JPH0117679B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing androst-4-ene-3,17-dione (hereinafter abbreviated as "4AD"). Specifically, the present invention relates to a method for producing 4AD using a mutant microorganism.

A method for producing 4AD from sterols using microorganisms belonging to the genus Mycobacterium (hereinafter abbreviated as "M.") is already known. One method is to use a chelating agent or an inhibitor such as nickel or cobalt.
Publication No. 52-145593, Special Publication No. 145593-
It is described in Publication No. 11998, etc. Another method is a method using a mutant strain, which is described in US Pat.

As a result of intensive research to develop a new production method that accumulates large amounts of androstane compounds, the present inventors have discovered a mutant strain of a microorganism belonging to the genus M. that is different from the microorganisms produced by known methods. It was discovered that androstane compounds including 4AD can be accumulated more than sterols by using
The application was filed as No. 124188.

However, the two strains exemplified in that application are derived from sterols and androsta-1,4-diene-3,17-dione (hereinafter abbreviated as "ADD").
The objective was to selectively produce 4AD, which is suitable for obtaining 4AD as the main product, but among the androstane compounds, it is more suitable for the production of many male hormones, anabolic hormones, and the antihypertensive diuretic spironolactone. It was insufficient.

Recently, the present inventors have endeavored to isolate a mutant strain that selectively produces 4AD, and have arrived at the present invention.

That is, the gist of the present invention is to dehydrate androst-4-ene-3,17-dione at the 1,2 positions using sterols as a substrate in a mutant strain belonging to M. parafortuitum complex. Androstase selected from the group consisting of 9-hydroxylase, 17-position reductase,
The present invention relates to a method for producing 4AD, which comprises culturing a microorganism in which a 4-ene-3,17-dione-degrading enzyme is deactivated or deleted.

The present invention will be explained in detail below.

The microorganism used in the method of the present invention is a mutant strain belonging to M. paraphutitutum complex.

The so-called type culture microorganisms (hereinafter referred to as "wild strains") belonging to the conventionally known M. paraphuthuitum complex have enzymes that decompose androstane-based compounds, and are capable of degrading chelating agents, nickel, and cobalt. Under special conditions such as in the presence of inhibitors such as sterols, ADD,
In addition, 4AD and 17β-hydroxyandroster-1,4-dien-3-one (=1-
It produces androstane-based compounds such as dehydrotestosterone (hereinafter abbreviated as "DHT").

However, under normal culture without inhibitors
No literature has yet been found that shows the formation of androstane compounds such as 4AD.

In contrast, the mutant strain used in the method of the present invention is clearly different from the wild strain in that it produces more 4AD than sterols under normal culture conditions in the absence of chelating agents or inhibitors such as nickel and cobalt. distinguished.

In addition, it is described in Japanese Patent Application No. 124188/1972.
M. Paraformitum Complex MCI−
Bacterium No. 0801 and MCI-0802 are clearly distinguished from each other in that they produce a larger amount of 4AD than ADD under the same culture conditions.

The mutant strain used in the method of the present invention can be obtained by isolating a strain that grows slowly or does not grow at all on an agar medium containing 4AD as the sole carbon source after mutation treatment. Therefore, used in the present invention
Mutant strains of M. paraphutitutum complexus grow much more slowly than the parent strain on media with 4AD as the sole carbon source. for example,
When the parent strain and the mutant strain are cultured at the same time with the same amount of bacteria in a medium with 4AD as the sole carbon source, the number of bacteria of the mutant strain will normally be the same as that of the parent strain when the parent strain enters the exponential (logarithmic) growth phase. less than half the number.

The main reason why such strains accumulate 4AD is that 4AD oxidation/reductase is missing or reduced compared to the parent strain, which interrupts metabolism at 4AD and accumulates 4AD.

That is, even if enzymes with originally low activity remain as they are, since there is no remaining 4AD oxidation/reductase that expresses strong activity, the decomposition of sterols stops at 4AD, and 4AD accumulates.

The enzyme that is deleted or decreased is 4AD 1,2 position dehydrogenase (4AD→
ADD), 9α-hydroxylase (4AD→9α−OH−
4AD) [9α-OH-4AD represents 9α-hydroxyandrost-4-ene-3,17-dione] and 17-position reductase (4AD→testosterone).

Even though the amount of 4AD oxidation/reductase remains unchanged compared to the parent strain, the expression of the enzyme is suppressed, and the reaction equilibrium shifts toward 4AD, so 4AD accumulates.

Because the enzymatic reaction at a certain stage that constitutes the sterol decomposition pathway is suppressed, the sterol decomposition pathway changes to the 4AD-mediated route, accelerating the decomposition of 4AD and accumulating 4AD.

etc. are possible.

An example of such a mutant strain is M. paraphutitutum complexus MCI-0807. This strain uses the parent strain M. paraphotitutum complexus ATCC 25790 [= M. neoaurum ATCC 25790] and is treated with ultraviolet light or N-
Obtained by repeated treatment with methyl-N'-nitro-N-nitrosoguanidine.

This strain has been deposited with the National Institute of Microbial Technology under the following number.

Microorganism M. paraphotitutum complex MCI−
No. 0807 Bacterial Science Institute No. 4476 To explain the parent strain of this bacterium in detail, the parent strain
ATCC No. 25790 was identified by the International Study Group on Mycobacterial Classification, which brought together 13 experts on M.
Working Group on Mycobacterial
As a result of joint research in M. taxonomy, it has been determined that it belongs to the M. paraphotitutum complex. [Refer to Hajime Saito, Michio Takamura et al., International Journal of Systematic Bacteriol., Vol. 27, pp. 75-85 (1977, USA)] This bacterial strain ATCC25790 is A new species of M. aurum, initially named Tsukamura #309 and using Tsukamura #358 (ATCC23366) as the reference strain.
It was included in the village. [Michio Tsukamura, Medicine and Biology, Vol. 72, pp. 270-273, 1966] Later, Mr. Tsukamura used Tsukamura #3503 (ATCC No. 25795) as the reference strain, and identified M. as a different species of M. aurum. . Neoaurum (neoaurum) founded Tsukamura, but
ATCC25790 was thought to belong to M. aurum. [Michio Takamura, Medicine and Biology, vol. 85, 229-
233 pages, 1972] However, according to the recent literature by Saito, Tsukamura et al. (mentioned above), ATCC No. 25790 is considered to be a member of M. neoaurum rather than M. aurum.
[See figure 1 on page 77 of Saito, Tsukamura et al., cited above] Furthermore,
In the same literature, regarding the classification of the genus M.
Juhasz) and “Kanazawa Strains”
("Kanazawa strains") are extremely closely related to each other and cannot be distinguished at this stage, so it has been concluded that these five species should be treated collectively as M. parafoetitutum complex rather than being separated. .

Takamura et al. reached almost the same conclusion in another literature,
M. aurum and M. neoaurum are considered to be members of the M. parafoethuitum complex or subspecies of M. parafouthuitum. [Journal of General Microbiology 98
See Vol. 511-517 (1977, UK)] The general properties of M. paraphotitutum complex and a comparison with closely related species are given by Hajime Saito (Tuberculosis,
50, p. 402, 1975) Saito et al. (ibid., vol. 75, 80)
~81 pages, 1977) and Tsukamura et al. (ibid., p. 515,
(1977).

As mentioned above, it is appropriate that the taxonomic position of the parent strain ATCC 25790 [Tsukamura #309] belongs to M. paraphutitutum complex, in accordance with the conclusions of the highest authority in this field. .

M. paraphotuitum complexus is a Gram-positive, acid-fast, non-motile, non-spore-forming bacillus that is rapidly growing, darkly colored.
It belongs to the group of mycobacteria (growing scotochromogenic mycobacteria) or Runyon.

On the other hand, the mutant strain MCI-0807 isolated from the parent strain ATCC No. 25790 differs from the parent strain in that the former forms rough colonies on agar medium, and the latter forms smooth colonies. There were no other mycological differences, and the parent strain was as described above.
Mutant strain MCI-0807 belongs to M. paraphotuitum complex.

Naturally, therefore, this mutant strain is similar to M. sp. NRRL, which was disclosed as a 4AD-producing mutant strain in U.S. Pat.
B-3805 [This strain was identified as M. vaccae in JP-A-52-105289]
It is a new bacterium different from that of JP 52-105289.
It is clearly different from M. fortuitum and M. phlei to which the ADD and 4AD producing mutant strains NRRL B-8153 and B-8154 belong, which were described in the publication.

For a comparison of these taxa, see “Bergey’s Manual of
Determinative Bacteriology ed.8”, 695-696
Page, Williams & Wilkins Co., 1974, USA),
Saito (ibid., p. 402, 1975), Saito et al. (ibid., p. 402, 1975), Saito et al.
80-81, 1977) and Tsukamura et al. (ibid., p. 515,
(1977).

In addition, MCI-0807 has taxonomic properties with NRRL B-3805, as well as ADD, 4AD, and 9α-OH-.
The difference is that the decomposition ability of 4AD differs, and while the former grows to a small extent using these as the only carbon source, the latter does not.

In this way, mutant strain MCI-0807 is considered to belong to M. paraphutitutum complexus, and it is clear that it is taxonomically different from known 4AD-producing mutant strains. It is.

As mentioned above, this bacterium grows to a small extent with 4AD as the only carbon source, and the fact that it produces a small amount of 4AD using ADD as a substrate and a small amount of ADD using 4AD as a substrate shows that the difference between 4AD←→ADD is It seems that the enzyme system of the molecule is not completely inactivated.

The sterols used as substrates in the present invention include various sterols, their C-3 esters, ether derivatives, or oxidized intermediates thereof. Various sterols are C-3 of the perhydrocyclopentanophenanthrene nucleus.
a hydroxyl group, usually a double bond at C-5,
C-17 has a chain type side chain having 8 to 10 carbon atoms, and in some cases C-7, C-8(10), C-9(11)
etc. may have a double bond.

Examples of such various sterols include cholesterol, stigmasterol, campesterol, β-sitosterol, ergosterol, brassicasterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dihydroagnosterol, α-sitosterol, and the like. It will be done. Preferred sterols are cholesterol, campesterol and β-sitosterol.

C-3 ester derivatives of 3β hydroxyl groups of various sterols and inorganic acids such as sulfuric acid or organic acids such as fatty acids can also be used as raw materials in the method of the present invention.

Examples of such C-3 ester derivatives include cholesteryl oleate, cholesteryl palmitate, cholesteryl sulfate, and the like.
Furthermore, C-3 ether derivatives obtained by, for example, a method of adding an alkylene oxide to the 3β hydroxyl group of various sterols can also be used as raw materials in the method of the present invention.

Examples of such C-3 ether derivatives include polyoxyethylene cholesteryl ether.

Wool wax containing C-3 ester derivatives of the various sterols mentioned above, lanolin, wool alcohol containing cholesterol obtained by hydrolysis of lanolin, and C-3 obtained by reacting wool alcohol with ethylene oxide. It goes without saying that polyoxyethylene lanolin alcohol ether, which is an ether derivative, can also be used as a raw material in the method of the present invention.

Sterol-containing natural and processed products, such as alkaline-washed dark oils from fish and squid oils, as well as deodorized scums of vegetable oils, deodorized sludge, and tall oil, are likewise used as raw materials in the process of the invention.

Additionally, oxidized intermediates of various sterols or their C-3 ester or ether derivatives can also be used as substrates in the process of the invention. Such oxidation intermediates include various sterols, their C-3 esters, and C-3-ether derivatives of 4-ene-3-
one or 1,4-dien-3-one derivatives, specifically, for example, cholest-4
-en-3-one, cholesta-1,4-diene-
3-one, cholesta-4,22-dien-3-one, and the like.

A carbon source, a nitrogen source, and an inorganic substance are appropriately added to the medium used in the method of the present invention.

Examples of carbon sources include n-paraffin,
Hydrocarbons such as α-olefin and xylene; Alcohols such as methanol, ethanol, glycerin, propanediol, butanediol, and higher alcohols; Organic acids and their salts such as succinic acid, acetic acid, and higher fatty acids; glycerides,
Fats and oils: sugars such as starch, maltose, sucrose, glucose, and rhamnose. Natural nutrient sources including carbon sources, nitrogen sources and other nutritional substances include:
Molasses including high test molasses, refined molasses and xylose molasses; bagasse, corn cob, alpha alpha, corn stable liquor, daytailer's soluble, flavor liquid, fishmeal, bran, meat extract, yeast, yeast extract, boteto extract, malt extract, Gluten, peptone, glutamate, asparagine, glycine, casein, casein decomposition products, skim milk, soybean meal, rapeseed meal, sesame meal, flaxseed meal,
Examples include vegetable oil meal such as cottonseed meal, and oilseeds and oil fruits such as whole soybeans, rapeseed, sesame, linseed, and cottonseed.

Inorganic substances include nitrogen sources such as ammonium sulfate and ammonium chloride; potassium and phosphorus sources such as dipotassium hydrogen phosphate;
Salts of iron, copper, magnesium, manganese, cobalt, zinc, calcium, etc.; natural products such as molasses, and ash. Other vitamins can also be added if necessary.

The composition of the medium is selected depending on the type of bacteria used, but a carbon source, nitrogen source, potassium, phosphorus, and magnesium are essential medium components.

If an antifoaming agent is required, a known antifoaming agent may be added. Specific examples include polyoxyalkylene glycol, but it is not always necessary to add an antifoaming agent.

Surfactants are effective as emulsifiers for sterols, and are preferably added to the culture medium. As the surfactant, nonionic and anionic surfactants are preferred. Specific examples include polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, polyethylene glycol monostearate, and the like.

The culture temperature is usually 20 to 40℃;
The optimum temperature is around 30-35℃.

The pH of the medium is usually adjusted to 5-10, but 6-9
is preferred. Since the microorganism used in the present invention belongs to the genus M., it can withstand a pH of around 10, as is well known.

Although sterols, which are raw materials for the method of the present invention, are generally sterilized together with the medium, they may be added to the medium after the start of culture. It is also possible to add in portions. In this case, it is added as is after dry sterilization or moist heat sterilization, or it is added as a solution after dissolving dimethylformamide, etc.
Alternatively, it can be finely dispersed and suspended by ultrasonication and added as a suspension. Further, it is preferable to add a surfactant at the same time because it promotes emulsification of substrate sterols and the like.

Cultivation time is not particularly limited, but usually starts from about 2 or 3 days after adding raw material sterols, etc.
4AD production increases rapidly. After that, the amount of 4AD produced gradually increases with the culture time, but culturing for more than 20 days has little industrial significance.

After completion of the culture, 4AD accumulated in the culture solution can be collected, separated and purified by known methods. For example, 4AD is extracted from a culture solution with several times the amount of an organic solvent that is not easily miscible with water, such as ethyl acetate, and the solvent is distilled off from the extract. , repeat reconsolidation from tetrahydrofuran, cyclohexane, hexane, acetone, etc., or in some cases, use porous resin, silica gel, alumina, etc. as an adsorbent, and use petroleum ether, benzene, chloroform, ether, acetone, methanol/ethyl acetate, etc. Purified 4AD can be obtained by column chromatography using as eluent.

U.S. patent No. 4AD main producing mutant strain
M. species described in specification No. 3759791
NRRL B-3805 has a sterol concentration of 0.1%.
4AD production of 0.4 mg/ml or less is only disclosed, and it was described in Japanese Patent Application Laid-open No. 105289/1989.
The two mutant strains producing ADD and 4AD are both 4AD
There is no mention of yield.

On the other hand, according to the present invention, it is possible to produce 4AD with a high yield with few by-products, and it is also highly effective in that a proportionate yield can be obtained at a high substrate concentration that is not found in conventional methods.

The present invention will be explained in more detail in the following examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof.

In addition, in the following examples, sterols and
Qualitative and quantitative determination of androstane compounds such as 4AD was performed by gas chromatography. Also, in the following examples, percentages are by weight.

Example 1 Glucose 1.0%, meat extract 1.0%, peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
Dispense into ml shoulder flasks, steam sterilize at 120℃ for 15 minutes, and then remove M. paraphritum complex.
One platinum loopful of MCI-0807 was inoculated, and the seed was cultured for 48 hours at 30°C under conditions of reciprocating shaking at 120 reciprocations/min and an amplitude of 7 cm. Add 2 ml of this seed culture solution to 4.0% crushed whole soybeans,
_NaNO3 0.2%, _{K2HPO4 0.2} %, _MgSO4・_7H2O0.1
%, cholesterol 0.8% and water (PH7.0, steam sterilized at 120℃ for 20 minutes)
Inoculate five 500 ml shoulder flasks containing The main culture is carried out at 30° C. under the conditions of reciprocating shaking at 120 cycles/min and an amplitude of 7 cm. The culture is stopped 156 hours after the start of the culture, and all the culture solutions are combined and extracted twice with ethyl acetate 2.

The extracts were combined to remove insoluble matter such as bacterial cells, and the steroid content was analyzed using gas chromatography. The results showed that 1.25 g of 4AD and 0.05 g of ADD were produced. Several other androstane compounds were also produced, but they were all less than 0.05 g.

Example 2 Glucose 1.0%, meat extract 1.0%, peptone 1.0
% and 500 ml of seed medium (PH7.2) consisting of water
Dispense into ml shoulder flasks, steam sterilize at 120°C for 15 minutes, and then remove M. paraformitum complex.
One platinum loopful of MCI-0807 was inoculated and cultured for 48 hours at 30°C under reciprocating shaking conditions of 120 reciprocations/min and an amplitude of 7 cm. Add 20ml of this seed culture solution to 2.0% defatted ground soybean powder,
Yeast 2.0%, lecithin 1.0%, NaNO ₃ 0.2%,
800 ml of main culture medium consisting of K ₂ HPO ₄ 0.2%, MgSO ₄ 7H ₂ O 0.1%, cholesterol 1.0% and water
(pH 7.0, steam sterilized at 120°C for 20 minutes) into a 6-capacity shoulder flask. The main culture is carried out at 30°C under reciprocating shaking conditions of 100 cycles/min and an amplitude of 7 cm.Culture is stopped 192 hours after the start of culture, and the culture solution is extracted with 2.4 ml of ethyl acetate. This extract contains
4AD1.73g and ADD0.09g were generated.

Example 3 In Example 2, instead of the substrate cholesterol, a 2:1 mixture of β-sitosterol and campesterol, stigmasterol, cholest-4-en-3-one, cholesta-1,4-dien-3-one and Example 2 is repeated except that cholesteryl oleate is used. The amount of the main product 4AD accumulated is shown in Table 1.

Table 1 4AD (g) β-sitosterol + campesterol 1.21 stigmasterol 0.43 cholest-4-en-3-one 0.94 cholesta-1,4-dien-3-one 0.25 cholesteryl oleate 0.09 Reference example MCI-0807 and Its parent strain ATCC25790
An example of growth with No. 1 bacteria in a medium with 4AD as the sole carbon source is as follows.

4AD0.2%, _NaNO3 0.2%, _{K2HPO4 0.1} %,
Medium consisting of KCl 0.05%, MgSO ₄ 7H ₂ O 0.01% (PH 7.0) (sterilized at 120°C for 20 minutes, 10ml/21mm x 210
mm test tube) and ATCC25790 bacteria and MCI-0807.
10 ⁶ bacteria were inoculated and cultured at 30°C on a test tube shaker. ATCC No. 25790 clearly showed growth of 10 ⁷ to 10 ⁸ /ml after 5 days of culture, but MCI-
Bacteria No. 0807 hardly multiplied and the number of bacteria was less than 10 ⁶ /ml.

In addition, 2% agar was added to the above 4AD sole carbon source medium.
was added to make a 3 mm thick plate medium in a sterile dish. A bacterial suspension containing 50 to 100 bacteria was applied to this. Bacterium ATCC25790 forms clear colonies after 4 days, but Bacterium MCI-0807 hardly forms colonies and does not grow.

Claims (1)

[Scope of Claims] 1. A mutant strain belonging to Mycobacterium parafortuitum complex that uses sterols as a substrate to produce androst-4-ene-3,17-
An androst-4-ene-3,17-dione degrading enzyme selected from the group consisting of dione 1,2-position dehydrogenase, 9-position hydroxylase, and 17-position reductase is activated or deleted. androst-4-en-3,17-, which is characterized by culturing microorganisms that
Zeon manufacturing method. 2. In the method described in claim 1,
A method characterized in that the sterols are selected from the group consisting of sterols, their C-3 ester derivatives, their C-3 ether derivatives or their oxidized intermediates. 3. In the method according to claim 1 or 2, the microorganism is obtained by artificial mutation and androst-4-ene-3,17
- A method characterized in that the strain exhibits reduced growth compared to the parent strain in a medium containing dione as the sole carbon source. 4. The method according to any one of claims 1 to 5, wherein the mutant strain belongs to Mycobacterium neoaurum. 5. In the method according to any one of claims 1 to 6, the mutant strain is Mycobacterium paraphytitutum complex.
A method characterized by using MCI-0807 bacteria.