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PubMed2001

Enhancement of cephalosporin C production by cultivation of Cephalosporium acremonium M25 using a mixture of inocula.

AIMS: To enhance the productivity of Cephalosporin C (CPC) by cultivation of Cephalosporium acremonium M25 using a mixture of inocula. METHODS AND RESULTS: Inoculum age was classified into three stages (early, intermediate and late) by image analysis. A mixture of inocula, according to the inoculum ages, was used for efficient production of CPC in the main culture. The most effective mixing ratio of inocula for CPC production in shake flasks was a 3 : 7 volume ratio of early- and late-stage inocula. This was also the case in a 1.5 l stirred-tank reactor. CPC productivity was enhanced by about 32% and 34% when using an inoculum mixture in the shake flask and 1.5 l stirred-tank reactor, respectively. CONCLUSION: The morphological characteristics of C. acremonium M25 in the seed culture were quite different according to inoculum age. The compromise of different ages of inoculum showed better production of CPC. SIGNIFICANCE AND IMPACT OF THE STUDY: The productivity of CPC was enhanced considerably when using mixed inocula. The results of this study can be applied to fungal cultures for efficient production of various metabolites.

Acremonium↗
PubMed1978

Cell-free ring expansion of penicillin N to deacetoxycephalosporin C by Cephalosporium acremonium CW-19 and its mutants.

To examine microbiological ring expansion of penicillin N to a cephalosporin, we obtained five mutants of Cephalosporium acremonium blocked in beta-lactam antibiotic biosynthesis from 2500 survivors of mutagenesis. In submerged fermentation, mutants M-0198, M-0199, and M-2351 produced no beta-lactam antibiotic (type A), whereas mutants M-1443 and M-1836 formed penicillin N but not cephalosporin C (type B). Cell-free extracts of type A mutants converted penicillin N to a cephalosporin; those of type B mutants did not. The product of the cell-free reaction was identified as deacetoxycephalosporin C by thin-layer chromatography, paper chromatography, paper electrophoresis, and enzyme tests. These data strongly support our hypothesis that penicillin N is an intermediate of cephalosporin biosynthesis.

Acremonium↗
PubMed1996

Functional analysis of conserved histidine residues in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis.

The isopenicillin N synthase of Cephalosporium acremonium (cIPNS) involves a catalytically important non-heme iron which is coordinated credibly to histidine residues. A comparison of the IPNS genes from various microbial sources indicated that there are seven conserved histidine residues. These were individually replaced by leucine residues through site-directed mutagenesis, and the sites of mutation were confirmed by DNA sequencing. The seven mutant genes were cloned separately into the vector pET24d for expression in Escherichia coli BL21 (DE3), and the proteins were expressed as soluble enzymes. All the resulting mutant enzymes obtained have mobilities of approximately 38 kDa, identical with the wild-type enzyme on SDS-polyacrylamide gel electrophoresis, and were also reactive to cIPNS antibodies. The enzymes were purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography, and these were analyzed for enzyme activity. A group of mutant enzymes, H49L, H64L, H116L, H126L, and H137L, were found to be enzymatically active with reduced activities of 16-93.7%, indicating that they are not essential for catalysis. Two of the mutant enzymes, H216L and H272L, were found to have lost their enzymatic activity completely, indicating that both His-216 and His-272 are crucial for catalysis. It is suggested that these histidines are likely to serve as ligands for binding to the non-heme iron in the IPNS active site. Alignment of the amino acid sequence of IPNS to related non-heme Fe(2+)-requiring enzymes indicated that the two essential histidine residues correspond to two invariant residues located in highly homologous regions. The conservation of the two closely located histidine residues indicates the possible conservation of similar iron-binding sites in these enzymes.

Acremonium↗
PubMed1998

Cephalosporin C production by Cephalosporium acremonium: the methionine story.

More than 40 years ago, it was reported that methionine markedly stimulated production of cephalosporin C by Cephalosporium acremonium. Over the years, many hypotheses were put forth to explain this phenomenon. The accumulating evidence strongly supported the concept that methionine stimulates by inducing enzymes of the biosynthetic pathway such as delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase. This mechanism has been strengthened by the finding that transcription of the genes encoding the above enzymes is markedly enhanced by growth with methionine. An effect of methionine in the fermentation unrelated to the titer stimulation is its contribution of the sulfur atom to the cephalosporin molecule. Methionine also stimulates mycelial fragmentation; the relationship between this effect on hyphal differentiation and the induction of the cephalosporin synthases remains to be elucidated.

Acremonium↗
PubMed2002

Microbial transformation of (+)-androsta-1 ,4-diene-3,17-dione by Cephalosporium aphidicola.

Fermentation of (+)-androsta-1,4-diene-3,17-dione ([structure: see text]) with Cephalosporium aphidicola for 8 days yielded oxidative and reductive metabolites, androst-4-ene-3,17-dione ([structure: see text]), 17beta-hydroxyandrosta-1,4-diene-3-one ([structure: see text]), 11alpha-hydroxyandrosta-1,4-diene-3,17-dione ([structure: see text]), 11alpha-hydroxyandrost-4-ene-3,17-dione ([structure: see text]), 11alpha,17beta-dihydroxyandrost-4-ene-3-one ([structure: see text]) and 11alpha,17beta-dihydroxyandrosta-1,4-diene-3-one ([structure: see text]). The fermentation of [structure: see text] with Fusarium lini also yielded metabolites [structure: see text]. The structures of these metabolites were elucidated on the basis of spectroscopic techniques.

Acremonium↗
PubMed2005

Two new dihydroisocoumarins from the fungus Cephalosporium sp. AL031.

Two new dihydroisocoumarins, 3,4-dihydro-3-heptantrienyl-8-hydroxyl-6-hydroxymethyl-5-methylisocoumarin and 6-acetoxymethylene-3,4-dihydro-3-heptantrienyl-8-hydroxyl-5-methylisocoumarin were isolated from a culture broth of a strain of the fungus, Cephalosporium sp. AL031. Their structures were elucidated by interpretation of their spectroscopic data.

Acremonium↗
PubMed1998

Involvement of a third histidine in the ferrous active site of isopenicillin N synthase of Cephalosporium acremonium repudiated by recombinant double histidine mutants.

Site-directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential histidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking histidine at positions 216 and 272 clearly supports the essential role these two histidines play in iron binding. However, nuclear magnetic resonance (NMR) studies have indicated that there could be three histidine residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS genes containing mutations at two histidine codons were created by in vitro cloning of fragments from the expression vectors bearing the respective cIPNS genes each with a single histidine mutation at positions H49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre of cIPNS, then one of the constructed double histidine mutants would have lost its enzymatic activity. However, analysis of the cIPNS activities of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third histidine can be repudiated.

Acremonium↗
PubMed2000

Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.

Acremonium↗
PubMed1984

Cerulenin resistance in a cerulenin-producing fungus. II. Characterization of fatty acid synthetase from Cephalosporium caerulens.

Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems of various microorganisms and animal tissues. This antibiotic specifically blocks the activity of beta-ketoacyl thioester synthetase (condensing enzyme) by binding to the functional cysteine-SH in the active center of the condensing enzyme domain (the peripheral SH-group). However, fatty acid synthetase from C. caerulens is much less sensitive to cerulenin than fatty acid synthetases from other sources. The properties of C. caerulens synthetase were investigated and compared to those of Saccharomyces cerevisiae synthetase, which is sensitive to the antibiotic. The molecular weight of the enzymically active form of C. caerulens synthetase was 2.53 X 10(6). The enzyme consisted of two multifunctional proteins, alpha and beta, which are arranged in a complex, alpha 6 beta 6. The synthetase was inactivated by iodoacetamide. At 0 degrees C and pH 7.15, the second-order rate constant of k = 15.6 M-1 X s-1 was obtained for the inactivation by iodoacetamide. This value was about 15 times greater than that for S. cerevisiae synthetase. Treatment of C. caerulens synthetase with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity. When S. cerevisiae synthetase was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide (Kawaguchi, A., Tomoda, H., Nozoe, S., Omura, S., & Okuda, S. (1982) J. Biochem. 92, 7-12). In the case of C. caerulens synthetase, on the other hand, malonyl-CoA decarboxylase activity was induced by iodoacetamide even after the preincubation of the enzyme with cerulenin.(ABSTRACT TRUNCATED AT 250 WORDS)

Acremonium↗
PubMed1985

Protoplast fusion as a tool for genetic analysis in Cephalosporium acremonium.

Protoplasts of nutritionally complementary strains of Cephalosporium acremonium were fused and plated onto media which supressed the growth of both parents. The regenerating colonies were used for genetic analysis and were found to be of two types, stable haploid recombinants and unstable heterozygotes (aneuploids and/or diploids). Analysis of these colonies provided evidence for eight linkage groups and a relatively high rate of mitotic crossing-over. The gene order for three of the markers on one linkage group was also determined.

Acremonium↗
PubMed2005

Morphology and kinetics studies on cephalosporin C production by Cephalosporium acremonium M25 in a 30-l bioreactor using a mixture of inocula.

AIMS: In this study, the relationship between morphology and cephalosporin C (CPC) production in a 30-l bioreactor culture of Cephalosporium acremonium M25 using a 3:7 seed mixture was investigated. In addition, the kinetic model was established and applied. METHODS AND RESULTS: CPC production was performed in a 30-l bioreactor using a 3:7 seed mixture. It was recognized that a 3:7 seed mixture was able to reduce lag phase and enhance CPC production. The maximum CPC production and cell mass were 1.96 and 81.5 g l-1 respectively. Through a morphology study by observation using image analysis, it was concluded that changes of morphological features predicted the progressive production of CPC and that a morphology study could be useful in monitoring the CPC fermentation by C. acremonium M25. In the kinetics study, a kinetic model of CPC fermentation was developed and applied. The proposed model could adequately describe the fermentation of C. acremonium M25 in a 30-l bioreactor. CONCLUSIONS: CPC productivity was improved by using a 3:7 seed mixture in a 30-1 bioreactor. The changes in morphological features showed a very similar tendency with CPC production. A kinetic model of CPC fermentation was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study suggest that the use of a 3:7 seed mixture inocula has considerable possibilities for improving CPC productivity if applied to industrial scale fermentations. Through morphology and kinetics study, the kinetic model to describe the morphological differentiation and CPC production by C. acremonium M25 was established.

Acremonium↗
PubMed1994

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity.

Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe(2+)-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli. When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.

Acremonium↗
PubMed1997

Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is instrumental in the catalytic conversion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to a bioactive intermediate isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. It has recently been shown that this reaction is dependent on a conserved aspartate, D214, in a bacterial Streptomyces jumonjinensis IPNS. Thus, this study was carried out to provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremonium IPNS (cIPNS). Initially, alteration of the aspartate residue to generate the mutant D218L cIPNS protein was achieved by site-directed mutagenesis. Subsequent enzyme assays indicated that the catalytic property of the mutant protein was lost, attesting to the need for the corresponding conserved aspartate to maintain IPNS functionality. It is also evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consideration when site-directed mutant proteins are analysed for catalytic function.

Acremonium↗
PubMed1998

Mutational evidence for the role of serine-283 in Cephalosporium acremonium isopenicillin N synthase.

Creation of isopenicillin N from delta-(L-alpha-aminodipyl)-L-cysteinyl-D-valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non-heme iron-containing dioxygenase. A tripeptide R-X-S motif which consists of arginine-281 and serine-283 (Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site-directed mutagenesis arginine-281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine-283 in cIPNS was also altered by site-directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the eliminatin of the mutant's substrate-binding capability similar to that of arginine-281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R-X-S motif in the functionality of cIPNS.

Acremonium↗
PubMed1999

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.

Acremonium↗
PubMed1973

Antiviral effects of aphidicolin, a new antibiotic produced by Cephalosporium aphidicola.

Aphidicolin is an antibiotic of novel structure produced by the mold Cephalosporium aphidicola. It is a potent inhibitor of cellular deoxyribonucleic acid synthesis, and it also strongly inhibits the growth of herpes simplex virus both in tissue culture and in the rabbit eye. Aphidicolin is active against iododeoxyuridine-resistant herpes virus, and does not itself readily induce the formation of drug-resistant strains of herpesvirus.

Acremonium↗
PubMed1973

Synthesis of cephalosporin C from sulfate by mutants of Cephalosporium acremonium.

The innate ability of Cephalosporium acremonium to use methionine preferentially over sulfate for synthesis of cephalosporin C can be influenced through mutation. Mutants of C. acremonium with altered capacity to utilize sulfate for synthesis of antibiotic were isolated and partially characterized with respect to the uptake of sulfate and the regulation of arylsulfatase.

Acremonium↗
PubMed1974

Synthesis of deactoxycephalosporin C by a mutant of Cephalosporium acremonium.

A number of Cephalosporium acremonium mutants blocked in the synthesis of cephalosporin C were investigated for accumulation of other beta-lactam compounds. The non-cephalosporin C producers were isolated after exposing the superior cephalosporin C-producing strain M8650-4 to ultraviolet light (268 nm). One of the blocked mutants, MH63, accumulated deacetylcephalosporin C (0.4 mg/ml), deacetoxycephalosporin C (1.5 mg/ml), and penicillin N (2.7 mg/ml). In contrast, the parent of MH63 produced high levels of cephalosporin C as well as deacetylcephalosporin C (2.2 mg/ml) and penicillin N (1.0 mg/ml), but only traces of deacetoxycephalosporin C (about 0.1 mg/ml). Deacetoxycephalosporin C was isolated from the mutant strain and identified by ultraviolet light, nuclear magnetic resonance, bioactivity spectrum, and co-migration with authentic standard in three chromatography systems.

Acremonium↗