Note: Descriptions are shown in the official language in which they were submitted.
1
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
CA 02108113 2005-12-O1
2
' 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.
CA 02108113 2005-12-O1
3
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.
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4
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
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6
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
7
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
8
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~
9
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,
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218113
_ ' 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
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11
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).
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2~~8_~1~
12
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
CA 02108113 2003-03-25
~I~811
13
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
CA 02108113 2003-03-25
_ 2~~g~1~
14
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
CA 02108113 2003-03-25
.,,.
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
CA 02108113 2003-03-25
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' 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~~~~
17
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~~~
TABLE 1
ORF # Start locationEnd locationLength Size of
ORF
(bp) (bp) (bp) (aa residues)
1* 109 1764 1656 552
2 2216 3937 1722 574
3 3940 5481 1542 514
4 5654 6595 942 314
6611 7588 978 326
6 7895 9076 1182 394
7 9241 10 908 1668 556
8* 10 998 12 296 1299 433
9* 12 622 13 365 744 248
13 769 14 995 1227 409
* Asterisks denote ORFs which are oriented in the opposite direction.
