Method for production of geranylgeraniol and analogous compounds thereof by microorganisms

Abstract

The present invention provides a method for producing geranylgeraniol and analogous compounds thereof, which comprises culturing yeast cells (ascomycetes and deuteromycetes), bacterial cells, actinomycete cells or filamentous fungus cells, all of which are capable of producing geranylgeraniol and analogous compounds thereof, in a medium to produce and accumulate geranylgeraniol and analogous compounds thereof in the cells and/or in the extracellular environment; and then collecting these compounds. The present invention enables inexpensive mass production of geranylgeraniol and analogous compounds thereof by using microorganisms capable of producing geranylgeraniol, farnesol and/or nerolidol useful as biosynthetic intermediates of terpenes, carotenoids and/or steroids.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method for production of geranylgeraniol and analogous compounds thereof using microorganisms.

BACKGROUND OF THE INVENTION

[0002] Geranylgeraniol and farnesol are believed to be produced in organisms through hydrolysis of geranylgeranyl pyrophosphate and farnesyl pyrophosphate with a phosphatase. Geranylgeranyl pyrophosphate is a pyrophosphate ester of geranylgeraniol, which is yielded by condensation between isopentenyl pyrophosphate and farnesyl pyrophosphate or condensation between three molecules of isopentenyl pyrophosphate and dimethyl aryl pyrophosphate. Geranylgeranyl pyrophosphate is metabolized into a diterpene compound (e.g., gibberellin) by cyclization, into a carotenoid compound via phytoene formed by tail-to-tail condensation, or into polyprenylpyrophosphate by head-to-tail condensation with isopentenyl pyrophosphate. On the other hand, farnesyl pyrophosphate is yielded by condensation between isopentenyl pyrophosphate and geranyl pyrophosphate or condensation between two molecules of isopentenyl pyrophosphate and dimethyl aryl pyrophosphate. Farnesyl pyrophosphate is metabolized into a sesquiterpene compound by cyclization, into steroid and triterpene compounds via squalene formed by tail-to-tail condensation, or into polyprenylpyrophosphate or dolichol by head-to-tail condensation with isopentenyl pyrophosphate. It is also metabolized into a prenylated protein when linked to a cysteine residue of a specific protein such as Ras protein or G protein. Thus, a series of geranylgeraniol derivatives, including geranylgeraniol, geranylgeranyl pyrophosphate and precursors thereof, i.e., farnesyl pyrophosphate, farnesol, geranyl pyrophosphate or geraniol, are dominant compounds as biosynthetic intermediates of terpenes, carotenoids or steroids. In addition, geranylgeraniol and analogous compounds thereof are important for use in the production of perfume, a taxane compound having an anti-tumor activity (Japanese Patent Application No. 8-227481), a hair tonic (Japanese Patent Application No. 8-180449), a therapeutic agent for osteoporosis (Japanese Patent Application No. 9-294089) and the like.

[0003] In the production of the geranylgeraniol derivatives stated above, there has been reported a technique for producing geranylgeraniol and/or geranylgeranyl pyrophosphate by culturing plant cells belonging to Euphorbiaceae under light irradiation (Japanese Patent Application Laying-Open (kokai) No. 9-238692), and a technique for allowing an erg mutant of Saccharomyces cerevisiae to produce and secrete farnesol [Curr. Genet., 18, 41-46 (1990)], but no naturally occurring cell has been reported to produce geranylgeraniol and/or farnesol. Among these prior techniques, in the technique using plant cells, light irradiation is essential to culture the cells and mass production is difficult because of expensive medium ingredients. Also, in the technique using the erg mutant, it is difficult for this mutant to survive in nature because it cannot synthesize ergosterol essential for yeast growth and it therefore requires the addition of expensive ergosterol to a medium. In contrast, if there is found a wild-type strain producing geranylgeraniol and/or farnesol that can be grown at a higher speed than plant cells and in an inexpensive medium without addition of any specific ingredient, such a strain would enable mass production of intermediates for the above effective substances and therefore would be very useful in industry.

SUMMARY OF THE INVENTION

[0004] Accordingly, the object of the present invention is to provide a method for inexpensive mass production of geranylgeraniol and analogous compounds thereof using a microorganism capable of producing geranylgeraniol and analogous compounds thereof.

[0005] Our research efforts were directed to achieving the above object, and we have performed screening on a wide spectrum of microorganisms, particularly those belonging to identified species, to find yeast cells (ascomycetes and deuteromycetes), bacterial cells, actinomycete cells and filamentous fungus cells capable of producing geranylgeraniol, farnesol and/or nerolidol, thereby finally completing the invention.

[0006] Namely, the present invention provides a method for producing geranylgeraniol and/or farnesol, which comprises culturing geranylgeraniol- and/or farnesol-producing cells belonging to any one of the following genera: Saccharomyces,

[0007] Saccharomycopsis,

[0008] Saccharomycodes,

[0009] Schizosaccharomyces,

[0010] Wickerhamia,

[0011] Debaryomyces,

[0012] Hansenula,

[0013] Hanseniaspora,

[0014] Lypomyces,

[0015] Pichia,

[0016] Kloeckera,

[0017] Candida,

[0018] Zygosaccharomyces,

[0019] Ogataea,

[0020] Kuraishia,

[0021] Komagataella,

[0022] Yarrowia,

[0023] Williopsis,

[0024] Nakazawaea,

[0025] Kluyveromyces,

[0026] Torulaspora,

[0027] Citeromyces,

[0028] Waltomyces,

[0029] Bacillus,

[0030] Staphylococcus,

[0031] Pseudomonas,

[0032] Micrococcus,

[0033] Exiguobacterium,

[0034] Mucor,

[0035] Ambrosiozyma,

[0036] Cystofilobasidium,

[0037] Metschnikowia,

[0038] Trichosporon,

[0039] Xanthophyllomyces,

[0040] Bullera,

[0041] Fellomyces,

[0042] Filobasidium,

[0043] Holtermannia,

[0044] Phaffia,

[0045] Rhodotorula,

[0046] Sporidiobolus,

[0047] Sporobolomyces,

[0048] Zygoascus,

[0049] Haloferax,

[0050] Brevibacterium,

[0051] Leucosporidium,

[0052] Myxozyma,

[0053] Trichosporiella, and

[0054] Alcaligenes

[0055] in a medium to produce and accumulate geranylgeraniol and/or farnesol in the cells and/or in the extracellular environment; and then collecting geranylgeraniol and/or farnesol.

[0056] Also, the present invention provides a method for producing nerolidol, which comprises culturing nerolidol-producing cells belonging to any one of the following genera:

[0057] Saccharomyces,

[0058] Cryptococcus,

[0059] Candida,

[0060] Streptomyces,

[0061] Nocardia,

[0062] Cystofilobasidium,

[0063] Rhodotorula,

[0064] Willopsis, and

[0065] Haloferax

[0066] in a medium to produce and accumulate nerolidol in the cells and/or in the extracellular environment; and then collecting nerolidol.

[0067] This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application Nos. 2000-401266 and 2001-376173, which are priority documents of the present application.

DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention will be described below in more detail.

[0069] In the present invention, a microorganism fermentation method is used to produce geranylgeraniol and analogous compounds thereof. As used herein, analogous compounds of geranylgeraniol refers to geranylgeranyl pyrophosphate and compounds produced in association with the synthesis thereof, i.e., geranylgeranyl monophosphate, farnesyl pyrophosphate, farnesyl monophosphate, farnesol, geranyl pyrophosphate, geranyl monophosphate, geraniol, nerolidol, geranyllinalool, linalool and the like.

[0070] In the present invention, any microorganism may be used for geranylgeraniol production, so long as it has the ability to produce geranylgeraniol. Examples include yeast strains belonging to any one of Saccharomyces, Saccharomycopsis, Saccharomycodes, Schizosaccharomyces, Wickerhamia, Debaryomyces, Hansenula, Hanseniaspora, Pichia, Kloeckera, Candida, Zygosaccharomyces, Ogataea, Kuraishia, Komagataella, Yarrowia, Williopsis, Nakazawaea, Kluyveromyces, Cryptococcus, Torulaspora, Bullera, Rhodotorula, Willopsis, Kloeckera and Sporobolomyces; filamentous fungus strains belonging to Mucor; archaebacterial strains belonging to Haloferax; or bacterial strains belonging to Alcaligenes.

[0071] Specific examples of geranylgeraniol-producing cells will be presented below:

[0072] (1) Saccharomyces:

[0073] Saccharomyces cerevisiae strains ATCC 12341, IFO 0565, IFO 0222, IFO 0216, ATCC 9080, IFO 1346, ATCC 204660, IFO 0538 and IFO 0210, Saccharomyces ellipsoideus strain 4102 (Kyoto Univ.), Saccharomyces sake strain Kyokai No.2, Saccharomyces rosei strain IFO 0252, and Saccharomyces kluyveri strain IFO 1892;

[0074] (2) Saccharomycopsis:

[0075] Saccharomycopsis fibuligera strains IFO 0106, IFO 1665, IFO 1774 and IFO 0107;

[0076] (3) Saccharomycodes:

[0077] Saccharomycodes ludwigii strain IFO 0339;

[0078] (4) Schizosaccharomyces:

[0079] Schizosaccharornyces octosporus strain IAM 4842 and Schizosaccharomyces pombe strain IFO 0346;

[0080] (5) Wickerhamia:

[0081] Wickerhamia fluorescens strain IFO 1116;

[0082] (6) Debaryomyces:

[0083] Debaryomyces hansenii var. fabryi strain IFO 0794, Debaryomyces castellii strain IFO 1359, and Debaryomyces vanrijiae var. vanrijiae JCM 2169;

[0084] (7) Hansenula:

[0085] Hansenula polymorpha strain 4327 (Kyoto Univ.);

[0086] (8) Hanseniaspora:

[0087] Hanseniaspora valbyensis strain IFO 0115;

[0088] (9) Pichia:

[0089] Pichia membranaefaciens strain IFO 0128, Pichia aganobii strain 4261 (Kyoto Univ.), Pichia naganishii strain IFO 1670, Pichia silvicola strain IFO 0807, and Pichia anomala strains IFO 0118, IFO 0569 and IFO 0707;

[0090] (10) Kloeckera:

[0091] Kloeckera japonica strain IFO 0151;

[0092] (11) Candida:

[0093] Candida krusei strain IFO 0013, Candida kefyr strain IFO 0706, Candida tenuis strain IFO 0716, Candida solani strain IFO 0762, Candida glabrata strains IFO 0005 and IFO 0622, Candida albicans strain IFO 1060, Candida zeylanoides strain IFO 0719, Candida catenulata strain IFO 0720, Candida cariosilignicola strain IFO 1910, Candida stellata strain IFO 0701, and Candida utilis strain IFO 0619;

[0094] (12) Zygosaccharomyces:

[0095] Zygosaccharomyces rouxii strain IFO 0487, and Zygosaccharomyces japanicus strain IFO 0595;

[0096] (13) Ogataea:

[0097] Ogataea glucozyma strain IFO 1472, and Ogataea polymorpha strain IFO 1475;

[0098] (14) Kuraishia:

[0099] Kuraishia capsulata strain IFO 0974;

[0100] (15) Komagataella:

[0101] Komagataella pastoris strain IFO 0948;

[0102] (16) Yarrowia:

[0103] Yarrowia lopolytica strain IFO 0717;

[0104] (17) Williopsis:

[0105] Williopsis saturnus var. saturnus strains IFO 0125 and PFO 0941, and Williopsis saturnus strain IFO 0895;

[0106] (18) Nakazawaea:

[0107] Nakazawaea holstii strain IFO 0980;

[0108] (19) Kluyveromyces:

[0109] Kluyveromyces marxianus strains IFO 0617 and IFO 0288, Kluyveromyces thermotolerans strain IFO 0662, and Kluyveromyces lactis strain IFO 0648;

[0110] (20) Torulaspora:

[0111] Torulaspora delbrueckii strain IFO 0422;

[0112] (21) Cryptococcus:

[0113] Cryptococcus humicolus strain IFO 1527; and

[0114] (22) Mucor:

[0115] Mucor javanicus strain IFO 4570;

[0116] (23) Bullera:

[0117] Bullera pseudoalba strain IFO 10179;

[0118] (24) Rhodotorula:

[0119] Rhodotorula minuta strain IFO 0715, and Rhodotorula rubra strain IFO 0870;

[0120] (25) Sporobolomyces:

[0121] Sporobolomyces salmonicolor strain IFO 0374;

[0122] (26) Haloferax:

[0123] Haloferax volcanii strain IFO 14742; and

[0124] (27) Alcaligenes:

[0125] Alcaligenes faecalis strain IFO 13111.

[0126] In the present invention, any microorganism may be used for farnesol production, so long as it has the ability to produce farnesol. Examples include yeast strains belonging to any one of Saccharomyces, Saccharomycopsis, Saccharomycodes, Schizosaccharomyces, Wickerhamia, Debaryomyces, Hanseniaspora, Lypomyces, Pichia, Candida, Ogataea, Kuraishia, Komagataella, Yarrowia, Kluyveromyces, Torulaspora, Zygosaccharomyces, Williopsis, Citeromyces, Waltomyces and Cryptococcus; bacterial strains belonging to any one of Bacillus, Staphylococcus, Pseudomonas, Micrococcus and Exiguobacterium; filamentous fungus strains belonging to Mucor; or microbial strains belonging to any one of Ambrosiozyma, Cystofilobasidium, Metschnikowia, Trichosporon, Xanthophyllomyces, Bullera, Fellomyces, Filobasidium, Holtermannia, Phaffia, Sporidiobolus, Sporobolomyces, Zygoascus, Leucosporidium, Myxozyma, Trichosporiella, Haloferax and Brevibacterium.

[0127] Specific examples of farnesol-producing cells will be presented below:

[0128] (1) Saccharomyces:

[0129] Saccharomyces cerevisiae strains IFO 1346, ATCC 204660, IFO 0258, IFO 0262, IFO 0538, IFO 0565, IFO 0210 and IFO 2347, Saccharomyces unisporus strain IFO 0215, Saccharomyces sake strain Kyokai No.2, Saccharomyces ellipsoideus strain 4102 (Kyoto Univ.), Saccharomyces rosei strain IFO 0252, Saccharomyces logos strain 4101 (Kyoto Univ.), Saccharomyces dairensis strain IFO 0285, Saccharomyces bayanus strains IFO 0539 and IFO 0613, Saccharomyces kluyveri strain IFO 1892, and Saccharomyces paradoxus strain IFO 0259;

[0130] (2) Saccharomycodes:

[0131] Saccharomycodes ludwigii strain IFO 0339;

[0132] (3) Schizosaccharomyces:

[0133] Schizosaccharomyces pombe strains IFO 0346, IFO 0638 and IFO 0358, and Schizosaccharomyces octosporus strain LAM 4842;

[0134] (4) Hanseniaspora:

[0135] Hanseniaspora valbyensis strain IFO 0115;

[0136] (5) Debaryomyces:

[0137] Debaryomyces hansenii strain IFO 0023, Debaryomyces hansenii var. fabryi strain IFO 0749, Debaryomyces castellii strain IFO 1359, and Debaryomyces vanrijiae var. vanrijiae strain JCM 2169;

[0138] (6) Lypomyces:

[0139] Lypomyces starkeyi strain IFO 0678;

[0140] (7) Pichia:

[0141] Pichia aganobii strain 4261 (Kyoto Univ.), Pichia naganishii strain IFO 1670, and Pichia anomala strains IFO 0118, IFO 0569, IFO 0963, IFO 707 and IFO 0146;

[0142] (8) Candida:

[0143] Candida utilis strains IFO 0626 and IFO 0619, Candida albicans strains IFO 0579 and IFO 1060, Candida zeylanoides strain IFO 0719, Candida glabrata strains IFO 0005, IFO 0622 and IFO 0741, Candida cariosilignicola strain IFO 1910, Candida stellata strain IFO 0701, Candida solani strain IFO 0762, Candida intermedia strain IFO 0761, Candida krusei strain IFO 0941 and Candida tenuis strain IFO 0716;

[0144] (9) Wickerhamia:

[0145] Wickerhamia fluoresces strain IFO 1116;

[0146] (10) Kuraishia:

[0147] Kuraishia capsulata strain IFO 0974;

[0148] (11) Komagataella:

[0149] Komagataella pastoris strain IFO 0948;

[0150] (12) Ogataea:

[0151] Ogataea glucozyma strain IFO 1472 and Ogataea polymorpha strain IFO 1475;

[0152] (13) Yarrowia:

[0153] Yarrowia lopolytica strain IFO 0717;

[0154] (14) Kluyveromyces:

[0155] Kluyveromyces marxianus strains IFO 0288 and IFO 0617, Kluyveromyces thermotolerans strain IFO 0662, and Kluyveromyces lactis strain IFO 0648;

[0156] (15) Torulaspora:

[0157] Torulaspora delbrueckii strain IFO 0422;

[0158] (16) Zygosaccharomyces:

[0159] Zygosaccharomyces rouxii strains IFO 0487 and IFO 0686, Zygosaccharomyces japanicus strain IFO 0595, and Zygosaccharomyces fermentati strain IFO 0021;

[0160] (17) Williopsis:

[0161] Williopsis saturnus var saturnus strain IFO 0941, Williopsis califormica strain IFO 0800 and Willopsis saturnus strain IFO 0895;

[0162] (18) Citeromyces:

[0163] Citeromyces matritensis strain IFO 0954;

[0164] (19) Waltomyces:

[0165] Waltomyces lipoder strain IFO 0673;

[0166] (20) Cryptococcus:

[0167] Cryptococcus humicolus strain IFO 1527;

[0168] (21) Bacillus:

[0169] Bacillus amyloliquefaciens strain IFO 3022 and Bacillus pumilus FO 3030

[0170] (22) Staphylococcus:

[0171] Staphylococcus epidermidis strain IFO 3762;

[0172] (23) Pseudomonas:

[0173] Pseudomonas sp. strain 876 (Kyoto Univ.);

[0174] (24) Micrococcus:

[0175] Micrococcus luteus strain IFO 3067;

[0176] (25) Exiguobacterium:

[0177] Exiguobacterium acetylicum strain IFO 12146;

[0178] (26) Mucor:

[0179] Mucor javanicus strain IFO 4570;

[0180] (27) Ambrosiozyma:

[0181] Ambrosiozyma platypodis strain IFO 10752;

[0182] (28) Cystofilobasidium:

[0183] Cystofilobasidium infirmominiatum strain IFO 1057;

[0184] (29) Leucosporidium:

[0185] Leucosporidium scottii strain IFO 1924;

[0186] (30) Metschnikowia:

[0187] Metschnikowia lunata strain IFO 1605;

[0188] (31) Myxozyma:

[0189] Myxozyma lipomycoides strain IFO 10351;

[0190] (32) Trichosporon:

[0191] Trichosporon pullulans strain IFO 1232;

[0192] (33) Xanthophyllomyces:

[0193] Xanthophyllomyces dendrorhous strain IFO 10130;

[0194] (34) Bullera:

[0195] Bullera pseudoalba strain IFO 10179;

[0196] (35) Fellomyces:

[0197] Fellomyces penicillatus strain IFO 10119;

[0198] (36) Filobasidium:

[0199] Filobasidium capsuligenum strain IFO 1185, and Filobasidium uniguttulatum strain IFO 0699;

[0200] (37) Kloeckera:

[0201] Kloeckera corticis strain IFO 0633;

[0202] (38) Holtermannia:

[0203] Holtermannia corniformis strain IFO 10742;

[0204] (39) Phaffia:

[0205] Phaffia rhodozyma strain ATCC 66270;

[0206] (40) Saccharomycopsis:

[0207] Saccharomycopsis fermentans strain IFO 10772;

[0208] (41) Sporidiobolus:

[0209] Sporidiobolus samonicolar strain IFO 1035;

[0210] (42) Sporobolomyces:

[0211] Sporobolomyces salmonicolor strain IFO 0374;

[0212] (43) Trichosporiella:

[0213] Trichosporiella flavificans strain IFO 1573;

[0214] (44) Zygoascus:

[0215] Zygoascus hellenicus strain IFO 10184;

[0216] (45) Haloferax:

[0217] Haloferax volcanii strain IFO 14742; and

[0218] (46) Brevibacterium:

[0219] Brevibacterium linens strain IFO 12171.

[0220] In the present invention, any microorganism may be used for nerolidol production, so long as it has the ability to produce nerolidol. Examples include yeast strains belonging to Saccharomyces, Candida or Cryptococcus; actinomycete strains belonging to Streptomyces or Nocardia; or microbial strains belonging to any one of Cystofilobasidium, Rhodotorula, Willopsis and Haloferax.

[0221] Specific examples of nerolidol-producing cells will be presented below:

[0222] (1) Nocardia:

[0223] Nocardia asteroides strain IFO 3384 and Nocardia fusca strain IFO 14340;

[0224] (2) Streptomyces:

[0225] Streptomyces gardneri strain IFO 12865;

[0226] (3) Saccharomyces:

[0227] Saccharomyces unisporus strain IFO 0215, Saccharomyces cerevisiae strain IFO 0210, and Saccharomyces ellipsoideus strain 4102 (Kyoto Univ.);

[0228] (4) Candida:

[0229] Candida glabrata strains IFO 0005 and IFO 0741, Candida solani strain IFO 0762, and Candida krusei strain IFO 0941;

[0230] (5) Cryptococcus:

[0231] Cryptococcus humicolus strain IFO 1527;

[0232] (6) Cystofilobasidium:

[0233] Cystofilobasidium infirmominiatum strain IFO 1057;

[0234] (7) Rhodotorula:

[0235] Rhodotorula minuta strain IFO 0715, and Rhodotorula rubra strain IFO 0870;

[0236] (8) Willopsis:

[0237] Williopsis californica strain IFO 0800; and

[0238] (9) Haloferax:

[0239] Haloferax volcanii strain IFO 14742.

[0240] In addition to the above-listed microorganisms, further examples of microorganisms capable of producing geranylgeraniol, farnesol and/or nerolidol, which can be used in the present invention, include microbial strains belonging to any one of Dipodascus, Issatchenkia, Mortierella, Rhodosporidium, Tsukamurella, Yamadazyma, Bensingtonia, Botryozyma, Brettanomyces, Clavispora, Dekkera, Eremascus, Eremothecium, Erythrobasidium, Kloeckeraspora, Kockovaella, Kodamaea, Kurtzmanomyces, Lodderomyces, Malassezia, Mrakia, Nadsonia, Pachysolen, Saturnispora, Schizoblastosporion, Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Sympodiomyces, Sympodiomycopsis, Trigonopsis, Tsuchiyaea, Zygozyma and Aciculoconidium.

[0241] Culture of the microorganisms used in the present invention will be described in turn. In general, any medium may be used to culture the microorganisms, so long as it allows the growth of these microorganisms. Specific examples include YM medium, KY medium and F101 medium for culture of yeast cells (ascomycetes and deuteromycetes); and KB medium for culture of bacterial cells and actinomycete cells.

[0242] Any carbon compound may be used as a carbon source, so long as the microorganisms can assimilate it for growth.

[0243] As a nitrogen source, for example, an inorganic nitrogen source including ammonium sulfate, ammonium chloride or ammonium nitrate, or an organic nitrogen source including yeast extract, peptone or meat extract may be used. In addition to these, a medium may further contain minerals, metal salts, and/or vitamins, if necessary.

[0244] Culture conditions will vary depending on the types of microorganisms. In general, the culture may preferably be performed at a temperature of 20° C. to 40° C., more preferably 25° C. to 35° C., and at a pH of 5 to 9. The culture may also be performed under anaerobic or aerobic conditions according to the types of microorganisms, preferably performed under aerobic conditions with shaking or rotating because aerobic conditions permit a higher growth speed than anaerobic conditions.

[0245] However, it is naturally important to select culture conditions for maximum production of geranylgeraniol and analogous compounds thereof, according to the type of microorganism to be used and the composition of the medium.

[0246] To improve production of geranylgeraniol and analogous compounds thereof (hereinafter, collectively referred to as “geranylgeraniol analogs”) and to stimulate product secretion from cells, a medium may be supplemented with a sugar and/or a fat or oil.

[0247] Examples of a sugar able to be used in the present invention include glucose, sucrose and the like. Examples of a fat or oil able to be used in the present invention include soybean oil, fish oil, almond oil, olive oil and the like.

[0248] For example, a sugar may be added to a medium at a concentration of 1% to 10%, preferably 2% to 7%, while a fat or oil may be added to a medium at a concentration of 0.01% or more, preferably 1% or more. As used herein, the percentage (%) used to express a sugar content etc. is based on w/v (%).

[0249] A medium may further be supplemented with ergosterol along with a squalene synthase inhibitor in order to give a further improvement in production of geranylgeraniol analogs. Examples of a squalene synthase inhibitor include BSM-187745 (Toxiocology and applied pharmacology 145, 91-98 (1987)), SQAD (Japanese patent Application No.8-508245) and zaragozic acid. The squalene synthase inhibitor may be used at a concentration of 1 mg/L to 20 mg/L.

[0250] In the present invention, geranylgeraniol analogs may be produced in a batch manner or in a continuous manner using a bioreactor. Microorganism cells may be provided as such for geranylgeraniol analog production or may be pre-treated to give crushed cells, a culture solution, a crude enzyme, or a purified enzyme. Cultured cells or these pre-treated products may also be immobilized by an immobilization technique. The cells or pre-treated products are cultured to produce and accumulate geranylgeraniol analogs in the cells or culture supernatant, which are then collected.

[0251] To collect geranylgeraniol analogs from a culture supernatant fraction, a supernatant from which cells have been removed by centrifugation is treated with alkaline phosphatase in a buffer containing magnesium chloride, and then extracted with a solvent such as pentane or methanol.

[0252] To collect geranylgeraniol analogs from a cultured cell fraction, on the other hand, the cells collected by centrifugation are crushed, treated with alkaline phosphatase in a buffer containing magnesium chloride, and then extracted with a solvent such as pentane or methanol.

[0253] The above solvent extraction step may be performed in combination with a known purification technique such as chromatography, as needed.

[0254] The use of alkaline phosphatase in the extraction step is effective in improving farnesol and geranylgeraniol production because it allows hydrolysis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate present as precursors for farnesol and geranylgeraniol in the cells or culture solution. A preferred phosphatase is alkaline phosphatase derived from E. coli, but other phosphatases including potato-derived acid phosphatase or calf intestine phosphatase may also be used. Since most microorganisms possess an endogenous phosphatase, the organic solvent extraction step may also be performed without phosphatase treatment, although a slight decrease in production is observed.

[0255] In the production method of the present invention, geranylgeraniol analogs are detected by gas chromatography/mass spectrometry (GCIMS) using a commercially available column and then quantified from the ratio of peak area between each analog and 1-undecanol as an internal standard.

EXAMPLES

[0256] The present invention will be further described in the following examples. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the invention.

Example 1

Screening of Geranylgeraniol Analog-Producing Cells

[0257] (1) Strain

[0258] Screening was performed on about 930 strains purchased from ATCC, IAM, IFO and JCM as well as provided by Professor Shimizu (Kyoto University, Japan) to find geranylgeraniol analog-producing cells.

[0259] (2) Strain Storage

[0260] Strains were stored appropriately in the following three ways.

[0261] (i) Glycerol Stocks

[0262] 300 μl of autoclaved 50% glycerol (Nacalai) was added to 900 μl of a liquid culture solution and then stored at −80° C. and −20° C. A strain stored at −20° C. was plated after one week to confirm that the storage was succeeded.

[0263] (ii) Lyophilized Stocks

[0264] 2 ml of a liquid culture solution was transferred to a 2 ml Eppendorf tube and then centrifuged at 6000 rpm for 5 minutes to collect cells. The cells were suspended in 150 μl of 20% skim milk (Morinaga Milk Industry Co., Ltd.) solution autoclaved at 110° C. for 20 minutes. The suspension was transferred to a glass tube for lyophilization using a dry-sterilized Pasteur pipette and then frozen in a freezer at −80° C. After sufficiently drying overnight in a lyophilizer (Lyph Lock 1L; Labconco), the glass tube was sealed using a band burner GB20001 (Prince). A Tesler coil (model WTC-100; Wakaida Science Corporation) was used to confirm whether sealing had succeeded.

[0265] (iii) Paraffin-Overlaid Stocks

[0266] A 2% agar slant having the same composition as that of a liquid medium was prepared. Cells were streaked from a liquid culture solution onto the slant by using a disposable loop. The slant was allowed to stand at room temperature for one week. The slant confirmed to show sufficient cell growth was overlaid with autoclaved liquid paraffin (Nacalai) at a depth of about 1 cm and stored at 4° C.

[0267] (3) Preparation of Liquid Medium

[0268] Yeast cells were cultured in YM medium (Difco), or KY or F101 medium prepared as presented below. Bacterial cells or actinomycete cells were cultured in KB medium.

[0269] A plate was prepared from the same medium by addition of Bactoagar (Difco) at a final concentration of 2%.

[0270] KY Medium

[0271] The following ingredients were added to 1 L of deionized water, adjusted to pH 5.5 with 2N sodium hydroxide, and then adjusted to 1 L with deionized water, followed by autoclaving.

Malt Extract (Difco)  5 g
Yeast Extract (Difco) 5 g

[0272] F101 Medium

[0273] 200 g of diced potato was introduced into 500 ml of deionized water, boiled for 30 minutes and then filtered through gauze to remove an insoluble residue. The following ingredients were added to the resulting solution, and then adjusted to 1 L with deionized water, followed by autoclaving.

Yeast Extract (Difco) 30 g
Glucose (Nacalai)     15 g
Sucrose (Nacalai)     15 g

[0274] KB Medium

[0275] The following ingredients were added to 1 L of deionized water, adjusted to pH 7.0 with 2N potassium hydroxide, and then adjusted to 1 L with deionized water, followed by autoclaving.

Bactotryptone (Difco) 5 g
Yeast Extract (Difco) 5 g
Glucose (Nacalai)     1 g
KH2PO4 (Nacalai)      0.7 g
K2HPO4 (Nacalai)      0.3 g

[0276] (4) Liquid Culture

[0277] Each medium (20 ml) prepared above was separately introduced into a 100 ml baffled Erlenmeyer flask, which was then closed with a Morton closure and autoclaved at 121° C. and at 1.2 atm for 15 minutes. A loopful of cells was inoculated from the slant into the autoclaved media and then cultured at 30° C. for 3 days while rotating at 130 rpm.

[0278] (5) Extraction of Geranylgeraniol Analogs from Supernatant Fraction

[0279] 2.5 ml of the culture solution was transferred into a test tube (φ18 mm×125 mm), and centrifuged in a Beckman centrifuge GP at 1000 rpm for 5 minutes to give the supernatant, which was then transferred to another new test tube (φ18 mm×125 mm). 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride and 5 μl (2 units) of E. Coli alkaline phosphatase (Takara Shuzo Co., Ltd.) were added to the supernatant and heated to 65° C. for 30 minutes. After sufficiently cooling on ice, the treated supernatant was mixed well with 2 ml of pentane and 1 ml of methanol, and centrifuged in a Beckman centrifuge GP at 1000 rpm for 5 minutes to give the supernatant, which was then transferred to another new test tube. After evaporation of pentane and methanol in a draft chamber, the resulting residue was re-dissolved in 300 ml of pentane and filled into a vial for GC/MS.

[0280] (6) Extraction of Geranylgeraniol Analogs from Cell Fraction

[0281] (i) Extraction from Bacterial and Actinomycete Cells

[0282] 10 ml of the liquid culture solution was transferred into a 50 ml Coming tube and centrifuged in a Beckman refrigerated centrifuge (Avant J25-I) at 6000 rpm for 5 minutes to collect the cells. After the cells were suspended in 0.5 ml of deionized water, the suspension was transferred into a 10 ml conical bottom tube and crushed using an ultrasonic vibrator UC W-201 (Tokai electric Inc.) at 10° C. for 20 minutes by repeating the following cycle: crushing for 1 minute and allowing to rest for 30 seconds. The crushed cells were transferred into a test tube (φ)18 mm×125 mm) and mixed with 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride, followed by phosphatase treatment and extraction as in (5) above.

[0283] (ii) Extraction from Yeast Cells

[0284] 2.5 ml of the liquid culture solution was transferred into a test tube (φ18 mm×125 mm) and centrifuged in a Beckman centrifuge GP at 1000 rpm for 5 minutes to collect the cells. After the cells were suspended in 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride, the suspension was transferred into a glass tube for crushing. An equal volume of glass beads (Sigma; acid washedφ+=425-600 μm) was added to the tube and the cells were crushed using a Multi-Beads Schocker MB-200 (YASUI KIKAI) at 2500 rpm and at room temperature for 20 minutes. The whole content of the glass tube was transferred into a test tube (φ18 mm×125 mm), followed by phosphatase treatment and extraction as in (5) above.

[0285] (7) Analysis of Geranylgeraniol Analogs

[0286] Analysis was performed using an Agilent HP6890/5973 GC/MS system under the following conditions:

i) Inlet temperature:     250° C.
ii) Detector temperature: 260° C.
iii) MS zone temperatures:
MS Quad:                  150° C.
MS Source:                230° C.
iv) Scan parameters:
Low Mass:                 35
High Mass:                200
Threshold:                40
v) Injection parameters:
Mode:                     automatic injection
Sample volume:            2 μl
Washing:                  3 times with methanol and twice with hexane
Split ratio:              1:20
Column:                   Agilent HP-5MS
                          (0.25 mm × 30 m; film thickness of
                          0.25 μm)
Carrier gas:              helium at 1.0 ml/min
Solvent delay:            2 minutes
Oven conditions:          holding at 115° C. for 1.5 minutes
                          heating to 250° C. at 70° C./min, holding for
                          2 minutes
                          heating to 300° C. at 70° C./min, holding for
                          7 minutes
                          post time = 0
Internal standard:        1-undecanol/ethanol solution (1 μl/ml),
                          added to each vial in an amount of 10 μl
Inlet liner:              split/splitless liners
Analysis:                 After incorporation of TIC, 69 mass was
                          selected to integrate the peak area for each of
                          1-undecanol (RT = 3.39 min), nerolidol
                          (RT = 3.86 min), farnesol (RT = 4.23 min)
                          and geranylgeraniol (RT = 5.78 min). Each
                          substance was quantified from the ratio of
                          peak area between the substance and
                          undecanol as an internal standard.

[0287] (8) Results

[0288] Among screened strains, about one-third of 162 ascomycete strains and about one-tenth of 73 deuteromycete strains were confirmed to produce geranylgeraniol and/or farnesol, while three of 95 actinomycete strains were confirmed to produce nerolidol.

[0289] Tables 1, 2, 3 and 4 illustrate strains producing geranylgeraniol alone, strains producing both geranylgeraniol and farnesol, strains producing farnesol alone and strains producing nerolidol alone, respectively, along with yields thereof.

Example 2

Examination of Culture Conditions

[0290] Examination was performed on the following three strains: the two stains found to provide the highest production of geranylgeraniol in Example 1, Hanseniaspora valbyensis strain IFO 0115 and Saccharomycodes ludwigii strain IFO 0339, and Candida glabrata strain IFO 0005 found to provide high production of both geranylgeraniol and farnesol in Example 1. The results, including days of culture, medium composition, and changes in production with or without phosphatase treatment, are shown in Table 5 (supernatant fraction) and Table 6 (cell fraction). Each strain produces more farnesol and geranylgeraniol over the course of time. The addition of 5% glucose to the medium permits an increased production in the cell fraction. Further, the addition of 1% soybean oil along with glucose stimulates Candida glabrata strain to secrete farnesol and geranylgeraniol from the cells. Thus, although some differences are found among microorganisms, the use of the medium supplemented with 5% glucose and 1% soybean oil enables more farnesol and geranylgeraniol to be produced in and secreted from the cells.

[0291] Also, the supernatant fraction tends to contain more farnesol and geranylgeraniol in the absence of phosphatase treatment, whereas the cell fraction tends to contain more farnesol and geranylgeraniol when treated with phosphatase, thereby suggesting that farnesyl pyrophosphate and geranyl pyrophosphate are present in the cell fraction.

Example 3

Examination of Culture Conditions

[0292] Some of the strains tested in Example 1 were cultured in YM medium alone and in YM medium supplemented with 5% glucose and 1% soybean oil to examine farnesol and geranylgeraniol production. Tables 7 and 8 show the results obtained. The medium supplemented with 5% glucose and 1% soybean oil effected a significantly increased production.

Example 4

Examination of Culture Conditions

[0293] Each of the strains shown in Tables 9 and 10 was cultured in YM medium supplemented with 4 mg/L ergosterol and 0-20 mg/L squalene synthesis inhibitor (SQAD) or in YM medium supplemented with 5% glucose and 1% soybean oil along with 4 mg/L ergosterol and 0-20 mg/L squalene synthesis inhibitor (SQAD) to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. Tables 9 and 10 also show the results obtained.

[0294] Further, each of the strains shown in Tables 11 and 12 was cultured in YM medium supplemented with 1% soybean oil, 6% glucose, 4 mg/L ergosterol, and 0 mg/L or 20 mg/L squalene synthesis inhibitor (SQAD) to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. The results are shown in Table 11 (supernatant fraction) and Table 12 (cell fraction).

[0295] These tables indicate that the addition of soybean oil, glucose and a squalene synthesis inhibitor effects an increased production.

Example 5

Examination of Culture Medium Conditions

[0296] Each bacterial strain detected to produce farnesol was cultured in KB medium supplemented with 1% soybean oil, 4 mg/L ergosterol, and 0 mg/L or 20 mg/L squalene synthesis inhibitor (SQAD) to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. Table 13 shows the results obtained. This table indicates that the addition of a squalene synthesis inhibitor effects an increased production in bacterial strains as in the case of yeast strains.

[0297] Example 6

[0298] Under the culture conditions shown in Table 14 (e.g., days of culture, culture temperature, type of medium), various strains were cultured according to the same culture procedures as described in Example 1, followed by extraction of geranylgeraniol analogs from both cell and supernatant fractions. Analysis was performed on these fractions. Table 14 also shows the results obtained. LBO-SSI, YPDO-SSI, YMO-SSI, YMOL-SSI and HVO-SSI media used in this example were prepared as follows.

[0299] LBO-SSI Medium

[0300] The following ingredients were dissolved in 1 L of deionized water and then autoclaved. After the autoclaved medium was fully cooled, a filter-sterilized aqueous solution of squalene synthase inhibitor SQAD (2.5 mg/ml) was added to the medium to give a final concentration of 20 mg/L.

Yeast Extract (Difco) 5 g
Bactopeptone (Difco)  10 g
NaCl (Nacalai)        5 g
Glucose (Nacalai)     50 g
Soybean oil (Nacalai) 10 ml
Ergosterol solution   200 μl (200 μl of 20 mg/ml solution in
                      50% EtOH-50% Tergitol)

[0301] YPDO-SSI Medium

[0302] The following ingredients were dissolved in 1 L of deionized water and then autoclaved. After the autoclaved medium was fully cooled, a filter-sterilized aqueous solution of squalene synthase inhibitor SQAD (2.5 mg/ml) was added to the medium to give a final concentration of 20 mg/L.

Yeast Extract (Difco) 10 g
Bactopeptone (Difco)  20 g
Glucose (Nacalai)     50 g
Soybean oil (Nacalai) 10 ml
Ergosterol solution   200 μl (200 μl of 20 mg/ml solution in
                      50% EtOH-50% Tergitol)

[0303] YMO-SSI Medium

[0304] The following ingredients were added to YM medium (Difco), adjusted to 1 L with deionized water and then autoclaved. After the autoclaved medium was fully cooled, a filter-sterilized aqueous solution of squalene synthase inhibitor SQAD (2.5 mg/ml; Ltd.) was added to the medium to give a final concentration of 1 to 20 mg/L.

Glucose (Nacalai)     50 g
Soybean oil (Nacalai) 10 ml
Ergosterol (Nacalai)  4 mg (200 μl of 20 mg/ml solution in
                      50% EtOH-50% Tergitol)

[0305] YMOL-SSI Medium

[0306] This medium was prepared by adding 10 ml of olive oil (Nacalai) to YMO medium in the same manner as used for YMO-SSI preparation.

[0307] HVO-SSI Medium

[0308] The following ingredients were dissolved in 1 L of deionized water and then autoclaved. After the autoclaved medium was fully cooled, a filter-sterilized aqueous solution of squalene synthase inhibitor SQAD (2.5 mg/ml) was added to the medium to give a final concentration of 20 mg/L.

NaCl (Nacalai)        156 g
MgCl2□6H2O (Nacalai)  13 g
MgSO4□7H2O (Nacalai)  20 g
CaCl2□2H2O (Nacalai)  1 g
KCl (Nacalai)         4 g
NaHCO3 (Nacalai)      0.2 g
KBr (Nacalai)         0.5 g
Yeast Extract (Difco) 5 g
Glucose (Nacalai)     50 g
Soybean oil (Nacalai) 10 ml
Ergosterol solution   200 μl (200 μl of 20 mg/ml solution in
                      50% EtOH-50% Tergitol)

[0309] The present invention enables inexpensive mass production of geranylgeraniol and analogous compounds thereof by using microorganisms capable of producing geranylgeraniol, farnesol and/or nerolidol useful as biosynthetic intermediates of terpenes, carotenoids and/or steroids.

[0310] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

	TABLE 1
	Supernatant	Cell
	fraction	fraction
	Strains	(μg/L of culture solution)
Strain No.	Genus	FOH	GGOH	FOH	GGOH
ATCC	12341	Saccharomyces	0.0	0.0	0.0	14.4
		cerevisiae
K	4010	Saccharomyces sp.	0.0	0.0	0.0	9.1
K	4031	Saccharomyces sp.	0.0	0.0	0.0	7.0
K	4037	Saccharomyces sp.	0.0	0.0	0.0	5.8
K	4039	Saccharomyces sp.	0.0	0.0	0.0	5.5
K	4041	Saccharomyces sp.	0.0	0.0	0.0	8.4
IFO	0565	Saccharomyces	0.0	0.0	0.0	54.7
		cerevisiae
IFO	0222	Saccharomyces	0.0	0.0	0.0	3.3
		cerevisiae
IFO	0216	Saccharomyces	0.0	0.0	0.0	5.8
		cerevisiae
ATCC	9080	Saccharomyces	0.0	0.0	0.0	5.7
		cerevisiae
IFO	0106	Saccharomycopsis	0.0	0.0	0.0	3.4
		fibuligera
IFO	0125	Williopsis saturnus	0.0	0.0	0.0	5.4
		var. saturuns
IFO	0807	Pichia silvicola	0.0	0.0	0.0	1.2
IFO	0980	Nakazawaea holstii	0.0	0.0	0.0	5.2
K	4327	Hansenula	0.0	0.0	0.0	4.7
		polymopla
IFO	0151	Kloeckera japonica	0.0	0.0	0.0	10.2
IFO	0128	Pichia	0.0	0.0	0.0	11.0
		membranaefaciens
K	4261	Pichia aganobii	0.0	4.0	0.0	0.0
IFO	0941	Williopsis saturnus	0.0	0.0	0.0	6.7
		var. saturuns
IFO	0013	Candida krusei	0.0	0.0	0.0	16.5
IFO	0706	Candida kefyr	0.0	0.0	0.0	4.9
IFO	0716	Candida tenuis	0.0	0.0	0.0	8.7
IFO	0617	Kluyveromyces	0.0	0.0	0.0	25.2
		marxianus
IFO	0762	Candida solani	0.0	0.0	0.0	29.0
IFO	1060	Candida albicans	0.0	2.4	0.0	0.0
IFO	0720	Candida	0.0	0.0	0.0	25.9
		catenulata
K: strain maintained by Kyoto University
FOH: farnesol
GGOH: geranylgeraniol

[0311]

	TABLE 2
	Supernatant	Cell
	fraction	fraction
	Strains	(μg/L of culture solution)
Strain No.	Genus	FOH	GGOH	FOH	GGOH
K	4002	Saccharomyces sp.	4.4	0.0	0.0	10.9
K	4006	Saccharomyces sp.	4.2	0.0	4.3	6.7
K	4013	Saccharomyces sake	0.0	0.0	7.3	5.0
K	4015	Saccharomyces sake	7.9	0.0	6.6	7.9
K	4016	Saccharomyces sake	4.5	0.0	2.2	6.5
K	4017	Saccharomyces sake	4.3	0.0	7.9	7.9
K	4018	Saccharomyces sake	4.4	0.0	5.0	9.9
K	4020	Saccharomyces sake	0.0	0.0	2.8	5.4
K	4022	Saccharomyces sake	0.0	0.0	2.1	4.7
K	4023	Saccharomyces sake	0.0	0.0	5.2	11.5
K	4025	Saccharomyces sake	5.0	0.0	5.5	2.0
K	4026	Saccharomyces sake	0.0	0.0	2.6	7.6
K	4029	Saccharomyces sake	0.0	0.0	2.2	2.3
K	4030	Saccharomyces sp.	5.1	0.0	4.6	3.7
K	4036	Saccharomyces sp.	0.0	0.0	4.1	15.2
K	4045	Saccharomyces	0.0	0.0	44.9	18.6
		cerevisiae
K	4102	Saccharomyces	0.0	0.0	22.9	7.2
		ellipsoideus
K	4103	Saccharomyces	0.0	0.0	22.6	8.6
		cerevisiae
K	4104	Saccharomyces	0.0	0.0	15.8	31.7
		cerevisiae
IFO	0252	Saccharomyces	0.0	0.0	28.5	39.0
		rosei
IFO	0288	Kluyveromyces	0.0	0.0	11.3	22.6
		marxianus
Kyokai	No. 2	Saccharomyces sake	0.0	0.0	9.7	26.1
IFO	0422	Torulaspora	0.0	0.0	8.2	11.9
		delbrueckii
IFO	0487	Zygo -	0.0	0.0	4.9	14.3
		saccharomyces
		rouxii
IFO	1346	Saccharomyces	0.0	0.0	4.3	13.1
		cerevisiae
ATCC	204660	Saccharomyces	8.7	0.0	9.3	26.8
		cerevisiae
IFO	0339	Saccharomycodes	15.3	0.0	6.0	80.9
		ludwigii
IAM	4842	Schizo -	2.7	0.0	2.2	12.1
		saccharomyces
		octosporus
IFO	1116	Wickerhamia	18.7	0.0	4.2	23.0
		fluorescens
IFO	0794	Debaryomyces	4.8	2.7	7.7	11.5
		hansenii
		var. fabryi
IFO	1359	Debaryomyces	18.5	0.0	6.3	21.1
		castellii
JCM	2169	Debaryomyces	73.7	0.0	14.3	6.2
		vanrijiae
		var vanrijiae
IFO	0115	Hanseniaspora	13.6	0.0	7.4	103.5
		valbyensis
IFO	0118	Pichia anomala	0.0	0.0	2.2	15.3
IFO	0569	Pichia anomala	0.0	0.0	7.3	28.2
IFO	0941	Williopsis saturnus	4.7	0.0	69.8	78.7
		var. saturnus
IFO	1475	Ogataea	0.0	0.0	13.5	36.1
		polymorpha
IFO	1670	Pichia naganishii	1.2	2.1	0.0	0.0
IFO	0707	Pichia anomala	8.6	0.6	0.0	12.3
IFO	0719	Candida	10.2	0.0	4.4	20.3
		zeylanoides
IFO	0701	Candida stellata	10.3	0.0	0.0	4.9
IFO	0662	Kluyveromyces	13.7	0.0	10.4	9.2
		thermotolerans
IFO	0648	Kluyveromyces	16.0	0.0	6.3	72.9
		lactis
IFO	0005	Candida glabrata	35.7	5.4	4.5	28.3
IFO	0595	Zygo -	9.3	3.6	0.0	0.0
		saccharomyces
		japanicus
K: strain maintained by Kyoto University
FOH: farnesol
GGOH: geranylgeraniol

[0312]

	TABLE 3
	Supernatant	Cell
	fraction	fraction
	Strains	(μg/L of culture solution)
Strain No.	Genus	FOH	GGOH	FOH	GGOH
K	4003	Saccharomyces sp.	5.0	0.0	19.3	0.0
K	4004	Saccharomyces sp.	10.6	0.0	15.2	0.0
K	4011	Saccharomyces sake	0.0	0.0	9.0	0.0
K	4021	Saccharomyces sake	0.0	0.0	2.2	0.0
K	4101	Saccharomyces logos	0.0	0.0	18.4	0.0
IFO	0686	Zygosaccharomyces	0.0	0.0	10.1	0.0
		rouxii
IFO	0285	Saccharomyces	4.4	0.0	0.0	0.0
		dairensis
IFO	0262	Saccharomyces	7.1	0.0	0.0	0.0
		cerevisiae
IFO	0021	Zygosaccharomyces	5.4	0.0	0.0	0.0
		fermentati
IFO	0259	Saccharomyces	3.2	0.0	0.0	0.0
		paradoxus
IFO	0539	Saccharomyces bayanus	1.4	0.0	0.0	0.0
IFO	0613	Saccharomyces bayanus	2.3	0.0	0.0	0.0
IFO	0346	Schizosaccharomyces	4.4	0.0	16.8	0.0
		pombe
IFO	0358	Schizosaccharomyces	2.3	0.0	4.7	0.0
		pombe
IFO	0023	Debaryomyces hansenii	12.2	0.0	0.0	0.0
IFO	0954	Citeromyces matritensis	8.5	0.0	0.0	0.0
IFO	0974	Kuraishia capsulata	0.0	0.0	8.2	0.0
IFO	0963	Pichia anomala	0.0	0.0	9.6	0.0
IFO	0673	Waltomyces lipofer	10.1	0.0	0.0	0.0
IFO	0678	Lypomyces starkeyi	4.8	0.0	0.0	0.0
IFO	0579	Candida albicans	12.8	0.0	0.0	0.0
IFO	0626	Candida utilis	3.0	0.0	0.0	0.0
IFO	3022	Bacillus	4.1	0.0	0.0	0.0
		amyloliquefaciens
IFO	3030	Bacillus pumilus	6.0	0.0	0.0	0.0
IFO	3762	Staphylococcus	6.1	0.0	0.0	0.0
		epidermidis
K	876	Pseudomonas sp.	18.6	0.0	0.0	0.0
K: strain maintained by Kyoto University
FOH: farnesol
GGOH: geranylgeraniol

[0313]

	TABLE 4
	Supernatant	Cell
	fraction	fraction
	(μg/L of culture solution)
Strain No.	Genus	NE	FOH	GOH	NE	FOH	GOH
IFO	12865	Streptomyces	0.0	0.0	0.0	11.5	0.0	0.0
		gardneri
IFO	3384	Nocardia	0.0	0.0	0.0	14.2	0.0	0.0
		asteroides
IFO	14340	Nocardia	0.0	0.0	0.0	6.4	0.0	0.0
		fusca
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol

[0314]

	TABLE 5
		Supernatant fraction
	Culti-	(μg/L of culture solution)
		vation	Phosphatase-	Phosphatase-
	Medium	period	untreated	treated
Strain No.	composition	(days)	FOH	GGOH	FOH	GGOH
IFO	0005	YM	1	0.0	0.0	1.5	0.0
			2	0.0	0.0	0.0	0.0
			3	0.0	0.0	0.0	0.0
		YM + Glc	1	0.0	0.0	6.1	0.0
			2	0.0	0.0	9.5	0.0
			3	29.4	2.3	13.9	0.0
		YM + Glc +	1	0.0	0.0	0.0	0.0
		SBO
			2	135.6	92.0	69.9	31.5
			3	850.7	416.5	116.6	40.0
IFO	0115	YM	1	0.0	0.0	0.0	0.0
			2	0.0	0.0	0.0	0.0
			3	34.0	0.0	0.0	0.0
		YM + Glc	1	0.0	0.0	0.0	0.0
			2	11.3	0.0	0.0	0.0
			3	36.4	0.0	18.0	0.0
		YM + Glc +	1	0.0	0.0	0.0	0.0
		SBO
			2	24.0	0.0	5.1	0.0
			3	30.3	26.5	7.9	0.0
IFO	0339	YM	1	0.0	0.0	0.0	0.0
			2	8.7	0.0	0.0	0.0
			3	0.0	0.0	0.0	0.0
		YM + Glc	1	0.0	0.0	0.0	0.0
			2	16.8	0.0	17.7	0.0
			3	67.1	0.0	20.8	0.0
		YM + Glc +	1	0.0	0.0	0.0	0.0
		SBO
			2	11.7	5.3	0.0	0.0
			3	85.6	7.3	6.5	0.0
FOH: farnesol
GGOH: geranylgeraniol
YM: YM medium (Difco)
YM + Glc: YM medium (Difco) + 5% glucose
YM + Glc + SBO: YM medium (Difco) + 5% glucose + 1% soybean oil

[0315]

	TABLE 6
		Cell fraction (μg/L of
	Culti-	culture solution)
		vation	Phosphatase-	Phosphatase-
	Medium	period	untreated	treated
Strain No.	composition	(days)	FOH	GGOH	FOH	GGOH
IFO	0005	YM	1	39.4	0.0	0.0	0.0
			2	14.8	37.3	0.0	0.0
			3	11.4	41.7	51.2	0.0
		YM + Glc	1	8.4	0.0	0.0	0.0
			2	44.1	134.4	86.8	304.8
			3	104.4	323.9	202.4	772.6
		YM + Glc +	1	5.8	0.0	0.0	0.0
		SBO
			2	427.4	178.9	248.0	239.6
			3	835.3	363.7	1321.6	1176.2
IFO	0115	YM	1	0.0	0.0	0.0	0.0
			2	34.0	5.1	0.0	0.0
			3	21.1	45.6	0.0	63.7
		YM + Glc	1	0.0	0.0	0.0	0.0
			2	46.6	26.1	23.9	0.0
			3	54.2	108.5	78.0	491.8
		YM + Glc +	1	0.0	0.0	0.0	0.0
		SBO
			2	0.0	0.0	9.7	0.0
			3	61.3	122.0	69.2	286.1
IFO	0339	YM	1	18.0	0.0	0.0	0.0
			2	16.6	0.0	0.0	0.0
			3	19.5	48.3	0.0	71.0
		YM + Glc	1	0.0	0.0	0.0	0.0
			2	58.6	34.1	12.0	0.0
			3	62.3	173.8	67.8	464.8
		YM + Glc +	1	0.0	0.0	0.0	0.0
		SBO
			2	96.4	10.3	9.3	0.0
			3	61.6	103.7	27.9	135.6
FOH: farnesol
GGOH: geranylgeraniol
YM: YM medium (Difco)
YM + Glc: YM medium (Difco) + 5% glucose
YM + Glc + SBO: YM medium (Difco) + 5% glucose + 1% soybean oil

[0316]

TABLE 7
Medium composition: YM medium (Difco)
	Supernatant	Cell
	fraction	fraction
	Strains	(μg/L of culture solution)
Strain No.	Genus	FOH	GGOH	FOH	GGOH
K	4104	Saccharomyces	0.0	0.0	15.8	31.7
		cerevisiae
IFO	0252	Saccharomyces rosei	0.0	0.0	28.5	39.0
IFO	0565	Saccharomyces	0.0	0.0	0.0	54.7
		cerevisiae
IFO	0941	Williopsis saturnus	4.7	0.0	69.8	78.7
		var. saturnus
IFO	1475	Ogataea	0.0	0.0	13.5	36.1
		polymorpha
IFO	0648	Kluyveromyces	6.0	0.0	6.3	72.9
		lactis
K: strain maintained by Kyoto University
FOH: farnesol
GGOH: geranylgeraniol

[0317]

TABLE 8
Medium composition: YM medium (Difco) + 5%
glucose + 1% soybean oil
	Supernatant	Cell
	fraction	fraction
		Culti-	(μg/L of
	Strains	vation	culture solution)
Strain No.	Genus	days	FOH	GGOH	FOH	GGOH
K	4104	Saccharomyces	3	281.0	127.7	241.1	161.6
		cerevisiae	6	338.2	186.9	155.3	132.2
IFO	2052	Saccharomyces	3	220.0	98.0	305.9	140.2
		rosei	6	381.2	176.6	193.1	122.5
IFO	0565	Saccharomyces	3	49.3	16.0	88.0	78.2
		cerevisiae	6	51.9	34.1	248.5	214.2
IFO	0941	Williopsis	3	54.8	22.6	255.3	169.3
		saturnus var.	6	88.3	33.1	363.2	220.9
		saturnus
IFO	1475	Ogataea	3	60.9	69.1	113.5	127.2
		polymorpha	6	19.8	14.4	60.8	95.9
IFO	0648	Kluyveromyces	3	28.5	37.8	61.8	91.1
		lactis	6	120.3	124.5	159.8	211.5
K: strain maintained by Kyoto University
FOH: farnesol
GGOH: geranylgeraniol

[0318]

TABLE 9
Medium composition: YM medium (Difco) + 4
mg/L ergosterol + 0-20 mg/L SQAD
	Supernatant	Cell
	fraction	fraction
	(μg/L of culture solution)
Strain No.	SQAD	Days	NE	FOH	GGOH	NE	FOH	GGOH
IFO 0215	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Saccharo -	1		0.0	0.0	0.0	0.0	0.0	0.0
myces	20		0.0	3.2	0.0	0.0	0.0	0.0
unisporus	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 0538	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Saccharo -	1		0.0	0.0	0.0	0.0	0.0	0.0
myces	20		0.0	0.0	0.0	0.0	8.1	0.0
cerevisiae	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 0622	0	3	0.0	3.1	0.0	0.0	1.9	30.4
Candida	1		0.0	3.5	0.0	0.0	1.4	24.3
glabrata	20		0.0	243.5	0.0	0.0	212.0	57.0
	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	5.5	40.5
IFO 0717	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Yarrowia	1		0.0	1.3	0.0	0.0	0.0	0.0
lopolytica	20		0.0	39.7	66.5	0.0	39.7	63.5
	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	2.1	0.0	0.0	1.5	0.0
	20		0.0	10.6	0.0	0.0	10.6	0.0
IFO 0948	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Komag -	1		0.0	0.0	0.0	0.0	0.0	0.0
ataella	20		0.0	3.4	2.3	0.0	3.4	1.8
	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 0974	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Kuraishia	1		0.0	0.0	0.0	0.0	0.0	0.0
capsulata	20		0.0	0.0	0.0	0.0	0.0	0.0
	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 1472	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Ogataea	1		0.0	0.0	0.0	0.0	0.0	0.0
glucozyma	20		0.0	0.0	18.3	0.0	0.0	17.5
	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	6.8
IFO 1892	0	3	0.0	0.0	0.0	0.0	0.0	8.9
Saccharo -	1		0.0	0.0	0.0	0.0	0.0	16.7
myces	20		0.0	0.0	0.0	0.0	0.0	38.5
kluyeri	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 1910	0	3	0.0	0.0	0.0	0.0	0.0	0.0
Candida	1		0.0	0.0	0.0	0.0	0.0	0.0
cario -	20		0.0	0.0	0.0	0.0	0.0	5.5
silignicola	0	7	0.0	0.0	0.0	0.0	0.0	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
IFO 0005	0	3	0.0	0.0	0.0	0.0	0.0	12.0
Candia	1		0.0	0.0	0.0	0.0	0.0	4.3
glabrata	20		0.0	33.8	2.7	0.0	0.0	53.7
	0	7	0.0	0.0	0.0	0.0	20.5	0.0
	1		0.0	0.0	0.0	0.0	0.0	0.0
	20		0.0	0.0	0.0	0.0	0.0	0.0
I: strain purchased from IFO
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol
SQAD: squalene synthesis inhibitor
0: none
1: 1 mg/L
20: 20 mg/L

[0319]

TABLE 10
Medium composition: YM medium (Difco) + 5% glucose + 1%
soybean oil + 4 mg/L ergosterol + 0-20 mg/L SQAD
	Supernatant fraction	Cell fraction
	(μg/L of culture solution)
Strain No.	SQAD	Days	NE	FOH	GGOH	NE	FOH	GGOH
IFO 0215	0	3	16.3	8.2	0.0	0.0	0.0	0.0
Saccharomyces	1		12.2	5.2	0.0	0.0	0.0	0.0
unisporus	20		67.4	68.4	0.0	0.0	16.9	0.0
	0	7	19.6	14.8	0.0	0.0	0.0	0.0
	1		27.3	17.5	0.0	0.0	0.0	0.0
	20		466.6	433.7	21.6	8.6	255.6	0.0
IFO 0538	0	3	0.0	139.3	59.8	0.0	0.0	0.0
Saccharomyces	1		0.0	102.9	46.2	0.0	15.5	0.0
cerevisiae	20		65.9	9087.5	331.6	0.0	1465.9	51.0
	0	7	0.0	426.4	246.7	0.0	38.0	0.0
	1		0.0	319.5	241.0	0.0	188.8	85.6
	20		216.2	34241.4	1568.8	18.4	5812.1	266.2
IFO 0622	0	3	0.0	106.1	112.3	0.0	28.8	33.6
Candida	1		9.4	149.2	119.7	0.0	9.7	0.0
glabrata	20		422.2	12186.9	327.2	0.0	331.6	9.3
	0	7	11.6	378.3	408.5	0.0	43.7	59.2
	1		17.2	530.0	231.5	0.0	133.3	58.5
	20		1256.9	22147.1	862.7	138.0	2750.6	170.5
IFO 0717	0	3	0.0	8.7	0.0	0.0	6.7	0.0
Yarrowia	1		2.4	100.1	19.1	0.0	165.3	0.0
lopolytica	20		19.0	983.3	17.8	0.0	882.1	0.0
	0	7	0.0	95.3	608.0	0.0	0.0	38.4
	1		19.0	405.4	546.8	0.0	0.0	0.0
	20		156.7	6812.9	76.2	0.0	1025.1	41.8
IFO 0948	0	3	0.0	73.5	78.9	0.0	13.8	42.3
Komagataella	1		0.0	82.2	71.5	0.0	22.9	28.4
pastoris	20		17.2	2956.2	132.5	0.0	608.4	80.1
	0	7	0.0	101.6	83.5	0.0	4.3	11.0
	1		0.0	569.8	235.7	0.0	53.4	28.2
	20		34.2	5513.3	360.4	0.0	537.5	84.5
IFO 0974	0	3	0.0	24.8	11.1	0.0	3.3	0.0
Kuraishia	1		0.0	1061.9	46.1	0.0	16.1	7.5
capsulata	20		0.0	2022.7	262.0	0.0	149.2	34.8
	0	7	0.0	167.5	236.3	0.0	3.2	8.5
	1		0.0	713.7	275.7	0.0	24.8	21.2
	20		35.9	8268.5	680.4	0.0	362.1	59.7
IFO 1472	0	3	0.0	34.8	79.8	0.0	7.4	20.4
Ogataea	1		0.0	40.5	86.0	0.0	3.7	9.5
glucozyma	20		0.0	808.8	278.7	0.0	41.1	37.5
	0	7	0.0	63.2	138.8	0.0	0.0	0.0
	1		0.0	93.8	114.7	0.0	0.0	0.0
	20		0.0	1832.0	513.0	0.0	73.7	52.6
IFO 1892	0	3	0.0	70.4	46.1	0.0	3.7	0.0
Saccharomyces	1		0.0	51.4	23.0	0.0	2.3	0.0
kluyeri	20		0.0	62.9	30.7	0.0	16.5	0.0
	0	7	0.0	158.5	71.4	0.0	0.0	0.0
	1		23.5	188.3	101.7	0.0	5.0	0.0
	20		44.1	935.3	126.4	0.0	227.8	27.8
IFO 1910	0	3	0.0	11.0	44.7	0.0	0.0	0.0
Candida	1		0.0	20.9	69.3	0.0	0.0	0.0
cariosilignicola	20		0.0	124.9	127.1	0.0	8.1	19.6
	0	7	0.0	51.7	152.9	0.0	0.0	0.0
	1		0.0	257.7	367.3	0.0	10.3	37.4
	20		0.0	14378.1	2997.2	0.0	845.5	205.0
IFO 0005	0	3	22.9	552.1	496.1	0.0	50.5	75.2
Candia	1		260.0	6784.9	818.5	8.6	462.0	86.5
glabrata	20		2384.0	37513.9	1022.9	423.9	9405.0	306.5
	0	7	77.1	1676.1	1434.6	0.0	41.2	51.2
	1		546.0	11219.5	1377.8	9.2	331.7	77.1
	20		6208.9	68495.6	3228.1	139.2	2682.5	217.6
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol
SQAD: squalene synthesis inhibitor
0: none
1: 1 mg/L
20: 20 mg/L

[0320]

TABLE 11
Medium composition: YM or KB or KY medium + 1% soybean oil +
6% glucose + 4 mg/L ergosterol
	Supernatant fraction (μg/L)
Medium	Strain No	SQAD	NE	FOH	GGOH
YM	IFO 0107	0	0.0	10.8	155.2
	Saccharomycopsis	20	0.0	9.7	386.5
	fibuligera
KB	K 0876	0	0.0	29.0	0.0
	Pseudomonas sp	20	0.0	7.8	0.0
YM	IFO 1665	0	0.0	4.7	211.5
	Saccharomycopsis	20	0.0	94.5	4214.9
	fibuligera
YM	IFO 1744	0	0.0	0.0	155.5
	Saccharomycopsis	20	0.0	41.1	3870.1
	fibuligera
KB	K 2103	0	0.0	10.9	0.0
	Norcadia asteroides	20	0.0	0.0	0.0
KY	IFO 4570	0	0.0	0.0	36.5
	Mucor Javanicus	20	0.0	38.8	343.6
KY	K 4003	0	30.1	511.9	694.7
	Saccharomyces Hafe	20	4980.0	56541.1	3603.4
	logos van Laer
KY	K 4045	0	148.1	870.4	766.8
	Saccharomyces	20	24606.3	37772.7	2590.6
	cerevisiae
KY	K 4102	0	17.5	541.1	711.3
	Saccharomyces	20	18160.8	50245.8	3207.1
	ellipsoideus
KY	K 4103	0	56.4	753.1	1072.8
	Saccharomyces	20	20930.8	53814.6	4589.9
	cerevisiae
KY	K 4104	0	19.5	569.2	546.0
	Saccharomyces	20	23620.7	54713.2	2654.4
	cerevisiae
KY	IFO 0565	0	0.0	411.7	535.2
	Saccharomyces	20	839.9	45723.6	2216.6
	cerevisiae
KY	IFO 0210	0	37.5	685.5	362.8
	Saccharomyces	20	25251.0	48795.6	1627.2
	cerevisiae
KY	IFO 0346	0	0.0	196.2	278.9
	Schizosaccharomyces	20	757.6	45282.1	1153.6
	pombe
KY	IFO 1475	0	0.0	236.9	462.7
	Ogataea polymorpha	20	0.0	5643.0	1195.1
KY	JCM 2169	0	0.0	809.8	241.2
	Debaryomyces vanrijiae	20	129.5	20359.5	2236.0
	var vanrijiae
KY	IFO 0339	0	0.0	164.4	864.3
	Saccharomycodes	20	131.0	28498.6	1483.8
	ludwigii
KY	IFO 0115	0	0.0	254.9	493.6
	Hanseniaspora	20	182.6	26807.1	1217.7
	valbyensis Valbyensis
KY	IFO 0648	0	0.0	136.4	433.8
	Kluyveromyces lactis	20	348.6	31785.7	3343.8
KY	IFO 0005	0	192.8	861.5	909.0
	Candida glabrata	20	15504.1	44573.8	2237.1
KY	IFO 0762	0	37.0	274.7	384.4
	Candida solani	20	1702.8	6574.7	619.1
KY	IFO 1527	0	0.0	16.6	24.7
	Cryptococcus humicolus	20	0.0	49.6	33.4
KY	IFO 1116	0	0.0	199.3	315.7
	Wickerhamia fluorescens	20	73.2	12200.1	1181.6
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol
SQAD: squalene synthesis inhibitor  0: none  20: 20 mg/L
Treated with phosphatase

[0321]

TABLE 12
Medium composition: YM or KB or KY medium + 1% soybean oil +
6% glucose + 4 mg/L ergosterol
	Cell fraction (μg/L)
Medium	Strain No.	SQAD	NE	FOH	GGOH
YM	IFO 0107	0	0.0	8.1	24.6
	Saccharomycopsis	20	0.0	4.8	79.9
	fibuligera
KB	K 0876	0	0.0	0.0	0.0
	Pseudomonas sp. H21	20	0.0	0.0	0.0
YM	IFO 1665	0	0.0	8.2	21.7
	Saccharomycopsis	20	0.0	20.8	228.9
	fibuligera
YM	IFO 1744	0	0.0	0.0	42.2
	Saccharomycopsis	0	0.0	29.8	819.1
	fibuligera
KB	K 2103	0
	Norcadia asteroides	20
KY	IFO 4570	0	13.4	22.7	17.5
	Mucor Javanicus	20	0.0	15.6	61.7
KY	K 4003	0	0.0	36.9	52.4
	Saccharomyces Hafe	20	291.8	7433.0	710.6
	logos van Laer
KY	K 4045	0	0.0	69.3	65.8
	Saccharomyces	20	3022.6	7893.0	534.5
	cerevisiae
KY	K 4102	0	0.0	36.4	76.3
	Saccharomyces	20	1350.0	6358.2	396.5
	ellipsoideus
KY	K 4103	0	0.0	591.7	39.4
	Saccharomyces	20	1073.6	5528.9	369.6
	cerevisiae
KY	K 4104	0	0.0	51.6	79.2
	Saccharomyces	20	2409.9	11464.9	656.2
	cerevisiae
KY	IFO 0565	0	0.0	40.2	54.7
	Saccharomyces	20	57.3	5071.6	333.5
	cerevisiae
KY	IFO 0210	0	0.0	119.4	89.6
	Saccharomyces	20	1698.7	4355.2	83.2
	cerevisiae
KY	IFO 0346	0	0.0	29.7	82.6
	Schizosaccharomyces	20	83.1	4826.5	159.4
	pombe
KY	IFO 1475	0	0.0	31.4	126.0
	Ogataea polymorpha	20	19.8	1196.1	402.7
KY	JCM 2169	0	0.0	67.2	54.3
	Debaryomyces vanrijiae	20	23.9	2807.0	492.6
	var vanrijiae
KY	IFO 0339	0	0.0	41.6	114.7
	Saccharomycodes	20	0.0	3217.2	199.4
	ludwigii
KY	IFO 0115	0	0.0	25.2	76.6
	Hanseniaspora	20	15.1	2823.5	185.8
	valbyensis Valbyensis
KY	IFO 0648	0	0.0	20.9	84.6
	Kluyveromyces lactis	20	14.5	1128.6	225.8
KY	IFO 0005	0	0.0	34.9	51.7
	Candida glabrata	20	1193.0	4710.9	279.6
KY	IFO 0762	0	16.2	90.5	126.0
	Candida solani	20	231.3	962.5	350.0
KY	IFO 1527	0	7.8	16.0	0.0
	Cryptococcus humicolus	20	21.2	160.9	51.9
KY	IFO 1116	0	0.0	15.0	86.5
	Wickerhamia fluorescens	20	32.6	4262.8	500.7
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol
SQAD: squalene synthesis inhibitor  0: none  20: 20 mg/L
Treated with phosphatase

[0322]

TABLE 13
Medium composition: KB medium + 1% soybean oil + ergosterol
	Supernatant fraction	Cell fraction
	(μg/L of culture solution)
Strain No.	SQAD	NE	FOH	GGOH	NE	FOH	GGOH
IFO 3032	0	0.0	11.6	0.0	0.0	0.0	0.0
Baccilus	20	0.0	22.4	0.0	0.0	0.0	0.0
Amyloliquefaciens
IFO 3030	0	0.0	21.9	0.0	0.0	0.0	0.0
Baccilus pumilus	20	0.0	36.6	0.0	0.0	6.0
IFO 3762	0	0.0	6.3	0.0	0.0	11.2	0.0
Staphylococcus	20	0.0	121.4	0.0	0.0	292.4	0.0
Epidermidis
IFO 3067	0	0.0	5.1	0.0	0.0	0.0	0.0
Micrococcus	20	0.0	57.3	0.0	0.0	11.8	0.0
Lutenus
IFO 12146	0	0.0	0.0	0.0	0.0	0.0	0.0
Exiguobacterium	20	0.0	115.7	0.0	0.0	10.4	0.0
Acetylicum
NE: nerolidol
FOH: farnesol
GGOH: geranylgeraniol
SQAD: squalene synthesis inhibitor  0: none  20: 20 mg/L
Treated with phosphatase

[0323]

TABLE 14
Production of nerolidol (NOH), farnesol (FOH) and geranylgeraniol (GGOH) (mg/L of culture solution)
	Day 3	Day 10
Strains	No.	NOH	FOH	GGOH	NOH	FOH	GGOH	Temp.	Medium
Alcaligenes faecalis	IFO 13111	0.00	0.01	0.11	0.00	0.04	0.09	30	LBO-SSI
Brevibacterium divaricatum	NRRL 2311	0.00	0.00	0.02	0.00	0.00	0.00	30	LBO-SSI
Brevibacterium fuscum	IFO 12127	0.00	0.02	0.00	0.00	0.00	0.00	30	LBO-SSI
Brevibacterium linens	IFO 12171	0.00	0.46	0.00	0.00	0.08	0.00	30	LBO-SSI
Candida catenulata	IFO 0720	0.04	8.10	0.34	0.04	8.61	0.47	30	YMO-SSI
Candida fragicola	IFO 1574	0.03	4.91	0.76	0.04	8.95	1.31	30	YMO-SSI
Candida krusei	IFO 0013	0.04	6.99	0.29	0.06	13.65	0.86	30	YMO-SSI
Candida lambica	IFO 1146	0.02	4.53	0.49	0.04	0.59	1.24	30	YMO-SSI
Candida maltosa	IFO 1977	0.00	6.74	0.19	0.08	9.03	0.58	30	YMO-SSI
Candida mycoderma	IFO 0164	0.02	5.45	0.20	0.03	4.69	0.17	30	YMO-SSI
Candida parapsilosis	IFO 0708	0.00	1.76	0.17	0.01	5.13	0.25	30	YMO-SSI
Candida rugosa	IFO 0591	0.00	0.90	0.08	0.00	4.20	0.19	30	YMO-SSI
Candida succiphila	IFO 1911	0.00	2.44	0.25	0.00	0.00	0.00	30	YMO-SSI
Candida tropicalis	IFO 0006	><><><	0.17	9.04	0.00	30	YMO-SSI
Candida zeylanoides	IFO 0719	0.00	1.43	0.29	0.00	7.38	1.24	30	YMO-SSI
Cryptococcus albidus	IFO 0881	0.00	0.15	0.03	0.02	2.41	0.45	30	YMO-SSI
Cryptococcus glutinis	IFO 1125	0.04	6.64	1.48	0.35	3.70	3.29	24	YMO-SSI
Dipodascus ovetensis	IFO 1201	0.00	4.23	0.14	0.04	8.68	0.95	30	YMO-SSI
Haloferax volcanii	IFO 14742	2.12	39.11	1.04	4.14	57.73	2.49	30	HVO-SSI
Hanseniaspora valbyensis	IFO 1758	0.00	0.33	0.04	0.04	2.40	0.12	30	YMO-SSI
Issatchenkia orientalis	IFO 1279	0.00	2.22	0.15	0.03	7.68	0.52	30	YMO-SSI
Kloeckera africana	IFO 0868	1.09	10.90	0.81	1.10	6.29	0.17	30	YMO-SSI
Kloeckera apiculata	IFO 0151	0.00	0.05	0.02	1.04	10.73	0.86	30	YMO-SSI
Kluyveromyces marxianus	IFO 0617	0.09	14.48	0.87	0.13	16.29	1.71	30	YMO-SSI
Kuraishia capsulata	IFO 0974	0.00	0.91	0.17	0.00	3.25	0.42	30	YMO-SSI
Mortierella ramanniana	ATCC 24786	><><><	0.00	0.76	0.13	24	YMO-SSI
Nakazawaea holstii	IFO 0980	0.01	1.06	0.18	0.02	5.85	0.38	30	YMO-SSI
Pichia capsulata	IFO 0984	0.00	0.63	0.04	0.04	4.17	0.11	30	YMO-SSI
Pichia henricii	IFO 1477	0.00	3.20	0.12	0.00	3.24	0.00	30	YMO-SSI
Pichia holstii	IFO 0980	0.04	6.22	0.22	0.04	2.79	0.65	30	YMO-SSI
Pichia naganishii	IFO 1670	0.00	2.14	0.22	0.02	9.28	0.96	30	YMO-SSI
Pichia rhodanensis	IFO 1272	0.03	3.34	0.91	0.46	26.57	10.29	30	YMO-SSI
Pichia saitoi	IAM 4945	0.34	10.78	0.51	0.28	24.31	1.38	30	YMO-SSI
Rhodosporidium toruloides	IFO 8766	0.88	6.59	2.47	0.72	2.47	2.52	24	YMO-SSI
Rhodotorula aurantinaca	IFO 0951	0.01	3.14	0.18	0.03	6.11	0.50	30	YMO-SSI
Rhodotorula rubra	IFO 0870	0.03	1.62	1.02	0.16	2.32	1.61	30	YMO-SSI
Saccharomycopsis fibuligera	IFO 0105	0.00	0.06	1.68	0.00	5.71	4.79	30	YMO-SSI
Saccharomycopsis lipolytica	IFO 1209	0.05	9.48	0.16	0.21	16.46	0.83	30	YMO-SSI
Schizosaccharomyces octosporus	IAM 4842	0.00	1.24	0.03	0.00	1.79	0.08	30	YMO-SSI
Staphylococcus aureus	IFO 3060	0.00	0.06	0.00	0.00	0.05	0.00	30	YMO-SSI
Torulaspora delbrueckii	IFO 1626	0.04	2.33	0.15	0.06	5.50	0.36	30	YMO-SSI
Trichosporon cutaneum	IFO 1198	0.00	10.23	0.69	0.00	0.14	0.09	30	YMO-SSI
Tsukamurella paurometabolum	IFO 12160	0.00	0.07	0.00	0.00	0.06	0.00	30	YMO-SSI
Yamadazyma farinosa	IFO 0193	0.00	1.27	0.18	0.00	2.36	0.82	30	YMO-SSI
Yerroiwa lipolytica	IFO 0746	0.06	9.66	0.15	0.16	13.41	0.36	24	YMO-SSI
Zygosaccharomyces japonicus	IFO 0595	0.05	0.77	0.05	0.10	2.17	0.15	30	YMO-SSI
	Day 3	Day 10	Temp.
Strains	IFO No.	NOH	FOH	GGOH	NOH	FOH	GGOH	(° C.)	Medium
Ambrosiozyma ambrosiae	10835	0.0	0.1	1.5	0.0	0.0	0.7	24	YPDO-SSI
Ambrosiozyma monospora	10751	0.0	1.0	0.3	0.0	4.8	1.4	24	YMO-SSI
Ambrosiozyma philentoma	1847	0.0	0.9	0.2	0.0	16.8	0.8	24	YMO-SSI
Ambrosiozyma platypodis	10752	0.0	1.7	0.1	0.0	48.6	1.4	24	YMO-SSI
Bensingtonia intermedia	10178	0.0	1.5	0.7	0.0	2.9	5.2	24	YMO-SSI
Botryozyma nematodophila	10830	0.0	2.5	0.3	0.0	5.6	2.8	24	YPDO-SSI
Brettanomyces anomalus	0627	0.0	18.0	0.4	0.6	16.7	0.0	24	YMO-SSI
Brettanomyces bruxellensis	0797	0.0	4.7	0.5	0.0	8.2	0.0	24	YMO-SSI
Brettanomyces custersianus	10735	0.0	0.1	1.4	0.0	20.0	0.6	24	YMO-SSI
Bullera crocea	10113	0.0	7.4	0.5	0.0	16.1	0.5	17	YMO-SSI
Bullera sinensis	10756	0.1	0.3	1.0	0.1	1.7	2.3	24	YMO-SSI
Citeromyces matritensis	0651	0.1	2.0	0.4	0.0	0.7	0.0	24	YMO-SSI
Clavispora lusitaniae	10059	0.0	1.0	0.3	0.1	9.2	9.1	24	YMO-SSI
Cystofilobasidium infirmominiatum	1057	5.7	14.9	2.6	10.8	48.4	3.6	24	YMO-SSI
Debaryomyces occidentalis	1842	0.0	0.7	0.2	0.0	0.4	4.1	24	YMO-SSI
Dekkera bruxellensis	1590	0.0	3.7	0.2	0.0	15.7	0.0	24	YMO-SSI
Dipodascus armillariae	10804	0.0	4.2	0.1	0.0	3.3	0.2	24	YMO-SSI
Dipodascus tetrasperma	10810	0.0	1.7	0.8	0.0	9.8	2.5	24	YMO-SSI
Eremascus albus	10811	0.0	0.0	4.8	0.0	0.0	3.6	24	YMO-SSI
Eremascus fertilis	0691	0.0	0.0	0.0	0.0	0.4	0.0	24	YMO-SSI
Eremothecium gossypii	1355	0.0	0.8	0.0	0.0	4.4	0.9	24	YMO-SSI
Erythrobasidium hasegawianum	1058	0.0	0.5	1.0	0.0	0.0	6.8	24	YMO-SSI
Hanseniaspora guilliermondii	1411	0.0	1.4	0.1	0.0	1.3	0.0	24	YMO-SSI
Hanseniaspora uvarum	10833	0.1	15.3	2.9	0.4	11.1	0.8	24	YPDO-SSI
Kloeckeraspora vineae	1415	0.4	3.4	0.3	0.7	1.2	0.0	24	YMO-SSI
Kockovaella imperatae	10522	0.0	3.2	3.5	0.0	19.5	6.2	24	YMO-SSI
Kodamaea ohmeri	0202	0.0	7.7	1.6	0.1	13.7	4.7	24	YMO-SSI
Kurtzmanomyces nectairei	10118	0.0	0.1	0.1	0.0	0.0	0.0	24	YMO-SSI
Leucosporidium scottii	1924	0.2	32.8	1.3	0.7	5.2	0.0	24	YMO-SSI
Lodderomyces elongisporus	1676	0.1	14.8	2.0	0.1	15.7	0.0	24	YMO-SSI
Malassezia furfur	0656	0.0	4.3	0.1	0.0	6.9	0.1	30	YMDO-SSI
Metschnikowia hawaiiensis	10791	0.0	0.0	0.3	0.0	0.0	0.2	24	YPDO-SSI
Metschnikowia krissii	1677	0.0	1.3	0.1	0.0	21.0	0.0	24	YMO-SSI
Metschnikowia lunata	1605	0.0	5.1	1.0	0.3	31.9	5.9	24	YMO-SSI
Metschnikowia pulcherrima	0863	0.0	10.5	0.2	0.1	15.4	0.3	24	YMO-SSI
Mrakia frigida	1926	0.5	25.3	0.3	0.3	11.3	0.1	12	YMO-SSI
Myxazyma lipomycoides	10351	0.7	23.3	5.4	1.3	35	1.1	24	YMO-SSI
Nadsonia commutata	10029	0.0	0.1	0.0	0.0	0.3	0.0	17	YMO-SSI
Pachysolen tannophilus	1007	0.0	0.1	0.1	0.0	1.9	1.4	24	YMO-SSI
Pichia burtonii	10837	0.0	3.0	1.1	0.0	7.5	1.7	24	YMO-SSI
Pichia misumaiensis	10221	0.3	20.8	1.8	1.0	14.5	2.0	24	YMO-SSI
Pichia ofunaensis	10709	0.0	0.1	0.1	0.0	0.0	0.0	24	YMO-SSI
Pichia pijperi	1290	0.5	5.1	0.6	0.5	6.4	0.5	24	YMO-SSI
Saccharomyces transvaalensis	1625	0.1	0.0	0.0	0.0	0.0	0.0	30	YMO-SSI
Sacoharomycodes sinensis	10111	0.1	0.4	0.0	0.6	0.8	0.1	30	YMO-SSI
Saccharomycopsis fibuligera	10829	0.0	0.0	4.1	0.0	0.0	2.8	24	YPDO-SSI
Saccharomycopsis javaensis	1848	0.0	0.0	0.0	0.0	0.0	0.6	24	YMO-SSI
Saccharomycopsis schoenii	10683	0.1	5.3	0.4	0.5	28.3	2.3	24	YMO-SSI
Sacaharomycopsis synnaedendra	1604	0.0	2.8	1.3	0.0	1.2	0.0	24	YMO-SSI
Saccharomycopsis vini	1748	0.0	3.9	0.4	0.0	23.2	6.4	24	YMO-SSI
Saturnispora zaruensis	1384	0.0	0.2	0.1	0.0	3.0	1.3	24	YMO-SSI
Schizoblastosporion kobayasii	1644	0.0	4.0	0.9	0.0	2.8	4.7	24	YMO-SSI
Schizoblastosporion starkeyi-	10842	0.0	0.9	0.2	0.0	1.1	0.8	24	YPDO-SSI
hennic
Sporopachydermia cereana	10013	0.0	0.6	0.2	0.2	0.1	1.2	24	YMO-SSI
Stephanoascus ciferii	1854	0.0	0.9	0.1	0.0	3.3	0.0	24	YMO-SSI
Sterigmatomyces elviae	1843	0.0	4.5	0.5	0.1	10.5	1.8	24	YMO-SSI
Sterigmatomyces halophilus	1488	0.0	0.0	0.0	0.8	0.3	0.0	24	YMO-SSI
Sterigmatosporidium polymorphum	10121	0.0	2.4	0.1	0.0	15.7	1.3	24	YMO-SSI
Sympodiomyces parvus	10132	0.0	3.7	0.1	0.0	3.0	0.0	17	YMO-SSI
Sympodiomycopsis paphiopedili	10750	0.0	1.3	1.0	0.0	0.8	1.0	24	YMO-SSI
Trichosporon brassicae	1584	0.0	13.0	0.7	0.0	13.2	0.0	24	YMO-SSI
Trichosporon pullulans	1232	0.2	10.9	0.2	0.3	30.5	1.1	17	YMO-SSI
Trigonopsis variabilis	0755	0.0	0.0	0.0	0.0	0.1	0.2	24	YMO-SSI
Tsuchiyaea wingfieldii	10204	0.0	15.3	0.7	0.0	17.5	1.4	24	YMO-SSI
Wickerhamilla domercqiae	1857	0.0	1.7	0.0	0.0	5.2	0.0	24	YMO-SSI
Xanthophyllomyces dendrorhous	10130	0.1	30.0	0.8	0.5	33.8	2.8	24	YMO-SSI
Zygozyma oligophaga	10360	0.1	6.4	5.3	0.8	24.3	5.6	24	YMO-SSI
	Day 3	Day 9	Temp.
Strains	No.	NOH	FOH	GGOH	NOH	FOH	GGOH	(° C.)	Medium
Aciculoconidium aculeatum	IFO 10124	0.0	21.3	1.4	0.0	26.4	2.3	24	YMO-SSI
Bullera pseudoalba	IFO 10179	0.0	11.0	2.0	0.0	40.7	15.0	24	YMO-SSI
Candida albicans	IFO 1060	0.2	108.8	3.5	0.8	32.9	4.7	30	YMO-SSI
Candida glabrata	IFO 0741	0.3	19.6	0.6	3.2	70.1	4.2	30	YMO-SSI
Candida guillermondii	IFO 0566	0.0	3.9	0.8	0.0	4.2	1.1	30	YMO-SSI
Candida intermedia	IFO 0761	0.0	56.2	2.5	0.1	87.0	6.2	30	YMo-SSI
Candida kefyr	IFO 0706	0.3	28.2	2.0	0.7	15.8	2.9	30	YMO-SSI
Candida krusei	IFO 0941	0.5	37.9	4.0	3.7	7.8	8.0	30	YMO-SSI
Candida tenuis	IFO 0716	0.0	2.2	0.2	0.0	31.2	2.2	30	YMO-SSI
Candida utilis	IFO 0619	0.2	42.2	5.5	0.8	52.5	11.1	30	YMO-SSI
Cryptococcus humicola	IFO 0753	0.2	6.3	2.0	0.0	0.3	3.3	30	YMO-SSI
Cryptococcus terreus	IFO 0727	0.0	1.2	0.1	0.2	1.9	0.3	30	YMO-SSI
Debaryomyces castellii	IFO 1359	0.0	11.4	1.1	0.0	26.8	4.6	30	YMO-SSI
Fellomyces penicillatus	IFO 10119	0.0	2.9	0.3	0.1	45.7	4.4	24	YMO-SSI
Filobasidium capsuligenum	IFO 1185	0.0	51.0	1.1	0.2	106.6	3.6	24	YMO-SSI
Filobasidium uniguttulatum	IFO 0699	0.0	28.7	1.3	0.4	85.4	8.9	24	YMO-SSI
Kloeckera corticis	IFO 0633	0.4	42.8	2.3	1.2	62.1	8.2	30	YMO-SSI
Holtermannia corniformis	IFO 10742	0.0	25.4	2.8	1.1	50.7	8.4	24	YMO-SSI
Kluyveromyces marxianus	IFO 0617	0.0	16.6	0.8	0.4	35.1	5.0	30	YMO-SSI
Phaffia rhodozyma	ATCC 66270	0.0	2.2	0.1	0.5	108.7	5.8	24	YMO-SSI
Pichia anomala	IFO 0146	0.2	34.8	2.6	0.2	7.4	4.3	30	YMO-SSI
Pichia fabianii	IFO 1254	0.0	14.3	1.5	0.2	0.1	4.2	30	YMO-SSI
Pichia farinosa	IFO 1003	0.0	3.2	0.6	0.0	11.1	1.8	30	YMO-SSI
Pichia jadinii	IFO 0987	0.0	23.1	2.0	0.1	24.6	8.3	30	YMO-SSI
Pichia polymorpha	IFO 0195	0.3	21.8	3.2	0.4	0.6	5.2	30	YMO-SSI
Pichia silvicola	IFO 0807	0.2	10.6	1.6	0.8	29.0	4.2	30	YMO-SSI
Rhodotorula glutinis	IFO 0695	0.0	3.8	0.2	0.0	3.4	0.3	30	YMO-SSI
Rhodotorula minuta	IFO 0715	0.3	6.6	12.0	3.4	5.0	0.7	30	YMO-SSI
Rhodotorula rubra	IFO 0870	0.3	5.5	10.8	3.5	4.4	26.4	30	YMO-SSI
Saccharomyces cerevisiae	IFO 0258	0.0	22.3	0.6	0.4	77.5	1.9	30	YMO-SSI
Saccharomyces cerevisiae	IFO 2347	0.0	18.8	0.7	0.1	36.5	1.4	30	YMO-SSI
Saccharomycodes ludwigii	IFO 10036	0.0	3.3	0.2	0.1	15.5	0.4	24	YMO-SSI
Saccharomycopsis fermentans	IFO 10772	0.1	41.4	1.9	0.3	37.9	3.1	24	YMO-SSI
Sporidiobolus samonicolar	IFO 1035	0.1	7.9	1.1	0.1	42.3	6.4	30	YMO-SSI
Sporobolomyces salmonicolar	IFO 0374	0.1	28.8	3.4	0.5	35.7	14.4	30	YMO-SSi
Trichosporiella flavificans	IFO 1573	0.0	0.0	0.0	0.2	31.5	3.0	24	YMO-SSI
Trichosporon penicillatum	IFO 2171	0.0	1.7	0.6	0.1	1.7	0.0	30	YMO-SSI
Williopsis californica	IFO 0800	10.1	95.7	4.7	10.5	90.1	5.2	24	YMO-SSI
Willopsis saturnus	IFO 0895	0.5	59.1	9.0	1.6	69.2	13.1	30	YMO-SSI
Yamadazyma farinosa	IFO 0459	0.0	6.2	0.8	0.0	20.8	2.2	30	YMO-SSI
Zygoascus hellenicus	IFO 10184	0.0	3.9	0.1	0.0	32.4	2.7	24	YMO-SSI

Claims

1. A method for producing geranylgeraniol and/or farnesol, which comprises culturing geranylgeraniol- and/or farnesol-producing cells belonging to any one of the following genera: Saccharomyces, Saccharomycopsis, Saccharomycodes, Schizosaccharomyces, Wickerhamia, Debaryomyces, Hansenula, Hanseniaspora, Lypomyces, Pichia, Kloeckera, Candida, Zygosaccharomyces, Ogataea, Kuraishia, Komagataella, Yarrowia, Williopsis, Nakazawaea, Kluyveromyces, Torulaspora, Citeromyces, Waltomyces, Bacillus, Staphylococcus, Pseudomonas, Micrococcus, Exiguobacterium, Mucor, Ambrosiozyma, Cystofilobasidium, Metschnikowia, Trichosporon, Xanthophyllomyces, Bullera, Fellomyces, Filobasidium, Holtermannia, Phaffia, Rhodotorula, Sporidiobolus, Sporobolomyces, Zygoascus, Haloferax, Brevibacterium, Leucosporidium, Myxozyma, Trichosporiella, and Alcaligenes in a medium to produce and accumulate geranylgeraniol and/or farnesol in the cells and/or in the extracellular environment; and then collecting geranylgeraniol and/or farnesol.

2. A method for producing nerolidol, which comprises culturing nerolidol-producing cells belonging to any one of the following genera: Saccharomyces, Cryptococcus, Candida, Streptomyces, Nocardia, Cystofilobasidium, Rhodotorula, Willopsis, and Haloferax in a medium to produce and accumulate nerolidol in the cells and/or in the extracellular environment; and then collecting nerolidol.