MXPA98010758A - Process for the fermentative production of deacylated cephalosporins - Google Patents

Abstract

The present invention discloses a process for the production of N-deacylated cephalosporin compounds via the fermentative production of their 7-acylated counterparts.

Description

PROCESS FOR THE FERMENTATIVE PRODUCTION OF DESCRIBED CEPHALOSPORINS FIELD OF IJSÍVENTION The present invention relates to the field of fermentative production of N-deacylated cephalosporin compounds, such as 7-ADCA.

Iu BACKGROUND OF THE INVENTION Β-Iactam antibiotics are the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominent ones are penicillins and cephalosporins. These compounds are naturally producible by the filamentous fungi Peni cil l i um chrysogen um and Acremoni um chrys ogen um, respectively. __U As a result of classical strain improvement techniques, the production levels of antibiotics in Peni ci l l um chrysogen um and Acremoni um chrysogen um have increased dramatically in the past decades. With the growing knowledge of the biosynthetic pathways REF: 28989 leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools have become available for the improvement of production strains and for the in vivo derivatization of the products. Most of the enzymes involved in the biosynthesis of β-lactams have been identified and their corresponding genes have been cloned, as described by Ingolia and Queener, Med. Res. Rev., 9 (1989), 245-264 (bosynthesis route and enzymes), and Aharonowitz, Cohen, and Martin, Ann. Rev. Microbiol. 46 (1992), 461-495 (gene cloning). The first two steps in the biosynthesis of penicillin in P. chrysogenum are the condensation of the three amino acids of L-5-amino-5-carboxypentanoic acid (L-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) in the tripeptide LLD-ACV, followed by the cyclization of this tripeptide to form isopenicillin N. This compound contains the typical structure of β-lactam. These first two steps in the biosynthesis of penicillins are common in fungi and bacteria that produce penicillin, cefamycin and cephalosporin.

The third . This step involves the exchange of the hydrophilic side chain of D-a-aminoadipic acid of isopenicillin N by L-5-amino-5-carboxypentanoic acid, through the action of the enzyme acyltransferase (AT). The AT-mediated enzymatic exchange reaction takes place within a cellular organelle, the microbody, as described in European Patent EP-A-0448180. In cephalosporin producing organisms, the third step is the isomerization of isopenicillin N a .penicillin N by an isomerase, after which the five-member ring structure characteristic of penicillins is expanded by the expandase enzyme to the six-membered ring characteristic of cephalosporin. The only directly fermented penicillins of industrial importance are penicillin V and penicillin G, produced by the addition of hydrophobic side chain precursors, phenoxyacetic acid or phenylacetic acid, respectively, during the fermentation of P, chrys ogen um, whereby the side chains of the natural β-lactams are replaced with phenoxyacetic acid or phenylacetic acid.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (eg, cephalexin) are made from penicillins by a number of chemical conversions. Cephalosporin C, by far the most important initial material in this respect, is very soluble in water at any pH, which implies of this process prolonged and expensive isolation processes using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted to therapeutically useful cephalosporin by a number of chemical and enzymatic conversions. The intermediate of cephalosporin 7-ADCA, is currently produced by the derivatization of penicillin G. The steps necessary to produce 7-ADCA involve the expansion of the annular structure of penicillin, from five members to a ring structure of 6 members of the cephalosporin. Recently, fermentative processes have been described to obtain 7-ADCA. In European Patent No. EP-A-0532341, it was shown that the application of a raw material of adipata (5-carboxypentanoate) results in the formation of a penicillin derivative with an adipyl side chain, namely the adiphenyl acid. 6-aminopenicillanic. This incorporation is due to the fact that acyltransferase has proven to have broad substrate specificity (Behrens et al., J. Biol. Chem. 175 (1948), 751-809, Cole, Process, Biochem. 1 (1966), 334- 338; Ballio et al., Nature 185 (1960) 97-99). In addition, when the adipate is fed to a strain of P. chrysogen um recombinant, which expresses an expandasa, the adipil-6-APA is expanded to its corresponding derivative of cephalosporin. Finally, the removal of the side chain of adipyl is suggested, producing 7-ADCA as a final product. European Patent Application EP-A-0540210 describes a similar process for the preparation of 7-ACA, including the extra steps of converting the 3-methyl group of the ADCA ring to the 3-acetoxymethyl group of ACA. Documents O95 / 04148 and WO95 / 04149 describe a raw material of certain dicarboxylic acids containing a thio group, to a strain of P. chrysogen um, expresses expandasa, resulting in the incorporation of these precursors to the penicillin backbone and subsequent expansion to the corresponding derivatives of 7-ADCA. In general, however, it is thought that an expandase that can provide the crucial link between the biosynthesis of penicillin N and cephalosporin has a narrow specificity (Maea et al., Enzyme and Microbial Technology (1995) 17: 2 ^ 1-234; Bald in et al., J. Chem. Soc. Chem. Commun. 374-375, 1987), preventing the possibility to catalyze the oxidative expansion of the penicillin N ring with non-natural side chains. The present invention describes a process for the fermentative production of cephalosporin compounds using novel side chain precursors, which has several advantages over existing processes, advantages with respect to yield and with respect to a decreased level of by-products.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes a process for the production of an N-deacylated cephalosporin compound comprising the steps of: * the fermentation of a microbial strain capable of producing β-lactam and expressing acyltransferase, as well as expandase activity, and optionally activity of acetyltransferase and / or hydroxylase, in the presence of a side chain precursor according to the formula (I) HOOC-.X-COOH (1 where X is (CH-) m-CH-A- (CH) po (CH2) mC = C- (CH2) n, where m and each individually are 0, 1, 2 or 3 and m + n = 2 or 3, and A is CH or N, or X is (CH2) P-CH = CH-CH = C- (CH2) q, where p and q each are individually 0 or 1 and p + q = 0 or 1, or a salt, ester or amide thereof, said side chain precursor producing an acyl-6-APA derivative incorporating said precursor, the acyl-6-APA derivative being expanded in itself to the acyl-7 derivative - corresponding ACA, which is optionally reacted afterwards to the acyl-7-ADAC or acyl-7-ACA derivative, and * the recovery of the acyl-7-cephalosporin derivative from the fermentation broth * the deacylation of the acyl-7-cephalosporin derivative , and * the recovery of the crystalline N-deacylated cephalosporin compound.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a process for the production of N-deacylated cephalosporin derivatives (7-ADCA, 7-ADAC or 7-ACA) by means of the fermentative production of their N-acylated counterparts, by applying a feed of new chain precursors side. By using these precursors, new N-acylated cephalosporin derivatives are formed. According to the invention, the fermentation of a microbial strain capable of producing β-lactam and expressing the acyltransferase, as well as expandase activity, and optionally hydroxylase or hydroxylase as well as acetyltransferase activity, in the presence of a dicarboxylic acid having one or two unsaturated bonds, leads to an improved incorporation of said side chain precursor into the cephalosporin backbone. As a result, low levels of undesired derivatives of acyl-6-APA are detectable in the process of the invention. Furthermore, the present invention shows that an improved yield of the N-acylated cephalosporin derivative on an unsaturated precursor is obtained, compared to the yield on the adipic acid. The precursor of the side chain according to the present invention has a structure according to formula (I): H00C-X-C00H 1) where X is (CH1) m-CH = A- (CH1) not (CH2) ra-C = C- (CH1) n, where yn each individually are 0, 1, 2 or 3 and A is CH or N, or X is (CHz) P-CH = CH-CH = C- (CH2) q, where P and q each individually are 0 or 1 and p + q = 0 or 1. According to the invention, fermentation in the presence of the unsaturated precursor of the formula (I), or a salt, ester or amide thereof, produces an acyl-6-APA derivative incorporating said precursor.

The acyl-6-APA derivative is subsequently expanded in itself to the corresponding acyl-7-ADCA derivative. In particular, the present invention discloses that an acyl-6-APA compound having the precursor of the invention incorporated is an efficient substrate for the subsequent expansion reaction. The amount of the acyl-6-APA product which is formed in the process of the invention is subsequently less than the amount of the by-product adipyl-6-APA formed in the process for the production of adiply-7-ADCA, using the precursor adipic acid side chain. In a preferred embodiment of the invention, a compound of the formula (I) is used as a side chain precursor, wherein m and n are 1 and A is CH. More preferably, the compound according to formula (1) is trans-β-hydromuconic acid. The present invention shows that the acyl-6-APA compound containing a trans-β-muconyl side chain is expanded very efficiently to the corresponding 7-ADCA derivative, since nothing or only a small amount of 6-APA derivative is detected. In addition, the yield of the N-acylated cephalosporin derivative on this precursor is shown to be improved compared to the yield on adipic acid. The microbial strains that are used in the process of the invention are strains that are capable of producing β-lactam and which express acyltransferase as well as expandase activity.

Optionally, said microbial strains may additionally express hydroxylase or hydroxylase plus acetyltransferase activity. The first strains make possible the production of acyl-7-ADCA derivatives, while the last strains make possible the production of acyl-7-ADAC or acyl-7-ACA derivatives. Examples of such microbial strains include penicillin-producing strains provided with an expression cassette that provides expression of expandase and cephalosporin-producing strains provided with an expression cassette that provides acyltransferase expression. The genes of the expandase which are conveniently used can originate from Acremoni um chrysogen um, Streptomyces cl a vuli gerus, Streptomyces an tibi ó ti cos or Nocardi a l actamdurans.

The acyltransferase gene can originate from P. chrysogen um, P. nal gi ovense or A nor sweet ans.

In a preferred embodiment, a strain of penicillin-producing fungi is used which expressly expresses the expandase. More preferably, a fungus of the genus Aspergi llus or Peni cilli um is used, more preferably, a strain of Peni cillim chrysogenum, P. Chrysogenum strain Penlabs P14-B10, DS 18541 (deposited in CBS under accession number 455, 95) is an example of a suitable host for the expression of expandase. The construction of the recombinant strains expressing the expandase is within the knowledge of the skilled person. Examples of expression cassettes which can be used for the construction of recombinant fungal strains expressing the expandase are described in European Patent EP-0532341, Crawford et al. (Biotechnol. 13 (1995), 58-62) and International Patent Application W095 / 04148. Care must be taken in selecting a transformed strain which has a sufficiently high level of expandase expression. Such transformants can for example be selected by testing for their ability to produce adiply-7-ADCA as described by Crawford et al. (Supra).

In a different embodiment, the cephalosporin producing strain is used which recombinantly expresses the acyltransferase, for example a strain of Acremoni um chrysogen um producing acyltransferase. A strain of A. chrys ogen um which recombinantly expresses acyltransferase will thereby produce an acyl-7-ACA derivative, since such a strain natively expresses hydroxylase and acetyltransferase. These preferred modalities will greatly contribute to reducing the amount of penicillin byproducts, which are not tolerated in the final 7-ADCA product by the regulatory authorities. The present invention further describes a process for the recovery of an acyl-7-cephalosporin derivative from the fermentation broth of a microbial fermentation according to the invention, using specific solvents, for example, the recovery of an acyl derivative. 7-ADCA from the fermentation broth of a strain of P. chrysogen um that expresses the expandasa. Specifically, the acyl-7-cephalosporin derivative is recovered from the fermentation broth by extracting a filtrate from the broth with an organic solvent not immiscible with water, at a pH of less than about 4.5 and retro-extracting it with water to a pH between 4.and 10. The broth is filtered and an organic solvent not immiscible with water is added to the filtrate. The pH is adjusted in order to extract the acyl-7-cephalosporin derivative from the aqueous layer. The pH range has to be lower than 4.5; preferably between 4 and 1, more preferably between 2, and 1. In this manner, the acyl-7-cephalosporin derivative is separated from many other impurities present in the fermentation broth. Preferably, a smaller volume of organic solvent is used, for example half the volume of the solvent relative to the volume of the aqueous layer, giving a concentrated solution of the. acyl-7-cephalosporin derivative, to achieve the reduction of volumetric flow rates. A second possibility is the extraction of the complete broth at a pH of 4 Q lower. Preferably, the broth is extracted between pH 4 and 1 with an organic solvent not miscible with water. Any solvent that does not interfere with the cephalosporin molecule can be used. Suitable solvents are, for example, butyl acetate, ethyl acetate, methyl isobutyl ketone, co or butanol alcohols, etc. Preferably, 1-butanol or isobutanol are used. After this, the acyl-7-cephalosporin derivative is again extracted with water at a pH between 4 and 10, preferably between 6 and 9.

Again, the final volume can be reduced. The recovery can be carried out at temperatures between 0 and 50 ° C, and preferably at ambient temperatures. The acyl-7-cephalosporin derivatives produced by the process of the invention are conveniently used as an intermediate for the chemical synthesis of the semisynthetic cephalosporin, since the 7-amino group is suitably protected by the presence of an acyl side chain, appropriate Finally, the derivatives of acyl-7-cephalosporin are deacylated in an enzymatic process in a single step, using a suitable enzyme, for example, Pseudomonas acylase.

SE83. Preferably, an immobilized enzyme is used, in order to make possible the use of the enzyme repeatedly. The methodology for the preparation of such particles and the immobilization of the enzymes has been described extensively in European Patent EP-A-0222462. The pH of the aqueous solution has a value of, for example, pH 4 to pH 9, at which the reaction of the degradation of the cephalosporin is minimized and the desired conversion with the enzyme is optimized. In this way, the enzyme is added to the aqueous cephalosporin solution while maintaining the pH at the appropriate level for example, by the addition of an inorganic base, such as potassium hydroxide solution, or the application of a potassium hydroxide resin. cation exchange When the reaction is complete the immobilized enzyme is removed by filtration. Another possibility is the application of the immobilized enzyme in a fixed or fluidized bed column or the use of the enzyme in solution or eliminating the products by membrane filtration. Subsequently, the reaction mixture is acidified in the presence of an organic solvent immiscible with water. After adjusting the pH to about 0.1 to 1.5, the layers are separated and the pH of the aqueous layer is adjusted from 2 to 5. The crystalline cephalosporin compound is then filtered.

The deacylation can be carried out chemically as is known in the prior art, for example by means of the formation of an imino-chloride side chain, by the addition of phosphorus pentachloride at a temperature below 10 ° C and subsequently isobutanol. at room temperatures or lower.

Example 1 Fermentation of recombinant P. chrys ogen um P. chrys ogen um strain Partí abs P14-B10, deposited in CBS under accession number 455.95, a used as the host strain for the constructs of the expression cassette of the expandase. The expression cassette used that contains the expandase gene under the transcriptional and translational regulation signals of the IPN3 gene of the P gene. chyrs ogen um is described in Crawford et al. (supra). Transformation and culture conditions are as described in Crawford et al. (Supra). The transformants are purified and analyzed for the expression of the expandase enzyme by testing their ability to produce adiply-7-ADCA as described by Crawford et al. (Supra). The Acyl-7-ADCA producing transformants are inoculated to 2, 106 conidia / pil in a seeding medium consisting of (g / 1): glucose 30, Pharmamedia (cottonseed meal), 10; Corn Infusion Solids, 20; (NH.S04, 20; CaCO5; 5; KH_P0 ,, 0.5; lactose, 10; yeast extract, 10 at a pH of 5.6 before sterilization.) The seed culture (20 ml in a closed Erlemeyer 250 ml flask). with a cotton plug) incubated at 25 ° C at 220 rpm After 48 hours, 1 ml was used to inoculate 15 ml of the production medium consisting of (g / 1), KH_POj, 0.5, K_SO_, 5; (NH SO ,, 17.5; lactose, 140; Pharmamedia, 20; CaCO., 10; butter oil, 10 at a pH of 6.6 before sterilization.) After inoculation with the seed culture, a solution of reserve to 20% of the precursor of choice, adjusted to pH 6.5 with KOH, to the fermentation broth to reach a final concentration of 0.5% to 2.0% The production culture is grown at 25 ° C and 220 rpm for 168 hours in a Erle eyer 250 ml flask closed with a milk filter The evaporated water is replaced every day At the end of the production fermentation, the mycelium is removed by centrifugation or filtration and analyzed in ac ± -l-6-APA and acyl-7-AD.C? by HPLC.

Example 2 Analysis of fermentation products The fermentation products from the transformed Peni ci l i um strains were analyzed by high performance liquid chromatography (HPLC). The HPLC system consisted of the following elements: P1000 solvent distribution system (TSP), Marathón basic model sampler (Spark Holland) (injection volume 3), UV150 (TSP) variable wavelength detector UV150 (TSP) (set at 260 nm) and a PCI 000 data system (TSP). The stationary phase was a packed YMC column ODS AQ 150 * 4.6 mm. The mobile phase consisted of an 84% pH 6.0 phosphate buffer, to which 0.17% tetrabutylammonium acid sulfate and 16% acetonitrile had been added.

The products were quantified by comparison to a standard curve of the expected acyl-7-ADCA.

Example 3 Production of N-acylated derivatives of penicillin and cephalosporin fermentation of P chrysogen um according to Example 1, in the presence of different concentrations of adipic acid (AA) or trans-β-hydromuconic acid (THMA). The N-acylated ß-lactam products were analyzed by HPLC according to Example 2. HPLC analysis revealed that THMA is incorporated into the cephalosporin backbone in the presence of this precursor, e.g. -β-Hydromuconyl-7-ADCA. From the results as described in Table 1 it appears that trans-β-hydromuconyl-6-APA is no longer detectable when fermentation occurs in the presence of THMA. In addition, the performance of trans-β-hydromuconyl-7-ADCA is improved in comparison with adiply-7-ADCA, so that when higher concentrations of the precursor are applied.

Table 1. Amount of acyl-6-APA and acyl-7-ADCA formed by feeding AA and THMA in different concentrations.

Expression in relation to the amount of acyl-7- ADCA formed with a feed of 0.5% adipate, whose value is adjusted to 100%. nd = not detectable.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (8)

1. A process for the production of a N-deacylated cephalosporin compound comprising the steps of: * the fermentation of a microbial strain capable of producing β-lactam and expressing acyltransferase, as well as expandase activity, and optionally acetyltransferase activity and / or hydroxylase, in the presence of a side chain precursor according to J- CL _.V_ _._ LLU ._. A \ _) HOOC-X-COOH (1 exi where is \ ^. £ X /: H = A- (CH (CH, • C = C- (CH, where m and each individually are 0, 1, 2 or 3 and m + n = 2 or 3, and A is CH or N, X is (CH;).-.- CH = CH-CH = C- (CH). Where p and q each individually are 0 or 1 and p + q 0 or 1, or a salt, ester or amide of the The said acrylate-6-APA derivative incorporating said precursor, said acyl-6-APA derivative being expanded by itself to the corresponding acyl-7-ACA derivative, which is then optionally reacted to the acyl derivative. -7-ADAC or acyl-7-ACA, and * the recovery of the acyl-7- ^ -cephalosporin derivative from the fermentation broth * the -decylation of the acyl-7-cephalosporin derivative, and * the recovery of the cephalosporin compound N- deacid crystalline.

2. The process according to claim 1, characterized in that an unsaturated side chain precursor of the formula (1) is used wherein m and n are 1 and A is CH.

3. The process according to claim 2, characterized in that the unsaturated side chain precursor is trans-β-hydromuconic acid.

4. The process according to any of claims 1 to 3, characterized in that the microbial strain is a penicillin-producing strain provided with an expression cassette that provides expression of expandase.

5. The process according to claim 4, characterized in that the penicillin-producing strain is Peni cilli um chrysogen u.

6. The process according to claim 4 or 5, characterized in that the crystalline cephalosporin compound is 7-ADCA.

7. The process according to any of claims 1 to 3, characterized in that the microbial strain is a cephalosporin-producing strain provided with an expression cassette that provides ac expression:

8. The process according to claim 7, characterized in that the cephalosporin producing strain is Acremoni um chrysogen um. process according to claim 7 or 8, characterized in that the crystalline cephalosporin compound is 7-ADCA or 7-ACA.