Biosynthesis of penicillin N and cephalosporin C. Antibiotic production and other features of the metabolism of a Cephalosporium sp.

Abstract

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.

Formation and Properties of Protoplasts from Antibiotic-producing Strains of Penicillium chrysogenum and Cephalosporium acremonium

“…49,137. The maintenance and growth of the Cephalosporium in shake flasks were as described by Smith et al (1967).…”

“…49,137. The maintenance and growth of the Cephalosporium in shake flasks were as described by Smith et al (1967).The strain of Penicillium chrysogenum (c21) was obtained from Glaxo Research Ltd. Lyophilized cultures were stored in ampoules. They were revived by the addition of I ml distilled water and transferred to the surface of agar slopes for 7 to 14 days at 27 "C (Gailey, Stefaniak, Olson & Johnson, 1946).…”

A spectrophotometric assay for deacetoxycephalosporin C synthase

“…This was performed as described [3,7] using supersensitive E. coli ESS 21/30. In this procedure DAC as well as DAOC was determined in the assay, but the relative amount of the former was very low under the conditions used.…”

“…Studies on DAOC synthase have been based on the hole plate bioassay [7], TLC, HPLC or NMR analysis of products after enzyme incubations. All these methods are discontinuous and therefore unsatisfactory for initial rate determinations.…”

Genetic and Biochemical Studies of Mutants of Penicillium chrysogenum Impaired in Penicillin Production

“…1 paper using 7.5 mg of the performic acid-oxidized sample. Electrophoresis was carried out in pH 1.8 buffer [20% (w/v) acetic acid containing 2 % (w/v) formic acid] at 70 V cm-l as described by Smith et al (1967) on an apparatus similar to that of Katz et al (1959). Paper chromatograms were run in butan-1-ol/acetic acid/water (4: 1 :4, by vol.)…”