Cephamycin fermentation

Occurrence, Fermentation, and Biosynthesis. Although a large number of Streptomjces species have been shown to produce carbapenems, only S. cattkja (2) and S. penemfaciens (11) have been reported to give thienamycin (2). Generally the antibiotics occur as a mixture of analogues or isomers and are often co-produced with penicillin N and cephamycin C. Yields are low compared to other P-lactams produced by streptomycetes, and titres are of the order of 1—20 p-g sohdusmL despite, in many cases, a great deal of effort on the optimization of the media and fermentation conditions. The rather poor stabiUty of the compounds also contributes to a low recovery in the isolation procedures. The fermentation and isolation processes for thienamycin and the olivanic acids has been reviewed in some detail (12). 

At present all of the cephalosporins ate manufactured from one of four P-lactams, cephalosporin C (2), penicillin V [87-08-17, penicillin G [113-98-4] and cephamycin C (8), which ate all produced in commercial quantities by fermentation (87). The manufacturing process consists of three steps fermentation, isolation, and chemical modification. 

Fermentation. The commercial P-lactam antibiotics which act as starting material for all of the cephalosporins ate produced by submerged fermentation. The organisms used for the commercial production of the penicillins and cephalosporins ate mutants of PenicU/in chTysogenum and Cephalosporium acremonium respectively (3,153,154). Both ate tme fungi (eucaryotes). In contrast, the cephamycins ate produced by certain species of procaryotic Streptomyces including Streptomyces clavuligerus and Streptomyces lipmanii (21,103). 

Isolation. Isolation procedures rely primarily on solubiHty, adsorption, and ionic characteristics of the P-lactam antibiotic to separate it from the large number of other components present in the fermentation mixture. The penicillins ate monobasic catboxyHc acids which lend themselves to solvent extraction techniques (154). Pencillin V, because of its improved acid stabiHty over other penicillins, can be precipitated dkecdy from broth filtrates by addition of dilute sulfuric acid (154,156). The separation process for cephalosporin C is more complex because the amphoteric nature of cephalosporin C precludes dkect extraction into organic solvents. This antibiotic is isolated through the use of a combination of ion-exchange and precipitation procedures (157). The use of neutral, macroporous resins such as XAD-2 or XAD-4, allows for a more rapid elimination of impurities in the initial steps of the isolation (158). The isolation procedure for cephamycin C also involves a series of ion exchange treatments (103). 

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

The cephamycins comprise a set of beta lactamase resistant fermentation products that consist of cephalosporins that include a methoxy group on the carbon bearing the amine. These agents were not suitable as drugs in their own right because of their poor biopharmaceutical properties. This was due to the circumstance that the natural products, like the original cephalosporins, occurred as their highly... 

For bioprocessing purposes, increase in the stability of biocatalysts is quite often achieved by immobilization of cells or enzymes [57-60], This technology is an attractive alternative to the use of expensive free enzymes and cofactors, and can coordinate multistep enzymatic processes into a single operation. Furthermore, fermentative biosynthesis of cephamycin C using immobilized cells of S. clavuli-gerus NRRL 3585 was accomplished by Freeman and Aharonowitz [61], Jensen et al. [62] reported on the immobilization of P-lactam synthesizing enzymes from the same wild-type culture. None of these early studies used penicillin substrates other than the normal intermediate penicillin N, such as penicillin G. 

One approach has been to modify the structure of the yS-lactam antibiotic so as to increase its stability and thus confer resistance to yS-lactamases, whilst retaining its inhibitory activity against transpeptidases. This was achieved by introducing sterically crowded 6-acyl substituents, as in methicillin (1) and the isoxazolyl penicillins (2) and (3) [20], or by the introduction of a methoxy substituent to the a-face of the -lactam, as in cefoxitin (9) [21] and temocillin (11) [22]. The two latter compounds were a direct result of the discovery of the y5-lactamase stable cephamycins (10), isolated by fermentation of Streptomyces in 1970 by Merck and Lilly [23]. 

In 1972, cephamycin C, a member of a family of antibiotics structurally closesy related to cephalosporin C, was isolated. The cephamycins were produced in submerged fermentation in a wide variety of media by one or more of eight different species of Streptomyces, including Streptomyces lactamdurans. To cephamycin C was assigned the structure 82 [135]. Cephamycin C and other members of this family... 

Figure 4.26 Structure of fermentation-derived penicillins and cephems penicillin F 67, penicillin G 68, dihydropenicillin F 69, penicillin K 70, isopenicillin N 71, penicillin X 72, penicillin N 73, penicillin KPN 74, penicillin V 75, isopenicillin RIT-2214 76 cephalosporin C 77, desacetoxycephalosporin C 78, deacetylcephalosporin C 79, cephalosporin PA-41937 80, 7-demethoxycephamycin C 81, cephamycin C 82, deacetoxycephamycin C 83, and deacetyl-cephamycin C 84. Some structures are of fermentation-derived cephabacins.

The discovery of cephamycins in streptomycetes fermentation broth and the announcement of their close structural relationship to cephalosporin C spurred efforts in many laboratories to develop chemical methods for methoxylating C-6 penicillins and C-7 cephalosporins. This chemistry engendered yet other methods for introducing a wide variety of substituents at this position. A number of 7-methoxycephalosporins possess interesting and useful biological activity. On the other hand, sporadic attempts to modify cephalosporins at C-2 proved more difficult and have not provided many biologically active compounds. 

In conclusion, with one notable exception, cephamycins appear to be products of streptomycetes. Cephamycins A and B are relatively common, being produced by a wide range of streptomycete species. Cephamycin C, on the other hand, produced by a streptomycete species and Nocardia, occurs relatively rarely. Other cephamycin-type compounds are coproduced in either cephamycin A and B or cephamycin C fermentations and are relatively uncommon. 

The fermentation of S. clavuligerus and S. lipmanii for cephamycin C production is described in detail by Nagarajan (1973). The following procedures are those used in the isolation of cephamycin C from 5. lactamdurans NRRL 3802. 

Arai et al. (1975) reported on the production of cephamycin C from Streptomyces jumonjinensis. With the following fermentation medium, titers of 40 pg/ml were achieved ... 

Fermenting S. lactamdurans, Nakao et al. (1975) reported titers of cephamycin C of about 2200 p.g/ml in the following medium after a 3-day incubation at 28°C ... 

As reported for cephamycin C production (Inamine and Bimbaum, 1975), the yields of cephamycins A and B can be increased by addition to the fermentation medium of lysine, L-a-aminoadipic acid, or OL-a-aminoadipic acid, according to Hallada et al. (1975). These workers reported that S. griseus NRRL 3912 produced 427, 546, and 562 units/ ml of cephamycins A and B in the presence of 0, 0.05, and 0.1% l-lysine-HCl, respectively, in the following medium ... 

Fermentation broth containing cephamycins A and B was acidified at harvest to obtain maximum stability of the antibiotics. The broth was filtered, and 4000 liters of filtrate containing about 80 pg cephamycin/ml was passed through a 380-liter bed of Amberlite XAD-2 (Rohm and Haas). The resin bed was washed with water and then eluted with 60% aqueous methanol. The rich eluate, 800 liters, was concentrated under reduced pressure to 160 liters and adjusted to pH 3.5 with aqueous ammonia. This procedure gave 40% yield of 2% purity. One half of this concentrate was diluted to 120 liters and absorbed on a 22.5-liter bed of Amberlite IRA-68 resin in the chloride cycle. The activity was recovered by elution with 200 liters of a pH 7.5 solution of I M sodium nitrate and 0.1 M sodium acetate. The eluate was adjusted to pH 3 and adsorbed on a 45-liter bed of Amberlite XAD-2 resin to separate the product from salts. The column was washed with water, eluted with 320 liters of 25% aqueous acetone and then concentrated under reduced pressure to 17.4 liters. The concentrate was adjusted to pH 4.0 with aqueous ammonia and freeze-dried. The yield was 620 g of cephamycin, about 13% pure, for a calculated recovery of 125%. 

Figure 5 shows the isolation methods used by Fukase et al. (1976) for the separation of cephamycins A and B and C-280IX from a fermentation of Streptomyces heteromorphus. Each of the compounds was obtained in 70-80% purity as a monosodium salt. The ultraviolet (UV) spectra of these three compounds are shown in Fig. 6. Some characteristic properties of C-2801X are shown in Table II.

---