MXPA98010757A - 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 CEFALOSPORI AS DESACILADAS r FIELD OF THE INVENTION The present invention relates to the field of fermentative production of N-deacylated cephalosporin compounds, such as 7-ADCA.

BACKGROUND OF THE INVENTION Β-lactam 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 produced naturally by the filamentous fungi Peni cilli um chrysogen um and Acremoni um chrysogen um, respectively. As a result of classical strain improvement techniques, the production levels of antibiotics in Peni cilli um chrysogenum and Acremoni um chrys ogen um have increased dramatically in the past decades. With the growing knowledge of biosynthetic pathways REF. 28990 leading to penicillins and cephalosporins, and to the advent of recombinant DNA technology, new tools have become available for the improvement of production strains and for the ivati zation of the compounds. Most of the enzymes involved in the β-lactam biosynthesis have been identified and their corresponding genes have been cloned, as described by Ingolia and Queener, Med. Res. Rev. 9 (1989), 245-264 (biosynthesis 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. chrysogen um are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (La-aminoadipic acid (A), L-cysteine (C) and L-valine (V) within 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. This step involves the exchange of the side chain of the hydrophilic Da-aminoadipic acid of isopenicillin N by L-5-amino-5-carboxypentanoic acid through the action of the enzyme aci 1 transferase (AT). by AT 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 ispenicillin N ap enicilin N by an epimerase, after which the five-member ring structure characteristic of penicillins is expanded by the expandase enzyme to the six-member ring characteristic of cephalosporins. 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 natural ß-lactams are replaced with phenoxyacetic acid or phenylacetic acid. Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (for example 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, thus implying prolonged and costly isolation processes, using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted to therapeutically useful cephalosporins by a number of chemical and enzymatic conversions. The cephalosporin intermediate, 7-ADCA is currently produced by chemical derivatization of penicillin G. The chemical steps necessary to produce 7-ADCA involve the expansion of the penicillin ring structure, from 5 members to an annular structure of cephalosporin, of 6 members. Recently, fermentative processes have been described to obtain 7-ADCA. In European Patent EP-A-0532341 the application of a feed material of an adipate (5-carboxypentanoate) is shown to result in the formation of a penicillin derivative with an adipyl side chain, namely adipic acid 1- 6-aminopenicillanic. This incorporation is due to the fact that acyl transferase 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 the P strain. chrys ogen um recombinant that expresses an expandase, ia adipyl-6-APA is expanded to its inactive cephalosporum derivative. 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 AC. Documents O95 / 04148 and O95 / 04149 describe a feedstock of certain dicarboxylic acids containing a thio group, with a chain length of 6 or 7 atoms to a strain of P. chrys ogen um expressing expandasa, resulting in the incorporation of these precursors into 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 penicillin N and cephalosporin, biosynthesis has a narrow specificity (Maea et al., Enzyme and Microbial Technology (1995) 17: 231-234; Baldwin et al. collaborators, J. Chem. Soc. Chem. Commun. 374-375, 1987), preventing the possibility to catalyze the oxidative expansion of the ring of penicillin N with the non-natural side chains. It has now surprisingly been found that a dicarboxylic acid feed material with a chain length that is longer than 7 carbon atoms produces β-lactam derivatives incorporating a side chain with a chain length of 6 or 7 atoms.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes 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 expressing aci 1 trans ferase, as well as expse activity, optionally acetyl transferase / or hydroxylase activity, in the presence of a precursor of. side chain according to the formula (1) HOOC-X- (CH £) n-COOH 1) where n &8-.a even number of at least 2, X is (CHU -A- (CH) jr where p q each independently are 0, 1, 2, 3 or 4, A is CH = CH, C = C, CHB, C = 0, O, S, NH, where the nitrogen is optionally substituted or the sulfur optionally oxidized, B is hydrogen, halogen, alkoxy of 1 to 3 carbon atoms, hydroxyl, or methyl optionally substituted, with the proviso that p + q must be 2 or 3, when A is CH = CH or C = C, or p + q must be 3 or 4, when A is CHB, C = 0, 0, S or NH, or a salt, ester or amide thereof, said side chain precursor producing an acyl-6-APA derivative, the acyl group having a structure according to formula (2) H00C-X-C0- (2) wherein X is as defined above, the acyl-6-APA derivative is expd in itself to the corresponding acyl-7-ADCA derivative, optionally further reacted with the acyl-7-ADAC or acyl-7-ACA derivative , the recovery of the acyl-7- cephalosporin derivative from the fermentation broth, the deacylation of the acyl-7-cephalosporin derivative, the recovery of the crystalline compound of 7- cephalosporin.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a process for the production of N-deacylated cephalosporin compounds (7-ADCA, 7-ADAC or 7-ACA) via the fermentative production of their acylated counterparts, by applying a feed of novel side chain precursors. The present invention surprisingly shows that the fermentation of a microbial strain capable of producing β-lactam expressing acyl transferase, as well as expse activity in the presence of a dicarboxylic acid having a chain length that is greater than 7 atoms, results in the formation of an acyl-7-ADCA derivative incorporating an acyl group with a chain length of 6 or 7 atoms, respectively. According to the invention, the 7-acylated ceporosin derivatives additional to acyl-7-ADCA, for example acyl-7-ADAC or acyl-7-ACA, respectively, are produced by a microbial strain capable of producing β-lactam expressing acyltransferase, as well as expse, if said microbial strain additionally expresses hydroxylase or hydroxylase plus acetyl trans ferase activity, respectively. The dicarboxylic acid to be used in the process of the invention has a structure according to formula (1): HOOC-X- (CH;) n-COOH (1) wherein n is an even number of at least 2, X is (CHU -A- (CH2) q, where p q each independently are 0, 1, 2, 3 or 4, with the proviso that p + q = 2, 3 or 4, A is CH = CH, C ^ C, CHB, C = 0, O, S, NH, the nitrogen being optionally substituted or the sulfur being optionally oxidized, B is hydrogen, halogen, alkoxy of 1 to 3 carbon atoms, hydroxyl, optionally substituted methyl C. According to the invention, the fermentation of the microbial strain in the presence of a side chain precursor of according to formula (1), or a salt, an ester or an amide, thereof, results in the formation of an acyl-7-cephalosporin derivative, wherein the acyl group has a structure according to the formula ( 2) : HOOC-X-CO- (2) where X is as defined above. To obtain an acyl-7-cephalosporin derivative with an acyl group having a chain length of 6 or 7 atoms, respectively, p + q must be 2 or 3, respectively, when A is CH = CH or CsC, or p + q must be 3 or 4, respectively, when A is CHB, C = 0, 0, S, NH, the nitrogen is optionally substituted or the sulfur is optionally oxidized, and b is as defined above. In this way, a fermentation of a microbial strain capable of producing β-lactam and expressing acyltransferase, as well as expandase activity in the presence of a precursor compound according to formula (1), produces an acyl-6-APA derivative with an acyl group according to formula (2), which subsequently expands in itself to produce the corresponding acyl-7-ADCA derivative. In other words, said precursor compound according to formula (1) is metabolized by the microbial strain, yielding an acid group of the formula (2). Said acyl group is subsequently incorporated into the ß-lactam backbone by means of the reaction mediated by aci 1trans ferase. The upper limit for the chain length of the precursor compound according to formula 1, for example, the upper value of n, is not critical. The upper limit will be mainly determined by the efficiency by which said precursor is metabolized by the microbial strain. Conveniently, the precursor may have a longer chain length, which is similar to the longer chain length of a fatty acid which can still be metabolized by the microbial strain. In one embodiment of the invention, dicarboxylic acids are used, which produce an adipyl-7-ADCA derivative after fermentation in the presence of said dicarboxylic acid. Suitable dicarboxylic acids for producing adipi lo-7-ADCA have a structure according to formula (1), where n is an even number of at least 2, and X is (CH 2) -A- (CH; ) .., where p is 1 and q is 2 and A is CH. Preferably, said dicarboxylic acid which produces adiply-7-ADCA is suberic acid or sebic acid (n = 2 or 4, respectively). In still another embodiment of the invention, dicarboxylic acids are used which produce an acyl-7-ADCA derivative containing a thio group in the acyl group according to formula (2). Suitable dicarboxylic acids for producing such acyl-7-ADCA compounds have a structure according to formula (1), wherein n is an even number of at least 2, and X is (CH2) PA- (CH2) q, wherein A is S. Preferably, p and q are 1, 2 or 3 and p + q = 3 or 4. More preferably, p is 1 and q is 2, op is 2 and q is 1 or 2. In two other embodiments of the invention , dicarboxylic acids are used which produce the new acyl-7-cephalosporin derivatives. Preferably, dicarboxylic acids are used which produce a pimelyl-7-ADCA derivative after fermentation in the presence of said dicarboxylic acid. Suitable dicarboxylic acids for producing pi 1-7-ADCA have a structure according to formula (1), wherein n is an even number of at least 2, and X is (CH2) PA- (CH2) q, where p and q are 2 and A is CH; . Preferably, said dicarboxylic acid which produces pimelyl-7-ADCA is azelaic acid (n = 2). In addition, dicarboxylic acids are used which produce an acyl-7-ADCA derivative containing an unsaturated bond in the acyl group, according to formula (2). Suitable dicarboxylic acids for producing such acyl-7-ADCA compounds have a structure according to formula (1), wherein n is an even number of at least 2, and X is (CH2) PA- (CH2) q , where A is CH = CH or C = C. Preferably, A is CH = CH and p and q are both 1. The trans isomer of the latter compound is thus more preferred. The microbial strains which are utilizable in the process of the invention are strains which are capable of producing β-lactam and which express the acyltransferase as well as the expandase activity. Optionally, said microbial strains may additionally express hydroxylase or hydroxylase plus acetyl transferase 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 for the expression of expandase and the cephalosporin-producing strains provided with an expression cassette that provides for the expression of acyltransferase. The expandase genes that are conveniently used may originate from Acremoniumum chrysogenum, Streptomyces vulba gerus, Streptomyces an tibi oti cus or Nocardl a la ctamdurans. The acryltransferase gene may originate from P. chrysogen u, P. nal gi or ven se o A. nor sweet ans.

In a preferred embodiment, a strain of penicillin-producing fungus is used, which recombinantly expresses the expandase. More preferably, a fungus of the genus Aspergillus or Penicillus is used, more preferably a strain of Penicillusumum chrysogenum. P. chrysogen um strain Panlabs 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 person skilled in the art. Examples of expression cassettes that can be used for the construction of recombinant fungi strains expressing expandase are described in European Patent EP-A-05323 1, Crawford et al. (Biotechnol.13 (1995), 58-62) and WO95 / 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 their ability to produce adiply-7-ADCA as described by Crawford et al. (S upra).

In a different embodiment, a cephalosporin producing strain is used, which recombinantly expresses acyltransferase, for example a strain of Acremoni um chrysogen um producing acyltransferase. A strain of A. chrysogenum which recombinantly expresses acyltransferase will thereby produce an acyl-7-ACA derivative, since such a strain natively expresses hydroxylase and acetyltransferase. The present invention further describes a process for the preparation 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-7 derivative. - ADCA, such as adipyl-, pimel-il-, 2- (carboxyethylthio) acetyl-, 3- (carboxymethylthio) propionyl- or trans-β-hydromuconyl-7- ADCA, from the fermentation broth of a strain of P. chrysogen um that expresses the expandasa. Specifically, a. 7-acylated cephalosporin derivative is recovered from the fermentation broth by extracting the filtrate from the broth with an organic solvent immiscible with water, at a pH of less than about 4.5 and retroretracting it with water at a pH between 4 and 10. The broth is filtered and an organic solvent immiscible with water is added to the filtrate. The pH is adjusted in order to extract the 7-acylated cephalosporin derivative from the aqueous layer. The pH range must be less than 4.5; preferably between 4 and 1, more preferably between 2 and 1. In this way, the 7-acylated cephalosporin derivative is separated from many other impurities present in the fermentation broth. Preferably, a smaller volume of the 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 7-acylated cephalosporin derivative, thereby achieving the reduction of the speeds of volumetric flow. A second possibility is the extraction of the complete broth at a pH of 4 or less. Preferably, the broth is extracted between pH 4 and 1 with an organic solvent immiscible with water. Any solvent that does not interfere with the cephalosporin molecule can be used. Suitable solvents are, for example, butyl acetate, ethyl acetate, methylisobutyl-tone, alcohols such as butanol, etc. Preferably, 1-butanol or isobutanol is used. The 7-acylated cephalosporin derivative is subsequently back-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 7-acylated cephalosporin derivatives produced by the process of the invention are conveniently used as an intermediate for the chemical synthesis of the sythetized cephalosporins., since the 7-amino group is suitably protected by the presence of an appropriate acyl side chain. Alternatively, the 7-acylated cephalosporin derivatives are deacylated in a one-step enzymatic process, using a suitable enzyme, for example the acylation of Ps e udomon a s SE83. Preferably, an immobilized enzyme is used, in order to be able to use 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 degradation reaction 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 by, for example, the addition of an inorganic base, such as a potassium hydroxide solution, or the application. of a cation exchange resin. 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 using the enzyme in solution and removing 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 approximately 0.1-1.5, the layers are separated and the pH of the aqueous layer adjusted from 2 to 5. The crystalline N-deacylated cephalosporin 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 iminochloride side chain, by addition of phosphorus pentachloride at a temperature below 10 ° C and subsequently isobutanol at ambient temperatures or minors.

Example 1 Fermentative production of acyl-7-ADCA P. chrys ogen um strain Panlabs P14-B10, deposited in CBS under accession number 455.95, is used as the host strain for constructions of expandase expression cassettes. The expression cassette used that contains the expandase gene under the transcriptional and transcriptional regulation signals of the IPNS gene of P. chrys ogen um, is described in Crawford et al. (s upra). Transformation and culture conditions are as described in Crawford et al. (S upra). 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. (S upra). The transformants producing acyl-7-ADCA. are inoculated to 2,106 conidia / ml in a seeding medium consisting of (g / 1): glucose, 30; Pharmamedia (cottonseed meal), 10; Corn Extract Solids, 20; (NH4) 2S04, 20; CaCO3, 5; KH2P04, 0.5; lactose, 10; yeast extract, 10 at a pH of 5.6 before sterilization. The seed culture (20 ml in a 250 ml Erlenmeyer flask closed with a cotton plug) is incubated at 25 ° C at 220 rpm. After 48 hours, 1 ml is used to inoculate 15 ml of production medium consisting of (g / 1): KH2P04, 0.5; K2S04, 5; (NH? .SOj, 17.5; lactose, 140; Pharmamedia, 20; CaC03, 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% The production culture is grown at 25 ° C and 220 rpm for 168 hours in an Erlenmeyer flask. 250 ml 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 the acyl-7-ADCA is analyzed by high performance liquid chromatography (HPLC) ).

Example 2 Production analysis of acyl-7-ADCA Fermentation products were analyzed from the transformed strains of Peni ci l li um by high performance liquid chromatography (HPLC). The HPLC system consisted of the following components: P1000 solvent distribution system (TSP), automatic sample marathon model (Spark Holland) (injection volume 3), variable wavelength detector UV150 (TSP) (adjusted) at 260 nm) and a PCI 000 data system (TSP). The stationary phase was a column YMC pack ODS AQ 150 * 4.6 m. The mobile phase consisted of an 84% phosphate buffer at pH 6.0, 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 Identity of acyl-7-ADCA products A strain of P, chrysogen um expressing recombinase expandase, was cultured according to Example 1 in the presence of the following precursors: adipic acid, suberic acid, sebacic acid, pimelic acid and azelaic acid. The analysis according to Example 2 of the fermentation products of these fermentations showed that fermentation in the presence of adipic acid, suberic acid and sebacic acid resulted in the formation of adiply-7-ADCA, while pimelyl-7-ADCA was formed in the case in which pimelic acid or azelaic acid were added. When high concentrations of suberic acid were used during fermentation (2.0% instead of 0.5%), a small but significant amount of suberil-7-ADCA was detected together with adiply-7-ADCA.

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 (11)

RE I IND ICATIONS

1. A process for the production of a N-deacylated cephalosporin compound, characterized in that it comprises 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 formula (1) HOOC-X- (CH;) "-COOH (1) where n is an even number of at least 2, and X is (CH;) rA- (CH :), where p and q each independently are 0, 1, 2, 3 or 4, and A is CH = CH, C = C, CHB, C = 0, O, S, NH, wherein the nitrogen is optionally substituted or the sulfur optionally oxidized, and B is hydrogen, halogen, alkoxy of 1 to 3 carbon atoms, hydroxyl, or optionally substituted methyl, with the proviso that p + q must be 2 or 3, when A is CH = CH or C = C, or p + q must be 3 or 4, when A is CHB, C = 0, O, S or NH, or a salt, ester or amide thereof, said side chain precursor producing an acyl-6-APA derivative, the acyl group having a structure according to formula (2) HOOC-X-CO- (2) wherein X is as defined above, the acyl-6-APA derivative is expanded in itself to the corresponding acyl-7-ADCA derivative, and optionally further reacted with the acyl-7-ADAC or acyl-7-ACA derivative , * 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 compound of 7- cephalosporin.

2. The process according to claim 1, characterized in that a side chain precursor is used according to the formula (1), where n is an integer of at least 2, and X is (CH2) P-A- (CH2) q, where p is 1, q is 2 and A is CH23.

The process according to claim 2, characterized in that the side chain precursor is suberic acid or sebacic acid.

4. The process according to claim 1, characterized in that a side chain precursor is used according to formula (1) wherein n is an even number of at least 2, and X is (CH? -A- (CH; J, where p and q are 2 and A is CH2.

5. The process according to claim 4, characterized in that the side chain precursor is azelaic acid.

6. The process according to any of claims 1 to 5, characterized in that the microbial strain is a penicillin-producing strain provided with an expression cassette that provides expression of expandase.

7. The process according to claim 6, characterized in that the penicillin-producing strain is Peni ci l l um chrysogen um.

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

9. The process according to any of claims 1 to 5, characterized in that the microbial strain is a cephalosporin-producing strain provided with an expression cassette that provides acyltransferase expression.

13. The process according to claim 9, characterized in that the cephalosporin producing strain is Acremoni um chrysogen u.

11. The process according to claim 9 or 10, characterized in that the crystalline cephalosporin compound is 7-ACA.