MXPA99005545A - Process for the production of penicillin g or v, cephalosporin g or v, and derivatives thereof - Google Patents

Abstract

The present invention discloses a fermentative process for the production of penicillin G or V or cephalosporin G or V derivatives wherein certain derivatives of phenylbutyric acid are used as a side chain precursor. In said phenylbutyric acid derivatives, the acyl chain is extended by pairs of carbon atoms and certain substituents are present at the&ohgr;- and/or&ohgr;-1 position. The corresponding phenoxybutyric acid derivatives are used to produce penicillin V or cephalosporin V derivatives. Optionally, the penicillin or cephalosporin G or V compounds are deacylated to produce their deacylated counterparts.

Description

- - PROCESS FOR THE PRODUCTION OF PENICILLIN G O V, CEFALOSPORINE G O V, AND DERIVATIVES FROM THEM.

FIELD OF THE INVENTION.

The present invention relates to the field of fermentative beta-lactam production.

BACKGROUND OF THE INVENTION.

Beta-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 fungus peni cilli um chrysogenum and chrysogenum respectively.

As a result of classical breeding strains, production levels of antibiotics in penicilli um chrysogenum and Acrenonium chrysogenum have increased dramatically during the past decades. With the growing knowledge of the biosynthetic pathways that lead to penicillins and ref: 30495 cephalosporins, and with the advent of recombinant DNA technology, new tools have been made available to improve the production of strains for in vivo derivation of the compounds.

Most of the enzymes involved in beta-lactam biosynthesis have been identified and their respective genes have been cloned, as described by Ingolia and Queener, Med. Res. Rev. 9 (1989), 245-264 (biosynthesis and enzymes pathway). ), and Aharonowits, Cohen and Martin, Ann. Rev. Microbiol. 46 (1992), 461-495 (gene cloning).

The first two steps in penicillin biosynthesis in P. chrysogenum are the condensation of the three amino acids, L-5-amino-5-carboxypentanoic acid (L-alpha-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) in the LLD-ACV tripeptide, followed by the cyclization of this tripeptide to form isopenicillin N. This compound contains the typical beta-lactam structure.

- - These first two steps in penicillin biosynthesis are common in the fungi and bacteria that produce penicillin, cefamycin, and cephalosporin.

The third step involves the exchange of the hydrophilic side chain of D-alpha-aminoadipic acid to L-5-amino-5-carboxypentanoic acid, by a hydrophobic side chain, by the action of the enzyme acyltransferase (AT). The AT-mediated enzymatic exchange reaction takes place inside a cell organelle, the micro body, as described in EP-A-0448180.

In organisms that produce cephalosporin, the third step is the isomerization of isopenicillin N to penicillin N by an epimerase, after which the five-member ring structure characteristic of penicillins is expanded by the expandase enzyme to a ring of Six characteristic members of the cephalosporins.

The only fermented penicillins directly of industrial interest are penicillin V and penicillin G, which are produced by the addition of phenoxyacetic acid precursors of the hydrophobic side chain, respectively, during the fermentation of P. chrysogenum, thereby replacing the side chains of natural beta-lactams with phenoxyacetic acid or phenylacetic acid.

Along with phenylacetic acid, phenylbutyric acid and certain derivatives, which motivate the production of penicillin G, although these acids produce penicillin G with lower efficiency than phenylacetic acid (Behrens et al., J. Biol. Chem. 175 (1948) , 793-809, Arnstein and Grant, Bacteriol, Rev. 20 (1956), (133-147).

It is surprisingly shown by the present invention that specific derivatives of phenylbutyric acid generate the production of penicillin G with even greater efficiency than with phenylacetic acid.

DESCRIPTION OF THE INVENTION The present invention discloses that certain phenylbutyric acid derivatives, ie, phenylbutyric acid derivatives where the acyl chain is extended by pairs of carbon atoms and where specific substituents are present at the position? and / or β1 of the acyl chain, are advantageously used as side chain precursors in the fermentative production of penaemia or N-phenylacetyl compounds.

The present invention further discloses that the use of phenoxy derivatives of the specified phenylbutyric acid derivatives leads to the production of penam or cefen N-phenoxyacetyl compounds.

Specifically, the present invention describes a process for the fermentative production of penfem or penam N-phenylacetyl or N-phenoxyacetyl penam or cephem compounds, where fermentation occurs in the presence of a phenylalkanoic acid? and / or (? -1) -substituted as a side chain precursor, said phenylalkanoic acid? - and / or (? -1) -substituted has a structure in accordance with formula 1: where -Ri and R3 are selected from the group consisting of -OH, = 0 or -H, where -Ri and R3 can be the same or different, with the proviso that -Ri and R3 are not both -H ó = 0, -R2 or R4, respectively, is -H if -Rx or -R3, respectively, is -OH or -H, -R? or R4, respectively, is not present if -Ri or R3 respectively, is = 0, -R5 is -OR6 or -NH2, wherein R6 is selected from the group consisting of -H, -CH3 or CH2CH3, m is 0 or 1, - n is an odd number from 1 to 15, the carbon chain optionally contains one or more double bonds.

It is understood that the upper limit in the length of the carbon chain of phenylalkanoic acid is mainly determined by the efficiency at which the fatty acyl group is attacked by beta-oxidation. Suitably, a length chain of up to about 18 carbon atoms can be used, implying that n is an odd number between 1 to 15. Preferably, n is an odd number between 1 to 9, more preferably 1 to 5. More preferably , n is 1.

Preferably, -Ri or -R3 is either = 0 or -OH implying that -R2 or -R4 is whichever is not present or -H, -R3 and R4 are -H, -R5 is -OH, m is 0 or 1 and n is 1. Better still -Rx is any of = 0 or -OH implying that -R2 or -H whatever is not present, -R3 and R4 are -H, -R5 is -OR6, where - R6 is -H, m is 0 or 1 and n is 1. Most preferably, 3-benzoylpropionic acid is used as a precursor of the side chain, for example Ri is = 0, R2 is not present, R3 and R are - H, R5 is -OH, m is 0 and n is 1. - - 3-Benzoylpropionic acid is a preferred side chain precursor in the process of the invention, since this compound is conveniently synthesized from relatively inexpensive constituents (Sommerville and Alien, Org. Synth Coll. Vol. II (1943), 81-83.

When a strain of P. chrysogenum is used in the process of the invention, penicillin G or V is produced. it is shown that 3-benzoylpropionic acid derived from phenylbutyric acid generates the production of penicillin G with greater efficiency than phenylbutyric acid and, more importantly, with a higher efficiency than phenylacetic acid.

The present invention additionally contemplates the production of cephalosporin derivatives G or V in a fermentation process by applying the precursors according to the invention, by the use of recombinant strains that produce penam or cephem, ie, recombinant strains P. chrysogenum or Acremoni um. Depending on the specific recombinant strain which is used in the fermentation process according to the invention, different compounds of cephalosporin G or V are produced.

The cephalosporin G or V desasetoxy derivatives are produced by, for example, a recombinant strain of P. chrysogenum expressing an expandase, i.e., a strain of P. chrysogenum provided with an expression cassette comprising an expandase gene (see EP 0532341 or O95 / 04149 describing strains of P. chrysogenum expressing an expandase). In consideration, document 096/38580 is relevant, since this document describes that penicillin G can be expanded in vivo in a strain of P. chrysogenum expressing an expandase.

If a recombinant strain of P. chrysogenum expressing an expandase is provided with one or more expression cassettes further comprising relevant cephalosporin biosynthetic genes, such as a gene encoding hydroxylase and / or a germ encoding acetyl transferase, they produce other derivatives of cephalosporin G or V instead of deacetoxy compounds. Alternatively, cephalosporin derivatives G - - or V are produced in place of the desacetoxy compounds using a recombinant P. chrysogenum strain expressing an acyltransferase gene.

The process of the invention is carried out by the fermentation of a suitable strain that produces penam- or cephem-, ie, a fungal strain as defined above, in a suitable fermentation medium. The fermentation conditions used are not critical to the present invention, provided that the fermentation occurs in the presence of phenyl or phenoxybutyric acid derivatives according to formula 1 as a precursor of the side chain. For example, the conditions of the fermentation can be applied as described in EP 0532341.

Subsequent to the fermentation process, the penicillin derivative G or V or cephalosporin G or V produced by fermentation is recovered from the fermentation broth using any suitable technology known to the skilled artisan.

- - Optionally, the derivative of penicillin G or V or cephalosporin G or V can be deacylated, that is, 6-aminopenicilanic acid (6-APA), or cephalosporin, 7-aminodesacetoxycephalosporanic acid (7-ADCA), 7-aminodesacetylcephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Deacylation is carried out in any suitable medium. Preferably, the deacylation is carried out in a one-step enzymatic process, using a suitable enzyme. the enzymes suitable for the deacylation of penicillin G derivatives or cephalosporin G are the acylases of E. coil or A. faecali s and for the deacylation of penicillin V compounds or cephalosporin V the acylases of a fungal source, such as the Fusari um . Preferably, an immobilized enzyme is used, so that the use of the enzyme is possible repeatedly.

In a preferred embodiment of the invention, P. chrysogenum is fermented in a suitable culture medium in the presence of 3-benzoylpropionate as a precursor of the side chain. After separation of the biomass, the fermentation broth is analyzed by the presence of penicillins, using HPLC and / or NMR by protons. La - 1 - penicillin G is shown to be the only penicillins present in the fermentation broth. In addition, 3-benzoylpropionate shows surprisingly that it produces penicillin G more efficiently than phenylacetate does.

Production of penicillin G using 3-benzoylpropionic acid as the precursor of the side chain.

Strains used. Penicilli um chrysogenum isconsin 54-1255 (ATCC 28089) Solutions Precursor solution a: 10% precursor (P / V) of 6.5 adjusted to a pH of 6.5 with 1 M KOH, and filter sterilization before use.

Growth conditions A two-stage fermentation of the strain is used P. chrysogenum Wisconsin 54-1255 in a flask flasks for the production of penicillins. The sowing stage begins with the addition of 2 * 108 spores to a 50ml / 500ml flask of a medium composed of (g / 1): glucose, 30; (NH4) 2S04, 10, KH2P04, 10; trace element solution I (MgS0 7H20, 25, FeS04, 7H20, 10, CuS04, 5H20, 0.5, ZnS04, 7H20, 2, Na2S04, 50, MnS04 H20, 2, CaCl2 2H20, 5), 10 (ml / 1 ) (pH 6.5 before sterilization).

The seeded culture is incubated for 48-72 hours at 25-30 ° C and subsequently used to inoculate 10-20 volumes of a production medium containing (g / 1): lactose, 80; maltose, 20; CaS04, 4; urea, 3; MgSO4 7H20, 2; KH2P04, 7; NaCl, 0.5; (NH4) 2S04.6; FeS04 7H20, 0.1; the trace element solution II (CuS0 5H20, 0.5, ZnS047H20, 2, MnS0 H20, 2, Na2S04, 50); (pH before sterilization 5.5-6.0). The selection precursor (solution) is added at the indicated concentration. The incubation is then continued for another 120 hours.

The ability of peni cilli um chrysogenum to use different side chain precursors for the production of penicillin was examined. Phenylacetic acid, butyric acid, phenylbutyric acid and acid 3-benzoylpropionic were evaluated at final concentrations - 0.04% and 0.08% (w / v). At the end of the production stage, the culture filtrates were collected and examined by H-NMR.

When the precursor was not added, the main beta-lactams that accumulated in the medium were 6-aminopenicilanic acid and isopenicillin N. The addition of either phenylacetic acid, phenylbutyric acid or 3-benzoylpropionic acid resulted in the single production of penicillin. G (Table 1). The highest production of penicillin G was obtained using 0.08% 3-benzoylpropionic acid (w / v). table 1 production of penicillin with different side chain precursors 1 determined by H-NMR. 2 relative to the production of penicillin a concentration of phenylacetic acid of 0.40% (w / v).

- It is noted that in relation to this date, the best method known to the applicant, to put into practice the aforementioned invention is that it is clear from the manufacture of the objects to which it refers. having described the invention as above, the content of the following is claimed as property:

Claims (8)

1. A process for the production of a penam or cefem N-phenylacetyl or N-phenoxyacetyl compound optionally including deacylation of penam or cephem N-phenylacetyl or N-phenoxyacetyl compound, characterized in that it comprises the steps of: * the fermentation of a penam producing strain or cefem- in a suitable fermentation medium in the presence of a precursor of the side chain according to formula 1: where -Ri and -R3 are selected from a group consisting of OH, = 0 or -H, - - where -Ri and -R3 can be the same or different, with the proviso that -Ri and -R3 are not both -H or = 0, -R2 or -R4, respectively, is -H if -Ri or -R3 / respectively, is -OH or -H, -R2 or -R4, respectively, is not present if -Ri or -R3, respectively , is = 0, -R5 is -ORß or -NH2, in which Re is selected from the group consisting of -H, -CH3 or CH2CH3, m is O or 1, n is an odd number between 1 and 15, the string carbon optionally contains that number or more double bonds, and recover the N-phenylacetyl compound produced or N-phenoxyacetyl penam or cefem or cefem compound from the fermentation broth, and * optionally the deacylation of N-phenylacetyl or N-phenoxyacetyl penam or cefem produced and recovered the corresponding deacylated compound of penam or cephem.

2. A process according to claim 1, characterized in that it comprises producing N-phenylacetyl penam or cephem wherein the fermentation is developed using a side chain precursor according to formula 1 where m is 0.

3. A process according to claim 2 characterized in that the fermentation is carried out using a side chain precursor according to formula 1 where Ri is = 0, R3 and R4 are -H, R5 is -OH, m is O and n is 1.

4. A process according to claim 1, characterized in that it comprises producing N-phenoxyacetyl penam or cephem where the fermentation is carried out using a side chain precursor according to formula 1 where m is 1.

5. A process according to any of claims 1-4, characterized in that it comprises producing a N-phenylacetyl or N-phenoxyacetyl penam compound, where the penam or cephem producing strain is penicillium um chrysogenum and the N-phenylacetyl compound or N-phenoxyacetyl is penicillin G or V.

6. A process according to claim 5, characterized in that penicillin G or V is deacylated producing 6-APAN. - -

7. A process according to any of claims 1-4, producing the compound N-phenylacetyl or N-phenoxyacetyl cephem, characterized in that the strain that produces penam or cefem is a recombinant strain of peni cilli um chrysogenum expressing an expandase and the compound N-phenylacetyl or phenoxyacetyl cefem is the derivative of cephalosporin G or V.

8. A process according to claim 7, characterized in that the derivative of cephalosporin G or V is deacylated producing 7-ADCA, or 7-ADAC or 7-ACA