Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1999-09-29
2001-05-22
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S106000, C435S136000, C435S886000, C514S210030, C540S349000
Reexamination Certificate
active
06235506
ABSTRACT:
This invention relates to a process for increasing the production of clavulanic acid and other clavams including those with a strong beta-lactamase inhibitory activity from organisms having the appropriate biosynthetic pathways.
Micro-organisms, in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins and penicillins.
Clavulanic acid is an important beta-lactamase inhibitor which is a key ingredient of the antibiotic sold under the name AUGMENTIN (Trade Mark of SmithKline Beecham plc). The commercial method by which clavulanic acid is produced is via fermentation of
Streptomyces clavuligerus.
A suitable fermentation medium for producing clavulanic acid is described in UK Patent Specification No. 1,508,977.
Whilst clavulanic acid and other clavams can be prepared in acceptable amounts by existing methods there remains a need for improving the titre of clavulanic acid in the fermentation broth so that the product can be produced more economically. One way in which the problem can be addressed is to seek mutant strains of
Streptomyces clavuligerus
ATCC 27064 or other clavulanic acid-producing micro-organisms which give rise to higher titre. To date relatively little has been published regarding process improvements by which a higher titre of clavulanic acid can be achieved by varying the reaction conditions in the fermentor.
It is customary to include ammonia as a source of nitrogen as manipulation of the nitrogen input to the fermentation is critical to clavulanic acid yield. However we have found that fermentation methods for producing clavulanic acid are particularly sensitive to the concentration of ammonia in the system.
According to the present invention there is provided a method for preparing clavams by fermentation of a clavam-producing organism in a suitable medium, characterised in that the amount of ammonia in the fermentation is kept at a low level during the fermentation in order to avoid repression of one or more key enzymes by ammonia.
By a low level we mean less than about 50 ug/ml. The level of ammonia should be maintained below 50 ug/ml for sufficient time, suitably between 1 and 10 hours, to allow derepression and biosynthesis of the key ammonia-repressible enzymes. Under customary fermentation conditions (with ammonia levels exceeding 100 ug/ml) it has been found that the ammonia acts to repress a number of enzymes involved in the metabolism of nitrogen including urease which catalyses the conversion of urea to ammonia and carbon dioxide (See V. Bascaran et al. J. Gen Microbiol. 1989 vol 135 pp 2465 to 2474). However the latter studies were not linked to antibiotic titre and made no suggestion for any link with improvement in clavam or clavulanic acid production in particular.
A surprising finding in the work leading up to the present invention was that urea accumulates in clavulanic acid fermentation broths. Urea is produced from the clavulanic biosynthetic process but there may well be other sources of this urea.
It has been found in accordance with a further aspect of the present invention that if the urea which can build up during clavulanic acid production is caused to react with a urease enzyme titres of clavulanic acid are considerably improved, for example in the order of 10%.
Accordingly in a further aspect of the invention there is provided a method for preparing clavams by fermentation of a clavam-producing micro-organism in a suitable medium characterised in that urea produced during the fermentation process is caused to react with a urease enzyme.
The urease can be either intrinsic (endogenous) or extrinsic (exogenous), that is to say can be caused to be produced by the clavulanic acid producing micro-organism (intrinsic urease) or added to the system (extrinsic urease).
If it is desired to use extrinsic urease the enzyme may be obtained from any suitable source and added to the fermentation reaction. If the urease is intrinsic it can, in one aspect of the invention, be liberated by derepressing expression of urease by the clavulanic acid producing organism.
Such derepression may be caused, for example, by adjustment of the pH by any suitable base other than ammonia. In this method the concentration of ammonia in the fermentation is preferably lowered until derepression occurs. Such derepression may suitably be monitored by studying changes in the pH profile of the reaction medium (see examples below).
In the above method the pH control agent can be any suitable base other than ammonia, for example an alkali metal hydroxide such as sodium hydroxide. Once derepression has occurred and urease has been expressed it is advantageous to control pH in the usual way in order to ensure a sufficient nitrogen supply. Optionally however, a pH regulant other than ammonia may be used throughout the fermentation.
Ammonia is generally preferred as a feed in the typical fermentation as it has the dual purpose of pH regulation and introduction of nitrogen to the fermentation. It also acts as an ionic counterbalance to the clavulanate accumulating. Whilst the advantage gained by derepression of the ammonia-repressible enzymes can be achieved by the process of using an alternative pH regulant during part of the fermentation, it can also be achieved by using an alternative pH regulant and then introducing the required nitrogen to the fermentation in the batch or by feeding an ammonium salt.
In a further method of derepressing urease an adjustment may be made to the amount of complex nitrogen source batched or fed to the fermentation. Ammonia released by deamination of the complex nitrogen source will repress urease. Therefore an adjustment to the amounts included in the fermentation or their release characteristics will affect urease derepression.
A further method of increasing clavulanic acid titre is to add to the fermentation reaction any suitable compound which directly or indirectly affects the concentration of ammonia or urea in the fermentation.
One way in which this can be achieved is to add a compound such as a zeolite capable of adsorbing ammonia and so reducing its concentration. Suitable ammonia adsorbing reagents include those capable of precipitating ammonia as a complex salt, for example as ammonium magnesium phosphate.
A further way in which the concentration of urea may be decreased is to add a compound to the fermentation medium which directly or indirectly increases the expression of urease from the clavulanic acid producing organism.
Genetic manipulation or strain improvement methods may also be used to increase the levels of intrinsic urease produced by the clavulanic acid producing micro-organism. For example strain improvement [e.g. by mutagenesis and subsequent selection on specific media such as those including methylammonium (Micro-biological Reviews; 1989; 53, 85-108) can be used to prepare a mutant strain of clavulanic acid producing micro-organism which is constituitive for urease expression, i.e. urease is not subject to significant repression in the presence of ammonia. Similar results may be obtained by targeted genetic manipulation techniques, including manipulation of the urease gene so as to affect its regulation, for example causing concerted expression with an enzyme involved in the clavulanic acid biosynthetic pathway.
The above methods have the advantage that substantial increases in clavulanic acid titre can be achieved. The clavulanic acid may be separated and purified by standard techniques.
The following examples illustrate the invention. In the examples, unless otherwise indicated the methods and standard techniques used are as given in Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition.
REFERENCES:
patent: 4110165 (1978-08-01), Cole et al.
patent: 5185139 (1993-02-01), Krishnamurthy et al.
patent: 5869299 (1999-02-01), Baggaley et al.
patent: 1508977 (1978-04-01), None
Reading et al. Animicrobial Agents Chemother. vol. 11, No. 5, pp. 852-857, 1977.*
Brana et al. Can. J. Microbiol. vol. 31, pp. 736-743, 1985.*
Shen et al. J.
Holms William Henry
Jeffkins Paul Alan
Mousdale David Michael
Valentine Brian Peter
Kerekes Zolton
Kinzig Charles M.
SmithKline Beecham p.l.c.
Tate Christopher R.
Venetianer Stephen
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