MXPA99007257A - Novel compounds - Google Patents

Abstract

Novel bacterial genes, microorganisms and processes for improving the manufacture of 5R clavams, e. g. clavulanic acid.

Description

NOVEL COMPOUNDS DESCRIPTIVE MEMORY The present invention relates to novel bacterial genes and methods for improving the manufacture of clavamas, for example clavulanic acid. The present invention also provides novel organisms capable of producing increased amounts of clavulanic acid. The microorganisms, in particular Streptomyces sp. they produce a number of antibiotics including clavulanic acid and other clavamas, cephalosporins, polyketides, cefamycins, tunicamycin, holomicin and penicillins. There is considerable interest in being able to manipulate the absolute and relative amounts of these antibiotics produced by the microorganism and accordingly there have been a number of studies that investigate the metabolic and genetic mechanisms of the biosynthetic pathways [Domain, A.L. (1990) "Biosynthesis and regulation of beta-lactam antibiotics." In: 50 years of Penicillin applications, history and trends]. Many of the enzymes that carry out the various steps in the metabolic pathways and the genes that encode these enzymes are known. The clavamas can be arbitrarily divided into two groups depending on their annular stereochemistry (clavamas 5S and 5R). The biochemical pathways for biosynthesis have not yet been completely elucidated. 5R and 5S clavams, but it has been suggested that it is derived from the same starting units (a three-carbon compound not yet identified [Townsend, CA and Ho, MF (1985) J. arm. chem. soc. 107 (4) , 1066-1068 and Elson, sw and Oliver, RS (1978) J. Antibiotics XXXI No.6, 568] and arginine [Valentine, BP et al. (1993) J. Am Chem. Soc. 15, 1210-1211] and it shares some common intermediates [Iwata-Reuyl, D. and CA Townsend (1992) J. Am. Chem. Soc. 114: 2762-63, and Jane, JW et al. (1993) Bioorg, Med. Chem. Lett. : 2313-16] Some examples of 5S clavames include clavama-2 (C2C) carboxylate, 2-hydroxymethylclavama (2HMC), 2- (3-alanyl) clavama, valclavama and clavamine acid [GB 1585661, Rohl, F. others, Arch. Microbiol. 147: 315-320, US 4,202,819.] There are, however, few examples of 5S clavamas and until now the best known is the beta-lactamase inhibitor clavulanic acid which is produced by the fermentation of Streptomyces clavuligerus.The combination is combined of clavulanic acid, in the form of potassium clavulanate, with beta-lactam-amoxicillin in the antibiotic AUGMENTIN (Trade Mark SmithKIine Beecham). Because of this commercial interest, research into the understanding of clavama biosynthesis has focused on the biosynthesis of clavama 5R, clavulanic acid, by S. clavuligerus. A number of enzymes and their genes associated with the biosynthesis of clavulanic acid have been identified and published. Some examples of such publications include Hodgson, J. E. et al., Gene 166, 49-55 (1995), Aidoo, K.A. and others, Gene 147, 41-46 (1994), Paradkar, A. S. and others, J. Bact. 177 (s), 1307-14 (1995). In contrast, nothing is known about the biosynthesis and genetics of the different 5S clavames of clavinic acid which is a precursor of clavulanic acid produced by clavinic acid synthase in the biosynthesis of clavulamic acid in S. clavuligerus. Some gene cloning experiments have identified that S. clavuligerus contains two isoenzymes of clavinic acid synthase, almost and cas2 [Marsh, E.N. and other Biocehmistry 31, 12648-657, (1992)] both contributing to the production of cavulanic acid under certain nutritional conditions [Paradkar, A. S. et al., J. Bact. 177 (S), 1307-14 (1995)]. Chlamine synthase acid activity has been detected in microorganisms that produce clavulanic acid, ie S. jumonjinesis [Vidal, CM, Es 550549m (1987)] and S. katsurahamanus [Kitano, K, et al., JP 53-104796, (1978 )] as well as S. antibiotics, producer of the 5S clavama, valclavama [Baldwin, JE and others, Tetrahedron Letts. 35 (17), 2783-86, (1994)]. The last document also reported that S. antibiotics have proclamamynic acid amidinohydrolase activity, another enzyme that is known to be involved in the biosynthesis of clavulanic acid. It has been reported that all other genes identified in S. clavuligerus are required as involved in the biosynthesis of the clavam, for the biosynthesis of clavulanic acid [Hodgson, J.E. and others, Gene 166, 49-55 (1995), Aidoo, K.A. and others Gene 147, 41-46 (1994)] and until now no one has been reported that is specific for the biosynthesis of 5S clavamas.

We have now identified certain genes that are specific for the 5S clavama biosynthesis, as exemplified by C2C and 2HMC in S. clavuligerus, According to the above, the present invention provides DNA comprising one or more genes that are specific for the clavama biosynthesis. 5S in S. clavuligerus and that are not essential for the biosynthesis of clavama 5R) for example clavulanic acid. By "gene" as used herein is also included any regulatory region required for the function or expression of genes. In a preferred aspect, the DNA is as identified in SEQ ID NO: 1. Preferably, the DNA comprises the nucleotide sequences indicated in SEQ ID NO: 1, designated as orfup3m orfup2, orfdwnl, orfdwn2 and orfdwn3. The present invention also provides proteins encoded by said DNA. The present invention also provides vectors comprising the DNA of the invention and hosts containing such vectors. Surprisingly, it has been found that, when at least one of the genes according to the invention is effective, the amount of clavulanic acid produced by the organism is increased. Accordingly, the invention also provides methods for increasing the amount of clavulanic acid produced by a suitable microorganism. In one aspect of the invention, the identified genes can be manipulated to produce an organism capable of producing increased amounts of clavam, suitably clavulanic acid. The findings of this work also allow for an improved procedure for the identification of organisms with higher production of clavulanic acid, which comprises the preliminary analysis of organisms with low or no production of 5S clavam (for example by CLAR and / or clavam analysis as described in the examples herein). Suitably, the 5S clavam genes of the present invention can be obtained by conventional cloning methods (such as PCR) based on the sequences provided herein. The function of the gene can be interfered with or eliminated / suppressed by genetic techniques, such as gene dissociation [Aidoo, K.A. et al., 81994), Gene, 147, 41-46], random mutagenesis, site-directed mutagenesis and antisense RNA. In a further aspect of the invention, plasmids containing no more defective genes are provided, preferably the plasmids pCEC060, pCEC061, pDES3, pCEC056 and pCEC057, described below. Genes can be made defective in a variety of ways, for example by insertion of a DNA fragment encoding an antibiotic resistance gene in which it completely cancels the activity of that gene. Alternatively, other strategies have been employed to produce defective genes, including the insertion of DNA that does not code for an antibiotic resistance gene, the location of part of the gene, the location of the entire gene or the creation of the nucleotide sequence of the gene by addition and / or substitution of one or more nucleotides. The defective genes according to the invention can be defective to different degrees. They may be defective in the sense that their activity is completely canceled or a proportion of the original activity can be retained. Suitably, the plasmids of the invention are used to transform an organism, such as S. clavuligerus, for example strain ATCC 27064 (which corresponds to S. clavuligerus NRRL 3585). Suitable transformation methods can be found in relevant sources including: Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Hopwood, D.A. and golds (1985), Genetic Manipulation of Sreptomyces. A Cloning Manual, and Paradkar, A.S. and Jensen, S.E. (1995), J. Bacteriol, 177 (S). 1307-1314. Industrial strains of the species S. clavuligerus are used industrially to produce clavulanic acid (potassium clavulanate). Within the British and United States pharmacopoeias for potassium clavunalate (British Pharmacopoeia 1993, Addendum 1994, p1362-3 and U.S. Pharmacopeia Official Monographs 1995, USP 23 NF18 p384-5), the amounts of 5S clavame and toxic clavama-2 carboxylate are specifically controlled. Therefore, in a further aspect of the invention an organism capable of producing high amounts of clavulanic acid is provided, but has been rendered incapable of forming C2C, or capable of producing high amounts of clavulanic acid, but capable of forming only two levels of C2C.

Suitably, the organism that produces clavulanic acid contains one or more defective clavama genes and is preferably strains 56-1 A, 56-3A, 57-2B, 57-1C, 60-1 A, 60-2A, 60-3A, 61-1A, 61-2A, 61- 3A and 61-4A, of S. clavuligerus, described below. Such organisms are suitable for the production of clavulanic acid without the production of 5S clavam, clavama-2 carboxylate or with significantly reduced production of clavama-2 carboxylate.

EXAMPLES In the examples, all methods are as in Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual (2nd Edition), or Hopwood, D.A. (1985) Genetic Manipulation of Streptomyces. A Cloning Manual, and Paradkar, A.S. and Jensen, S.E. (1995) J. Bacteriol. 177 (5): "1307-1314, unless otherwise stated.

I. DNA SEQUENCING OF THE STREPTOMYCES CHROMOSOME CLAVULIGERUS TO THE END 5 'AND TO THE END 3' OF THE CAS1 GENE OF CLAVAMINATO SINTASA A. Almost isolating To isolate the chromosomal DNA fragments of Streptomyces clavuligerus encoding the gene for the isozyme clavaminate synthase I (almost), an oligonucleotide probe RMO1 was synthesized based on the nucleotides 9-44 of the gene almost previously sequenced (Marsh, E.N., Chang, M.D.T. and Townsend, C.A. (1992) Biochemistry 31: 12648-12657). Oligonucleotides were constructed using conventional methods in an Applied Biosystems 391 DNA synthesizer. The 36-part RMO1 sequence was synthesized antiparallel to that published by Marsh et al. (1992, right there), RM01 was re-labeled with 32P using conventional techniques. for labeling at the ends of the DNA oligonucleotides (Sambrook et al., 189 therein), and was used to analyze a cosmid bank of Streptomyces clavuligerus genomic DNA by Southern hybridization as described by Stahl and Amman (En: Nucleic acid techniques) bacterial systematics, Ed. E. Stackebrandt and M. Goodfellow, Toronto: John Wiley and Sons, pp. 205-248, 1991). The DNA genomic bank of S. clavuligerus, prepared in the cosmid pLAFR3, was as described by Doran, J.L et al., (1990), J. Bacteriol. 172 (9), 4909-4918. Colonies spots from the S. clavuligerus cosmid bank were incubated overnight with radiolabelled RMOI at 60 ° C in a solution consisting of 5 x SSC, 5 x Denhardt's solution and 0.5% SDS (1 x SDS: NaCl a 0.15 M + Na3 citrate, 0.015 M, 1 x Denhardt's solution: 0.02% BSA, 0.01% Ficoll and 0.02% PVP). The spots were then washed at 68 ° C for 30 minutes in a 0.5 x SSC + 0.1% SDS solution. A cosmid clone, 10D7, which hybridized strongly to RM01 was isolated and gave hybridization signals following digestion with the restriction endonucleases Sacl and EcoRI that were compatible with the hybridization signals. detected in similar experiments with digestions of genomic DNA of S. Clavuligerus.

B. DNA sequencing of the nearly flanking S. clavuliazerus chromosome A partial restriction map of cosmid 10D7 was generated, using the restriction endonucleases Sacl, Ncol, and Kpnl. Southern hybridization experiments between RM01 and various DNA digestions of 10D7 indicated that Sacl was most likely located at one end of the 7 kb Sacl-Sacl DNA subfragment. This fragment consisted of the almost open reading frame and approximately 6 kb of DNA towards the 5 'end. The 7 kb fragment of the Sacl digestion of 10D7 was then subcloned into the phagemid vector pBluescriptlI SK + (2.96 kb, Stratagene), thus generating the recombinant plasmid pCEC007. To facilitate the procedure of sequencing the chromosome to the 5 'end of almost, a 3 kb Ncol-Ncol subfragment of the 7 kb Sacl-Sacl fragment was subcloned into pUC120 (3.2 kb; Vieirra and Messing, Methods Enzymol. 3-11, 1987) in both orientations, generating the recombinant plasmids pCEC026 and pCEC0.27. The 3 kb subfragment consists of the amino terminal coding portion of about and about 2.6 kb of DNA towards the 5 'end. Nested and overlapping deletions were created in both pCEC026 and pCEC0.27 using exonuclease II and S1 digestion nuclease (Sambrook et al. 1989 there and the DNA sequence of the 3 kb Ncol-Ncol fragment was determined in both chains by the dideoxy chain termination method (Sanger, F., Nicklen, S and Coulson, AR ( 1977), Proc. Nati, Acad. Sci. USA 74: 5463-5467), using the Taq dye-deoxy3 terminator kit and an Applied Biosystems 373A sequencer, to determine the chromosome DNA sequence immediately towards the 3 'end of almost , a 4.3 kb Kpnl-EcoRI DNA fragment of the cosmid clone 10D7 was subcloned into pBluescriptlI SK +, generating pCEC018.From pCEC018, a 3.7 kb Sacl-Sacl subfragment was cloned into pSL1180 (3422 kb, Pharmacia); Sacl ends of this fragment partially overlapped the TGA stop codon of almost, the other was encoded in the vector Both orientations of the 3.7 kb fragment were obtained during subcloning and the resulting recombinant plasmids were designated pCEC023 and pCEC024. to nests and overlaps in both plasmids and the DNA sequence of the 3.7 kb fragment was determined in both chains. In SEQ ID NO: 1, the nucleotide sequence of the S. clavuligerus chromosome generated in those experiments is shown, including and flanking the sequence of almost.

II. FUNCTIONAL ANALYSIS OF OPEN READING FRAMES THAT FLANK CAS1 Computer analysis of the DNA sequence towards the 5 'end of almost predicted the presence of two complete open reading frames and an incomplete open reading frame. The three open reading frames were located in the DNA chain opposite almost and were oriented in the opposite direction. The first open reading frame, orfupl, was located 579 bp towards the 5 'end of almost and encoded a 344 amino acid (aa) polypeptide. The second open reading frame, orfup2, was located 437 bp beyond the 3 'end of orfup 1 and encoded a 151 aa polypeptide. Beyond orfup 2 is orfup 3. The start codon of orfup 3 overlaps the stop codon by translation of orfup 2, suggesting that the two open reading frames are coupled by translation. No stop codon for translation for orfup 3 was located in the Nocl-Nocl fragment of 3 kb. A similar analysis of the DNA sequence towards the 3 'end of almost predicted the presence of two complete open reading frames and an incomplete open reading frame. Two of the open reading frames were located in the DNA chain opposite to and almost oriented towards almost. The third open reading frame was located in the same chain as almost and was therefore oriented away from it. The first open reading frame towards the 3 'end, orfdwnl, was located at 373 bp to the 3 'end of almost and encoded a 328 aa polypeptide. The second open reading frame, orfdwn2, was located 55 bp towards the 5 'end of orfdwnl and encoded a polypeptide of 394 aa. At 315 bp towards the 5 'end of orfdwn2 and on the opposite chain was orfdwn3. Since no stop codon was observed for orfdwn3 in the 3.7 kb fragment, it encoded an incomplete polypeptide of 219 aa.

Gene dissociation of the open reading frames of orfup and orfdwn To assess the possible functions of the open reading frames that flanked almost in the biosynthesis of clavulanic acid and other clavamas produced by S. clavuligerus, inactivation or deletion mutants were created of insertions for gene replacement. The method used for dissociation and replacement of genes was essentially as described by Paradkar and Jensen (1995 right there).

A. orfupl A 1.5 kb Ncol-Ncol fragment having the apramycin resistance gene (aprr), constructed as described by Paradkar and Jensen (1995 right there), was treated with Klenow fragment to generate shaved ends (Sambrook and others 1989). right there) and ligated to pCEC026 that was digested with BsaBI and treated equally with Klenow fragment. PCEC026 possessed a BsaBI site indicated within orfupl at 636 bp of the translation start codon. The ligation mixture was used to transform the cells Competent E. coli GM2163 (obtainable from New England Biolabs, USA., Marinus, M.G. et al., M GG (1983) vol 122, p288-9) in resistance to apramycin. Of the resulting transformants, two clones containing the plasmids pCEC054 and pCEC055 were isolated; by restriction analysis, it was found that pCEC054 possessed the aprr fragment in the same orientation as orfupl, while pCEC055 possessed it in the opposite orientation. To introduce pCEC054 to S. Clavuligerus, plasmid DNA was engineered with BamYW and HindWl the high copy number Streptomyces vector plJ486 (6.2 kb, Ward et al., 81986) Mol was ligated. Gen. Genet. 203: 468-478). The ligation mixture was then used to transform competent E. coli GM2163 cells into apramycin resistance. Of the resulting transformants, a clone having the promiscuous plasmid pCEC061 was isolated. This plasmid was then used to transform S. Clavuligerus NRRL 3585. The resulting transformants were subjected to two successive sporulation cycles in non-selective media and then replicated to the antibiotic-containing media to identify apramycin-resistant transformants and sensitive to thiostrepton. From this procedure, four putative mutants (61-1 A, -2A, -3A and -4A) were chosen for further analysis. To confirm that these putative mutants dissociated in orfupl, DNA was prepared from the isolated components 61-1 A and 61-2A, digested with Sacl and subjected to Southern blot analysis. Southern blot results were compatible with a double cross that had occurred and demonstrated that these mutants are true replacement mutants by dissociation in orfupl. The mutants 61-1A, -2A, -3A and -4A were grown in soybean meal medium and the culture supernatants were examined by CLAR for the production of clavulanic acid and clavama. It was previously reported about the composition of the soybean meal medium and the method for examining clavamas by CLAR (Paradkar and Jensen, 1995 there), except that the regulator that operated for the CLAR analysis consisted of 0.1 M NaH2P04 + methanol. 6%, pH 3.68 (adjusted with glacial acetic acid). The CLAR analysis indicated that none of the mutants produced detectable levels of clavama-2 carboxylate or a hydroxymethylclavama-2. In addition, when the culture supernatants were analyzed biologically with respect to Bacillus sp. ATCC 27860, using the method of Pruess and Kellett (1983, J. Antibiot 36: 208-212), none of the mutants produced detectable levels of alanylclavama. In contrast, the HPLC analyzes of the culture supernatants terminated with the mutants seemed to produce higher levels of clavulanic acid, when compared with the wild-type ones (Table 1).

TABLE 1 Titration of clavulanic acid (CA) from orfupl mutants in shake flask tests Deletion of orfupl A cloning experiment was undertaken to create the deletion of 654 nucleotide genes between the AatW sites of orfupl. PCR products were generated using the primer oligonucleotides listed below and pCEC061 described above as template. The original nucleotide sequence was altered to incorporate a Pst \ oligo 11 and a Sphl to oligo 14 site.

Pair of nucleotides 1 used to generate the product PCR 1 Initiator 11: 5 'dCTGACGCTGCAGGAGGAAGTCCCGC 3' Initiator 12: 5 'dCGGGGCGAGGACGTCGTCCCGATCC 3' Pair of nucleotides 2 using general pair the product PCR2 Initiator 13: 5 'dGAGCCCCTGGACGTCGGCGGTGTCC 3' Initiator: 14: 5 'dGACGGTGCATGCTCAGCAGGGAGCG 3' Standard PCR reactions were carried out using the PTC-200 Peltier thermal cycler, from GRI (Felsted, Dunmow, Essex, CM6 3LD). The PCR 1 product was generated using primers 11 and 12. This product is approximately 1 kb and contains the carboxy terminus of orfup? from the second site > 4afll and regions towards the 3 'end. The PCR 2 product is generated using the primers 13 and 14. The product ester is about 1.1 Kb and contains the amino terminus of the first site -4-lll and the regions towards the 5'-end. The PCR 2 product was ligated to pCR-Script amp SK (+) digested by Srfl according to the reduction analysis (Strategene Ltd, Cambridge Science Park, Milton Road, Cambridge CB4 4GF). A ligation mixture was used to transform supercompetent E. coli XL1-Blue MRF 'Kan epicurean cells (obtainable from Strategene) into ampicillin resistance (according to manufacturers' instructions). Plasmid DNA was isolated from the Resulting transformants and DNA restriction analysis revealed that 7 clones containing the plasmid to which the PCR2 product had bound had been ligated. One of these plasmids was designated as PDES1. The PCR1 product was digested with Psil and AatW and the resulting DNA was fractionated by agarose gel electrophoresis. It was excised from 1 kb and eluted using the Sephaglas band preparation kit (Pharmacia, St Albans, Herts, ALI 3 AW). The isolated fragment was then ligated into pDES1 digested by AatW and Psil. The ligation mixture was used to transform the competent E. coli KL1-blue cells (obtainable from Strategene) into ampicillin resistance (according to manufacturers' instructions). Plasmid DNA was isolated from the resulting transformants and restriction analysis revealed that a clone contained the plasmid to which the PCR product had been ligated. This plasmid was designated pDES2. To introduce pDES2 to S. clavuligerus, the plasmid was modified further to contain an origin of replication that could work in Streptomyces. to achieve this, pDES2 plasmid DNA was digested with EcoRI and HindW and ligated to the high copy number Streptomyces vector plJ486 (6.2Kb: ward et al., (1986) Mol.Gen.genet 203: 468-478) also digested with EcoR \ and HidW. The ligation mixture was used to transform the E. coli competent cells (JM109) into ampicillin resistance. Plasmid DNA was isolated from the resulting transformants and restriction analysis revealed that 6 clones possessed pDES2 containing plJ486. One of the plasmids was designated as pDES3. The plasmid was used pDES3 to transform a strain of S. clavuligerus in which the oplpl gene had already been dissociated by insertion of the apramycin resistance gene (as described above). Triostrepton resistant transformants were selected and then these transformants were subjected to three cycles of sporulation in non-selective media and analyzed for the loss of apramycin resistance. From this procedure, 45 mutants that had lost resistance to apramycin were identified. These were then analyzed by CLAR, which confirmed that these strains, such as the dissociating 61-1A, 61-2A, 61-3A and 61-4A de oriup were unable to produce carboxylates of clavama-2 and 2-hydroxymethyl-clavama, when they were fermented under conditions in which these clavamas are normally produced.

B orfdwnl and orfdwn2 Was a deletion / replacement mutant created in orfdwn? and orfdwnl, first digesting pCEC018 (7.3 kb) with Nco \ and releasing a subfragment of 1 kb that contained most of the orfwn? and a portion of orfdwnl. The digestion was fractionated by electrophoresis with agarose-gel and the 6.3 kb fragment was excised and eluted from the gene. This fragment was then ligated to a DNA fragment Nco \ -Nco \ that had apr1"was used to transform E. coli XLI-blue in apramycin resistance A clone was obtained from this experiment, but the restriction analysis of the plasmid resulting recombinant revealed that two copies of apramycin resistance fragment they had ligated to the deletion plasmid. To remove the additional copy of the fragment api ^ the plasmid was digested with? / Cabbage and self-ligated. The ligation mixture was used to transform E. co // GM2163 into apramycin resistance. Of the transformants, two clones containing the plasmids pCEC052 and pCEC053 both having only one copy of the aprr fragment were isolated; pCEC052 had the fragment oriented api-1"inversely with respect to oridwnl and 2, whereas pCEC053 possessed the aprr fragment inserted in the same orientation as orfdwn and 2. A promiscuous plasmid of pCEC052 ligand pCECE052 digested by BamYW was constructed with similarly digested plJ486 and transforming E. coli GM2163 into apramycin resistance.From this experiment, a clone containing the promiscuous plasmid pCEC060 was isolated.This plasmid was used to transform S.clavuligerus 3585 wild-type in resistance to apramycin and triestrepton. subjected the resulting transformants to two sporulation cycles under non-selective conditions and then coated the antibiotic-containing media to identify apramycin-resistant colonies, sensitive to triostrepton. Three putative mutants (60-1A.-2A and -3A) were chosen for further analysis. To establish the identity of these putative mutants, genomic DNA was isolated from strain 60-1 A and 60-2A and Sacl or BstEII were digested and subjected to Southern blot analysis. The hybridization bands generated from this experiment were compatible with both strains that had experienced a double-crossing event, demonstrating that these mutants are true replacement mutants by dissociation in orfdwn1 / 2. When these were grown in soybean meal medium and the culture supernatants were analyzed by HPLC, none of the mutants produced detectable levels of clavama-2 or 2-hydroxymethylclavama carboxylate. A biological analysis of the culture supernatants revealed that the mutants stopped producing also detectable levels of alanylclavama. As with orfup 1 mutants, the orfdwn 1/2 mutants are capable of producing higher levels than the wild-type clavulanic acid (Table 2).

TABLE 2 Titration of clavulanic acid (CA) from orfdwn1 / 2 mutants in shake flask tests orfdwn3 To dissociate orfdwn3, pCEC023 (consisting of a 3.7 kb fragment of DNA towards the 3 terminus of almost subcloned to pSI11809) was digested with Ncol and then self-ligated. After transforming E. coli with the ligation mixture, a clone having the plasmid pCEC031 was isolated. This plasmid retained only the 1.9kb Ncol-EcoRI fragment encoding a portion of orfdwn2 and the incomplete orfdwn3. An examination of the DNA sequence revealed that pCEC031 possesses a unique BstEII site at 158 bp from the translation start site of Orfdwn3. Therefore, pCEC031 was digested with BstEII, treated with Klenow fragment to create shaved ends and ligated then to a shaved cassette of apramycin resistance. The ligation mixture was used to transform E. coli GM2183 into apramycin resistance and ampicillin resistance. Two transformants containing respectively pCEC050 and pCEC051 were selected. Restriction analysis revealed that the apramycin resistance cassette is oriented in the same orientation as an orfdw3 in pCEC050 and in the opposite orientation in pCEC051. These two plasmids were then digested with Hindlll and ligated to PIJ486 digested similarly. The ligation mixtures were then used to transform E.Coli GM2163 into resistance to apramycin and ampicillin. Promiscuous plasmids pCEC056 (pCEC050 + plJ486) and pCEC057 (pCEC051 + plJ486) were isolated from the resulting transformants. Both plasmids S. Clavuligerus NRRI 3585 were transformed. One transformant from each experiment was selected with transformants and subjected to successive sporulation cycles in non-selective media and then coated with replicate to medium containing antibiotic to indicate apramycin-resistant transformants. and sensitive to triestreptone. From this procedure, two putative mutants were isolated from the progeny of each primary transformant 56-1 A and 56-3A for pCEC056, and 57-IC and 57-2B for pCEC057). To establish the identity of these putative mutants, genomic DNA was isolated from these strains and digested with Sacl or Acc65l and subjected to Southern blot analysis. The hybridization bands generated by this experiment were compatible with both strains that had experienced a double-crossing event, demonstrating that these mutants are true displacement mutants by dissociation in an orfdwn3. When these were grown in soybean meal medium and the culture supernatants were analyzed by HPLC, none of the mutants produced detectable levels of clavama-2 or 2-hydroxymethylclavama carboxylate. A biological analysis of the culture supernatants revealed that the mutants have also stopped producing detectable levels of alanylclavama. As with the orfup and orfdwn 1/2 mutants, the orfdwn 3 mutants are capable of producing higher levels than the wild-type clavulanic acid (Table 3).

TABLE 3 Titration of clavulanic acid (CA) from orfdwn3 mutants in shake flask tests The application discloses the following sequences of neuroketidos and amino acids: SEQ ID No: 1 - a DNA sequence of 7193 bp SEQ ID No: 2 - the sequence of orfup3 SEQ ID No: 3 - the sequence of orfup2 SEQ ID No: 4 - the sequence of orfupl SEQ ID No: 5 - the sequence of orfdwnl SEQ ID No: 6 - the sequence of orfdwn2 SEQ ID No: 7 - the sequence of orfdwn3 SEQ ID No: 8 - the sequence of the primer 11 SEQ ID No: 9 - the sequence of the primer 12 SEQ ID No: 10 - the sequence of the primer 13 SEQ ID No: 11 - the sequence of the primer 14 SEQ ID No: 12 - the DNA sequence of the open reading frame of CAS SEQ ID NO: 13 - the predicted partial amino acid sequence of the polypeptide encoded by orfup3 SEQ ID No: 14 - the predicted amino acid sequence of the polypeptide encoded by orfup2 SEQ ID No: 15 - the predicted amino acid sequence of the polypeptide encoded by orfupl SEQ ID No: 16 - the predicted amino acid sequence of the polypeptide encoded by orfdwnl SEQ ID No: 17 - the predicted amino acid sequence of the polypeptide encoded by orfdwn2 SEQ ID No: 18 - the predicted amino acid sequence of the polypeptide encoded by orfdwn3 SEQ ID No : 19 - the amino acid sequence of CAS LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: SmithKIine Beecham pie et al (ii) TITLE OF THE INVENTION: Novel compounds (iii) NUMBER OF SEQUENCES: 19 (iv) DOMICILE TO RECEIVE CORRESPONDENCE: (A) RECIPIENT: SmithKine Beecham (B) STREET: Two, New Horizons Court, Great West Road (C) CITY: Brentford (D) STATE: (E) ) COUNTRY: UNITED KINGDOM (F) POSTAL CODE: TW8 9EP (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM Compatible (C) OPERATVO SYSTEM: TWO (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: ( B) DATE OF PRESENTATION: (viii) INFORMATION OF THE POWDER / AGENT: (A) NAME: Valentine, Jill B (B) REGISTRATION NUMBER: (C) REFERENCE NUMBER: P31731 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE : 0181-9752000 (B) TELEFAX: 0181-9756294 (C) TELEX: (2) INFORMATION OF THE SEQUENCE SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7193 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple -D- TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: CCATGGCGGG CGGCGGCTGC CCCGGAGCCT CGGCCGGACC GGTGACCAGG ACCACCCCGG 60 TGGGATAGTG GCCCGCCACC CGGCGCAGCA GACTCCCGGA CACGGACCCG TGGGTGTGCG 120 CGGAAAGGCC CGGAGGCCGG GTCACAGCCA CGGGTAACGC GCGGTGTCCT TGCCCGCGTA 180 ATCGGGGTCC AGATAGACGA AGGCCCGGTG GACGAGGAAG TCCCGCACCT CGTAGACCGT 240 GCACCAGCGC CCGGCGGCCC ACTCGGGGTC ACCCGCCCGC CACGGCCCGT CCCGGTGCTC 300 ACCGTGGGTG GTGCCCTCCG CGGCGAGGAG TTCGGTCCCG GTCAGAATCC AGTTGACGGA 360 CCACAGATGG TGGGTGATCG AGCGGATGGT GCCCCCGAGG TCGTCGAAGA GCCGGGCGAT 420 CTCGGACTTG CCCCGGGCCA GACCCCACTT GGGGAAGAAG AAGACCGCGT CCTCGGCGAA 480 GTAGTCGATC GCGGGGGTGC CGTCGCTGCC GACGCCGCCG TTGTCGAACG CCTTGAAGTA 540 CGCGGTGATG ACCGCCTTGC GCTGCTCGTC CGTCATACCG GCCGATGCCA CGGACATGAA 600 ACGACCTCCA GAGATTCCGG GTGGCTGTGC TGGGGCTGCG GAAGGGGTGT CCCCCGCGAA 660 , - GGACGGCGGA CGCCGCGGAC GCCGCGGCCG TCTCCCCGGC GGACGGGTCC CAGCGTCCTG 720 ° GAGAGGGCTT GGCGGCGGCT TGACGCCGTG CTGTCCCGCG GCTTGCGGAA CGCGAAGTAC 780 CGGCCAGCGT ACGGGCGTTG CACCGGACGT GTACGCCGGT CGGGACCCCT CGTACCCCCG 840 GAGCCGGCCG ACCCCGGCGG CTCCGGGGGT ACGGACGCGC CGGACCGGCC CGAGCGAGCC 900 GGACGGGTCG GACGGTGCGC GTGGTTCCGG TGTGTCGGAC AGCTCGGACG GACCGGACGG 960 TGCGCGTGGT TCCGGTGTGT CGGACAGCTC GGACGGGTCG GACGGTGCGC GTGGTTCCGG 1020 CACGCCGGAC GGGTCAGTTG CCGATCATGG CGAGCAATGC CGGGGTGTAC CGCTCCCCGG 1080 ACACCGGGTG GGAGATCGCG GCCGTCACCT CCGCGAGGGA CCGGTCGTCC AGCCGGATCG 1140 AGGCGGCGGC GAGATTGTCC GCGAGATGGG CCGGGTTCGC GGTGCCCGGG ATCGGGACGA 1200 CGTCCTCGCC CCGGTGGTGC AGCCAGGCGA GCGCGAGCTG TGCCAGGGTC AGCCCCAGAC 1260 CGTCCGCGAC CGGGCGCAGC CGGTGCAGCA ACGAGCGGTT GCGCGCGAGG GCCGGAGCGC 1320 TGAACCGGGG CTGGCCCCGG CGGAAGTCCT CGTCCCCCAG ATCGTCGGTG GTGCGGATGG 1380 TGCCGGTGAG AAAACCCCGT CCCAGAGGGG CGTAAGCGAC GATCCCGATC CCCAGCTCCC 1440 GGCAGACGGG CACCACCTCG TCCTCGATCC CGCGCGACCA CAGGCTCCAC TCGCTCTGCA 1500 - | 0 CCGCCGTCAC CGGGTGCACC GCGTCCGCCC GGCGCAGCGT GGCCGCGGAG GGCTCGGAGA 1560 GACCGAGCCT GCGGACCTTG CCCTCGCGCA CCAGCTCGGC CACCGCACCC ACGGTCTCCT 1620 CGATCGGCAC CGCCGGGTCC GTCCAGTGCT GGTAGTACAG GTCGATGCGG TCGGTGCCGA 1680 GACGACGCAG GGACCGTTCG CAGGCCGCGC GGACGTAGGA CGGCTCGCCG CACAAGCCCT 1740 GGGAGGCGCC GTCGGACGAG CGCACCATGC CGAACTTGGT GGCGATCAGC ACCTCGTCCC 1800 GGCGGCCCGC GACCGCCCGT CCGAGCAGCT CCTCACCGGC GCCGAGCCCC TGGACGTCGG 1860 CGGTGTCCAG CAGGGTGACC CCGGCGTCGA CGGCGGCGCG GATGGTGGCC GTCGCCCGGG 1920 CGCGGTCCGG GCGTCCGTAG AAGTCGGTGG TCGGCAGGCA GCCGAGCCCC TGGGCACTGA 1980 CCGGAAGGTC CCGCAGGGCG CGGACCGGCG GACGCGGAAC CGCGGCGGAC ACGGAACCGG 2040 CCGGGGACTC GGGCGGAGAG CGGGACATAC GGAACCTCCA CAGGCGGAGC CGGGAACGGG 2100 ACGAGGGCGA GGACGGGACG GAACGAAGGA GAGGACGGGA CGGACAGCAC GGACGGGACG 2160 GACGGAACGG AGTCGGGAAC CGGGGGGGGT GACCGGAACC GGGCCGTCCT TGGCCCTCCC 2 20 CCGTCCTCCC CGCCATCCGC CGTTCTCCCC CGTTCCCTCT CCCGTCCTCC AGCCAACACC 2280 GCCGCCCTTT CCAAGCGCTT GACACGGCAC CGACAGCCGC CGCCGGGCGC CCGATGGGGA 2340 CCCGTGCCCG CCGGTGAGCG GCGGTGAGCG CCGGTACGGG ACCCCACGCG CCGCCGCCCG 2400 GGCGCCCGCC AGGGCCCGCG CGGCCACCCC GGCCCGCCCC GGCCGGAGCG GCGATCCGGG 2460 CCGCTCGCTG CAAGAGGAAC ATCCACAGCC GCACAAGGAG CGCTCCGCAC AGTGGGCACC 2520 ACGTCCGCCC CGTCCCCCAC ACCGTGGCCG GTCCCCACCG GACAGCACAG CACCGCACAG 2580 CACCACATCG CACGGCACAG CACAGCACCA CCGGCACGAG GAACCAAGGA AAGGAACCAC 640 ACCACCATGA CCTCAGTGGA CTGCACCGCG TACGGCCCCG AGCTGCGCGC GCTCGCCGCC 2700 CGGCTGCCCC GGACCCCCCG GGCCGACCTG TACGCCTTCC TGGACGCCGC GCACACAGCC 2760 GCCGCCTCGC TCCCCGGCGC CCTCGCCACC GCGCTGGACA CCTTCAACGC CGAGGGCAGC 2820 GAGGACGGCC ATCTGCTGCT GCGCGGCCTC CCGGTGGAGG CCGACGCCGA CCTCCCCACC 2880 ACCCCGAGCA GCACCCCGGC GCCCGAGGAC CGCTCCCTGC TGACCATGGA GGCCATGCTC 2940 GGACTGGTGG GCCGCCGGCT CGGTCTGCAC ACGGGGTACC GGGAGCTGCG CTCGGGCACG 3000 GTCTACCACG ACGTGTACCC GTCGCCCGGC GCGCACCACC TGTCCTCGGA GACCTCCGAG 3060 ACGCTGCTGG AGTTCCACAC GGAGATGGCC TACCACCGGC TCCAGCCGAA CTACGTCATG 3120 0 CTGGCCTGCT CCCGGGCCGA CCACGAGCGC ACGGCGGCCA CACTCGTCGC CTCGGTCCGC 3180 AAGGCGCTGC CCCTGCTGGA CGAGAGGACC CGGGCCCGGC TCCTCGACCG GAGGATGCCC 3240 TGCTGCGTGG ATGTGGCCTT CCGCGGCGGG GTGGACGACC CGGGCGCCAT CGCCCAGGTC 3300 AAACCGCTCT ACGGGGACGC GGACGATCCC TTCCTCGGGT ACGACCGCGA GCTGCTGGCG 3360 CCGGAGGACC CCGCGGACAA GGAGGCCGTC GCCGCCCTGT CCAAGGCGCT CGACGAGGTC 3420 ACGGAGGCGG TGTATCTGGA GCCCGGCGAT CTGCTGATCG TCGACAACTT CCGCACCACG 3480 CACGCGCGGA CGCCGTTCTC GCCCCGCTGG GACGGGAAGG ACCGCTGGCT GCACCGCGTC 3540 TACATCCGCA CCGACCGCAA TGGACAGCTC TCCGGCGGCG AGCGCGCGGG CGACGTCGTC 3600 GCCTTCACAC CGCGCGGCTG AGCTCCCGGG TCCGACACCG CGCGGCTGAA CCCACGGTCC 3660 GGGGCCCACG GTCCGGCACC GCGCGGCTGÁ GCCCCCGGGT CCGGCAGCGG GCGGCTGAAC 3720 CCCCGCCCCG GGCCACCGCC CGACCGCCCC CGCGCACCGG ACGCGCCCGC CTGTACGGCG 3780 GTCCCGCCCG GGCCCGTACA CCTGAAGCGC CCGGCGGACC GCCGCCCCGC CGGGGGACGG 3840 ACAGAGCCGG GTGCGGGAGG ACGTCCTCCC GCACCCGGCT CCCACCGTTC CGCACCGACC 3900 GCACCCGACC GTGCCGCAGG CGCCACCGGC ACCGCACCGC CCGCGCCGGC AGCCACCACA 3960 GGCGCCACGC CGCCCGCACG GTGCCCGCGC TGCTCAGCCC CCGTCCACCG GGCTGTCCAG 4020 CAGCCGCCGC AGCGCGCCCC CGATGAACTC CCGGTCGGCG GCCGACCCCC CGGACCCCGC 4080 GAGATGCCCC CACACTCCCG GGATCACCTC CAGCGAGGCA TACGGCAGCA GATCGGCCAC 4140 CCGCTTCTCG TCCTCGACGG CGAAACACAC GTCCAGGGCG CCCGGCAGCA CCACGGCCCG 4200 CGCCGTGACG GAGGCCAGCG CCGCCTCGAC GCTCCCCCCG GCCCCGGGTG TCGCCCCCAC 4260 , - ATCCGTGTTC TCCCAGGTGC GCACCATGGT GAGCAGATCC GCGGCGCCGG GCCCGGAGAG 4320 ^ GAAGACCTGC TCCCAGAAGC CGGTGAGGTA CTCCTCGCGG GTGGCGAAAC CCAGCTCCCG 4380 GTGGGCACGG CGGGCCCAGA AGGAACGCGA GGTCCCCCAC CCGGCGAACA CCCGGCCCGC 4440 CGCCTTCCGC CCCCGCTCCC CGGCGTCGGC GCTGAGCGCC GCGGCCAGAC CGGACAGCAG 4500 GACCAGGCTG TGCGGGCTGC TCACCGGCGC CCCGCAGATC GGGGCGATCC GGCGCACCAT 4560 CCCCGGATGC GACACGGCCC ACTGGTAGGC GTGGGCCGCG CCCATCGACC AGCCCGTGAC 4620 CAGGGCCAGT TCCCGTACCC CCAGCTCCTC GGTGAGCAGC CGGTGCTGCG CCGCGACATT 4680 GTCCTGCGGA GTGATCAGCG GAAAGCGGGA CCCCGACGGG TGGTTGCCGG GCGAGCTGGA 4740 GACCCCGTTG CCGAAGAGTC CGGCGGTGAC GACGCAGTAC CGCCGGGTGT CCAGCGGCAG 4800 CCCCGCACCG ATCAGCCAGT CGTACCCGGT GTGGTCCCGG CCGAAGAACG ACGGACAGAG 4860 CACCACGTTC GTCCCGTCGG CGTTCGGCGT GCCGTACATG GCGTAACCGA TCCGGGCGTC 4920 CCGCAGGACC TCCCCGTCCA GCAACGGCAG TTCGTCGATC TCGAATATGC GGCATTCCAC 4980 CGCTGACCTC CTTGTTCGAT CCCCCCGGAC AACAGGTCGG TCGTGGCCGG AGACTCAGAG 5040 CCAGTTGGGG GCGATCTCGG TGGCCCACAG CTCCAGGCTG CGCAGCTGGA CATCGTGCGG 5100 0 GATCAGCCCG GAGTACTGGC ACTGGAGCAG ATACTCCGGA TCGTGCCGCT CCACCAGCTT 5160 CTCGATCATG CGGTTGATGT CGTCCGGGGT GCCGACCCAC TCCAGCCCCC GGTCGACCAG 5220 GGTCTTGTAG TCCGAGCCGA TCGGACCCGT CTCGCCGGTC GCGCGCAGCG CCTCGGTGAA 5280 GCCCATGGGG CCGAACCAGT TCTCGAAGAT GAAGCCGCCG CCGCGGGACG CCCAGTGGTG 5340 GGCCTCGCCG GAGTCCCGGG AGACCAGGAC GTCCTTCATC ACCCCGACCC GCTCGCCCCG 5400 CCGCAGGGTG CCGTGGCCCG CCGCCTCGGC CTCCTCCCGG TAGATGTCCA TCAGCCGGGC 5460 GACGATCTGG TCGTCGGTGT TCATCAGGAT CGGCACCACG CCCTCCCGGG CACAGAACCG 5520 GAACGTGTCC TCACTGAAGC TGAACGGCTG GAAGACGGGC GGGTGGGGGC GCTGGTAGGG 5580 CTTGGGCGCG ATGCCCACCT CGCGGATGAC GCCGTTCTCG TCGAGGCCCC GGCCGTAGCG 5640 GCGCACCGCC TCGTAGGGGA ACTCCAGGTC CGGCACCGGG ATCGTCCACT GCTCCCCGGA 5700 GTGGGTGAAC GTCTCGGTCG TCCACGCCTT CTTGATGATC TCCCAGTGCT CCTCGAAGAG 5760 GGCACGATTG CGCCGGTCCC GCTCCCCGGC GTCGGACAGG GTGCCGCCGA CCCCGTACAC 5820 CTGCCCCATG ATGTCGGCCC AGCGCTTCTG GAACCCGCGC GCGATCCCGA CGAAGGCGCG 5880 GCCCCGGGTC ATGTGGTCGA GCATCGCCAG ATCCTCGGCC AGCCGCAGCG GATTGTGCAG 5940 CGGCAGGACG TTGGCCATCT GGCCGACCCG GATGTGCCGG GTCTGCATGC CGAGGTAGAG 6000 CCCCAGCATG ATCGGGTTGT TGGAGACCTC GAAACCCTCG GTGTGGAAGT GGTGCTCGGT 6060 GAAGGACAGT CCCCAGTAGC CGAGTTCGTC GGCCGCCTGC GCCTGCCGGG TGAGCTGCCG 6120 GAGCATGTTC TGGTAGTTCT GCGGATTGAC CCCCGCCATA CCCCGCTGGA CCTGCGCATG 6180 ACTGCCGACC GTTGGCAGAT AGAAGAGAAT GGACTTCACC CTGGCTCCTC CGGTTCGCGG 6240 CGCCCTCCAT TGACGTGCGC CGAAAGCGGC TCGACCGTCC CACTCCGCCC TTGAGTTCCG 6300 TCTGACGCCG CGCCAGTCGG CGGGCCGTCC GCCGGGGTGC CCGCCGGGGT CCGCACCCGC 6360 CGGACGGCAC GGCGCGCACC GCGCGCGCGG CGCTTCGGGG CACCGGGCTC GACGGGGTGC 6420 TCAGCGGGAC GTCCAACGGA AGGCAAGCCC CCGTACCCAG CCTGGTCAAG GCGCTCATCG 6480 CCATTCCCTG AGGAGGTCCC GCCTTGACCA CAGCAATCTC CGCGCTCCCG ACCGTGCCCG 6540 GCTCCGGACT CGAAGCACTG GACCGTGCCA CCCTCATCCA CCCCACCCTC TCCGGAAACA 6600 CCGCGGAACG GATCGTGCTG ACCTCGGGGT CCGGCAGCCG GGTCCGCGAC ACCGACGGCC 6660 GGGAGTACCT GGACGCGAGC GCCGTCCTCG GGGTGACCCA GGTGGGCCAC GGCCGGGCCCC 6720 AGCTGGCCCG GGTCGCGGCC GAGCAGATGG CCCGGCTGGA GTACTTCCAC ACCTGGGGGA 6780 CGATCAGCAA CGACCGGGCG GTGGAGCTGG CGGCACGGCT GGTGGGGCTG AGCCCGGAGC 6840 CGCTGACCCG CGTCTACTTC ACCAGCGGCG GGGCCGAGGG CAACGAGATC GCCCTGCGGA 6900 TGGCCCGGCT CTACCACCAC CGGCGCGGGG AGTCCGCCCG TACCTGGATA CTCTCCCGCC 6960 GGTCGGCCTA CCACGGCGTC GGATACGGCA GCGGCGGCGT CACCGGCTTC CCCGCCTACC 7020 ACCAGGGCTT CGGCCCCTCC CTCCCGGACG TCGACTTCCT GACCCCGCCG CAGCCCTACC 7080 GCCGGGAGCT GTTCGCCGGT TCCGACGTCA CCGACTTCTG CCTCGCCGAA CTGCGCGAGA 7140 CCATCGACCG GATCGGCCCG GAGCGGATCG CGGCGATGAT CGGCGAGCCG ATC 7193 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 145 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other, - (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: OR GTGACCCGGC CTCCGGGCCT TTCCGCGCAC ACCCACGGGT CCGTGTCCGG GAGTCTGCTG 60 CGCCGGGTGG CGGGCCACTA TCCCACCGGG GTGGTCCTGG TCACCGGTCC GGCCGAGGCT 120 CCGGGGCAGC CGCCGCCCGC CATGG 145 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 453 base pairs (B ) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear 0 (Ü) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: ATGTCCGTGG CATCGGCCGG TATGACGGAC GAGCAGCGCA AGGCGGTCAT CACCGCGTAC 60 TTCAAGGCGT TCGACAACGG CGGCGTCGGC AGCGACGGCA CCCCCGCGAT CGACTACTTC 120 GCCGAGGACG CGGTCTTCTT CTTCCCCAAG TGGGGTCTGG CCCGGGGCAA GTCCGAGATC 180 GCCCGGCTCT TCGACGACCT CGGGGGCACC ATCCGCTCGA TCACCCACCA TCTGTGGTCC 240 GTCAACTGGA TTCTGACCGG GACCGAACTC CTCGCCGCGG AGGGCACCAC CCACGGTGAG 300 CACCGGGACG GGCCGTGGCG GGCGGGTGAC CCCGAGTGGG CCGCCGGGCG CTGGTGCACG 360 GTCTACGAGG TGCGGGACTT CCTCGTCCAC CGGGCCTTCG TCTATCTGGA CCCCGATTAC 420 GCGGGCAAGG ACACCGCGCG TTACCCGTGG CTG 453 (2) SEQUENCE INFORMATION SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1032 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: ATGTCCCGCT CTCCGCCCGA GTCCCCGGCC GGTTCCGTGT CCGCCGCGGT TCCGCGTCCG 60 CCGGTCCGCG CCCTGCGGGA CCTTCCGGTC AGTGCCCAGG GGCTCGGCTG CCTGCCGACC 120 ACCGACTTCT ACGGACGCCC GGACCGCGCC CGGGCGACGG CCACCATCCG CGCCGCCGTC 180 GACGCCGGGG TCACCCTGCT GGACACCGCC GACGTCCAGG GGCTCGGCGC CGGTGAGGAG 240 CTGCTCGGAC GGGCGGTCGC GGGCCGCCGG GACGAGGTGC TGATCGCCAC CAAGTTCGGC 300 ATGGTGCGCT CGTCCGACGG CGCCTCCCAG GGCTTGTGCG GCGAGCCGTC CTACGTCCGC 360 GCGGCCTGCG AACGGTCCCT GCGTCGTCTC GGCACCGACC GCATCGACCT GTACTACCAG 420 CACTGGACGG ACCCGGCGGT GCCGATCGAG GAGACCGTGG GTGCGGTGGC CGAGCTGGTG 480 CGCGAGGGCA AGGTCCGCAG GCTCGGTCTC TCCGAGCCCT CCGCGGCCAC GCTGCGCCGG 540 GCGGACGCGG TGCACCCGGT GACGGCGGTG CAGAGCGAGT GGAGCCTGTG GTCGCGCGGG 600 ATCGAGGACG AGGTGGTGCC CGTCTGCCGG GAGCTGGGGA TCGGGATCGT CGCTTACGCC 660 CCTCTGGGAC GGGGTTTTCT CACCGGCACC ATCCGCACCA CCGACGATCT GGGGGACGAG 720 GACTTCCGCC GGGGCCAGCC CCGGTTCAGC GCTCCGGCCC TCGCGCGCAA CCGCTCGTTG 780 CTGCACCGGC TGCGCCCGGT CGCGGACGGT CTGGGGCTGA CCCTGGCACA GCTCGCGCTC 840 GCCTGGCTGC ACCACCGGGG CGAGGACGTC GTCCCGATCC CGGGCACCGC GAACCCGGCC 900 CATCTCGCGG ACAATCTCGC CGCCGCCTCG ATCCGGCTGG ACGACCGGTC CCTCGCGGAG 960 GTGACGGCCG CGATCTCCCA CCCGGTGTCC GGGGAGCGGT ACACCCCGGC ATTGCTCGCC 1020 ATGATCGGCA AC 1032 (2) SEQUENCE INFORMATION SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 984 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: GTGGAATGCC GCATATTCGA GATCGACGAA CTGCCGTTGC TGGACGGGGA GGTCCTGCGG 60 GACGCCCGGA TCGGTTACGC CATGTACGGC ACGCCGAACG CCGACGGGAC GAACGTGGTG 120 CTCTGTCCGT CGTTCTTCGG CCGGGACCAC ACCGGGTACG ACTGGCTGAT CGGTGCGGGG 180 CTGCCGCTGG ACACCCGGCG GTACTGCGTC GTCACCGCCG GACTCTTCGG CAACGGGGTC 240 TCCAGCTCGC CCGGCAACCA CCCGTCGGGG TCCCGCTTTC CGCTGATCAC TCCGCAGGAC 300 AATGTCGCGG CGCAGCACCG GCTGCTCACC GAGGAGCTGG GGGTACGGGA ACTGGCCCTG 360 GTCACGGGCT GGTCGATGGG CGCGGCCCAC GCCTACCAGT GGGCCGTGTC GCATCCGGGG 420 ATGGTGCGCC GGATCGCCCC GATCTGCGGG GCGCCGGTGA GCAGCCCGCA CAGCCTGGTC 480 CTGCTGTCCG GTCTGGCCGC GGCGCTCAGC GCCGACGCCG GGGAGCGGGG GCGGAAGGCG 540 GCGGGCCGGG TGTTCGCCGG GTGGGGGACC TCGCGTTCCT TCTGGGCCCG CCGTGCCCAC 600 CGGGAGCTGG GTTTCGCCAC CCGCGAGGAG TACCTCACCG GCTTCTGGGA GCAGGTCTTC 660 CTCTCCGGGC CCGGCGCCGC GGATCTGCTC ACCATGGTGC GCACCTGGGA GAACACGGAT 720 GTGGGGGCGA CACCCGGGGC CGGGGGGAGC GTCGAGGCGG CGCTGGCCTC CGTCACGGCG 780 CGGGCCGTGG TGCTGCCGGG CGCCCTGGAC GTGTGTTTCG CCGTCGAGGA CGAGAAGCGG 840 GTGGCCGATC TGCTGCCGTA TGCCTCGCTG GAGGTGATCC CGGGAGTGTG GGGGCATCTC 900 GCGGGGGTCCG GGGGGTCGGC CGCCGACCGG GAGTTCATCG GGGGCGCGCT GCGGCGGCTG 960 CTGGACAGCC CGGTGGACGG GGGC 984 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1182 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (x) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: GTGAAGTCCA TTCTCTTCTA TCTGCCAACG GTCGGCAGTC ATGCGCAGGT CCAGCGGGGT 60 ATGGCGGGGG TCAATCCGCA GAACTACCAG AACATGCTCC GGCAGCTCAC CCGGCAGGCG 120 CAGGCGGCCG ACGAACTCGG CTACTGGGGA CTGTCCTTCA CCGAGCACCA CTTCCACACC 180 GAGGGTTTCG AGGTCTCCAA CAACCCGATC ATGCTGGGGC TCTACCTCGG CATGCAGACC 240 CGGCACATCC GGGTCGGCCA GATGGCCAAC GTCCTGCCGC TGCACAATCC GCTGCGGCTG 300 GCCGAGGATC TGGCGATGCT CGACCACATG ACCCGGGGCC GCGCCTTCGT CGGGATCGCG 360 CGCGGGTTCC AGAAGCGCTG GGCCGACATC ATGGGGCAGG TGTACGGGGT CGGCGGCACC 420 CTGTCCGACG CCGGGGAGCG GGACCGGCGC AATCGTGCCC TCTTCGAGGA GCACTGGGAG 480 ATCATCAAGA AGGCGTGGAC GACCGAGACG TTCACCCACT CCGGGGAGCA GTGGACGATC 540 CCGGTGCCGG ACCTGGAGTT CCCCTACGAG GCGGTGCGCC GCTACGGCCG GGGCCTCGAC 600 GAGAACGGCG TCATCCGCGA GGTGGGCATC GCGCCCAAGC CCTACCAGCG CCCCCACCCG 660 CCCGTCTTCC AGCCGTTCAG CTTCAGTGAG GACACGTTCC GGTTCTGTGC CCGGGAGGGC 720 GTGGTGCCGA TCCTGATGAA CACCGACGAC CAGATCGTCG CCCGGCTGAT GGACATCTAC 780 CGGGAGGAGG CCGAGGCGGC GGGCCACGGC ACCCTGCGGC GGGGCGAGCG GGTCGGGGTG 840 ATGAAGGACG TCCTGGTCTC CCGGGACTCC GGCGAGGCCC ACCACTGGGC GTCCCGCGGC 900 GGCGGCTTCA TCTTCGAGAA CTGGTTCGGC CCCATGGGCT TCACCGAGGC GCTGCGCGCG 960 ACCGGCGAGA CGGGTCCGAT CGGCTCGGAC TACAAGACCC TGGTCGACCG GGGGCTGGAG 1020 TGGGTCGGCA CCCCGGACGA CATCAACCGC ATGATCGAGA AGCTGGTGGA GCGGCACGAT 1080 CCGGAGTATC TGCTCCAGTG CCAGTACTCC GGGCTGATCC CGCACGATGT CCAGCTGCGC 1140 AGCCTGGAGC TGTGGGCCAC CGAGATCGCC CCCAACTGGC TC 1182 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 660 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: GTGCCCGGCT CCGGACTCGA AGCACTGGAC CGTGCCACCC TCATCCACCC CACCCTCTCC 60 GGAAACACCG CGGAACGGAT CGTGCTGACC TCGGGGTCCG GCAGCCGGGT CCGCGACACC 120 GACGGCCGGG AGTACCTGGA CGCGAGCGCC GTCCTCGGGG TGACCCAGGT GGGCCACGGC 180 CGGGCCGAGC TGGCCCGGGT CGCGGCCGAG CAGATGGCCC GGCTGGAGTA CTTCCACACC 240 TGGGGGACGA TCAGCAACGA CCGGGCGGTG GAGCTGGCGG CACGGCTGGT GGGGCTGAGC 300 CCGGAGCCGC TGACCCGCGT CTACTTCACC AGCGGCGGGG CCGAGGGCAA CGAGATCGCC 360 CTGCGGATGG CCCGGCTCTA CCACCACCGG CGCGGGGAGT CCGCCCGTAC CTGGATACTC 420 TCCCGCCGGT CGGCCTACCA CGGCGTCGGA TACGGCAGCG GCGGCGTCAC CGGCTTCCCC 480 GCCTACCACC AGGGCTTCGG CCCCTCCCTC CCGGACGTCG ACTTCCTGAC CCCGCCGCAG 540 CCCTACCGCC GGGAGCTGTT CGCCGGTTCC GACGTCACCG ACTTCTGCCT CGCCGAACTG 600 CGCGAGACCA TCGACCGGAT CGGCCCGGAG CGGATCGCGG CGATGATCGG CGAGCCGATC 660 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other -xi > DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CTGACGCTGC AGGAGGAAGT CCCGC 25 (2) SEQUENCE INFORMATION SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: CGGGGCGAGG ACGTCGTCCC GATCC 25 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: GAGCCCCTGG ACGTCGGCGG TGTCC 25 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: GACGGTGCAT GCTCAGCAGG GAGCG 25 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 972 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other -? i. DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: ATGACCTCAG TGGACTGCAC CGCGTACGGC CCCGAGCTGC GCGCGCTCGC CGCCCGGCTG 60 CCCCGGACCC CCCGGGCCGA CCTGTACGCC TTCCTGGACG CCGCGCACAC AGCCGCCGCC 120 TCGCTCCCCG GCGCCCTCGC CACCGCGCTG GACACCTTCA ACGCCGAGGG CAGCGAGGAC 180 GGCCATCTGC TGCTGCGCGG CCTCCCGGTG GAGGCCGACG CCGACCTCCC CACCACCCCG 240 AGCAGCACCC CGGCGCCCGA GGACCGCTCC CTGCTGACCA TGGAGGCCAT GCTCGGACTG 300 GTGGGCCGCC GGCTCGGTCT GCACACGGGG TACCGGGAGC TGCGCTCGGG CACGGTCTAC 360 CACGACGTGT ACCCGTCGCC CGGCGCGCAC CACCTGTCCT CGGAGACCTC CGAGACGCTG 420 CTGGAGTTCC ACACGGAGAT GGCCTACCAC CGGCTCCAGC CGAACTACGT CATGCTGGCC 480 TGCTCCCGGG CCGACCACGA GCGCACGGCG GCCACACTCG TCGCCTCGGT CCGCAAGGCG 540 CTGCCCCTGC TGGACGAGAG GACCCGGGCC CGGCTCCTCG ACCGGAGGAT GCCCTGCTGC 600 GTGGATGTGG CCTTCCGCGG CGGGGTGGAC GACCCGGGCG CCATCGCCCA GGTCAAACCG 660, CTCTACGGGG ACGCGGACGA TCCCTTCCTC GGGTACGACC GCGAGCTGCT GGCGCCGGAG 720 GACCCCGCGG ACAAGGAGGC CGTCGCCGCC CTGTCCAAGG CGCTCGACGA GGTCACGGAG 780 GCGGTGTATC TGGAGCCCGG CGATCTGCTG ATCGTCGACA ACTTCCGCAC CACGCACGCG 840 , - CGGACGCCGT TCTCGCCCCG CTGGGACGGG AAGGACCGCT GGCTGCACCG CGTCTACATC 900 ° CGCACCGACC GCAATGGACA GCTCTCCGGC GGCGAGCGCG CGGGCGACGT CGTCGCCTTC 960 ACACCGCGCG GC 972 (2) SEQUENCE INFORMATION SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein 0 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: Met Thr Arg Pro Pro Gly Leu Ser Ala His Thr His Gly Ser Val Ser 1 5 10 15 Gly Ser Leu Leu Arg Arg Val Ala Gly His Tyr Pro Thr Gly Val Val 20 25 30 Leu Val Thr Gly Pro Wing Glu Wing Pro Gly Gln Pro Pro Pro Wing Met 35 40 45 (2) SEQUENCE INFORMATION SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 151 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: Met Ser Val Wing Wing Wing Gly Met Thr Asp Glu Gln Arg Lys Ala Val 1 5 10 15 lie Thr Ala Tyr Phe Lys Wing Phe Asp Asn Gly Val Gly Ser Asp 20 25 30 Gly Thr Pro Ala lie Asp Tyr Phe Ala Glu Asp Wing Val Phe Phe Phe 35 40 45 Pro LYS TrP G1Y Leu Wing Ar9 G1Y LYS Ser Glu Ile Wing Ar9 Leu phe 50 55 60 Asp Asp Leu Gly Gly Thr lie Arg Ser lie Thr His His Leu Trp Ser 65 70 75 80 Val Asn Trp lie Leu Thr Gly Thr Glu Leu Leu Wing Ala Glu Gly Thr 85 90 95 Thr His Gly Glu His Arg Asp Gly Pro Trp Arg Wing Gly Asp Pro Glu 100 105 110 Trp Wing Wing Gly Arg Trp Cys Thr Val Tyr Glu Val Arg Asp Phe Leu 115 120 125 Val His Arg Wing Phe Val Tyr Leu Asp Pro Asp Tyr Wing Gly Lys Asp 130 135 140 Thr Ala Arg Tyr Pro Trp Leu 145 150 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 344 amino acids (B) TYPE: amino acid (C) TYPE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: Met Ser Arg Ser Pro Pro Glu Ser Pro Wing Gly Ser Val Ser Ala Ala 1 5 10 15 Val Pro Arg Pro Pro Val Arg Ala Leu Arg Asp Leu Pro Val Ser Wing 20 25 30 Gln Gly Leu Gly Cys Leu Pro Thr Thr Asp Phe Tyr Gly Arg Pro Asp 40 45 Arg Ala Arg Ala Thr Ala Thr He Arg Ala Ala Val Asp Ala Gly Val 50 55 60 Thr Leu Leu Asp Thr Wing Asp Val Gln Gly Leu Gly Wing Gly Glu Glu 65 70 75 80 Leu Leu Gly Arg Ala Val Ala Gly Arg Arg Asp Glu Val Leu He Wing 85 90 95 Thr Lys Phe Gly Met Val Arg Ser Ser Asp Gly Wing Ser Gln Gly Leu 100 105 lys Cys Gly Glu Pro Ser Tyr Val Arg Ala Wing Cys Glu Arg Ser Leu Arg 115 120 125 Arg Leu Gly Thr Asp Arg He Asp Leu Tyr Tyr Gln His Trp Thr Asp 130 135 140 Pro Ala Val Pro He Glu Glu Thr Val Gly Ala Val Ala Glu Leu Val 145 150 155 160 Arg Glu Gly Lys Val Arg Arg Leu Gly Leu Ser Glu Pro Ser Ala Ala 165 170 175 Thr Leu Arg Arg Wing Asp Wing Val His Pro Val Thr Wing Val Gln Ser 180 185 190 Glu Trp Ser Leu Trp Ser Arg Gly He Glu Asp Glu Val Val Pro Val 195 200 205 Cys Arg Glu Leu Gly He Gly He Val Wing Tyr Wing Pro Leu Gly Arg 210 215 220 Gly Phe Leu Thr Gly Thr He Arg Thr Thr Asp Asp Leu Gly Asp Glu 225 230 235 240 AsP phe Arg Arg Gly Gln Pro Arg Phe Ser Ala Pro Ala Leu Ala Arg 245 250 255 Asn Arg Ser Leu Leu His Arg Leu Arg Pro Val Wing Asp Gly Leu Gly 260 265 270 Leu Thr Leu Ala Gln Leu Ala Leu Ala Trp Leu His His Arg Gly Glu 275 280 285 Asp Val Val Pro He Pro Gly Thr Ala Asn Pro Ala His Leu Ala Asp 290 295 300 Asn Leu Ala Ala Ala Ser He Arg Leu Asp Asp Arg Ser Leu Ala Glu 305 310 315 320 Val Thr Ala Ala He Ser His Pro Val Ser Gly Glu Arg Tyr Thr Pro 325 330 335 Ala Leu Leu Ala Met He Gly Asn 340 (2) SEQUENCE INFORMATION SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 328 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: Met Glu Cys Arg He Phe Glu He Asp Glu Leu Pro Leu Leu Asp Gly 1 5 10 15 Glu Val Leu Arg Asp Wing Arg He Gly Tyr Wing Met Tyr Gly Thr Pro 20 25 30 Asn Wing Asp Gly Thr Asn Val Val Leu Cys Pro Ser Phe Phe Gly Arg 40 45 Asp His Thr Gly Tyr Asp Trp Leu He Gly Wing Gly Leu Pro Leu Asp 50 55 60 Thr Arg Arg Tyr Cys Val Val Thr Wing Gly Leu Phe Gly Asn Gly Val 65 70 75 80 Being Being Pro Gly Asn His Pro Being Gly Being Arg Phe Pro Leu He 85 90 95 Thr Pro Gln Asp Asn Val Wing Wing Gln His Arg Leu Leu Thr Glu Glu 100 105 110 Leu Gly Val Arg Glu Leu Ala Leu Val Thr Gly Trp Ser Met Gly Ala 115 120 125 Wing His Wing Tyr Gln Trp Wing Val Ser His Pro Gly Met Val Arg Arg 130 135 140 He Ala Pro He Cys Gly Ala Pro Val Ser Ser Pro His Ser Leu Val 145 150 155 160 Leu Leu Ser Gly Leu Ala Ala Ala Leu Ser Ala Asp Ala Gly Glu Arg 165 170 175 Gly Arg Lys Wing Wing Gly Arg Val Phe Wing Gly Trp Gly Thr Ser Arg 180 185 190 Ser Phe Trp Wing Arg Arg Wing His Arg Glu Leu Gly Phe Wing Thr Arg 195 200 205 Glu Glu Tyr Leu Thr Gly Phe Trp Glu Gln Val Phe Leu Ser Gly Pro 210 215 220 Gly Wing Wing Asp Leu Leu Thr Met Val Arg Thr Trp Glu Asn Thr Asp 225 230 235 240 V l G1Y Ala thr Pr ° Giy Wing Gly Gly Ser Val Glu Ala Wing Leu Wing 245 250 255 Ser Val Thr Ala Arg Ala Val Val Leu Pro Gly Ala Leu Asp Val Cys 260 265 270 Phe Wing Val Glu Asp Glu Lys Arg Val Wing Asp Leu Leu Pro Tyr Wing 275 280 285 Ser Leu Glu Val He Pro Gly Val Trp Gly His Leu Ala Gly Ser Gly 290 295 300 Gly Ser Ala Ala Asp Arg Glu Phe He Gly Gly Ala Leu Arg Arg Leu 305 310 315 320 Leu Asp Ser Pro Val Asp Gly Gly 325 (2) SEQUENCE INFORMATION SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:, - (A) LENGTH: 394 amino acids "(B) TYPE: amino acid (C) TYPE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: Met Lys Ser He Leu Phe Tyr Leu Pro Thr Val Gly Ser His Wing Gln 1 5 10 15 Val Gln Arg Gly Met Wing Gly Val Asn Pro Gln Asn Tyr Gln Asn Met 20 25 30 Leu Arg Gln Leu Thr Arg Gln Wing Gln Wing Wing Asp Glu Leu Gly Tyr 0 35 40 45 Trp Gly Leu Ser Phe Thr Glu His His Phe His Thr Glu Gly Phe Glu 50 55 60 Val Ser Asn Asn Pro He Met Leu Gly Leu Tyr Leu Gly Met Gln Thr 65 70 75 80 Arg His He Arg Val Gly Gln Met Ala Asn Val Leu Pro Leu His Asn 85 90 95 Pro Leu Arg Leu Ala Glu Asp Leu Ala Met Leu Asp His Met Thr Arg 100 105 110 Gly Arg Ala Phe Val Gly He Ala Arg Gly Phe Gln Lys Arg Trp Wing 115 120 125 Asp He Met Gly Gln Val Tyr Gly Val Gly Gly Thr Leu Ser Asp Ala 130 135 140 Gly Glu Arg Asp Arg Arg Asn Arg Ala Leu Phe Glu Glu His Trp Glu 145 150 155 160 He He Lys Lys Wing Trp Thr Thr Glu Thr Phe Thr His Ser Gly Glu 165 170 175 Gln Trp Thr He Pro Val Pro Asp Leu Glu Phe Pro Tyr Glu Wing Val 180 185 190 Arg Arg Tyr Gly Arg Gly Leu Asp Glu Asn Gly Val He Arg Glu Val 195 200 205 Gly He Wing Pro Lys Pro Tyr Gln Arg Pro His Pro Pro Val Phe Gln 210 215 220 Pro Phe Ser Phe Ser Glu Asp Thr Phe Arg Phe Cys Wing Arg Glu Gly 225 230 235 240 Val Val Pro He Leu Met Asn Thr Asp Asp Gln He Val Wing Arg Leu 245 250 255 Met AsP Ile tyr Ar9 Glu Glu Wing Glu Wing Wing Gly His Gly Thr Leu 260 265 270 Arg Arg Gly Glu Arg Val Gly Val Met Lys Asp Val Leu Val Ser Arg 275 280 285 Asp Ser Gly Glu Wing His His Trp Wing Ser Arg Gly Gly Gly Phe He 290 295 300 Phe Glu Asn Trp Phe Gly Pro Met Gly Phe Thr Glu Ala Leu Arg Ala 305 310 315 320 Thr Gly Glu Thr Gly Pro He Gly Ser Asp Tyr Lys Thr Leu Val Asp 325 330 335 Arg Gly Leu Glu Trp Val Gly Thr Pro Asp Asp He Asn Arg Met He 340 345 350 Glu Lys Leu Val Glu Arg His Asp Pro Glu Tyr Leu Leu Gln Cys Gln 355 360 365 Tyr Ser Gly Leu He Pro His Asp Val Gln Leu Arg Ser Leu Glu Leu 370 375 380 Trp Ala Thr Glu He Ala Pro Asn Trp Leu 385 390 (2) SEQUENCE INFORMATION SEQ ID NO: 18: '(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 220 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 18: Met Pro Gly Ser Gly Leu Glu Ala Leu Asp Arg Ala Thr Leu He His 1 5 10 15 Pro Thr Leu Ser Gly Asn Thr Wing Glu Arg He Val Leu Thr Ser Gly 25 30 Ser Gly Ser Arg Val Arg Asp Thr Asp Gly Arg Glu Tyr Leu Asp Ala 40 45 Wing Wing Val Leu Gly Val Thr Gln Val Gly His Gly Arg Wing Glu Leu 50 55 60 Wing Arg Wing Wing Wing Gln Gln Met Wing Arg Leu Glu Tyr Phe His Thr 65 70 75 80 Trp Gly Thr He Ser Asn Asp Arg Ala Val Glu Leu Ala Ala Arg Leu 85 90 95 Val Gly Leu Ser Pro Glu Pro Leu Thr Arg Val Tyr Phe Thr Ser Gly 100 105 110 Gly Ala Glu Gly Asn Glu He Ala Ala Leu Arg Met Ala Arg Leu Tyr His 115 120 125 His Arg Arg Gly Glu Be Wing Arg Thr Trp He Leu Ser Arg Arg Ser 130 135 140 Ala Tyr His Gly Val Gly Tyr Gly Ser Gly Gly Val Thr Gly Phe Pro 145 150 155 160 Ala Tyr His Gln Gly Phe Gly Pro Ser Leu Pro Asp Val Asp Phe Leu 165 170 175 Thr Pro Pro Gln Pro Tyr Arg Arg Glu Leu Phe Wing Gly Ser Asp Val 180 185 190 Thr Asp Phe Cys Leu Wing Glu Leu Arg Glu Thr He Asp Arg He Gly 195 200 205 Pro Glu Arg He Wing Wing Met He Gly Glu Pro He 210 215 220 (2) INFORMATION OF THE SEQUENCE SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 324 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: protein r- (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: O Met Thr Ser Val Asp Cys Thr Ala Tyr Gly Pro Glu Leu Arg Ala Leu 1 5 10 15 Ala Ala Arg Leu Pro Arg Thr Pro Arg Ala Asp Leu Tyr Ala Phe Leu 20 25 30 Asp Ala Ala His Thr Ala Ala Ala Ser Leu Pro Gly Ala Leu Ala Thr 35 40 45 Ala Leu Asp Thr Phe Asn Ala Glu Gly Ser Glu Asp Gly His Leu Leu 50 55 60 Leu Arg Gly Leu Pro Val Glu Wing Asp Wing Asp Leu Pro Thr Thr Pro 65 70 75 80 Be Ser Thr Pro Wing Pro Glu Asp Arg Ser Leu Leu Thr Met Glu Wing 85 90 95 0 Met Leu Gly Leu Val Gly Arg Arg Leu Gly Leu His Thr Gly Tyr Arg 100 105 110 Glu Leu Arg Ser Gly Thr Val Tyr His Asp Val Tyr Pro Ser Pro Gly 115 120 125 Wing His His Leu Ser Ser Glu Thr Ser Glu Thr Leu Leu Glu Phe His 130 135 140 Thr Glu Met Wing Tyr His Arg Leu Gln Pro Asn Tyr Val Met Leu Wing 145 150 155 160 Cys Ser Arg Ala Asp His Glu Arg Thr Ala Ala Thr Leu Val Ala Ser 165 170 175 Val Arg Lys Ala Leu Pro Leu Leu Asp Glu Arg Thr Arg Ala Arg Leu 180 185 190 Leu Asp Arg Arg Met Pro Cys Cys Val Asp Val Ala Phe Arg Gly Gly 195 200 205 Val Asp Asp Pro Gly Wing He Wing Gln Val Lys Pro Leu Tyr Gly Asp 210 215 220 Wing Asp Asp Pro Phe Leu Gly Tyr Asp Arg Glu Leu Leu Wing Pro Glu 225 230 235 240 Asp Pro Wing Asp Lys Glu Wing Val Wing Wing Leu Ser Lys Wing Leu Asp 245 250 255 Glu Val Thr Glu Wing Val Tyr Leu Glu Pro Gly Asp Leu Leu He Val 260 265 270 Asp Asn Phe Arg Thr Thr His Wing Arg Thr Pro Phe Ser Pro Arg Trp 275 280 285 Asp Gly Lys Asp Arg Trp Leu His Arg Val Tyr He Arg Thr Asp Arg 290 295 300 Asn Gly Gln Leu Ser Gly Gly Glu Arg Wing Gly Asp Val Val Wing Phe 305 310 315 320 Thr Pro Arg Gly

Claims (24)

NOVELTY OF THE INVENTION CLAIMS

1. - DNA that comprises one or more specific genes for the biosynthesis of clavama 5S in S. Clavuligerus and that is not essential for the biosynthesis of clavama 5R.

2. DNA according to claim 1 as identified in (SEQ ID NO: 1);

3. DNA according to claim 1, which has the sequences or substantially the sequences determined as orfup3, orfup2, orfupl, orfdwnl, orfdwn2 or orfdwn3, which correspond respectively to SEQ ID NO: 2 to 7.

4.- DNA according to claim 1, which has the sequence or substantially the sequence determined as SEQ ID NO: 4.

5.- S. clavuligerus comprising DNA corresponding to an open reading frame that flanks almost, DNA that has been dissociated or otherwise made defective.

6.- Clavuligerus conformation with claim 5, further characterized in that the open reading frame is orfup3, orfup2, orfup 1, orfdwn 1, orfdwn2 or orfdwn3.

7.- A procedure to improve the production of 5R clavama in a suitable microorganism that comprises the manipulation of DNA as defined in claim 1 or 2 and its inclusion in said microorganism.

8. A method according to claim 7, further characterized in that said suitable organism is S. clavuligerus.

9. A method to improve the production of clavama 5R in S. clavuligerus which comprises dissociating or otherwise rendering defective the almost flanking DNA regions.

10. A method according to any of claims 7 to 9, further characterized in that said DNA corresponds to the open reading frames orfup3, orfup2, orfupl, orfdwn2 or orfdwn3.

11. A method according to any of claims 7 to 10, further characterized in that said clavam 5R is clavulanic acid.

12. A method for identifying a microorganism suitable for the high production of clavama 5R comprising a preliminary screening for microorganisms with low or no production of clavama 5S.

13. A method according to claim 12, further characterized in that the microorganism is S. clavuligerus.

14. A method according to claim 12 or 13, further characterized in that clavama 5R is clavulanic acid.

15. - A method according to any of claims 12 or 14, further characterized in that one or more specific genes for the production of 5S clavamas are defective.

16. A microorganism selected from the group consisting of: a) a microorganism that is capable of the production of 5R clavam and of the low or zero production of 5S clavama obtainable by the method of any of claims 7 to 15; b) a microorganism that is capable of the production of 5R clavam and of the low or no production of 5S clavama obtemible by the method of claim 12 which is capable of producing clavulanic acid, but which does not produce clavama-2 carboxylate and / or 2-hydroxymethylclavama; and c) a microorganism obtained by the method of claim 12 which is strain 56-1 A, 56-3A, 57-2B, 1C, 60-1 A, 60-2A, 60-3A, 61-1A, 61 -2A, 61-3A or 61-4A.

17.- Clavulanic acid obtainable by the fermentation of a microorganism as defined in claim 16.

18. Clavulanic acid according to claim 17, which is free of clavama-2 carboxylate, or has significantly reduced levels thereof. .

19. Clavulanic acid according to claim 18 in the form of its potassium salt.

20.- Clavulanic acid which is free of 5S clavam, or has significantly reduced levels thereof.

21. - Clavulanic acid which is free of clavama-2 carboxylate, or has significantly reduced levels thereof.

22. A composition comprising potassium clavulanate according to claim 19, in combination with a beta-lactam antibiotic.

23. A composition according to claim 22, wherein the beta-lactam antibiotic is amoxicillin.

24. A process for the preparation of a composition comprising potassium clavulanate and amoxicillin, which process comprises producing clavulanic acid from a microorganism according to claim 12 and then converting it to the potassium salt and combining the salt of potassium with amoxicillin.