Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Reexamination Certificate
1998-05-11
2001-01-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
C435S193000, C435S252300, C435S252330, C435S320100, C435S471000, C435S476000, C435S477000, C536S023100, C536S023200
Reexamination Certificate
active
06171814
ABSTRACT:
The present invention relates to processes for increasing the production of clavulanic acid and other clavams including those with strong &bgr;-lactamase inhibitory activity from producing organisms. The present invention also provides novel organisms capable of producing increased amounts of these clavams.
Microorganisms, in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins, cephamycins, tunicamycin, holomycin and penicillins. There is considerable interest in being able to manipulate the absolute and relative amounts of these antibiotics produced and accordingly there have been a large number of studies investigating the metabolic and genetic mechanisms of these pathways [Domain, A. L. (1990) “Biosynthesis and regulation of beta-lactam antibiotics.” In: 50 years of Penicillin applications, history and trends]. Many of the enzymes which carry out the various steps in the metabolic pathways and the genes which code for these enzymes are known.
In the cephalosporin metabolic pathway in, for example,
Strepromyces clavuligerus
three important antibiotics can be produced namely penicillin N, O-carbamoyldeacetylcephalosporin C and cephamycin C.
These antibiotics are synthesised from the tripeptide precursor d-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) [J. F. Martin et al (1990)., “Clusters of genes involved in Penicillin and cephalosporin biosynthesis” In: 50 years of penicillin applications history and trends].
The recognised first dedicated step in the biosynthesis of both penicillins and cephalosporins in
S. clavuligerus
involves the enzyme lysine -&egr;-amino transferase (LAT). The nucleotide sequence and derived amino acid sequence of
S. clavuligerus
lat gene is known [Tobin, M. B et al., (1991) J. Bacteriol, 173, 6223-6229].
U.S. Pat. No. 5,474,912 (published Dec. 12, 1995) describes a process for producing increased amounts of cephalosporins in
S. clavuligerus
by inserting one or more copies of a LAT gene into the chromosome of the organism. Although effects on &bgr;-lactam antibiotics are claimed, only effects on products of the cephalosporin pathway in
S. clavuligerus
are disclosed ie. no effects on clavam production were measured or described.
Clavulanic acid and other clavams are derived from a 3 carbon compound [Townsend, C. A. and Ho, M. F. (1985) J. Am. Chem. Soc. 107 (4), 1066-1068 and Elson, S. W and Oliver, R. S. (1978) J. Antiobiotics XXXI No.6, 568] and arginine [Valentine, B. P et al (1993) J. Am Chem. Soc. 15, 1210-1211].
The gene clusters which determine the biosynthesis of cephalosporins and clavulanic acid, although adjacent to each other on the
S. clavuligerus
chromosome [Ward, J. M. and Hodgson, J. E (1993) FEMS Microbiol. Lett. 110, 239-242] do not share common biosynthetic structural genes. Therefore there are no apparent biochemical links between the pathways.
Surprisingly we have found that when at least one step in the cephalosporin pathway of an organism is interfered with the amount of clavams produced by the organism is increased.
Accordingly the present invention provides a process for increasing the amount of clavam produced by an organism having both a clavam pathway or a portion thereof and a cephalosporin pathway or a portion thereof by interfering with the conversion of L-lysine to L-&agr;-aminoadipic acid in the cephalosporin pathway.
Preferably the clavam has &bgr;-lactamase inhibitory activity and more preferably the clavam is clavulanic acid.
In a preferred aspect of the invention the process of conversion of L-lysine to L-&agr;-aminoadipic acid is interfered with by altering the function of the LAT enzyme or the lat gene. For example the enzyme can be inhibited or otherwise blocked (for example by using a non-metabolisable substrate or analog).
Suitably the LAT gene can be obtained by conventional cloning methods (such as PCR) based on the published sequence. The function of the gene can be interfered with or eliminated/deleted by genetic techniques such as gene disruption [Aidoo, K. A. et al., (1994), Gene, 147, 4146]., random mutagenesis, site directed mutagenesis and antisense RNA.
Mahro, B. and Demain, A (1987), Appl.Microbiol. Technol. 27, 272-275 described a spontaneous mutant strain of
S. clavuligerus
(NP1) which did not produce cephamycin and was demonstrated to be defective in lat, acvs and ipns enzyme activities. H. Yu et al (1994) [Microbiology, 140,3367-3377] demonstrated that the activity of these three enzymes can be recovered by transforming the mutant with a fragment of DNA encoding the entire LAT gene (lat) and the upstream half of the acvs gene (pcbAB). However there was no teaching of any effect in this mutant in relation to the production of clavulanic acid or other clavams.
In a further aspect of the invention there are provided plasmids containing a defective lat gene, preferably the plasmids pCF002 and p486latap described below. Suitably the plasmids are used to transform an organism such as
S. clavuligerus
eg strain ATCC 27064 (equivalent to
S. clavuligerus
NRRL 3585).
In a further aspect of the invention there is provided an organism containing a defective lat gene.
REFERENCES:
patent: 5474912 (1995-12-01), Sherman et al.
patent: 5705340 (1998-01-01), Rasmussen et al.
patent: 5756326 (1998-05-01), Martin et al.
patent: 0349121 (1990-03-01), None
patent: WO A 9418326 (1994-08-01), None
Romero, et al., “Isolation and Biochemical Characterization ofStreptomyces clavuligerusMutants in the Biosynthesis of Clavulanic Acid and Cephamycin C”,Appl. Microbiol. Biotechnol., 27:510-516.
J.M. Wards et al., “The Biosynthetic Genes for Clavulanic Acid and Cephamycin . . . ”, FEMS Microbiology Letters (1993) vol. 110, pp. 239-242.
H. Yu et al., “Possible Involvement of the Lat Gene in the Expression . . . ”, Microbiology (1994), vol. 140, No. 12, pp. 3367-3377.
Holms William Henry
Mosher Roy Henry
Paradkar Ashish Sudhakar
Achutamurthy Ponnathapu
Kerekes Zoltan
Kinzig Charles M.
Moore William W.
SmithKline Beecham p.l.c.
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