HK1020356A - Process for the fermentative production of deacylated cephalosporins - Google Patents

Description

Process for the fermentative production of deacylated cephalosporins

Technical Field

The present invention relates to N-deacylated cephalosporins such as: field of fermentative production of 7-ADCA

Background

Beta-lactam antibiotics are the most important group of antibiotic compounds and have a long history of clinical application. Prominent representatives of this group are penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi Penicillium chrysogenum (Penicillium chrysogenum) and acremonium chrysogenum, respectively.

The production levels of antibiotics in penicillium chrysogenum and Acremonium chrysogenum have been greatly increased over the past decades by classical strain improvement techniques. With the increasing knowledge of the biosynthetic pathways for penicillins and cephalosporins and the advent of recombinant DNA technology, new tools for increasing strain yield and in vivo derivatization of compounds have emerged.

Most of the enzymes involved in β -lactam biosynthesis have been identified and their corresponding genes have been cloned. Such as described in Lnggolia and Queener in the review of medical research (Med. Res. Rev.)9(1989),245-264 (biosynthetic pathway and enzymes), and Aharonowitz, Cohen and Martin in Ann.Rev.Microbiol.46(1992),461-495 (Gene clone).

The first two steps of penicillin biosynthesis in penicillium chrysogenum are three amino acids: l-5-amino-5-carboxypentanoic acid (L-C α -aminoadipic acid) (A), L-cysteine (C) and L-valine (V) are condensed to form the tripeptide LLD-ACV, which is subsequently cyclized to form isopenicillin N. This compound contains the typical beta-lactam structure.

The first two steps of penicillin biosynthesis are identical in fungi and bacteria producing penicillin, cephamycin and cephalosporin.

The third step involves the exchange of the hydrophilic D-alpha-aminoadipic acid side chain of isopenicillin N with L-5-amino-5-carboxypentanoic acid by the action of an Acyltransferase (AT). The enzymatic exchange reaction by AT regulation takes place in the organelle microsomes of the cell, as described in EP-A-0448180.

In cephalosporin-producing organisms, the third step is the isomerization of isopenicillin N to penicillin N by epimerase, whereupon the five-membered ring structures characteristic of penicillins form the six-membered ring structures characteristic of cephalosporins by the action of expandase.

The commercially significant penicillins produced only by direct fermentation are penicillin V and penicillin G, respectively by adding hydrophobic side chain precursor substances during fermentation of penicillium chrysogenum: phenoxyacetic acid or phenylacetic acid, whereby the phenoxyacetic acid or phenylacetic acid replaces the side chains of the natural beta-lactams.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (e.g. cephalexin) are derived from penicillins by a number of chemical transformations. Cephalosporin C, which is by far the most important starting material in this respect, is readily soluble in water at any pH, which implies the use of cumbersome and expensive column chromatography techniques for longer and more costly isolation procedures. Cephalosporin C obtained in this way has to be converted by a large number of chemical and enzymatic conversions to form other cephalosporins of therapeutic value.

The intermediate 7-ADCA of cephalosporin is currently produced by chemical derivatization of penicillin G. This necessary chemical step for the production of 7-ADCA involves the expansion of the five-membered ring of penicillin into the six-membered ring of cephalosporin.

Recently, fermentation processes have been disclosed to obtain 7-ADCA.

The use of adipic acid (5-carboxypentanoic acid) as starting material in EP-A-0532341 has shown that it is possible to form ｃA penicillin derivative having adipoyl side chains, namely: adipoyl-6-aminopenicillanic acid. This integration is due to the broad substrate specificity that has been demonstrated for acyltransferases (Behrens et al, J. Bid. chen. 175(1948), 751-809; Cole, Biochemical methods (Process Biochem.)1(1966), 334-338; Ballio et al, Nature 185(1960), 97-99). In addition, when adipic acid is added to a recombinant P.chrysogenum strain expressing an expandase, adipoyl-6-APA is expanded to form its corresponding cephalosporin derivative. Finally, it is proposed to eliminate the adipoyl side chain to produce 7-ADCA as the final product.

Patent application EP-A-0540210 describes ｃA similar process for the preparation of 7-ACA, comprising an additional step of converting the 3-methyl group of the ADCA ring into the 3-acetoxymethyl group of ACA.

WO95/04148 and WO95/04149 disclose the addition of a specific dicarboxylic acid source containing a sulphur group to a Penicillium chrysogenum strain expressing an expandase enzyme (expandase) such that these precursor species are integrated onto the penicillin backbone and subsequently expanded to form the corresponding 7-ADCA derivative.

However, it is generally accepted that expandases which provide crucial bonds in the biosynthesis of penicillin N and cephalosporins should have a narrow specificity (Maea, et al, enzyme and microbial technology (enzyme and Micoobiol. technology) (1995)17: 231-.

It has now been surprisingly found that a dicarboxylic acid starting material having a carbon chain length of greater than 7 carbon atoms produces a β -lactam derivative having incorporated side chains of 6 or 7 atoms in length.

Summary of The Invention

The invention discloses a method for preparing an N-deacylated cephalosporin compound, which comprises the following steps:

fermenting a strain of a microorganism capable of producing β -lactam and expressing acyltransferase and expandase activities and optionally acetyl transferase and/or hydroxylase activities in the presence of a side chain precursor material according to formula (1):

HOOC-X-(CH₂)_n-COOH (1)

wherein

n is an even number of at least 2, and

x is (CH)₂)_p-A-(CH₂)_qWherein

p and q are each independently 0, 1, 2, 3 or 4, and A is CH = CH, C ≡ C, CHB, C = O, O, S, NH, the N atom is optionally substituted or the S atom is optionally oxidized, and B may be H, halogen, C_1-3Alkoxy, hydroxy or optionally substituted methyl, with the proviso that p + q =2 or 3 should be satisfied when a is CH = CH or C ≡ C, and p + q =3 or 4 should be satisfied when a is CHB, C = O, O, S or NH,

alternatively, the side chain precursor species may yield an acyl-6-APA derivative in the presence of a salt, ester or acyl form of the precursor species. The acyl group has a structure of formula (2)

HOOC-X-CO- (2)

Wherein X is as described above, and wherein,

the acyl-6-APA derivative is subjected to in-situ ring expansion to form a corresponding acyl-7-ADCA derivative, and is selectively further reacted to generate acyl-7-ADAC or acyl-7-ACA derivative,

recovering the acyl-7-cephalosporin derivative from the fermentation medium,

deacylating the acyl-7-cephalosporin derivative, and

recovery of crystals of 7-cephalosporin compound

Detailed Description

The invention discloses a method for preparing an N-deacylated cephalosporin compound (7-ADCA,7-ADAC or 7-ACA), which is realized by applying a novel side chain precursor material to carry out fermentation production on an acylated counterpart of the compound.

Surprisingly, the present invention provides that microbial strains which produce beta-lactams and which express acyltransferase and expandase activities, can be fermented in the presence of dicarboxylic acids having a carbon chain length of greater than 7 carbon atoms to form an acyl-7-ADCA derivative in which the acyl group is incorporated with a carbon chain length of 6 or 7 carbon atoms, respectively.

According to the invention, if the microorganism strain used which produces beta-lactam and which expresses acyltransferase and expandase activities additionally expresses hydroxylase or hydroxylase plus acetyltransferase activity, respectively, then in addition to the production of acyl-7-ADCA, additionally 7-acylated cephalosporin derivatives, namely acyl-7-ADAC or acyl-7-ACA, respectively, are produced.

The dicarboxylic acids used in the process of the present invention have the structure of formula (1):

HOOC-X-(CH₂)_n-COOH (1)

wherein

n is an even number of at least 2, and

x is (CH)₂)p-A-(CH₂) q is wherein

p and q are each independently 0, 1, 2, 3 or 4, and should satisfy p + q =2, 3 or 4, and

a is CH = CH, C ≡ C, CHB, C = O, O, S, NH, the N atom is optionally substituted or the S atom is optionally oxidized, and B is H, halogen, C_1-3Alkoxy, hydroxy or optionally substituted methyl.

According to the invention, the microbial strain is fermented in the presence of a precursor substance of formula (1) or in the form of a salt, ester or amide thereof, resulting in the formation of an acyl-7-cephalosporin derivative, wherein the acyl group has the structure of formula (2):

HOOC-X-CO- (2)

wherein X is as described above.

In order to obtain acyl-7-cephalosporin derivatives of acyl groups with a chain length of 6 or 7 carbon atoms, respectively, when a is CH = CH or C ≡ C, p + q should be 2 or 3, respectively, and when a is CHB, C = O, O, S, NH, p + q should be 3 or 4, respectively, the N atom is optionally substituted or the S atom is optionally oxidized, and B is as described above.

Thus, fermentation of a strain of a microorganism which produces beta-lactam and which expresses acyltransferase and expandase activities in the presence of a precursor compound of formula (1) gives an acyl-6-APA derivative with an acyl group of formula (2), which derivative is subsequently in situ expanded to form the corresponding acyl-7-ADCA derivative. In other words, the precursor substance (represented by formula (1)) is metabolized by the microorganism strain to produce an acyl group of formula (2). The acyl group is then incorporated into the β -lactam backbone under acyltransferase mediated action.

The upper limit of the carbon chain length of the precursor compound represented by the formula (1), i.e., the upper limit value of n, is not critical. The upper limit is determined primarily by the efficiency of the microbial strain that metabolizes the precursor substance. Conveniently, the maximum carbon chain length that the precursor species can carry is similar to the longest carbon chain length of the fatty acids that the microbial strain is capable of metabolizing.

In one embodiment of the invention, a dicarboxylic acid is used, which in the presence of the dicarboxylic acid ferments to produce an adipoyl-7-ADCA derivative. Suitable dicarboxylic acids for the production of adipoyl-7-ADCA have the structure shown in formula (1) wherein n is an even number of at least 2; and X is (CH)₂)p-A-(CH₂) q, wherein p is 1 and q is 2, and A is CH₂. Preferably, the adipoyl-7-ADCA producing dicarboxylic acid is suberic acid or sebacic acid (n is 2 or 4, respectively).

In another embodiment of the invention, a dicarboxylic acid is used which produces an acyl-7-ADCA derivative containing an acyl group having a sulfur group as shown in formula (2). Suitable dicarboxylic acids for producing such acyl-7-ADCA compounds have the structure shown in formula (1), wherein n is an even number of at least 2 and X is (CH)₂)p-A-(CH₂) q, wherein A is S. Preferably, p and q are 1, 2 or 3, and p + q =3 or 4. Most preferably, p is 1 and q is 2, or p is 2 and q is 1 or 2.

In two further embodiments of the invention, dicarboxylic acids are used which yield novel acyl-7-cephalosporin derivatives.

First, a dicarboxylic acid is used, and fermentation in the presence of the dicarboxylic acid produces a pimeloyl-7-ADCA derivative. Suitable dicarboxylic acids for the production of pimeloyl-7-ADCA have the structure shown in formula (1), wherein n is an even number of at least 2 and X is (CH)₂)p-A-(CH₂) q, wherein p and q are 2, and A is CH₂. Preferably, the pimeloyl-7-ADCA producing dicarboxylic acid is azelaic acid (n = 2).

In addition, dicarboxylic acids are used which produce an acyl-7-ADCA derivative containing an acyl group having an unsaturated bond as shown in formula (2). Suitable dicarboxylic acids for producing such acyl-7-ADCA compounds have the structure shown in formula (1), wherein n is an even number of at least 2 and X is (CH)₂)p-A-(CH₂) q, wherein a is CH = CH or C ≡ C. Preferably, a is CH = CH, and both p and q are 1. Most preferred is the trans isomer of the latter compound.

The microbial strain used in the method of the invention is a strain that produces beta-lactam and expresses acyltransferase and expandase activities. Optionally, the microorganism strain may additionally express hydroxylase or hydroxylase plus acetyltransferase activity. The former strain produces acyl-7-ADCA derivatives, while the latter produces acyl-7-ADAC or acyl-7-ACA derivatives.

Examples of such microbial strains include penicillin producing strains with an expression cassette providing for expression of expandase and cephalosporin producing strains with an expression cassette providing for expression of an acyltransferase.

The expandase gene which is easy to apply can be derived from Acremonium chrysogenum, Streptomyces clavuligerus, Streptomyces antibioticus or Nocardia lactamdurans. The acyltransferase gene may be derived from Penicillium chrysogenum (P. chrysogenum), Penicillium naegiovense (P. nalgiovense) or Aspergillus nidulans (A. nidulans).

In a preferred embodiment, a penicillin producing fungal strain recombinantly expressing expandase is used. More preferably, a fungus of the genus Aspergillus (Aspergillus) or Penicillium (Penicillium) is used. Most preferably, a strain of Penicillium chrysogenum is used. The Penicillium chrysogenum strain Panlabs P14-B10, DS18541 (deposited at CBS under accession No. 455.95) is an example of a suitable host for expandase expression.

The construction of strains which recombinantly express an expandase is within the knowledge of a person skilled in the art. Examples of expression cassettes which can be used for the construction of fungal strains for the recombinant expression of expandases are disclosed in EP-A-0532341, Crawford et al, (Biotechnology, (Biotechnol.)13(1995),58-62) and WO 95/04148. In selecting transformed strains care should be taken to select a strain with sufficiently high expandase expression levels. Such transformants can be selected by testing their ability to produce adipoyl-7-ADCA. As described in Crawford et al, supra.

In another embodiment, a cephalosporin-producing strain that recombinantly expresses an acyltransferase is used, for example: an acyltransferase-producing Acremonium chrysogenum strain. Thus, an A.chrysogenum strain recombinantly expressing an acyltransferase can produce an acyl-7-ACA derivative, since such a strain expresses both a hydroxylase and an acetyltransferase itself.

The invention further describes a process for fermenting a fermentation medium with a specific solvent from a microorganism according to the invention, for example: process for the recovery of acyl-7-cephalosporin derivatives from the fermentation medium of P.chrysogenum strains expressing expandase, for example: acyl-7-ADCA derivatives such as: adipoyl, pimeloyl, 2- (carboxyethylthio) acetyl, 3- (carboxymethylthio) propionyl or trans-beta-hydrohexadienedioyl-7-ADCA.

In particular, the 7-acylated cephalosporin derivative is recovered from the fermentation broth by first extracting the fermentation broth filtrate with a water-immiscible organic solvent at a pH below about 4.5 and then back extracting the organic phase with an aqueous solution having a pH between 4 and 10.

The filtrate obtained after filtration of the culture medium is added with a water-immiscible organic solvent. The pH of the filtrate was adjusted for extraction of 7 acylated cephalosporin derivatives from the aqueous layer. The pH range must be below 4.5; preferably between 4-1, more preferably between 2-1. This allows the 7-acylated cephalosporin derivative to be separated from many other impurities in the fermentation medium. Preferably, a smaller volume of organic solvent is used, for example: the volume of the solvent is equivalent to half the volume of the aqueous layer, so that a concentrated solution of the 7-acylated cephalosporin derivative is obtained, which allows a reduction in the volume flow rate. A second possibility is to extract the entire medium at a pH of 4 or less. The culture medium is preferably extracted using a water-immiscible organic solvent at a pH between 4 and 1.

Any solvent that does not affect the cephalosporin molecule can be used. Suitable solvents are, for example: butyl acetate, ethyl acetate, methyl isobutyl ketone, alcohols such as butanol, and the like. Preferably n-or isobutanol.

Thus, the 7-acylated cephalosporin derivatives may be back-extracted with an aqueous solution having a pH between 4 and 10, preferably between 6 and 9. The final volume decreases again. The recovery can be carried out at a temperature between 0 and 50 ℃ and preferably at room temperature.

The 7-acylated cephalosporin derivatives produced by the process of the present invention are conveniently used as an intermediate in the chemical synthesis of semi-synthetic cephalosporins, since the presence of the appropriate acyl side chain substantially protects the 7-amino group.

Furthermore, 7-acylated cephalosporin derivatives can be deacylated in a one-step enzymatic process using a suitable enzyme, such as Pseudomonas SE83 acyltransferase.

In order to reuse the enzyme, preferably an immobilized enzyme is used. The methodology of such particle preparation and enzyme immobilization is well elucidated in EP-A-0222462. The pH of the aqueous solution is, for example, from pH4 to pH9, which reduces the degradation reaction of the cephalosporin and is optimal for the desired enzymatic conversion. Thus, the enzyme is added while the pH of the aqueous cephalosporin solution is maintained at an appropriate level. The pH can be maintained, for example, by: adding an inorganic base such as: KOH solution, or by using a cation exchange resin. After the reaction is complete, the immobilized enzyme can be removed by filtration. Another possibility is to apply the immobilized enzyme in a fixed or fluidized bed of columns or to use the immobilized enzyme in solution and remove the product by membrane filtration. Subsequently, the reaction mixture is acidified in the presence of a water-immiscible organic solvent. After adjusting the pH to about 0.1-1.5, the two phases are separated and the pH of the aqueous layer is adjusted to 2-5. The N-deacylated cephalosporin crystals are obtained by filtration.

The deacylation can also be carried out chemically, as known in the art, for example: this can be achieved by the formation of the side chain of the imminochloride by addition of phosphorus pentachloride at a temperature below 10 ℃ followed by the addition of isobutanol at room temperature or lower.

Example 1

Fermentative production of acyl-7-ADCA

The Penicillium chrysogenum strain Panlabs P14-B10 (deposited at CBS under accession number 455.95) was used as host strain for the expandase expression cassette construct.

The expression cassette used as described by Crawford et al (supra) contains an expandase gene under the transcriptional and translational regulatory signals of the Penicillium chrysogenum IPNS gene. Transformation and culture conditions were as described in Crawford et al, supra. Transformants were purified and expandase expression was analyzed by testing their ability to produce adipoyl-7-ADCA as described by Crawford et al, supra.

acyl-7-ADCA-producing transformants at 2X 10⁶The conidia/ml were inoculated into a seed medium containing (g/l): glucose, 30; pharmamedia (cottonseed meal), 10; 20 parts of corn steep liquor; (NH)₄)₂SO₄20, 20; calcium carbonate, 5; KH (Perkin Elmer)₂PO₄0.5; lactose, 10; yeast extract, 10, medium pH 5.6 before sterilization.

The seed medium (20ml in 250ml Erlemeyer flasks with cotton stoppers) was incubated at 220 rpm at 25 ℃. After 48 hours, 1ml was inoculated onto 15ml of production medium. The culture medium contains (g/l) KH₂PO₄,0.5；K₂SO₄,5；(NH₄)₂SO₄17.5; lactose, 140; pharmamedia, 20; calcium carbonate, 10; lard, 10, medium pH 6.6 before sterilization.

A stock solution of 20% of the selected precursor substance (pH adjusted to 6.5 with KOH) was added to the seed medium inoculated production medium to a final concentration of 0.5%.

The production medium was incubated in a 250ml Erlemeyer flask closed with a milk filter at 220 rpm for 168 hours at 25 ℃. Every other day, evaporated water was replenished.

After the end of the fermentation process, the mycelium was removed by centrifugation or filtration and analyzed for acyl-7-ADCA by HPLC.

Example 2

Analysis of acyl-7-ADC production

The fermentation product from the transformed Penicillium strain was analyzed by High Performance Liquid Chromatography (HPLC). The HPLC system consisted of the following components: p1000 solvent delivery system (TSP), basic marathon model autosampler (Spark Holland) (sample volume 3), UV150(TSP) variable wavelength detector (set at 260nm) and a PC1000 data system. The stationary phase was a YMC pack ODS AQ column (150X 4.6 mm). The mobile phase is composed of the following materials: 84% phosphate buffer with 0.17% tetrabutylammonium hydrogen sulfate added and 16% acetonitrile at pH 6.0. The fermentation product can be quantified by comparison with the expected standard curve for acyl-7-ADCA.

Example 3

Identification of acyl-7-ADCA products

In the presence of the following precursor materials: adipic acid, suberic acid, sebacic acid, pimelic acid and azelaic acid, a Penicillium chrysogenum strain recombinantly expressing an expandase was cultivated according to example 1.

Analysis of these fermentation products obtained by fermentation in the presence of adipic acid, suberic acid and sebacic acid according to the method of example 2 revealed the formation of adipoyl-7-ADCA and the formation of pimeloyl-7-ADCA upon administration of pimelic acid or azelaic acid.

If suberic acid (2.0% instead of 0.5%) was used at high concentrations during the fermentation, a small but significant amount of suberoyl-7-ADCA could be detected in addition to adipoyl-7-ADCA.

Claims (11)

1. A process for the preparation of an N-deacylated cephalosporin compound comprising the steps of:

fermenting a strain of a microorganism capable of producing β -lactam and expressing acyltransferase and expandase activities and optionally acetyl transferase and/or hydroxylase activities in the presence of a side chain precursor material according to formula (1):

HOOC-X-(CH₂)_n-COOH (1)

wherein

n is an even number of at least 2, and

x is (CH)₂)p-A-(CH₂) q is wherein

p and q are each independently 0, 1, 2, 3 or 4, and A is CH = CH, C ≡ C, CHB, C = O, O, S, NH, the N atom is optionally substituted or the S atom is optionally oxidized, and B is H, halogen, C_1-3Alkoxy, hydroxy or optionally substituted methyl, with the proviso that p + q =2 or 3 should be satisfied when a is CH = CH or C ≡ C, and p + q =3 or 4 should be satisfied when a is CHB, C = O, O, S or NH,

or in the presence of a salt, ester or amide form of the precursor species, the side chain precursor species may yield 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, and wherein,

the acyl-6-APA derivative is subjected to in-situ ring expansion to form a corresponding acyl-7-ADCA derivative, and is selectively further reacted to generate acyl-7-ADAC or acyl-7-ACA derivative,

recovering the acyl-7-cephalosporin derivative from the fermentation medium,

deacylating the acyl-7-cephalosporin derivative, and

recovering the crystals of the 7-cephalosporin compound.

2. The method of claim 1, wherein a side chain precursor of formula (1) is used, n being an even number of at least 2 and X being (CH)₂)p-A-(CH₂) q, wherein p is 1, q is 2, and A is CH₂。

3. The method of claim 2, wherein the side chain precursor material is suberic acid or sebacic acid.

4. The method of claim 1, wherein a side chain precursor of formula (1) is used, n being an even number of at least 2 and X being (CH)₂)p-A-(CH₂) q, wherein p and q are 2, and A is CH₂。

5. The method of claim 4, wherein the side chain precursor species is azelaic acid.

6. The process of any one of claims 1 to 5, wherein the microbial strain is a penicillin producing strain providing an expression cassette for expression of expandase.

7. The process of claim 6 wherein the penicillin producing strain is Penicillium chrysogenum.

8. The process of claim 6 or 7, wherein the crystalline cephalosporin compound is 7-ADCA.

9. The method of any one of claims 1-5, wherein the microbial strain is a cephalosporin-producing strain providing an expression cassette for expression of the acyltransferase.

10. The method of claim 9 wherein the cephalosporin producing strain is Acremonium chrysogenum.

11. The process of claim 9 or 10, wherein the crystalline cephalosporin compound is 7-ACA.