Process for the Production of Lipstatin and Tetrahydrolipstatin

Europaisches Patentamt (19) European Patent Office

Office europeen des brevets (11) EP 0 803 576 A2

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) IntCI.6: C12P 17/02 29.10.1997 Bulletin 1997/44 //(C12P17/02, C12R1:04) (21) Application number: 97106445.6

(22) Date of filing: 18.04.1997

(84) Designated Contracting States: • Stohler, Peter AT BE CH DE DK ES Fl FR GB GR IE IT LI LU NL 44l5Lausen(CH) PTSE • Weber, Wolfgang 79639 Grenzach-Wyhlen (DE) (30) Priority: 26.04.1996 EP 96106598 (74) Representative: Mane, Jean et al (71) Applicant: F. HOFFMANN-LA ROCHE AG F.Hoffmann-La Roche AG 4070 Basel (CH) Patent Department (PLP), 1 24 Grenzacherstrasse (72) Inventors: 4070 Basel (CH) • Bacher, Adelbert 85748 Garching (DE)

(54) Process for the production of lipstatin and tetrahydrolipstatin

(57) A process for the fermentative production of lipstatin, which process comprises

a) aerobically cultivating a micro-organism of the order of actinomycetes which produces lipstatin, in an aqueous culture medium which is substantially free of fats and oils, and which contains suitable carbon and nitrogen sources and inorganic salts, until the initial growth phase is substantially finished and sufficient cell mass has been produced, and

b) adding to the broth linoleic acid, optionally together with caprylic acid, [wherein part or the totality of the linoleic acid and/or of the caprylic acid can be replaced by the corresponding ester(s) and/or salt(s)], and N-formyl-L-leucine or preferably L-leucine, the linoleic acid or its ester(s) or salt(s) being stabilized by an antioxidant.

Lipstatin can be isolated from the broth and hydro- genated to tetrahydrolipstatin.

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Description tion. The biosynthetic pathway leading to lipstatin is sub- The invention relates to a novel fed-batch process ject to a series of general control mechanisms, such as for the fermentative production of lipstatin, which proc- nitrogen repression. The addition of readily available ess comprises 5 nitrogen sources, especially amino acids and their derivatives, including L-leucine or N-Formyl-L-leucine, a) aerobically cultivating a micro-organism of the to the medium strongly represses the formation of lip- order of actinomycetes which produces lipstatin, in statin. In the present invention this is overcome by start- an aqueous culture medium which is substantially ing the addition of these amino acids only after the free of fats and oils, and which contains suitable 10 biosynthetic enzymes have been formed and thus are carbon and nitrogen sources and inorganic salts, no longer repressed on a genetical level. In addition, L- until the initial growth phase is substantially finished leucine or N-formyl-L-leucine is added in such a way, and sufficient cell mass has been produced, and that their concentration in the broth remains low. Conveniently, the actinomycete producing lipstatin b) adding to the broth linoleic acid, optionally 15 is grown in a suitable aqueous basal medium containing together with caprylic acid, [wherein part or the one ore more carbon sources, such as starch, starch totality of the linoleic acid and/or of the caprylic acid hydrolysates and sugars, e.g., glucose and/or sucrose, can be replaced by the corresponding ester(s) glycerol, phospholipids, as well as one or more nitrogen and/or salt(s)], and N-formyl-L-leucine or preferably sources, such as soybean flour, cotton seed flour, corn L-leucine, the linoleic acid or its ester(s) or salt(s) 20 steep powder or corn steep liquor and yeast extract. being stabilized by an antioxidant. Both carbon and nitrogen sources are supplied in such amounts that an abundant growth is enabled, typically in Lipstatin, a fermentative process for its production, a range of 5 to 50 grams of each carbon source and of a process for its isolation from the broth and a process each nitrogen source per liter of medium. Further, for its hydrogenation to tetrahydrolipstatin (THL, orlistat, 25 macro- and micro-elements are added to the medium. an anti-obesity agent) are described in US Pat. They might be contained in complex media components 4,598,089. or added as inorganic salts. The organism producing lipstatin as described in The aqueous culture medium is substantially free of US Pat. 4,598,089 is Streptomyces toxytricini Preo- fats and oils and contains less than 10 grams of triglyc- brazhenskaya & Sveshnikova (see Bergey's Manual of 30 erides per liter of medium. Conveniently, linoleic acid Determinative Bacteriology, 8th edition, page 811). It and/or caprylic acid and/or their esters or salts are fed at was deposited on June 14, 1983, at the Agricultural such a rate as to be freely available in the broth but so Research Culture Collection, Peoria, III. under the des- that their accumulation is prevented, particularly at a ignation NRRL 15443. The process of the instant inven- rate of 10 to 1000, preferably of 100 to 300 mg per liter tion can be performed with this organism or with any 35 and hour. The feeding of linoleic acid and caprylic acid other strain derived of it, such as a mutant strain and/or their salts or of their esters is preferably con- selected for a better productivity. It can also be per- ducted so that their concentration in the broth remains formed with other lipstatin producing organisms of the inferior to 1000, preferably inferior to 300 mg per liter, order actinomycetes, either belonging to the family of and discontinued so that the broth is practically free of streptomycetes or belonging to another family within the 40 said fatty acid(s) and/or its esters or salts before lipstatin order of actinomycetes. is isolated. Conveniently, the linoleic acid and caprylic When applying a fermentation system as described acid are added to the broth in a ratio of 1 to 10, prefera- in US Patent 4,598,089, the fermentation broth after cul- bly 1 .5 to 3 parts per weight of linoleic acid being added tivation contains lipstatin in very small amounts of a few for 1 part of caprylic acid. Conveniently, N-formyl-L-leu- milligram per liter and it is difficult to isolate it by eco- 45 cine or preferably L-leucine is added to the broth at a nomically and technically feasible methods. rate of 1 to 100, preferably 5 to 50 mg per liter of broth The present invention provides an improved proc- per hour, so that its concentration remains less than 25 ess for the fermentative production of lipstatin, occur- millimolar. ring in the fermentation broth with a higher Examples of salts and esters which can be substi- concentration by using the fed-batch process described 50 tuted for a part (or for the totality) of the linoleic acid or above. In the first step a) of this process the cells of the of its mixture with caprylic acid are alkaline or alkaline lipstatin producing micro-organism are grown in a basal earth metal salts, e.g. sodium, potassium, calcium or medium. In the second step b) of this process, to this magnesium salts, and lower alkyl esters, e.g. methyl basal medium certain components are added, which esters, or glycerides. either serve directly as biochemical precursors or 55 In order to prevent the oxidation of linoleic acid [or undergo a short biochemical conversion and then serve of its ester(s) or salt(s)] it is mixed with an antioxidant, as precursors of the biosynthetic pathway. By this sys- such as ascorbyl palmitate, tocopherol, lecithin, or mix- tem the micro-organism is enabled to synthesise the tures thereof, and/or a radical trapping agent, such as desired product, lipstatin, in a much higher concentra- BHA (tert.-butyl-4-hydroxy-anisol) or BHT (2,6-ditert.-

2 3 EP 0 803 576 A2 4 butyl -p-cresol). toxytricini strain NRRL 15443 and subsequently The invention further relates to a process for the incubated under shaking at 27°C for 24 hours. production of tetrahydrolipstatin, which process comprises b) 1 00 ml of this seed culture is used to inoculate a 5 fermentor with a vessel size of 1 4 I containing 8 I of a) aerobically cultivating a micro-organism of the a production medium containing per liter: 32 g of order of actinomycetes which produces lipstatin, in defatted soybean flour, 20 g of glycerol, 14 g of lec- an aqueous culture medium which is substantially ithin, 0.25 ml of polypropylene glycol as an antifoam free of fats and oils, and which contains suitable agent, whereas the pH is adjusted to 7.4 with NaOH carbon and nitrogen sources and inorganic salts, 10 28 %. The medium contains less than 5 grams per until the initial growth phase is substantially finished liter of triglycerides. and sufficient cell mass has been produced, c) After a growth phase of 47 hours the feeding is b) adding to the broth linoleic acid, preferably started. It consists of the fatty acids linoleic acid together with caprylic acid, [wherein part or the 15 and caprylic acid, whereby linoleic acid is stabilized totality of the linoleic acid and/or of the caprylic acid by the addition of 0.2 % (w/w) of an antioxidant can be replaced by the corresponding ester(s) (RONOXAN A) consisting of 70% (w/w) of lecithin, and/or salt(s)], and N-formyl-L-leucine or preferably 25% of ascorbyl palmitate and 5% of tocopherol. L-leucine, the linoleic acid or its ester(s) or salt(s) These fatty acids are added at a rate of 136 to 190 being stabilized by an antioxidant, 20 mg per liter and hour, and the rate of addition is adjusted in such a way that the concentration of c) isolating lipstatin from the broth and hydrogenat- each linoleic acid and caprylic acid remains below ing lipstatin to tetrahydrolipstatin. 70 mg per liter. Totally added in the course of the fermentation are 108 grams of linoleic acid and 54 The isolation of the lipstatin from the fermentation 25 g of caprylic acid. The L-leucine is added at a rate broth can be carried out according to methods which of 14.4 mg per liter and hour, as an aqueous solu- are known per se and which are familiar to any person tion containing 80 g of L-leucine per I of feed, the skilled in the art. For example, it can be carried out as pH being adjusted to 1 1 with NaOH 28 % . A total of follows: 10.2 grams of L-leucine is added. After completion of the fermentation, the fermenta- 30 tion broth is centrifuged. The resulting cell mass can The culture medium after seeding with the afore- then be treated with a lower alkanol such as methanol mentioned seed culture and while feeding with the or ethanol, and extracted with the same solvent. The aforementioned fatty acids and L-leucine is incubated centrifugate can be extracted with a suitable organic under stirring, and aeration at a rate of 4 I of air per solvent (e.g. with methylene chloride or ethyl acetate). 35 minute to keep the culture aerobic. The pH is main- The material produced from the extracts contains the tained in a range of 6.1 to 7.3 by the automated addition desired lipstatin and can be enriched and purified by of sulfuric acid or sodium hydroxide solution. The dis- chromatographic methods, e.g. as described in US Pat. solved oxygen concentration is prevented to be less 4,598,089. than 1 0% of the saturation concentration by adjusting The hydrogenation of lipstatin to tetrahydrolipstatin 40 the stirrer speed. At harvest time, the culture is practi- can be carried out according to methods which are cally free of linoleic acid and caprylic acid. The titer of known per se, e.g. as described in US Pat. 4598 089, in lipstatin, i.e. its concentration in the culture medium, is the presence of a suitable catalyst. Examples of cata- 1 50 mg/l after an incubation period of 1 38 hours. lysts which can be used are palladium/carbon, platinum oxide, palladium and the like. Suitable solvents are, for 45 Example 2 example, lower alcohols such as methanol and ethanol. The hydrogenation is preferably carried out at low The same seed culture as in Example 1a) above is hydrogen pressures and at room temperatures. used to inoculate a 14 I fermentor with a production medium as described in US Pat. 4,598,089, namely the Example 1 so production medium N 7 in Example 1 thereof. This medium contains, per 8 liters: 80 g of potato starch, 40 a) A seed culture is prepared consisting of the fol- g of glucose, 80 g of ribose, 40 g of glycerol, 16 of pep- lowing pre-culture medium: 10 g of defatted soy tone, 1 60 g of soybean flour, 1 6 g of ammonium sulfate. flour, 10 g of glycerol, 5 g of yeast extract and water An antifoam agent (0.25 ml of polypropylene glycol pro to make 1 I. The pH is adjusted to 7.0 with NaOH 28 55 liter of medium) is added as in example 1b) above. The % , giving a pH of 6.8 after sterilisation. 100 ml of pH is adjusted to 7.0 with NaOH 28% before steriliza- this medium is filled into a 500 ml Erlenmeyer flask, tion. Incubation is carried out aerobically, while stirring closed with a cotton plug and sterilised. It is then at 400 rpm and with an aeration rate of 4 I of air per inoculated with a loopful of spores of Streptomyces minute.

3 5 EP 0 803 576 A2 6

After incubation, a concentration of lipstatin of less which process comprises than 10 mg/l was found in the culture medium. a) aerobically cultivating a micro-organism of Claims the order of actinomycetes which produces lip- 5 statin, in an aqueous culture medium which is 1 . A process for the fermentative production of lipsta- substantially free of fats and oils, and which tin, which process comprises contains suitable carbon and nitrogen sources and inorganic salts, until the initial growth a) aerobically cultivating a micro-organism of phase is substantially finished and sufficient the order of actinomycetes which produces lip- 10 cell mass has been produced, statin, in an aqueous culture medium which is substantially free of fats and oils, and which b) adding to the broth linoleic acid, preferably contains suitable carbon and nitrogen sources together with caprylic acid, [wherein part or the and inorganic salts, until the initial growth totality of the linoleic acid and/or of the caprylic phase is substantially finished and sufficient is acid can be replaced by the corresponding cell mass has been produced, and ester(s) and/or salt(s)], and N-formyl-L-leucine or preferably L-leucine, the linoleic acid or its b) adding to the broth linoleic acid, optionally ester(s) or salt(s) being stabilized by an antioxi- together with caprylic acid, [wherein part or the dant, totality of the linoleic acid and/or of the caprylic 20 acid can be replaced by the corresponding c) isolating lipstatin from the broth and hydro- ester(s) and/or salt(s)], and N-formyl-L-leucine genating lipstatin to tetrahydrolipstatin. or preferably L-leucine, the linoleic acid or its ester(s) or salt(s) being stabilized by an antioxidant. 25

2. A process as in claim 1 , wherein the micro-organism of the order of actinomycetes is of the family of streptomycetes, and the aqueous culture medium, substantially free of fats and oils, contains less than 30 10 grams of triglycerides per liter of medium.

3. A process as in claim 1 or 2, wherein the linoleic acid or its mixture with caprylic acid and/or their esters or salts are added at such a rate as to be 35 freely available in the broth but so that their accumulation is prevented, preferably at a rate of 10 to 1000, specially of 100 to 300 mg per liter and hour.

4. A process as in claim 3, wherein the addition of lino- 40 leic acid and caprylic acid and/or their salts or of their esters is conducted so that their concentration in the broth remains inferior to 1000, preferably inferior to 300 mg per liter, and discontinued so that the broth is practically free of said fatty acid(s) and/or 45 its esters or salts before lipstatin is isolated.

5. A process as in claim 3 or 4, wherein linoleic acid and caprylic acid are added to the broth in a ratio of 1 to 10, preferably 1 .5 to 3 parts per weight of lino- so leic acid being added for 1 part of caprylic acid.

6. A process as in claim 3, 4 or 5, wherein N-formyl-L- leucine or preferably L-leucine is added to the broth at a rate of 1 to 100, preferably 5 to 50 mg per liter 55 of broth per hour, so that its concentration remains less than 25 millimolar.

7. A process for the production of tetrahydrolipstatin,