Wild-type P. chrysogenum is capable of producing penicillin compounds, however this organism is unable to produce cephalosporin compounds.

Ceph-3-em compounds (such as CPC) are produced by organisms like Acremonium chrysogenum (A. chrysogenum) (Aharonowitz, Y. , Cohen, G. , Martin, J. F. Annu. Rev. Microbio !. 46: 461-495) Penicillin compounds have a penam core structure characterized by a five-membered ring, whereas cephalosporin compounds are characterized by a six- membered ring in the ceph-3-em core. The key enzyme needed to convert a penam core into the corresponding ceph-3-em core is DAOC synthase, which enzymatically expands the five-membered ring into a six-mernbered ring.

This DAOC synthase enzyme is lacking in wild-type P. chrysogenum.

An essential step in the production of CPC in A. chrysogenum is the conversion of isopenicillin N into penicillin N by the socalled isopenicillin N isomerase enzyme system. This latter enzyme system is lacking in P. chrysogenum as well. Uhlan et al. recently have reported the identification of the isopenicillin N isomerase of A. chrysogenum (Ullan RV, Casqueiro J, Banuelos O, Fernandez FJ, Gutierrez S, Martin JF. 2002. J Biol Chem 277 (48) : 46216-25). According to this report, the isopenicillin N isomerase activity of A. chrysogenum resides in the joint action of a number of gene products (Fig. 1). Two of the relevant genes were identified as cefD1 and cefD2.

Blocking of the expression of either of these two genes impaired the formation of penicillin N in A. chrysogenum.

The structure of these genes has been elucidated as well (Ullan et al, 2002, supra).

The enzyme encoded by the cefD1 gene shows high similarity to long chain acyl-CoA synthetases and is considered to be responsible for the activation of isopenicillin N to isopenicillyl-CoA.

The enzyme encoded by the cefD2 gene has high similarity to a- methylacyl-racemases and 2-arylpropionyl-CoA epimerases suggesting that after activation of isopenicillin N to isopenicillyl-CoA epimerisation to penicillyl-CoA occurs via a similar mechanism.

However, a thioesterase which would establish the hydrolysis of penicillyl-CoA to form penicillin N was not identified by Ullan et al. In view of the required energy efficiency of the cells this should be expected to be an enzyme specific for the stereochemical configuration of penicillyl-CoA.

Hence, it seems that further information on a third enzyme essential for the conversion of isopenicillin N into penicillin N in A. chrysogenum is still lacking.

In view thereof it is highly surprising that according to the present invention it was found that a full epimerase activity could be established in P. chrysogenum by transforming this filamentous fungus by DNA encoding the enzymes cefD1 and cefD2 of the isopenicillin N epimerase system of A. chrysogenum.

According to a preferred embodiment of the present invention P. chrysogenum strains are transformed with the genes encoding the cefD1 and cefD2 enzymes of A. chrysogenum. Thus transformed strains are capable to produce penicillin N.

In a further embodiment of the present invention P. chrysogenum strains are transformed with the genes encoding the cefD1 and cefD2 enzymes of A. chrysogenum as well as with a gene encoding an enzyme with DAOC synthase activity. Thus transformed strains are capable to produce DAOC.

In a further embodiment of the present invention P. chrysogenum strains are transformed with the genes encoding the cefD1 and cefD2 enzymes of A. chrysogenum as well as with a gene or genes encoding enzyme with DAOC synthase activity and DAC synthase activity. Thus transformed strains are capable to produce DAC.

In a still further embodiment of the present invention P. chrysogenum strains are transformed with the genes encoding the cefD1 and cefD2 enzymes of A. chrysogenum, with a gene or genes encoding enzymes with DAOC synthase activity and DAC synthase activity as well as with a gene encoding an enzyme having acetyltransferase activity. Thus transformed strains are capable to produce cephalosporin C (CPC)

A further embodiment of the present invention relates to a bioprocess for preparing a cephalosporin derivative comprising the steps of a) maintaining in a culture medium capable of sustaining its growth a strain of P. chrysogenum which produces isopenicillin N; b) carrying out the following enzymatic conversion by in situ expression of the corresponding at least one gene: i) the isopenicillin N is in situ converted into penicillin N by isopenicillin N epimerase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the isopenicillin N epimerase enzyme system comprising the cefD1 and cefD2 genes of A. chrysogenum capable of accepting said isopenicillin N as a substrate, whereupon as a result of its expression, said isopenicillin N produced by said strain is also thereafter in situ converted into penicillin N ii) the penicillin N is in situ ring-expanded to form the corresponding desacetoxycephalosporin C (DAOC) by DAOC synthase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the DAOC synthase enzyme capable of accepting said penicillin N as a substrate, whereupon as a result of its expression, said penicillin N produced by said strain is also thereafter in situ ring-expanded to form DAOC.

A further embodiment of the present invention relates to a bioprocess for preparing a cephalosporin derivative comprising the steps of a) maintaining in a culture medium capable of sustaining its growth a strain of P. chrysogenum which produces isopenicillin N; b) carrying out the following enzymatic conversion by in situ expression of the corresponding at least one gene: i) the isopenicillin N is in situ converted into penicillin N by isopenicillin N epimerase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the isopenicillin N epimerase enzyme system comprising the cefD1 and cefD2 genes of A. chrysogenum capable of accepting said isopenicillin N as a substrate, whereupon as a result of its expression, said isopenicillin N produced by said strain is also thereafter in situ converted into penicillin N ii) the penicillin N is in situ ring-expanded to form the corresponding DAOC by DAOC synthase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the DAOC synthase enzyme capable of accepting said penicillin N as a substrate, whereupon as a result of its

expression, said penicillin N produced by said strain is also thereafter in situ ring-expanded to form DAOC iii) the 3-methyl side chain of said DAOC is in situ hydroxylated to yield DAC by DAC synthase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the DAC synthase enzyme capable of accepting said DAOC as a substrate, whereupon as a result of its expression, said DAOC produced by said strain is also thereafter in situ hydroxylated to form DAC.

A further embodiment of the present invention relates to a bioprocess for preparing cephalosporin derivative comprising the steps of a) maintaining in a culture medium capable of sustaining its growth a strain of P. chrysogenum which produces isopenicillin N; b) carrying out the following enzymatic conversion by in situ expression of the corresponding at least one gene: i) the isopenicillin N is in situ converted into penicillin N by isopenicillin N epimerase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the isopenicillin N epimerase enzyme system comprising the cefd, and cefD2 genes of A. chrysogenum capable of accepting said isopenicillin N as a substrate, whereupon as a result of its expression, said isopenicillin N produced by said strain is also thereafter in situ converted into penicillin N ii) the penicillin N is in situ ring-expanded to form the corresponding DAOC by DAOC synthase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the DAOC synthase enzyme capable of accepting said penicillin N as a substrate, whereupon as a result of its expression, said penicillin N produced by said strain is also thereafter in situ ring-expanded to form DAOC iii) the 3-methyl side chain of said DAOC is in situ hydroxylated to yield DAC by DAC synthase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the DAC synthase enzyme capable of accepting said DAOC as a substrate, whereupon as a result of its expression, said DAOC produced by said strain is also thereafter in situ hydroxylated to form DAC iv) DAC is in situ acetylated to yield CPC, by acetyltransferase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the activity of the acetyltransferase enzyme capable of accepting

said DAC as a substrate, whereupon as a result of its expression, said DAC produced by said strain is also thereafter in situ acetylated to form CPC.

Preferably the P. chrysogenum strain used in the instant bioprocess has a non-functional acyltransferase In a preferred bioprocess the DNA encoding the activity of the DAOC synthase, DAC synthase and/or acetyl-CoA : DAC acetyltransferase enzymes is derived from A. chrysogenum.

Alternatively in a bioprocess according to the present invention a single bifunctional DAOC synthase/DAC synthase enzyme is used. Such a bifunctional enzyme may be derived e. g. from A. chrysogenum.

A further embodiment of the present invention is a genetically transformed P. chrysogenum comprising DNA encoding an enzyme having isopenicillyl-CoA synthase activity, and DNA encoding an enzyme having isopenicillyl- CoA-racemase activity and DNA encoding an enzyme having DAOC synthase activity, and optionally comprising DNA encoding an enzyme having DAC synthase activity, and further optionally comprising DNA encoding an enzyme having acetyl-CoA : DAC acetyltransferase activity.

Preferably this P. chrysogenum strain has a non-functional acyltransferase This transformed P. chrysogenum strain can be transformed with DNA encoding the cefD1 and cefD2 enzymes of A. chrysogenum as well as with DNA encoding an enzyme with DAOC synthase activity, optionally also with DNA encoding an enzyme with DAC synthase activity and optionally also with DNA encoding an enzyme with acetyl-CoA : DAC acetyl transferase activity.

The DNA encoding the DAOC synthase enzyme, DAC synthase enzyme, or acetyl-CoA : DAC acetyl transferase enzyme for use according to the present invention can be obtained from micro-organisms reported to contain this DNA and which are available from culture collections or it may be obtained from micro- organisms isolated from appropriate natural sources.

Examples of microorganisms reported to contain the DAOC synthase enzyme are A. chrysogenum (cefEF), Streptomyces clavuligerus (cefE), Nocardia lactamdurans (cefE), Lysobacter lactamgenus (cefE).

Examples of microorganisms reported to contain the DAC synthase enzyme are A. chrysogenum (cefEF), S. clavuligerus (cefF), N. lactamdurans (cefF), Lysobacter L. (cefF).

An example of a microorganism reported to contain the acetyl- CoA : DAC acetyl transferase enzyme is A. chrysogenum (cefG).

The DNA for use according to the present invention can be obtained from the source organism by methods known in the art.

Transformation of host cells, for example of P. chrysogenum or other fungi can, in general, be achieved by different means of DNA delivery, like PEG-Ca mediated protoplast uptake, electroporation or particle gun techniques, and selection of transformants. See for example Van den Hondel and 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 et a/., supra). Selection markers of both homologous (P. chrysogenum derived) and heterologous (non-P. chrysogenum derived) origin have been described (Gouka et a/., J. Biotechnol. 20 (1991) 189-200).

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

The DNA sequence encoding the DAOC synthase activity, the DAC synthase activity and the acetyl-CoA : DAC acetyl transferase activity are introduced into and expressed in this way in P. chrysogenum, for instance in strain Wisconsin 54-1255 (deposited at ATCC under accession number 28089). Other strains of P. chrysogenum, including mutants of strain Wisconsin 54-1255, having an improved beta-lactam yield, are also suitable. Examples of such high-yielding strains are the strains CBS 455.95, Panlabs P2 and ASP-78.

Furthermore, the cefG gene together with the cetE and cefF or cefEF gene are placed under the transcriptional and translational control of heterologous or homologous control elements, preferably under control of fungal gene control elements. Those elements can be obtained from cloned fungal genes like the P. chrysogenum IPNS or pcbC gene, the (3-tubulin gene, the Aspergillus nidulans gpdA gene, or the A. niger glaA gene.

The cefD1 and cefD2 genes can come to expression under their respective native transcriptional and translational control elements, however, other control elements suitable for expression in P. chrysogenum can be employed as well.

Preferably use is made of fungal transcriptional and translational control elements, such as those from cloned fungal genes like the P. chrysogenum IPNS or pcbC gene, the p-tubu) in gene, the A. nidulans gpdA gene, or the A. niger glaA gene.

The cephalosporin derivatives prepared according to the process of the present invention can be isolated and further purified according to methods known in the art.

Brief description of the Figures: Fig. 1. : Schematic representation of the enzymatic conversion of isopenicillin N into penicillin N in A. chrysogenum.

Fig. 2.: Detailed map of the p43EFG plasmid. K, Kpnl ; X, Xhol ; S, Sall ; EV, EcoRV; E, EcoRl ; P, Pstl ; H, Hindlil ; Xb, Xbal ; Scl, Sacl. The ble (phleomycin/bleomycin resistance) cassette is expressed from the pcbC promoter (pr pcbC). tcyc1, transcriptional terminator of the S. cerevisiae cyc1 gene. pBSK (+) corresponds to pBluescript SK (+).

Fig. 3.: Presence of intact copies of p43EFG in different P. chrysogenum transformants. (A) Map of the p43EFG plasmid ; the bands of non-reorganized copies of the cefEF and cefG genes are shown. (B) Southern blot hybridization of genomic DNAs digested with Sall using a probe of the cefG gene : lane 1, TA98; Lane 2 TA2. (C) Southern blot hybridization of genomic DNAs digested with Xbal using a probe of the cefEF gene: lane 1, TA98; lane 2 TA2. The size of the hybridization bands (in kb) is indicated on the right.

Fig. 4.: Detailed map of the pCD1+2 plasmid. K, Kpnl ; X, Xhol ; S, Sall ; EV, EcoRV; E, EcoRl ; P, Pstl ; H, Hindlll ; Xb, Xbal ; Scl, Sacl. Other gene designations are as in the legend of Fig. 2.

Fig. 5.: Presence of intact copies of pCD1+2 in different P. chrysogenum transformants. (A) Map of the pCD1+2 plasmid showing the bands of non- reorganized copies of the cefD1 and cefD2 genes. (B) Southern blot hybridization of genomic DNAs digested with Spel/Clal using a probe of the bidirectional promoter of the cefD1-cefD2 genes: lane 1, TA98 ; lane 2, TA64; lane 3, TA 71 and lane 4, TA2. The size (in kb) of the hybridizing band is shown on the right side.

Fig. 6.: (A) Production of total p-iactams in cultures of TA98, TA2 and Wis 54-1255.

(B) Production of cephalosporins in cultures of TA98, TA2 and Wis 54-1255.

Fig. 7.: HPLC chromatograms for determination of DAC and CPC in cell-free extracts of strains TA2 (upper panel) and TA98 (lower panel) grown in CPM medium for 72 hours. The peaks corresponding to DAC and CPC in transformant TA98 are indicated by arrows

Example 1 Transformation of a P. chrysogenum strain with the genes cefD1, cefD2, cefEF and cefG A. MATERIALS AND METHODS a. Strains and media P. chrysogenum Wisconsin 54-1255 is a low penicillin production strain containing a single copy of the penicillin gene cluster. P. chrysogenum npe6 pyrG is a Wis 54-1255 derivative obtained by nitrosoguanidine treatment that lacks acyl-CoA : isopenicillin N acyltransferase (Fernández FJ, Gutiérrez S, Velasco J, Montenegro E, Marcos AT, Martin JF (1994) Molecular characterization of three loss- of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of P. chrysogenum. J. Bacteriol. 176: 4941-4948. ) and is also a pyrG mutant obtained as described previously (Diez B, Alvarez E, Cantoral JM, Barredo JL, Martin JF (1987) Isolation and characterization of pyrG mutants of P. chrysogenum by resistance to 5'- fluoroorotic acid. Curr. Genet. 12: 277-282. ). P. chrysogenum npe10 is a deletion mutant that lacks the penicillin gene cluster (Fierro et al., 1995 PNAS 92: 6200-6204 "The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences" ; Fierro F, Montenegro E, Gutiérrez S, Martin JF (1996a) Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence. Appl. Microbiol.

Biotechnol. 44: 597-604) P. chrysogenum spores were obtained from plates of PW medium (Fierro et al 1996a, supra) grown for 5 days at 28°C. Seed cultures were initiated by inoculating fresh spores during 18-24 h in CIM (Complex Inoculum Medium): corn steep solids 20 g/L; sucrose 20 g/L; yeast extract 10 g/L; CaCO3 5 g/L. Cultures in CPM medium (Complex Production Medium: Pharmamedia 20 g/L; lactose 50 g/L (NH4) 2SO4 4 g/L; CaCO3 5 g/L); were inoculated with 5% of seed cultures and incubated in a orbital shaker (250 rpm, 25°C). b. Transformation of P. chrysogenum protoplasts Protoplasts of P. chrysogenum were obtained as described by Fierro et al. (Fierro F, Gutiérrez S, Díez B, Martin JF (1993) Resolution of four chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome 11 (9.6 Mb) in P. notatum and chromosome I (10.4 Mb) in P. chrysogenum. Mol. Gen. Genet. 241: 573-578). Transformation was performed according to the procedures of Cantoral et al. (Cantoral JM, Diez B, Barredo JL,

Alvarez E, Martin JF (1987) High-frequency transformation of P. chrysogenum.

BiolTechnology 5: 494-497) and Diez et al. (supra). Transformant clones were selected by complementation of the uridine auxotrophy. c. Nucleic acid isolation and Southern blotting Small amounts of total DNA from P. chrysogenum were isolated from mycelium grown in MPPY medium (Fierro et al, 1993, supra), following the protocol described by Casqueiro et al. (Casqueiro J, Bañuelos O, Gutiérrez S, Hijarrubia MJ, Martin JF (1999) Intrachromosomal recombination in P. chrysogenum : gene conversion and deletion events. Mol. Gen. Genet. 261: 994-1000). Total RNAwas isolated with the RNeasy Kit (Qiagen). Total DNA digested with restriction enzymes were separated by agarose gel electrophoresis and blotted onto Nylon membranes (Hybond NX, Amersham) (Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning : a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Probes were labeled with [a32P] dCTP by nick-translation and purified by filtration through Wizard minicolumns (Promega). Hybridizations were carried out as described by Sambrook et al. (supra).

B. Construction of P. chrysogenum TA2 : integration of the cefEF and cefG genes of A. chrvsoaenum Protoplasts of P. chrysogenum npe6 pyrG were transformed with the integrative plasmid p43EFG (Figure 2). This plasmid bears the cefEF under the control of its own promoter. The p43EFG plasmid also bears the cDNA encoding region of the cefG under the control of the pcbC gene promoter from P. chrysogenum and the ble gene conferring resistance to phleomycin as selection marker. After transformation, a Southern blot hybridization (Fig. 3) was performed to select one strain with non- reorganized copies of the exogenous added copies of the cefEF and cefG genes. To study the presence of the cefG gene the genomic DNA of the transformants was digested with Sall. Results showed that transformant P. chrysogenum TA2 (Fig 3, lane 2) showed a 2.1 kb hybridization band that corresponds to a non-reorganized copy of the cefG gene. To study the integration of cefEF gene the genomic DNA of the transformants was digested with Xbal. Results showed that transformant TA2 (Fig 3, lane 2) showed a 2.7 kb hybridization band that corresponds to a non-reorganized copy of the cefEF gene.

C. Construction of P. chrysogenum TA98: integration of cefD1 and cefD2 genes of A. chrysogenum TA2 is a pyrG-strain. TA2 was co-transformed with pCD1+2 (Fig 4), that contains the cefD1 and cefD2 genes under its own promoter, and pBG that bears the pyrG gene of P. chrysogenum. Transformants were selected by protothrophy (complementation of the pyrG mutation in the host strain) in Czapek medium.

A Southern blot with the genomic DNA of some of the transformants was performed to select one with non-reorganized copies of the cefD1 and cefD2 genes. The genomic DNA was digested with Clal/Spel. Results (Fig 5) showed that transformants P. chrysogenum TA64, TA71 and TA98 had a non-reorganized copy of he cefD1 and cefD2 genes. The TA98 transformant was selected for further studies.

D. Production of 0-lactams and cephalosporins in TA98 Fermentations in complex penicillin production medium CPM (without phenylacetic acid) were performed with P. chrysogenum strains Wis 54-1255, TA2 and TA98. Results showed (Fig. 6) that TA2 produced a low level of p-iactams while both Wis 54-1255 and TA98 showed a similar level in the production of (3-lactams. The quantification of cephalosporins titers by bioassay showed that only strain TA98 containing all cephalosporin biosynthesis genes was able to produce cephalosporins while in P. chrysogenum strain Wis 54-1255 and TA2 no detectable amounts of cephalosporins (CPC, DAC nor DAOC) were formed.

E. HPLC analysis of culture broth of P. chrysogenum TA98 The intracellular production of DAC and CPC in P. chrysogenum TA2 and P. chrysogenum TA98 grown in CPM (without phenylacetic acid) was studied.

Results showed (Fig. 7) formation of DAC and CPC in transformant TA98 inside the cells.