MICROORGANISM HAVING ABILITY TO CONVERT STEROL INTO ANDROST-4-ENE-3,17-DIONE/ANDROSTA-1,4-DIENE-3,17-DIONE AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD The present invention relates to a microorganism, Mycobacterium fortuitum EUG-119, having an excellent ability to convert sterol into androst-4-ene-3,17-dione and androsta-1, 4-diene-3,17-dione, a method for preparation of the microorganism and use thereof, and more particularly, to a microorganism having four or more times higher conversion rate than that of previously known microorganisms, a method for preparation of the microorganism and use thereof in preparing androst-4- ene-3,17-dione and androsta-1, 4-diene-3,17-dione.

PRIOR ART Steroids, which are secretary hormones released from the adrenal cortex, the testicle, the ovary or the placenta, the corpus luteum are synthesized from cholesterol. They are classified into about five classes according to their physiological activities, as follows : sex hormones of androgen (androsterone, testosterone, etc.) and estrogen (estradiol, etc.) playing a critical role in the development of secondary sex characteristics in men and women, respectively, gestogen (progesterone, etc.) stimulating and maintaining pregnancy, glucocorticoid (cortisone, hydrocortisone, etc.) stimulating gluconeogenesis and increasing liver glycogen levels by catabolism of proteins, and mineralcorticoid (deoxycorticosterone, aldosterone, etc.) playing a important role in maintaining the balance of electrolytes and water in a body.

Levels of the above hormones become unbalanced in a body owing to increased stresses and exposure to environmental hormones in accordance with the advance of civilization, resulting in occurrence of many diseases, and widespread use of the steroid hormones is conducted for therapy of the diseases. In particular,

synthesized estrogens are essentially used in artificial fertilization and therapy of sterile patients, and glucocorticoids play a role in relieving the pain caused by various inflammations, such, as iriditis, arthritis and the like. In addition, Addison's disease, which is fatal, can be treated by administration of deoxycorticosterone and hydrocortisone.

There have been conducted a variety of researches into in vitro synthesis of steroid hormones to meet the increased demands as described above, and one of them relates to production of steroid hormone precursors using a microorganism.

Mamoli and Vercellone (Ber. 70470 and Ber. 702079,1937) reported reduction of 17-ketosteroids to 17-p-hydroxysteroid by fermentation of yeast. Also, Peterson and Murray in U. S. Pat. No. 2,602,769 disclosed a method for producing lla- hydroxylation of progesterone using the fungus of genus Rhizopus, and Kraychy in U. S. Pat. No. 3,684,657 disclosed a process for androst-4-ene-diene-3,17-dione, androsta-l, 4-diene-3,17-dione, and 20a-hydroxymethylpregna-1, 4-dien-3-one from a steroid comprising a 17-alkyl using Mycobacterium species.

To achieve mass production of steroid hormones, there were attempts to isolate microorganisms using sterols as a sole carbon source and to modify structure of the sterol used as a substrate for fermentation, and also, to enhance an acquisition rate of steroids with the use of chemical additives capable of preventing degradation of sterol nucleus, such as a metal, a metal absorbent and a metal reducing agent, have made great progress (Marsheck, et. al., Applied microbiology. 23 (1). 72-77, 1972). Moreover, there were also performed researches for improving productivity of steroid precursors, in which mutagenesis was performed on microorganisms isolated from soil through physical and chemical treatments, and particularly, Upjohn in U. S. Pat. No. 4,293,644 described a method for yield-up androstN4-ene- 3, 17-dione (hereinafter referred to as AD) predominantly and a small amount of androsta-1, 4-diene-3,17-dione (hereinafter referred to as ADD) from a variety of sterols by a mutant strain from Mycobacterium (ATCC 29472).

In accordance with increased demands for steroid hormone medicines, there is a need for mass production of the above mentioned hormone precursors, especially AD and ADD, which are important precursor compounds for in vitro

synthesis of steroids. However, the previously known microorganisms have low productivity in synthesizing AD and ADD. With this regard, it is urgently required to develop microorganisms having a high conversion rate of sterols into AD and ADD.

DISCLOSURE OF THE INVENTION As a result of many trials by the inventors of the present invention, it was found that a Mycobacterium fortuitum EUG-ll9 mutant strain of Mycobacterium fortuitum (ATCC 29472), has an excellent conversion efficiency of sterols into AD and ADD, and the present invention was accomplished on the basis of the result.

Accordingly, it is an object of the present invention to provide a mutant microorganism having an excellent conversion efficiency of sterols into AD and ADD.

It is another object of the present invention to provide a method for preparing a mutant microorganism having an excellent conversion efficiency of sterols into AD and ADD.

It is still another object of the present invention to provide a method for preparing AD and ADD using a mutant microorganism having an excellent conversion efficiency of sterols into AD and ADD.

To achieve the above objects, the present invention provides a method for preparing Mycobacte7-ium fortuitu7n EUG-119, deposited in the Korean Culture Center of Microorganisms (KCCM) with accession No. KCCM-10259, comprising the steps of : (a) culturing Mycobacterium fortuitum ATCC 29472 strain in a sterol- containing culture medium ; (b) treating the cultured Mycobacteriumfortuiium with nitrosoguanidine (NTG); and (c) growing the nitrosoguanidine-treated bacteria in a medium supplemented with sterol or AD and ADD at a concentration of 0.1-2.0 g/L, whereby mutant strains growing rapidly in a sterol-adding medium and slowly in an AD and ADD-adding medium can be selected.

A method for preparing AD and ADD of the present invention comprises steps of culturing the mutant microorganism, Mycobacterium fortuitunz EUG-119, in a sterol-containing liquid medium and recovering AD and ADD from the cultivated medium.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a HPLC chromatogram of AD and ADD in the cultured medium of Mycobacterium fortuitum EUG-l l 9.

BEST MODES FOR CARRYING OUT THE INVENTION In accordance with the present invention, there was used Mycobacterium fortuitum (ATCC 29472), which is capable of transforming to steroids from cholesterols by fermentation.

To prepare Mycobacteriunz fortuitum EUG-119 of the present invention, at first, Mycobacterium fortuitum strain (ATCC 29472) was grown to O. D. 0.6 to 0.8, and then treated with nitrosoguanidine (NTG) at an amount capable of killing 99.9% of organisms. NTG, which is a compound commonly used in producing mutant strains of microorganisms, induces mutations through substitution of GC pair with AT pair during DNA replication by methylation at C6 of guanine. The amount of NTG is 300 to 400 ag per 5 ml of medium, and the preferred amount is 330 u. g per 5 ml. In addition, the mutation can be accomplished by UV irradiation, INH (Isoniazid) and other mutagenic compounds.

From the NTG-treated strains, there were selected mutant strains having both a high growth rate on a sterol-adding solid medium and a low growth rate on a AD and ADD-adding solid medium, and finally, a mutant strain Mycobacterium fortuitum EUG-119 having the highest conversion rate was obtained through the flask cultivation.

Preferably, sterol or AD and ADD used in the strain selection is added to a medium at an amount of 0.1 to 2.0 g/L, and preferably, 0.4 to 0.6 g/L. Sterol can be selected from the group consisting of sitosterol, cholesterol, stigmasterol and campesterol, and it is preferable that the sterol is from cholesterol.

Mycobacterium fortuitum EUG-119 prepared by the method as described above was deposited in Korean Culture Center of Microorganisms with accession No. KCCM 10259 on April 14,2001.

In an embodiment of the present invention, in which the finally selected mutant strain, Mycobacterium fortuitum EUG-119 was incubated and its conversion rate of cholesterol into AD and ADD was analyzed, the mutant strain showed about 2.3 times higher conversion rate in a small scale cultivation and about 4.2 times higher in a large scale cultivation, than a wild type strain. AD and ADD were recovered at a purification yield of about 80 % from 2 L of the mutant strain- cultivated medium by common separation and purification processes. It was found that, 2 days after cultivation, the mutant strain produced higher concentrations of AD and ADD than the wild strain.

Accordingly, Mycobacterium fortuitum EUG-119 of the present invention can be used for mass production of AD and ADD. Such a method comprises the steps of culturing Mycobacteriu2 fortuitum EUG-119 in a sterol-containing liquid medium and recovering AD and ADD from the cultivated medium.

When Mycobacterium fortuitum EUG-l l9 of the present invention is used in preparing AD and ADD used as a precursor for steroid hormone synthesis, AD and ADD can be obtained at a high yield.

The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawing. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.

EXAMPLE 1 Preparation of mutant strains

To induce mutation, Mycobacterium fortuitum strain (ATCC 29472) was inoculated in YNG medium, and cultured upon reaching O. D. 0.6 to 0.8,5 ml of the cultured medium was centrifuged, obtaining a cell pellet. The obtained pellet was washed twice with sterilized 0.1 M sodium citrate buffer (pH 5.6) containing 0.5 % Tween 80, and resuspended in 5 ml of the buffer, and NTG was added to the cell suspension at a concentration of 330 pg/ml. NTG-treated cell suspension was incubated with shaking at 37°C for 90 min, and then centrifuged, obtaining a cell pellet and the obtained cell pellet was washed three times with a sterilized 0.1 M sodium phosphate buffer (pH 7.0) and resuspended in the buffer. The final cell suspension was loaded onto a SM1 solid medium and incubated at 37°C for 3-4 days to form colonies.

EXAMPLE 2 Selection of mutant strains Colonies obtained from Example 1 were inoculated in MS1, MS1+cholesterol, MS1+ADD (or AD) and YNG solid media and incubated at 30°C for 3 days to select a strain having both high growth rate in cholesterol- containing medium (MSl+cholesterol medium) and a low growth rate in ADD (or AD)-containing medium, and the selected strain was designated as Mycobacterium fortuitum EUG-119. The MS1 medium, a minimum inorganic medium, was used as a control, and compositions of the media are shown in Table 1.

TABLE 1 Culture medium (%) Ingredient YNG SM1 MS1 SM4 (Fermentation medium) Yeast extract 0. 1 0. 5 Nutritional broth 0. 8 Glycerol 0. 5 1- Tween 80 0.1 - - 0. 01 NaCl-0. 0005 K2HPO4 - 0. 05 0.02 0.04 KH2PO4 - - - 0.08 NH4CI0. 0. 1 MgS047H200. 05 0.25 0.2 FeC136H20 0. 005 NH4NO3 - - 0.1 - FeS047H200. 0001 0.0005 Ammonium acetate---0. 15 ZnS047H20---0. 0002 MnCI44H20---0. 00005 Glucose Agar-1. 5 1. 5- H 7.0 - - 7. 5

EXAMPLE 3 Investigation for conversion rate of cholesterol into AD and ADD in M. fortuitum EUG-119 strain Mutant strain, M fortuitum EUG-119, selected from Example 2 was pre- cultured in 5 ml of YNG medium, and then cultivated at 30°C for 120 hours at 200 rpm in 100 ml SM4 fermentation medium (refer to Table 1) containing 1 g glucose, 0.5 g yeast extract, 0.01 g Tween 80 and various inorganic salts. Cholesterol was dissolved with acetone, because it is not dissolved well in culture media, and cholesterol suspension in acetone was added to the medium at an amount of 0.1 g cholesterol per 100 ml. After the incubation, cultured media were extracted with ethyl ether and petroleum ether and then dissolved with 2-propanol, and used in an analysis of amounts of AD and ADD produced by Ad fortuitum EUG-119 strain from cholesterol, which was carried out by high pressure liquid chromatography (HPLC). A conversion rate of cholesterol into AD and ADD was represented as yield calculated by a ratio of molar concentration of AD and ADD and molar concentration of added cholesterol. Also, there were investigated produced amounts of AD and ADD and a conversion rate of wild strain, M. fortuitum (ATCC 29472). The results are shown in Table 2.

TABLE2 Wild strain M ortuitum EUG-119 Yield (Molar %) 27-64 Produced amounts of AD and ADD (mg/L) 199 472 Note: Yield = Molar concentration of produced AD and ADD/ Molar concentration of added cholesterol As shown in Table 2, mutant strain, Ad fortuitum EUG-1 19 showed about 2.3 times higher conversion rate of cholesterol into AD and ADD than wild strain, M fortutum ATCC 29472.

EXAMPLE 4 Mutant strain pre-cultured in 100 ml medium, prepared by the same method as Example 3, was inoculated into a 5 L fermentor containing sterilized 2.5 L medium (pH 8.0) and incubated at 1 vvm (aeration flow rate) for 120 hours at 30 °C with shaking at 600 rpm. Cholesterol dissolved in acetone was added to at an amount of 5 g/L. Produced amounts of AD and ADD and conversion rate of cholesterol into AD and ADD were analyzed by the same method as Example 3, and the results are shown in Table 3.

TABLE 3 Wild strain A {fortHitum EUG-119 Yield(Molar %) 14 59 Produced amounts of AD and ADD (mg/L) 510 2, 177 As shown in Table 3, the mutant strain, Ad fortuitum EUG-119, exhibited about 4.2 times higher conversion rate than the wild strain.

EXAMPLE 5 AD and ADD were purified from the culture broth prepared in Example 4.

2.5 L of the culture broth was adjusted to pH 3.0 and centrifuged at 5. 000 rpm for 10 min at 4°C. The resulting biomass pellet was extracted by suspension in 70% acetone and filtered, and after evaporation of acetone, held at 5-10°C, allowing precipitation of hormones. The resulting precipitate was filtered and dried at 55°C, and the dried precipitate was added with hexane to remove remaining cholesterol, and then filtered and dried again, and the resulting crude hormone intermediates were recovered. Recovery yields for hormone intermediates are shown in Table 4.

TABLE 4 Weight (g) of biomass Yield (%) Culture broth 5. 2 100 After acetone extraction 4. 6 88 Crude hormones 4. 2 81

As shown in Table 4, purification yield of hormone intermediates was about 80% from the 2.5 L culture broth of the mutant strain, Ad fortuitum EUG- 119.

EXAMPLE 6 The hormone intermediates produced by a wild type of Ad fortuitum and a mutant strain, Ad fortuitum EUG-119, were measured for concentration using HPLC. The culture broth medium containing the wild type or the mutant type of Ad fortuitum, was extracted twice with four volumes of a mixture of diethyl ether and petroleum ether in a ratio of 1 : 1, and the solvents were then evaporated. The extracted hormones were suspended in isopropyl alcohol for quantitative analysis, with resort to HPLC utilizing Pegasil ODS (4.6 x 250,5 µm, 120Å, Senshu Pak, Japan) as a column under conditions of a flow rate of 1.0 ml/min, 2,700 psi and 250 nm, and the mobile phase was analyzed using 50% THF (refer to Fig. 1).

As a result, it was found that, from 2 days after culturing, the mutant strain, M. fortuitum EUG-119, produced AD and ADD in higher levels than the wild strain, M. fortuitum ATCC 29472.

EXAMPLE 7 Mycological properties of mutant strain, Mycobacteriunz fortuitum EUG- 119, were investigated using the known methods, and are as follows.

M. fortuitum EUG-119 can grow at 30-35°C, utilizing sugars including glucose, polysaccharides, glycerol, fatty acids, sterols, and the like as a carbon source. Also, M. fortuitum EUG-119, which is a Gram (+) strain, contain abundant fats in its cell wall and grows forming yellow colonies.

As a whole, M. fortuitum EUG-119 of the present invention shows similar morphological and physical properties to the known M. fortuitum ATCC 29472 strain, but has an excellent ability to convert sterol into AD and ADD by its fermentation action.

INDUSTRIAL APPLICABILITY As described above, Mycobacterium fortuitum EUG-119 of the present invention has an excellent conversion efficiency of sterol to AD and ADD, which can be used as hormone precursors, in comparison with the previously known microorganisms, especially M. fortuitum ATCC 29472. Accordingly, M. fortuitum EUG-119 of the present invention is greatly useful for mass production of steroid hormones.

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOST OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURE INTERNATIONALFORM - To, NL) h Fig Science Inc. 1 (-)-7, Samjung-dung. Ohjung-gu, Bucheon, Kyunggi, 421-150 RECEPT IN THE CASE OF AN ORIGINAL issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page 1, ! DENT) F ! CAT ! nN OF THE MICROORGAMJSM IdcntiftC. utj'on rcforc-ncf Mn by the Accession number tivr'n by the DtCPO. TOR : NTERNADON/U. DRP () S ! TA] <Y/\ ! ; THOmTY : Mycnbactet-ium fr. rCcciLurr EUG-1 t9 KCCM-1025 2. SCI) j : NT ! PfC DESCRIPTtON AD/OR'PROPOSED TAXONOMfC DES ! GNA'f' ! ON 'Il7r micrcurt, raiism ifcwl. ificd uridcr 1 abwc was accompaniel by : -2 a scicntitic cicsscriyf n D 2) pnafioSGC ! laxcmumic dc : sinalion (Mzl-k wi1h tl cross where appiicailc) kECF : It'T 11ND lic.'CL)''7'lIMC This Int. crr'xont Dfpasit. Hry Auhhouity Hc. ccpt. s the microorcanism idcntifi'.'. d undt')'I Hhne, Whici ! W} ts rccied hy it'nn Apr. M. 2001. (date of the origins) dspotiit)' IV, 1NTERNI' !'iUNI_ C>FPC51'I'IHtY nllTHUR ('Y N : : ICcJrettal C lrx Cenltsr ol ixlicrotllganisms SignaturuS) nf pUlrsUncs) ilavins hc puwe. r tu represent, tf InfprnnHoM ! Dc'pnKitHry Address : 361-221, Yurim rs Hongie 1-dOng, Authurily of Qf * ; S¢eodacmlll5-gts g i-) HHdaemun-gu U-!- SliOUL 12O-091 Uatc : Apr. ll, 200 Republic rBf f KorLLi . A- 1 Where Ruel 6.4(d) applics, such date is the dae on which the status of international depositary authority was acquired : where a deposit made puiside the Budapest Treaty after the acquisition of the stauts of i nternational depositary authority is converted into a deposit under the Budaper Treaty, such date is the date on which the microorganism was received by the international depositary @uthouity.