Cephalosporins fermentation

Capturing desired products from aqueous reaction mixtures (in a form useful for further synthesis steps) using monomeric agents is well known. We successfully applied such a technology to the extractive esterification of cephalosporin derivatives from a filtered cephalosporin fermentation broth65 and a solution of a 3-... 

ADCA (cephalosporins) Fermentation and biocatalysis metabolic engineering, acylase... 

The oil has been suggested as an antifoaming agent in penicillin and cephalosporin fermentations. ... 

Antibiotics. Solvent extraction is an important step in the recovery of many antibiotics (qv) such as penicillin [1406-05-9] streptomycin [57-92-17, novobiocin [303-81-1J, bacitracin [1405-87-4] erythromycin, and the cephalosporins. A good example is in the manufacture of penicillin (242) by a batchwise fermentation. Amyl acetate [628-63-7] or -butyl acetate [123-86-4] is used as the extraction solvent for the filtered fermentation broth. The penicillin is first extracted into the solvent from the broth at pH 2.0 to 2.5 and the extract treated with a buffet solution (pH 6) to obtain a penicillin-rich solution. Then the pH is again lowered and the penicillin is re-extracted into the solvent to yield a pure concentrated solution. Because penicillin degrades rapidly at low pH, it is necessary to perform the initial extraction as rapidly as possible for this reason centrifugal extractors are generally used. 

Pharmaceutical. Ion-exchange resins are useful in both the production of pharmaceuticals (qv) and the oral adrninistration of medicine (32). Antibiotics (qv), such as streptomycin [57-92-17, neomycin [1404-04-2] (33), and cephalosporin C [61-24-5] (34), which are produced by fermentation, are recovered, concentrated, and purified by adsorption on ion-exchange resins, or polymeric adsorbents. Impurities are removed from other types of pharmaceutical products in a similar manner. Resins serve as catalysts in the manufacture of intermediate chemicals. 

AH cephalosporins found in nature (Tables 1 and 2) have the D-a-aminoadipic acid 7-acyl side chain (21). AH of these compounds can be classified as having rather low specific activity. A substantial amount of the early work in the cephalosporin area was unsuccessfiiHy directed toward replacing the aminoadipic acid side chain or modifying it appropriately by fermentation or enzymatic processes (6,22). A milestone ia the development of cephalosporins occurred in 1960 with the discovery of a practical chemical process to remove the side chain to afford 7-ACA (1) (1). Several related processes were subsequendy developed (22,23). The ready avaHabHity of 7-ACA opened the way to thousands of new semisynthetic cephalosporins. The cephalosporin stmcture offers more opportunities for chemical modification than does that of penicillins There are two side chains that especiaHy lend themselves to chemical manipulation the 7-acylamino and 3-acetoxymethyl substituents. 

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). 

Superior penicillin producing cultures ate capable of producing in excess of 30 mg/mL of penicillin G (154). Cephalosporin producing strains, however, generally grow poorly and cephalosporin C production is not as efficient as is that of penicillin. Factors such as strain maintenance, strain improvement, fermentation development, inoculum preparation, and fermentation equipment requkements ate discussed in the hterature (3,154). 

The P-lactam antibiotics ate produced by secondary metaboHc reactions that differ from those responsible for the growth and reproduction of the microorganism. In order to enhance antibiotic synthesis, nutrients must be diverted from the primary pathways to the antibiotic biosynthetic sequences. Although most media for the production of penicillins and cephalosporins are similar, they ate individually designed for the specific requkements of the high yielding strains and the fermentation equipment used. 

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). 

In spite of the considerable progress in developing methods for total synthesis, this route to cephalosporins cannot compete with fermentation or penicillin rearrangement (see Sections 5.10.4.1 and 2) for the industrial production of cephalosporin antibiotics. While total synthesis does provide access to nuclear analogs not readily obtainable from fermentation products, none of the totally synthetic materials have displayed sufficient advantages to Warrant their development as new drug products (b-81MI51000). 

In the relatively few years since the preparation of the previous volume in this series, the explosion of synthetic and clinical experimentation on the semi and totally synthetic antibacterial p-lactam antibiotics has continued, providing a rich body of literature from which to assemble this chapter. The search for utopiasporin, the perfect cephalosporin, continues. The improvements in. spectrum and clinical properties achieved to date, however, are largely incremental and have been achieved at the price of substantially higher costs to the patient. Nonetheless, these newer compounds are truly remarkable when compared with the properties of the fermentation-derived substances from which they have sprung. 

Biotechnological processes may be divided into fermentation processes and biotransformations. In a fermentation process, products are formed from components in the fermentation broth, as primary or secondary metabolites, by microorganisms or higher cells. Product examples are amino acids, vitamins, or antibiotics such as penicillin or cephalosporin. In these cases, co-solvents are sometimes used for in situ product extraction. 

After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ion-exchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

Semisynthesis. This means that part of the moleeule is produced by a fermentation proeess using the appropriate microorganism and the produet is then further modified by a ehemical process. Many penicillins and cephalosporins (seetion 2) are produced in this w. ... 

It is possible to convert penicillin V or benzylpenieillin to a cephalosporin by chemical ring expansion. The first-generation cephalosporin cephalexin, for example, can be made in this way. Most cephalosporins used in clinical practice, however, are semisynthetics produced from the fermentation product cephalosporin C. 

Manufacturing processes for cephalosporin C and benzylpenicilhn are broadly similar. In common with mai other antibiotic fermentations, no specific precursor feed is necessary for cephalosporin C. There is sufficient acetyl group substrate for the terminal acetyltransferase reaction available fiom the organism s metabolic pool. 

Bruggink (1996) has given an account of how the production of cefalexin, which is the largest cephalosporin in the market, can be converted from a ten-step process based on benzaldehyde and penicillin into a six-step process where biocatalysis is involved in three steps. The wastewater stream, containing 30-40 kg of unwanted materials in the conventional process, has been substantially reduced. Similarly, Van Loon et al. (1996) have given details of fermentation processes for cleaner and cheaper compared to the process practised so far. 

The p-lactams, mainly penicillins and cephalosporins, are by production volume the most important class of antibiotics worldwide, enjoying wide applicability towards a range of infectious bacteria. Most of the key molecules are semi-synthetic products produced by chemical modification of fermentation products. Production of these molecules has contributed significantly to the development of large-scale microbial fermentation technology, and also of large-scale biocatalytic processing. 

Semi-synthetic penicillins are accessed from 6-aminopenicillanic acid, (6-APA), derived from fermented penicillin G. Starting materials for semi-synthetic cephalosporins are either 7-aminodesacetoxycephalosporanic acid (7-ADCA), which is also derived from penicillin G or 7-aminocephalosporanic acid (7-ACA), derived from fermented cephalosporin C (Scheme 1.10). These three key building blocks are produced in thousands of tonnes annually worldwide. The relatively labile nature of these molecules has encouraged the development of mild biocatalytic methods for selective hydrolysis and attachment of side chains. 

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). ... 

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