# Patent

## Title
Luciferases, fluorescent proteins, nucleic acids encoding the luciferases

## Abstract

This is a divisional application of application Ser. No. 08/790,462, filed Jan. 29, 1997, now abandoned, which is a continuation-in-part of application Ser. No. 08/567,801, filed Dec. 6, 1995, now abandoned, which is a continuation of application Ser. No. 08/134,018, filed Oct. 8, 1993, now abandoned.

## Background
This invention relates to methods for the production of the antibiotic, clavulanic acid.

Clavulanic acid is a broad spectrum beta-lactamase inhibitor and is an important antibiotic for the treatment of infectious diseases. It is produced commercially by the gram-positive mycelial prokaryote Streptomyces clavuligerus, which also produces the .beta.-lactam antibiotics penicillin N, desacetoxy cephalosphorin C and cephamycin C. Until recently, however, the pathway employed for clavulanic acid biosynthesis was much less well understood than the pathways leading to these other antibiotics.

Without knowledge of the pathway for clavulanic acid biosynthesis, it was not possible to isolate the genes coding for the key enzymes and to manipulate these genes to increase antibiotic yield or permit production of the antibiotic in heterologous systems.

One of the earliest enzymes of the pathway to be purified and characterised was clavaminic acid synthase. Two isozymes have now been identified and characterised (Marsh et al., (1992), Biochem., vol. 31, pp. 12648-657).

European Patent Application 0349121 describes a 6.8 kb Bgl II restriction fragment isolated from S. clavuligerus that encodes a portion of the genetic information involved in clavulanic acid synthesis. No further characterization of this fragment was performed nor was the utility of this fragment determined.

Marsh et al (1992, Biochem. vol 31, pp. 12648-657) cloned and sequenced two isozymes of clavaminate synthase cs1 and cs2 separated by 28 kb, however their location relative to the cephamycin and penicillin biosynthetic clusters was not determined. Ward and Hodgson (1993, FEMS Microbiol. Lett. vol 110, pp. 239-242) reported on the occurrence of a biosynthetic gene cluster for clavulanic acid. Aidoo et al (1994, Gene vol 147, pp. 4146) cloned and sequenced cla the gene encoding a protein (possibly proclavaminic acid amidinohydrolase) involved in clavulanic acid production. They reported that this gene was upstream from and adjacent to an ORF with the identical sequence of cs2. Paradkar and Jensen (1995, J Bacteriol, vol 177, pp. 1307-1314) further analyzed a 6.6 kb Bgl II fragment from S. clavuligerus comprising cs2 and through gene disruption experiments demonstrated its role in clavulanic acid synthesis.

Until the work of the present inventors, the complete complement of genes required for clavulanic acid synthesis had not been identified.

The present inventors have now isolated, cloned and sequenced a 15 kb DNA fragment from S. clavuligerus which encodes 10 ORFs. Within this 15 kb of DNA lies an 11.6 kb EcoRI fragment which codes for eight proteins and enables the production of clavulanic acid by transformants of non-clavulanic-producing organisms. This 11.6 kb fragment includes 8 complete ORFs (ORFs 2 to 9), two of which have been previously characterized (Marsh et al 1992, Paradkar and Jensen 1995). ORF1, which is incomplete, is not involved in clavulanic acid synthesis, ORF4 encodes the CLA protein and ORF5 is cs2, encoding one of the isozymes of clavaminate synthase. The function of the other remaining ORFs within this 11.6 kb fragment or their role in clavulanic acid synthesis is unknown.

An isolated genomic DNA molecule of 15 kb is provided comprising the nucleotide sequence set out in FIG. 2. This DNA molecule comprises 10 ORFs, eight of which are involved in clavulanic acid synthesis. A process is provided for producing clavulanic acid in a transformant of a non-clavulanate-producing host.

The present invention provides isolated DNA molecules having the nucleotide sequence of SEQ ID NOS: 15, 16, 19, 20, 21 and 22.

Furthermore, the present invention is directed to DNA molecules comprising the nucleotide sequences that encode the amino acid sequence found in FIGS. 11, 12, 15, 16, 17 and 18 which corresponds to SEQ IN NOS: 4, 5, 8, 9, 10 and 11.

This invention also embraces DNA molecules comprising the nucleotide sequences encoding the amino acid sequences of FIGS. 11, 12, 15, 16, 17 and 18. These amino acid sequences correspond to the expression products of ORFs 2, 3, 6, 7, 8 and 9.

This invention is directed to isolated proteins having the amino acid sequence of FIGS. 11, 12, 15, 16, 17 and 18. These amino acid sequences correspond to the expression products of the ORFs 2, 3, 6, 7, 8 and 9 as defined in SEQ ID NOS: 4, 5, 8, 9, 10 and 11.

This invention is also directed to recombinant vectors that comprise DNA molecules as defined above where hosts that have been transformed with these recombinant vectors.

This invention is also directed to the process for producing clavulanic acid in a non-clavulantic acid producing host that comprises transforming the host with a DNA molecule as defined above and culturing the host under suitable conditions to produce clavulanic acid.

Furthermore, this invention is directed to processes for enhancing clavulanic acid production in a clavulanic acid producing host comprising transforming the host with a DNA molecule as defined above.

The invention, as exemplified by a preferred embodiment, is described with reference to the accompanying drawings in which:

FIG. 1 shows the N terminal amino acid sequence of CLA (amino acid residues 1-25 of SEQ ID NO: 6), the potential codon corresponding with this sequence (SEQ ID NO: 24), and the nucleotide sequence of a probe (SEQ ID NO: 25) directed to the underlined region of the sequence. The nucleotide sequence of the actual DNA sequence is defined in SEQ ID NO: 1, specifically nucleotides 5687-5710 of SEQ ID NO: 1, 3665-3678 of SEQ ID NO: 13, or 34-57 of SEQ ID NO: 17.

FIG. 2 (2-1 to 2-10) shows the nucleotide sequence (Sequence ID No.:1) of a 15 kb genomnic DNA fragment from S. clavuligerus. The sequences of the ten ORFs within the fragment are shown in upper case letters and the intergenic regions are shown in lower case letters. The locations of the beginning and end of each ORF are also indicated directly above the nucleotide sequence. Asterisks above the sequence indicate the EcoRl sites which mark the beginning and end of the portion of the DNA sequence which contains all the genetic information for clavulanic acid synthesis.

FIG. 3 shows the location of the open reading frames downstream from pcbC.

FIG. 4 shows a partial restriction map of the DNA sequence of FIG. 2 in the region surrounding cla (ORF4).

FIG. 5 shows a shuttle vector used for disruption of the cla gene.

FIG. 6 shows a photograph of an agar plate bearing cultures of S. lividans transformants.

FIG. 7 shows a Southern blot of NcoI digests of genomic DNA from five presumptive mutants (lanes 1-5) and from wild-type S. clavuligerus (lane 6). Panel A: membranes probed with cla-specific probe. Panel B: membranes probed with tsr-specific probe.

FIG. 8 shows restriction enzyme maps of S. clavuligerus DNA inserts in cosmids. A. Restriction enzyme map of cosmid K6L2. B. Partial restriction enzyme map of cosmid K8L2. C. Restriction map of cosmids K6L2 and K8L2 indicating location of pcbC gene in relation to cla. D. The 2.0 kb NcoI fragment encompassing the cla gene used in generating nested deletions for sequencing. Abbreviations: Ba, BamHI; B,BGlII; E,EcoR1; K,KpnI; N, NcoI; S,SalI; and Sm,SmaI.

FIG. 9 shows the deduced amino acid sequence (Sequence ID No.:3) of ORF1 of FIG. 2.

FIG. 10 shows the deduced amino acid sequence (Sequence ID No.:4) of ORF2 of FIG. 2.

FIG. 11 shows the deduced amino acid sequence (Sequence ID No.:5) of ORF3 of FIG. 2.

FIG. 12 shows the deduced amino acid sequence (Sequence ID No.:6) of ORF4 of FIG. 2.

FIG. 13 shows the deduced amino acid sequence (Sequence ID No.:7) of ORF5 of FIG. 2.

FIG. 14 shows the deduced amino acid sequence (Sequence ID No.:8) of ORF6 of FIG. 2.

FIG. 15 shows the deduced amino acid sequence (Sequence ID No.:9) of ORF7 of FIG. 2.

FIG. 16 shows the deduced amino acid sequence (Sequence ID No.:10) of ORF8 of FIG. 2.

FIG. 17 shows the deduced amino acid sequence (Sequence ID No.:11) of ORF9 of FIG. 2.

FIG. 18 shows the deduced amino acid sequence (Sequence ID No.:12) of ORF10 of FIG. 2.

FIGS. 19 to 23. The upper panel shows the results of Southern hybridizations using the probes and gene fragments as set forth in the restriction maps of the lower panel.

FIG. 19 shows the construction of the orf-2 mutant.

FIG. 20 shows the construction of the orf-3 mutant.

FIG. 21 shows the construction of the orf-6 mutant.

FIG. 22 shows the construction of the orf-8 mutant.

FIG. 23 shows the construction of the orf-9 mutant.

FIGS. 24(A), (B) and (C), high pressure liquid chromatography (HPLC) analysis of clavulanic acid in culture supernatants. Culture supernatants from 96 hour Starch-Asparagine medium-grown cultures of either wild type S. clavuligerus, or one of the gene disruption mutants were analyzed by HPLC. (A)Retemion time of a clavulanic acids standard; (B) Wild type culture supernatant showing a peak due to clavulanic acid eluting with a retention time of 6.5 min. (C) Gene disruption mutant culture supernatant (ORF8 mutant). The same HPLC profile was seen for all of the other disruption mutants, including ORF2, ORF3, or ORF9, indicating that none of the mutants produced clavulanic acid under these culture conditions.

Production of penicillin and cephamycin antibiotics in S. clavuligerus starts with the conversion of lysine to .alpha.-aminoadipic acid (Madduri et al., (1989), J. Bacteriol., v. 171, pp. 299-302; (1991), J. Bacteriol., v. 173, pp. 985-988). .alpha.-Aminoadipic acid then condenses with cysteine and valine to give .delta.-(L-.alpha.-aminoadipyl)-L cysteinyl-D-valine (ACV) by the action of aminoadipyl cysteinyl-valine synthetase (ACVS). ACV is converted by isopenicillin N synthase (IPNS) to isopenicillin N, and, through a series of reactions, to desacetoxycephalosporin C and ultimately to cephamycin C (Jensen et al., (1984), Appl. Microbiol. Biotechnol., v. 20, pp 155-160).

The ACVS of S. clavuligerus has been purified and partially characterized by three separate groups, and estimates of its molecular weight vary from 350,000 to 500,000 Da (Jensen et al., (1990) J. Bacteriol., v. 172, pp. 7269-7271; Schwecke et al., (1992), Eur. J. Biochem., v. 205, pp. 687-694; Zhang and Demain, (1990), Biotech Lett., v. 12, pp. 649-654). During their purification, Jensen et al. observed a 32,000 Da protein which co-purified with ACVS despite procedures which should remove small molecular weight components. It has now been found that this protein is not related to ACVS but rather to clavulanic acid biosynthesis. It has been designated CLA .

In accordance with one embodiment of the invention, the present inventors have identified, cloned and sequenced the gene (cla) encoding this protein.

In accordance with a further embodiment of the invention, the inventors have cloned and sequenced a 15 kb stretch of genomic DNA from S. clavuligerus which includes the cla gene. Within this 15 kb sequence, the inventors have identified an 11.6 kb DNA fragment which, when introduced into the non-clavulanate producer S. lividans as described in Example 4, enabled that species to produce clavulanic acid. This indicates that the 11.6 kb fragment contains all the genetic information required for clavulanate production.

As will be understood by those skilled in the art, the identification of the DNA sequence encoding the enzymes required for clavulanate synthesis will permit genetic manipulations to modify or enhance clavulanate production. For example, clavulanate production by S. clavuligerus may be modified by introduction of extra copies of the gene or genes for rate limiting enzymes or by alteration of the regulatory components controlling expression of the genes for the clavulanate pathway.

Heterologous organisms which do not normally produce clavulanate may also be enabled to produce clavulanate by introduction, for example, of the 11.6 kb DNA sequence of the invention by techniques which are well known in the art, as exemplified herein by the production of S. lividans strains capable of clavulanate synthesis. Such heterologous production of clavulanic acid provides a means of producing clavulanic acid free of other contaminating clavams which are produced by S. clavuligerus.

Suitable vectors and hosts will be known to those skilled in the art; suitable vectors include pIJ702, pJOE829 and pIJ922 and suitable hosts include S. lividans, S. parvulus, S. griseofulvus, S. antibioticus and S. lipmanii.

Additionally, the DNA sequences of the invention enable the production of one or more of the enzymes of the clavulanate pathway by expression of the relevant gene or genes in a heterologous expression system.

The DNA sequences coding for one or more of the pathway enzymes may be introduced into suitable vectors and hosts by conventional techniques known to those skilled in the art. Suitable vectors include pUC118/119 and pET-11 and suitable hosts include many organisms, including E. coli strains such as MV1193 and BL21(DE3).

An oligonucleotide probe (SEQ ID NO: 25) based on the N-terminal amino acid sequence of CLA (amino acid residues 1-25 of SEQ ID NO: 6); was constructed as shown in FIG. 1 and was used to isolate the gene coding for the protein from S. clavuligerus, as described in Example 1.

The gene was found to be located in the S. clavuligerus chromosome about 5.7 kb downstream of pcbC, the gene which encodes isopenicillin N synthase. The gene contains a 933 bp open reading frame (ORF), encoding a protein of molecular weight 33,368. The deduced amino acid sequence was compared to database sequences and showed greatest similarity to enzymes associated with arginine metabolism, notably agmatine, ureohydrolase and arginases.

When an internal fragment of the cla gene was labelled and used to probe restriction endonuclease digests of genomic DNA from a variety of other Streptomyces and related species, evidence of homologous sequences was seen only in other clavulanic acid or clavam metabolite producers, including Streptomyces jumonjinensis, Streptomyces lipmanii and Streptomyces antibioticus. No cross reactivity was seen to the .beta.-lactam producing species Nocardia lactamdurans, Streptomyces griseus or Streptomyces cattleya, nor to any of a variety of other Streptomyces species which do not produce .beta.-lactam compounds, including S. fradiae ATCC 19609, S. venezuelae 13s and S. griseofulvus NRRL B-5429.

Disruption of the cla gene, as described in Example 3, led to loss of the ability to synthesise clavulanic acid.

A 15 kb DNA sequence extending downstream from pcbC was cloned and sequenced as described in Example 5. The nucleotide sequence is shown in FIG. 2. When this SEQ ID No.: 1 sequence information was analysed for percent G+C as a function of codon position (Bibb et al., (1984), Gene, v. 30, pp. 157-166), ten complete ORFs were evident, as shown in FIG. 3. ORF 4 corresponds to cla. ORF 1, 7 & 8 are oriented in the opposite direction to pcbC. ORFs 2-6 and ORF 10 are all oriented in the same direction as pcbC. ORFs 2 and 3, and ORFs 4 and 5 are separated by very short intergenic regions suggesting the possibility of transcriptional and translational coupling. Table 1 summarises the nucleotide sequences and lengths of ORFs 1-10. Start       End Location    Location  Length    Size of ORF SEQ ID ORF# (bp)        (bp)      (bp)      (aa residues) NO. 1* 1764         109 1656      552         14 2       2216        3937      1722      574         15 3       3940        5481      1542      514         16 4       5654        6595       942 314         17 5       6611        7588       978 326         18 6       7895        9076      1182      394         19 7       9241        10908  1668      556         20 8* 12296  10998  1299      433         21 9       13365  12622   744 248         22 10  13769  14995  1227      409         23 *ORFs which are oriented in the opposition direction.

When the predicted amino acid sequences of proteins encoded by ORFs 1-10 were compared to protein sequence databases, some similarities were noted in addition to the already mentioned similarity between CLA and enzymes of arginine metabolism. ORF 1 (SEQ ID No.: 14) showed a low level of similarity to penicillin binding proteins from several different microorganisms which are notable for their resistance to .beta.-lactam compounds.

An EcoRI fragment of the 15 kb DNA sequence, containing 11.6 kb DNA, was cloned into a high copy number shuttle vector and introduced into S. lividans, as described in Example 4. Of seventeen transformants examined, two were able to produce clavulanic acid, indicating that the 11.6 kb fragment contains all the necessary genetic information for clavulanic acid production.

This 11.6 kb fragment encompasses ORF 2 to ORF 9 of the 15 kb DNA sequence.

ORF 2 (SEQ ID No.: 15) shows a high degree of similarity to acetohydroxyacid synthase (AHAS) enzymes from various sources. AHAS catalyses an essential step in the biosynthesis of branched chain amino acids. Since valine is a precursor of penicillin and cephamycin antibiotics, and valine production is often subject to feedback regulation, it is possible that a deregulated form of AHAS is produced to provide valine during the antibiotic production phase. Alternatively, an AHAS-like activity may be involved in clavulanic acid production. While the presently recognized intermediates in the clavulanic acid biosynthetic pathway do not indicate a role for AHAS, the final step in the biosynthetic pathway, conversion of clavaminic acid to clavulanic acid, requires NADPH, and either pyruvate or .alpha.-ketobutyrate as well as other cofactors (Elson et al., (1987), J. Chem. Soc. Chem. Commun., pp. 1739-1740). It is striking that these same substrates and cofactors are required for AHAS activity. Perhaps the conversion of clavaminate to clavulanate actually involves several steps, one of which is catalyzed by an AHAS-like activity. ORFs 3 (SEQ ID No.: 16) and does not show a significant similarity to any proteins in the data bases. ORF 6 (SEQ ID No.: 19) shows similarity to ornithine acetyltransferase. Ornithine has been suggested to be the immediate precursor of a 5-C fragment of the clavulanic acid skeleton, but the details of the reaction required for the incorporation of ornithine are unknown. ORF 7 (SEQ ID No.: 20) shows weak similarity to protein XP55 from S. lividans, and a lower level of similarity to oligopeptide binding proteins from various other species. Similarly, ORF 8 (SEQ ID No. 21) shows weak similarity to several transcription activator proteins, and ORF 9 (SEQ ID No.: 22) shows weak similarity to ribitol 5 P0.sub.4 dehydrogenase-type enzymes. ORF 10 (SEQ ID NO: 23) shows a high similarity to cytochrome P450 type enzymes from other Strepomyces species.

ORF5 has now been identified as the gene for clavaminate synthase II (Marsh (1993) supra).

When a plasmid isolated from one of the two clavulanic acid-producing transformants was retransformed into S. lividans, about 40-45% of the resulting colonies were able to produce clavulanic acid, as shown in FIG. 6.

Bacterial Strains, Vectors and Growth Conditions.

Streptomyces clavuligerus NRRL 3585, Stretomyces jumonjinenisis NRRL 5741, Streptomyces lipmanii NRRL 3584, Streptomyces griseus NRRL 3851, Nocardia lactamdurans NRRL 3802 and Streptomyces cattleya NRRL 3841 were provided by the Northern Regional Research Laboratories, Peoria, Ill. Streptomyces antibioticus ATCC 8663 and Streptomyces fradiae ATCC 19609 were obtained from the American Type Culture Collection, Manassas, Va. Streptomyces lividans strains 1326 and TK24 were provided by D. A. Hopwood (John lnnes Institute, Norwich, U.K.), Streptomyces venezuelae 13s and Streptomyces griseofuscus NRRL B-5429 were obtained from L. C. Vining (Department of Biology, Dalhousie University, Halifax, N.S.). Cultures were maintained on either MYM (Stuttard (1982) J. Gen. Microbiol., v. 128, pp. 115-121) or on a modified R5 medium (Hopwood et al. (1985) in "Genetic Manipulation of Streptomyces: a laboratory manual", John Innes Foundation, U.K.) containing maltose instead of glucose and lacking sucrose (R5-S). Escherichia coli MV1193 (Zoller and Smith (1987) Methods in Enzymology, v. 154, pp. 329-349), used as recipient for all of the cloning and subcloning experiments, was grown in Luria Broth (LB; Sambrook et al. (1989) in "Molecular Cloning: a laboratory manual", Cold Spring Harbour, N.Y.) or on LB agar (1.5%) plates containing ampicillin (50 .mu.g/mL) or tetracycline (10 .mu.g/mL). The cloning vectors pUC118 and pUC119 (Vieira and Messing (1987) Methods in Enzymology, v. 153, pp. 3-11) were provided by J. Vieira (Waksman Institute of Microbiology, Rutgers University, Piscataway, N.J.). The plasmid vector pJOE829 was generously provided by J. Altenbuchner (University of Stuttgart, Stuttgart, Germany). The plasmid pIJ702 was obtained from the American Type Culture Collection, Manassas, Va. Restriction enzymes were purchased from Boehringer Mannheim, and used according to the manufacturers' specifications.

Separation of CLA from ACVS

CLA was previously characterized as a 32,000 Da molecular weight protein present in preparations of highly purified ACVS (Jensen et al. (1990), supra). The small size of CLA suggested that its co-purification with ACVS resulted from a physical association between the two proteins.

ACVS and CLA were resolved by applying a 0.2 ml sample of purified ACVS containing CLA onto a Superose 6 HR 10/30 (Pharmacia), which was equilibrated and eluted in 0.1 M MOPS buffer, pH 7.5 containing 0.05 M KCl, 1 mM dithiothreitol, and 20% glycerol, at a flow rate of 0.25 ml/min.

Comparison of the CLA retention time with those of molecular weight standards indicated that the native molecular weight of CLA was in excess of 270 kDa. The difference in molecular weight between native and denatured forms of CLA suggests that the native protein exists as an oligomer of eight identical subunits.

Isolation of Gene (cla) for CLA

N-terminal amino acid sequence information for CLA was obtained by electrophoretically transferring the protein from SDS polyacrylamide gels onto Immobilon membranes (Millipore Ltd.,) and submitting the material to the Protein Microsequencing Laboratory (University of Victoria,) for analysis. Information obtained for 25 amino acids at the N-terminus (amino acid residues 1-25 of SEQ ID NO: 6) was used to prepare a 24 mer oligonucleotide probe (SEQ ID NO: 25) with 8-fold degeneracy to the amino acid sequence underlined in FIG. 1. The amino acids in brackets indicate ambiguities in the N terminal sequence. The actual DNA sequence from the cloned fragment is indicated in FIG. 2 and SEQ ID No: 1.

The probe was designed as an 8-fold degenerate mixture of oligonucleotides to take into consideration the biased codon usage of streptomyces Wright and Bibb (1992), Gene, v. 113, pp. 55-65).). End-labelled probe was then used to screen a cosmid library of S. clavuligerus genomic DNA fragments.

A library of S. clavuligerus genomic DNA fragments (15-22 kb size fractionated fragments) was constructed as previously described (Doran et al. (1990), J. Bacteriol, v. 172, pp. 4909-4918). Using the cosmid vector pLAFR3. A collection of 1084 isolated E. coli colonies contained recombinant cosmids was screened for the presence of cla using the 24-mer mixed oligonucleotide probe (FIG. 1) which had been end-labelled with [.gamma.-.sup.32 P]dATP and polynucleotide kinase (Boehringer Mannheim). Colony hybridization and subsequent washing was performed as described by Sambrook et al., (1989), at 55.degree. C. with a final wash in 0.2.times.SSC (I.times.SSC, 0.15M NaCl and 0.015M sodium citrate) and 0.1% SDS.

Five colonies which gave strong hybridization signals were isolated from the panel of 1084 clones, and restriction analysis showed that the positive clones contained overlapping fragments of DNA. Two clones, K6L2 and K8L2, with sequences that spanned about 40 kb of the S. clavuligerus genome, were chosen for further analysis. Clone K8L2 contained about 22 kb of S. clavuligerus genomic DNA and included a portion of cla and all of the pcbC gene which encodes IPNS in the penicillin/cephamycin biosynthetic pathway. A restriction map of K6L2 is shown in FIG. 9. Within the approximately 27 kb of DNA contained in K6L2, the oligonucleotide probe hybridized to a 2.0 kb NcoI fragment which was subsequently found to contain the entire cla gene. Hybridization studies, restriction mapping and DNA sequence analysis revealed that cla was situated 5.67 kb downstream of the pcbC gene of S. clavuligerus (FIG. 9).

DNA Sequencing and Analysis

Ordered sets of deletions were generated (Henikoff, 1984) extending across the cla region of the 2.0 kb NcoI fragment (FIG. 9C). The deletion generated fragments were sequenced in both orientations by the dideoxynucleotide chain termination method of (Sanger et al. (1977), P.N.A.S., v. 74, pp. 5463-5467) using Sequenase (version 2.0) DNA polymerase (United States Biochemical Corporation). Areas of compression in the sequence band pattern were relieved by carrying out reactions using 7-deaza-dGTP in place of dGTP. The nested deletion fragments resided either in pUC118 or pUC119, and were sequenced using the commercially available universal primers.

The nucleotide sequence data were analyzed for the presence of restriction sites, open reading frames (ORFs) and codon usage by the PC-Gene programme (Intelligenetics Corp.). Similarly searches were accomplished with the FASTA program searching the GenPept database (release number 71) available through GenBank (Pearson and Lipman (1988), P.N.A.S., v. 85, pp. 2444-2448).

An ORF at 939 bp with a potential ribosome site 9 bp from the GTG start codon was found which encoded a putative protein with a molecular weight of 33,368 Da. This value is in close agreement to the molecular weight estimated for CLA by SDS-PAGE (Jensen et al., 1990). The analysis of percent G+C as a function of codon position (FRAME analysis), using the algorithm of Bibb et al., (1984), indicated the presence of a typical streptomycete ORF (data not shown) with a G+C content of 70%. Computer aided data base searches for sequences similar to cla revealed a high degree of similarity to agmatine ureohydrolase (40.5% identity over 291 amino acids) and somewhat lower similarity to arginases (29.6% identity over 135 amino acids to arginases from yeast and rat) as shown in FIG. 7. The S. clavuligerus CLA sequence was aligned with the E. coli AUH sequence by the FASTA program described above. The AUH sequence had previously been aligned with the three ARG sequences (Szumanski & Boyle (1990), J. Bacteriol., v. 172, pp. 538-547). Identical matches in two or more sequences are indicated with upper case letters.

DNA Hybridization

Genomic DNA preparations from various Streptomyces species were isolated as described by Hopwood et al. (1985). For interspecies DNA hybridization analysis, 2.0 .mu.g amounts of genomic DNA preparations were digested with NcoI for 16 h, and electrophoresed in 1.0% agarose gels. The separated DNA fragments were then transferred onto nylon membranes (Hybond-N, Amersham) and hybridized with a cla specific probe prepared by labelling an internal 459 bp SalI fragment (FIG. 1) with [.alpha..sup.32 P]DAPT by nick translation. Hybridization was done as decribed by Sambrook et al., (1989). Hybridization membranes were washed twice for 30 min in 2.times.SSC; 0.1% SDS and once for 30 min in 0.1.times.SSC; 0.1% SDS at 65.degree. C.

Sequences Homologous to cla in Other Streptomycetes

Three of six producers of .beta.-lactam antibiotics, S. clavuligerus, S. lipmanii and S. jumonjinesis showed positive hybridization signals whereas S. cattleya, S. griseus, and N. lactamdurans did not (data not shown). None of the nonproducing strains examined, S. venezuelae, S. lividans, S. fradiae, S. antibioticus and S. griseofuscus gave any signal. All of the streptomycetes that gave positive signals were producers of clam-type metabolites (Elson et al., 1987).

Disruption of the Genomic cla Gene

A 2.0 kb NcoI fragment that contained the entire cla gene was digested at its unique KpnI site and the ends made blunt by treatment with the Klenow fragment of E. coli DNA polymerase I. A thiostrepton resistance gene (tsr), isolated as a 1085 bp BclI fragment from pIJ702 and cloned into the BamHI site of pUC118 was excised as a SmaI/XbaI fragment and the ends made blunt as above and ligated into the KpnI site of cla. The ligation mixture was introduced into E. coli MV1193 and the transformants screened for the presence of the tsr gene by colony hybridization (Sambrook et al., 1989).

Replacement of the chromosomal cla gene by a copy disrupted by the insertion of tsr, at an internal KpnI site, was achieved by double recombination. Successful gene replacement was apparent when the 2.0 kb NcoI fragment which carries cla in the wild type organism was replaced by a 3.0 kb NcoI fragment due to the insertion of the 1.0 kb tsr gene in the mutants. Pour of the five mutants tested showed the expected increase in the size of the NcoI fragments, and the larger NcoI fragments also hybridized with a tsr specific probe. The fifth mutant was apparently a spontaneous theostrepton resistant mutant.

Antibiotic Assay

The agar diffusion assay was used for determining both penicillin/cephamycin and clavulanic acid production. S. clavuligerus strains to be assayed were grown in 10 ml. amounts of Trypticase Soy Broth (TSB; Baltimore Biological Laboratories) medium with 1.0% starch for 48 h. The cultures were washed twice with 10.3% sucrose and once with MM (Jensen et al. (1982), J. Antibiot., v. 35, pp. 483-490) and the mycelium resuspended in 10.0 mL of MM. Two milliliters of washed cell suspension was inoculated into 100 mL of MM and incubated at 28.degree. C. for 48 h. The cultures were harvested by centrifugation, and the supernatants were assayed for both penicillin/cephamycin and clavulanic acid using bioassay procedures described previously (Jensen et al. (1982), supra).

All of the resulting colonies with disrupted cla genes grew equally well on minimal medium and complex media and produced as much penicillin and cephamycin as did the wild-type, but produced no clavulanic acid (data not shown). HPLC analysis of cell supernatants confirmed the inability of the disrupted cla mutants to synthesize any clavulanic acid (data not shown).

Protoplast Formation and Transformation

E. coli competent cell preparation and transformation were as described by Sambrook et al., (1989). Protoplasts of S. clavuligerus were, prepared, transformed and regenerated as described by Bailey et al. (1984), Bio/Technology, v. 2, pp. 808-811, with the following modifications. Dextrin and arginine in the regeneration medium were replaced by starch and sodium glutamate respectively. Protoplasts were heat shocked at 43.degree. C. for 5 min prior to the addition of DNA. Standard procedures were used for protoplasting and transformation of S. lividans (Hopwood et al. (1985)).

The 11.6 kb EcoR1 fragment from K6L2 (FIG. 9) was cloned into the EcoR1 site of pCAT-119. pCAT-119 is derivative of pUC119 which was prepared by insertionally inactivating the ampicillin resistance gene of pUC119 by the insertion of a chloramphenicol acetyltransferase gene (Jensen et al. (1989), Genetics & Molec. Biol. of Ind. Microorg., pp. 239-245 Ed. Hershberger, Amer. Soc. Microbiol). The PCAT-119 plasmid carrying the 11.6 kb fragment was then digested with PstI and ligated to the Streptomyces plasmid pIJ702, which had also been digested with PstI. The resulting bifunctional plasmid carrying the 11.6 kb insert was capable of replicating in either E. coli (with selection for chloramphenicol resistance) or in S. lividans (with selection for thiostrepton resistance). The ligation mixture was transformed to E. coli. Plasmid DNA was isolated from several of the chloramphenicol resistant transformants and analyzed by agarose gel electrophoresis to ensure that the proper plasmid construct was obtained. This isolated plasmid material from E. coli was then transformed into S. lividans as described by Hopwood and transformants were selected by plating onto R2YE medium containing thiostrepton at a concentration of 50 .mu.g/ml.

Thiostrepton resistant S. lividans transformants carrying the bifunctional plasmid with the 11.6 kb insert were patched onto MYM agar plates and allowed to incubate for 48 h at 28.degree. C. before they were overlayered with molten soft nutrient agar containing penicillin G at a concentration of 1 .mu.g/ml and inoculated with Staphylococcus aureus N-2 as indicator organism (Jensen, 1982). (S. aureus N-2 was obtained form the Department of Microbiology Culture Collection, University of Alberta. Any organism which produces a .beta.-lactamase sensitive to clavulanic acid may be used as indicator organism.) Zones of inhibition which appeared around the S. lividans colonies upon incubation overnight at 30.degree. C. were evidence of clavulanic acid production. Clavulanic acid-producing colonies were found amongst these initial S. lividans transformants at a frequency of about 12%. When plasmid DNA was isolated from one of these clavulanic acid-producing transformants and re-introduced into S. lividans, the frequency of clavulanic acid production in these 2nd round transformants was about 40-45%. FIG. 6 shows a photograph of an agar plate bearing 2nd. round transformants. Zones of inhibition are seen as clear areas in the agar; these appear on the photograph as dark circular areas.

Sequencing of 15 kb DNA Fragment

Ordered sets of deletions were generated as described in Example 1 using fragments of the DNA insert from the cosmid clone K6L2 (FIG. 9) and subcloned into the E. coli plasmids pUC118 and pUC119. Overlapping fragments were chosen which extended from the end of the pcbC gene downstream for a distance of about 15 kb ending at the BglII site. The deletion generated fragments were sequenced in both orientations as described in Example 1. The sequence is shown in FIG. 2 and SEQ ID NO: 1.

Gene Disruption of the 12 kb Fragment

Gene disruption experiments were conducted on the various open reading frames (ORF) to determine if a particular ORF was involved in clavulanic acid production or not. In order to determine the role of the various ORFs in clavulanic acid biosynthesis, mutants disrupted in one of the ORFs were constructed by a gene replacement procedure based on that described in Paradkar and Jensen (Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314).

In all cases, the apramycin-resistance gene cassette (apr) was used to disrupt the genes encoded within the 12-kb DNA fragment. Before use, unless otherwise indicated, the apr-cassette was modified by adding NcoI restriction sites to both ends. This modification of the apr-cassette has been described in Paradkar and Jensen (Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314). Since four NcoI sites are present within the 12 kb fragment and present within ORFs 3, 5, 6, and 8, respectively, insertion of the apr-cassette within the NcoI sites created a series of plasmids (called pCATL2) with disruptions in ORFs 3, 5, 6, or 8. The plasmids containing these fragments are referred to as pCATL2orf3, pCATL2orf5, pCATL2orf6, and pCATL2orf8, respectively. The construction of pCATL2orf5 has been previously disclosed (Paradkar and Jensen, Journal of Bacteriology, March 1995, Vol. 177, no. 5, pages 1307 to 1314). From each of these plasmids, a smaller fragment carrying only the disrupted gene and some flanking sequence (see details below) was subcloned and finally inserted into the Streptomyces vector pIJ486 (obtained from David A Hopwood, John Innes Institute) for transformation into S. clavuligerus. All routine manipulations involved in subcloning were done in Escherichia coli using standard cloning vectors, such as pUC119, pBluescript SK+ etc. Since ORF2 and ORF9 do not contain NcoI sites, the disruptions of these ORFs were done in a slightly different manner. The preparation of ORF2, ORF3, ORF6, ORF8 and ORF9 are described in more detail below.

Construction of the Insertional Mutants

1. ORF2 Mutant

NotI-NcoI linker oligonucleotides were added to both ends of the apr gene cassette, and then this modified apr gene fragment was inserted into the NotI site within ORF2 carried on a 2.1-kb EcoRI-BglII fragment. Subsequently, the EcoRI-BglII fragment carrying the disrupted ORF2 was inserted into pIJ486.

Genomic DNA isolated from the wild type (wt) and from an ORF2 mutant was digested with EcoRI and NcoI, and then probed with a 2.1-kb EcoRI-BglII fragment (FIG. 20). The 2.7 kb hybridizing fragment present in the wild type has been replaced with a 1.8 kb fragment in the mutant. A 0.85 kb fragment is also expected to hybridize to the probe in the mutant, but owing to the conditions of electrophoresis under which the gel was that smaller fragment was not retained on the gel. The sizes of the remaining hybridizing fragments are consistent with the replacement of the wild type ORF2 with the apr-disrupted ORF2.

2. ORF3 Mutant

The apr cassette (with NcoI sites on each end) was inserted into the NcoI site within ORF3 carried on a 4 kb EcoRI-KpnI fragment. The fragment carrying the disrupted ORF3 was then subcloned into pIJ486.

Genomic DNA from the wild type (wt) and from an ORF3 mutant was digested separately with BglII, and with KpnI, and probed with a 0.6-kb BglII-NcoI fragment (FIG. 21). In the BglII digests, the 6.5 kb hybridizing fragment present in the wild type has been replaced with 8 kb fragment in the mutant, while in the KpnI digests, the 5.8 kb fragment has been replaced with a 4.5 kb fragment. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF3 with the apr-disrupted ORF3.

3. ORF6 Mutant

An 8-kb BglII fragment carrying the disrupted ORF6 was subcloned from pCATL2orf6 into pIJ486.

Genomic DNA from the wild type (wt) and from an ORF6 mutant was digested with BglII, and probed with a 1.6-kb NruI fragment (FIG. 22). The 6.5 kb hybridizing fragment present in the wild type has been replaced with an 8 kb fragment in the mutant. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF6 with the apr-disrupted ORF6.

4. ORF8 Mutant

A 2.9 kb BglII-EcoRI fragment carrying the disrupted ORF8 was subcloned from pCATL2orf8 into pIJ486.

Genomic DNA from the wild type (wt) and from an ORF8 mutant was digested with BglII, and also with both BglII and KpnI, and probed with a 1.3 kb BglII-NcoI fragment (FIG. 23). In the BglII digests, the 1.9 kb hybridizing fragment present in the wild type has been replaced with a 3.4 kb fragment in the mutant, while in the BglII/KpnI digests, the 1.9-kb fragment has been replaced with a 1.3 kb fragment. The sizes of these hybridizing fragments are consistent with the replacement of the wild type ORF8 with the apr-disrupted ORF8.

5. ORF9 Mutant

The apr gene cassette was first cloned as an EcoRI-PstI fragment into the E. coli vector Pbluescript, and re-isolated as an EcoRV-SmaI fragment. This fragment was then inserted into the MscI site present in ORF9 contained within a 2.4 kb BglII fragment. Subsequently, the 4 kb fragment carrying the disrupted ORF9 was inserted into pIJ486.

Genomic DNA from the wild type (wt) and an ORF9 mutant was digested with BglII, and with both BglII and EcoRI, and probed with a 0.6-kb BglII-SstI fragment (FIG. 24). In the BglII digests, the 2.4 kb hybridizing fragment present in the wild type has been replaced with a 3.9 kb fragment in the mutant, while in the BglII-EcoRI digests, the 1-kb fragment has been replaced with a 0.6 kb fragment. In BglII-EcoRI digests of the ORF9 mutant genomic DNA another 3.3-kb EcoRI-BglII fragment might have been expected to hybridize to the probe, but failure to see this fragment can be attributed to the fact that the probe has only approximately 50 bp of sequence homologous to the expected fragment. Since the post-hybridization washes were carried out under stringent conditions this small amount of homology might not be sufficient to give a hybridizing band. Nevertheless, the sizes of other hybridizing fragments are consistent with the replacement of the wild type ORF9 with the apr disrupted ORF9.

The Effect of Mutations Within the ORF's of the 12 kb Fragment on Clavulanic Acid Synthesis

1. Bioassay

Clavulanic acid production in S. lividans transformants was bioassayed by the method of Jensen et al., (Industrial Microorganisms:Basic and Applied Molecular Genetics, 1993, Chapter 22, Edited by R. H. Baltz, G. D. Hegemam, P. L. Skatrud, Published by The American Society for Microbiology, Washington, D.C.). Basically the method involves patching select transformants onto agriplates, and after a 48 hours incubation period at 28.degree. C., overlayering these organisms with a .beta.-lactamase-producing indicator organism, together with penicillin G, at a concentration of 1 .mu./ml. The results are summarized below: Strain             No Penicillin Penicillin G (1 .mu./ml) wild type          0 mm        28 mm ORF2               0 mm        0 mm ORF3               0 mm        0 mm ORF6               0 mm        0 mm ORF8               0 mm        0 mm ORF9               0 mm        0 mm

If a zone of inhibition is obtained in the presence of penicillin but not in the absence thereof or if the size of the zones in the presence of penicillin is greater than the zones without penicillin, it is indicative of a .beta.-lactamase inhibitory activity of clavulanic acid. The bioassay thus showed that transformants containing mutants with insertion in any one of the ORF2, ORF3, ORF6, ORF8 or ORF9 failed to produce .beta.-lactamase inhibitory activity of clavulanic acid, thus indicating that each of these ORFs are involved in the production of clavulanic acid.

2. HPLC Analysis

The amount of clavulanic acid produced by each transformant was also quantitated in each sample by HPLC. This assay was done to confirm the presence or absence of clavulanic acid production, within the cultured supernatants produced from each gene disruption mutant.

Culture Conditions for the Growth of Wild Type and the Mutants for Clavulanic Acid Production

Spores of transformants containing insertional mutations within either ORF2, ORF3, ORF6, ORF8 or ORF9 were first inoculated into 20 mL Trypticase Soy Broth containing 1% starch, and the culture was grown shaking for 48 hours at 28.degree. C. which served as a seed culture. The seed culture was then used at 1% inoculum to inoculate 20 ml Starch Asparagine medium the composition of which has been described earlier (Paradkar and Jensen (1995), J Bacteriol Vol. 177, pp. 1307-1314), and the culture was grown in the same conditions as the seed culture. Supernatants (0.1 mL) were obtained from 96 hour cultures, and derivatized with 25 .mu.L of imidazole reagent. Subsequently, a 50 .mu.L aliquot of the derivatized sample was analyzed by HPLC.

The high pressure liquid chromatography assay was conducted as described in Foulstone and Reading (1982, Antimicrob. Agents Chemother. 22:753-762).

Analysis was performed with a model M-45 pump, model 712 WISP automated sample injector, and model 480 variable-wavelength UV detector, all from Millipore Waters (Mississauga, Ontario, Canada). Samples (100 .mu.l) were mixed with imidazole reagent (25 .mu.l) and incubated at room temperature for 15 min to form imidazole-derivatized clavulanic acid. Derivatized samples (50 .mu.l) were analyzed on a reverse-phase column (.mu.Bondapak-C18) with an isocratic buffer system consisting of 0.1 M KH.sub.2 PO.sub.4 -6% methanol, PH 3.2 (adjusted with H.sub.3 PO.sub.4). Under these conditions, authentic clavulanic acid has a retention time of 6.5 min.

The HPLC chromatograms of culture supernatants from transformants containing an insertional mutation within ORF2, ORF3, ORF6, ORF8 or ORF9 and of the wild type were obtained. An example of these results is illustrated in FIG. 25, which represents the HPLC profile of wild type (B) or the ORF8 insertion mutant (C). As can be seen from FIG. 25, clavulanic acid was detected in the supernatant of the wild type as a peak, at a retention time of 9.1 minutes. An authentic sample of clavulanic acid also gave a retention time of approximately 9.1 minutes (FIG. 25 (A). The amount of clavulanic acid produced by the wild type was determined by integrating the clavulanic acid peak in the wild type supernatant.

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

## Claims

1. An isolated protein having an amino acid sequence selected from the group consisting of SEQ ID NOS: 4, 5, 8, 9, 10 and 11.

2. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO:4.

3. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO:5.

4. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO:8.

5. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO:9.

6. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO:10.

7. The isolated protein of claim 1 having the amino acid sequence of SEQ ID NO: 11.