CA2108113C - Dna sequence encoding enzymes of clavulanic acid biosynthesis - Google Patents
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Abstract
DNA sequences are provided which encode the enzymes required for clavulanic acid synthesis. A process is provided for producing clavulanic acid in a transformant of a non-clavulanate-producing host.
Description
DNA SEQUENCE ENCODING ENZYMES OF CLAVULANIC ACID
BIOSYNTHESIS
This invention relates to methods for the production of the antibiotic, clavulanic acid.
Background of the Invention 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 clavuli eg rus, which also produces the ~3-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 identifie=d and characterised (Marsh et al., (1992), Biochem., vol. 31, pp. 12648-657).
European Patent Application 0349121 describes a DNA
restriction fragment encoding a portion of the genetic information involved in clavulanic acid synthesis but provides no sequence information.
Unti:L 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 an 11.6 kb genomic DNA sequence from S. clavuliaerus which codes for eight proteins and enables the production of clavulanic ' acid by transformants of non-clavulanic-producing organisms.
Summary of the Invention An isolated genomic DNA molecule is provided comprising the nucleotide sequence set out in Figure 2.
A process is provided for producing clawlanic acid in a transformant of a non-clawlanate-producing host.
Description of Drawings The invention, as exemplified by a preferred embodiment, is described with reference to the accompanying drawings in which:
Figure 1 shows the N terminal amino acid sequence of CLA and the nucleotide sequence of a probe directed to the underlined region of the sequence.
Figure 2 (2-1 to 2-10j shows the nucleotide sequence of a 15 kb genomic DNA fragment from S. clawliaerus. 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 coRi sites which ~0.ark the beginning and end of the portion of the DNA sequence which contains all the genetic information for claw lanic acid synthesis.
Figure 3 shows the location of the open reading frames downstream from pcbC.
Figure 4 shows a partial restriction map of the DNA
sequence of Figure 2 in the region surrounding cla (ORF4j.
Figure 5 shows a shuttle vector used for disruption of the cla gene.
Figure 6 shows a photograph of an agar plate bearing cultures of S. lividans transformants.
BIOSYNTHESIS
This invention relates to methods for the production of the antibiotic, clavulanic acid.
Background of the Invention 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 clavuli eg rus, which also produces the ~3-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 identifie=d and characterised (Marsh et al., (1992), Biochem., vol. 31, pp. 12648-657).
European Patent Application 0349121 describes a DNA
restriction fragment encoding a portion of the genetic information involved in clavulanic acid synthesis but provides no sequence information.
Unti:L 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 an 11.6 kb genomic DNA sequence from S. clavuliaerus which codes for eight proteins and enables the production of clavulanic ' acid by transformants of non-clavulanic-producing organisms.
Summary of the Invention An isolated genomic DNA molecule is provided comprising the nucleotide sequence set out in Figure 2.
A process is provided for producing clawlanic acid in a transformant of a non-clawlanate-producing host.
Description of Drawings The invention, as exemplified by a preferred embodiment, is described with reference to the accompanying drawings in which:
Figure 1 shows the N terminal amino acid sequence of CLA and the nucleotide sequence of a probe directed to the underlined region of the sequence.
Figure 2 (2-1 to 2-10j shows the nucleotide sequence of a 15 kb genomic DNA fragment from S. clawliaerus. 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 coRi sites which ~0.ark the beginning and end of the portion of the DNA sequence which contains all the genetic information for claw lanic acid synthesis.
Figure 3 shows the location of the open reading frames downstream from pcbC.
Figure 4 shows a partial restriction map of the DNA
sequence of Figure 2 in the region surrounding cla (ORF4j.
Figure 5 shows a shuttle vector used for disruption of the cla gene.
Figure 6 shows a photograph of an agar plate bearing cultures of S. lividans transformants.
Figure 7 shows an alignment of the amino acid sequence of CLA (S. clavuli9 erus CLA) with those of E.
Coli agmatine ureohydrolase (E. Coli AUH), yeast arginase (yeast ARG), rat arginase (rat ARG) and human arginase (human ARG).
Figure 8 shows a Southern blot of NcoT digests of genomic DNA from five presumptive mutants (lanes 1-5) and from wild-type S. clavuliqerus (lane 6). Panel A
membranes probed with cla-specific probe. Panel B
membranes probed with tsr-specific probe.
Figure 9 shows restriction enzyme maps of S.
clavuliaerus DNA inserts in cosmids. A. Restriction enzyme map of cosmid K6L2. B. Partial restriction enzyme map of cosmid KBL2. C. Restriction map of cosmids K6L2 and K8L2 indicating location of pcbC gene in relation to cla. D. The 2.o kb NcoI fragment encompassing the cla gene used in generating nested deletions for sequencing. Abbreviations: Ba, Ba_~ mHI;
B,B~3clII; E,F,coRl; K,KunI; N, NcoI; S,SalI; and Sm,SmaI.
Figure 10 shows the deduced amino acid sequence of ORF1 of Figure 2.
Figure 11 shows the deduced amino acid sequence of oRF2 of Figure 2.
Figure 12 shows the deduced amino acid sequence of ORF3 of Figure 2.
Figure 13 shows the deduced amino acid sequence of ORF4 of Figure 2.
Figure 14 shows the deduced amino acid sequence of ORF5 of Figure 2.
Figure 15 shows the deduced amino acid sequence of ORF6 of Figure 2.
Figure 16 shows the deduced amino acid sequence of ORF7 of Figure 2.
Figure l7 shows the deduced amino acid sequence of ORF8 of Figure 2.
Figure 18 shows the deduced amino acid sequence of ORF9 of Figure 2.
Coli agmatine ureohydrolase (E. Coli AUH), yeast arginase (yeast ARG), rat arginase (rat ARG) and human arginase (human ARG).
Figure 8 shows a Southern blot of NcoT digests of genomic DNA from five presumptive mutants (lanes 1-5) and from wild-type S. clavuliqerus (lane 6). Panel A
membranes probed with cla-specific probe. Panel B
membranes probed with tsr-specific probe.
Figure 9 shows restriction enzyme maps of S.
clavuliaerus DNA inserts in cosmids. A. Restriction enzyme map of cosmid K6L2. B. Partial restriction enzyme map of cosmid KBL2. C. Restriction map of cosmids K6L2 and K8L2 indicating location of pcbC gene in relation to cla. D. The 2.o kb NcoI fragment encompassing the cla gene used in generating nested deletions for sequencing. Abbreviations: Ba, Ba_~ mHI;
B,B~3clII; E,F,coRl; K,KunI; N, NcoI; S,SalI; and Sm,SmaI.
Figure 10 shows the deduced amino acid sequence of ORF1 of Figure 2.
Figure 11 shows the deduced amino acid sequence of oRF2 of Figure 2.
Figure 12 shows the deduced amino acid sequence of ORF3 of Figure 2.
Figure 13 shows the deduced amino acid sequence of ORF4 of Figure 2.
Figure 14 shows the deduced amino acid sequence of ORF5 of Figure 2.
Figure 15 shows the deduced amino acid sequence of ORF6 of Figure 2.
Figure 16 shows the deduced amino acid sequence of ORF7 of Figure 2.
Figure l7 shows the deduced amino acid sequence of ORF8 of Figure 2.
Figure 18 shows the deduced amino acid sequence of ORF9 of Figure 2.
Figure 19 shows the deduced amino acid sequence of ORF10 of Figure 2.
Detailed description of the Inyention Production of penicillin and cephamycin antibiotics in S. clavuliaerus starts with the conversion of lysine to a-aminoadipic acid (Madduri et al., (1989), J.
Bacteriol., v. 171, pp. 299-302; (1991), J. Bacteriol., v. 173, pp. 985-988). a-Aminoadipic acid then condenses with cysteine and valine to give d-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) by the action of aminoadipyl-cysteinyl-valine synthetase (RCVS). 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 RCVS 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; Sehwecke 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 c0-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. clavuliqerus which includes the cla gene. Within this 15 kb sequence, the inventors have identified an 11.6 kb DNA fragment which, ~1_(~~113 when introduced into the non-clavul<~nate 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 5 for clavulanate production.
As wall 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.
clavuliqerus 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 tec:hniques 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 c:lavulanic acid free of other contaminating clavams which are produced by S.
clavulicterus .
Suitable vectors and hosts will be known to those skilled in the art; suitable vectors include pIJ702, pJOE829 and pIJ922 and suitable hosta include S.
lividans, S. parvulus, S. griseofulvus, S. antibioticus and S. lipmanii.
Additionally, the DNA sequence: 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 technique's 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 oligonucleatide probe based on the N-terminal amino acid sequence of CLA was constructed as shown in Figure 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 locatE~d in the S.
clavuliqerus chromosome about 5.7 kb downstream of pcbC, the gene which encodes isopenicillin N synthase. The gene contains a 933 by 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 ~umonjinensis, Streptomyces lipmanii (7) and Streptom~ces antibioticus. No cross reactivity was seen to the a-lactam producing species Nocardia lactamdurans, Streptomyces a~riseus or Streptomyce:~ cattleya, nor to any of a variety of other Streptomyces species which do not produce R-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 Figure 2. When this 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 ~10~~~.13 shown in Figure 3. ORF 4 corresponds to cla. ORF 1,7 &
Detailed description of the Inyention Production of penicillin and cephamycin antibiotics in S. clavuliaerus starts with the conversion of lysine to a-aminoadipic acid (Madduri et al., (1989), J.
Bacteriol., v. 171, pp. 299-302; (1991), J. Bacteriol., v. 173, pp. 985-988). a-Aminoadipic acid then condenses with cysteine and valine to give d-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) by the action of aminoadipyl-cysteinyl-valine synthetase (RCVS). 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 RCVS 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; Sehwecke 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 c0-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. clavuliqerus which includes the cla gene. Within this 15 kb sequence, the inventors have identified an 11.6 kb DNA fragment which, ~1_(~~113 when introduced into the non-clavul<~nate 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 5 for clavulanate production.
As wall 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.
clavuliqerus 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 tec:hniques 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 c:lavulanic acid free of other contaminating clavams which are produced by S.
clavulicterus .
Suitable vectors and hosts will be known to those skilled in the art; suitable vectors include pIJ702, pJOE829 and pIJ922 and suitable hosta include S.
lividans, S. parvulus, S. griseofulvus, S. antibioticus and S. lipmanii.
Additionally, the DNA sequence: 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 technique's 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 oligonucleatide probe based on the N-terminal amino acid sequence of CLA was constructed as shown in Figure 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 locatE~d in the S.
clavuliqerus chromosome about 5.7 kb downstream of pcbC, the gene which encodes isopenicillin N synthase. The gene contains a 933 by 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 ~umonjinensis, Streptomyces lipmanii (7) and Streptom~ces antibioticus. No cross reactivity was seen to the a-lactam producing species Nocardia lactamdurans, Streptomyces a~riseus or Streptomyce:~ cattleya, nor to any of a variety of other Streptomyces species which do not produce R-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 Figure 2. When this 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 ~10~~~.13 shown in Figure 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 i~he 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.
When the predicted amino acid sequences of proteins encoded by ORFs 1 - to were compared to protein sequence databases, some similarities were nested in addition to the already mentioned similarity between CLA and enzymes of arginine metabolism. ORF 1 showed a low level of similarity to penicillin binding proteins from several different microorganisms which are notable for their resistance to ,~-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 them 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 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 passible that a deregulated form of AHAS is produced to provide valine curing 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 cl.avulanic acid, requires NADPH, and either pyruvate or a-ketobutyrate a:~ 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 and 5 do not show a significant similarity to any proteins in the data bases. ORF 6 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 o~_-nithine are unknown.
ORF 7 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 shows weak similarity to several transcription activator proteins, and ORF 9 shows weak similarity to ribitol 5 P04 dehydrogenase-type enzymes. ORF 10 shows a high similarity to cytochrome P450 type enzymes from other Strepomyces species.
ORFS 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 Figure 6.
21(~811~
When the predicted amino acid sequences of proteins encoded by ORFs 1 - to were compared to protein sequence databases, some similarities were nested in addition to the already mentioned similarity between CLA and enzymes of arginine metabolism. ORF 1 showed a low level of similarity to penicillin binding proteins from several different microorganisms which are notable for their resistance to ,~-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 them 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 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 passible that a deregulated form of AHAS is produced to provide valine curing 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 cl.avulanic acid, requires NADPH, and either pyruvate or a-ketobutyrate a:~ 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 and 5 do not show a significant similarity to any proteins in the data bases. ORF 6 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 o~_-nithine are unknown.
ORF 7 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 shows weak similarity to several transcription activator proteins, and ORF 9 shows weak similarity to ribitol 5 P04 dehydrogenase-type enzymes. ORF 10 shows a high similarity to cytochrome P450 type enzymes from other Strepomyces species.
ORFS 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 Figure 6.
21(~811~
EXAMPLES
Example 1 Bacterial strains, vectors and growth conditions.
Streptomyces clavulig~erus NRRL 3585, Streptomyces jumoniinenisis NRRL 5741, Streptom~ces lipmanii NRRL 3584, Streptom_y_ces griseus NRRL 3851, Nocardia lactamdurans NRRL 3802 and Streptomyces cattleya NRRL
3841 were provided by the Northern Regional Research Laboratories, Peoria, I1. Streptomyces antibioticus ATCC
8663 and Streptomyces fradiae ATCC :L9609 were obtained from the American Type Culture Collection, Rockville, MD.
Streptom~ces lividans strains 1326 and TK24 were provided by D.A. Hopwood (John Innes Institute, Norwich, U.K.), Streptom~ces venezuelae 13s and Stre~tomyces qriseofuscus 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 Streptom~ces . 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 (1.50) plates containing ampicillin (50 ~,g/mL) or tetracycline (10 ~,g/mL). The cloning vectors pUC118 and pUC119 (Vieira and Messing (1987) Methods in Enzymology, v. 153, pp. 3-11) were provided by ,:~. Vieira (Waksman Institute of Micrabiology, Rutgers University, Piscataway, N,J.). The plasmid vecaor pJOE829 was generously provided by J. Altenbuchner (University of Stuttgart, Stuttgart, Germany). The plasmid pIJ702 was obtained from the American Type Culture Collection, _ ' Rockville, MD. 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.
RCVS and CLA were resolved by applying a 0.2 ml sample of purified RCVS 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 KC1, I 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 eaccess 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 was used to prepare a 24-mer oligonucleotide probe with 8-fold degeneracy to the amino acid sequence underlined in Figure 1. The amino acids in brackets indicate ambiguities in the N-terminal sequence. The actual DNA sequence from the cloned fragment is indicated in Figure 1.
* trade-marks The probe was designed as an B-fold degenerate mixture of oligonucleotides to take into consideration the biased codon usage of Streptomyces (Bibb et al., 1984, Wright and Bibb (1992), Gene, v. 113, pp. 55-65).).
S End-labelled probe was then used to screen a cosmid library of S. clavuliaerus genomic DNA fragments as described in Materials and Methods.
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 containing recombinant cosmids was screened for the presence of cla using the 24-mer mixed oligonucleotide probe (Fig. 1) y which had been end-labelled with [y-s2P]dATP and polynucleotide kinase (Boehringer Mannheim). Colony hybridization and subsequent washing was performed as described by Sambrook et al., MOLECU1;AR CLONING: A
LABORATORYMANUAL, 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), at 55°C with a final wash in 0.2X SSC (I7C 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. clavuli ea rus genomic DNA and included a portion of cla and all of the gcbC 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. clavuliaerus (Fig. 9).
2~~8_~1~
ANA sequencing and analysis Ordered sets of deletions were generated (Henikoff, S (1984) Gene vol. 28(3) pp351-359) 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 inthe to sequence band pattern were relieved by carrying out reactions using 7-dea2a-dGTP in place of dGTP. The nested deletion fragments resided either in pUC118 or pUCll9, and were sequenced using the commercially available universal primers (Vieira and Messing, 1987).
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.). Similarity 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 of 939 by with a potential ribosome site 9 by 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, S.E, et al (1990) Journal of Bacteriology vol. 172 pp7269~-7271). The (FRAME analysis), using the algorithm of Bibb, M.J. et al (1984) .
Gene vol. 30 pp157-166, indicated the presence of a typical , 3 o 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 Figure 7. The S. clavuliaerus CLA sequence was ,;
aligned with the E. cola AUH sequence by the FASTA w * trade-marks ~I~811 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.
Example 2 DNA hybridization Genomic DNA preparations from various Stre~tomyces species were isolated as described by Hopwood, D.A. et al (1985) °°Genetic manipulation of Streptomyces - A Laboratory Manual"
published by the John times Foundation. For interspecies DNA
hybridization analysis, 2.0 pg amounts of genomic DNA preparations were digested with NcoI for 16h, 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 labeling an internal 459 by SalI fragment (Fig. 1) with [a 3ZP]dATP by nick translation. Hybridization was done as described by Sambrook et al., 0.989). Hybridization membranes were washed twice for 30 min in 2X SSC; 0.1% SDS and once for min in O.1X SSC; 0.1% SDS at 65°C.
,~gauer~res homoloaous to cla in other Strentomycetes Three of six producers of S-lactam antibiotics, S.
25 &lavuliaerus, ~. l~,pmanii and S. iumoniinensis showed positive hybridization signals whereas S. cattleya, griseus, and N. Iactamdurans did not (data not shown).
None of the nonproducing strains examined, S _ venezuelae, Ss, lividans, S. fradiae, S, antibioticus and S.
30 qriseofuscus gave any signal. All of the streptomycetes that gave positive signals were producers of clavam-type metabolites (Elson et al., 1987) Example 3 Disruption of the c~enomic cla aene A 2.0 kb NcoI fragment that contained the entire cla gene was digested at its unique Kp_nI site and the ends * trade-mark _ 2~~g~1~
made blunt by treatment with the Klenow fragment of E.
coli DNA polymerase I. A thiostrepton resistance gene (tsr), isolated as a 1085 by BclI fragment from pIJ702 and cloned into the BamriI site of pUC118 was excised as a SmaI/XbaI fragment and the ends made blunt as above and ligated into the KnnI 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), Supra.
Replacement of the chromosomal cla gene by a copy disrupted by the insertion of tsr, at or internal KpnI
site, was achieved by double recombination. Successful gene replacement was apparent when the 2.0 kb coI
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. Four of the five mutants tested showed the expected increase in the size of the coI -fragments, and the larger NcoI fragments also hybridized with a tsr specific probe. The fifth mutant was apparently a spontaneous theostrepton resistant mutant.
Antibiotic Assav The agar diffusion assay was used for determining both penicillin/cephamycin and clavulanic acid production. S. clavuliaerus strains to be assayed were grown in 10 ml. amounts of Trypticase Soy Broth (TSB;
Baltimore Biological Laboratories) medium with 1.0%
starch for 48h. The cultures were washed twice with 10.3% sucrose and once with ICI (Jensen et al. (1982), J.
Antibiot., Supra, v.35, pp 483-490) and the mycelium resuspended in 10.0 mL of MM. Two millilitres of washed cell suspension was inoculated into 100 mL of MM and incubated at 28°C for 48h. The cultures were harvested by centrifugation, and the supernatants were assayed for both penicillin/cephamycin and clavulanic acid using .,,.
21~811~3 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 5 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).
l0 ~,xample 4 ~roto~last formation and transformation E. coli competent cell preparation and transformation were as desCrlbed by Sambrook et al., Supra, 15 (1989). Protoplasts of S, clavyig~erus were, prepared, transformed and regenerated as described by Bailey et al.
(1984), Bio/Technology, v. 2, pp. 808-811, with the ~oliow~ng modifications. Dextrin and arginine in the regeneration medium were replaced by starch and sodium glutamate respectively. Protoplasts were heat shocked at 43°C for 5 min prior to the addition of DNA. Standard procedures were used for protoplasting and transformation of S. 1i r~'.dans (Hopwood et al. (1.985) Supra).
The 11.6 kb EcoRl fragment from K6L2 (Fig. 9) was ~.
cloned into the EcoRl site of pCAT-119. pCAT-119 is y derivative of pUCil9 which was prepared by insertionally inactivating the ampicillin resistance gene of pUC119 by the insertion of a chloramphenicol acetyltiansferase 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 Pstl and ligated to the Streptomyces plasmid pIJ702, which had also been digested with PstI. The resulting bifunctional plasmid carrying '..
the ll.skb insert was capable of replicating in either E.
cpli (with selection for chloramphenicol resistance) or in S. lividans (with selection for thiostrepton - 21t~8~.~3 ' 16 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, Supra, and transformants were selected by plating onto R2YE medium containing thiostrepton at a concentration of 50 ,ug/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 48h at 28°C before they were overlayered with molten soft nutrient agar containing penicillin G at a concentration of 1 ~g/ml and inoculated with Staphylococcus aureus N-2 as indicator organism (Jensen, 1982). (S. aureus N-2 was obtained from the Department of Microbiology Culture Collection, University of Alberta.
Any organism which produces a ~-lactamase sensitive to clavulanic acid may be used as indicator organism.) Zones of inhibition which appeared around the S. livida~s colonies upon incubation overnight at 30°C were evidence -of clavulanic acid productiuon. Clavulanic acid-producing colonies were found amongst these initial S.
~ividans transformants at a frequency of about 12%. When , plasmid DNA was isolated from one of these clavulanic acid-producing transformants. and re-introduced into 5~., lividans, the frequency of clavulanic acid production in these 2nd round transformants was about 40-45%. Figure 6 shows a photograph of an agar plate bearing 2nd. round transformants. Zones of inhibition are seen as clear y areas in the agar; these appear on the photograph as dark circular areas.
~zo~~~~
Example 5 Seduencing of 15 kb DNA fra mint Ordered sets of deletions were generated as described in Examp:Le 1 using fragments of the DNA insert from the cosmid clone K6L2 (Figure 9) and subcloned into the E. coli plasmids pUC118 andpUC119. Overlapping fragments were chosen which extended from the end of the pcbC gene downstream for a distance of about 15 kb ending at the BalII site. The deletion generated fragments were sequenced in both orientations as described in Example 1.
The sequence is shown in Figure 2.
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.
~~os~~~
ORF # Start locationEnd locationLength Size of ORF
(bp) (bp) (bp) (aa residues) 1* 109 1764 1656 552 8* 10 998 12 296 1299 433 9* 12 622 13 365 744 248 * Asterisks denote ORFs which are oriented in the opposite direction.
Example 1 Bacterial strains, vectors and growth conditions.
Streptomyces clavulig~erus NRRL 3585, Streptomyces jumoniinenisis NRRL 5741, Streptom~ces lipmanii NRRL 3584, Streptom_y_ces griseus NRRL 3851, Nocardia lactamdurans NRRL 3802 and Streptomyces cattleya NRRL
3841 were provided by the Northern Regional Research Laboratories, Peoria, I1. Streptomyces antibioticus ATCC
8663 and Streptomyces fradiae ATCC :L9609 were obtained from the American Type Culture Collection, Rockville, MD.
Streptom~ces lividans strains 1326 and TK24 were provided by D.A. Hopwood (John Innes Institute, Norwich, U.K.), Streptom~ces venezuelae 13s and Stre~tomyces qriseofuscus 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 Streptom~ces . 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 (1.50) plates containing ampicillin (50 ~,g/mL) or tetracycline (10 ~,g/mL). The cloning vectors pUC118 and pUC119 (Vieira and Messing (1987) Methods in Enzymology, v. 153, pp. 3-11) were provided by ,:~. Vieira (Waksman Institute of Micrabiology, Rutgers University, Piscataway, N,J.). The plasmid vecaor pJOE829 was generously provided by J. Altenbuchner (University of Stuttgart, Stuttgart, Germany). The plasmid pIJ702 was obtained from the American Type Culture Collection, _ ' Rockville, MD. 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.
RCVS and CLA were resolved by applying a 0.2 ml sample of purified RCVS 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 KC1, I 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 eaccess 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 was used to prepare a 24-mer oligonucleotide probe with 8-fold degeneracy to the amino acid sequence underlined in Figure 1. The amino acids in brackets indicate ambiguities in the N-terminal sequence. The actual DNA sequence from the cloned fragment is indicated in Figure 1.
* trade-marks The probe was designed as an B-fold degenerate mixture of oligonucleotides to take into consideration the biased codon usage of Streptomyces (Bibb et al., 1984, Wright and Bibb (1992), Gene, v. 113, pp. 55-65).).
S End-labelled probe was then used to screen a cosmid library of S. clavuliaerus genomic DNA fragments as described in Materials and Methods.
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 containing recombinant cosmids was screened for the presence of cla using the 24-mer mixed oligonucleotide probe (Fig. 1) y which had been end-labelled with [y-s2P]dATP and polynucleotide kinase (Boehringer Mannheim). Colony hybridization and subsequent washing was performed as described by Sambrook et al., MOLECU1;AR CLONING: A
LABORATORYMANUAL, 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), at 55°C with a final wash in 0.2X SSC (I7C 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. clavuli ea rus genomic DNA and included a portion of cla and all of the gcbC 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. clavuliaerus (Fig. 9).
2~~8_~1~
ANA sequencing and analysis Ordered sets of deletions were generated (Henikoff, S (1984) Gene vol. 28(3) pp351-359) 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 inthe to sequence band pattern were relieved by carrying out reactions using 7-dea2a-dGTP in place of dGTP. The nested deletion fragments resided either in pUC118 or pUCll9, and were sequenced using the commercially available universal primers (Vieira and Messing, 1987).
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.). Similarity 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 of 939 by with a potential ribosome site 9 by 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, S.E, et al (1990) Journal of Bacteriology vol. 172 pp7269~-7271). The (FRAME analysis), using the algorithm of Bibb, M.J. et al (1984) .
Gene vol. 30 pp157-166, indicated the presence of a typical , 3 o 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 Figure 7. The S. clavuliaerus CLA sequence was ,;
aligned with the E. cola AUH sequence by the FASTA w * trade-marks ~I~811 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.
Example 2 DNA hybridization Genomic DNA preparations from various Stre~tomyces species were isolated as described by Hopwood, D.A. et al (1985) °°Genetic manipulation of Streptomyces - A Laboratory Manual"
published by the John times Foundation. For interspecies DNA
hybridization analysis, 2.0 pg amounts of genomic DNA preparations were digested with NcoI for 16h, 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 labeling an internal 459 by SalI fragment (Fig. 1) with [a 3ZP]dATP by nick translation. Hybridization was done as described by Sambrook et al., 0.989). Hybridization membranes were washed twice for 30 min in 2X SSC; 0.1% SDS and once for min in O.1X SSC; 0.1% SDS at 65°C.
,~gauer~res homoloaous to cla in other Strentomycetes Three of six producers of S-lactam antibiotics, S.
25 &lavuliaerus, ~. l~,pmanii and S. iumoniinensis showed positive hybridization signals whereas S. cattleya, griseus, and N. Iactamdurans did not (data not shown).
None of the nonproducing strains examined, S _ venezuelae, Ss, lividans, S. fradiae, S, antibioticus and S.
30 qriseofuscus gave any signal. All of the streptomycetes that gave positive signals were producers of clavam-type metabolites (Elson et al., 1987) Example 3 Disruption of the c~enomic cla aene A 2.0 kb NcoI fragment that contained the entire cla gene was digested at its unique Kp_nI site and the ends * trade-mark _ 2~~g~1~
made blunt by treatment with the Klenow fragment of E.
coli DNA polymerase I. A thiostrepton resistance gene (tsr), isolated as a 1085 by BclI fragment from pIJ702 and cloned into the BamriI site of pUC118 was excised as a SmaI/XbaI fragment and the ends made blunt as above and ligated into the KnnI 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), Supra.
Replacement of the chromosomal cla gene by a copy disrupted by the insertion of tsr, at or internal KpnI
site, was achieved by double recombination. Successful gene replacement was apparent when the 2.0 kb coI
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. Four of the five mutants tested showed the expected increase in the size of the coI -fragments, and the larger NcoI fragments also hybridized with a tsr specific probe. The fifth mutant was apparently a spontaneous theostrepton resistant mutant.
Antibiotic Assav The agar diffusion assay was used for determining both penicillin/cephamycin and clavulanic acid production. S. clavuliaerus strains to be assayed were grown in 10 ml. amounts of Trypticase Soy Broth (TSB;
Baltimore Biological Laboratories) medium with 1.0%
starch for 48h. The cultures were washed twice with 10.3% sucrose and once with ICI (Jensen et al. (1982), J.
Antibiot., Supra, v.35, pp 483-490) and the mycelium resuspended in 10.0 mL of MM. Two millilitres of washed cell suspension was inoculated into 100 mL of MM and incubated at 28°C for 48h. The cultures were harvested by centrifugation, and the supernatants were assayed for both penicillin/cephamycin and clavulanic acid using .,,.
21~811~3 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 5 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).
l0 ~,xample 4 ~roto~last formation and transformation E. coli competent cell preparation and transformation were as desCrlbed by Sambrook et al., Supra, 15 (1989). Protoplasts of S, clavyig~erus were, prepared, transformed and regenerated as described by Bailey et al.
(1984), Bio/Technology, v. 2, pp. 808-811, with the ~oliow~ng modifications. Dextrin and arginine in the regeneration medium were replaced by starch and sodium glutamate respectively. Protoplasts were heat shocked at 43°C for 5 min prior to the addition of DNA. Standard procedures were used for protoplasting and transformation of S. 1i r~'.dans (Hopwood et al. (1.985) Supra).
The 11.6 kb EcoRl fragment from K6L2 (Fig. 9) was ~.
cloned into the EcoRl site of pCAT-119. pCAT-119 is y derivative of pUCil9 which was prepared by insertionally inactivating the ampicillin resistance gene of pUC119 by the insertion of a chloramphenicol acetyltiansferase 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 Pstl and ligated to the Streptomyces plasmid pIJ702, which had also been digested with PstI. The resulting bifunctional plasmid carrying '..
the ll.skb insert was capable of replicating in either E.
cpli (with selection for chloramphenicol resistance) or in S. lividans (with selection for thiostrepton - 21t~8~.~3 ' 16 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, Supra, and transformants were selected by plating onto R2YE medium containing thiostrepton at a concentration of 50 ,ug/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 48h at 28°C before they were overlayered with molten soft nutrient agar containing penicillin G at a concentration of 1 ~g/ml and inoculated with Staphylococcus aureus N-2 as indicator organism (Jensen, 1982). (S. aureus N-2 was obtained from the Department of Microbiology Culture Collection, University of Alberta.
Any organism which produces a ~-lactamase sensitive to clavulanic acid may be used as indicator organism.) Zones of inhibition which appeared around the S. livida~s colonies upon incubation overnight at 30°C were evidence -of clavulanic acid productiuon. Clavulanic acid-producing colonies were found amongst these initial S.
~ividans transformants at a frequency of about 12%. When , plasmid DNA was isolated from one of these clavulanic acid-producing transformants. and re-introduced into 5~., lividans, the frequency of clavulanic acid production in these 2nd round transformants was about 40-45%. Figure 6 shows a photograph of an agar plate bearing 2nd. round transformants. Zones of inhibition are seen as clear y areas in the agar; these appear on the photograph as dark circular areas.
~zo~~~~
Example 5 Seduencing of 15 kb DNA fra mint Ordered sets of deletions were generated as described in Examp:Le 1 using fragments of the DNA insert from the cosmid clone K6L2 (Figure 9) and subcloned into the E. coli plasmids pUC118 andpUC119. Overlapping fragments were chosen which extended from the end of the pcbC gene downstream for a distance of about 15 kb ending at the BalII site. The deletion generated fragments were sequenced in both orientations as described in Example 1.
The sequence is shown in Figure 2.
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.
~~os~~~
ORF # Start locationEnd locationLength Size of ORF
(bp) (bp) (bp) (aa residues) 1* 109 1764 1656 552 8* 10 998 12 296 1299 433 9* 12 622 13 365 744 248 * Asterisks denote ORFs which are oriented in the opposite direction.
Claims (31)
1. An isolated genomic DNA molecule comprising the nucleotide sequence of Figure 2.
2. An isolated DNA molecule having the nucleotide sequence of nucleotides 2033 to 13636 of Figure 2.
3. An isolated DNA molecule having the nucleotide sequence of nucleotides 2216 to 3937 of Figure 2.
4. An isolated DNA molecule having the nucleotide sequence of nucleotides 3940 to 5481 of Figure 2.
5. An isolated DNA molecule having the nucleotide sequence of nucleotides 5654 to 6595 of Figure 2.
6. An isolated DNA molecule having the nucleotide sequence of nucleotides 7895 to 9076 of Figure 2.
7. An isolated DNA molecule having the nucleotide sequence of nucleotides 9241 to 10908 of Figure 2.
8. An isolated DNA molecule having the nucleotide sequence of nucleotides 10998 to 12296 of Figure 2.
9. An isolated DNA molecule having the nucleotide sequence of nucleotides 12622 to 13365 of Figure 2.
10. An isolated DNA molecule having the nucleotide sequence of nucleotides 13769 to 14995 of Figure 2.
11. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 11.
12. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 12.
13. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 13.
14. An isolated DNA molecule comprising a nucleotide seguence encoding the amino acid sequence of Figure 15.
15. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 16.
16. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 17.
17. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 18.
18. An isolated DNA molecule comprising a nucleotide sequence encoding the amino acid sequence of Figure 19.
19. An isolated protein having the amino acid sequence of Figure 11.
20. An isolated protein having the amino acid sequence of Figure 12.
21. An isolated protein having the amino acid sequence of Figure 13.
22. An isolated protein having the amino acid sequence of Figure 15.
23. An isolated protein having the amino acid sequence of Figure 16.
24. An isolated protein having the amino acid sequence of Figure 17.
25. An isolated protein having the amino acid sequence of Figure 18.
26. An isolated protein having the amino acid sequence of Figure 19.
27. A recombinant vector comprising a DNA molecule in accordance with any of claims 1 to 18.
28. A cell transformed with a recombinant vector comprising a DNA molecule in accordance with claim 27.
29. A host transformed with a recombinant vector comprising a DNA molecule in accordance with claim 2 wherein the host is a Streptomycete.
30. A host in accordance with claim 29 which is S.
lividans.
lividans.
31. A process for producing clavulanic acid in S.lividans comprising transforming the host with a DNA molecule in accordance with claim 2 and culturing the host under suitable conditions to produce clavulanic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2108113 CA2108113C (en) | 1993-10-08 | 1993-10-08 | Dna sequence encoding enzymes of clavulanic acid biosynthesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2108113 CA2108113C (en) | 1993-10-08 | 1993-10-08 | Dna sequence encoding enzymes of clavulanic acid biosynthesis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2108113A1 CA2108113A1 (en) | 1995-04-09 |
| CA2108113C true CA2108113C (en) | 2006-12-05 |
Family
ID=4152430
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2108113 Expired - Fee Related CA2108113C (en) | 1993-10-08 | 1993-10-08 | Dna sequence encoding enzymes of clavulanic acid biosynthesis |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2108113C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9702218D0 (en) * | 1997-02-04 | 1997-03-26 | Smithkline Beecham Plc | Novel product |
| ES2131001B1 (en) * | 1997-06-16 | 2000-04-01 | Antibioticos Sau | PROCEDURE TO INCREASE THE PRODUCTION OF CLAVULANIC ACID THROUGH THE EXPRESSION OF REGULATORY AND BIOSYNTHETIC GENES OF STREPTOMYCES CLAVULIGERUS. |
| GB9815666D0 (en) * | 1998-07-17 | 1998-09-16 | Smithkline Beecham Plc | Novel compounds |
| AU7983400A (en) * | 1999-09-16 | 2001-04-17 | Johns Hopkins University, The | Improvement of clavulanic acid production |
| JP2005508189A (en) * | 2001-11-07 | 2005-03-31 | スミスクライン ビーチャム パブリック リミテッド カンパニー | New molecule |
| GB0130351D0 (en) * | 2001-12-19 | 2002-02-06 | Isis Innovation | Cyclisation process |
| GB0308696D0 (en) | 2003-04-15 | 2003-05-21 | Glaxo Group Ltd | New process |
-
1993
- 1993-10-08 CA CA 2108113 patent/CA2108113C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2108113A1 (en) | 1995-04-09 |
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