WO2007078263A2

WO2007078263A2 - The strain streptomyces toxytricini lipstatin-producing microorganism and preparation of lipstatin with inscribed strain

Info

Publication number: WO2007078263A2
Application number: PCT/SK2006/050008
Authority: WO
Inventors: Ivan Varga; Blanka Gondova; Jana Gajdosikova; Ludmila Kovacova
Original assignee: Biotika A.S.
Priority date: 2006-01-03
Filing date: 2006-12-18
Publication date: 2007-07-12
Also published as: SK22006A3; WO2007078263A3

Abstract

Technical solution is related to industry production microorganism Streptomyces toxytricini CCM 7349 and method of lipstatin biotechnology preparation by this strain. Production strain listed above with use of two step vegetative inoculum produce lipstatin in concentration from 3,0 to 3,2 g/kg fermentation broth after 180 till 230 hours of cultivation under temperature at 28°C, under optimal mixing above 450 rpm, aeration 1 till 1,25 vvm and optimal addition of linoleic acid and 8% solution of L-Leucine.

Description

The strain Streptomyces toxytricini lipstatin-produdng microorganism and preparation of lipstatin with inscribed strain.

Technical field

Patent is related to the strain Streptomyces toxytricini stored in the Czek Collection of Microorganism in Brno under the number CCM 7349, and biotechnology preparation of lipstatin with inscribed strain in concentration 3,0 till 3,2 g/kg of fermentation broth after 180 till 230 hours of cultivation in production broth at 28⁰C.

Background art

The strain Streptomyces toxytricini CCM 7349 produce lipstatin during aerobically cultivating conditions. The basic building unit is β-laktam circle with two aliphatic rests of fatty acids with 6 - 14 carbon atoms.. One of the side chains contain two double bonds and one hydroxyl group esterified as N-Formyl-leucine. By catalitic hydrogenation of lipstatin is able to get tetrahydrolipstatin (orlistat). Microorganism Streptomyces toxytricini is able to utilized fats or fatty acids to acetyl-CoA. When during fermentation biosynthese of lipstatin occurs, condensation of C14 and C8 fatty acids is started which becomes from fats and long chain fatty acids. Linoleic acid contain the same number of double bonds as lipstatin chain and it is the basic building unit of lipstatin. Claisen condensation of 3-hydroxy- 5,8-tetradekadienoyl-CoA and oktanoyl-CoA is formed basic intermediate ester of 5-hydroxy-2-hexyl- 3-0X0-7,10-hexadekadien acid. In the next step take place catalytic hydrogenation of ester group, after which formation of β-laktam circle followes. Esterification of the side chain occurs in present of leucine.

Orlistat (tetrahydrolipstatin) molecular weight 495,74 g/mol, is active compound on reduction of body weight. It have effect on the place where fats split up eg. in stomach and small intestine. Its action pancreatic enzyme called lipase is stoped, inhibit absorbtion of fats from food till one third. This phenomenon is used in treatment of obesity, and can be used as prevention against hyperlipidemia and arteriosclerosis. Taxonomy of bacterial strain Streptomyces species discribes International Streptomyces Project in Journal of Fermentation Technology Vol2, p.52, 1974. The strain Streptomyces toxytricini Preobrazhenskaya & Sveshnikova is described in Bergey's Manual of Determinative Bacteriology, 8th Edition, page 811.

U. S. Patent 4,598,089 and Eur.Pat.Appl. 129748 describe the method of lipstatin and orlistat preparation by biotechnology way with specific strain Streptomyces toxytricini NRRL 15443. This patent describe preparation of two step vegetative inoculum which is necessary for seeding of production step.

Production medium contain mixture of potato starch, glucose, ribose and glycerine as main carbon sources. Pepton, soya flour and ammonium sulphate are mentioned as a sources of nitrogen. During production step cultivation temperature of cultivation is keeped at 28⁰C, aeration 1vvm and stirring 150 rpm. U. S. Patent under publication number US2002/0110873 A1 mention lipstatin production during conditions which are described in patent U. S. Patent 4,598,089 and with usage of the strain Streptomyces toxytricini NRRL 15443 with production of lipstatine 9106 mg/l after 184 hours of cultivation.

Weibel et al. describes fermentation process of lipstatin ( Journal of Antibiotics VoI XL, No.8 pp.1086 - 1091 ) a E.Hochuli describes chemical structure of lipstatin ( Journal of Antibiotics VoI XL,No.8 pp.1081 - 1085).

EP 0 803 567 A2 describes fermentation process of lipstatin preparation under help of precursors, which are linoleic acid, caprylic acid and N-formyl-L-leucine or L-Leucine. The yield of fermentation is low because of toxicity of the both acids. In comparison with U. S. Patent 6,844,174 use production medium containing free fatty acids and natural oils in fermentation process, synthetic oils or their mixture. As a results is liberation of fatty acids from oils during fermentation process in dependence on pH of production media and high residual concentration of oil at the end of fermentation. Folowing problem is question of emulsification of production media with suitable emulsifier such as Lecithin, Triton X-100, Brij 35 S. Disadvantage is, that by increasing of emulsifier, viscosity of production media is increased and transfer of oxygen into the fermentation broth is worst, which has negative influence on lipstatin production.

Patent under publication number WO 03/048335 describes lipstatin fermentation by flask cultivation in production media which contain soya flour and cotton flour, glycerine as carbon source, polypropylenglykol as antifoam agent and emulsifier lecithin. Fermentation takes 6 till 7 days with maximum production 1 ,700 till 1 ,800 mg/l. In middle-size 4 liters of fermentation media reach lipstatin production by the same technology 1 ,500 till 1 ,600 mg/l after 144 till 168 hours of cultivation.

Under publication number WO 2004/003212 A1 is described lipstatin preparation on different starch substrates with maximum lipstatin production 1486 mg/ kg of substrate.

Disclosure of the invention

Patent is related to the strain of microorganism Streptomyces toxytricini CCM 7349 and biotechnology lipstatin preparation without usage of emulsifier in production broth, and with usage of precursors, which are linoleic acid and L-Leucine. The strain Streptomyces toxytricini CCM 7349 produce lipstatin in concentration 3,0 till 3,2 g/kg of fermentation broth after 180 till 230 hours of cultivation in production media at 28⁰C under use of two step inoculum.

Cultivation of the strain Streptomyces toxytricini CCM 7349 being used in the fermentation process, which is related to the patent take place in four steps:

1. Sporulation of the culture

2. I. inoculation step

3. II. inoculation step

4. Production step Sporulation of the culture

For quality of inoculum is important good sporulation ( during 5 till 10 days at 28⁰C spores of culture of the strain occurs, colonies are in a shape of a circle slitly concave till wrinkly, aerial mycelium is brown-pink color with white zone at the periphery of the colony with size 5 till 10 mm). For sporulation of the culture is used sporulation broth with following composition: dextrine 4g/l, yeast extract 4g/l, malt extract 10 g/l, CaCO₃ 2 g/l, agar 20 g/l and destiled water to 1000 ml

I. inoculation step

Cultivation of the vegetative inoculum of I. inoculation step take place at 28⁰C 24 hours. Quality of inoculum is characterised by following properties:

- Small pelets, fibrous

- biomass 14 till 16%

Inoculation medium of the I. step contain only one carbon substrate with following composition: soya flour 10 g/l, glycerine 10 g/l, yeast extract 5 g/l, drinking water to 1000 ml.

II. inoculation step

Cultivation of the vegetative inoculum of II. inoculation step take place at 28⁰C 12 till 16 hours. Quality of inoculum is characterised by following properties:

- great pelets, fibrous

- biomass 20 az 26%

Inoculation medium of the II. step contain only one carbon substrate with following composition: soya flour 30 g/l, yeast extract 5g/l, glycerine 20 g/l, (NhU)₂SO₄ 2 g/l, CaCO₃ 6 g/l, antifoam Slovanik 1 g/l.

Production step a.) For testing and colonies selection of production strain Streptomyces toxytricini CCM 7349 with best production is used production medium with following composition: soya flour 48 g/l, glycerine 30 g/l, CaCO₃ 5g/l, yeast extract 1 g/l, metylester of soya bean oil 40 g/l, L-Leucine 5 g/l, MgSO₄.7H₂O 0,05 g/l, drinking water to 1000 ml.

Production medium contain as a precursor of lipstatin untoxic metylester of soya bean oil in comparison with linoleic acid, which concentration above 1 g/l affect toxical on production microorganism. Cultivation take place at temperature 28⁰C at 250 rpm 72 hours with lipstatin production 0,3 till 0,45 g/kg. b.) For middle-size preparation of lipstatin with production microorganism Streptomyces toxytricini CCM 7349 is used production medium with following composition: soya flour 48 g/l, glycerine 50 g/l, yeast extract 1 g/l, CaCO₃ 5 g/l, KH2PO4 0,1 g/l, MgSO₄JH₂O 0,3 g/l, FeSO₄JH₂O 0,03 g/l, MnSO₄.4H₂O 0,04 g/l, ZnSO₄JH₂O 0,005g/l, soya bean oil 40 g/l, antifoam Slovanik 1g/l. Cultivation take place in usage two step inoculum at cultivation temperature 28⁰C with lipstatin production 3,0 till 3,2 g/kg after 180 till 230 hours. The pH during cultivation is maintained in a range of 6 to 8, the optimal pH in a range of 6,7 to 7,3 by addition of 28% solution of sodium hydroxide or 20% solution of sulfuric acid.

During synthesis of lipstatin is used as a precursor linoleic acid with holding concentration 0,2 till 1 g/l of the fermentation broth together with 8% solution of L-Leucine with pH 11. The feeding of linoleic acid and L-Leucine is started at the end of the growth phase in 34. hour of cultivation, which is characterised by utilisation of start carbon source accompanied by increasing concentration of dissolved oxygen in fermentation broth. Linoleic acid and L-Leucine is added continuously in ratio 1 :0,8. The whole amount of added L-Leudne during cultivation was 3265 g on volume 40 I of fermentation broth.

Production medium use as a source of carbon substrate glycerine and soya bean oil, as a source of organic nitrogen and phosphate use soya flour and yeast extract. Advantage of the cultivation is, that is not neccessary to added emulsifiers into the fermentation broth as it is mentioned in U. S. Patent 6,844,174 for provide of viskosity. In middle-size production step with volume 40 I of production media, cultivation take place in 10 times higher volume as it is mentioned in WO 03/048335 with transfer characteristics aeration above 1vvm and stirring above 450 rpm, holding concentration of dissolved oxygen in fermentation broth is not less than 30%. Mentioned parameters are real achievable in big volume size.

Advantage of the cultivation with the strain Streptomyces toxytricini CCM 7349 is, that by elimination of emulsifier from basic composition of production media lead to improvement of oxygen transfer to the cells of production strain.

Description of the strain: The strain Streptomyces toxytricini CCM 7349

Morphology: - substrate mycellium of the strain Streptomyces toxytricini CCM 7349 on sporulation broth is darkbrown color, aerial mycellium is brown-pink color. Unit colonies of the strain Streptomyces toxytricini are circle, slitly concave till wrinkly, brown-pink color with white zone at the periphery of colony, with average 5 till 10 mm.

Optimal pH of the cultivation: from 6 to 8

Utilisation of carbon substrate: good utilisation of glycerine, glucose, dextrin, worse utilisation of saccharose, lactose

Optimal cultivation temperature: 28 ± 1⁰C

Precursors: Linoleic acid, L-Leucine, metylester of soya bean oil

Examples of embodiments

Example 1 a) Preparation of spore material

The spore suspension of the strain Streptomyces toxytricini is puted on the agar plate with composition: dextrin 4g/l, yeast extract 4g/l, malt extract 10 g/l, CaCO₃ 2 g/l, agar 20 g/l a destiled water into 1000 ml. The strain is cultivated 28⁰C, from 5 to 10 days.

b) inoculation step

The loop of spores of the strain Streptomyces toxytricini is used for seeding of 100 ml inoculation broth in 500 ml Erlenmayer flask. Composition of inoculation broth is : soya flour 10 g/l, glycerine 10 g/l, yeast extract 5 g/l, drinking water into 1000 ml. Optimal pH after sterilization 30 minutes 120 ⁰C is pH from 6,8 to 7,2. Inoculum is cultivated 28⁰C, from 24 to 26 hours. Prepared vegetative inoculum will be used for seeding of production step.

c) production step

Vegetative inoculum prepared in Erlenmayer flask is seeded in amount from 1% to 5% into 60 ml of production media in Erlenmayer flask with composition: soya flour 48 g/l, glycerine 30 g/l, CaCO₃ 5g/l, yeast extract 1 g/l, metylester of soya bean oil 40 g/l, L-Leucine 5 g/l, MgSO₄.7H₂O 0,05 g/l and drinking water into 1000 ml. Production media is sterilized 30 minutes, 12O⁰C. Optimal pH is from 6,8 to 7,4. After 72 hours of cultivation on rotary shaker with stirring from 180 to 250 rpm and temperature 28⁰C concentration of lipstatin is measured by HPLC method, its concentration occurred from 0,3 g/kg to 0,45 g/kg.

Example 2 a) Preparation of spore material

b) inoculation step

The loop of spores of the strain Streptomyces toxytricini is used for seeding of 100 ml inoculation broth in 500 ml Erlenmayer flask. Composition of inoculation broth is : soya flour 10 g/l, glycerine 10 g/l, yeast extract 5 g/l, drinking water into 1000 ml. Optimal pH after sterilization 30 minutes 120 ⁰C is pH from 6,8 to 7,5. Inoculum is cultivated 28⁰C, from 24 to 26 hours. Prepared vegetative inoculum will be used for seeding of production step.

c) production step

Vegetative inoculum prepared in Erlenmayer flask is seeded in amount from 1% to 5% into production step in 70 I fermenter with volume 40 I production broth with composition: soya flour 48 g/l, glycerine 50 g/l, yeast extract 1 g/l, CaCO₃ 5 g/l, KH2PO4 0,1 g/l, MgSO₄JH₂O 0,3 g/l, FeSO₄JH₂O 0,03 g/l, MnSO₄.4H₂O 0,04 g/l, ZnSO₄JH₂O 0,005g/l, soya bean oil 40 g/l, antifoam Slovanik 1 g/l. During cultivation are kept optimal cultivation conditions. Concentration of pO2 in broth is keeping above 30%, during aeration from 1 to 1 ,25 vvm, mixing above 450 rpm and temperature 28⁰C. Optimal pH during cultivation is kept from 6,8 to 7,2 with 20% NaOH solution or 28% H₂SO₄ solution. Addition of linoleic acid and L-Leucine start from 34. hour of cultivation in ratio 1 :0,8. Optimal concentration of linoleic acid during fermentation is keeping from 0,2 to 1 ,0 g/l by feeding of linoleic acid. During cultivation 8% solution of L-Leucine with pH 11 is added into production media. Average dosage of L-Leucine solution is 17g/hour, average dosage od linoleic acid is 21 g/hour into working volume. Cultivation is stopped, when decrease of linoleic acid concentration below 0,2 g/l is not observed. Concentration of lipstatin in fermentation broth is from 1 ,8 to 2,3 g/kg after 180 or 200 hours of cultivation.

Example 3 a) Preparation of spore material

b) inoculation step I.

The loop of spores of the strain Streptomyces toxytricini is used for seeding of 100 ml inoculation broth in 500 ml Erlenmayer flask. Composition of inoculation broth is : soya flour 10 g/l, glycerine 10 g/l, yeast extract 5 g/l, drinking water into 1000 ml. Optimal pH after sterilization 30 minutes 120 ⁰C is pH from 6,8 to 7,2. Inoculum is cultivated 28⁰C, from 24 to 26 hours. Preparated vegetative inoculum will be used for seeding of production step.

c) inoculation step II.

Vegetative inoculum from I. inoculation step is seeded with amount of 1 % to 3% into 12 I inoculation broth in 30 I fermenter. Composition of inoculation broth is: soya flour 30 g/l, yeast extract 5g/l, glycerine 20 g/l, (NH₄)₂SO₄ 2 g/l, CaCO₃ 6 g/l, antifoam Slovanik 1 g/l. Optimal cultivation conditions are: temperature 28⁰C, aeration from 0,5 to 1 vvm, mixing 350 rpm, and concentration of dissolved oxygen in broth above 60% saturation. Inoculum is cultivated at 28⁰C , from 12 to 16 hours during conditions mentioned hereby.

d) production step

Vegetative inoculum from II. inoculation step is seeded into production broth as in example 2 c).

After 180 or 230 hours of cultivation concentration of lipstatin is from 3,0 to 3,2 g/kg of fermentation broth. Industrial applicability

The strain of microorganism Streptomyces toxytricini CCM 7349, which is subject of the patent, is able to use for biotechnology preparation of pharmaceutical active substance lipstatin, hydrogenation of its in down-stream process occures transformation to orlistat (tetrahydrolipstatin), which is successfully used for treatment of obesity, cardiovascular deseases, decrease of blood pressure, decrease of cholesterol or treatment of diabetes mellitus type 2.

Claims

1. The strain of microorganism Streptomyces toxytricini CCM 7349, characterized by, that produce lipstatin.

2. Method of lipstatin production characterized by, that it conclude cultivation of the strain Streptomyces toxytricini CCM 7349 according to the patent right in production broth for suitable cultivation conditions.

3. Method of lipstatin production according to the patent right 2, characterized by, that production broth contain as a precursors lenoleic acid and L-Leudne in ratio 1 : 0,8.

4. Method of lipstatin production according to the patent right 3, characterized by, that lenoleic acid and L-Leucine is added continuously from 34. hour of cultivation during which time concentration of linoleic acid in production broth is keep up from 0,2 to 1 ,0 g/l.

5. Method of lipstatin production according to the patent right 2 till 4, characterized by, that the strain of microorganism Streptomyces toxytricini CCM 7349 according to the patent right 1 is cultivated in production broth containing glycerine and soya bean oil as a carbon sources, lenoleic acid with L- Leucine as a source of precursors, under cultivation temperature 28⁰C, pH in range 6,7 till 7,3 during which time produce lipstatin in concentration 3,0 till 3,2 g/kg after 180 till 230 hours of cultivation.

6. Method of lipstatin production according to the patent right 2 till 5, characterized by, that production broth does not contain emulsifier.