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 lip- statin, 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. CM < CO r»- LO CO o CO o Q_ LU Printed by Rank Xerox (UK) Business Services 2.14.18/3.4 1 EP 0 803 576 A2 2 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 com- prises 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.