Biosynthesis of Cephalosporin

OTT, J. L. (Eli Lilly and Co., Indianapolis, Ind.), C. W. GODZESKI, D. PAVEY, J. D. FARRAN, AND D. R. HORTON. Biosynthesis of cephalosporin C. I. Factors affecting the fermentation. Appl. Microbiol. 10:515-523. 1962.-A series of complex and synthetic media have been developed that are suitable for the production of cephalosporin C and cephalosporin N by a mutant strain of Cephalosporium (C.M.I. 49,137). DL-Methionine increased the yield of both antibiotics, with more effect on cephalosporin N. L-Cystine had a greater enhancing effect on formation of cephalosporin C than on formation of cephalosporin N in synthetic media; serine increased yields of cephalosporin C under certain conditions. Disaccharides or polysaccharides appeared to be the best source for carbohydrates. No evidence was found for precursor action such as is found in penicillin fermentations. The ability of resting cells to produce antibiotic was demonstrated. Cephalosporin C was first reported by Newton and Abraham (1955) in crude preparations of cephalosporin N. Abraham and co-workers have described the isolation, properties, and structure of cephalosporin C (Newton and Abraham, 1956; Abraham and Newton, 1956a, b, 1961; Jeffery, Abraham, and Newton, 1960). Cephalosporin N and a series of other solvent-extractable antibiotics designated as cephalosporin P were reported by Burton and Abraham (1951) to be produced by a strain of Cephalosporium (C.M.I. 49,137). Another culture, identified as C. salmosynnematum Roberts, was shown to produce synnematin B (Gottshall et al., 1951; Olson, Jennings, and Junek, 1953). This antibiotic was later shown to be identical with cephalosporin N (Abraham et al., 1953, 1955). The perfect stage of this fungus was demonstrated by Grosklags and Swift (1957), and was classified as Emericellopsis salmosynnemata. They also reported that many species produced antibiotic activity similar to cephalosporin N. Kavanagh, Tunin, and Wild (1958a) confirmed these observations of the production of cephalosporins N and P. Fermentation studies on the production of cephalosporin N have been reported by a number of workers (Gottshall et al., 1951; Pisano, Olson, and San Clemente, 1954; Bhuyan and Johnson, 1958; Kavanagh, Tunin, and Wild, 1958b; Harvey and Olson, 1958; Nara and Johnson, 1959). Very little has been published on Cephalosporium C.M.I. 49,137, from which cephalosporin C was isolated (Crawford et al., 1952; Florey et al., 1956; Kavanagh et al., 1958a, b). The studies reported here were carried out with a mutant strain from Cephalosporiumn C.M.I. 49,137, which produced cephalosporins C, N, and P. Several media suitable for the production of cephalosporins C and N will be described. MATERIALS AND METHODS A strain of Cephalosporium (C.M.I. 49,137; mutant 8650), obtained from E. P. Abraham, was used throughout this investigation. The mutant produced cephalosporin N, cephalosporin C, and antibiotics of the cephalosporin P type. The culture was preserved by lyophilization in bovine serum. Stock slants were prepared from lyophilized cells on a modified LePage and Campbell (1946) medium. The medium was prepared at one-tenth the original strength, except for use of 2 % agar and the addition of 1 % calcium chloride; slants were prepared with 20 ml of medium in 29 X 162 mm tubes. Slants were incubated at 25 C for 14 days and remained productive after several months at 4 C. The surface growth from a slant was suspended in 12 ml of sterile water; each flask of seed medium was inoculated with 2.0 ml of this suspension. Fermentations were carried out in 250-ml wide-mouth Erlenmeyer flasks with cotton stoppers. Separate flasks were used for each time period of sampling. In most cases, duplicate or triplicate flasks were taken for assay. A rough estimation of mycelial growth was obtained by measuring packed solids after centrifugation of a fermentation sample at 1,500 X g for 10 min in a graduated conical centrifuge tube. Mycelial weights were not determined. The pH of the fermentations was determined with a pH meter. Data on packed solids and pH are not reported with every experiment. The pH of the fermentations varied from 6.5 to 8.2 at the time of harvest. With all supplements tested, the packed solids and pH of the fermentation flasks were quite similar to those of control flasks unless otherwise noted. Antibiotic assay. Antibiotic activity was determined by 515 on A uust 4, 2017 by gest ht://aem .sm .rg/ D ow nladed fom