JPH09107982A - Biologically pure cultured substance of microorganism, method for forming lactone, method for forming diol, method for forming compound and method for forming cyclic ether - Google Patents

Abstract

PURPOSE: To obtain a culture product comprising a diol for a synthetic raw material, etc., of a perfume-relating compound, sclareolide, by culturing new specific bacteria in an aqueous nutritious medium containing sclareol, episclareol, etc., under an aerobic condition.

CONSTITUTION: A culture product comprising a diol having a structure expressed by formula III is obtained by culturing new microorganisms comprising Bensingtonia-ciliata (ATCC20919) and Cryptococcus laurentii (ATCC20920) in an aqueous nutrition medium containing sclareol expressed by formula I and/or episclareol expressed by formula II under an aerobic condition at pH 2.5-9.0, 12-33°C and 0.1-130g/l compound concentration. The diol is used as a synthetic intermediate, etc., of a sclareolide which is a lactone compound expressed by formula IV and useful as a perfume-relating compound.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel microorganism and the production of a perfume-related compound using the same.

[0002]

PRIOR ART,

[Chemical formula 10] Sclareolide of the formula, which has its own fragrance but is an important substance used in perfumes,

[Chemical formula 11] Was found as a valuable intermediate in the production of ether compounds.

[0003] US Patent No. 4,798,799 describes the compounds related to the present application as follows. formula,

[Chemical formula 12] Are useful as intermediates in the production of sclareolide and as precursors for the ether compounds. Also, the formula,

[Chemical formula 13] Is described as being useful as an intermediate in the production of the diol.

Indeed, this US patent describes the microorganism Hyphoz.
yma roseoniger ATCC 20604 is the formula,

[Chemical formula 14] Are capable of converting sclareol, as well as compounds including the cyclic ethers, to the diols, and discloses the use of cultures of this microorganism.

However, this prior art includes the following equation (i):

[Chemical formula 15] Reaction through a microorganism to obtain lactone in relatively high yield, formula (ii),

[Chemical formula 16] Reaction of Bensingtonia ciliata, ATCC 20919 or Cryp
performing through a characteristic method using tococcus laurentii ATCC 20920; and (iii)

[Chemical formula 17] There is no teaching or suggestion that the reaction of Cryptococcus laurentii ATCC 20920 would be microbial.

[0006] Furthermore, the microorganisms of the present invention are all novel.

[0007]

SUMMARY OF THE INVENTION The present invention relates to biologically pure cultures of the following microorganisms. Cryptococcus albidus, ATCC 20918, Bensingtonia ciliata, ATCC 20919, Cryptococcus laurentii, ATCC 20920, and Cryptococcus albidus, ATCC 20921

In another embodiment, the present invention is directed to a culture of the following microorganisms: Cryptococcus albidus, ATCC 20918, Bensingtonia ciliata, ATCC 20919, Cryptococcus laurentii, ATCC 20920, and Cryptococcus albidus, ATCC 20921

[0009] Each of these cultures, under aeration conditions in an aqueous nutrient medium, has the following structure:

[Chemical formula 18] The following structure of sclareolide

[Chemical formula 19] Either can be produced as follows. Cryp
tococcus albidus, ATCC20918 and Cryptococcus albidu
s, ATCC 20921 has the following structure:
l] and

[Chemical formula 20] From a mixture with episclareol of the following structure

[Chemical formula 21] Sclareolide of the following structure

[Chemical formula 22] Can produce also Bensingtonia ciliat
a, ATCC 20919 and Cryptococcus laurentii, ATCC 2092
0 is sclareol of the following structure,

[Chemical formula 23] From episclareol of the following structure

[Chemical formula 24] A diol of the following structure

[Chemical formula 25] Can be produced.

In yet another embodiment, the invention also relates to a mixture produced by (individually) culturing the following microorganisms under aeration conditions in an aqueous nutrient medium. ATCC
20918, Cryptococcus albidus, ATCC 20919, Bensingto
nia ciliata, ATCC 20920, Cryptococcus laurentii, ATC
C 20921, Cryptococcus albidus, and above

In still another embodiment, the present invention provides (1) the following structure

[Chemical formula 26] And / or of the following structure

[Chemical formula 27] A sclareolide having the following structure, characterized by culturing either a microorganism such as Cryptococcus albidus of ATCC 20918 or Cryptococcus albidus of ATCC 20921 under aeration conditions in an aqueous nutrient medium containing one or more compounds.

[Chemical formula 28] The process of the production method.

In yet another embodiment, the invention relates to a process for producing a diol having the structure:

[Chemical formula 29] (a) ATCC 20919, Bensingtonia ciliata, or (b) AT
Any of microorganisms such as CC 20920 and Cryptococcuslaurentii, (1) Sclareol having the following structure

[Chemical formula 30] (2) Episclareol having the following structure

[Chemical formula 31] (3) Acetate with the following structure

[Chemical formula 32] Characterized by culturing under an aerated condition in an aqueous nutrient medium having one or more compounds selected from the group consisting of:

A still further embodiment of the invention provides a method for producing a cyclic ether of the following formula by culturing Cryptococcus laurentii, ATCC 20920 in an aqueous nutrient medium containing episclareol and / or sclareol under aeration conditions. It is characterized by doing.

[Chemical formula 33] Here are the possible reactions:

[Chemical formula 34] as well as,

[Chemical formula 35]

The conversion process of the compound has the following structure

[Chemical formula 36] Cryptococcus albidus of ATCC 20918, ATCC 20919 in an aqueous nutrient medium in the presence of one or two or all of the compounds.
Bensingtonia ciliata, ATCC 20920 Cryptococcus la
urentii, or Cryptococcus albidus of ATCC 20921.

Thus, these compounds can be used alone or as a mixture containing any number of the above compounds.

Thus, Cryptococcus albid of ATCC 20918
When the reaction is performed using us, or Cryptococcus albidus of ATCC 20921, the following reaction can be caused.

[Chemical formula 37] And / or

[Chemical formula 38]

When the reaction is carried out using Bensingtonia ciliata of ATCC 20919 or Cryptococcus laurentii of ATCC 20920, the following reaction can be caused.

[Chemical Formula 39] And / or

[Chemical formula 40]

The form of the microorganism used is not critical. The microorganism can be used as a culture (suspension) containing the cells and a nutrient solution suitable for the cells, or in the form of cells suspended in a buffer. The cells or their extracted enzymes can be immobilized on a suitable solid support, and they can be used for compound conversion.

The suspension culture mixture is prepared by inoculating a suitable aqueous nutrient medium with the microorganism. Suitable nutrient media include those that contain a nitrogen source, inorganic salts, growth factors, suitable substrates, and optionally other carbon sources. Suitable types of carbon sources for carrying out the method of the present invention include, for example, glucose, galactose, L-sorbose, maltose,
Sucrose, cellobiose, trehalose, L-arabinose, L-rhamnose, ethanol, glycerol,
L-erythritol, D-mannitol, lactose,
Melibiose, raffinose, melezitose, starch, D-xylose, D-sorbitol, a-methyl-
There are D-glucoside, lactic acid, citric acid, and succinic acid. Suitable nitrogen sources include, for example, nitrogen-containing organic substances such as peptone, meat extract, yeast extract, corn starch liquor, casein, urea, amino acids, or nitrogen-containing inorganic substances such as nitrates, nitrites, inorganic ammonia salts. There is a compound. Suitable inorganic salts are, for example, magnesium, potassium, calcium or sodium phosphates.
The nutrients of the above medium can optionally be supplemented with one or more of the B vitamins and / or one or more trace minerals such as Fe, Mo, Cu, Mn and B. Vitamins and trace minerals are not needed when small amounts of yeast extract are added to the medium. When bacterial contamination becomes a problem, chloroamphenico
l] or an antibiotic such as chlorotetracycline is preferably added.

The microorganism can be cultured as a static culture or as a submerged culture under aeration conditions (eg, stirring culture, culture using a fermentor). Some can be carried out at a pH in the range of about 2.5 to 9.0, but preferably in the range of about 3.0 to 7.5, and most preferably in the range of about 3.0 to 6.5. The pH value can be adjusted by adding an inorganic or organic acid such as hydrochloric acid, acetic acid, oxalic acid, or by adding a base such as sodium hydroxide, ammonium hydroxide, or phosphate. Alternatively, it can be adjusted by adding a buffer such as phthalate. Incubation temperatures must be maintained between about 12 ° C and about 33 ° C, more preferably between about 15 ° C and 30 ° C, optimally between about 18 ° C and 28 ° C.

The method according to the present invention comprises a compound having the following structure:

[Chemical formula 41] And / or

[Chemical formula 42] This is preferably performed by adding one or a mixture of the above as a single carbon source to the nutrient medium at the start of the culture. Alternatively, during culture or when the carbon source is depleted, the substrate may be added in combination with another carbon source, such as dextrose. The only limitation on the substrate concentration in the medium is that the culture can be effectively exposed to air. However, the substrate concentration is preferably from about 0.1 g / l to about 13 g / l.
0 g / l, more preferably from about 0.5 g / l to 120 g / l, optimally from about 2.5 g / l to about 100 g / l. Conversion of the compound can be optimally performed under any of the above circumstances.

Total time for compound conversion (after initial culture period)
Depends on the composition of the nutrient medium and the substrate concentration.
In general, culture using a shake flask is performed for about 12 hours to about 26 hours.
It takes 4 hours. However, when fermentors are used, the incubation time is reduced to about 48 hours or less.

The conversion of the compounds is carried out using microbial cells isolated from the culture, or with an extraction enzyme isolated from the cells in a manner well known in the art. In this case, the conversion of the compounds is expediently carried out in various aqueous nutrient media, for example in buffer solutions, physiological saline solutions, in fresh nutrient media or in water. The isolated cells or extracted enzymes are immobilized on a solid support to achieve conversion of the desired compound. The conversion of the substrate compound is also affected by mutants of this organism (microorganism). Such mutants can be obtained simply by well-known methods, for example, by exposing the cells to ultraviolet light or X-rays or known mutagens, such as, for example, acridine orange.

The substrate may be as a powder or TWEEN 80 ™ (polyoxyethylene sorbitan monooleate)
As a solution in an emulsifier, or as a solution in an emulsifier, or as a solution in a hydrophilic solvent such as acetone, methanol, ethanol, ethylene glycol, or dioxane. Surfactants or dispersants can also be added to the aqueous suspension of the substrate, or the substrate can be emulsified using ultrasound.

A readily available defoamer such as silicone oil (eg, UCON), polyalkylene glycol derivatives, corn oil, or soybean oil can be used to suppress foaming.

The conversion of the substrate is carried out by gas-liquid chromatography (G
LC), thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC), infrared spectrum (IR), and nuclear magnetic resonance
It can be monitored using standard analytical methods such as (NMR). If rapid disappearance of the substrate is observed, more substrate can then be added to maximize the ability of the microorganism to convert the compound. Generally, this process is terminated when most of the substrate has disappeared from the medium. Depending on the type of microorganism used, a compound of the following structure,

[Chemical formula 43] Or a compound of the following structure

[Chemical formula 44] It can be recovered from the aqueous nutrient medium. The compound of the following structure

[Chemical formula 45] As described in U.S. Pat.No. 4,798,799 at column 8, line 52, line 53, which is cyclized to a compound of the following structure (incorporated herein by reference): You.

[Chemical formula 46] Compounds of the following structure

[Chemical formula 47] Also, it may be used because of its aroma value, or it can be used for a compound having the following structure

[Chemical formula 48] It may be reduced through the following reaction.

[Chemical formula 49]

The formation of a compound of the following structure

[Chemical formula 50] First, a compound with the following structure

[Chemical formula 51] Reduced to a diol of the structure

[Chemical formula 52] The diol is then reclosed to produce a compound of the following structure:

[Chemical formula 53] This is due to the following reaction.

[Chemical formula 54] and

[Chemical formula 55]

Preferably, the reduction reaction is

[Chemical formula 56] Reducing agents such as VITRIDE®-U.S. Pat.
It is carried out in the presence of an inert solvent such as toluene using hydrogenated bis (2-methoxyethoxy) aluminium hydroxide as described in 7,895. This reaction is preferably carried out at a temperature of about 70 ° C. to about 90 ° C. for about 1 hour to about 3 hours.

The ring closure reaction, ie,

[Chemical formula 57] Preferably, the reaction is carried out under basic conditions such as aqueous potassium hydroxide or aqueous alkali metal hydroxide such as aqueous sodium hydroxide. The details will be described in Example 8 described later. Isolation and purification of the following compounds from fermentation broth

[Chemical Formula 58] Or

[Chemical formula 59] It can be performed by conventional techniques such as filtration, centrifugation, solvent extraction, distillation, crystallization and the like.

The compound having the following structure is

[Chemical formula 60] U.S. Pat.No. 4,798,799, column 8, lines 58-68, U.S. Pat.No. 4,798,799, column 9, lines 1-2, according to commonly used cyclization methods well known to those skilled in the art, Can be converted to a compound of the structure

[Chemical formula 61]

Each of the microorganisms used in the present invention was isolated from soil samples obtained from various geographic locations.
Each strain was deposited with the American Type Culture Collection [ATCC] under the following accession numbers. Cryptococcus albid
us, ATCC 20918; Bensingtonia ciliata, ATCC 20919; Cr
yptococcus laurentii, ATCC 20920; and Cryptococcus
albidus, ATCC 20921

Bensingtonia ciliata and Cryptococcus lau
Both microorganisms of rentii were examined at Central Bureau Vauxmere Cultures (CBS), which gave them the following names: Lecythophere hoffmannii (van Beijma), W. Gams (synonym Phialophora hoffmannii) because according to CBS this is a filamentous fungus.

Cryptococcus albidus, ATCC 20918 is also CBS
CBS was used to clarify this culture as Cryptococcus albidus.
var. albidus (Saito) Skinner. Cryptococcu
S. albidus, ATCC 20918, is described as follows. Morphology: Growth in liquid medium revealed monopolar budding cells. A thin film appeared on the liquid surface, while heavy sediment was observed. Growth on solid agar was single cells, with white to slightly pinkish, very shiny, bright, gooey, round, well-defined colonies. No pseudohyphae was formed on cornmeal agar. Physiology and biochemistry: carbon fixation: carbon fixation: (proliferation) (proliferation) glucose + D-ribose unknown galactose + L-rhamnose + L-sorbose + D-glucosamine + maltose + ethanol unknown sucrose + erythritol-cellobiose + glycerol unknown trehalose + Adonitol (ribitol) + lactose + dulcitol (galactitol) + melibiose-D-mannitol + raffinose + D-sorbitol (glucitol) + meletitose + a-methyl-D-glucoside + inulin-salicin + D-soluble starch-inositol-D Xylose-lactate-L-arabinose-citrate-D-arabinose-succinic acid + grow at 30 ° C. + grow at 37 ° C.-growth without vitamins-albu Tin splitting + Nitrogen assimilation: NH4NO3 + KNO3 + NO2 + ethylamine + enzyme (acid formation) glucose-galactose-maltose-sucrose-lactose-raffinose-melibiose-inulin-cellobiose-meletitose-starch-trehalose

Bensingtonia ciliata, ATCC 20919 is described as follows. Morphology: On yeast maintenance broth (ATCC medium # 200), cells are spherical with 1-3 buds per cell. On solid medium the cells become filamentous with the produced spores produced. The colonies are salmon tan, flat, dull, but have well-defined boundaries. Cornmeal agar (ATCC medium
# 307), a true mycelium was observed after 3 weeks. Physiology: carbon fixation: glucose + D-ribose-galactose + L-rhamnose + L-sorbose + D-glucosamine + maltose + ethanol weak sucrose + erythritol + cellobiose + glycerol + trehalose + adnitol lactitol (ribitol) lactitol ) Weak melibiose + D-mannitol weak raffinose + D-sorbitol (glucitol) weak meletitose + a-methyl-D-glucoside weak inulin-salicin weak soluble starch + inositol-D-xylose + lactic acid-L-arabinose + citric acid -Arabinose-succinic acid weak vitamin-free growth-nitrogen assimilation: NH4 NO3 weak growth at high temperature: 30 ° C weak /-37 ° C-

Taxonomy: Bensingtonia ciliata CT Ingold (See Note below) Fungi Imperfecti Fungus of ejected spores (spores that are vigorously repelled) This ejected spore is colorless, 2 × 5μ, almost 8 × 5μ oval in liquid medium, with a pointed tip and a flat base.
The ejected spores germinate by forming budding spores such as yeast. The budding spores form a typical yeast colony while repeating sporulation. See FIG. Some shoot spores germinate with the formation of short spore-producing hyphae and the repeated release of spores, resulting in an overall mycelial colony. See FIG.

Evaluation from the following medium: ATCC medium # 307 Cornmeal agar (Difco 0386) and half strength cornmeal agar # 200 Yeast malt agar (Difco 0712) # 331 Neurospora agar (Difco0321) # 1245 YEPD # 324 Malt extract agar (Difco 0024) # 336 Potato dextrose agar # 343 V-8 juice agar Notes 1. Ingold, CT (1986) Bensingtonia ciliata
Gen. et.sp.nov., Ballistoporic Fungus. Trans. B
r. Mycol. Soc. 86 (2): 325-328 Precautionary Statement 2. Ingold, CT (1988) Bensingtonia ciliata
Further observations on Trans. Br. Mycol. Soc. 91 (1):
162-166

Cryptococcus laurentii, ATCC 20920 is described as follows. Physiology and biochemistry: carbon fixation: (growth) glucose + D-ribose + weak galactose + L-rhamnose + L-sorbose + weak D-glucosamine variable maltose + ethanol + sucrose + erythritol + cellobiose + glycerol + trehalose + trehalose (Ribitol) + weak lactose + weak dulcitol (galactitol) variable melibiose + D-mannitol + raffinose + D-sorbitol (glucitol) + meletitose + a-methyl-D-glucoside + inulin + salicin + soluble starch + inositol + D-inositol Xylose + lactic acid + L-arabinose + citric acid + D-arabinose + weak succinic acid +

Nitrogen assimilation: NH 4 NO 3 + KNO 3 + NO 2 + ethylamine + growth without vitamins + digestion of arbutin + fermentation (gas generation) glucose-galactose-maltose-sucrose-lactose-raffinose-melibiose-inulin-cellobiose-meleptitol − Trehalose −

Morphology: pink gooey colonies, round germinating cells, heavy sediment in flasks; Dalmau
Thin mycelium formed on the dish. Reference: CP Kurtzman, Mycologia 65; p.388-395, 19
73

Cryptococcus albidus, ATCC 20921 is described as follows. Physiology and biochemistry: carbon fixation: (proliferation) glucose + D-ribose-galactose + L-rhamnose + L-sorbose + D-glucosamine + maltose + ethanol variable sucrose + erythritol-cellobiose + glycerol variable trehalose + adnitol variable Lactose + dulcitol (galactitol) + melibiose-D-mannitol + raffinose + D-sorbitol (glucitol) + meletitose + a-methyl-D-glucoside + inulin-salicin + soluble starch + inositol + D-xylose-L-xylose-L Arabinose + citric acid-D-arabinose + succinic acid +

Nitrogen assimilation: NH 4 NO 3 + KNO 3 + NO 2 + ethylamine + growth without vitamins + arbutin fission +

Fermentation (gas generation): glucose-galactose-maltose-sucrose-lactose-raffinose-melibiose-inulin-cellobiose-meleticose-starch-trehalose-

Morphology: tan, gooey colonies. Round germinating cells. Heavy sediment, pseudohypha in flask
Body or true mycelium.

[0044]

EXAMPLES FIG. 1 shows Cryptococcus albidus (ATCC 2091).
8) is a photograph of a thin-layer chromatography developed spot of the product of Example 1 with respect to pH when using 8), and a sclareolide having the following structure:

[Chemical formula 62] With a diol intermediate having the following structure,

[Chemical formula 63] It is produced from sclareol (substrate) having the following structure.

[Chemical formula 64]

FIG. 2 shows the GC-MS of the starting material of Example 3.
FIG. The peak denoted by reference numeral 21 is a peak of sclareol having the following structure.

[Chemical formula 65] The peak indicated by reference numeral 20 is a peak of a compound having the following structure, that is, an internal standard compound.

[Chemical formula 66]

FIG. 3 is a nuclear magnetic resonance [NMR] spectrum of the reaction product of Example 2 which is a compound having the following structure.

[Chemical formula 67]

FIG. 4 is a nuclear magnetic resonance spectrum of the reaction product of Example 3 having the following structure.

[Chemical formula 68]

FIG. 5 is a nuclear magnetic resonance spectrum of the compound having the following structure according to Example 9.

[Chemical formula 69]

The following examples are provided to illustrate presently preferred embodiments of the invention and are not intended to limit the scope of the invention in any way. Weight is in grams, temperature is in degrees Celsius, pressure is unless otherwise noted
Shown in units of mm / Hg.

Example 1 Cryptococcus albidus (Saito
Effect of pH when converting sclareol to sclareolide using [Skinner var. Albidus]) (ATCC 20918) Reaction:

[Chemical formula 70] and

[Chemical formula 71] The following medium was prepared. NH4NO3 0.1% KH2PO4 0.1% MgSO4.7H2O 0.05% Yeast extract 0.2%

Each 100 ml of medium and 1 g of sclareol in TWEEN80 ™ (TWEEN80: sclareol =
11 flasks containing 2: 1). Each flask is
Grow on dextrose at 150 rpm at 25 ° C. 24
Inoculated with 5 ml of time culture. Products and substrates were monitored by thin layer chromatography against known standard compounds. Here, "substrate" refers to a compound having the following structure,

[Chemical formula 72] Compound with the following structure

[Chemical formula 73] And sclareol, which is a mixture of 80:20.
"Intermediate" refers to a compound having the following structure.

[Chemical formula 74] "Product" refers to sclareolide, a compound having the structure:

[Chemical formula 75]

[0052]

[Table 1]

FIG. 1 shows Cryptococcus albidus (ATCC 2091).
It is the spot of the thin layer chromatography eluate of the product using 8).

Example 2 Formation of a Diol Intermediate Reaction:

[Chemical formula 76] and

[Chemical formula 77] While screening 10 soil samples brought in from Greenwood Forest, Burnegat Township, NJ, several flasks showed thin layer chromatography spots corresponding to compounds of the following structure.

[Chemical formula 78] The following medium was prepared. NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.05% Yeast extract 0.2% Dextrose 1.0%

In a 500 ml flask, 100 ml of medium and 50:50 sclareol powder: TWEEN 80 ™
1 g of the mixture of This flask is
Inoculated with 400 microliters of an isolate of ngtonia ciliata, ATCC 20919. After one week at 25 ° C. and 150 rpm, the product formed was extracted with 330 ml of ethyl acetate and the extract was dried over anhydrous sodium sulphate. Solvent was removed on a rotary evaporator. The residue was dissolved in warm hexane and ethyl acetate. The extract was spontaneously evaporated over 24 hours, at which time pure crystals (350 mg) of the compound of the following structure were obtained.

[Chemical formula 79]

FIG. 3 is a nuclear magnetic resonance spectrum of the compound having the following structure.

[Chemical formula 80]

Example 3 Cryptococcus albidus (Sait
o [Skinner var. albidus]), formation of sclareolide using ATCC20918 Reaction:

[Chemical formula 81] and

[Chemical formula 82] Medium NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.05% Extracted yeast 0.2% Defoamer 10.0g d-H2O 8.5l Fermented body temperature: 25 ° C Aeration: 1.0l Stirring: 430 rpm The pH is controlled at 5.8 with 25% NaOH. Duration: 4 days Preparation of substrate: 500 g sclareol, 250 g TWEEN 80 and 1
125 g of water was blended and stirred for 5 minutes to form an emulsion. 1 fermenter with the above medium
The solution was sterilized at 21 ° C for 30 minutes and cooled to 25 ° C. This fermenter is Cryptococcus albidus (Saito [Skinner va
r. albidus]) was inoculated with 300 ml of ATCC 20918 24-hour growing bacteria. 600 g of sclareol emulsion is added during the inoculation and 600 g of emulsion is
Added at hours, 48 and 72 hours. After 96 hours, the contents of the fermenter were filtered through a 400 mesh sieve. The crude solid product thus obtained was dissolved in IPA, filtered, and the solution was concentrated and crystallized to obtain 430 g of pure sclareolide.

FIG. 2 is a GC-MS spectrum of the initial reaction mixture in Example 3. The peak indicated by reference numeral 21 is the peak of sclareol, and the peak of the compound having the following structure is 80: 2
0 mixture.

[Chemical formula 83] The peak indicated by reference numeral 20 is a peak of an internal standard compound having the following structure.

[Chemical formula 84] FIG. 4 is a nuclear magnetic resonance spectrum of sclareolide produced according to this Example 3 having the following structure.

[Chemical formula 85]

Example 4: Cryptococcus albidus, ATCC
Production of sclareolide from sclareol using 20918 The same media and parameters used in Example 3 were used. However, the method of substrate preparation and addition was changed. 160 g of sclareol powder and 80 g of TWEEN 80 ™ were added to the medium prior to sterilization. Another substrate is finely ground sclareol (2 parts) and TWEE
It was prepared by mixing N 80 (1 part) to form a paste. 225 g portion of this paste was applied 24 hours after inoculation,
Added at 8 hours, 72 hours. 655g in total,
A crude sclareolide having a purity of 67.34% was obtained.

Example 5: Cryptococcus albidus, ATCC
Production of sclareolide from sclareol using 20921 The same media and parameters used in Example 4 were used. However, the amount of substrate and agitation varied. 150 g of sclareol and 75 g of TWEEN80 ™ were added to the medium prior to sterilization. Paste-like substrate 24
Only 1 g was added 24 hours after inoculation. Stir 43
Started at 0 rpm and then increased to 630 rpm. A total of 491 g of crude sclareolide with a purity of 44% (94.7% mol / mol conversion) was obtained.

Example 6 Production of Sclareolide from Sclareol, Diol Intermediate, and Diol Acetate Reaction:

[Chemical formula 86] and

[Chemical formula 87]

The following medium was prepared. NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.05% Extracted yeast 0.2% Dextrose 0.5%

A 10 l fermenter was used under the following operating conditions: Temperature: 25 ° C pH: 6.0 Stirring: 430 rpm Sterilization: 30 minutes at 121 ° C

This fermenter was used in the same medium at 25 ° C. and 150 ° C.
100 ml of shake flask culture for 48 hours grown at rpm
Was inoculated. After 24 hours of growth, each issue in the issuer was aliquoted to 200 ml and centrifuged at 10,000 rpm for 10 minutes in a chilled centrifuge. The cells in each tube were washed twice with Butterfield's phosphate-buffer.

Preparation of Buffer Solution Stock solution: 34.0 g of monopotassium hydrogen phosphate Distilled water 500.0 ml Adjust pH to 7.2 with about 175 ml of 1.0N (normal) sodium hydroxide solution, dilute to 1 liter and store. . Diluent: Dilute 1.25 ml stock solution to 1 liter with distilled water. Prepare the diluted blind solution in a suitable container. 121 ° C
Sterilize for 15 minutes.

The cells are then harvested in 100 ml of this buffer. The pH is adjusted to 6 and the mixture is adjusted to 5
Transferred to a 00 ml flask.

Compounds tested: a. Compound 1 = sclareol in TWEEN80 (1: 2)
Paste b. Compound 2 = acetate of the following structure in TWEEN80:
What was mixed in 1

[Chemical formula 88] c. Compound 3 = diol (1: 1) paste of the following structure mixed in TWEEN80

[Chemical formula 89]

A 300 ml flask containing 100 ml of buffer and cells (resting cells) were placed in a shaker incubator at 25 ° C. and 150 rpm and were subjected to thin-layer chromatography to a standard known compound for 24 hours, 48 hours,
At and 72 hours, the samples were analyzed.

In Table 2 below, P is a product, a sclareolide having the following structure:

[Chemical formula 90] S is a substrate having the following structure and one of the compounds having a ratio of 80:20.

[Chemical formula 91] and

[Chemical formula 92] I is an intermediate which is a compound having the following structure:

[Chemical formula 93] T means a very small amount.

[0070]

[Table 2]

Example 7: Bensingtonia ciliata, ATCC
Production of a diol intermediate from sclareolide using 20919 An experiment by the same method as in Example 2 was performed with the following specifications. Microorganism: Bensingtonia ciliata, ATCC 20919 Medium: NH4NO3 40.0 g Extracted yeast 40.0 g KH2PO4 20.0 g MgSO4.7H2O 10.0 g Sclareol 160.0 g TWEEN 80 80.0 g d-H2O 19.0 l Fermenter parameters: temperature 25 Aeration: 2 l / min Stirring: 300 rpm pH: Controlled to 6.0 with 25% NaOH Duration: 15 days Fermenter with the above medium is 121 ° C.
For 30 minutes and cooled to 25 ° C. This fermenter was grown for 48 hours in Bensingtonia ciliata, ATCC 20
Inoculated with one liter of 919 culture. After 15 days of incubation, the substrate was product. The contents of the fermenter were sieved through a 400 mesh screen and filtered. The crude solid product thus obtained was air-dried. 10
1.7 g of product were obtained.

Example 8: Decahydro-3a, 6,6,9
Preparation of a-tetramethylnaphtho [2-1-b] furan Reaction:

[Chemical formula 94] and

[Chemical formula 95]

In a 1 l three-necked reaction vessel equipped with a mechanical stirrer, thermometer, dropping funnel and reflux condenser, 12
4 ml of VITRIDE ™ {sodium bis (2-methoxyethoxy) aluminium hydride} was added. This VITR
500 ml of toluene was added to the IDE with stirring.
50 g of sclareolide of the following structure prepared by the method of Example 6 was dissolved in 120 ml of toluene.

[Chemical formula 96] The sclareol / toluene solution was then added dropwise via a dropping funnel to the reaction mixture over a half hour period. At this time, the temperature of the reaction mixture may be increased to 55 ° C.
The reaction mixture was then heated to 80 C for 2 hours.
The reaction mixture was then worked up by slowly adding 600 ml of a 5% aqueous sodium hydroxide solution while cooling with ice. The toluene layer was separated, washed with a saturated aqueous sodium chloride solution, and washed with equal volumes of an aqueous solution of methyl alcohol and saturated sodium bicarbonate. Next, the toluene layer was concentrated to obtain a solid having a weight of 43.34 g (yield: 86%). The solid thus obtained has the following structure.

[Chemical formula 97] 1.5 g of this compound was added to 1 ml of potassium hydroxide in 30 ml of water.
It was mixed with 2.0 g of solution. The mixture was placed in a flask equipped with a heater, stirrer, and reflux condenser and refluxed at 80 ° C. for 3 hours. After 3 hours, the product has a steam temperature of 162 to 164 ° C. and a pressure of 15 mm /
Fractional distillation over Hg. Afforded a fairly pure compound of the structure

[Chemical formula 98]

Example 9: Formation of cyclic ether Reaction:

[Chemical formula 99] and

[Chemical formula 100] The following medium was prepared. Ingredients Parts by weight NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.05% Extracted yeast 0.2% Dextrose 1.0%

A 500 ml flask was charged with 100 ml of medium and 1.0 g of a 50:50 mixture of sclareol powder / TWEEN800. The flask was inoculated with 400 microliters of a isolate of Cryptococcus laurentii, ATCC 20920. After one week at 150 rpm at 25 ° C., the resulting product was extracted with 330 ml of ethyl acetate, and the extract was dried over anhydrous sodium sulfate. The solvent was distilled on a rotary evaporator. The residue was dissolved in warm hexane and ethyl acetate. The organic layer was spontaneously evaporated for 24 hours to obtain pure crystals (350 mg) of a compound having the following structure.

[Chemical formula 101]

[Brief description of the drawings]

1 is a thin layer chromatographic development spot of the product of Example 1 against pH when using Cryptococcus albidus (ATCC 20918).

FIG. 2 is a GC-MS spectrum of a starting material of Example 3. FIG. 3 shows Example 2 which is a compound having the following structure.
2 is a nuclear magnetic resonance [NMR] spectrum of the reaction product of.

FIG. 4 is a nuclear magnetic resonance spectrum of the reaction product of Example 3 having the following structure.

FIG. 5 is a nuclear magnetic resonance spectrum of a compound having the following structure according to Example 9.

[Chemical formula 102]

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[Procedure amendment]

[Submission date] April 9, 1996

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 Cryptococcus albidus
FIG. 5 is a thin layer chromatography developed spot of the product of Example 1 versus pH when using (ATCC 20918).

FIG. 2 is a GC-MS spectrum of a starting material of Example 3.

FIG. 3 is a nuclear magnetic resonance [NMR] spectrum of a reaction product of Example 2 which is a compound having the following structure.

FIG. 4 is a nuclear magnetic resonance spectrum of the reaction product of Example 3 having the following structure.

FIG. 5 is a nuclear magnetic resonance spectrum of a compound having the following structure according to Example 9.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication (C12N 1/20 C12R 1:01) (C12P 17/06 C12R 1:01) (72) Inventor James A. Morris, New Jersey, USA 07719, Wall, West Court 1406 (72) Inventor Arthur E. Downey 07036, New Jersey, USA 07036, Morristown Road 135

Claims (5)

[Claims] 1. A 1 selected from the group consisting of the following bacteria:
Biologically pure culture of microorganisms: (a) Bensingtonia ciliata, ATCC 20919 and (b) Cryptococcus laurentii, ATCC 20920, wherein the microorganism is a sclareol of the following structure under aeration conditions in an aqueous nutrient medium: sclareol] [Chemical formula 1] And / or an episclare of the following structure [episclare
ol] [Chemical formula 2] A diol having the following structure is represented by [Chemical formula 3] It is characterized by being able to produce. 2. The following diol is represented by the following chemical formula: A compound having the following structure under aeration conditions: [Chemical Formula 5] And [Chemical Formula 6] 1. A method of converting at least one of the above compounds in an aqueous nutrient medium containing 1 or 2 and producing a recoverable amount. This method is based on Bensingtonia ciliata, ATCC 20918, and Cry.
ptococcus albidus, ATCC 20919, and Cryptococcus
It is characterized in that a microorganism selected from the group consisting of laurentii and ATCC 20920 is cultured in the above medium. 3. A compound selected from the group of compounds having the following structures: embedded image And [Chemical formula 8] Cryptococcus laurentii, ATCC 20920 is cultivated under aeration conditions in an aqueous nutrient medium containing one or more of the above compounds, and at least one of the above compounds is converted and recovered in a recoverable amount. ] How to produce. 4. The culture is in an aqueous nutrient medium containing an additional carbon source: (i) pH: about 2.5 to about 9.0 (ii) Temperature: about 12 ° C. to about 33 ° C. (iii) Compound concentration A process according to claims 2 or 3 carried out under the conditions of about 0.1 to about 130 g / l. 5. The method according to claim 4, wherein the additional carbon source is dextrose.