1. Field of the Invention
The present invention is in the field of synthesis methods for the preparation of commercial cephalosporin antibiotics, of which there are presently a significant number, these therapeutic agents now being in their fourth generation. The large variety of side chains to be found in commercial cephalosporins and the significant economic importance of the cephalosporins has placed increased importance on achieving more economic and efficient methods of preparing key intermediates which permit ready synthesis of the various cephalosporins.
One of these key intermediates is 7-aminodesacetoxy cephalosporanic acid (7-ADCA), which may be represented by the following formula: ##STR1## Currently, 7-ADCA is produced from penicillin G and requires four or five chemical steps to expand the penicillin ring system from 5 members to the 6-membered ring which characterizes cephalosporins. As is typical of totally chemical synthesis, this process has serious disadvantages. Among these are the requirements of a multi-step and complex process, expensive reagents, significant quantities of process by-products resulting in effluent treatment problems, and purification of a highly impure starting material before chemical treatment begins. Consequently, there has been an ongoing search for a microbiological or fermentative process which would achieve enzymatic ring expansion and side chain cleavage to provide 7-ADCA on a more economic basis than the chemical process currently in use.
Accordingly, the present invention is particularly in the field of preparing the key cephalosporin intermediate 7-ADCA, and more particularly, in the field of bioprocesses for the preparation of 7-ADCA.
To date, the search for a successful bioprocess for making 7-ADCA has largely proved futile, certainly with respect to one of commercial scale. For example, while it has been possible to prepare 6-amino penicillanic acid (6-APA) by direct fermentation and/or by enzymatic treatment of penicillin G, leaving only ring expansion necessary to give 7-ADCA, it has been found that, unfortunately, the Cephalosporium or Streptomyces enzymes which carry out ring expansion in the normal metabolic pathways of these microorganisms do not accept 6-APA as a substrate. These enzymes, which are collectively referred to in the art as the DAOCS or expandase enzyme, are defined as enzymes which catalyze the expansion of penam ring structures found in penicillin-type molecules to ceph-3-em rings, as found in the cephalosporins. Hereafter, these enzymes will be referred to as "the expandase enzyme".
A substrate on which the expandase enzyme does operate is penicillin N, which upon ring expansion, gives deacetoxy cephalosporin C (DAOC). Here, it is only necessary to cleave the (D)-a-aminoadipoyl side chain to give 7-ADCA, but this side chain has proven stubbornly resistant to enzymatic cleavage, giving only unacceptably low yields.
In accordance with the present invention it has been possible to achieve an efficient bioprocess wherein a penicillin compound (having an adipoyl side chain) is produced by a novel fermentation process in high titers, said penicillin compound being an acceptable substrate for the expandase enzyme system which is produced in situ by the same microorganism which produces the penicillin compound, having been transformed to express said expandase enzyme system. The expandase enzyme then operates to ring expand the penicillin compound to a cephalosporin compound in high yields. And, importantly in the second critical step, the side chain of the penicillin compound, now a cephalosporin compound, is removable by another enzyme system in surprisingly high yields. The unexpected result of this unique bioprocess which comprises the present invention is the production of 7-ADCA in surprisingly high yields.
2. Brief Description of the Prior Art
Cantwell et al., in Curr Genet (1990) 17:213-221, have proposed a bioprocess for preparing 7-ADCA by ring expansion of penicillin V followed by enzymatic hydrolysis of the resulting deacetoxycephalosporin V to form 7-ADCA. This proposal is based on the availability of a cloned penicillin N expandase gene (cefE) from S. clavuligerus, Kovacevic et al., J. Bacteriol. (1989) 171:754-760 and U.S. Pat. No. 5,070,020. However, since the expandase operates on penicillin N, its natural substrate, but not on penicillin V, the proposal requires genetic engineering to produce a modified expandase gene which can ring-expand the penicillin V. The required modification was not achieved by Cantwell et al., however, and they only succeeded in transforming Penicillium chrysogenum with the cef E gene from Streptomyces clavuligerus and getting low-level expression of the DAOCS (expandase) enzyme.
The expandase enzyme has been well studied in the art, both with respect to its activity and its genetic sequence. For example, in Wolfe U.S. Pat. Nos. 4,510,246 and 4,536,476, cyclase, epimerase and ring expansion enzymes were isolated separately from a cell free extract of prokaryotic .beta.-lactam producing organisms, including Streptomyces clavuligerus, to provide stable enzyme reagents. EP-A-0 366 354 describes an isolated and purified expandase enzyme from S. clavuligerus which is characterized, including by a terminal residue and amino acid composition, and is said to have a molecular weight of about 34,600 Daltons. This is in contrast, however, to the molecular weight of 29,000 assigned to what would appear to be the same enzyme in U.S. Pat. No. 4,536,476. EP-A-0 233 715 discloses isolation and endonuclease restriction map characterization of the expandase enzyme obtained from S. clavuligerus, transformation and expression in a host of said enzyme, and demonstration of ring expansion of penicillin N substrate using said enzyme. U.S. Pat. No. 5,070,020 discloses the DNA sequence encoding the expandase enzyme obtained from S. clavuligerus and describes the transformation of a P. chrysogenum strain with an expression vector containing said DNA sequence, thereby obtaining expression of the expandase enzyme. While it is suggested that this enzyme is useful for the expansion of substrates other than penicillin N, there is no actual demonstration of such an expansion.
The work described above has focused on the expandase enzyme derived from prokaryotic S. clavuligerus. This same enzyme, or at least an enzyme apparently having the same ring expansion activity, is also expressed by strains of eukaryotic Cephalosporium acremonium (also referred to as Acremonium chrysogenum). However, in such strains expandase activity is expressed by a bifunctional gene (cefEF), which also expresses the DACS (hydroxylase) activity whose natural function is to convert the desacetoxycephalosporanic acid (DAOC) product of the expandase enzyme to deacetyl cephalosporin C (DAC). The result is a single, but bifunctional expandase/hydroxylase enzyme. While there have been efforts to separate the activities of these two gene products, none have yet been successful. For example, EP-A-0 281 391 discloses the isolation and DNA sequence identification of the DAOCS/DACS gene obtained from C. acremonium ATCC 11550 together with the corresponding amino acid sequences of the enzymes. A Penicillium is transformed and expresses the enzymes, however, the attempted conversion of penicillins G and V to the corresponding cephalosporins is never demonstrated. Further, despite a suggestion that genetic engineering techniques provide a ready means to separate the genetic information encoding DAOCS from DACS and separately express them, no actual demonstration of such separation is set forth.
The DAOCS/DACS (expandase/hydroxylase) enzyme of C. acremonium has also been well studied in the art, both with respect to its activity and its characteristics and genetic sequence. For example, in Demain U.S. Pat. No. 4,178,210; 4,248,966; and 4,307,192 various penicillin-type starting materials are treated with a cell-free extract of C. acremonium which epimerizes and expands the ring to give a cephalosporin antibiotic product. Wu-Kuang Yeh U.S. Pat. No. 4,753,881 describes the C. acremonium enzyme in terms of its isoelectric point, molecular weights, amino acid residues, ratio of hydroxylase to expandase activities and peptide fragments.
The prior art discussed above deals with only a single aspect of the present invention, i.e., the transformation of a P. chrysogenum strain with the gene expressing the expandase enzyme and obtaining expression of that enzyme. The art, however, has only used the expressed enzyme to ring-expand penicillin N, not penicillins G and V. Even in that case, penicillin N has a 7-position side chain which cannot be cleaved enzymatically to give 7-ADCA, as in the method of the present invention. The present invention relies on the surprising discovery that an adipoyl side chain can be efficiently added by a P. chrysogenum strain, that the expandase enzyme expressed in situ can use that compound efficiently as a substrate for ring expansion to adipoyl 7-ADCA, and that the adipoyl side chain can then be efficiently removed by yet another enzyme to give 7-ADCA. While various isolated fragments of the present invention may be found in the prior art, there has been no suggestion that they be combined to give the unexpected results obtained with the method of the present invention.
For example, production of 6-adipoyl penicillanic acid is known in the art; see Ballio, A. et al., Nature (1960) 185, 97-99. The enzymatic expansion of 6-adipoyl penicillanic acid on an in vitro basis is also known in the art. See Baldwin et al., Tetrahedron (1987) 43, 3009-3014; and EP-A-0 268 343. And, enzymatic cleavage of adipoyl side chains is also known in the art; see Matsuda et al., J. Bact. (1987) 169, 5815-5820.
The adipoyl side chain has the following structure: COOH--(CH.sub.2).sub.4 --CO--, while a side chain of closely related structure is that of glutaryl, having the following formula: COOH--(CH.sub.2).sub.3 --CO--. The enzymatic cleavage of glutaryl side chains is known in the art. See, e.g., Shibuya et al., Agric. Biol. Chem. (1981) 45, 1561-1567; Matsuda and Komatsu, J. Bact. (1985) 163, 1222-1228; Matsuda et al., J. Bact. (1987) 169, 5815-5820; Jap. 53-086084 (1978-Banyu Pharmaceutical Co. Ltd.); and Jap. 52-128293 (1977-Banyu Pharmaceutical Co. Ltd.).
Also, EPA-A-0 453 048 describes methods for improving the adipoyl-cleaving activity of the glutaryl acylase produced by Pseudomonas SY-77-1. By substituting different amino acids at certain locations within the alpha-subunit, a three to five times higher rate of adipoyl cleavage (from adipoyl-serine) was observed. It should be noted that although EP-A-0 453 048, apparently, demonstrates an acylase with improved activity towards adipoyl-side chains, it does not describe any ways (either chemical or through a bioprocess in any way analogous to that described in the instant specification) in which an adipoly-cephalosporin might be generated in the first place.
Where a (D)-a-aminoadipoyl side chain is present, it is known in the art to first enzymatically remove the amino group and shorten the side chain with a (D)-aminoacidoxidase, leaving a glutaryl (GL-7) side chain, with removal of the glutaryl side chain by a second enzyme (glutaryl acylase). Such a two-step cleavage is disclosed in Matsuda U.S. Pat. No. 3,960,662; EP-A-0 275 901; Jap. 61-218057 (1988-Komatsu, Asahi Chemical Industry Co.); WO 90/12110 (1990-Wong, Biopure Corp.); and Isogai et al., Bio/Technology (1991) 9, 188-191.