JPH0120878B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing androstane compounds from sterols or derivatives thereof by fermentation using the conversion ability of microorganisms. Conventionally, androsta-1,4- Diene-3,17-dione (hereinafter referred to as ADD)
), androst-4-ene-3,17
-dione (hereinafter abbreviated as 4AD), 17β-hydroxyandrosta-1,4-dien-3-one (dehydrotestosterone) (hereinafter abbreviated as DHT), or 17β-hydroxyandrost-1,4-dien-3-one (dehydrotestosterone) (hereinafter abbreviated as DHT)
4-en-3-one (testosterone) (hereinafter
It is already known to obtain androstane-based compounds (hereinafter abbreviated as androstane-based compounds) in which the A ring is dehydrogenated, such as (abbreviated as TSN). When sterols are oxidized using microorganisms that can decompose them, androstane compounds are produced as metabolic intermediates until complete decomposition. However, androstane compounds produced as intermediate metabolites are more susceptible to oxidation, and it is virtually impossible to accumulate them in a medium at a high concentration. Methods of inhibiting the oxidation of the androstane-based compounds generated therein include a method of converting them in the presence of heavy metal ions such as nickel, cadmium, and cobalt (Japanese Patent Publication No. 17951/1983), 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 (Japanese Patent Publication No. 10862/1986) is known. As another method, a method using a mutant strain in which one of the androstane compound degrading enzymes possessed by the wild strain is deleted or has low activity is known (Japanese Unexamined Patent Publication No. 105289/1989). ing. An object of the present invention is to provide actinomycetes belonging to the genus Pseudonocardia, which have not been widely used in steroid conversion reactions, as new converting bacteria. Conventionally, microorganisms that have the ability to produce androstane compounds from sterols include the genus Arthrobacter, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, and the genus Microbacterium. The existence of genera such as Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Seratia, and Streptomyces is known. However, the existence of actinomycetes belonging to the genus Pseudonochaldeia is completely unknown. The present inventors conducted various studies on methods for culturing actinomycetes using methanol as the main carbon source, and as a result, they found that they prefer to assimilate methanol, and the bacterial cell yield relative to methanol is also good.
In addition, actinomycetes were discovered in nature that have the ability to convert sterols into androstane compounds with high activity, leading to the completion of the present invention. Regarding the actinomycetes of the genus Pseudonocardia that can assimilate methanol, Pseudonocardia methanolignica (Pseudonocardia methano
-lignica) and Pseudonocardia yukotes (Japanese Unexamined Patent Publication No. 55-96091). These species of the genus Pseudonochaldeia, which are able to preferentially assimilate methanol, have extremely distinctive morphological and physiological properties. The present invention was made against this technical background. The present inventors have widely used the genus Seudonochaldeia in the natural world, established an isolation method, and succeeded in isolating a large number of strains. The actinomycetes used in the present invention are Pseudonocardia var. LM-145 (Pseudonocardia var.
methanolignica variety LM-145) (Feikoken Bacteria No. 6057) and Pseudonochaldeia methanolignica variety LM-1258
(Pseudonocardia methanolignica variety LM
-1258) (Feikoken Bibori No. 6058) is a genus of Pseudonochaldeia. Next, the present inventors isolated and collected Pseudonochaldeia methanolignica variety LM-145 (hereinafter simply LM-145) to be used in the method of the present invention.
(sometimes abbreviated as 145), Pseudonochaldeia methanolignica variety LM-
The mycological properties of two strains of 1258 (hereinafter sometimes simply referred to as LM-1258) will be described. It should be noted that among mycological properties, those not specifically described by bacterial species are properties and growth conditions common to each bacterial species. A. Morphological characteristics Vegetative hyphae develop well on both synthetic and natural media, and abundant white aerial hyphae are attached on glucose-asparagine agar plates. When observed under a microscope, aerial hyphae consist of 10 or more long spore chains that branch irregularly, sometimes elongating in a zigzag pattern, and their tips are straight. As a result of observation using a scanning electron microscope, spores are 0.4 to 0.6×
It has a cylindrical or oblong shape of 1.0 to 1.8 microns. In addition, crestal growth is observed in the process of forming so-called blast spores. The surface of the spore is smooth. No sclerotia, sporangia and zoospores are found. B Properties on various media Properties on various media were observed after culturing at 37°C for 14 days unless otherwise specified. (1) Seuculose/nitrate agar medium: Good growth, with white aerial mycelia attached. The back side shows a pale yellow color and no soluble pigment is produced. LM
-145 strain has slightly inferior growth. (2) Asparagine-glucose agar medium: Abundant growth, with faint white aerial mycelia growing around the thick cover. The back side shows a pale yellow color and no soluble pigment is produced. (3) Glycerin/asparagine agar medium: Produces abundant growth and forms a thick cover. LM−
The 1258 strain causes swelling and wrinkles. Faint white aerial hyphae grow on the periphery, and the underside is deep pale yellow, and no soluble pigment is produced. (4) Starch/inorganic salt agar medium: Medium growth, powdery white aerial mycelia are grown on one side, the back side is ivory-colored, and no soluble pigment is produced. (5) Tyrosine agar medium: It grows abundantly, forms a cover and swells, wrinkles and cracks form. White aerial mycelium grows on one side, and the underside is pale yellow. The LM-1258 strain exhibits a pale yellow color with a hint of brown. Soluble dyes are not produced together. (6) Nutrient agar medium: Abundant growth, forming faint white aerial hyphae. The back side is yellow and no soluble pigment is produced. (7) Yeast/malt agar medium: Abundant growth, cracks appear on the colony surface,
White aerial mycelium grows on the whole surface. The underside is pale yellow and does not produce soluble pigments. (8) Oatmeal agar medium: Medium growth, abundant white aerial mycelium. The underside is ivory to yellow in color and produces no soluble pigment. (9) Glucose-peptone-gelatin medium (28
℃): Abundant growth, bulges and wrinkles. Aerial mycelium does not grow. No soluble dyes are produced. (10) Pridham-Godelive agar medium: Medium growth, with white aerial mycelia growing all over. The underside is ivory to pale yellow. No soluble dyes are produced. (11) Peptone yeast iron agar medium: Abundant growth, showing mounded growth, wrinkled and cracked. There is no aerial mycelium, the underside is pale yellow, and no soluble pigment is produced. C Physiological properties (1) Growth temperature range: 25-47℃ (LM-145 strain) 25-45℃ (LM-1258 strain) (2) Optimal growth temperature: 37-44℃ (LM-145 strain) 35-45℃ 43°C (LM-1258 strain) Both strains show growth at 43°C as good or better than at 37°C. (3) Liquefaction of gelatin: Positive (4) Hydrolysis of starch: Negative (5) Coagulation of skim milk: Negative (6) Peptonization of skim milk: Positive (7) Production of melanin-like pigment Tyrosine agar medium: Negative Peptone・Yeast/iron agar medium: Negative (8) Nitrate reducing ability: Positive (9) Carbon source assimilation ability: (Pridham-Godelive agar medium) (a) Assimilated: L-arabinose, D-
xylose, D-glucose, D-fructose, inositol, L-rhamnose,
D-mannitol, methanol, ethanol, n-paraffin (b) Non-assimilable substances: seuculose, raffinose (10) Chemical composition of cell wall [method of Rittibaria et al. (International Journal of Systematic
Bacteriology Vol 20, 435-443 (1970). (a) Main component of cell wall: Diaminopimelic acid is meso-type and does not contain glycine, but contains arabinose and galactose. (b) Cell wall type: Cell wall type: Whole cell sugar pattern:
Type A D Isolation source: Soil As mentioned above, since the above two strains have cell wall composition and sugar composition of type A and type A, they belong to the genus Mycobacrium and Nocardia.
Genus, Micropolyspora,
Genus Pseudonocardia,
It is thought to belong to the genus Thermomonospora. However, in its morphology, 10 or more long spore chains are irregularly branched, and the spores are cylindrical or oblong, with straight tips. Pseudonocardia, because its spores are characterized by the formation of blastopores.
It is appropriate to consider it as a genus (Arch.Fur
Microbiology Vol, 26, 373-414 (1957)).
A known bacterial species of the genus Seudonochaldeia is Seudonochaldeia thermophila ATCC-19285.
(Pseudonocardia thermophila ATCC−19285),
Syudonochaldeia spinosa ATCC−25924
(Pseudonocardia spinosa ATCC−25924), Pseudonocardia spinosa ATCC−
31181 (Pseudonocardia Fastidiosa ATCC−
31181), Pseudonochaldeia methanolignica
ATCC−31596 (Pseudonocardia methanolignica
ATCC-31596), Pseudonocardia yukotes ATCC-31597 (Pseudonocardia yukotes)
ATCC-31597), Pseudonocardia SP.AM-3696
(Japanese Unexamined Patent Publication No. 115894, 1982) is known. The strain used in the present invention has a growth temperature range of 25 to 47°C, can grow using methanol and n-paraffin as a single carbon source, and can liquefy gelatin and peptonize skim milk. Since it is positive, it has very similar mycological properties to S. methanolignica (see JP-A-55-96091). However, since there were large differences in cholesterol decomposition activity and ADD and 4AD production, it was recognized as a variant of Pseudonocardia methanolignica, and Pseudonocardia methanolignica var. LM-145 (Pseudonocardia methanolignica var. LM-145)
variety LM-145), Pseudonochaldeia methanolignica variety LM-1258
(Pseudonocardia methanolignica variety LM
−1258). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under microbial accession number 6057 (LM-145) and 6058 (LM-1258). The strains used in the present invention include the above-mentioned 2
Bacterial strains can be cited as preferred examples, but any bacterium belonging to the genus Pseudonochaldeia that accumulates androstane compounds can be used. As for the composition of the medium used, any medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic salt, and other nutrient sources required by the bacteria used as shown in the examples. Carbon sources include hydrocarbons such as n-paraffin, α-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, starch, and maltose. Examples include sugars such as sucrose, glucose, and rhamnose. carbon source,
Natural sources of nutrients, including nitrogen sources and other nutrients, include various types of molasses, cornstarch liquor,
Examples include flavor liquid, fish meal, meat extract, yeast, yeast extract, potato extract, and malt extract. Inorganic substances include dipotassium phosphate, sodium phosphate,
Magnesium sulfate etc. can be used. In addition, vitamins can be added as necessary. The composition of the medium is selected depending on the type of bacterial strain used, but a carbon source, nitrogen source, potassium, phosphorus, and magnesium are essential components of the medium. If an antifoaming agent is required, a known antifoaming agent may be added. Surfactants are effective as emulsifiers for sterols, and are preferably added to the culture medium. Specific examples of the surfactant include polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, and polyethylene glycol monostearate. The culture temperature is usually 25 to 50℃;
The optimum temperature is around 37-43℃. The pH of the medium is usually adjusted to 5-9, but it should be 6.5-7.5.
is preferred. Sterols used as raw materials in the present invention include cholesterol, stigmasterol, campesterol, sitosterol, ergosterol, brassicasterol, fucosterol, lanosterol, agnosterol, dihydrolanosterol, dihydroagnosterol, and the like. Preferred sterols are cholesterol,
campesterol and sitosterol.
Also alkaline washed dark oils from fish oil and squid oil,
Furthermore, sterol-containing natural and processed products, such as deodorized scum of vegetable oils, deodorized sludge, and tall oil, are likewise 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 or 1,4-dien-3-one derivatives of various sterols, and specifically, for example, cholest-4-en-
3-one, cholesta-1,4-dien-3-one, cholesta-4,22-dien-3-one, 22,
23-bisnorcholar-5-cholenic acid-
3β-ol, 22,23-bisnorchola-1,4-
These include diene-3-one-22-oic acid, pregnenolone, and progesterone. In the present invention, it is preferable to add an oxidation inhibitor of androstane compounds in order to accumulate the androstane compounds in the medium at a high concentration. As such an oxidation inhibitor, for example, salts such as nickel, cobalt, cadmium, etc. or chelating agents are used. Examples of chelating agents include 2,
2'-dipyridyl, 1,10-phenanthroline, 8
-Hydroxyquinoline, cuberone, isonicotinic acid hydrazitone, orthophenylenediamine,
Examples include sodium N,N-diethyldithiocarbamate. It is also possible to use two or more of these chelating agents in combination. The addition of sterols or their 4-en-3-one or 1,4-dien-3-one derivatives as raw materials is carried out after bacterial cell growth. Its concentration is usually 0.5 to 5 g/1 to 1 to 5 g per medium volume.
A range of 3g/ is preferred. The oxidation inhibitor is added simultaneously with the addition of the substrate or several hours after the addition of the substrate. The concentration of the oxidation inhibitor added is selected depending on the type and medium composition;
A suitable amount is 1×10 ⁻¹ to 1×10 ⁻³ mol based on the amount of normal medium. Cultivation is performed under aerobic conditions and lasts for 16-120 hours. After the conversion reaction is completed, a commonly known method is used to collect the target androstane compound from the culture solution. For example, after the reaction is completed, the liquid is extracted with an organic solvent such as ethyl acetate, chloroform, or n-hexane, and after removing the solvent from the extract, it is adsorbed on a column packed with an adsorbent such as silica gel or alumina, and then petroleum ether , elute and fractionate using a solvent such as benzene, chloroform, ethyl acetate, ether, methanol, or ethanol. The androstane compound eluted in this manner is obtained by concentrating the eluate, distilling off the solvent, and then crystallizing it from an organic solvent such as ethyl acetate or benzene. It can also be obtained by appropriately concentrating the extract, precipitating and removing some of the impurities with n-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, sterols, their derivatives, and androstane compounds were qualitatively and quantitatively determined by thin layer, chromatography, infrared absorption, nuclear magnetic resonance, mass spectrometry, gas chromatography, and high performance liquid chromatography. Analysis confirmed. Example 1 100 ml of a seed medium (PH7.0) consisting of 10 g of glucose, 5 g of polypeptone, 3 g of yeast extract, 1 g of malt extract, and 1 part of water was dispensed into a 500 ml flask with a shoulder.
After sterilization at 121°C for 15 minutes, one platinum loop of Pseudonochaldeia methanolignica variety LM-145 was inoculated. Seed culture was performed by shaking at 40°C for about 16 hours under ventilation. Glucose 10g, Ammonium chloride 4g, Potassium phosphate 7g, Sodium phosphate dihydrate 3
g, magnesium sulfate 0.5g, ferric chloride hexahydrate 1mg, copper sulfate pentahydrate 0.1mg, zinc sulfate heptahydrate 0.1mg, manganese sulfate heptahydrate 0.1mg, cobalt chloride hexahydrate Seeds obtained as above were placed in a 500 ml shouldered flask containing 100 ml of main culture medium (PH 7.0) containing 1 mg of yeast extract, 0.03 g of yeast extract, 1 g of polypeptone, 1 portion of water and sterilized for 15 minutes at 121°C. Inoculate 2 ml of culture. The main culture was carried out at 40℃, and 18 hours after the start of culture, sterols suspended in 2 ml of water were added.
Add 100 mg, and then add 0.1 ml after 24 hours from the start of culture.
2,2'-dipyridyl dissolved in ml methanol
15.62mg was added. The culture was stopped 42 hours after the addition of 2,2'-dipyridyl, the culture solution was extracted with ethyl acetate, and the amounts of ADD and 4AD produced were measured by gas chromatography and high performance liquid chromatography. Sterols include cholesterol, stigmasterol, a 2:1 mixture of β-sitosterol and campesterol, and cholesterol-4.
Table 1 shows the results when -en-3-one and cholesta-1,4-dien-3-one were used.

[Table] Example 2 Eudonochaldeia methanolignica variety LM-, which was seed cultured in the same manner as in Example 1.
A medium in which 2 ml of the culture solution of 145 was substituted with 1% methanol for the glucose in the main culture medium shown in Example 1.
Inoculate 100ml. Culture was carried out at 40°C, and 200 mg of cholesterol was added 48 hours after the start of culture, and 15.62 mg of 2,2'-dipyridyl was added 54 hours after the start of culture. The culture was stopped 16 hours after the addition of 2,2'-dipyridyl. After that, the production amounts of ADD and 4AD were measured in the same manner as in Example 1, and it was found that ADD
It produced 18 mg and 9.5 mg of 4AD. Example 3 Seed culture and main culture of Pseudonochaldeia methanolignica var. LM-1258 were carried out according to the method shown in Example 1, and 100 mg of cholesterol was added 18 hours after the start of culture, and further cultured for 24 hours.
At hour 15.62 mg of 2,2'-dipyridyl was added.
The culture was stopped 72 hours after the addition of 2,2'-dipyridyl, and the amount of androstane compounds in the ethyl acetate extract was measured.
16.4mg of DHT, 4.4mg of DHT, and 3.9mg of TSN were produced.

Claims (1)

[Scope of Claims] 1. A microorganism of the genus Eudonocardia having the ability to convert sterols or a dehydrogenated derivative in the A ring thereof into an androstane compound is cultivated, 1. A method for producing an androstane compound using a microorganism, the method comprising converting a dehydrogenated derivative into an androstane compound and collecting the same. 2 The microorganism is Pseudonochaldeia methanolignica var. LM-145 or Pseudonochaldeia methanolignica var. LM-145
-1258. 3 Androstane-based compounds are
1,4-diene-3,17-dione, androst-4-ene-3,17-dione, 17,β-hydroxyandrost-1,4-dien-3-one,
17, β-hydroxyandrost-4-ene-3
3. The method according to claim 1 or 2, characterized in that - on.