WO1998027221A1 - Procede de preparation de penicilline g ou v, de cephalosporine g ou v et de leurs derives - Google Patents

Procede de preparation de penicilline g ou v, de cephalosporine g ou v et de leurs derives Download PDF

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Publication number
WO1998027221A1
WO1998027221A1 PCT/EP1997/007153 EP9707153W WO9827221A1 WO 1998027221 A1 WO1998027221 A1 WO 1998027221A1 EP 9707153 W EP9707153 W EP 9707153W WO 9827221 A1 WO9827221 A1 WO 9827221A1
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WO
WIPO (PCT)
Prior art keywords
penam
penicillin
producing
phenoxyacetyl
phenylacetyl
Prior art date
Application number
PCT/EP1997/007153
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English (en)
Inventor
Roelof Ary Lans Bovenberg
Original Assignee
D.S.M. N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by D.S.M. N.V. filed Critical D.S.M. N.V.
Priority to JP52734198A priority Critical patent/JP2001506131A/ja
Priority to EP97954433A priority patent/EP0944729A1/fr
Priority to BR9713579-8A priority patent/BR9713579A/pt
Priority to AU58586/98A priority patent/AU5858698A/en
Publication of WO1998027221A1 publication Critical patent/WO1998027221A1/fr

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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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin

Definitions

  • the present invention relates to the field of fermentative Mactam production.
  • /M_actam antibiotics constitute the most important group of antibiotic compounds, with a long 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
  • 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- ⁇ -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.
  • L- ⁇ -aminoadipic acid A
  • L-cysteine C
  • L-valine V
  • This compound contains the typical /Mactam structure.
  • the third step involves the exchange of the hydrophilic D- ⁇ -aminoadipic acid side chain of L-5-amino-5-carboxypentanoic acid by a hydrophobic side chain, by the action of the enzyme acyltransferase (AT).
  • AT acyltransferase
  • the enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody, as has been described in EP-A-04481 80.
  • 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.
  • 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 /Mactams with phenoxyacetic acid or phenylacetic acid.
  • 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 ⁇ and/or ⁇ - 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.
  • 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 ⁇ - and/or ( ⁇ /-1 )-substituted phenylalkanoic acid as a side chain precursor, said ⁇ - and/or ( -1 )-substituted phenylalkanoic acid having a structure according to formula 1 :
  • 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 /?-oxidation.
  • n is an odd number from 1 up to 1 5.
  • n is an odd number from 1 up to 9, more preferably from 1 up to 5.
  • 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 ( 1 943), 81 -83).
  • 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.
  • recombinant penam or cephem- producing strains i.e. recombinant P. chrysogenum or Acremonium chrysogenum strains.
  • 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 WO95/04149 disclosing expandase- expressing P. chrysogenum strains).
  • 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 WO95/04149 disclosing expandase- expressing P. chrysogenum strains).
  • WO96/38580 is relevant since this document discloses that penicillin G can be expanded in vivo in an expandase-expressing P. chrysogenum strain.
  • 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.
  • 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.
  • a suitable penam- or cephem-producing strain i.e. a fungal strain as defined above
  • 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.
  • fermentation conditions can be applied such as disclosed in EP 0532341 .
  • 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.
  • 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.
  • 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.
  • an immobilized enzyme is used, in order to be able to use the enzyme repeatedly.
  • 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.
  • Precursor-solution 10 % (w/v) precursor adjusted to pH 6.5 with 1 M KOH, filter-sterilized before use.
  • a two-stage fermentation of the P. chrysogenum Wisconsin 54-1 255 strain in shake flasks was used for the production of penicillins.
  • the seed stage was initiated by adding 2 * 10 8 spores to 50ml/500ml flask of medium composed of (g/l): glucose, 30; (NH 4 ) 2 SO 4 , 10; KH 2 PO 4 , 10; trace element solution I (MgSO 4 .7H 2 O, 25; FeSO 4 .7H 2 O, 10; CuSO 4 .5H 2 O, 0.5; ZnSO 4 .7H 2 O, 2; Na 2 SO 4 , 50; MnSO 4 .H 2 O, 2; CaCI 2 .2H 2 O, 5), 10 (ml/I) (pH before sterilization 6.5).
  • medium composed of (g/l): glucose, 30; (NH 4 ) 2 SO 4 , 10; KH 2 PO 4 , 10; trace element solution I (MgSO 4 .7H 2
  • the seed 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/l): lactose, 80; maltose, 20; CaSO 4 , 4; urea, 3; MgSO 4 .7H 2 O, 2; KH 2 PO 4 , 7; NaCI, 0.5; (NH 4 ) 2 SO 4 , 6; FeSO 4 .7H 2 O, 0.1 ; trace element solution II (CuSO 4 .5H 2 O, 0.5; ZnSO 4 .7H 2 O, 2; MnSO 4 .H 2 O, 2; Na 2 SO 4 , 50);(pH before sterilization 5.5-6.0).
  • the precursor of choice solution 1
  • the incubation is then continued for another 1 20 hours.
  • 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.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Cephalosporin Compounds (AREA)

Abstract

L'invention concerne un procédé de fermentation servant à préparer de la pénicilline G ou V ou des dérivés de céphalosporine G ou V et consistant à utiliser certains dérivés d'acide phénylbutyrique en tant que précurseur de chaîne latérale. Dans lesdits dérivés d'acide phénylbutyrique, la chaîne acyle est allongée par des paires d'atomes de carbone et certains substituants sont présents à la position φ- ou φ-1. On utilise les dérivés correspondants d'acide phénoxybutyrique afin de préparer de la pénicilline V ou des dérivés de céphalosporine V. On effectue éventuellement la désacylation des composés de pénicilline ou de céphalosporine G ou V afin de préparer leurs homologues désacylés.
PCT/EP1997/007153 1996-12-16 1997-12-15 Procede de preparation de penicilline g ou v, de cephalosporine g ou v et de leurs derives WO1998027221A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP52734198A JP2001506131A (ja) 1996-12-16 1997-12-15 ペニシリンｇ又はｖ、セファロスポリンｇ又はｖ、及びその誘導体の製造方法
EP97954433A EP0944729A1 (fr) 1996-12-16 1997-12-15 Procede de preparation de penicilline g ou v, de cephalosporine g ou v et de leurs derives
BR9713579-8A BR9713579A (pt) 1996-12-16 1997-12-15 Processo para a produção de penicilina g ou v, cefalosporina g ou v e derivados das mesmas.
AU58586/98A AU5858698A (en) 1996-12-16 1997-12-15 Process for the production of penicillin g or v, cephalosporin g or v, and derivatives thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP96203544.0 1996-12-16
EP96203544 1996-12-16

Publications (1)

Publication Number Publication Date
WO1998027221A1 true WO1998027221A1 (fr) 1998-06-25

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PCT/EP1997/007153 WO1998027221A1 (fr) 1996-12-16 1997-12-15 Procede de preparation de penicilline g ou v, de cephalosporine g ou v et de leurs derives

Country Status (8)

Country Link
EP (1) EP0944729A1 (fr)
JP (1) JP2001506131A (fr)
KR (1) KR20000057579A (fr)
AU (1) AU5858698A (fr)
BR (1) BR9713579A (fr)
ID (1) ID22234A (fr)
PL (1) PL333912A1 (fr)
WO (1) WO1998027221A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4208481A (en) * 1978-11-16 1980-06-17 E. R. Squibb & Sons, Inc. Use of phenylalkanes as precursors in benzylpenicillin fermentation
US4250258A (en) * 1979-12-20 1981-02-10 E. R. Squibb & Sons, Inc. Use of phenoxyalkanes as precursors in penicillin V fermentation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4208481A (en) * 1978-11-16 1980-06-17 E. R. Squibb & Sons, Inc. Use of phenylalkanes as precursors in benzylpenicillin fermentation
US4250258A (en) * 1979-12-20 1981-02-10 E. R. Squibb & Sons, Inc. Use of phenoxyalkanes as precursors in penicillin V fermentation

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ARNSTEIN H.R.V. & GRANT P.T.: "The metabolism of the penicillia in relation to penicillin biosynthesis.", BACTERIOLOGICAL REVIEWS, vol. 20, no. 3, September 1956 (1956-09-01), BALTIMORE, USA, pages 133 - 147, XP002064219 *
BEHRENS O. ET AL.: "Biosynthesis of penicillins III. Preparation and evaluation of precursors for new penicillins.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 175, 1948, pages 771 - 792, XP002064220 *
BEHRENS O.K. ET AL.: "Biosynthesis of penicillins IV. New crystalline biosynthetic penicillins.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 175, 1948, pages 793 - 809, XP002064154 *
CLARKE H.T., JOHNSON J.R. & ROBINSON R.: "The chemistry of penicillin.", 1949, PRINCETON UNIVERSITY PRESS, PRINCETON, NEW JERSEY, XP002064155, 614 *
FLOREY H.W. ET AL: "ANTIBIOTICS", 1949, OXFORD UNIVERSITY PRESS, LONDON, XP002064156 *

Also Published As

Publication number Publication date
PL333912A1 (en) 2000-01-31
EP0944729A1 (fr) 1999-09-29
AU5858698A (en) 1998-07-15
KR20000057579A (ko) 2000-09-25
ID22234A (id) 1999-09-23
JP2001506131A (ja) 2001-05-15
BR9713579A (pt) 2000-03-14

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