FERMENTATIVE PRODUCTION OF CLAVULANIC ACID UNDER CONTROLLED PHOSPHATE CONDITIONS

Abstract: An amended process for the fermentative preparation of clavulanic acid has been provided whereby the level of free phosphate in the fermentation medium is controlled both at the start of the fermentation and/or during the fermentation.

Description: FERMENTATIVE PRODUCTION OF CLAVULANIC ACID UNDER CONTROLLED PHOSPHATE CONDITIONS The present invention relates to a method for the production of a clavulanic acid producing microorganism in a suitable medium. Clavulanic acid is an important 3-lactamase inhibitor. Clavulanic acid is produced from various strains of microorganism, for example the strains belonging to the genus Streptomyces such as S . clav ligerus ATCC 27064, S . Jumonjinensis (UK patent 1,563,103), S . Katsurahamanus IFO 13716 (JP patent 83,009,679) and Streptomyces sp . P6621 FER 2804 (JP 55,162,993) . Clavulanic acid can be prepared in acceptable amounts by existing methods. However, there remains a need for improving the titre of clavulanic acid in the fermentation broth. Regarding obtaining process improvements by varying the reaction conditions in the fermentor it is already known that the fermentation can be advantageously carried out continuously (GB 1508977) ; batch wise but with the carbon source added fed-batch (EP-182522) and with low levels of ammonia and urea (WO 96/18743) . Surprisingly, it has been found that fermentation methods for producing clavulanic acid are particularly sensitive to the concentration of phosphate in the medium and that controlling the phosphate level during batch, fed- batch or continuous fermentation of a clavulanic acid producing microorganism causes a higher titre of clavulanic acid in the fermentation broth. For the batch wise fermentation of clavulanic acid it has been found to be critical to apply an amount of nitrogen source within a certain range. For the continuous and fed-batch ways of fermentation it has turned out to be of crucial importance to maintain the concentration of phosphate present in the fermentation broth within certain ranges. The present invention provides a method for preparing clavulanic acid and other clavams by fermentation of a clavam-producing organism (e.g. Streptomyces clavuligerus) in a suitable medium characterised in that the amount of phosphate in the fermentation is controlled either at the beginning and/or during the fermentation in order to avoid repression of one or more key enzymes by phosphate. By controlled at the beginning of the fermentation a phosphate [P0 4 ] level in the batch medium at the start of the fermentation has been meant that is between 0.5 g/1 to 2.5 g/1. Phosphate levels higher or lower than this range in the batch medium have shown to adversely affect clavulanic acid production. The understanding of the profound influence of batch phosphate level on clavulanic acid production evolved in a strategy of feeding phosphate to the culture during the fermentation, which is the essence of an aspect of the present invention. By controlling free phosphate level between 0.1 g/1 and 1.2 g/1, preferably 0.2 - 0.8, or between 0.5 g/1 - 1.2 g/1, most preferably between 0.6 g/1 - 1.2 g/1, including or excluding the extreme values, through adjusting phosphate feed rates a significant increase of more than 10% in clavulanic acid production was observed. The advantage of a somewhat higher concentration in phosphate will result in a better buffering capacity which is especially important for production at large scale. According to another aspect of the invention there is provided a method for preparing clavulanic acid by fermentation of a clavam-producing organism in a suitable medium characterized in that, besides phosphate, a nitrogen source is added to the fermentation process whereby the level of the nitrogen source especially in the form of free ammonia is kept low (<100 mg/1) and while that of phosphate is maintained between 0.1 and 1.2 g/1, especially between 0.1 and 1.0 g/1 during the feeding course of the fermentation. In another embodiment of the present invention there is provided a method for preparing clavulanic acid by fermentation of a clavam-producing organism in a suitable medium characterized in that, besides phosphate and a nitrogen source, a suitable carbon source is added, continuously or intermittently, to the fermentation process whereby the level of carbon source is kept between 0.5 and 10 g/1 while that of nitrogen source is kept low (<100 mg/1) and that of phosphate between 0.1 and 1.2 g/1, especially between 0.1 and 1.0 g/1 during the feeding course of the fermentation. Clavulanic acid production in such a fermentation system was found to be appreciably high. From the encouraging results obtained from the experiments conducted in the cadre of the above invention, it can be envisaged that such a phosphate feeding strategy can also be imposed on a continuous culture process for the production of clavulanic acid. In a continuous culture process, complete medium composition is added to the culture while simultaneously the same volume that is fed to the culture is removed from the fermentor. In a classical continuous culture fermentation the volume in the fermentor remains almost constant throughout the course of the process. In a semi continuous process medium feed is added continuously while the removal of the culture from the fermentor is carried out intermittently, maintaining the volume constant. Therefore, a continuous cultivation process should be designed where the residual phosphate concentration in the culture is maintained between a range, say 0.1 to 1.2 g/1, especially 0.1 to 1.0 g/1. Such a continuous cultivation process may be either nitrogen- limited or carbon-limited. Of course, the recovery of the impure clavulanic acid solution as formed by the fermentative process of the present invention as well as the subsequent conversion thereof into a pharmaceutically acceptable salt by methods known in the art do form an aspect of the present invention. One of the most advantageous procedures is the conversion of the impure clavulanic acid into an amino salt thereof by adding the corresponding amino salt forming compound as for instance N,N,N' ,N' -tetramethylethylenediamine, l,3-bis(di- methylamino) -2-propanol , t-butylamine, t-octylamine, benzhydrylamine and bis (2- (dimethyl-amino) ethyl) ether and reacting said amine clavulanate with a non-toxic pharmaceutially acceptable salt as for instance postassium ethylhexanoate to form the corresponding purified salt, for instance potassium clavulanate. The following examples are only to be considered as illustration of the present invention. Example 1 Influence of phosphate on batch fermentation The following medium was prepared: Pharmamedia 25.0 g Soluble starch 20.0 g Bactopeptone 5.0 g Yeast extract 2.5 g Distilled water 1000 ml The pH was adjusted to 7.0 and 100 ml aliquots were placed in 500 ml Erlenmeyer flasks and sterilized (30 min. at 121°C) . The shake flasks were then seeded with a 1 ml vegetative suspension of S . clavuligerus NRRL 3585 (or mutants thereof) culture and incubated for 48 h at 27°C under constant orbital agitation. 100 ml of the culture was employed to inoculate the 6 1 of production medium in a 10 1 glass fermentor. The production medium had the following composition: Soybean meal 10 . 0 g Glycerol 30 . 0 g NZ-Amine ® 12 . 8 g Calcium chloride 0 . 2 g Iron sulphate 0 . 3 g Potassium phosphate (KH 2 P0 4 ) 0 . 7 - 3 . 5 g Magnesium sulphate 0 . 3 g Distilled water 1000 ml Antifoam f ew drops NZ-Amine ® has been obtained from Sheffield. The medium was sterilized at 121°C for 30 minutes, after adjusting the pH to 7.0 with NaOH and H 2 S0 4 . 100 ml of the shake flask culture was aseptically transferred to the glass fermentor and thereafter the fermentation started at a constant pH of 7.0. The fermentation was conducted at 28°C under constant stirring and optimal aeration conditions. The fermentation was terminated after glycerol was totally consumed from the culture (between 80 and 120 h) . Clavulanic acid was found to decrease significantly if the phosphate concentration in the production batch medium was increased above 3 g/1 or decreased below 0.5 g/1. The most optimum concentration is found to be strain dependent. Example 2 Maintaining phosphate level in the fed-batch fermentation The seed culture (as described in Example 1) was used to inoculate the production medium with the following composition: Glycerol 14 . 0 g NZ-Amine ® 7 . 5 g Calcium chloride 0 . 2 g Iron sulphate 0 . 2 g Potassium phosphate (KH 2 P0 4 ) 1 . 7 g Magnesium sulphate 0 . 4 g Trace element salt soln. 1 . 2 ml Distilled water 1000 ml Anti foam few drops After inoculation, the fermentation was started under constant agitation and optimal aeration. The fermentation was conducted at 28°C and pH 7.0. After approximately 48 h, two feed flows were started comprising of the following compositions : Feed I Glycerol 124.0 g Citric acid 8.0 g di-Potassium phosphate (K 2 HP0 4 ) 8.8 g Iron sulphate 1.1 g Calcium chloride 0.8 g Magnesium sulphate 1.5 g Trace element salt soln. 4.5 ml Distilled water 1000 ml Feed II Ammonia solution (25%) 66.3 g Distilled water 1000 ml The free residual phosphate concentration was regularly monitored and stringently maintained between 0.1 and 1.0 g/1 after the start of Feed I by appropriately adjusting the flow rate of Feed I . Residual glycerol level in the fermentation was also monitored and maintained between 2.0 and 20 g/1 during the course of the fermentation. Ammonia levels were stringently maintained below 100 mg/1 during the fermentation by appropriately adjusting the flow rate of Feed II. The fermentation was terminated after approximately 168 h. Significant high levels of clavulanic acid was obtained by employing this method.

Claims: Claims 1. A method for the production of clavulanic acid by the fermentation of a clavulanic acid producing microorganism in a suitable medium, characterized in that the conditions in the fermentor are such that the level of phosphate is controlled at the start of the fermentation and/or during the fermentation. 2. A method for the production of clavulanic acid according to claim 1, characterized by a batch fermentation with a starting concentration of phosphate of 0.5-2.5 g/1. 3. A method according to claim 2, characterized by a starting concentration of phosphate of 0.8-1.8 g/1. 4. A method according to claim 3, characterized by a starting concentration of phosphate of 1.0-1.5 g/1. 5. A method according to claim 1, characterized by a continuous fermentation whereby the level of phosphate during the fermentation is kept between 0.1 and 1.2 g/1. 6. A method according to claim 1, characterized by a fed-batch addition of phosphate wherein the level of phosphate in the fermentation broth is kept between 0.1 and 1.2 g/1. 7. A method according to claim 6 wherein the level of phosphate in the fermentation broth is kept between 0.5 and 1.2 g/1. 8. A method according to claim 7 wherein the level of phosphate in the fermentation broth is kept between 0.6 and 1.2 g/1. 9. A method according to claim 6, wherein the level of phosphate in the fermentation broth is kept between 0.2 - 0.8 g/1. s 10. A method according to anyone of claims 6 - 9, characterized by controlling the level of a suitable nitrogen source. 11. A method according to claim 10, characterized by a o fed-batch addition of the nitrogen source. 12. A method according to anyone of the claims 6 - 11, characterized by controlling the level of the carbon source. s 13. A method according to claim 12, characterized by fed-batch addition of the carbon source. 14. A method for the purification of clavulanic acid characterized by the isolation of impure clavulanic acid o from the fermentation broth obtained by the process of claims 1 - 13 and subsequent purification of said impure clavulanic acid. 15. A method according to claim 14, and subsequent 5 conversion to a salt thereof by converting the impure clavulanic acid into an amine salt thereof by adding the corresponding amine salt forming compound and reacting said amine clavulanate with a non-toxic pharmaceutically acceptable salt to form the corresponding purified salt of 0 clavulanic acid.

Inventors: GUPTA PRASHANT, GUPTA, PRASHANT
Applicants: GIST BROCADES BV, GUPTA PRASHANT, GIST-BROCADES B.V., GUPTA, PRASHANT
Application year: 1998
Main classification: C12P17
IPC codes: C12P17