Further studies on microbiological ring-expansion of penicillin N.

Abstract

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:

Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N

“…The LLD-tripeptide cyclization reaction of crude lytic enzyme extracts was found to be stimulated, like ring-expansion activity (10,11), by Fe2", Triton X-100, and sonication. The optimum FeSO4 concentration for cyclization was 80 t,M as compared to 40 ,uM for ring-expansion (10).…”

“…The optimum FeSO4 concentration for cyclization was 80 t,M as compared to 40 ,uM for ring-expansion (10). Other differences between the two activities include lack of stimulation of the cyclization reaction by ascorbate and adenosine 5'-triphosphate.…”

“…O'Sullivan et al (9) isolated the product of the cell-free conversion and determined that the side chain of the penicillin was L-a-aminoadipic acid. Because oxidation of deacetoxycephalosporin C to deacetylcephalosporin C is stimulated by ferrous ions, ascorbate, and a-ketoglutarate (12) and because oxidative ring-expansion of penicillin N to deacetoxycephalosporin C is stimulated by ferrous ions and ascorbate (4,10) we checked the effect of these additives on the oxidative ring-closure reaction. We also tested the effect of Triton X-100, which, we found, stimulates ring-expansion by these same cell-free extracts (11).…”

Naturally Occurring β-Lactams

“…[U-14 C]-Glycerol was fed to a S. clavuligerus fermentation and the resulting 14C-Iabelled clavulanic acid was isolated as the crystalline p-bromobenzyl ester (293). This was degraded with dibenzylamine in methanol to yield the three ~-lactam carbon atoms in the form of the crystalline aminoacrylate (294). When the specific radioactivities of the starting material and degradation product were compared it was found that '" 95% of the radioactivity was retained in the degradation product.…”

Interference by methionine on valine uptake in Acremonium chrysogenum

“…The biosynthesis of cephalosporin C in Acremonium chrysogenum is a multistep metabolic pathway through which a linear molecule, b-(L-cx-aminoadipyl)-L-cysteinyl-Dvaline (ACV), which is derived by nonribosomal condensation of three amino acids (1,7,16,17), is cyclized to isopenicillin N (14,21) and subsequently expanded to deacetoxycephalosporin (25,27). A fourth amino acid, methionine, plays an important regulatory role in the biosynthesis of this antibiotic (5,12,22; G. A. Miller, B. K. Kelly, and G. C. F. Newton, British patent 759624,1956).…”