WO2004113508A1 - Production d'intermediaires de la cephalosporine dans penicillium chrysogenum - Google Patents

Production d'intermediaires de la cephalosporine dans penicillium chrysogenum Download PDF

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Publication number
WO2004113508A1
WO2004113508A1 PCT/NL2004/000433 NL2004000433W WO2004113508A1 WO 2004113508 A1 WO2004113508 A1 WO 2004113508A1 NL 2004000433 W NL2004000433 W NL 2004000433W WO 2004113508 A1 WO2004113508 A1 WO 2004113508A1
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Prior art keywords
chrysogenum
dac
daoc
synthase
enzyme
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PCT/NL2004/000433
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English (en)
Inventor
Ricardo Ullan
Santiago Gutiérrez MARTIN
Javier Casquerio Blanco
Juan-Francisco Martin Martin
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DSM IP Assets BV
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DSM IP Assets BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/06—Cephalosporin C; Derivatives thereof

Definitions

  • the present invention is concerned with a process for the production of a ceph-3-em compound selected from the group consisting of desacetoxycephalosporin C (DAOC), deacetylcephalosporin C (DAC) and cephalosporin C (CPC) in Penicillium chrysogenum ⁇ P. chrysogenum).
  • DAOC desacetoxycephalosporin C
  • DAC deacetylcephalosporin C
  • CPC cephalosporin C
  • 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. Microbiof. 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-membered 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.
  • Ullan 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 ⁇ - 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.
  • P. chrysogenum strains are transformed with the genes encoding the cefD1 and cefD2 enzymes of A. chrysogenum.
  • transformed strains are capable to produce penicillin N.
  • 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.
  • transformed strains are capable to produce DAOC.
  • 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.
  • transformed strains are capable to produce DAC.
  • 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.
  • transformed strains are capable to produce cephalosporin C (CPC)
  • CPC cephalosporin C
  • a further embodiment of the present invention relates to a bioprocess reparing 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 cefD 1 and cefD 2 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.
  • DAOC desacetoxycephalosporin C
  • a further embodiment of the present invention relates to a bioprocess reparing 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 cefDi and cefD ⁇ 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 reparing 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 c fD ⁇ and cefD 2 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.
  • chrysogenum strain used in the instant bioprocess has a non-functional acyltransferase
  • the DNA encoding the activity of the DAOC synthase, DAC synthase and/or acetyl-CoA:DAC acetyltransferase enzymes is derived from A. chrysogenum.
  • a single bifunctional DAOC synthase/DAC synthase enzyme is used.
  • 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.
  • this P. chrysogenum strain has a non-functional acyltransferase
  • This transformed P. chrysogenum strain can be transformed with
  • 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 microorganisms isolated from appropriate natural sources.
  • chrysogenum (cefEF)
  • Streptomyces clavuligerus (cefE)
  • Nocardia lactamdurans (cefE)
  • Lysobacter lactamgenus (cefE).
  • DAC synthase enzyme examples include A. chrysogenum (cefEF), S. clavuligerus (cefF), N. lactamdurans (cefF), Lysobacter L (cefF).
  • 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.
  • PEG-Ca mediated protoplast uptake like PEG-Ca mediated protoplast uptake, electroporation or particle gun techniques, and selection of transformants.
  • Van den Hondel and Punt “Gene and Transfer and Vector Development for Filamentous Fungi”
  • the application of dominant and non-dominant selection markers has been described (Van den Hondel et al., 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 (1991) 189-200).
  • 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.
  • 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 cbC gene, the ⁇ -tubulin gene, the Aspergillus nidulans gpdA gene, or the A. niger glaA gene.
  • 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.
  • control elements suitable for expression in P. chrysogenum can be employed as well.
  • fungal transcriptional and translational control elements such as those from cloned fungal genes like the P.chrysogenum IPNS or pcbC gene, the ⁇ -tubulin 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, Kpn ⁇ ; X, Xtjol; S, Sal ⁇ ; EV, EcoRV; E, EcoRI; P, Psfl; H, Hind ⁇ ; Xb, Xba ⁇ ; Scl, Sad
  • the ble (phleomycin/bleomycin resistance) cassette is expressed from the pcbC promoter (pr c ⁇ C).
  • tcyd transcriptional terminator of the S. cerevisiae cyc 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 Sal ⁇ using a probe of the cefG gene: lane 1 , TA98; Lane 2 TA2.
  • C Southern blot hybridization of genomic DNAs digested with Xba ⁇ using a probe of the cefEF gene: lane 1 , TA98; lane
  • Fig. 4. Detailed map of the pCD1+2 plasmid. K, Kpn ⁇ ; X, Xho ⁇ ; S, Sa/I; EV, EcoRV; E, EcoRI; P, Psfl; H, HindWY, Xb, Xba ⁇ ; Scl, Sad. 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.
  • P. chrysogenum Wisconsin 54-1255 is a low penicillin production strain containing a single copy of the penicillin gene cluster.
  • P. chrysogenum npe ⁇ pyrG is a Wis 54-1255 derivative obtained by nitrosoguanidine treatment that lacks acyl-CoA:isopenicillin N acyltransferase (Fernandez FJ, Gutierrez 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.
  • chrysogenum npe * ⁇ 0 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, Gutierrez 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; CaCO 3 5 g/L. Cultures in CPM medium (Complex Production Medium: Pharmamedia 20 g/L; lactose 50 g/L (NH ) 2 SO 4 g/L; CaCO 3 5 g/L); were inoculated with 5% of seed cultures and incubated in a orbital shaker (250 rpm, 25°C).
  • CIM Complex Inoculum Medium
  • Protoplasts of P. chrysogenum were obtained as described by Fierro et al. (Fierro F, Gutierrez S, Diez B, Martin JF (1993) Resolution of four chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (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. Bio/Technology 5:494-497) and Diez et al. (supra). Transformant clones were selected by complementation of the uridine auxotrophy.
  • 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.
  • 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.
  • TA2 is a pytG " 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 pytG gene of P. chrysogenum. Transformants were selected by protothrophy (complementation of the pyrG mutation in the host strain) in Czapek medium.

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Abstract

L'invention porte sur un bioprocessus de production d'un composé ceph-3-em sélectionné dans le groupe comprenant désacétoxycéphalosporine C, désacétylcéphalosporine C et céphalosporine C dans P. chrysogenum. L'invention porte également sur une souche de P.chrysogenum qui, en raison de la transformation génétique, est capable de produire le composé ceph-3-em. Cette souche de P. chrysogenum se caractérise par sa capacité convertir l'isopénicilline N en pénicilline. Cette capacité est notamment établie par l'introduction de gènes codant une acyl-CoA synthétase et une CoA racémase de A. chrysogenum.
PCT/NL2004/000433 2003-06-20 2004-06-17 Production d'intermediaires de la cephalosporine dans penicillium chrysogenum Ceased WO2004113508A1 (fr)

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NL03076927.7 2003-06-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110627816A (zh) * 2019-09-10 2019-12-31 丽珠集团新北江制药股份有限公司 一种从头孢菌素c发酵液中分离纯化脱乙酰氧基头孢菌素c的方法

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EP0281391A1 (fr) * 1987-03-04 1988-09-07 Eli Lilly And Company Vecteurs recombinants d'expression d'ADN et composés d'ADN codant pour la déacétoxycéphalosporine C synthétase et la déacétylcéphalosporine C synthétase
EP0566897A2 (fr) * 1992-04-07 1993-10-27 Hoechst Aktiengesellschaft Le gène complet (cefG) codant pour l'acétyl-CoA: déacétylcéphalosporine C acétyltransférase de Cephalosporium acremonium, son isolation et usage
WO2000037671A2 (fr) * 1998-12-22 2000-06-29 Dsm N.V. Production in vivo amelioree de cephalosporines
WO2004026902A1 (fr) * 2002-09-17 2004-04-01 Sandoz Ag Methode de production de cephalosporine c

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0281391A1 (fr) * 1987-03-04 1988-09-07 Eli Lilly And Company Vecteurs recombinants d'expression d'ADN et composés d'ADN codant pour la déacétoxycéphalosporine C synthétase et la déacétylcéphalosporine C synthétase
EP0566897A2 (fr) * 1992-04-07 1993-10-27 Hoechst Aktiengesellschaft Le gène complet (cefG) codant pour l'acétyl-CoA: déacétylcéphalosporine C acétyltransférase de Cephalosporium acremonium, son isolation et usage
WO2000037671A2 (fr) * 1998-12-22 2000-06-29 Dsm N.V. Production in vivo amelioree de cephalosporines
WO2004026902A1 (fr) * 2002-09-17 2004-04-01 Sandoz Ag Methode de production de cephalosporine c

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Title
CANTWELL C ET AL: "ISOLATION OF DEACETOXYCEPHALOSPORIN C FROM FERMENTATION BROTHS OF PENICILLIUM CHRYSOGENUM TRANSFORMANTS: CONSTRUCTION OF A NEW FUNGAL BIOSYNTHETIC PATHWAY", PHILOSOPHICAL TRANSACTIONS. ROYAL SOCIETY OF LONDON. BIOLOGICAL SCIENCES, ROYAL SOCIETY, LONDON, GB, vol. 248, no. 1323, 22 June 1992 (1992-06-22), pages 283 - 289, XP002049879, ISSN: 0962-8436 *
FERNANDEZ FRANCISCO J ET AL: "Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum", JOURNAL OF BACTERIOLOGY, vol. 176, no. 16, 1994, pages 4941 - 4948, XP008024600, ISSN: 0021-9193 *
ULLAN RICARDO V ET AL: "A novel epimerization system in fungal secondary metabolism involved in the conversion of isopenicillin N into penicillin N in Acremonium chrysogenum.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 48, 29 November 2002 (2002-11-29), pages 46216 - 46225, XP002261579, ISSN: 0021-9258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110627816A (zh) * 2019-09-10 2019-12-31 丽珠集团新北江制药股份有限公司 一种从头孢菌素c发酵液中分离纯化脱乙酰氧基头孢菌素c的方法

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