Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.
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.
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 FeSO, 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°C, respectively. a-Ketoglutarate and MnCl2 showed no marked effect on the reaction, 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.In our earlier studies' 21, we demonstrated the enzymatic conversion of penicillin N to deacetoxycephalosporin C using cell-free extracts of Cephalosporium acremonium CW-19 and its antibiotic-negative mutant M-0198. The extracts catalyzing this ring-expansion reaction were prepared by lysing C. acrenionium protoplasts3'". The activity was relatively weak, however, and other investigators have had difficulty carrying out the reaction. HoOK et al." discovered that a cofactor mixture of ferrous sulfate, ascorbate, and a-ketoglutarate (a-KG) stimulated the reaction. We have further optimized the cofactor requirements and markedly increased the conversion's rate and extent. These experiments are described in the present paper along with additional data concerning the ring-expansion reaction.We expect that, with these modifications, the system will be widely used by antibiotic investigators.
Materials and MethodsCulture C. acremoniumn strain M-0198 (NRRL 11418), the 8-lactam-negative mutant of strain CW-19'', was used. Maintenance and growth methods were as previously described') with the exception noted in the next paragraph.
GrowthIn previous studies'), individual fermentation flasks were prepared and inoculated with 5 ml of seed culture. In the present work, the procedure was modified to improve reproducibility between flasks. The basal medium and sugars were autoclaved (separately) and combined in a large sterile flask, and the inoculum added.After thorough mixing, individual 45-m1 portions were aseptically * Present address:
This report describes an improved procedure for production of cytochalasin E and tremorgens by solid substrate, agitated fermentation ofAspergillus clavatus on pearled barley.
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