, and the site of Arg-Gly(22-23) was cleaved, which is a specific site for trypsin-like protease.

Acremonium↗
PubMed2001

A hybrid neural network algorithm for on-line state inference that accounts for differences in inoculum of Cephalosporium acremonium in fed-batch fermentors.

One serious difficulty in modeling a fermentative process is the forecasting of the duration of the lag phase. The usual approach to model biochemical reactors relies on first-principles, unstructured mathematical models. These models are not able to take into account changes in the process response caused by different incubation times or by repeated fedbatches. To overcome this problem, we have proposed a hybrid neural network algorithm. Feedforward neural networks were used to estimate rates of cell growth, substrate consumption, and product formation from on-line measurements during cephalosporin C production. These rates were included in the mass balance equations to estimate key process variables: concentrations of cells, substrate, and product. Data from fed-batch fermentation runs in a stirred aerated bioreactor employing the microorganism Cephalosporium acremonium ATCC 48272 were used. On-line measurements strongly related to the mass and activity of the cells used. They include carbon dioxide and oxygen concentrations in the exhausted gas. Good results were obtained using this approach.

Acremonium↗
PubMedDate not supplied

Site-directed mutagenesis of proline-285 to leucine in Cephalosporium acremonium isopenicillin-N-synthase affects catalysis and increases soluble expression at higher temperatures.

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N-synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline-285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

Acremonium↗
PubMedDate not supplied

Replacement of tyrosine-197 and the corresponding tyrosine-195 to isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

Acremonium↗
PubMed1975

Occurrence of a new cephalosporoate in a culture broth of a Cephalosporium acremonium mutant.

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.

Acremonium↗
PubMed1975

Effect of methionine on cephalosporin C and penicillin N production by a mutant of Cephalosporium acremonium.

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.

2-Aminoadipic Acid↗
PubMed1976

RIT 2214, a new biosynthetic penicillin produced by a mutant of Cephalosporium acremonium.

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.

2-Aminoadipic Acid↗
PubMed1977

Sulfur metabolism of a mutant of Cephalosporium acremonium with enhanced potential to utilize sulfate for cephalosporin C production.

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.

Acremonium↗
PubMed1984

Repression of beta-lactam production in Cephalosporium acremonium by nitrogen sources.

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.

Acremonium↗
PubMed1984

Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of beta-lactams in Cephalosporium acremonium.

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.

Acremonium↗
PubMed1987

Effect of ammonium as nitrogen source on production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase by Cephalosporium acremonium C-10.

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+).

Acremonium↗
PubMed1991

Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. Evidence for the presence of phosphopantothenate in ACV synthetase.

delta-(L-alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase was isolated and partially characterised from Cephalosporium acremonium CO728 and Streptomyces clavuligerus. The purification procedure resulted in a 745- and 277-fold increase in specific enzyme activity, respectively. Both enzymes had similar apparent molecular masses of ca. 300 kdaltons by SDS-polyacrylamide electrophoresis, under reducing and denaturing conditions, and in excess of 600 kdaltons in the native state by gel filtration. Attempts to obtain an N-terminal amino acid sequence of ACV synthetase from C. acremonium were unsuccessful, hence internal amino acid sequence data were obtained after tryptic digestion of the protein. Phosphopantothenic acid was shown to be associated with the enzyme from both sources, which suggests the possible involvement of pantothenate as a 'swinging arm' in the formation of the tripeptide ACV.

Acremonium↗