The present invention relates to a process for the production of cephalosporins and in particular for the production of 7-ACA or a derivative thereof comprising the steps of fermenting a P. chrysogenum strain being transformed with an expression construct comprising a nucleotide sequence encoding an expandase, a hydroxylase and an acetyltransferase in the presence of a suitable acyl side chain precursor, or a salt or ester thereof, such that an N-acylated 7-ACA compound is produced, N-deacylating the thus produced N-acylated 7-ACA compound and, optionally, acylating the free amino group and/or substituting the 3'acetate group with a side chain suitable to form a cephalosporin antibiotic.

Semi-synthetic routes to prepare cephalosporins mostly start from fermentation products such as penicillin G, penicillin V and cephalosporin C, which are converted to the corresponding p- ! actam nuctei, for instance in a manner as is disclosed in K. Matsumoto, Bioprocess. Techn., 16,, 67-88 (1993), J. G. Shewale & H. Sivaraman, Process Biochemistry, August 1989, 146-154, T. A. Savidge, Biotechnology of Industrial Antibiotics (Ed. E. J.

Vandamme) Marcel Dekker, New York, 1984, or J. G. Shewale et al., Process Biochemistry International, June 1990,97-103. The obtained p-tactam nuclei are subsequently converted to the desired antibiotic by coupling to a suitable side chain, as has been described in inter alia EP 0 339 751, JP 53005185 and CH 640 240. By making different combinations of side chains and ß- lactam nuclei, a variety of penicillin and cephalosporin antibiotics may be obtained.

The cephalosporin nuclei 7-amino desacetoxycephalosporanic acid (7- ADCA) and 7-amino cephalosporanic acid (7-ACA) are known to be the most

important intermediates for the production of antibiotics used in the pharmaceutical industry.

Cephalosporin C is by far the most important starting material for preparation of 7-ACA as well as for other therapeutically used cephalosporins.

However, cephalosporin C is very soluble in water at any pH, and this implies lengthy and costly isolation processes using cumbersome and expensive column technology to remove non-converted cephalosporin C from its product. In addition, the a-aminoadipoyl side chain of cephalosporin C is not very amenable to the enzymatical or chemical cleavage necessary to produce 7-ACA.

To overcome some of the drawbacks mentioned herein above, a fermentative process has been disclosed for the production of 7-ACA, involving fermentative production of certain N-substituted cephalosporins of which the side chain is easily removable by a simple enzymatical or chemical cleavage reaction.

The fermentative production of these N-substituted cephalosporins, such as adipoyl-7-ACA, is achieved by a recombinant Penicillium chrysogenum strain capable of expressing the enzyme activities desacetoxycephalosporin synthetase (also known as"expandase"), desacetylcephalosporin C synthetase ("hydroxylase") as well as cephalosporin C synthetase ("acetyltransferase") (EP 0 540 210).

In the in vivo production process of adipoyl-7-ACA using a recombinant P. chrysogenum strain it was observed that the precursor for adipoyl-7-ACA, adipoyl-7-amino desacetylcephalosporanic acid (adipoyl-7-ADAC), was present in substantial amounts as compared to adipoyl-7-ACA. Apparently, the acetyltransferase gene was not expressed in an amount sufficient to obtain a substantial conversion of adipoyl-7-ADAC to adipoyl-7-ACA.

The present invention discloses acetyltransferase expression constructs which are designed such as to obtain a high expression level of acetyltransferase. In this way, an increased amount of the precursor adipoyl- 7-ADAC is converted to adipoyl-7-ACA.

The documents describing the cloning and nucleotide sequence of the acetyltransferase gene from Acremonium chrysogenum (cefG) disclose a coding sequence starting either at the first (EP 0 437 378; Gutiérrez et al,, J.

Bacteriol. 174: 3056-3064 (1992)), the second (Mathison et al., Curr Genet.

23: 33-41 (1992)) or the third ATG (EP 0 450 758) of the acetyltransferase open reading frame (ORF). In addition, in an investigation on the efficiency of various promoters to express acetyltransferase in A. chrysogenum one of the contructs to be tested was a fusion construct comprising an acetyltransferase coding sequence starting at the second ATG (Gutierrez et al., Appl. Microbiol.

Biotechnol. 48: 606-614 (1997)).

None of the documents cited herein above disclose the advantage of the selection of a particular start codon of the cefG ORF to obtain efficient acetyltransferase expression in a recombinant P. chrysogenum strain for use in a fermentative process for the production of 7-ACA or a derivative thereof.

The present invention discloses a process for the production of 7-ACA or a derivative thereof comprising the steps of fermenting a P. chrysogenum strain being transformed with an expression construct comprising a nucleotide sequence encoding expandase, hydroxylase and acetyltransferase activity in the presence of a suitable acyl side chain precursor, or a salt or ester thereof, such that an N-acylated 7-ACA compound is produced, N-deacylating the thus produced N-acylated 7-ACA compound and, optionally, acylating the free amino group and/or substituting the 3'acetate group with a side chain suitable to form a cephalosporin antibiotic, characterised in that the nucleotide sequence. encoding the acetyltransferase is derived from A. chrysogenum and starts at the second ATG of said nucleotide sequence.

It is surprisingly found by the present invention that an expression construct comprising the acetyltransferase coding sequence from A. chrysogenum is expressed more efficiently when the coding sequence starts at the second ATG of the open reading frame (ORF) than when it starts at the first or the third ATG of said ORF. One of the effects of a more efficient

acetyltransferase expression is that the N-acylated 7-ADAC derivative is converted more efficiently to the N-acylated 7-ACA derivative.

In the process of the invention, a transformed P. chrysogenum strain is used expressing the three enzymatic activities of the cephalosporin biosynthetic pathway leading to the production of a 3'-acetylated cephalosporin compound.

Suitable sources for the genes encoding said three enzymatic activities are the bacteria Streptomyces clavuligerus or Nocardia lactamdurans for the expandase gene cefE and the hydroxylase gene cefF (see EP 0 341 892 for cefE and EP 0 465 189 for cerf) or the fungus A. chrysogenum for the bifunctional expandase/hydroxylase gene cefEF and the acetyltransferase gene cefG (see EP 0 281 391 and Coque et al., Mol. Gen. Genet. 236: 453- 458 (1993) for cefEF and EP 0 437 378 and EP 0 450 758 for cefG).

According to the present invention, the acetyltransferase enzyme activity is provided by the cefG gene as obtained from A. chrysogenum. In particular, the present invention shows that it is advantageous to use the second ATG of the cefG ORF as the start codon. The use of the second ATG of the cefG ORF as the start codon implicates that the acetyltransferase enzyme as used in the process of the present invention has an N-terminal amino acid sequence starting with methionine-leucine-arginine-aspartic acid- serine.

In the process of the invention, the genes encoding the three enzymatic activities expandase, hydroxylase and acetyltransferase may be provided with 5'and 3'regulatory sequences native to the genes in question or may be provided with regulatory sequences heterologous to said genes.

Examples of suitable 5'and 3'regulatory sequences, i. e. promoters and terminators, providing for recombinant gene expression in filamentous fungus host cells are mentioned in Van den Hondel et a/. (in: More Gene Manipulations in Fungi, Eds. Bennett and Lasure, 396-427 (1991)) or in Applied Molecular Genetics of filamentous fungi (Kinghorn, Turner (eds.), Blackie, Glasgow, UK, 1992). Preferred promoters are the Aspergillus niger glucoamylase promoter

or P. chrysogenum promoters derived from the genes encoding ACV synthetase, isopenicillin N synthase, acyltransferase, phosphoglycerate kinase or gene Y. Transcriptional terminators can be obtained from the same genes as well.

In one embodiment of the invention, a process for the fermentative production of 7-ACA or a derivative thereof is provided comprising the use of a P. chrysogenum strain transformed with expression constructs wherein the coding sequence of the gene encoding expandase, hydroxylase and/or acetyttransferase activity is fused to a promoter sequence which is heterologous for said coding sequence. Said heterologous promoter sequence for example is the IPNS (pcbC) promoter from P. chrysogenum.

In another embodiment of the invention, exact fusions between a promoter sequence of choice and the start codon of the coding sequence encoding expandase, hydroxylase and/or acetyltransferase activity are conveniently obtained using PCR technology.

Transformation of P. chrysogenum host cells can, in general, be achieved by different means of DNA delivery, like PEG-Ca mediated protoplast uptake, electroporation or particle gun techniques, and subsequent selection of transformants. See for example Van den Hondel en Punt, Gene and Transfer and Vector Development for Filamentous Fungi, in: Applied Molecular Genetics of Fungi (Peberdy, Laten, Ogden, Bennett, eds.), Cambridge University Press (1991). The application of dominant and non-dominant selection markers has been described (Van den Hondel, supra). Selection markers of both homologous (P. chrysogenum derived) and heterologous (non- P. chrysogenum derived) origin have been described (Gouka et al., J.

Biotechnol. 20 189-200 (1991)).

The application of the different transformant selection markers, homologous or heterologous, in the presence or absence of vector sequences, physically linked or not to the non-selectable DNA, in the selection of transformants are well known in the art.

Fermentation of a transformed P. chrysogenum strain may be done in any suitable fermentation medium known in the art, provided that fermentation occurs in the presence of a suitable side chain precursor. In this respect, a suitable side chain precursor is defined as an N-acyl side chain precursor leading to an N-acyl side chain of the fermentatively produced cephem compound, said N-acyl side chain being amenable to simple chemical or enzymatical removal. In particular, a suitable side chain precursor is a dicarboxylic acid, more particularly a dicarboxylic acid according to formula (1) HOOC-X- n-COOH (1) wherein n is an even number of at least 2, and X is (CH2) P-A- (CH2) q, wherein p and q each individually are 0,1,2,3 or 4, and A is CH=CH-CH=CH, CH=CH, C=C, CHB, C=O, O, S, NH, the nitrogen optionally being substituted or the sulphur optionally being oxidised, and B is hydrogen, halogen, C, 3 alkoxy, hydroxyl, or optionally substituted methyl, with the proviso that p+q should be 0 or 1 when A is CH=CH-CH=CH, p+q should be 2 or 3, when A is CH=CH or C=C or p+q should be 3 or 4, when A is CHB, C=O, O, S or NH, or a salt or ester thereof.

Examples of suitable side chain precursors according to Formula (1) are adipic acid, 3'-carboxymethylthiopropionic acid (WO 95/04148), 3,3'- thiodipropionic acid (WO 95/04149) or the side chain precursors as provided in WO 98/48034 or WO 98/48035. Preferred side chain precursors are adipic acid or trans-p-hydromuconic acid.

The N-acylated 7-ACA compound, for instance adipoyl-7-ACA, as obtained by fermentation in the presence of a suitable acyl side chain precursor, for instance adipic acid, may efficiently be recovered from the

fermentation medium through conventional recovery technology, for instance a simple solvent extraction process as follows: 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 cephalosporin 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 way the cephalosporin is separated from many other impurities present in the fermentation broth. Preferably a small volume of organic solvent is used, giving a more concentrated solution of the cephalosporin, so achieving reduction of the volumetric flow rates. A second possibility is whole broth extraction at a pH of 4 or lower. Preferably the broth is extracted at a pH between 4 and 1 with an organic solvent immiscible with water.

Any solvent that does not interfere with the cephaiosporin molecule can be used. Suitable solvents are, for instance, butyl acetate, ethyl acetate, methyl isobutyl ketone, alcools like butanol, etc..

Hereafter the cephalosporin is 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 temperatures between 0 and 10 °C.

The N-acylated cephalosporin derivatives produced by the process of the invention can conveniently be used as an intermediate for the chemical synthesis of semisynthetic cephalosporins, since the 7-amino group is adequately protected by presence of an appropriate acyl side chain.

Alternatively, the aqueous N-acylated cephalosporin solution thus obtained is treated with a suitable enzyme in order to remove the N-acyl, e. g. the adipoyl, side chain and obtain the desired 7-ACA.

Preferably, an immobilised enzyme is used, in order to be able to use the enzyme repeatedly. The methodology for the preparation of such particles and the immobilisation of the enzymes have been described extensively in EP 0 222 462. The pH of the aqueous solution has a value of, for example pH 4 to pH 9, at which the degradation reaction of cephalosporin is minimised and

the desired conversion with the enzyme is optimised. Thus, the enzyme is added to the aqueous cephalosporin solution while maintaining the pH at the appropriate level by, for instance, adding an inorganic base, such as a potassium hydroxide solution, or applying a cation exchange resin. When the reaction is completed the immobilised enzyme is removed by filtration.

Another possibility is the application of the immobilised enzyme in a fixed or fluidised bed column, or using the enzyme in solution and removing the products by membrane filtration. Subsequently, the pH of the aqueous solution is adjusted to a value between 2 and 5, preferably between 3 and 4.

The crystalline 7-ACA is then filtered off.

Suitable enzymes are, for instance, derived from a Pseudomonas SY77 micro-organism having a mutation in one or more of the positions 62,177, 178 and 179. Also enzymes from other Pseudomonas micro-organisms, preferably Pseudomonas SE83, optionally having a mutation in one or more of the positions corresponding to the 62,177,178 and 179 positions in Pseudomonas SY77, may be used.

The deacylation can also be carried out chemically as known in the art, for instance, via the formation of an iminochloride side chain, by adding phosphorus pentachloride at a temperature of lower than 10 °C and subsequently an alcohol like isobutanol at ambient temperatures or lower.

In one embodiment of the invention, the aqueous solution containing the N-acylated 7-ACA derivative or the 7-ACA as obtained after deacylation may be treated by a suitable acetylating agent to convert any (acyl)-7-ADAC which may be present in said aqueous solution to the corresponding (acyl)-7- ACA derivative. Said acetylation for instance can be done using acetic anhydride, for instance by the method as disclosed in US 5,221,739, or using a suitable lipase or esterase, for instance as disclosed in EP 667 396.

In a further step, the 7-ACA compound as obtained by the process of the invention is used as a starting compound in the preparation of a wide variety of cephalosporin antibiotics, end products as well as intermediates thereto. The free amino group of 7-ACA for instance may be acyiated with

any suitable side chain, using commonly known chemical or enzymatical coupling methods, resulting in an N-acylated 7-ACA derivative. In addition, substitutions at the 3'position may occur. Examples of the thus-produced cephalosporin compounds are cefotaxime, cefazolin, ceftriaxone, cefuroxime, cefprozil, ceftazidime and cefaclor.

EXAMPLE 1 Construction of plCG, WA, plCG2WA and plCG3WA for acetyltransferase expression in Penicillium chrysogenum The desacetylcephalosporin C acetyltransferase (cefG) expression cassette plCG, WA, which contains the wild type Acremonium chrysogenum cefG gene including the Penicillium chrysogenum pcbC promoter and penDE terminator, was constructed as described below. The N-terminal part of the cefG gene, i. e. starting at the first ATG of the ORF, was derived from A. chrysogenum chromosomal DNA in a PCR reaction using primers #1 and #2 (SEQ ID NO 1 and 2, respectively). The C-terminal part of the cefG gene was derived from the same template in a PCR reaction using primers #3 and #4 (SEQ ID NO 3 and 4, respectively). After a fusion PCR using primers #1 and #4 and the above fragments as template, a complete cefG gene (further indicated herein as the cefG, gene) was generated in which the internal Sfil and Hindlll sites were deleted and a novel Nsil site was created.

In the next step, the first part of the pcbC promoter was PCR-amplified using primers #5 and #6 (SEQ ID NO 5 and 6, respectively) and, after a fusion PCR using primers #5 and #4, introduced directly in front of the cefG, gene.

After digestion with PstllNsil a 1592 bp fragment was ligated to a 4.3 kb PstlINsA vector fragment of pISEWA-N (vector previously described in W098/46772) to yield the Penicillium transformation vector plCG, WA The N-terminal part of the cefG2 gene, i. e. starting at the second ATG of the ORF, needed for the construction of plCG2WA, was derived from

piCG1WA in a PCR reaction using primers p#5 and #7 (SEQ ID NO 7). The C- terminal part of the cefG2 gene was derived from the same template in a PCR reaction using primers #8 and #9 (SEQ ID NO 8 and 9, respectively). After a fusion PCR using primers #5 and #9 and the above fragments as template, a complete cefG2 gene was generated.

For the construction of plCG3WA, wherein the cefG gene starts at the third ATG, the same procedure was used as described for the construction of plCG2WA. For this construction the primers #5/#10 (SEQ ID NO 5/10, respectively) and #11/#9 (SEQ ID NO 11/9, respectiveiy) were used. <BR> <BR> <BR> <BR> <P> After digestion with the interna Pstl/Ncol sites the cefG21cefG3 fusion<BR> <BR> <BR> <BR> <BR> <BR> <BR> fragments were ligated to a PstllNcol vector fragment of plCG, WA yielding the Penicillium transformation vectors plCG2WA and plCG3WA.

EXAMPLE 2 Effect of different ATG start codon usage on the expression of acetyltransferase After Notl digestion of the different plCGWA constructs the isolated <BR> <BR> <BR> <BR> cefG fragments were introduced into P. chrysogenum by the Ca-PEG mediated protoplast transformation as described in EP 635 574.

The P. chrysogenum strain which was used has previously been transformed with an expression construct comprising the bifunctional <BR> <BR> <BR> <BR> expandase/hydroxylase coding sequence (cefEF) from A. chrysogenum under<BR> <BR> <BR> <BR> <BR> <BR> <BR> regulatory, control of the P. chrysogenum pcbC promoter and penDE terminator.

The fragments were co-transformed with amdS (EP 635 574), which enables P. chrysogenum transformants to grow on selection medium containing acetamide as sole nitrogen source. Transformants were purified by repeated cultivation on selective medium. Single stable colonies were used for further screening on the presence of the cefG gene by PCR. CefG positive

colonies were used for further screening of expression of cefG by measuring the capacity of the transformants to produce adipoyl-7-ACA.

To this end, transformants were inoculated in liquid medium as described in WO 95/04149, supplemented with 0.5-3 mg/ml sodium adipate as a side chain precursor for production tests. Filtrates of well grown cultures were analyse by HPLC and NMR for adipoyl-7-ACA production. The results presented in Table 1 clearly show an increased adipoyl-7-ACA production for transformants comprising cefG starting at the second ATG of the ORF<BR> (indicated as"ATG2"), as compared to the transformants comprising cefG starting at the first ATG (indicated as"ATG1") or the third ATG (indicated as "ATG3").

Table 1 Adipoyi-7-ACA productivity of P. chrysogenum transformants incorporating a cefG coding sequence starting at the first, second or third ATG. cefG start codon productivity (%) ATG 1 49 ATG2 100 ATG3 77