Europäisches Patentamt *EP000803576B1* (19) European Patent Office Office européen des brevets (11) EP 0 803 576 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12P 17/02 of the grant of the patent: // (C12P17/02, C12R1:04) 04.12.2002 Bulletin 2002/49 (21) Application number: 97106445.6 (22) Date of filing: 18.04.1997 (54) Process for the production of lipstatin and tetrahydrolipstatin Verfahren zur Herstellung von Lipstatin und Tetrahydrolipstatin Procédé de préparation de lipstatine et de tétrahydrolipstatine (84) Designated Contracting States: • Weber, Wolfgang AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL 79639 Grenzach-Wyhlen (DE) PT SE (74) Representative: Witte, Hubert, Dr. et al (30) Priority: 26.04.1996 EP 96106598 124 Grenzacherstrasse 4070 Basel (CH) (43) Date of publication of application: 29.10.1997 Bulletin 1997/44 (56) References cited: EP-A- 0 129 748 (73) Proprietor: F. HOFFMANN-LA ROCHE AG 4070 Basel (CH) • CHEMICAL ABSTRACTS, vol. 107, no. 21, 23 November 1987 Columbus, Ohio, US; abstract (72) Inventors: no. 196439, WEIBEL, E. K. ET AL: "Lipstatin, an • Bacher, Adelbert inhibitor of pancreatic lipase, produced by 85748 Garching (DE) Streptomyces toxytricini. I. Producing • Stohler, Peter organism, fermentation isolation and biological 4415 Lausen (CH) activity" XP002094769 & J. ANTIBIOT. (1987), 40(8), 1081-5 CODEN: JANTAJ;ISSN: 0021-8820, Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 803 576 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 0 803 576 B1 2 Description uct, lipstatin, in a much higher concentration. [0006] The biosynthetic pathway leading to lipstatin is [0001] The invention relates to a novel fed-batch proc- subject to a series of general control mechanisms, such ess for the fermentative production of lipstatin, which as nitrogen repression. The addition of readily available process comprises 5 nitrogen sources, especially amino acids and their de- rivatives, including L-leucine or N-Formyl-L-leucine, to a) aerobically cultivating a micro-organism of the or- the medium strongly represses the formation of lipstatin. der of actinomycetes which produces lipstatin, in an In the present invention this is overcome by starting the aqueous culture medium which is substantially free addition of these amino acids only after the biosynthetic of fats and oils, and which contains suitable carbon 10 enzymes have been formed and thus are no longer re- and nitrogen sources and inorganic salts, until the pressed on a genetical level. In addition, L-leucine or N- initial growth phase is substantially finished and suf- formyl-L-leucine is added in such a way, that their con- ficient cell mass has been produced, and centration in the broth remains low. [0007] Conveniently, the actinomycete producing lip- b) adding to the broth linoleic acid, optionally togeth- 15 statin is grown in a suitable aqueous basal medium con- er with caprylic acid, [wherein part or the totality of taining one ore more carbon sources, such as starch, the linoleic acid and/or of the caprylic acid can be starch hydrolysates and sugars, e.g., glucose and/or su- replaced by the corresponding ester(s) and/or salt crose, glycerol, phospholipids, as well as one or more (s)], and N-formyl-L-leucine or preferably L-leucine, nitrogen sources, such as soybean flour, cotton seed the linoleic acid or its ester(s) or salt(s) being stabi- 20 flour, corn steep powder or corn steep liquor and yeast lized by an antioxidant. extract. Both carbon and nitrogen sources are supplied in such amounts that an abundant growth is enabled, [0002] Lipstatin, a fermentative process for its produc- typically in a range of 5 to 50 grams of each carbon tion, a process for its isolation from the broth and a proc- source and of each nitrogen source per liter of medium. ess for its hydrogenation to tetrahydrolipstatin (THL, or- 25 Further, macro- and micro-elements are added to the listat, an anti-obesity agent) are described in US Pat. medium. They might be contained in complex media 4,598,089. components or added as inorganic salts. [0003] The organism producing lipstatin as described [0008] The aqueous culture medium is substantially in US Pat. 4,598,089 is Streptomyces toxytricini Preo- free of fats and oils and contains less than 10 grams of brazhenskaya & Sveshnikova (see Bergey's Manual of 30 triglycerides per liter of medium. Conveniently, linoleic Determinative Bacteriology, 8th edition, page 811). It acid and/or caprylic acid and/or their esters or salts are was deposited on June 14, 1983, at the Agricultural Re- fed at such a rate as to be freely available in the broth search Culture Collection, Peoria, Ill. under the desig- but so that their accumulation is prevented, particularly nation NRRL 15443. The process of the instant inven- at a rate of 10 to 1000, preferably of 100 to 300 mg per tion can be performed with this organism or with any oth- 35 liter and hour. The feeding of linoleic acid and caprylic er strain derived of it, such as a mutant strain selected acid and/or their salts or of their esters is preferably con- for a better productivity. It can also be performed with ducted so that their concentration in the broth remains other lipstatin producing organisms of the order actino- inferior to 1000, preferably inferior to 300 mg per liter, mycetes, either belonging to the family of streptomyc- and discontinued so that the broth is practically free of etes or belonging to another family within the order of 40 said fatty acid(s) and/or its esters or salts before lipstatin actinomycetes. is isolated. Conveniently, the linoleic acid and caprylic [0004] When applying a fermentation system as de- acid are added to the broth in a ratio of 1 to 10, preferably scribed in US Patent 4,598,089, the fermentation broth 1.5 to 3 parts per weight of linoleic acid being added for after cultivation contains lipstatin in very small amounts 1 part of caprylic acid. Conveniently, N-formyl-L-leucine of a few milligram per liter and it is difficult to isolate it 45 or preferably L-leucine is added to the broth at a rate of by economically and technically feasible methods. 1 to 100, preferably 5 to 50 mg per liter of broth per hour, [0005] The present invention provides an improved so that its concentration remains less than 25 millimolar. process for the fermentative production of lipstatin, oc- [0009] Examples of salts and esters which can be curring in the fermentation broth with a higher concen- substituted for a part (or for the totality) of the linoleic tration by using the fed-batch process described above. 50 acid or of its mixture with caprylic acid are alkaline or In the first step a) of this process the cells of the lipstatin alkaline earth metal salts, e.g. sodium, potassium, cal- producing micro-organism are grown in a basal medi- cium or magnesium salts, and lower alkyl esters, e.g. um. In the second step b) of this process, to this basal methyl esters, or glycerides. medium certain components are added, which either [0010] In order to prevent the oxidation of linoleic acid serve directly as biochemical precursors or undergo a 55 [or of its ester(s) or salt(s)] it is mixed with an antioxidant, short biochemical conversion and then serve as precur- such as ascorbyl palmitate, tocopherol, lecithin, or mix- sors of the biosynthetic pathway. By this system the mi- tures thereof, and/or a radical trapping agent, such as cro-organism is enabled to synthesise the desired prod- BHA (tert.-butyl-4-hydroxy-anisol) or BHT (2,6-ditert.- 2 3 EP 0 803 576 B1 4 butyl-p-cresol). of this medium is filled into a 500 ml Erlenmeyer [0011] The invention further relates to a process for flask, closed with a cotton plug and sterilised. It is the production of tetrahydrolipstatin, which process then inoculated with a loopful of spores of Strepto- comprises myces toxytricini strain NRRL 15443 and subse- 5 quently incubated under shaking at 27°C for 24 a) aerobically cultivating a micro-organism of the or- hours. der of actinomycetes which produces lipstatin, in an aqueous culture medium which is substantially free b) 100 ml of this seed culture is used to inoculate a of fats and oils, and which contains suitable carbon fermentor with a vessel size of 14 l containing 8 l of and nitrogen sources and inorganic salts, until the 10 a production medium containing per liter: 32 g of initial growth phase is substantially finished and suf- defatted soybean flour, 20 g of glycerol, 14 g of lec- ficient cell mass has been produced, ithin, 0.25 ml of polypropylene glycol as an antifoam agent, whereas the pH is adjusted to 7.4 with NaOH b) adding to the broth linoleic acid, preferably to- 28 %. The medium contains less than 5 grams per gether with caprylic acid, [wherein part or the totality 15 liter of triglycerides. of the linoleic acid and/or of the caprylic acid can be replaced by the corresponding ester(s) and/or salt c) After a growth phase of 47 hours the feeding is (s)], and N-formyl-L-leucine or preferably L-leucine, started.