FERMENTATION PROCESS TO PRODUCE CLAVULANIC ACID AT A LOW CONCENTRATION OF FREE AMINO ACIDS

Abstract: An improved method for the production of secondary metabolites by the fermentation of a <u>Streptomyces</u> strain has been disclosed. If the concentration of free amino acids is kept low, a surprisingly high yield is obtained.

Description: FERMENTATION PROCESS TO PRODUCE CLAVULANIC ACID AT A LOW CONCENTRAΗON OF FREE AMINO ACIDS Field of the invention The present invention relates to the field of the fe'mentative production of secondary metabolites from a Streptomyces strain on a 10 suitable medium comprising carbon and nitrogen sources. Background of the invention A large variety of different secondary metabolites is produced 15 fermentatively from Streptomyces microorganisms, as for instance β- lactams, polyketides and macrolides, such as clavulanic acid, pimaricine and erythromycine. Clavulanic acid is an important inhibitor of β- lactamases and is produced by various microbial strains belonging to the genus of Streptomycetes such as 5. clavuligerus ATCC 27064, S. 20 jumonjinensis (GB patent 1 5631 03), S. katsurahamanus IFO 1 371 6 FERM 3944 (JP patent 83009679B) and Streptomyces sp. P6621 FERM 2804 (JP patent application 551 62993A). Clavulanic acid can be produced in substantial amounts and production conditions are being optimized continuously in order to 25 increase the yield and the purity of the end product. The production of clavulanic acid has been optimized with respect to continuous fermentation (GB 1 508977), feeding of carbon to a batch process (EP- 1 82522), maintaining ammonia at low concentrations (WO 96/1 8743 and Romero J., Liras P. and Martin J.F., Appl. Microbiol. Biotechnol. ( 1 984), 30 Vol. 20, 31 8-325), reduction of phosphate concentration in fermentation media during growth and production (Romero ( 1 984), v.s., Lebrihi A., Germain P. and Lefebre G. ; Applied Microbiol. Biotechnology. ( 1 987), Vol. 26, 1 30-1 35 and International patent applications WO 97/1 91 87 a§d WO 97/391 37). With respect to carbon sources it was mentioned that triglycerides are the preferred carbon sources compared to glycerol (Butterworth (1 984); Biotechnology of Industrial antibiotics, E.J. Vandamme, 1 st edition, Marcel Dekker Inc. pages 225-235 and the patent application WO 97/1 91 87) . With respect to the nitrogen source a lot of research has been done by Brana A.F., Paiva N. and Demain A.L., JL of General Microbiology (1 986), Vol 1 32, 1 305-1 31 7. It was found that growth on media containing an amino acid is faster than growth on ammonia as sole nitrogen source. Furthermore, on a medium containing ammonia as the sole nitrogen source, the maximum specific growth rate is < 0.05 h "1 , Brana ( 1 986) v.s., and Aharonowitz Y. and Demain A.L., Can J. Microbiol. (1 979), Vol. 25, 61 -67, while the maximum specific growth rate is > 0.05 h "1 in media containing at least one amino acid like asparagine, aspartate, giutamate, glutamine, alanine, histidine, proline, threonine or arginine. Therefore, one would prefer the use of a medium containing one or more amino acids. In such media, the production of biomass takes less time compared to inorganic media containing only ammonia which is of course an advantage over a slower production process. Asparagine is described in the non-prepublished International application WO 98/371 79 as fermentation ingredient for the production of clavulanic acid and also for cephalosporin, e.g. cephamycin C production from Streptomyces clavuligerus (Aharonowitz Y. and Demain A.L., v.s.) . Besides this, giutamate was shown to repress clavulanic acid formation in a batch process (Romero (1 984), v.s.), one would not be directed to use giutamate or glutamine. Furthermore it is even more common to use proteins as source of amino acids as this is much cheaper than the individual ones. The disadvantage is obviously the heterogeneity of proteins and the irreproducible quality generally associated with these complex nutrients. For clavulanic acid production typical complex nitrogen sources applied are: fishmeal, soybean meal, peanut meal for S. clavuligerus (EP 0 1 82 522 B1 and WO 97/1 91 87), soybean meal for S. jumonjinensis (UK patent 1 5631 03), soybean meal and corn steep liquor for Streptomyces sp. P6621 FERM 2804 (WO 97/391 37 + JP patent application 551 62993), and soybean flour and cotton seed flour for S. katsurahamanus (JP patent 83009679B) . In an overview article it was reported that soybean protein was the most important protein for clavulanic acid production (Butterworth ( 1 984), v.s.) . Here we describe the surprisingly advantageous application of hydrolysed proteins preferably rich in giutamate and proline in the fermentation broth of a secondary metabolite producing Streptomyces and the particular advantage of gluten hydrolysate and casein hydrolysate in this respect. Moreover we describe the unexpected advantageous application of a source of amino acids, in particular giutamate as a feeding nutrient in a fed batch process for the production of these compounds. Description of the Figures Figure 1 : titre of clavulanic acid during a batch fermentation at low levels of free giutamate. Figure 2: titre of clavulanic acid during a fed batch fermentation at low levels of free giutamate. Summary of the invention The present invention provides a method for the production of secondary metabolites by the fermentation of such a secondary metabolite producing Streptomyces on a suitable medium by keeping the concentration of free amino acids lower than 5 g/l fermentation broth, preferably lower than 2.5 g/l and more preferably lower than 0.5 g/l, provided that a concentration of 4 g/l free asparagine supplied Jo a fermentation broth of Streptomyces clavuligerus has been excluded. This process is especially favourable for the production of -lactams, polyketides and macrolides, preferably clavulanic acid, pimaricine, erythromycine, nystatine or amphotericine. According to one aspect of the invention to maintain such low concentrations in the fermentation broth, one or more of the amino acids, preferably giutamate or proline. is applied in a fed-batch or continuous mode. According to another aspect of the invention, besides amino acids itselves, also protein hydrolysate, more preferably glutenhydrolysate or caseinhydrolysate and most preferably wheat glutenhydrolysate is applied in a batch, fed-batch, semi-continuous or continuous mode. Description of the invention The present invention describes the use of media poor in free amino acids, by the application of said amino acids in a fed batch or continuous mode or by the application of protein hydrolysate in any mode. For the present patent application a protein is defined as a polymer of amino acids with a size expressed by a Molecular Weight > 20,000 Dalton which has not been processed by any means to degrade the protein to smaller fragments. A protein hydrolysate is defined to be a polymer of amino acids with an average size between 300 Dalton and 20,000 Dalton and a free amino acid content of less than 30% of the total amino acids. These protein hydrolysates can be produced either by enzymatic or by chemical hydrolysis of the corresponding proteins. Alternatively the advantage of using hydrolyzed proteins is achieved by using strains improved for protease activity in a process using non- hydrolyzed proteins as raw materials. Furthermore, giutamate stands in the present application for glutam like compounds as giutamate and glutamine. In the present application a protein extract is defined to be a protein hydrolysate with a molecular weight lower than 300 Daitorx and wherein > 30% of the amino acids are present as free amino acids. When a protein or the hydrolysate thereof is defined as rich in giutamate it is meant that > 1 5% of the amino acid content consists of giutamate and glutamine. Proteins described as rich in giutamate are casein (21 %) and wheat gluten (35%). According to one aspect of the present invention it was surprisingly found that clavulanic acid production was especially high when a protein hydrolysate was included in the medium, especially when it was derived from wheat gluten or casein. As the production levels were reduced dramatically when protein extracts were used of other protein sources like yeast and corn, especially at high hydrolysis degree ( > 35%) compared to the peptides ( < 30% free amino acids), it is shown that protein hydrolysates should preferably contain less than 30% free amino acids, even more preferably less than 5 % and most preferably less than 1 % free amino acids. A protein hydrolysate preferably rich in giutamate can be used as well supplied in all forms of feeding, viz. batch, fed batch, continuous or semi-continuous. By semi-continuous feeding is meant the continuous addition of nutrients to the fermentation broth while intermittently a small volume of the broth is removed. The fermentation medium may either be a defined medium comprising (NH 4 ) 2 SO 4 , free amino acid, KH 2 PO 4 , MgSO 4 .7H 2 O, CaCI 2 .2H 2 O, 3-(N-morpholino), propanesulfonic acid, glycerol, sodium succinate and a solution of trace elements, with a low concentration of amino acid, or with a protein hydrolysate which results in a complex medium. Also the application of a complex medium as for instance flours from nuts, vegetables, seeds, cereals, grasses such as those useful in fermentation industry, soybean flour, lineseed flour, peanut flour, potato flour, sunflower, pea- or beanflour, cotton seed flour, wheat gluten, whole wheat, rice meal to which protein hydrolysate is added, wherein the concentration of free amino acid is low, is part of the invention. The medium may also contain mixtures of mentioned flours with mixtuRes of protein hydrolysates of various sources and of various peptide sizes as desired to achieve the optimal result. Besides the wild type strains, also microbial strains which are capable of being fermented in a chemically defined medium and/or improved by subjecting a parent strain of interest to a classical mutagenic treatment using physical means, such as UV irradiation, or a suitable chemical mutagen, such as N-methyl-N'-nitro-N-nitrosoguanidine or ethylmethane sulfonate may be used for the process of the present invention. The same does apply to a parent strain of interest to recombinant DNA technology, whereby the parent strain is transformed with a one or more functional genes of interest. Any assimilable carbon source may be added to the above said mixture, like sugars such as glucose, fructose, sucrose, maltose, lactose, or polysaccarides like starch, maltodextrines and inuline or other fructose polymers, triglycerides such as soybean oil, sunflour oil, olive oil, tri-oleate etc., (poly-) alcohols such as ethanol, propanol, glycerol, mannitol, or organic acids or a salt thereof such as acetate, propionate, succinate, adipate, malonate, citrate, lactate, gluconate etc. An inorganic nitrogen source may be added to the medium such as ammonia and/or nitrate or any of its salts. Ureum may be used as well. Furthermore also vitamins, and various sorts of inorganic anions such as sulphates, phosphates, chlorides, borates, molybdate, iodate or their salts and the cations potassium, sodium, zinc, manganese, magnesium, iron, copper, cobalt, nickel etc. may be added to the medium. A fermentation is started by inoculating from a preculture or inoculum fermentation at a volume of about 1 to 50% of the main fermentation medium, particularly from 5 to 20%. The process may last from about 24 to 400 hours and especially from 48 to 1 68 hours. The temperature will be kept between 20 and 40 °C, preferably between 25 and 35 ° C, and even more preferably between 26 and 30 ° C. The pH can be maintained at pH 6 to 8 by means of titration with an alkaline substance such as ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or an organic base like lysine, arginine and histidine and an acid substance, such as the anorganic acids like sulphuric acid and hydrochloric acid. Alternatively, an organic acid may be used such as giutamate, citrate, gluconate or acetate. The dissolved oxygen concentration may be controlled in the optimal range for the process by varying the oxygen concentration in the inlet gas, application of overpressure, modification of stirrerspeed and airflow. The range may vary between 0 and 1 00% of air saturation. The process may be carried out by controlling various non-growth limiting nutrients in their optimal concentrations. Dependent on the growth limiting nutrient of choice, these growth-non-limiting nutrients may contain any relevant carbon, nitrogen, phosphor, sulphur source or oxygen. Carbon dioxide should be kept at non-toxic concentrations by for instance increasing the airflow through the fermentor so that the carbon dioxide concentration in the outlet-gas is less than 5%, preferably less than 2.5%. The fermentation can be carried out in a batch, fed batch, or (semi-) continuous fermentation process mode. 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, 1 ,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 potassium ethylhexanoaie to form the corresponding purified salt, for instance potassium clavulanate. Example 1 Batch fermentations of clavulanic acid comparing the use of proteins, protein hydrolysates and protein extracts. Streptomyces clavuligerus ATCC27064 was improved for clavulanic acid production by means of several rounds of classic mutation (UV, nitroso guanidine (NTG)) and selection in shake flask cultures whereby clavulanic acid production was tested by imidazole methods as known in the art. The strain was conserved as vegetative mycelium grown for 48 hours in Tryptone Soytone Broth-medium (TSB-medium) at 28 ° C in a shaker incubator shaken at 280 rpm and stored frozen at -80 °C. 1 ml of the frozen mycelium was inoculated to 1 00 ml of a sterilized (30 minutes, 1 21 °C) preculture medium containing 5-20 g/l maltose.1 aq, 1 5-30 g/l bacto tryptone, 1 5-30 g/l bacto peptone, 1 -1 0 g/l bacto soytone, mono potassium phosphate 1 -5 g/l and 0.2 g/l synthetic antifoam. After 72 hours of cultivation at 27 to 28 °C, 2.5% of this preculture is transferred to a sterile production medium containing 2.5 g/l from a complex nitrogen source such as a protein, a protein hydrolysate and/or or protein extract. The production medium further contains 50- 1 00 g/l glycerol, 5-20 g/l soybean flour, 0.5-2 g/l mono potassium phosphate, a suitable trace element cocktail and 0.2 - 2 g/l synthetic antifoam. After 4 days of cultivation at 28 °C and adequate shaking, the cultures were harvested and assayed for clavulanic acid by means of standard HPLC- methods. When different nitrogen sources were compared in a batch process, it was surprisingly found that the application of protein hydrolysates increased the production of clavulanic acid with at least 1 0% compared to the use of proteins. The best protein hydrolysates were those derived from gluten, followed by casein- and soy-proteins respectively (see table 1 ). Table 1 . Clavulanic acid production with a mutant strain from S. clavuligerus ATCC 27064 using different complex nitrogen sources additional to soybean flour. Example 2 Fermentation of clavulanic acid at low levels of free giutamate in the medium Batch fermentation of clavulanic acid Streptomyces clavuligerus ATCC 27064 was precultivated for 26 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM on a medium containing 5 to 30 g/l glycerol, 5 to 30 g/l soy peptone, 2 to 6 g/l sodium chloride, and 0.5 to 3 g/l calcium carbonate. The preculture was inoculated at a volume of 1 0 % into 1 I of chemically defined medium containing 1 0-30 g/l glycerol, 0.5 to 3 g/l KH 2 PO 4 , 1 to 3 g/l (NH 4 ) 2 SO 4 , 1 5-25 g/l monosodium giutamate, 0.05 to 0.2 g/l FeSO 4 -7 H 2 O, 0.1 to 1 g/l MgSO 4 -7 H 2 O, 1 0-20 g/l 2-(N- morpholine) propane sulfonic acid (MOPS), 0.2-1 g/l basildon antifoam, and a suitable trace element solution. The pH was adjusted to 6.8 with 4N NaOH. The second preculture was cultivated for 20 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM. The second- preculture was inoculated at a volume of 3.3 % into 29 I of a chemically defined medium containing 1 0 to 30 g/l glycerol, 0.5 to 1 g/l KH 2 PO 4 , 0.5 to 3 g/l (NH 4 ) 2 SO 4 , 1 0-30 g/l monosodium giutamate, 0.05-0.1 5 g/l FeSO 4 -7 H 2 O, 0.1 to 1 g/l MgSO 4 -7 H 2 O, 0.1 to 1 g/l basildon antifoam, and a suitable trace elements solution. The fermentation was carried out at 30°C and the pH was controlled at 6.95 to 7.05 by titration with 4N NaOH and 4N H 2 SO 4 . The dissolved oxygen tension was maintained above 50 % of air saturation or regulated at 50 % of air saturation by the stirrer speed. Fed-batch fermentation of clavulanic acid Streptomyces clavuligerus ATCC 27064 was precultivated for 26 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM on a medium containing 5 to 30 g/l glycerol, 5 to 30 g/l soy peptone, 2 to 6 g/l sodium chloride, and 0.5 to 3 g/l calcium carbonate. The preculture was inoculated at a volume of 1 0 % into 1 I of chemically defined medium containing 1 0-30 g/l glycerol, 0.5 to 3 g/l KH 2 PO 4 , 1 to 3 g/l (NH 4 ) 2 SO 4 , 1 5-25 g/l monosodium giutamate, 0.05 to 0.2 g/l FeSO 4 -7 H 2 O, 0.1 to 1 g/l MgSO 4 -7 H 2 O, 1 0-20 g/l 2-(N- morpholine) propane sulfonic acid (MOPS), 0.2-1 g/l basildon antifoam, and a suitable trace element solution. The pH was adjusted to 6.8 with 4N NaOH. The second preculture was cultivated for 20 h at a temperature of 28°C and a start pH of 6.8 in a shaken incubator rotated at 220 RPM. The second preculture was inoculated at a volume of 4 % into 24 I of a chemically defined medium containing 5 to 1 5 g/l glycerol, 0.5-2 g/l K 2 HPO 4 , 0.5 to 3 g/l (NH 4 ) 2 SO 4 , 1 0 g/l monosodium giutamate, 0.05 to 0.1 5 g/l FeSO 4 .7H2O, 0.1 to 1 g/l MgS0 4 7H 2 O, 0.05 to 1 g/l basildon antifoam, and a suitable trace elements solution. The fermentation was carried out at 30°C and the pH was controlled at 6.95 to 7.05 by titration with 4N NaOH and 4N H 2 SO 4 . The dissolved oxygen tension was maintained above 50 % of air saturation or regulated at 50 % of air saturation by the stirrer speed. Five hours after the phosphate was exhausted from the batch medium a carbon feed containing 300 to 500 g glycerol/kg feed was added at a rate of 30-60 g/h and a nitrogen-phosphate-feed containing 50 to 1 00 g Na-glutamate/kg feed, 5 to 10 g (NH 4 ) 2 SO 4 / kg feed, and 5 to 1 0 g K 2 HPO 4 /kg feed was added at a rate of 50 to 1 50 g/h. Clavulanic acid and giutamate concentrations in the medium are demonstrated in figures 1 and 2 respectively for the batch process and the fed batch process. From figure 1 it can seen that clavulanic acid production starts when the giutamate concentration is dropping below 5 and even more preferably below 2.5 g/l and ends at 250-300 mg clavulanic acid /liter. Figure 2 shows that when giutamate is fed to the fermentor keeping the concentration very low ( < 1 g/L) after 30 hour^. clavulanic acid titers can increase to 500 mg/L in this experiment, giving a doubling compared to the batch experiment.

Claims: CLAIMS 1 . A method for the production of a secondary metabolite by the fermentation of a Streptomyces strain on a suitable medium comprising carbon and nitrogen sources, wherein the concentration of free amino acids, supplied to the secondary metabolite producing Streptomyces, is kept lower than 5 g/l fermentation broth, more preferably lower than 2.5 g/l and most preferably lower than 0.5 g/l with the proviso that a concentration of free asparagine of 4 g/l fermentation broth of Streptomyces clavuligerus has been excluded. 2. A method according to claim 1 wherein the secondary metabolite produced is a Mactam, polyketide or macrolide. 3. A method according to claim 2 wherein the secondary metabolite produced is clavulanic acid, pimaricine, erythromicine, nystatine or amphotericine. 4. A method according to claim 1 - 3 characterized by the application of proteinhydrolysate as a source of free amino acids, in a batch, fed-batch, semi-continuous or continuous mode. 5. A method according to claim 4 wherein the proteinhydrolysate is formed by the addition of proteins to a strain with an increased protease activity compared to the corresponding wild type strain. 6. A method according to claim 4 or 5, characterized by the application of a proteinhydrolysate rich in anyone of giutamate or proline. 7. A method according to claim 6, characterized by the application of glutenhydrolysate or caseinhydrolysate. 8. A method according to claim 7, characterized by the application of wheat glutenhydrolysate. 9. A method according to anyone of the claims 1 - 3, characterized by the application of free amino acids in a fed-batch, semi- continuous or continuous mode. 1 0. A method according to claim 9, characterized by the application of giutamate or proline. 1 1 . A method according to anyone of the claims 1 - 1 0, characterized by the production of clavulanic acid by the fermentation of Streptomyces clavuligerus. 1 2. A method for the production of clavulanic acid by the fermentation of Streptomyces clavuligerus on a suitable medium, characterized by the application of glutenhydrolysate or caseinhydrolysate. 1 3. A method for the further purification of clavulanic acid characterized by the purification of clavulanic acid obtained by the processes of claims 1 1 or 1 2. 1 4. Process for the purification of clavulanic acid according to claim 1 3 and subsequent conversion to a salt thereof characterized 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 clavulanic acid.

Inventors: DE LAAT WILHELMUS THEODORUS AN, KRABBEN PREBEN, DE LAAT, WILHELMUS, THEODORUS, ANTONIUS, MARIA, KRABBEN, PREBEN
Applicants: DSM NV, KRABBEN PREBEN, LAAT WILHELMUS THEODORUS ANTON, DE LAAT, WILHELMUS, THEODORUS, ANTONIUS, MARIA, DSM N.V., KRABBEN, PREBEN
Application year: 1999
Main classification: C12P17
IPC codes: C12P17