Process to Increase the Production of Clavam

Europäisches Patentamt *EP000832247B1* (19) European Patent Office

Office européen des brevets (11) EP 0 832 247 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: C12N 15/54, C12P 17/18, of the grant of the patent: C12N 1/20 24.11.2004 Bulletin 2004/48 // (C12N1/20, C12R1:465) (21) Application number: 96921954.2 (86) International application number: PCT/EP1996/002497 (22) Date of filing: 06.06.1996 (87) International publication number: WO 1996/041886 (27.12.1996 Gazette 1996/56)

(54) PROCESS TO INCREASE THE PRODUCTION OF CLAVAM ANTIBIOTICS VERFAHREN ZUR ERHÖHUNG DER PRODUKTION VON CLAVAM-ANTIBIOTIKA PROCEDE POUR AUGMENTER LA PRODUCTION D’ANTIBIOTIQUES CLAVAM

(84) Designated Contracting States: (56) References cited: AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC EP-A- 0 349 121 WO-A-94/18326 NL PT SE Designated Extension States: • FEMS MICROBIOLOGY LETTERS, vol. 110, 1993, SI pages 239-242, XP000601357 J.M.WARD AND J.E.HODGSON: "The biosynthetic genes for (30) Priority: 09.06.1995 GB 9511679 clavulanic acid and cephamycin production occur as a ’super-cluster’ in three (43) Date of publication of application: Streptomyces" cited in the application 01.04.1998 Bulletin 1998/14 • MICROBIOLOGY, vol. 140, no. 12, December 1994, pages 3367-3377, XP000601913 H.YU ET (73) Proprietors: AL.: "Possible involvement of the lat gene in the • SmithKline Beecham plc expression of the genes encoding ACV Brentford, Middlesex TW8 9GS (GB) synthetase (pcbAB) and isopenicillin N synthase • THE GOVERNORS OF THE UNIVERSITY OF (pcbC) in Streptomyces clavuligerus" cited in ALBERTA the application Edmonton, Alberta T6G 2E1 (CA) • MALMBERG ET AL: ’Precursor flux control through targeted chromosomal insertion of the (72) Inventors: lysine epsilon-aminotransferase (LAT) gene in • HOLMS, William, Henry Bioflux Limited Cephamycin C biosynthesis.’ JOURNAL OF 54 Dumbarton Road Glasgow G11 6AQ (GB) BACTERIOLOGY vol. 175, no. 21, November • PARADKAR, Ashish Sudhakar, 1993, pages 6916 - 6924 University of Alberta, • ROMERO J. ET AL: ’Isolation and biochemical Edmonton, Alberta T6G 2E1 (CA) characterization of Streptomyces-Clavuligerus • MOSHER, Roy Henry Universityof Alberta, mutants in the biosynthesis of clavulanic acid Edmonton Alberta T6G 2E1 (CA) and cephamycin C.’ APPLIED MICROBIOLOGY • Jensen, Susan E., Dr., Dept. of Biol. Sciences AND BIOTECHNOLOGY vol. 27, no. 5-6, 1988, Edmonton, Alberta T6G 2E9 (CA) pages 510 - 516

(74) Representative: White, Susan Mary GlaxoSmithKline Corporate Intellectual Property (CN9.25.1) 980 Great West Road Brentford, Middlesex TW8 9GS (GB)

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Description Microbiol. Lett. 110, 239-242] do not share common biosynthetic structural genes. Therefore there are no ap- [0001] The present invention relates to processes for parent biochemical links between the pathways. increasing the production of clavulanic acid and other [0008] Romero et al (1988) Appl. Microbiol. Biotech- clavams including those with strong β-lactamase inhib- 5 nol 27:510-516 describe a number of mutants of Strep- itory activity from producing organisms. The present in- tomyces clavuligerus and the effects of the mutations vention also provides novel organisms capable of pro- carried by these organisms on the biosynthesis of ce- ducing increased amounts of these clavams. phamycin C (a cephalosporin) and clavulanic acid. A [0002] Microorganisms, in particular Streptomyces number of these mutants were defective in enzymatic sp. produce a number of antibiotics including clavulanic 10 activities known to be essential for cephamycin C bio- acid and other clavams, cephalosporins, cephamycins, synthesis. For example the mutant strain ncc1 lacks LAT tunicamycin, holomycin and penicillins. There is consid- activity and also isopenicillin N synthetase (IPNS) activ- erable interest in being able to manipulate the absolute ity, both of which are absolutely required to synthesise and relative amounts of these antibiotics produced and cephamycin C. This mutant produced no cephamycin C accordingly there have been a large number of studies 15 and no clavulanic acid. However a mutant lacking IPNS investigating the metabolic and genetic mechanisms of alone, nce2, was found to retain the ability to produce these pathways [Domain, A.L. (1990) "Biosynthesis and clavulanic acid, at levels higher than that shown by the regulation of beta-lactam antibiotics. " In : 50 years of wild-type. Penicillin applications, history and trends]. Many of the [0009] Surprisingly we have found that when the LAT enzymes which carry out the various steps in the meta- 20 gene in the cephalosporin pathway of an organism is bolic pathways and the genes which code for these en- interfered with the amount of clavams produced by the zymes are known. organism is increased. [0003] In the cephalosporin metabolic pathway in, for [0010] Accordingly the present invention provides a example , Streptomyces clavuligerus three important process for increasing the amount of clavam produced antibiotics can be produced namely penicillin N, O-car- 25 by an organism having both a clavam pathway or a por- bamoyldeacetylcephalosporin C and cephamycin C. tion thereof and a cephalosporin pathway or a portion These antibiotics are synthesised from the tripeptide thereof by interfering with the conversion of L-lysine to precursor d-(L-a-aminoadipyl)-L-cysteinyl-D-valine L-α-aminoadipic acid in the cephalosporin pathway . (ACV) [J.F. Martin et al (1990)., "Clusters of genes in- [0011] Preferably the clavam has β-lactamase inhibi- volved in Penicillin and cephalosporin biosynthesis" In : 30 tory activity and more preferably the clavam is clavulanic 50 years of penicillin applications history and trends]. acid. [0004] The recognised first dedicated step in the bio- [0012] In a preferred aspect of the invention the proc- synthesis of both penicillins and cephalosporins in S. ess of conversion of L-lysine to L- α-aminoadipic acid is clavuligerus involves the enzyme lysine -ε - amino trans- interfered with by altering the function of the LAT en- ferase (LAT). The nucleotide sequence and derived 35 zyme or the lat gene. For example the enzyme can be amino acid sequence of S.clavuligerus lat gene is inhibited or otherwise blocked (for example by using a known [Tobin, M.B et al., (1991) J. Bacteriol, 173, non-metabolisable substrate or analog). 6223-6229]. [0013] Suitably the lat gene can be obtained by con- [0005] Malmberg et al (1993) J. Bacteriol. 175(21): ventional cloning methods (such as PCR) based on the p6916-6924 (and US patent 5,474,912 ( published 12 40 published sequence. The function of the gene can be December 1995)) describe a process for producing in- interfered with or eliminated/deleted by genetic tech- creased amounts of cephalosporins in S. clavuligerus niques such as gene disruption [Aidoo, K.A. et al., by inserting one or more copies of a LAT gene into the (1994), Gene, 147, 41-46], random mutagenesis, site chromosome of the organism. Although effects on β directed mutagenesis and antisense RNA. -lactam antibiotics are claimed, only effects on products 45 [0014] Mahro,B. and Demain, A (1987), Appl.Micro- of the cephalosporin pathway in S. clavuligerus are dis- biol. Technol.27, 272-275 described a spontaneous mu- closed ie. no effects on clavam production were meas- tant strain of S. clavuligerus (NP1) which did not pro- ured or described. duce cephamycin and was demonstrated to be defec- [0006] Clavulanic acid and other clavams are derived tive in lat, acvs and ipns enzyme activities. H. Yu et al from a 3 carbon compound [Townsend ,C.A. and Ho, M. 50 (1994) [Microbiology, 140,3367-3377] demonstrated F. (1985) J. Am. Chem. Soc. 107 (4), 1066-1068 and that the activity of these three enzymes can be recov- Elson, S. W and Oliver, R.S. (1978) J. Antibiotics XXXI ered by transforming the mutant with a fragment of DNA No.6, 568] and arginine [ Valentine , B.P et al (1993) J. encoding the entire lat gene (lat) and the upstream half Am Chem. Soc. 15, 1210-1211] . of the acvs gene (pcbAB). However there was no teach- [0007] The gene clusters which determine the biosyn- 55 ing of any effect in this mutant in relation to the produc- thesis of cephalosporins and clavulanic acid, although tion of clavulanic acid or other clavams. adjacent to each other on the S. clavuligerus chromo- [0015] In a further aspect of the invention there are some [Ward, J.M. and Hodgson, J.E (1993) FEMS provided plasmids containing a defective lat gene , pref-

2 3 EP 0 832 247 B1 4 erably the plasmids pCF002 and p486latap described EcoR1 and Kpn1 to liberate its 1.08 kb fragment insert. below. Suitably the plasmids are used to transform an The digest was fractionated by agarose gel electro- organism such as S. clavuligerus eg strain ATCC 27064 phoresis and the 1.08 kb fragment was eluted and ligat- (equivalent to S. clavuligerus NRRL 3585). ed to EcoR1 and Kpn1-digested pCF001A. The ligation [0016] In a further aspect of the invention there is pro- 5 mixture was used to transform MV1193 to apramycin re- vided an organism containing a defective lat gene. sistance. The resulting transformants were screened and a clone was isolated that contained a recombinant EXAMPLES plasmid pCF002 [see Fig 1(a)]which possessed the 1.08 kb 5'-lat fragment upstream of the 3'-lat fragment, [0017] In the examples all methods are as in Sam- 10 both in the same orientation, but separated from each brook, J., Fritsch, E.F. and Maniatis,T. (1989) Molecular other by the inversely oriented aprr fragment. The iden- Cloning A Laboratory Manual (2nd Edition) unless oth- tity of pCF002 was confirmed by restriction analysis; the erwise stated. results indicated that the aprrfragment was inserted at the Kpn1 site, within the lat gene at 852 bp from the start Example 1 - Assembling the disrupted lat gene 15 codon and 521 bp from the stop codon.

[0018] To start the process pA2/119 (Thesis by A. K. Example 2 - Introduction of the disrupted lat gene Petrich(1993) Transcriptional analysis of the Isopenicil- into S. clavuligerus lin N synthetase gene of Streptomyces clavuligerus; University of Alberta) was digested with KpnI to liberate 20 [0021] The disrupted lat gene was subcloned by di- its 0.688 kb insert. The digest was fractionated by aga- gesting pCF002 with EcoR1 and HindIII (to liberate the rose gel electrophoresis and the 0.688 kb fragment was DNA fragment carrying the disrupted lat gene) and ligat- eluted and Klenow-treated. The blunted fragment was ing the digest with similarly digested pIJ486 to create then ligated to HincII-digested pUC119 and used to p486latap [see Fig. 1(b)]. The ligation mixture was used transform E. coli MV1193 to ampicillin resistance. A 25 to transform S. lividans protoplasts to apramycin resist- transformant, containing the recombinant plasmid here- ance (Genetic Manipulation of Streptomyces. A Labo- inafter named pELK004 was isolated and its identity ratory manual (1985) D.A. Hopwood et al.). A clone was confirmed by restriction analysis. pELK004 was digest- selected that contained the recombinant plasmid ed with BamHI and KpnI and ligated to a BamHI-KpnI p486latap; restriction analysis was then used to confirm aprrfragment. The ligation mixture was used to trans- 30 the presence of the disrupted lat gene in p486latap. form E. coli MV1193 to apramycin resistance. The re- [0022] p486latap was used to transform protoplasts sulting transformants were screened and a recombinant of wild type S. clavuligerus (NRRL 3585) to apramycin plasmid pCF001A containing the aprrfragment insert- resistance [Bailey ,C.R and Winstanley,D.J J. Gen. ed upstream of , and inversely oriented to the 0.688-kb Microbiol. (1986) 132, 2945-2947]. Only one transform- 3'-lat fragment was isolated and confirmed by restriction 35 ant was obtained which grew up well when subcultured analysis. on MYM [Paradkar , A.S. and Jensen, S.E. (1995) J. [0019] In the next step pA2/119 was used as a tem- Bact. 177, 1307-1314] supplemented with thiostrepton plate in a PCR reaction to subclone the upstream region and apramycin. The transformant was then subcultured of the lat gene into pUC119. The DNA primers used were to IPS medium #3 agar (unsupplemented) [Paradkar the M13 reverse sequencing primer and a primer with 40 and Jensen, ibid]and allowed to sporulate at 28 degrees the DNA sequence 5' CATGCGGATCCCGTCGAC- C. Spores were harvested and then replated for single GAGCATATGC-3' under standard reaction conditions colonies on to ISP medium #3 agar and MYM agar, both [PCR technology Principles and Applications for DNA unsupplemented. These were allowed to sporulate and amplification, Ed. H A Ehrlich (1989)] and a cycle com- were then replica-plated successively on to MYM sup- position of 92 degrees C for 30 s, 55 degrees C for 30 45 plemented with thiostrepton, MYM supplemented with s and 72 degrees C for 60 s. PCR products were isolated apramycin and MYM (unsupplemented). After three by gel electrophoresis on a 1.25% agarose gel. The am- days at 28 degrees C colonies were apparent on the plified DNA fragment was gel purified, digested with re- apramycin supplemented and unsupplemented plates striction endonucleases EcoR1 and BamH1, and ligated but no colonies grew on the thiostrepton plates. Four of with similarly digested pUC119. Subsequent E. coli 50 the apramycin resistant, thiostrepton sensitive colonies MV1193 transformants were analysed by restriction en- (lat#2,lat#5, lat#7 and lat#11) were selected for further donuclease treatment followed by gel electrophoresis investigation. and DNA sequence analysis to ensure that the correct [0023] Because these isolates were aprr, thiosit was fragment was cloned and that no unintended mutations assumed that a double cross-over event had occurred were introduced during amplification by PCR. The 55 between p486latap and the S. clavuligerus chromo- pUC119 construction containing the DNA region up- some. This assumption was confirmed by Southern hy- stream of lat was named pL/119. bridisation analysis. [0020] The next step involved digesting pL1/119 with

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Example 3 - Cephamycin and clavulanic acid way or a portion thereof by inhibiting the conversion production by the lat-disrupted mutant of L-lysine to L-α-aminoadipic acid in the cephalosporin pathway and detecting said clavam. [0024] To determine the effect of the presumed lat disruption mutation on antibiotic production all four strains 5 2. A process according to claim 1 wherein the process (disruptants) were grown in starch-asparagine (SA) liq- of conversion is inhibited by altering the function of uid medium (Paradkar, A.S. and Jensen, S.E (1995) , J. the LAT, lysine ε-aminotransferase, enzyme or the Bacteriol. 177 , 1307-1314.) for 66 h at 28 degrees C. lat gene. The supernatants were adjusted to account for different growth rates and then bioassayed against E. coli ESS 10 3. A process according to claim 2 wherein the function (Paradkar and Jensen ibid.) and compared to similarly of the lat gene is altered by disrupting or eliminating adjusted supernatants from cultures of wild type S. cla- the gene. vuligerus. Everyone of the lat-disruptants produced very small zones of inhibition corresponding to approximate- 4. A process according to claim 1 to 3 wherein the cl- ly 0.045 ug/ml/cephalosporins; under similar conditions 15 avam has β-lactamase inhibitory activity. the wild type strain was producing 3.18 ug/ml/cephalosporins. 5. A process according to claim 4 wherein the clavam [0025] Samples of the same supernatants were bio- is clavulanic acid. assayed for clavulanic acid production against Klebsiel- la pneumoniae (Bailey, C.R. et al. (1984) Bio/Technolo- 20 6. A recombinant plasmid containing a lat gene from gy 2(9) 808-811. All four disruptants appeared to pro- Streptomyces clavuligerus disrupted by a genetic duce two to three times the amount of clavulanic acid insertion. as compared to the wild type as judged by the bioassay. [0026] Because all four of the lat disruptants seemed 7. A plasmid according to claim 6 selected from the to exhibit similar phenotypes with respect to clavulanic 25 recombinant plasmid designated pCF002 having a acid production, and all four had been derived from one disrupted lat gene and the configuration of restric- primary transformant, lat#2 was selected for further in- tion sites shown in Figure 1(a) and the recombinant vestigation. A time course experiment was carried out plasmid designated p486latap having a disrupted in which triplicate shake cultures of lat#2 and the wild lat gene and the configuration of restriction sites type S. clavuligerus were grown in SA medium at 28 de- 30 shown in Figure 1(b). grees C and sampled at 48h, 72 h and 99h after inocu- lation. Bioassay against K. pneumoniae showed that at 8. An organism having both a clavam pathway or a 48 h , lat#2 was producing approximately four times the portion thereof and a cephalosporin pathway or a amount of clavulanic acid as the wild type . However by portion thereof and containing a lat gene that has 99h the lat#2 cultures were producing a little over twice 35 been disrupted by a genetic insertion and intro- as much clavulanic acid as the wild type. A concurrent duced into the chromosome such that the chromo- bioassay of the same cultures for the same time periods somal lat gene is rendered non-functional. against E. coli ESS showed that lat#2 produced no detectable antibacterial activity. Wild type cultures pro- 9. An organism according to claim 8 which is S. cla- duced moderate but easily detectable amounts of ce- 40 vuligerus. phalosporins (maximum titre 6.5 ug/ml)

Summary Patentansprüche

[0027] A lat disruption mutant has been created. Ex- 45 1. Verfahren zur Steigerung der Menge von Clavam, periments have shown that it produces negligible das von einem Organismus produziert wird, der so- amounts of β-lactam antibiotics except clavulanic acid. wohl einen Clavam-Biosyntheseweg oder einen Of the latter it produces at least two to three times the Teil davon als auch einen Cephalosporin-Biosyn- amount of clavulanic acid produced by wild type S. cla- theseweg oder einen Teildavon hat, durch die Hem- vuligerus grown under shake flask conditions in Starch- 50 mung der Umwandlung von L-Lysin zu L-α-Ami- Asparagine medium. noadipinsäure im Cephalosporin-Biosyntheseweg, und zum Nachweis von Clavam.

Claims 2. Verfahren nach Anspruch 1, wobei der Umwand- 55 lungsvorgang durch eine Veränderung der Funktion 1. A process for increasing the amount of clavam pro- des LAT, Lysin ε-Aminotransferase, -Enzyms oder duced by an organism having both a clavam path- des lat-Gens gehemmt wird. way or a portion thereof and a cephalosporin path-

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3. Verfahren nach Anspruch 2, wobei die Funktion des mase. lat-Gens durch eine Inaktivierung oder Eliminierung des Gens verändert wird. 5. Procédé suivant la revendication 4, dans lequel le clavame est l'acide clavulanique. 4. Verfahren nach Anspruch 1 bis 3, wobei das Cla- 5 vam β-Lactamase-hemmende Aktivität hat. 6. Plasmide recombinant contenant un gène LAT pro- venant de Streptomyces clavuligerus présentant 5. Verfahren nach Anspruch 4, wobei das Clavam Cla- une rupture par une insertion génétique. vulansäure ist. 10 7. Plasmide suivant la revendication 6, choisi entre le 6. Rekombinantes Plasmid, enthaltend ein lat-Gen plasmide recombinant appelé pCF002 ayant un gè- aus Streptomyces clavuligerus, welches durch eine ne LAT présentant une rupture et la configuration genetische Insertion inaktiviert ist. de sites de restriction représentée sur la figure 1(a) et le plasmide recombinant désigné par p486latap 7. Plasmid nach Anspruch 6, ausgewählt aus dem re- 15 ayant un gène lat présentant une rupture et la con- kombinanten Plasmid mit der Bezeichnung figuration de sites de restriction représentés sur la pCF002, welches ein inaktiviertes lat-Gen und die figure 1(b). Konfiguration von Restriktionsschnittstellen, wie in Figur 1(a) gezeigt, hat, und dem rekombinanten 8. Organisme ayant à la fois une voie de formation de Plasmid mit der Bezeichnung p486latap, welches 20 clavame ou une de ses parties et une voie de for- ein inaktiviertes lat-Gen und die Konfiguration von mation de céphalosporine ou une de ses parties et Restriktionsschnittstellen, wie in Figur 1(b) gezeigt, contenant un gène lat qui a été soumis à une rup- hat. ture par une insertion génétique et introduit dans le chromosome de telle sorte que le gène lat chromo- 8. Organismus, welcher sowohl einen Clavam-Bio- 25 somique soit rendu non fonctionnel. syntheseweg oder einen Teil davon als auch einen Cephalosporin-Biosyntheseweg oder einen Teil da- 9. Organisme suivant la revendication 8, qui est S. cla- von hat und welcher ein lat-Gen enthält, das durch vuligerus. genetische Insertion inaktiviert und in das Chromo- som eingeführt worden ist, so daß das chromoso- 30 male lat-Gen nicht mehr funktionsfähig ist.

9. Organismus nach Anspruch 8, welcher S. clavuligerus ist. 35

Revendications

1. Procédé pour augmenter la quantité de clavame produite par un organisme ayant à la fois une voie 40 de formation de clavame ou une partie de celle-ci et une voie de formation de céphalosporine ou une partie de celle-ci, en inhibant la conversion de la L- lysine en acide L-α-aminoadipique dans la voie de formation de céphalosporine et en détectant ledit 45 clavame.

2. Procédé suivant la revendication 1, dans lequel le procédé de conversion est inhibé en modifiant la fonction de l'enzyme lysine-ε-aminotransférase 50 LAT ou du gène lat.

3. Procédé suivant la revendication 2, dans lequel la fonction du gène lat est modifiée par rupture ou sup- pression du gène. 55

4. Procédé suivant les revendications 1 à 3, dans lequel le clavame a une activité inhibitrice de β-lacta-

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