JPS6024720B2 - Method for producing androstane compounds using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing androstane compounds using microorganisms.

Specifically, the present invention relates to a method for producing androstane compounds using mutant microorganisms. A method for producing androstane compounds 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.
It is described in Japanese Patent No. 4908, Japanese Patent No. 46-17951, etc. Another method is to use mutant strains, as disclosed in U.S. Pat. No. 3,684,657 and U.S. Pat.
, No. 759, 791, each specification and 1975-10
It is stated in the 528th Publication etc. The inventors,
A new manufacturing method that accumulates large amounts of androstane compounds should be developed. The present invention was achieved by discovering that it is sufficient to use a mutant strain of a microorganism belonging to the genus that is different from the microorganism used in known methods.

In other words, the gist of the present invention is to use sterols as a substrate.
M. Paraphthalmia complex (parabr)
The present invention relates to a method for producing an androstane compound, which comprises culturing a mutant strain belonging to P. tuitum complex.

The present invention will be explained in detail below.

The microorganism used in the method of the present invention is M. It is a mutant strain that belongs to Parafittum complex.

Conventionally known M. Microorganisms belonging to the Grafauozytum cosoplex (hereinafter referred to as "wild strains") have an enzyme that decomposes androstane-based compounds, and they are Under such conditions, androstar 1,4-diene-3
, 17-dione (hereinafter abbreviated as "ADD"), androstole-4-ene-3,17-dione (hereinafter abbreviated as "Isketsu") and 178-hydroxyandros-1,4-dione (hereinafter abbreviated as "ADD").
-Dien-3-one (=1-dehydrotestosterone)
(hereinafter abbreviated as "DHT"), but no production of androstane compounds was observed in normal culture.

Therefore, the mutant strain used in the present invention is one in which any of the androstane compound degrading enzymes possessed by the wild strain is deleted or has low activity, and such mutant strains are treated with chelating agents or nickel. It is clearly distinguished from the wild strain in that it produces large amounts of androstane compounds under normal culture conditions in the absence of inhibitors such as cobalt.

Examples of such mutant strains include, for example, M. Parafittum complex bacteria MCI-0801 and MCI-0802 can be mentioned. Both of these two strains are M. Paraphatium complex A
TCC 2579 bacterium [=M. Neo Aurum (neo
aumm) ATCC No. 2579] was used as the parent strain, and treated with ultraviolet light or N-methyl-N'nitro-N-
Each was obtained by treatment with nitrosoguanidine. Both strains have been deposited with the National Institute of Microbial Technology under the following numbers.

Microorganisms M. /Graf Auto Tweet Complex MCI-08
No. 01 Bacterial Engineering Research Institute Bacteria No. 425 M. Paraphthalmology complex MCI-080
To explain in detail the parent strain of this bacterium, No. 2 Bacterium Microtechnical Research Institute No. 426, the parent strain ATCC2579 is M. Experts on 13
The International Research Group on Mycobacterial Classification
As a conclusion of the joint research of aIWorking Group Mycobac and riaITaxonomy),
M. /It is said to belong to the Graf O'ottitum complex.

[Hajime Saito, Michio Higashimura et al. International Journal of Systematic Bacterology (lnt.J.S.
$t. Bacteriol. ) Volume 27, pages 75-85 (
1977, USA)] This strain ATCC257
Bacterium No. 9 was first identified as Higashimura #309, then Higashimura #35.
A new species of M. chinensis with Bacterium No. 8 (ATCC No. 23366) as the reference strain. Aurum (ammm) was included in Higashi Village.

[Michio Higashimura, Medicine and Biology, 72 lids, pp. 270-273, 1966] After that, Mr. Tsukamura developed Higashimura #3503 bacterium (
ATCC No. 25795) was used as the reference strain, and M. M. as another species of Aurum. Neoammm Higashimura was founded, but ATCC No. 2579 was M. It was said to belong to Aurum. [Michio Higashimura, Medicine and Biology,
85, pp. 229-233, 1972] However, according to the recent literature by Saito, Higashimura et al. (cited above), ATCC25
Bacterium No. 79 is M. M. instead of Aurum. It is said to be a member of Neo Aurum.

[See Saito, Higashimura et al., supra, p. 77, Figure 1] Furthermore, in the same document, M. Regarding the classification of the genus M. Parahuottim Higashimura, M. Aurum Higashimura, M. Neo Aurum Higashimura, M. Djernhoferi Benitsuk and Kohaku (
Dier skin oferi B′′omicke & J ground as
z) and “Kanaza Trains” (“Kanaza
waStrains") are closely related to each other and cannot be distinguished at this stage, so it was concluded that these five species should not be separated but should be treated collectively as the M. paraforitum complex. Higashimura et al. Almost similar conclusions were reached in another literature, M. Aurum and M.
．． Neo Aurum is M. as a member of the paraphthalmia complex or M. It is said to be a subspecies of Parahuozytum.

[Journal Optical Internal Microbiology (J
oumaloofneral Microbiolog
y) Vol. 98, pp. 511-51, (1977, UK)] M. The general properties of the paraphaitum complex and its comparison with closely related species are discussed in Hajime Saito (Tuberculosis, 5 Ikari-gata, p. 402, 1973) and Saito et al. (ibid., 79 Suzume, 80).
~81, 1977) and Tsukamura et al. (supra, p. 515, 1977). As above, parent stock ATC
C25790 bacteria [Tsukamura #
The taxonomic position of M. 309] is based on the conclusion of the highest authority in this field. It is reasonable to assume that it belongs to the paraphoritum complex.

M. Paraphytium complex is a gram-positive, acid-fast, non-motile, non-spore-forming camphorbacterium, with rapid-growing, chromogenic acid-fast (rapid scales)
It belongs to group W mycobacteria of chromocrab nlcmycocteria) or Runyon. On the other hand, the mutant strains MCI-0801 and MCI-0802 isolated from the parent strain ATCC 25790 differ from the parent strain in that the former two have a rough surface on the ground of the agar tower.
) There were no mycological differences other than the fact that they formed colonies;
Furthermore, as mentioned above, the parent strain is M. Both mutant strains MCI-0801 and MCI-0802 belong to the M. parafolium complex. Belongs to the paraphthalmia complex.

Naturally, therefore, these two mutant strains are the same as those described in U.S. Pat.
7 as a 4AD- and ADD-producing mutant strain. Species (SP.

) NRRL B-3683 and NRRL B-
Bacterium No. 3805 [This strain was disclosed in Japanese Patent Application Laid-open No. 105,289/1989. It is a new bacterium that is different from the one previously identified as P. vaccae.
ADD and 4AD producing mutant strains NRRL B-8153 and NRRL B-8 described in Publication No. 28y
M. to which Bacterium No. 154 belongs. fonuit
um) and M. It is clearly different from phlei. A comparison of these taxa can be found in Bergey's Manual of the Eighth Edition (''
Dete skin iMtiVe Mari cteriolo number ed
．． 8'', pp. 695-696, William & Wilkins Co.
), 1974, USA)], Hajime Saito (ibid., 402)
(1975), Saito et al. (ibid., pp. 80-81, 197)
7) and Higashimura et al. (ibid., 515, 1977). In addition, MCI-0801 bacteria, MC
Bacterium I-0802 is NRRL B-3805, B-
In addition to their taxonomic properties, ADD, 4AD, and Niji-OH-KeiD differ from Bacterium No. 3683 in their degrading abilities, and the former grows using these as substrates, while the latter does not.

In this way, mutant strains MCI-0801 and MC
Both bacteria No. I-0802 are M. It is thought that it belongs to the parafittum complex, and it is clear that it is taxonomically different from the known ADD and essential D producing mutant strains. The androstane compound which is the object of the production method of the present invention has an androstane skeleton, has an oxygen atom or a hydroxy group as a substituent at C-17, and has a substituent containing a carbon atom at C-17. It is a general term for compounds that do not have

As the androstane compound, the above-mentioned ADD, 4
In addition to AD and DHT, testosterone, 5Q-androstane-3,6,17-trione (hereinafter referred to as "5Q-AT")
), 58-androst-1-en-3,1
7-dione (hereinafter abbreviated as "58-lAD"), 9Q
-Hydroxyandrost-4-ene-3,17-dione (hereinafter abbreviated as "9Q-OH-4AD"), 5Q-
Androstane-3,17-dione (hereinafter “5Q-AD”)
), 33-hydroxyandrost-5-
En-17-one (=dehydroepiandrosterone)
(hereinafter abbreviated as "DHA"), etc. Among these androstane compounds, ADD and oligocythemia are the main target compounds of the present invention. The sterols used as substrates in the present invention include various sterols, their C13 esters, ether derivatives, and oxidized intermediates thereof.

Various sterols have a hydroxyl group at C-3 of the perhydrocyclobentanophenanthrene nucleus, a double bond at C-5, and a side chain with 8 to 1 carbon atoms at C-17. However, in some cases C-7, C-8, C-9 (
11) etc. may have a double bond. Examples of such various sterols include cholesterol, stigmasterol, strengthbesterol, sitosterol, ergosterol, plasticacysterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dihydroagnosterol, and the like.

Preferred sterols are cholesterol, cambeth 7-lol and sitosterol. C-3 ester derivatives of various sterol 33 hydroxyl groups and non-carboxylic 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 alkylene oxide to the 38-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 fat (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- obtained by reacting wool alcohol with ethylene oxide. It goes without saying that polyoxyethylene lanolin alcohol ether, which is a 3-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.

Oxidized intermediates of various sterols or their C-3 ester or ether derivatives are also used as substrates in the process of the invention.

Such oxidation intermediates include various sterols and their C
-3 ester, C-3 ether derivative 4-ene-3
-one or 1,4-en-3-one derivatives, specifically, for example, cholest-4-en-3-one derivatives.
-one, cholester 1,4-dien-3-one, cholester 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.

Carbon sources include, for example, hydrocarbons such as n-paraffin, Q-olefin, and xylene; alcohols such as methanol, ethanol, glycerin, and higher alcohols; organic acids and their salts such as succinic acid, acetic acid, and higher fatty acids; Examples include glycerides, fats and oils such as soybean oil and cottonseed oil; and saccharides such as starch, maltose, sucrose, glucose, and rhamnose.

Natural nutritional sources, including carbon sources, nitrogen sources and other nutritional substances, include bran molasses, including hightest molasses, refined molasses and xylose molasses: bagasse, corncow alfalfa, corn staple liquor, distiller's solubles, Flavor liquid, fishmeal, bran, meat extract, yeast, yeast extract, potato extract, malt extract, gluten, veptone,
glutamate, asparagine, glycine, casein,
Casein decomposition products, skim milk, vegetable oil meal such as soybean oak, rapeseed meal, sesame oak, flaxseed meal, and cottonseed slime, and oilseeds and oil fruits such as whole soybean, rapeseed, sesame, flaxseed, and cottonseed are included. Inorganic substances include nitrogen sources such as ammonium sulfate and ammonium chloride; potassium and phosphorus sources such as dipotassium hydrogen phosphate; salts such as iron, copper, magnesium, manganese, cobalt, zinc, and calcium; and ash of natural products such as molasses. can give.

Other vitamins can also be added if necessary. The composition of the medium is selected depending on the type of bacteria used, but
Carbon sources, nitrogen sources, potassium, phosphorus and magnesium are essential as 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, sorpitan monopalmitate, polyethylene glycol monostearate, and the like. The culture temperature is usually 20 to 40 o'clock, but the optimum culture temperature is around 30 to 35 o'clock.
The pH of the medium is usually adjusted to 5-10, preferably 6-9.

The microorganism used in the present invention is M. Since it belongs to the genus, it is well known that it can withstand pH levels close to 1 needle. 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 it is after dry sterilization or moist heat sterilization, or it is added as a solution after being dissolved in dimethylformamide or the like, or it is added as a suspension after being finely dispersed by ultrasonication. Further, it is preferable to add a surfactant at the same time because it promotes emulsification of substrate sterols and the like. Although the culture time is not particularly limited, the production amount of androstane-based compounds usually increases rapidly from about the third day after addition of raw materials such as sprawl.

Thereafter, the production amount of androstane compounds gradually increases with the culture time, but culturing for more than 20 days has little industrial significance. After completion of the culture, the androstane compound accumulated in the culture solution can be collected, separated and purified by known methods.

For example, an androstane compound from a culture solution is mixed with several times the amount of water such as ethyl acetate, extracted with one volume of an organic solvent, the solvent is distilled off from the extract, and the resulting crude mixed androstane compound is extracted. The above products can be separated by column chromatography using a porous resin, silica gel, alumina, etc. as an adsorbent and petroleum ether, benzene, chloroform, ether, acetone, methanol/ethyl acetate, etc. as a dust reagent. Depending on the culture conditions, certain androstane compounds, such as ADD or 4
It is possible to use AD as a product, but in that case, if you remove the solvent from the solvent extract of the culture solution, dissolve it in cyclohexane or hexane, and repeat recrystallization, ADD can be obtained without the need for column chromatography. Alternatively, you can obtain pure crystals of 4AD. According to the present invention, it is possible to produce androstane-based compounds with few by-products in good yield, 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 the following examples, the sterol androstane compounds were qualitatively and quantitatively determined by gas chromatography.

Also, in the following examples, percentages are by weight.

Example 1 Glucose 1.0%, Meat Extract 1.0%, Beptone 1.0%.
100ml of seed medium (pH 7.2) consisting of 0% M. 1 platinum loop was inoculated with Charahu Occidentum Complex MCI-0801 bacteria, and 1 platinum loop was inoculated at 30°C.
The seeds were cultured for 4 hours under the conditions of 20 reciprocations/min and an amplitude of 7 times.

This seed culture solution 2' was mixed with 4.0% crushed whole soybeans, NaNo.
30.2%, K2HP040.2%, M$04.7 days 2
Ten shouldered flasks containing 500 ml of main culture medium (pH 7.0, steam sterilized for 2 minutes at 12000) consisting of 0.00.1% cholesterol, 1.0% cholesterol and water are inoculated. Main culture is 30 ○, 12 main cycles/min, amplitude 7
The culture was carried out under the same conditions of reciprocating bonito shaking, and the culture was stopped at the 15th hour after the start of culture, and all the culture solutions were combined and extracted twice with 2 sleeves of ethyl acetate. This extract was filtered to remove insoluble matter such as bacterial cells, and the amount of steroids was analyzed using gas chromatography.The results showed that ADDO. 75th evening, 4ADO. 13th evening, DH
T.O. 03 evening, plan StoSterone0.02 evening, 5
Q-ATO. 01 evening, 58-l ADO. It was produced in 2002.

As a result of thin layer chromatography, the formation of 9Q-OH-oligoemia and 5Q-AD was confirmed at 0.01 ta or less. Example 2 Glucose 1.0%, Meat Extract 1.0%, Beptone 1.0%.
Seed solution (pH 7.2) consisting of 0% and water was dispensed into a 500' shouldered flask, and 15
After steam sterilization for a minute, M. Inoculate 1 platinum loop of Parafittum complex MCI-10802 with 30q○
Seed culture was carried out for 4 hours under the conditions of a reciprocating rope of 20 reciprocations/min and an amplitude of 7 cycles.

20% of this seed culture solution, 2.0% ground whole soybeans, 2% yeast
．． 0%, glycerin 4.0%, NaN030.2%, K
2HP040.2%, MgS04・7 days 200.1%,
Main culture medium 8 consisting of cholesterol 1.0% and water
00 I (pH 7.0, steam sterilized for 20 minutes at 120o0)
Inoculate a 6-capacity shoulder flask containing. The main culture is 300
Performed under the conditions of low main vibration/min at 0 and reciprocating vibration with an amplitude of 7 skin.
After 18 m from the start of culture, the cut culture was stopped and the culture solution was extracted with 2.4 volumes of ethyl acetate. This extract contains androstane compounds, including ADD2.65 and 4ADO.
．． 55 evening, DHTO. 05 evening, testosterone 0.05
Evening is generated, and further 5Q-AT, 58-IAD, DH
A, 5Q-AD generated a trace amount (less than 0.01 ta).

Example 3 In place of the substrate cholesterol in Example 2, a 2:1 mixture of B-sitosterol and cambesterol, stigmasterol, cholester 4-en-3-one, cholester 1,4-dien-3-one and cholesteryl oleate Example 2 is repeated except that .

The accumulated main product ADD and the required amount are shown in Table 1. Table 1ADD 4AD (9) (9)

Claims (1)

[Scope of Claims] 1. An androgen which is characterized by culturing Mycobacterium paraformitum complex MCI0801 or MCI0802 using sterols as a substrate. A method for producing an androstane compound selected from the group consisting of star-1,4-diene-3,17-dione and androst-4-ene-3,17-dione. 2. The method according to claim 1, wherein the sterols are sterols, C-3 ester derivatives thereof, C-3 ester derivatives thereof,
-3 ether derivatives or oxidized intermediates thereof.