JPS58138396A - Production of androstanes by use of microorganism - Google Patents

Abstract

PURPOSE:A mutant of a microorganism in Pseudonocardia is cultured in a medium containing sterol in the absence of any chemical inhibiting the oxidation of androstanes to produce androstanes. CONSTITUTION:A mutant in Pseudonocardia, e.g., Pseudonocardia methanoglynica DIC-94125 or DIC-94127, is cultured in a medium containing sterols in the absence of any chemical inhibiting the oxidation of androstanes to convert the sterols into androstanes. As the sterols, are used sterols, C-3 ester derivatives of sterols, C-3 ether derivatives of sterols or their oxidative intermediates. The converted androstanes are androst-4-ene-3,17-dione and androsta-1,4-diene-3,17- dione.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing anthrostane compounds using microorganisms. More specifically, a mutant strain of a microorganism called Shoot Socardia XK, which has the ability to convert sterol compounds to anthrostane compounds and the ability to accumulate this compound in the medium in the absence of an oxidation inhibitor, was used. ,
The present invention relates to a method for producing an anthrostane compound using a sterol compound as a raw material.

Conventionally, cholesterol, stigmatol, ergosterol, campesterol, sitostol, and other nosterol compounds and their dehydrogenated compounds (hereinafter abbreviated as sterol compounds) are used as raw materials to produce and- star 1,4-diene-3,
17-dione (hereinafter abbreviated as ADD), and-st-4-ene-5,17-dione (hereinafter abbreviated as 4-D), etc., to obtain an anthrostane-based compound in which the mu ring is dehydrogenated. is already known. When microorganisms capable of degrading sterol compounds are used to degrade sterol compounds as raw materials, various anthrostane compounds are produced as metabolic intermediates until complete decomposition. however.

Such and-1 stane compounds produced as intermediate metabolites are susceptible to further oxidation in the metabolic system of scissor microorganisms.
It is virtually impossible to accumulate it in high concentrations in the culture medium. Methods of inhibiting the oxidation of the anthrostane-based compounds produced there include a method of converting them in the presence of heavy metal ions such as nickel, cadmium, and cobalt (% Publication No. 46-17951), and a method of chelating them with iron or copper. A method of culturing in the presence of a compound capable of forming a compound (
% Publication No. 42-10842) and the like are known.

Another method is to use a mutant strain in which one of the anthrostane compound degrading enzymes of the wild strain is deleted or has low activity (Japanese Patent Laid-Open No. 52-10528).
No. 9) is known.

Conventionally, as a microorganism that has the ability to convert sterol compounds into anthrostane compounds using mutant strains as raw materials, Arthrobacter (Mrthrobaat@
r) Genus, Brevibacterium
lum) genus, Corynebacterium (Coryn@ba@
t@rtum) genus, Mycobacterium (My@*ba@
t@riwum) genus and Nocardia (Ne*ar41
m) The existence of horses, etc. is known. However, the book
! The existence of the II mutant strain of a microorganism belonging to the genus Shadeno*ludia used by @Kan et al. is completely unknown, and there is no single document.

The purpose of the present invention is to develop siatesocardia (Ps・1−・ron・axis r4
An object of the present invention is to provide a method for producing an anthrostane compound using a sterol compound obtained by using a mutant strain of a microorganism belonging to the genus 1m) as a new sterol compound as a raw material.

As a result of intensive research aimed at devising a novel microbial production method for selectively accumulating a large amount of and-stan-based compounds, Motoya Myoja Misaki developed a microorganism belonging to the genus Donocardia. By conducting random mutations, we discovered a mutant strain that accumulates a large amount of anthrostane-based compounds in the nutrient solution using Sta-based compounds as raw materials, and achieved the present invention.

That is, the present invention provides a method for converting sterol compounds, such as sterols, their c-5-ester derivatives, C-3-ester derivatives, or their oxidized intermediates, into anthrostane-based compounds, such as androst-4-ene-3. , 17-dione (hereinafter abbreviated as 4-D), androsta-1,4-diene-3,17-dione (hereinafter abbreviated as Mu-DD), etc., and has the ability to convert anthrostane-based compounds into Mutant strains of microorganisms belonging to the genus Schietosocardia that have the ability to accumulate skeleton compounds in the medium in the absence of oxidation inhibitors, such as Schietocardia methanolignica DIC-94125 and Schietosocardia DIC-94125;
The present invention relates to a method for producing an anthrostane compound using a sterol compound as a raw material, which is characterized by culturing No. 94127 or the like.

The present invention will be explained in detail below.

The microorganism belonging to the genus Shadenocardia used in the present invention is preferably a mutant strain belonging to the genus Pseudonocardia.

This microorganism belonging to Pseudonocardia methanolignica has extremely excellent mycological properties in the fermentation industry.

In other words, it has an optimal growth temperature at 42°C, and has a wide range of characteristic physiological properties such as using methanol-1-paraffin as the sole growth source in addition to carbohydrates as a carbon source, and is highly effective against sterol compounds. It is a microorganism with high decomposition activity. Pseudonocardia methanolignica is a strain isolated from the natural world by the present inventors.
Type culture collection Amsrlaan
TFII@Cu1ttIr*C@11@ctlom
) KATCCAs 1596, or microbial industry l... It has been deposited with the Research Institute of Technology as Microtechnology Research Institute No. 4747, and its mycological properties are also published in the UK ffl patent application publication 2,0
The content is the same as that published in No. 42,548 and Japanese Patent Publication No. 56-55552, so the description thereof will be omitted.

Regarding the conversion of sterol compounds to anthrostane compounds by microorganisms of the genus Shadenocardia, see Pseudonocardia, Methanolignica variety L.
M-145 or Pseudonocardia mutanoligni or Variety strain LM-1258, under special conditions such as the presence of oxidation inhibitors such as chelating agents, ADD and 4AD from sterol compounds, and more! The present inventors were the first to clearly demonstrate that anthrostane compounds such as testosterone and dehydrotestosterone are produced in small amounts.

(% Ganshoto 5 (No. 5-141097)) On the other hand, the mutant strain used in the present invention does not contain sterol compounds under normal culture conditions in the absence of chelating agents or oxidation inhibitors such as nickel and cobalt. It is clearly distinguished from the wild strain Shadenocardia methanolignicum TCC-41596 in that it converts and accumulates ADD and 4AD% of anthrostane compounds as raw materials.

A preferred mutant strain for 41 used in the present invention is Shadenocardia methanolignica ATCC-5159
Bacterium No. 6 (simply abbreviated as MuTCC-31596 No. 6) was used as the parent strain and was subjected to appropriate mutagenic treatment such as drugs such as N-methyl-N'-nitro-N-nitronganidine or ultraviolet irradiation. After, for example, MUDD or 4A
The seeds were sown on a flat plate agar medium using D as the sole growth source, and a colony with good growth was taken.

Furthermore (after mutagenic treatment of this strain with a mutagenic agent,
For example, if you isolate a strain that does not grow at all on a plate agar medium with MuDD as the only growth source, or a strain that grows only weakly, you will find that it is more likely that it will contain MuDD, which is a steryl compound, or a strain that grows poorly. A microorganism is obtained that has the ability to convert into anthrostane-based compounds such as D and accumulate them in the culture medium.

Therefore, Shadenocardia methanolignica DIC-94125 used in the present invention, DIC-94
Bacterium No. 127 (DIC-94125, DIC-941
Bacterium No. 27 (abbreviated as Bacterium ru) is the parent strain ATCC-515.
It is obtained by a two-step mutation method in which Bacterium No. 96 is made into a strain that has acquired resistance to A0D or 4muD and high degrading activity, and then made into a strain lacking 4AD and ADD degrading enzymes.

Among the anthrostane-based compounds, the mutant strain obtained in this way has the property of selectively accumulating 4AD, for example. The reason for this is that the 4muD present in the main KIIi strain, imacholest 4-en-3 This is thought to be due to the deficiency of 1- and 2-position dehydrogenase and the deficiency or low activation of 9α-position hydroxylase.

Conventionally, known mutant strains that produce 4AD include the genus Nocardia (Published in Japanese Patent Publication No. 54-67098) and Kencopactericum (Japanese Patent Laid-open Publication No. 55-85394). However, in these strains, 4muD cannot be obtained as the main product from sterol compounds, and 4muD
It also produces many other by-products such as KADD. Mye@b Mye@b
a*t@ri1uIIparafortuitmas
wpl*x) M CI-0807 Manga (Japanese Patent Publication No. 5
5-10 Publication), MuDDgF)ill production was also observed, and 1 and 2 position dehydrogenase, which is a 4AD degrading enzyme, was completely inactivated [but not many male hormones, anabolic hormones, and antihypertensives]. It is insufficient for the purpose of selectively producing 4-mu-D4, which is more suitable for producing a decongestant spinolactone or an anti-inflammatory agent.

The strain of the present invention has been deposited with the Microbial Technology Research Institute under the following number. That is, as a microorganism that has the ability to convert and accumulate sterol compounds into ADDK, Shootsocardia methanolignica DIC-94125 Manga (Imitation Koken Bacteria Collection No. 6250) is used as a microorganism that has the ability to convert and accumulate sterol compounds into 4AD. A typical strain is Shoot Socardia methanolignica DIC-94127 (Feikoken Bacteria No. 6251).

To further describe the parent strains of these strains used in the present invention, ATCC-31596 was isolated from nature by the present inventors as an actinomycete that has the ability to assimilate methanol and has a high vitamin B content. Shingeki stock,
Abundant snow-white air threads grow on the glucose-asparagine Ginten plate medium, and when observed on each Mutual medium, they look very similar to Streptomyces (8tr@ptomye@s), but the scanning electron - When observed with a microscope, aerial hyphae are 1
0 or more long spore chains branch irregularly and extend in a zigzag shape, with straight tips, and the apical growth seen in the process of forming a so-called plastobore is observed. Furthermore, meso-DAP (cyanopimelic acid) is a main component of the cell wall.

It is different from the genus Streptomyces because it contains arabinose and galactose, and Nocardia (N@card1m) because it grows on aerial mycelia consisting of long spore chains.
) is also different from the genus.

Therefore, it was determined that it belongs to the genus Shadenocardia. These mycological properties were described by Bergei's Maeil op Determinative Pacteriology (Il@rg@y's M
annual @fD@t@rminative la*
t*r1el*gy) 8th edition, it was recognized that it is a new fungal species belonging to the genus Shadenocardia, so we named it Schietosocardia methanoligi. - New from No. 51596 (isolated mutant strain DIC-94125 and DIC-
No differences were observed between Bacterium No. 9A127 and the parent strain in terms of colony formation on the agar field and orchidological properties.

Mutant strain DIC-94125 and DIC-9A1
The difference between No. 27 So and Parent No. Bacteria TCC-3159+6 manga is:
Naturally, the former occurs when a sterol compound is used as the conversion substrate.

ADD, 4AD as anthrostane compounds in the medium
This is a distinctly different bacteria in that the former accumulates the latter, while the latter does not accumulate at all.

The strain used in the present invention is the mutant strain DIC- which belongs to Pseudonocardia methanolignica.
Bacteria No. 94125 Sono and DIC-9J127 can be cited as examples of favorable conditions, but any mutant strain of the genus Shadenocardia that has the ability to accumulate anthrostane compounds using sterol compounds as raw materials can be used. be able to.

The anthrostane compounds obtained from the present invention include, for example, eva-and-4-sto-4-ene-3,17-dione, and preferably star-1,4-diene-5,17-dione.

As for the medium composition used in the present invention, a medium containing a carbon source, a nitrogen source, an inorganic salt, and other nutrient sources required by the bacteria used as shown in Example 1I can be used.

Examples of carbon sources include 1-paraffin, α-olefin, hydrocarbons such as xylene, methanol, ethanol,
Examples include alcohols such as glycerin and higher alcohols, organic acids and salts thereof such as succinic acid, acetic acid, and higher fatty acids, and saccharides such as starch, maltose, silucose, glucose, and rhamnose. Natural nutritional sources including carbon sources, i1 sources and other nutritional substances include 1, for example various types of molasses, cornstarch liquor.

Flavor liquid, fish meal, meat extract, yeast, yeast extract, potato extract.

Examples include malt extract. Examples of inorganic substances include dipotassium phosphate, sodium phosphate, magnesium sulfate, and trace metals. In addition, vitamins, etc. may be added as necessary.

If an antifoaming agent is required, a known antifoaming agent may be added.

Surfactants are effective as emulsifiers for sterol compounds, and are preferably added to the culture medium. Examples include polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, and polyethylene glycol monostearate. can.

The culture temperature is usually 25 to 50°C, preferably 37°C.
A temperature around ~45°C is optimal.

The pH of the medium is usually adjusted to 5-9, preferably 65-z5.

Examples of sterol-based compounds used as raw materials in the present invention are evacholesterol, stigmasterol, campestyrol, sitosterol, ergosterol, brassicasterol, heptosterol, lanosterol, agnosterol, dihydranosterol, dihydrotetraagnosterol -L, etc. Preferred sterols are cholesterol, campestol and cytosteol.

Sterol-containing natural and processed products, such as alkali-washed dark oils from fish oil and squid oil, as well as deodorized scum of vegetable oils, deodorized azalea, and tall oil, are also used as raw materials in the process of the invention. Furthermore, oxidized intermediates of various sterols can also be used as raw materials in the present invention. Examples of such oxidation intermediates include 4-en-3-one and 1,4-dinin-3-one derivatives of the various sterols.
1,4-dien-5-one, cholesta-4,22-dien-3-one, 22゜25-bisnorchola-5-cholenic acid-! ! , /-ol, 22.25-bisnorchola-1,4-dien-3-one-22-oic acid, gregnelone, grogesterone, and the like.

The sterol compound that is the raw material for the method of the present invention is generally added to the medium after the start of culture, but it may also be sterilized together with the medium. It is also possible to add in portions. in this case.

It can be added as is after dry sterilization or moist heat sterilization, or it can be added as a solution by dissolving it in dimethylformamide, etc., or it can be finely dispersed and suspended by ultrasonication and added as a suspension. be done. Further, it is preferable to add a surfactant at the same time because emulsification of the raw material sterol compound etc. is promoted.

Although the culture time is not particularly limited, the culture is usually carried out for 5 to 20 days, with the amount of anthrostane compound gradually increasing with the culture time after adding the raw material sterol compound.

There is little industrial significance in culturing for more than 20 days.

After the conversion reaction is completed, a commonly known method is used to collect the target anthrostane compound from the culture solution. For example, after the reaction is complete, the solution is extracted with an organic solvent such as ethyl acetate, lyroform, or hexane, and the solvent is removed from the extract. (Adsorbed with 1 petroleum ether, benzene, 4 μform, ethyl acetate, ether, methanol,
Elute and fractionate using an organic solvent such as ethanol. The eluate of the anthrostane compound thus eluted is forged, the solvent is distilled off, and then the anthrostane compound is crystallized from an organic solvent such as ethyl acetate or benzene. Also, add appropriate extracts (
It can also be obtained by concentrating, precipitating and removing a portion of impurities with 1m-hexane, etc., and then repeating recrystallization.

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 compounds, their derivatives, ADD, and 4D were qualitatively and quantitatively determined using thin-layer chromatography, infrared absorption, nuclear magnetic resonance, mass spectrometry, gas chromatography, and high-performance liquid talomad. Confirmed by analysis using graphics.

5J! Example 1 20g glucose, 5g sodium phosphate dihydrate.

Seed medium (pH 70) 100 consisting of dipotassium phosphate 7N, peptone 5Ii, cornstap liquor 5g, water 11
Dispense 117 into a 500-shouldered flask and heat at 121°C.
After sterilization for 15 minutes, one platinum loop of V. Eutosocardia methanolignica DIC-94125 was inoculated. 40
The mixture was shaken at ℃ for about 48 hours under ventilation, and cultured in al.

This seed culture 2@I is inoculated into a 500811 shouldered flask containing a main culture medium 100- having the same composition as the seed medium.

The main culture was carried out at 40° C. under the conditions of reciprocating shaking at 115 reciprocations/min and an amplitude of 7 degrees, and 500 da of presterul suspended in water was added 48 hours after the start of the culture.

The culture was stopped 120 hours after the addition of cholesterol, and all the culture solutions were combined and extracted with 10G of ethyl acetate.

After combining this extract and filtering out insoluble matter such as bacterial cells, the steroid content is determined using Gas Gro! Analysis by togelafy and high performance liquid chromatography revealed that Mu DD1251f and 4 Mu D151f were produced. Other anthrostane compounds are not produced in small amounts, and cholesterol decomposition intermediates prest-4-en-3-one and cholesta-1,4-dien-3-one are produced at 95q and 25q, respectively.
I generated q.

Example 2 β-sitosterol and campesterol were used instead of raw cholesterol in Fruit 111Afg 1 :
1 Mixed n, Steel/FF Steal, Ergosterol,
Example 1 is repeated with the exception of Cholest-4-ene-3-one, Prestar 1,4-diene-3-one, and length. The amount of accumulated products is shown in Table 1.
No.), the culture time of the cholesterol-added bales was increased to 144
Example 1 was repeated using the same method except for the time.

As a result, 4-muD15411 was produced, and no other anthrostane compounds were produced.Cholest-4-en-3-one was the only cholesterol decomposition intermediate produced.
4wtp was generated.

! Il! ! Example 4 Except that in place of the raw material cholesterol in Example 3, β-sitosterol and campesterol 2S II & compound, sticmasterol, ergosterol, and nylst-4-en-5-one were used. Example 3 was repeated in all the same manner. As a result, the amount of 4muD accumulated is shown in Table 2.

Table 2

Claims (1)

[Claims] t. And has the ability to convert sterol compounds into methane compounds, and accumulates the converted and-stan compounds in the medium in the absence of an oxidation inhibitor. 1. A method for producing an anthrostane compound using a sterol compound as a raw material, the method comprising culturing a mutant strain of a microorganism belonging to the genus Shadenocardia that has the ability to 2 The sterol compound is a sterol and a sterol
2. The method according to claim 1, wherein the compound is selected from C-5 and c-3 ester derivatives and oxidized intermediates thereof. (5) Anthrostane compound b ζ androst-4-ene-5,17-dione, androsta-1,4-diene-3゜17-dione The method described in item 1 or 2. A mutant strain of a microorganism belonging to the genus Shoottscabudia is Pseudonocardia methanolignica DIC-944.
No. 25. or DIC-94127, the method according to claim 1, 2, or 3.