Virus-Like Particles in Cephalosporium acremonium

Cephalosporium acremonium cultures were studied for the presence of virus-like particles. Relatively few particles were found in the preparations, indicating that the number of particles present in these cells may be much lower than in Penicillium species.     

Extrachromosomal genetics of Cephalosporium acremonium

Summary A hybrid vector carrying a 1.9 kb ars from the mitochondrial DNA (mtDNA) of Cephalosporium acremonium was shown to be relatively stable in yeast even without selective pressure. Subcloning of parts of this 1.9 kb fragment indicated that ars activity (i.e., high transformation rate) is associated with a 675 bp HinfI-fragment. Sequence analysis of the ars-subfragment revealed several ars-typical features, a long open reading frame and, most interestingly, homology to a mitochondrial origin of replication.     

Effect of Methionine and Sulfate on the Metabolism of Cephalosporium acremonium

The metabolism of Cephalosporium acremonium grown in a complex medium supplemented with DL-methionine or inorganic sulfate was studied. More growth occurred in a sulfate medium than in a methionine medium. Methionine-grown cells had an increased rate of respiration, a higher rate of catabolism with acetate and glucose as substrate, and higher specific activities of certain respiratory enzymes than sulfate-grown cells. Labeled acetate and glucose were assimilated at a faster rate by methionine-grown cells than sulfate-grown cells. Taurine, cystathionine, and small quantities of four acidic compounds were present in the amino acid pool of methionine-grown cells, but they were not detected in the pool of sulfate-grown cells. The differences in metabolic activity of sulfate and methionine-grown cells are discussed in regard to cephalosporin C synthesis.     

Saprophytic behaviour of Cephalosporium maydis and C. acremonium

The competitive saprophytic ability (CSA) of Cephalasporium maydis was smaller at 30 C when measured by the agar-plate modification than by the original Cambridge method. The agar-plate method suggested that C. maydis was a less competitive saprophyte than C. acremonium although both were low in CSA.
C. acremonium grows and sporulates well on organic and synthetic media. C. maydis grows faster but is more exacting nutritionally and is less able to decompose cellulose or maize straw than C. acremonium . Neither fungus produced pectolytic culture filtrates and both were susceptible to antibiotics produced by soil micro-organisms.
C. maydis survived on maize straw much longer than C. acremonium . In field soils C. maydis colonized and survived in supplemented wheat bran poorly and not below the top 20 cm of soil.     

Pathogenic behaviour of Cephalosporium maydis and C. acremonium

Cephalosporium maydis infects young maize plants easily, but as plants age fewer are infected and none after approx. 50 days from sowing. The mesocotyl and seminal, fibrous and adventitious roots are attacked, especially when there is damage or much inoculum. Most penetration occurs where roots are elongating and emerge from the mesocotyl or from fibrous roots. At first the fungus grows superficially on roots, producing hyphae with short, brown, thick-walled, and swollen cells. After penetrating, the fungus spreads towards the xylem, where it grows slowly at first but after 5 weeks grows faster upwards.
C. acremonium causes black-bundle disease of maize. It seems to infect plants growing in unfavourable conditions but the details remain uncertain. The percentage of plants infected was not related to the amount of inoculum and the fungus may not be a primary parasite. The sterile culture filtrate of the fungus produces vascular discoloration and wilt of maize seedlings.     

Structure of a Cephalosporium acremonium mtDNA replicator

We have investigated the ARS (autonomously replicating sequence) activity of a 1.94 kb mitochondrial DNA fragment of Cephalosporium acremonium and found that several subfragments of this piece of mtDNA conferred the ARS phenotype. The nucleotide sequence of the fragment shows: (i) a high A + T content (72.5%); (ii) a perfect consensus ARS sequence (ATTTATATTTA) in the subfragment with the highest ARS activity; (iii) a large number of ARS consensus-related sequences in the other subfragments, even in one lacking ARS activity; (iv) several potential hairpin structures. One of them contains the perfect consensus ARS sequence.     

Lysine stimulation of cephalosporin C synthesis in Cephalosporium acremonium

Summary Lysine added to a defined medium at a concentration of 1 mg/ml enhances the capacity of Cephalosporium acremonium to produce cephalosporin C. The lysine effect was accompanied by a delayed differentiation to arthrospores followed by more extensive mycelial fragmentation. Higher lysine concentrations reduced the synthesis of cephalosporin C. The effect of lysine on cephalosporin C synthesis was not strain related and was not elicited by the other amino acids tested. Both lysine stimulation and inhibition were evidenct using resting cells. The lysine inhibition of cephalosporin C synthesis with resting cells was relieved by supplementing the medium with 1 to 2 mg/ml -aminoadipic acid. This reversal supports the hypothesis that lysine restricts the availability of -aminoadipic acid for -lactam biosynthesis.     

Characterization of the Cephalosporium acremonium ribosomal RNA genes

To investigate the organization of the ribosomal RNA genes in Cephalosporium acremonium, we cloned the whole r X DNA repeat in pBR322 and pNEO plasmids. Both the cloned and the genomic r X DNA fragments were characterized by restriction mapping. The r X DNA repeat unit was found to be 8.0 kb long and there was no significant heterogeneity among the individual repeats.     

Antibiotic Synthesis and Morphological Differentiation of Cephalosporium acremonium

In submerged cultures, Cephalosporium acremonium exists in four morphological forms: hyphae, arthrospores, conidia, and germlings. The phase of hyphal differentiation into arthrospores coincides with the maximum rate of β-lactam antibiotic synthesis. Furthermore, arthrospores, separated by density-gradient centrifugation, possess 40% greater antibiotic-producing activity than any other morphological cell type. In a series of mutants, each with an increased potential to produce β-lactam antibiotics, differentiation into arthrospores was proportional to the increased titer of these antibiotics. Thus, arthrospores exhibit enhanced synthesis of β-lactam antibiotics and appear to be a determining factor in high-yielding mutants. Since a non-antibiotic-producing mutant readily differentiated into arthrospores, antibiotic synthesis and cellular differentiation are not obligately related.     

Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium

NADP-isocitrate dehydrogenase [isocitrate:NADP(sup+) oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg(sup2+) is the true substrate. The V(infmax) depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK(infes) (pK of the enzyme-substrate complex) of 6.9 and a (Delta)H(infion) (activation enthalpy) of -2 (plusmn) 0.4 kcal mol(sup-1) (ca. 8 (plusmn) 2 kJ mol(sup-1)). The enzyme showed maximum activity at 60(deg)C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP(sup+) were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40(deg)C following a first-order kinetics. The presence of 12 mM Mg(sup2+) plus 10 mM dl-isocitrate led to 100% protection of enzyme activity against inactivation at 60(deg)C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg(sup2+) was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.     

Production of cephalosporin C by immobilized cells of Cephalosporium acremonium

Cephalosporium acremonium ATCC 48272 cells were immobilized on various adsorbents and in various entrapment matrices. The influence of the incubation period, the best immobilization technique and the optimum concentrations of the selected matrices were investigated. From the results of the repeated batch fermentation in shake flasks, a good level of antibiotic was maintained for a period of about 19 days using 4% calcium alginate and 1% glass wool as entrapment and adsorbent supports, respectively.     

Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.     

头孢菌素C产生菌的诱变育种及培养基优化

通过对顶头孢霉（Cephalosporium acremonium）FC-01进行诱变选育及特定种子培养基的优化,提高了头孢菌素C的发酵产量。分别采用紫外-氯化锂和钴-60（60Co）γ射线对FC-01进行诱变选育,筛选到高产菌株FC-1-4和FC-4-2,产量较出发菌株分别提高了26%和54.5%。运用Plackett-Burman设计方法和响应面法对种子培养基进行优化,头孢菌素C发酵效价较对照分别提高了34.7%和13.2%,优化后的种子培养基主要成分为玉米浆3.70%、葡萄糖2.62%和硫酸镁0.15%,得到的菌株及相应的种子培养条件已成功应用在160M³工业发酵罐生产中,具有重要的工业生产能力。     

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.