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

Background of the invention -Lactam antibiotics constitute the most important group of antibiotic compounds, with a iong history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum, respectively.

As a result of classical strain improvement techniques, the production levels of the antibiotics in Penicillium chrysogenum and Acremonium chrysogenum have increased dramatically over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools for the improvement of production strains and for the in vivo derivatization of the compounds have become available.

Most enzymes involved in fi-lactam biosynthesis have been identified and their corresponding genes been cloned, as is decribed 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. chrysogenum

are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-a-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclization of this tripeptide to form isopenicillin N. This compound contains the typical P-lactam structure.

These first two steps in the biosynthesis of penicillins are common in penicillin, cephamycin and cephalosporin producing fungi and bacteria.

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

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

The only directly fermented penicillins of industrial interest are penicillin V and penicillin G, produced by adding the hydrophobic side chain precursors phenoxyacetic acid or phenylacetic acid, respectively, during fermentation of P.

chrysogenum, thereby replacing the side chains of the natural -iactams with phenoxyacetic acid or phenylacetic acid.

Next to phenylacetic acid, phenylbutyric acid and certain derivatives give rise to the production of penicillin G, although said acids produce penicillin G with a much 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 give rise to the production of penicillin G with an even higher efficiency than phenylacetic acid.

Description of the invention The present invention discloses that certain derivatives of phenylbutyric acid, i.e. phenylbutyric acid derivatives wherein the acyl chain is extended by pairs of carbon atoms and wherein specific substituents are present at the w and/or w-l position of the acyl chain, are advantageously used as side chain precursors in the fermentative production of N-phenylacetyl penam or cephem compounds.

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

Specifically, the present invention discloses a process for the fermentative production of N-phenylacetyl or N-phenoxyacetyl penam or cephem compounds, wherein fermentation occurs in the presence of an w- and/or (Z-1)-substituted phenylalkanoic acid as a side chain precursor, said w- and/or (S-1)-substituted phenylalkanoic acid having a structure according to formula 1 wherein - -R, and -R3 are selected from the group consisting of -OH, =O or -H, where -R1 and -R3 can be the same or different, with the proviso that -R and -R3 are not both -H or =0, -R2 or -R4, respectively, is -H if -R1 or -R3, respectively, is -OH or -H, - -R2 or -R4, respectively, is not present if -R, or -R3, respectively, is =0, - -R, is -OR6 or -N H2, 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 up to 15, the carbon chain optionally containing on or more double bonds.

It is to be understood that the upper limit of the carbon chain length of the phenylalkanoic acid is mainly determined by the efficiency by which the fatty acyl group is attacked by fl-oxidation. Suitably, a chain length up to about 1 8 carbon atoms may be used, implicating that n is an odd number from 1 up to 1 5. Preferably, n is an odd number from 1 up to 9, more preferably from 1 up to 5. Most preferably, n is 1.

Preferably, -R1 or -R3 is either =0 or -OH implicating that -R2 or -R4 is either not present or -H, -R3 and -R4 are -H, -R5 is -OH, m is 0 or 1 and n is 1.

More preferably -R, is either =0 or -OH implicating that -R2 is either not present or -H, -R3 and -R4 are -H, -R5 is -OR6, wherein R6 is -H, m is 0 or 1 and n is 1. Most preferably, 3-benzoylpropionic acid is used as a side chain precursor, i.e. R1 is =O, R2 is not present, R3 and R4 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 cheap constituents (Sommerville and Allen, Org. Synth. Coll. Vol. II (1943), 81-83).

When using a P. chrysogenum strain in the process of the invention, penicillin G or V are produced.

It is shown that the phenylbutyric acid derivative 3-benzoylpropionic acid gives rise to the production of penicillin G with a much higher efficiency than phenylbutyric acid and, more importantly, with an even higher efficiency than phenylacetic acid.

The present invention additionally envisages the production of cephalosporin G or V derivatives in a fermentation process applying the precursors according to the invention, by using recombinant penam or cephem- producing strains, i.e. recombinant P. chrysogenum or Acremonium chrysogenum strains. Depending on the specific recombinant strain which is used in the fermentation process according to the invention, different

cephalosporin G or V compounds are produced.

Deacetoxy cephalosporin G or V derivatives are produced by, for instance, a recombinant expandase-expressing P. chrysogenum strain, i.e. a P. chrysogenum strain provided with an expression cassette comprising an expandase gene (see EP 0532341 or W095/04149 disclosing expandase- expressing P. chrysogenum strains). In that regard, W096/38580 is relevant since this document discloses that penicillin G can be expanded in vivo in an expandase-expressing P. chrysogenum strain.

If a recombinant expandase-expressing P. chrysogenum strain is provided with one or more expression cassettes comprising additional relevant cephalosporin biosynthetic genes, such as a gene encoding hydroxylase and/or a gene encoding acetyl transferase, other cephalosporin G or V derivatives than deacetoxy compounds are produced. Alternatively, cephalosporin G or V derivatives other than deacetoxy compounds are produced using an A.

chrysogenum strain recombinantly expressing an acyltransferase gene.

The process of the invention is carried out by fermentation of a suitable penam- or cephem-producing strain, i.e. a fungal strain as defined above, in a suitable fermentation medium. The fermentation conditions which are used are not critical for the present invention, provided that the fermentation occurs in the presence of a phenyl- or phenoxybutyric acid derivative according to Formula 1 as a side chain precursor. For instance, fermentation conditions can be applied such as disclosed in EP 0532341.

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

Optionally, the penicillin G or V or cephalosporin G or V derivative may be deacylated to form the corresponding deacylated penicillin, i.e. 6- aminopenicillanic acid (6-APA), or cephalosporin, e.g. 7-aminodeacetoxy- cephalosporanic acid (7-ADCA), 7-aminodeacetylcephalosporanic acid (7- ADAC) or 7-aminocephalosporanic acid (7-ACA). Deacylation is performed by any suitable means. Preferably, deacylation is performed in a one-step enzymatical process, using a suitable enzyme. Suitable enzymes for deacylation

of penicillin G or cephalosporin G derivatives are the acylases from E. coli or A. faecalis and for deacylation of penicillin V or cephalosporin V compounds the acylases from a fungal source, such as Fusarium. Preferably, an immobilized enzyme is used, in order to be able to use the enzyme repeatedly.

In a preferred embodiment of the invention, P. chrysogenum is fermented in a suitable culture medium in the presence of 3-benzoylpropionate as the side chain precursor. After separating off the biomass, the fermentation broth obtained is analyzed for the presence of penicillins, using HPLC and/or proton NMR. Penicillin G is shown to be the sole penicillin present in the fermentation broth. In addition, 3-benzoylpropionate is surprisingly shown to produce penicillin G more efficiently than phenylacetate does.

Example 1 Production of penicillin G using 3-benzoylpropionic acid as the side chain precursor Strains used Penicillium chrysogenum Wisconsin 54-1255 (ATCC 28089) Solutions Precursor-solution: 10 % (w/v) precursor adjusted to pH 6.5 with 1 M KOH, filter-sterilized before use.

Growth conditions A two-stage fermentation of the P.chrysogenum Wisconsin 54-1255 strain in shake flasks was used for the production of penicillins. The seed stage was initiated by adding 2 * 108 spores to 50ml/500ml flask of medium composed of (g/l): glucose, 30; (NH4)2SO4, 10; KH2PO4, 10; trace element solution I (MgSO4.7H2O, 25; FeSO4.7H2O, 10; CuSO4.5H2O, 0.5; ZnSO4. 7H2O, 2; Na2SO4, 50; MnSO4. H2O, 2; CaCl2.2H2O, 5), 10 (ml/l) (pH before sterilization

6.5).

The seed culture is incubated for 48-72 hours at 25-30 OC and subsequently used to inoculate 10-20 volumes of a production medium containing (g/l): lactose, 80; maltose, 20; CaSO4, 4; urea, 3; MgSO4.7H2O, 2; KH2PO4, 7; NaCI, 0.5; (NH4)2SO4, 6; FeSO4.7H2O, 0.1; trace element solution II (CuSO4.5H2O, 0.5; ZnS04.7H2O, 2; MnSO4.H2O, 2; Na2SO4, 50);(pH before sterilization 5.5-6.0). The precursor of choice (solution 1) is added to the indicated concentration. The incubation is then continued for another 1 20 hours.

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

When no precursor was added the main fi-lactams that accumulated in the medium were 6-aminopenicillanic acid and isopenicillin N. Addition of either phenylacetic acid, phenylbutyric acid or 3-benzoylpropionic acid resulted in the sole production of penicillin G (Table 1). The highest production of penicillin G was obtained using 0.08% (w/v) 3-benzoylpropionic acid.

Table 1 Penicillin production with different side-chain precursors Precursor Concn. Main -lactam Prodn.2 (w/v) products' (%) Phenylacetic acid 0.04% penicillin G 100 0.08% penicillin G 80 Penylbutyric acid 0.04% penicillin G 70 0.08% penicillin G 85 3-Benzoylpropionic acid 0.04% penicillin G 90 0.08% penicillin G 125 'determined by H-NMR 2relative to the production of penicillin G at an initial phenylacetic acid concentration of 0.04% (w/v)