JP2007190030A - Method for high-production of prenyl alcohol by microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To culture prenyl alcohol-producing microorganisms in a medium having increased sugar content in the presence of at least one substance selected from surfactants, fats-and-oils and terpenoids to increase the production quantity of prenyl alcohol and enable efficient secretion and production of the alcohol from the microbial cells. <P>SOLUTION: The present invention provides a high-production method of prenyl alcohol comprising the culture of prenyl alcohol-producing microorganisms in a medium having increased sugar content in the presence of at least one substance selected from surfactants, fats-and-oils and terpenoids to effect the formation and accumulation of prenyl alcohol in the microbial cells, the secretion of the alcohol from the cells and the collection of the secreted alcohol. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a high production method of prenyl alcohol using microorganisms.

  It is considered that geranylgeraniol and farnesol, which are typical prenyl alcohols, are produced by hydrolysis of geranylgeranyl pyrophosphate and farnesyl pyrophosphate by phosphatase in organisms. Geranylgeranyl pyrophosphate is a pyrophosphate ester of geranylgeraniol, and is obtained by condensation of isopentenyl pyrophosphate and fanesyl pyrophosphate or condensation of three molecules of isopentenyl pyrophosphate and dimethylaryl pyrophosphate. Geranylgeranyl pyrophosphate is condensed into diterpenes such as gibberellin by cyclization reaction to form phytoene after condensation at the tail and tail, and then to carotenoid, and condensed with isopentenyl pyrophosphate at the head and tail to polyprenyl pyrophosphate, etc. It is metabolized. Farnesyl pyrophosphate, on the other hand, is produced by the condensation of isopentenyl pyrophosphate and geranyl pyrophosphate or by the condensation of two molecules of isopentenyl pyrophosphate and dimethylaryl pyrophosphate. The cyclization reaction results in sesquiterpene, and the tail and tail condense squalene. Then, it is condensed into steroids and triterpenes, and with isopentenyl pyrophosphate at the head and tail to be metabolized to polyprenyl pyrophosphate and dolichol. In addition, it binds to cysteine of certain proteins such as Ras protein and G protein and is metabolized to prenylated protein. Thus, a series of geranylgeraniol derivatives such as geranylgeraniol, geranylgeranyl pyrophosphate, and their precursors farnesyl pyrophosphate, farnesol, geranyl pyrophosphate, geraniol, are intermediates in the biosynthesis of terpenes, carotenoids, steroids. It is a central compound as a body. In addition, geranylgeraniol and its related compounds are fragrances, production of taxanes having antitumor activity (Japanese Patent Application No. 8-227481), hair nourishing agents (Japanese Patent Application No. 8-180449), osteoporosis treatment agents (Japanese Patent Application No. 9- 294089) and other important uses.

  Regarding the geranylgeraniol derivative as described above, there is an example [Curr.Genet., 18,41-46 (1990)] in which the erg mutant of Saccharomycescerevisie secretes and produces farnesol. L) Not practical. In addition, an example of producing an arachidonic acid-containing lipid by adding a hydrocarbon, a fatty acid, an oil or the like to a culture medium of a mutant strain obtained by subjecting a microorganism capable of producing an arachidonic acid-containing lipid to mutation, is disclosed in JP 2000-69987 A. Although the added component is converted into arachidonic acid, that is, used as a precursor of the final product or consumed as a nutrient source, it does not have an effect of extracting the intracellular product. In addition, when producing useful substances, it may be necessary to increase the concentration of the synthetic energy and the sugar source as a raw material. However, when producing an oily substance by increasing the sugar concentration, the permeability of the cell membrane is poor, and the bacterial body Accumulation causes product inhibition and yields cannot be expected.

  Accordingly, an object of the present invention is to provide a method for efficiently secreting and producing prenyl alcohol produced and accumulated in the microbial cells using microorganisms capable of producing prenyl alcohol.

  As a result of repeated studies to solve the above problems, the present inventors have cultivated yeasts (ascomycetes, imperfect fungi), bacteria, actinomycetes, and filamentous fungi that can produce prenyl alcohol. While culturing in a medium with increased sugar concentration in the presence of at least one selected from active agents, fats and oils, and terpenoids, the prenyl alcohol accumulated in the cells is actively secreted outside the cells. It has been found that productivity itself can be improved by lowering the bacterial cell concentration. Furthermore, surprisingly, it was also found that the above-mentioned microbial bacteria that do not produce prenyl alcohol under normal culture conditions can be produced under the same conditions as described above, thereby producing the compound. It came to complete.

  That is, the present invention cultivates prenyl alcohol-producing bacteria belonging to any of the following genera in a medium having an increased sugar concentration in the presence of at least one selected from surfactants, fats and oils, and terpenoids. A high production method of prenyl alcohol, characterized in that alcohol is produced and accumulated in the microbial cells and secreted and collected outside the microbial cells.

Saccharomyces genus Saccharomycopsis genus Saccharomycodes genus Schizosaccharomyces genus Wickerhamia genus Debaryomyces genus Henula Pichia genus Kloeckera genus Candida genus Zygosaccharomyces genus Ogataea genus Kuraishia genus Komagataella genus Yarrowia Staphylococcus genus Pseudomonas genus Williopsis genus Nakazawaea genus Krivyveromyces genus Torulaspora genus Citeromyces genus Waltomyces genus (Micrococcus) genus Cryptococcus genus Exiguobacterium genus Nocardia genus Mucor genus Ambrosiozyma genus Cystofilobasidium genus Metschnikowia Trichosporon genus Xanthophyllomyces genus Bullera genus Ferromyces genus Filobasidium genus Hortelmannia genus Phaffia genus Rhodotorula genus Sporidiolus Sporobolomyces spp.Willopsis sp.Zygoascus sp. Less than the genus Alcaligenes The present invention will be described in detail.

  According to the present invention, when a prenyl alcohol-producing bacterium is cultured, the amount of prenyl alcohol produced is increased by using a medium having an increased sugar concentration in the presence of at least one selected from surfactants, fats and oils, and terpenoids. And can be efficiently secreted and produced outside the cells.

  In the present invention, prenyl alcohol, typically geranylgeraniol and its related compounds are produced by fermentation using microorganisms. The related compounds of geranylgeraniol referred to in the present invention are geranylgeranyl pyrophosphate and farnesyl pyrophosphate, farnesyl monophosphate, farnesol, geranyl pyrophosphate, geranyl monophosphate, geraniol, nerolidol, geranyl produced in connection with the synthesis thereof. It means linalool and linalool.

  The microorganisms used in the production of geranylgeraniol in the present invention include the genus Saccharomyces, the genus Saccharomycopsis, the genus Saccharomycodes, the genus Schizosaccharomyces, the genus Vickerhamia Genus Hansenula, Hanseniaspora, Pichia, Kloeckera, Candida, Zygosaccharomyces, Ogataea Kura, ), Genus Komagataella, Yarrowia, Willopsis, Nakazawaea, Kluyveromyces, Torulaspora or Cryptococcus (genus Cryptococcus) Genus, Rhodotorula (Rh Yeast belonging to any genus of the genus odotorula, Sporobolomyces, Kloeckera, Willoppsis, or the fungus belonging to the genus Mucor, the genus Haloferax Any bacterial strain belonging to the genus Algaigenes and Alcaligenes, which has the potential to produce geranylgeraniol.

Geranylgeraniol-producing bacteria are specifically listed below.
(1) Saccharomyces genus; Saccharomyces cerevisiae ATCC 12341, IFO 0565, IFO 0222, ATCC 64031, ATCC 76625, Kyoto University preservation strain 4104, Kyoto University preservation strain 4045, Kyoto University preservation strain 4103, Kyoto University preservation strain 4104, ATCC 9080, IFO 1346, ATCC 204660, IFO 0538, IFO 0210, Saccharomyces ellipsoideus Kyoto University Conservation Stock 4102, Saccharomyces sake Kyoto University Conservation Stock 4023, Association No. 2, Saccharomyces rosei IFO 0252 Saccharomyces vanka, Saccharomyces kluyver Laer Kyoto University stock 3009
(2) Saccharomycopsis genus; Saccharomycopsis fibuligera IFO 0106
(3) Saccharomycodes genus; Saccharomycodes ludwigii IFO 0339
(4) Schizosaccharomyces genus; Schizosaccharomyces octosporus IAM 4842, Schizosaccharomyces pombe IFO 0346, IFO 0638
(5) Wickerhamia genus; Wickerhamia fluorescens IFO 1116
(6) Debaryomyces genus; Debaryomyces hansenii var.fabryi IFO 0794, Debaryomyces castellii IFO1359, Debaryomyces vanrijiae var.vanrijiae JCM 2169
(7) Hansenula genus; Hansenula valbyensis, Hansenula saturnus, Hansenula beijerinckii, Hansenula matritensis, Hansenula capsulata, Hansenula polymorpha, Hansenula polymopla
(8) Hanseniaspora genus Hanseniaspora valbyensis IFO 0115
(9) Pichia genus; Pichia membranaefaciens IFO 0128, Pichia aganobii, Pichia naganishiiIFO 1670, Pichia silvicola IFO 0807, Pichia anomala IFO 0118, IFO 0569, IFO 0707, Pichia pastoris ATCC 20864
(10) Genus Kloeckera; Kloeckera japonica IFO 0151
(11) Candida genus; Candida krusei IFO 0013, Candida kefyr IFO 0706, Candida tenuis IFO 0716, Candida solani IFO 0762, Candida glabrata IFO 0005, IFO 0622, Candida albicans IFO 1060, Candida 0120, Candida 0120 , Candida cariosilignicola IFO 1910, Candida stellata IFO 0701, Candida utilis IFO 0619
(12) Zygosaccharomyces genus; Zygosaccharomyces rouxii IFO 0487, IFO 0686, Zygosaccharomyces japanicus IFO 0595
(13) Ogataea genus; Ogataea glucozyma IFO 1472, Ogataea polymorpha IFO 1475
(14) The genus Kurashia; Kurashia capsulata IFO 0974
(15) Genus Komagataella; Komagataella pastoris IFO 0948
(16) Yarrowia genus; Yarrowia lopolytica IFO 0717
(17) Williopsis genus; Willopsis saturnus var. Saturnus IFO 0125, IFO 0941, Willopsis saturnus IFO 0895
(18) Nakazawaea genus; Nakazawaea holstii IFO 0980
(19) Kluyveromyces genus; Kluyveromyces marxianus IFO 0617, IFO 0288, Kluyveromyces thermotolerans IFO 0662, Kluyveromyces lactis IFO 0648
(20) Torulaspora genus; Torulaspora delbrueckii IFO 0422
(21) Cryptococcus genus; Cryptococcus humicolus IFO 1527
(22) Mucor genus; Mucor javanicus IFO 4570
(23) Genus Bullera; Bullera pseudoalba IFO 10179
(24) Rhodotorula genus; Rhodotorula minuta IFO 0715, Rhodotorula rubra IFO 0870
(25) Sporobolomyces genus; Sporobolomyces salmonicolor IFO 0374
(26) Haloferax genus; Haloferax volcanii IFO 14742
(27) Alcaligenes genus; Alcaligenes faecalis IFO 13111

  In the present invention, microorganisms that can be used for the production of farnesol include the genus Saccharomyces, the genus Saccharomycodes, the genus Schizosaccharomyces, the genus Vickerhamia, the genus Debaryomyces, Spans (Hanseniaspora), Lipomyces, Pichia, Candida, Ogataea, Kuraishia, Komagataella, Yarrowia, Criveromyces (Kluyveromyces), Torulaspora, Zygosaccharomyces, Willopsis, Citeromyces, Waltomyces or Cryptococcus Yeast belonging to the genus Bacillus A bacterium belonging to any genus of the genus Staphylococcus, Pseudomonas, Micrococcus, Exiguobacterium, Actinomyces belonging to the genus Nocardia, Alternatively, the fungus belonging to the genus Mucor, the genus Ambrosiozyma, the genus Cystofilobasidium, the genus Metschnikowia, the genus Trichosporon, the genus Xanthophyllomyces , Genus Bullera, genus Fellomyces, genus Filobasidium, genus Holtermannia, genus Phaffia, genus Sporidiobolus, genus Sprobolomyces, Willopsis ) Genus, Zygoascus genus, leucosporidium genus, myxoza Any microorganism belonging to the genus Myxozyma, Trichosporiella, Haloferax, Brevibacterium, etc., which has the potential to produce farnesol. That's fine.

The farnesol-producing bacteria are specifically listed below.
(1) Saccharomyces genus; Saccharomyces cerevisiae IFO 1346, ATCC 204660, IFO 0258, IFO 0565, ATCC 64031, ATCC 76625, IFO 0262, IFO 0538, Kyoto University preservation strain 4104, Kyoto University preservation strain 4045, Kyoto University preservation Strain 4103, IFO 0210, IFO 2347, Saccharomyces unisporus IFO 0215, Saccharomyces sake association No. 2, Saccharomyces ellipsoideus Kyoto University Conservation Stock 4102, Saccharomyces rosei IFO 0252, Saccharomyces logos Kyoto University Conservation Stock 4101 IFO 0613, Saccharomyces kluyveri IFO 1892, Saccharomyces paradoxus IFO 0259, Saccharomyces Hafe logos van Laer Kyoto University Conservation 4003
(2) Saccharomycodes genus; Saccharomycodes ludwigii IFO 0339
(3) Schizosaccharomyces genus; Schizosaccharomyces pombe IFO 0346, IFO 0638, IFO 0358, Schizosaccharomyces octosporus JAM 4842
(4) Hanseniaspora genus; Hanseniaspora valbyensis IFO 0115
(5) Debaryomyces genus; Debaryomyces hansenii IFO 0023, Debaryomyces hansenii var.fabryi IFO 0749, Debaryomyces castellii IFO 1359, Debaryomyces vanrijiae var. Vanrijiae JCM 2169
(6) The genus Lypomyces; Lypomyces starkeyi IFO 0678
(7) Pichia genus; Pichia aganobii Kyoto University Conservation 4261, Pichia naganishii IFO 1670, Pichia anomala IFO 0118, IFO 0569, IFO 0963, IFO 707, IFO 0146, Pichia pastoris ATCC 20864
(8) Candida genus; Candida utilis IFO 0626, IFO 0619, Candida albicans IFO 0579, IFO 1060, Candida zeylanoides IFO 0719, Candida glabrata IFO 0005, IFO 0622, IFO 0741, Candida cariosilignicola IFO 1910 ella st. , Candida solani IFO 0762, Candida intermedia IFO 0761, Candida krusei IFO0941, Candida tenuis IFO 0716
(9) Wickerhamia genus; Wickerhamia fluoresces IFO 1116
(10) The genus Kurashia; Kurashia capsulata IFO 0974
(11) Komagataella genus; Komagataella pastoris IFO 0948
(12) Ogataea genus; Ogataea glucozyma IFO 1472, Ogataea polymorpha IFO 1475
(13) Yarrowia genus; Yerrowia lopolytica IFO 0717
(14) Kluyveromyces genus; Kluyveromyces marxianus IFO 0288, IFO 0617, Kluyveromyces thermotolerans IFO 0662, Kluyveromyces lactis IFO 0648
(15) Torulaspora genus; Torulaspora delbrueckii IFO 0422
(16) Genus Zygosaccharomyces; Zygosaccharomyces rouxii IFO 0487, IFO 0686, Zygosaccharomyces japanicus IFO 0595, Zygosaccharomyces fermentati IFO 0021
(17) Williopsis genus; Williamopsis saturnus var. Saturnus IFO 0941, Willopsis californica IFO0800, Willoppsis saturnus IFO 0895
(18) Citeromyces genus Citeromyces matritensis IFO 0954
(19) Waltomyces lipoder IFO 0673
(20) Cryptococcus); Cryptococcus humicolus IFO 1527
(21) Bacillus genus; Bacillus amyloliquefaciens IFO 3022, Bacillus pumilus IFO 3030
(22) Staphylococcus genus; Staphylococcus epidermidis IFO 3762
(23) Pseudomonas genus; Pseudomonas sp. Kyoto University Conserved Strain 876
(24) Micrococcus genus; Micrococcus luteus IFO 3067
(25) Exiguobacterium genus;
Exiguobacterium acetylicum IFO 12146
(26) Nocardia genus; Nocardia asteroides Kyoto University Conservation 2103
(27) Mucor genus; Mucor Javanicus IFO 4570
(28) Ambrosiozyma genus; Ambrosiozyma platypodis IFO 10752
(29) Cystofilobasidium genus; Cystofilobasidium infirmominiatum IFO 1057
(30) Leucosporidium genus; Leucosporidium scottii IFO 1924
(31) Metschnikowia genus; Metschnikowia lunata IFO 1605
(32) Myxozyma genus; Myxozyma lipomycoides IFO 10351
(33) Trichosporon genus; Trichosporon pullulans IFO 1232
(34) Xanthophyllomyces genus; Xanthophyllomyces dendrorhous IFO 10130
(35) Bullera pseudoalba IFO 10179
(36) Fellomyces genus; Fellomyces penicillatus IFO 10119
(37) Filobasidium genus; Filobasidium capsuligenum IFO 1185, Filobasidium uniguttulatum IFO 0699
(38) Genus Kloeckera; Kloeckera corticis IFO 0633
(39) Holtermannia; Holtermannia corniformis IFO 10742
(40) Phaffia genus; Phaffia rhodozyma ATCC 66270
(41) Saccharomycopsis genus; Saccharomycopsis fermentans IFO 10772
(42) Sporidiobolus genus; Sporidiobolus samonicolar IFO 1035
(43) Sporobolomyces genus; Sporobolomyces salmonicolor IFO 0374
(44) Trichosporiella genus; Trichosporiella flavificans IFO 1573
(45) Zygoascus genus; Zygoascus hellenicus IFO IFO 10184
(46) Haloferax genus; Haloferax volcanii IFO 14742
(47) Brevibacterium genus; Brevibacterium linens IFO 12171

  The microorganism used for the production of nerolidol in the present invention is a yeast belonging to the genus Saccharomyces or Candida, or a filamentous fungus belonging to the genus Mucor, Cystofilobasidium genus, Rhodotorula ( Any microorganism that belongs to the genus Rhodotorula, the genus Willoppsis, the genus Zygoascus, the genus Haloferax, and the like and may potentially have the ability to produce nerolidol.

Nerolidol-producing bacteria are specifically listed below.
(1) Saccharomyces genus; Saccharomyces unisporus IFO 0215, Saccharomyces cerevisiae Kyoto University stock 4040, Kyoto University stock 4103, Kyoto University stock 4104, IFO 0210, ATCC 64031, Saccharomyces Hafe logos van Laer Kyoto University stock , Saccharomyces elliposoideus Kyoto University Stock 4102
(2) Candida genus; Candida grabrata IFO 0622, IFO 0005, IFO 0741, Candida solani IFO 0762, Candida cariosilignicola IFO 1910, Candida krusei IFO 0941
(3) Mucor genus; Mucor Javanicus IFO 4570
(4) Cystofilobasidium genus; Cystofilobasidium infirmominiatum IFO 1057
(5) Rhodotorula genus; Rhodotorula minuta IFO 0715, Rhodotorula rubra IFO 0870
(6) Willopsis genus; Willopsis californica IFO 0800
(7) Haloferax genus; Haloferax volcanii IFO 14742

  In addition, bacteria that produce prenyl alcohol such as geranylgeraniol, farnesol, and / or nerolidol that can be used in the present invention include the genus Dipodascus, the genus Issatchenkia, the genus Mortierella, The genus Rhodosporidium, the genus Tsukamurella, the genus Yamadazyma, the genus Bensingtonia, the genus Botryozyma, the genus Brettanomyces, the genus Clavispora, the genus kk Genus (Eremascus), genus Eremothecium, genus Erythrobasidium, genus Kloeckeraspora, genus Kockovaella, genus Kodamaea, genus Kurtzmanomyces , Genus Malassezia, Malakia (Mr akia, Nadsonia, Pachysolen, Saturnispora, Schizoblastosporion, Sporopachydermia, Stephanoascus, (Sterigmatomyces), Sterigmatosporidium, Sympodiomyces, Sympodiomycopsis, Trigonopsis, Tsuchiyaea, Zygozyma, Zygozyma Examples include those belonging to the genus Acriculoconidium. The microorganism used in the present invention also includes a recombinant bacterium obtained by introducing a gene and modifying the bacterium collected from nature as described above.

  Next, culture of microorganisms used in the present invention will be described. The medium for culturing the microorganisms is usually a medium that can grow these microorganisms. Specifically, in the case of yeast (ascomycete yeasts, incomplete yeasts), YM medium, KY medium, F101 When the medium is bacteria, actinomycetes, or filamentous fungi, KB medium is exemplified.

  Any carbon source can be used as long as it is a carbon compound that can be assimilated and grown by cells. As the nitrogen source, for example, inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, and ammonium nitrate, and organic nitrogen sources such as yeast extract, peptone, and meat extract can be used. In addition to these, inorganic salts, metal salts, vitamins, and the like can be added as necessary.

  Culturing is preferably performed at a temperature of 20 to 40 ° C., more preferably 25 to 35 ° C. and a pH of 5 to 9, although it varies depending on the type of microorganism. Depending on the type of microorganism, either anaerobic or aerobic can be performed, but since the growth rate is high, shaking culture and rotary culture under aerobic conditions are preferred. However, as a matter of course, it is important to set the culture conditions so as to maximize the production amount of prenyl alcohol according to the microorganism used, the composition of the medium, and the like.

  In the present invention, in order to increase the production amount of prenyl alcohol and promote the secretion of the product substance outside the cells, the prenyl alcohol-producing bacterium is at least one selected from surfactants, fats and oils, and terpenoids. Cultivation is performed in a medium with an increased sugar concentration in the presence of seeds.

  In the present invention, “medium with increased sugar concentration” refers to a medium in which the amount of saccharide added to the medium is 2% to 7%. Examples of the saccharide include glucose and sucrose. In this specification, (%) used to indicate the addition ratio means w / v (%).

  Examples of the fats and oils used in the present invention include soybean oil, fish oil, almond oil, olive oil and the like, and the addition amount thereof is, for example, 0.01% or more, preferably 1% or more with respect to the medium. In addition, since the effect will not increase any more if the addition amount exceeds 3% or more, considering the cost, it is desirable to set it to 0.01 to 3%.

  The surfactant used in the present invention is preferably a nonionic surfactant, for example, polyethylene glycol type (higher alcohol ethylene oxide adduct, alkylphenol ethylene oxide adduct, fatty acid ethylene oxide adduct, polyhydric alcohol fatty acid ester ethylene Oxide adduct, higher alkylamine ethylene oxide adduct, fatty acid amide ethylene oxide adduct, polypropylene ethylene glycol ethylene oxide adduct, etc.), polyhydric alcohol type (fatty acid ester of sucrose, alkyl ether of polyhydric alcohol, etc.), or Silicone oil (dimethyl silicone, polyether-modified silicone oil, etc.) can be mentioned.

  Specifically, Taditol NP-40 (manufactured by Nakarai), cholic acid (manufactured by Nakarai), deoxycholic acid (manufactured by Nakarai), N-lauryl sarcosine (manufactured by Nakarai), sucrose monolaurate (manufactured by Nakarai), Triton X- 100 (manufactured by Nakarai), TritonX-305 (manufactured by Nakarai), Nonidet P-40 (manufactured by Iwai Chemical), Tween 20 (manufactured by Nakarai), Tween 80 (manufactured by Nakarai), Span 20 (manufactured by Nakarai), Span 85 (manufactured by Nakarai) ), CTAB (manufactured by Nacalai), nonyl-β-D-glucose (manufactured by Sigma), Adekapluronic L-61 (manufactured by Asahi Denka; PPG bonded with ethylene oxide), Adecanol LG-109 (manufactured by Asahi Denka; polyether type PPG), Adecanol LG-294 (Asahi Denka; polyether type PPG), Adecanol LG-295S (Asahi Denka; polyether type PPG), Adecanol LG-297 (Asahi Denka; Polye Tell-type PPG), Adecanol B3009A (made by Asahi Denka; oil and fatty acid ester), KS66 (made by Shin-Etsu Chemical), KS69 (made by Shin-Etsu Chemical), KS502 (made by Shin-Etsu Chemical), KM73 (made by Shin-Etsu Chemical) Chemical products), KM82F (manufactured by Shin-Etsu Chemical), and other commercial products can be used.

  The addition amount of the surfactant having no defoaming effect may be, for example, 0.005% to 1%, preferably 0.05% to 0.5%, and more than 1% with respect to the medium. Bubbles are generated, which is not preferable. In the case of a surfactant having an antifoaming effect, it may be added in excess of 1%.

  In the present invention, examples of terpenoids include squalene and tocopherol, and the added amount thereof may be, for example, 0.01% or more, preferably 1% or more, based on the medium. If the added amount exceeds 3%, the effect cannot be improved any more. Therefore, if considering the cost, 0.01 to 3% is desirable. Of the above additives, fats and oils are preferred, and fats and surfactants (particularly antifoaming surfactants) are used to increase the production of prenyl alcohol and promote the secretion of the product outside the cells. The combined use of the agent is more preferable.

  In the present invention, the process for producing prenyl alcohol can be performed either batchwise or continuously using a bioreactor. The microbial cells may be used for the production of prenyl alcohol as they are, or may be processed cells such as crushed cells, cell culture broth, crude enzyme, and purified enzyme. Moreover, the cultured microbial cells or the treated microbial cells may be immobilized by an immobilization method. By culturing such microbial cells or processed microbial cells, prenyl alcohol is produced and accumulated in the microbial cells or in the culture supernatant and collected.

  In order to collect prenyl alcohol from the culture supernatant fraction, after removing the cells by centrifugation, the resulting supernatant was treated by adding a buffer containing magnesium chloride and alkaline phosphatase, and then pentane, methanol, etc. Extract with the solvent. Moreover, in order to collect prenyl alcohol from the cultured cell fraction, the cells collected by centrifugation are crushed, treated with a buffer containing magnesium chloride and alkaline phosphatase, and then pentane, methanol, etc. Extract with the solvent. Moreover, well-known purification methods, such as a chromatography, can also be used together with said solvent extraction method suitably.

  When alkaline phosphatase is used during extraction, farnesyl pyrophosphate and geranylgeraniol pyrophosphate, which are present as precursors of farnesol and geranylgeraniol in the cells or culture solution, are hydrolyzed to increase the production of farnesol and geranylgeraniol. It is valid. The phosphatase is preferably E. coli-derived alcal phosphatase, but potato acid phosphatase, calf intestinal wall phosphatase, or the like may also be used. In addition, since most microorganisms have endogenous phosphatase, the production amount is slightly reduced, but organic solvent extraction may be performed without phosphatase treatment. In the production method of the present invention, prenyl alcohol is detected and quantified by commercially available gas chromatography / mass spectrometry (GC / MS), and quantified by the peak area ratio with respect to 1-undecanol as an internal standard.

  The present invention will be described in detail below with reference to typical examples, but these examples do not limit the scope of the present invention.

[Reference example]
(1) Preparation of liquid medium In each example, YM medium (manufactured by Difco) or KY medium prepared as follows was used for yeast culture. In addition, for culturing squalene synthase-deficient yeast, ergosterol was added to the YM medium for yeast culture. For bacteria and actinomycetes, KB medium was used. The plate was prepared by adding 2% Bactagar (Difco) to the same medium.

Ergosterol-supplemented YM medium Ergosterol (Sigma) 20 mg was suspended in an ethanol solution containing 50% Taditol (Nacalai), heated in a boiling water bath and completely dissolved, and the whole amount was YM broth (Difco) ) In addition to 1L, autoclaved.

The following KY medium was added to 1 L of deionized water, adjusted to pH 5.5 with 2N sodium hydroxide solution, made 1 L with deionized water, and autoclaved.
Malt Extract (Difco) 5g
Yeast Extract (Difco) 5g

The KB medium or lower was added to 1 L of deionized water, adjusted to pH 7.0 with 2N potassium hydroxide solution, made 1 L with deionized water, and autoclaved.
Bactotryptone (Difco) 5g
Yeast Extract (Difco) 5g
Glucose (Nacalai) 1g
KH ₂ PO ₄ (Nacalai) 0.7g
K ₂ HPO ₄ (Nacalai) 0.3g

(2) Extraction of prenyl alcohol from the supernatant fraction Place 2.5 ml of the culture solution into an 18φmm × 125mm test tube, centrifuge at 1000rpm for 5 minutes with a Beckman centrifuge GP centrifuge, and the supernatant into a new 18φmm × 125mm test tube. Moved. 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride and 5 μl (2 units) of Escherichia coli alkaline phosphatase (Takara Shuzo) were added and heated at 65 ° C. for 30 minutes. After sufficiently cooling on ice, 2 ml of pentane and 1 ml of methanol were added. After thorough mixing, the mixture was centrifuged at 1000 rpm for 5 minutes with a Beckman centrifuge GP centrifuge, and the supernatant was transferred to another new test tube. After evaporating the pentane / methanol solvent in a fume hood, it was redissolved in 300 ml of pentane and filled into a GC / MS vial.

(3) Extraction of prenyl alcohol from the bacterial cell fraction
1 In the case of bacteria / actinomycetes 10 ml of liquid culture solution was placed in a 50 ml Corning tube and centrifuged at 6000 rpm for 5 minutes in a Beckman cooling centrifuge (Avant J25-I) to collect the cells. The cells were suspended in 0.5 ml of deionized water, transferred to a 10 ml Spitz tube, and crushed with an ultrasonic cell crusher UC W-201 manufactured by Tokai Electric Co., Ltd. Repeat for 20 minutes). The sample was transferred to an 18φ mm × 125 mm test tube, 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride was added, and phosphatase treatment and extraction were performed in the same manner as (2).

2 In the case of yeast 2.5 ml of the liquid culture solution was put into a 18φ mm × 125 mm test tube, and centrifuged at 1000 rpm for 5 minutes in a Beckman centrifuge GPcentrifuge to collect the cells. After adding 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride, the cells were suspended, and transferred to a crushing glass tube. An equal amount of glass beads (Sigma acid washed 425φ-600 μm) was added and crushed with Yasui Kikai Multi-Beads Shocker MB-200 (conditions: room temperature, 2500 rpm, 20 minutes). The entire amount was transferred to a 18φmm × 125mm test tube, and phosphatase treatment and extraction were performed in the same manner as (2).

(4) Analysis of prenyl alcohol Using an HP6890 / 5973 GC / MS system manufactured by Agilent, analysis was performed under the following conditions.
1 Inlet temperature: 250 ° C
2 Detector temperature: 260 ° C
3 MS zone temperature MS Quad: 150 ℃
MS Source: 230 ℃
4 Scan parameters
Low Mass: 35
High Mass: 200
Threshold: 40
5 Injection parameters Mode: Automatic injection Sample volume: 2 μl
Number of washings: 3 times with methanol, 2 times with hexane Split ratio: 1:20
Column: HP-5MS (0.25 mm × 30 M, film thickness 0.25 μm) manufactured by Agilent
Carrier gas: Helium 1.0ml / min
Solvent delay: 2 min
Oven temperature rising condition: 115 ° C, hold for 1.5 minutes
Heated to 250 ° C at 70 / min and held for 2 minutes
Heated to 300 ° C at 70 / min and held for 7 minutes
Post time 0
Internal standard: 10 μl of 1-undecanol / ethanol solution (1 μl / ml) added to each vial Inlet liner: Split / splitless liner Analysis: After taking TIC, 69 masses were selected and 1-undecanol (RT = 3.39) min), nerolidol (RT = 3.86min), farnesol (RT = 4.23min), and lanylgeraniol (RT = 5.78min) peak areas were integrated. It quantified from the peak area ratio with respect to the internal standard undecanol.

[Example 1] (Effect of surfactant on prenyl alcohol secretion production)
(1) Strain A squalene synthase-deficient yeast (ATCC # 64031 strain) purchased from ATCC was selected for colony, and a strain with increased farnesol production was used.

(2) Preparation of medium Each of the following surfactants was added to the YM medium to which the above ergosterol was added, and sterilized in an autoclave.
(Surfactant)
Taditol NP-40 (Nacalai)
Triton X-100 (Nakarai)
Tween 20 (Nacalai)
Adecanol LG-109 (Asahi Denka)
Adeka Pluronic L-61 (Asahi Denka)
Triton X-305 (Nakarai)
Adecanol LG-295S (Asahi Denka)
Adecanol LG-297 (Asahi Denka)
Span 85 (Nakarai)
Adecanol B3009A (Asahi Denka)

(3) Liquid culture One platinum ear was inoculated from a slant into a 300 ml Erlenmeyer flask containing 50 ml of YM medium supplemented with ergosterol and rotationally cultured at 26 ° C. and 150 rpm. After culturing for 2 days, 50 μl was added to 5 ml of the medium prepared in (2) and cultured at 26 ° C. for 2 days in a 18φ mm × 150 mm test tube.

(4) Measurement of bacterial cell volume 100 μl of the culture solution was diluted with physiological saline, and OD660 nm was measured with a spectrophotometer.

(5) Extraction and analysis of prenyl alcohol from supernatant fraction It was performed according to the method described in Reference Example.

(6) Results FIG. 1 shows the effect of Taditol on geranylgeraniol and farnesol secretion production. In general, it can be seen that farnesol secretion increases at a higher concentration than the concentration (0.05%) used as the ergosterol dispersant in the YM medium containing ergosterol. This is the same as the amount of secretion per cell (OD660nm), so farnesol accumulated in the cell is not simply released outside the cell, but released outside the cell. This shows that productivity itself has also increased. On the other hand, geranylgeraniol is secreted by Taditol, but its productivity is hardly changed.

  FIG. 2 shows the result of comparing Triton X-100 or Tween20 with Taditol, but the same effect can be obtained with these surfactants. In particular, Triton X-100 also has good ergosterol solubility and greatly increases farnesol production.

  FIG. 3 is a result of examining the secretion effect in the same manner for several types of antifoaming agents for fermentation selected from the viewpoint of a surfactant with little foaming during jar culture and little influence on the growth of yeast. Although there are some differences depending on the antifoaming agent, the same secretory promoting effect as that of Taditol is observed. These are safer than nonionic surfactants used in laboratories such as Triton X-100 and have a defoaming action and are very practical.

  FIG. 4 shows the results of testing at various concentrations for Adecanol LG-109, which was good in FIG. When the concentration was in the range of 0 to 0.5%, the secretion amount increased with an increase in the addition amount.

[Example 2] (Effect of fats and oils on prenyl alcohol secretion production)
Squalene synthase in the same manner as in Example 1 using a medium in which fats and oils [almond oil, fish oil, soybean oil, olive oil (all manufactured by Sigma)] were added to the ergosterol-supplemented YM medium in a final concentration of 0.1%. Deficient yeast (ATCC # 64031 strain) was cultured, and the effect on the secretion production of farnesol was examined.

  FIG. 5 shows the results when cultured in a medium supplemented with almond oil, fish oil, and soybean oil. In order to add the ergosterol required by the same strain to the medium, 0.05% of tarditol is added as a dispersant in any system. For this reason, about 2/3 farnesol is secreted outside the cells even without the addition of fats and oils. On the other hand, in the systems to which oils and fats are added, the secretion amount of farnesol is 98% or more.

  FIG. 6 shows the results of a similar experiment conducted with increasing amounts of soybean oil and olive oil. In any of the systems to which the above fats and oils are added, the farnesol secretion amount increases, and the farnesol production amount increases by increasing the addition amount of fats and oils. Thus, the addition of fats and oils also showed the effect of promoting farnesol secretion like the surfactant.

[Example 3] (Effect of sugars on prenyl alcohol secretion production)
A squalene synthetase-deficient yeast (ATCC # 64031 strain) is cultured in the same manner as in Example 1 using a medium in which sugars [glucose or sucrose (both manufactured by Nakarai)] are added to the ergosterol-supplemented YM medium. The effect of farnesol on secretory production was investigated. Since the YM medium (Difco) contains 1% glucose from the beginning, the final concentration of saccharide is 1% glucose + added glucose or sucrose (0-5%).

  FIG. 7 shows the results when cultured in media containing various concentrations of glucose or sucrose. It can be seen that farnesol production increases as the sugar concentration increases. As the production amount increases, the proportion of farnesol accumulated in the microbial cells also increases. However, since the increase in the secretion amount is not significant, the secretion rate is not sufficiently high compared to the production rate. Moreover, since the result of the secretory production per microbial cell (O.D.660nm) is the same, the production of farnesol per microbial cell increases when the sugar concentration in the medium is increased.

[Example 4] (Effect of combined use of sugars, fats and surfactants on prenyl alcohol secretion production)
In the same manner as in Example 1 using a medium in which 6% glucose as a saccharide, 1% soybean oil as a fat and oil, and 0.1% TritonX-100 as a surfactant were added singly or in combination to the ergosterol-added YM medium. A squalene synthase-deficient yeast (ATCC # 64031 strain) was cultured, and its effect on secretory production of geranylgeraniol and farnesol was examined.

  FIG. 8 shows the result. When 1% soybean oil is added, farnesol in the cells is remarkably reduced, and the amount of farnesol secretion increases. When 6% glucose is added, the production amount of farnesol mainly in the microbial cells increases. Moreover, in the system to which 6% glucose and 1% soybean oil are added, the amount of secretion increases and farnesol in the cells is hardly seen. This effect is also seen with 0.1% Triton X-100, but it has less secretion effect than soybean oil. Furthermore, in the system to which 6% glucose, 1% soybean oil, and 0.1% Triton X-100 were added, both the secretion effect and the production amount were the same as in the case of the combination of glucose and soybean oil.

  On the other hand, geranylgeraniol was not detected at all in the bacterial cell fraction, but was detected outside the bacterial cell only in a system to which glucose and soybean oil or TritonX-100 were added. This indicates that secretion of geranylgeraniol is promoted by addition of these components, and the production amount is increased.

  FIG. 9 shows the results of examining the farnesol secretion effect of soybean oil in culture using a jar fermenter. FIG. 9A shows a culture profile in a system to which 5% glucose is added, but it can be seen that there is a time when the concentration of farnesol accumulated in the medium is maximized, and almost all of it is accumulated in the cells. This is because farnesol present in the microbial cells is degraded as glucose is depleted. On the other hand, FIG. 9 (B) shows the culture profile in the system to which 5% glucose and 1% soybean oil were added, but the farnesol production increased and 95% compared to the system to which only 5% glucose was added. The above is secreted outside the cells. Since the production amount does not decrease even in the later stage of the culture, it is considered that farnesol dissolves in soybean oil to reduce the decomposition amount and increase the production amount. In addition, compared with the results of FIGS. 1 to 8 so far, the amount of secretion in FIG. 9 (A) in which no fats and oils are added is low because the concentration of ergosterol in the culture solution is 4 mg / L, and accordingly the dispersant is used. This is because the carry-in of certain Taditol is 1/5, 0.01%.

[Example 5] (Effects of tocopherol and squalene on prenyl alcohol secretion production)
Effects of Saccharomyces cerevisiae ATCC 64031 strain and IFO 0538 strain on the secretory production of prenyl alcohol using the above-mentioned ergosterol-supplemented YM medium supplemented with tocopherol or squalene at the concentrations shown in Tables 1 and 2, respectively. I investigated. The results are shown in Tables 1 and 2. Saccharomyces cerevisiae strain IFO 0538 does not produce prenyl alcohol in the YM medium, which is a common yeast medium. However, it is produced when squalene is added. Moreover, the amount secreted out of the cells is larger than that of geranylgeraniol and farnesol in the cells (Table 2). Table 1 shows the case of Saccharomyces cerevisiae ATCC 64031 strain that originally produces farnesol. As the amount of tocopherol added increases, the amount of farnesol in the culture supernatant fraction increases.

[Example 6] (Effect of combined use of saccharides and fats and oils on prenyl alcohol secretion production)
Number of culture days, medium composition, and phosphatase when culturing Hanseniaspora valbyensis IFO 0115, Saccharomycodes ludwigii IFO 0339, and Candida glabrata IFO 0005 with high production of farnesol and geraniogeraniol without adding specific components to the culture medium Table 3 (culture supernatant fraction) and Table 4 (bacterial cell fraction) show changes in production amount depending on the presence or absence of treatment. In both strains, farnesol and geraniogeraniol production increases with the culture time, and when 5% glucose is added to the medium, the production amount in the cell fraction increases. Furthermore, when 1% soybean oil is added, Candida glabrata promotes secretion outside the cells. The Hanseniaspora valbyensis IFO 0115 and Saccharomycodes ludwigii IFO 0339 strains did not show a secretion-enhancing effect due to the addition of fats and oils in this study.The reason for this was that during sampling, oil layers with high and low concentrations of farnesol and geranylgeraniol were used. This is because they are separated and uniform sampling cannot be performed. However, as a result of changing to the test tube culture and treating the whole amount, the same secretion promoting effect was observed in these two strains (Tables 9 to 11).

  Thus, although there are differences depending on the microbial species, the production amount of farnesol and geranylgeraniol in the cell fraction and the production amount in the culture supernatant fraction by using a medium containing 5% glucose and 1% soybean oil It is possible to increase (the amount of secretion outside the cell).

  In addition, when compared with the presence or absence of phosphatase treatment, the amount of detection in the supernatant fraction was higher when not treated with phosphatase, and the amount of detection tends to increase when phosphatase treatment was performed in the bacterial fraction, so farnesyl pyrophosphate, It is considered that geranyl pyrophosphate is present in the bacterial cell fraction.

[Example 7] (Effect of combined use of saccharides and fats and oils on prenyl alcohol secretion production)
About some of the strains shown in Table 5, the production amounts when cultured in a test tube in YM medium and when cultured in a medium in which 5% glucose and 1% soybean oil are added to YM medium are shown in Table 5, respectively. Table 6 shows. In the medium supplemented with 5% glucose and 1% soybean oil, both the production amount in the bacterial cell fraction and the production amount in the culture supernatant fraction (secreted amount to the outside of the bacterial cell) increased significantly.

[Example 8] (Effect of combined use of sugars and fats and oils on prenyl alcohol secretion production)
Each strain shown in Tables 7 and 8 below is obtained by adding 4 mg / L ergosterol and 0-20 mg / L squalene synthesis inhibitor (SQAD) to 1 YM medium, or 5% glucose and 1% to 2 YM medium. Cultured in a test tube in a medium supplemented with soybean oil in a medium supplemented with 4 mg / L ergosterol and 0-20 mg / L squalene synthesis inhibitor (SQAD), and each of nerolidol, geranylgeraniol, farnesol The production was examined. The results are shown in Tables 7 and 8. It can be seen that the production amount in the bacterial cell fraction and the production amount in the culture supernatant fraction (secreted amount to the outside of the bacterial cell) are increased by the addition of soybean oil, glucose and squalene synthesis inhibitor.

[Example 9] (Effect of combined use of saccharides and fats and oils on prenyl alcohol secretion production)
Each strain shown in Tables 9 and 10 below is prepared by adding 1% soybean oil, 6% glucose, 4 mg / L ergosterol and 0 mg / L or 20 mg / L squalene synthesis inhibitor (SQAD) to YM or KB or KY medium. The culture was carried out in the added medium, and the production amounts of nerolidol, geranylgeraniol, and farnesol were examined in the same manner. The results are shown in Table 9 (culture supernatant fraction) and Table 10 (bacteria cell fraction).

  As a comparison, Table 11 shows the results obtained by culturing in YM medium to which no strain was added, and similarly examining the production amounts of nerolidol, geranylgeraniol, and farnesol. It can be seen that the production amount in the bacterial cell fraction and the production amount in the culture supernatant fraction (secreted amount to the outside of the bacterial cell) are increased by the addition of soybean oil, glucose and squalene synthesis inhibitor.

[Example 10] (Effects of combined use of surfactant, sugar and fats and oils on prenyl alcohol secretion production)
Each strain shown in Table 12 below was prepared by using medium A (medium supplemented with 0.1% adecanol, 4 mg / L ergosterol in YM medium) or medium B (0.1% adecanol, 4 mg / L ergosterol, 5% glucose, 3% in YM medium). Incubate for 4 days or 10 days at 30 ° C in a medium supplemented with soybean oil) and examine the production of nerolidol, geranylgeraniol, and farnesol (culture supernatant fraction and fungal fraction) in the same manner. The results are shown in the same table.

[Example 11] (Effect of combined use of saccharides and fats and oils on prenyl alcohol secretion production)
Bacteria in which farnesol was detected were cultured in a medium supplemented with 1% soybean oil, 4mg / L ergosterol, or squalene synthesis inhibitor (SQAD) 0 or 20mg / L in KB medium. Each production of geranylgeraniol and farnesol was examined. The results are shown in Table 13.

  As a comparison, Table 14 shows the results obtained by culturing in a KB medium to which the same strain is not added and examining the production amounts of nerolidol, geranylgeraniol, and farnesol in the same manner. In the case of bacteria, it can be seen that the production amount is increased by addition of a squalene synthesis inhibitor, as in the case of yeast.

[Example 12] (Mass production of geranylgeraniol using recombinant yeast)
Saccharomyces cerevisiae PRS 435 GGF / pRS434 GAP-HMG1 / YPH499 # 1 capable of high expression of HMG CoA reductase gene, fusion gene of geranylgeranyl pyrophosphate synthase and farnesyl pyrophosphate synthase was prepared according to the following procedure. Cultured on a jar fermenter.

(1) Acquisition of GGPS and HMG-CoAR1 genes from yeast Saccharomyces cerevisiae
Based on the information of S. cerevisiae-derived GGPS (ANU31632) and HMG-CoAR1 gene (ANM22002) in GenBank, the following primers that match the N-terminus and C-terminus were prepared and used to make a yeast cDNA library (Toyobo No. PCR was performed under the following conditions using .CL7220-1) as a template. A Perfectmutch manufactured by Stratage was used for PCR of the HMG-CoAR1 gene.
(Primer)
N-terminal for GGPS 5'-atg gag gcc aag ata gat gag ct-3 '(SEQ ID NO: 1)
C end 5'-tca caa ttc gga taa gtg gtc ta-3 '(SEQ ID NO: 2)
N-terminal 5'-atg ccg ccg cta ttc aag gga ct-3 'for HMG-CoAR1 (SEQ ID NO: 3)
C end 5'-tta gga ttt aat gca ggt gac gg-3 '(SEQ ID NO: 4)
(PCR conditions)
Denaturation 94 ℃, 45 seconds annealing 55 ℃, 1 min extension 72 ℃, 2 min

  Since the fragments were confirmed at the target positions (about 1.0kbp and 3.2kbp, respectively), these genes were then cloned into a pT7Blue T vector capable of TA cloning, and the entire region of GGPS and about 40% of HMG-CoAR1. Was determined. As a result, it was confirmed that the gene was completely identical to the GenBank sequence and was derived from S. cerevisiae. The vector containing the HMG-CoAR1 gene cloned in the pT7Blue T vector obtained here was named pT7HMG1.

(2) Construction of yeast expression vector pYES2 (production of pYES-GGPS6)
pYES2 (manufactured by Invitrogen) is a shuttle vector for yeast expression having ori of yeast 2 μmDNA as a replication origin and a GAL1 promoter inducible with galactose. The pT7BlueT vector in which the GGPS gene was cloned was treated with BamHI and SalI, the GGPS gene was taken out, introduced into the BamHI and XhoI sites of pYES2, and named pYES-GGPS6.

(3) Cloning of Yeast Saccharomyces cerevisiae Farnesyl Pyrophosphate Synthase Gene (FPS) ERG20 The FPP synthase gene ERG20 was cloned from Saccharomyces cerevisiae YPH499 (Stratagene) cDNA by PCR using the following primers.
Primer 1 (SCFPS1): 5'-ATG GCT TCA GAA AAA GAA ATT AG-3 '(SEQ ID NO: 5)
Primer 2 (SCFPS2): 5'-CTA TTT GCT TCT CTT GTA AAC TT-3 '(SEQ ID NO: 6)
A plasmid DNA prepared by purifying the amplified 0.9 kbp fragment by agarose gel electrophoresis and then T / A ligating to pT7Blue-T was designated as pT7ERG20.

(4) Construction of expression vector
(4-1) Construction of pRS405Tcyc and pRS404Tcyc (insertion of CYC1t fragment into pRS vector)
The CYC1 transcription terminator CYC1t fragment was prepared by PCR using the following combinations of primers.
(Primer)
1 CYC1t-XK
XhoI-Tcyc1FW: 5'-TGC ATC TCG AGG GCC GCA TCA TGT AAT TAG-3 (SEQ ID NO: 7)
KpnI-Tcyc1RV: 5'-CAT TAG GTA CCG GCC GCA AAT TAA AGC CTT CG-3 '(SEQ ID NO: 8)
2 CYC1tXA
XhoI-Tcyc1FW: 5'-TGC ATC TCG AGG GCC GCA TCA TGT AAT TAG-3 '(SEQ ID NO: 9)
ApaI-Tcyc1RV: 5'-CAT TAG GGC CCG GCC GCA AAT TAA AGC CTT CG-3 '(SEQ ID NO: 10)
(PCR conditions)
Template: pYES2 (Invitrogen) 0.1 μg
Primer: 50 pmol primer DNA
Reaction solution: 1 x pfu buffer with MgSO ₄ (Promega, Madison, WI),
10 nmol dNTP,
1.5 u Pfu DNA polymerase (Promega),
1μl perfect match polymerase enhancer (Stratagene)
50 μl solution containing
Reaction: 95 ° C for 2 minutes (95 ° C, 45 seconds, 60 ° C, 30 seconds, 72 ° C, 1 minute) x 30 cycles
72 ℃ 5 minutes, 4 ℃ stock

  The DNA amplified in 1 and 2 above was cleaved with XhoI and KpnI or XhoI and ApaI, respectively, and 260 bp DNA fragments were purified by agarose gel electrophoresis to obtain CYC1t-XK and CYC1tXA. CYC1t-XK was inserted into the XhoI-KpnI site of pRS405 (Stratagene), and CYC1tXA was inserted into the XhoI-ApaI site of pRS404 (Stratagene) to obtain pRS405Tcyc and pRS404Tcyc, respectively.

(4-2) Preparation of TDH3p (Preparation of transcription promoter)
Saccharomyces cerevisiae YPH499 (Stratagene) genomic DNA was prepared using the “Gen Toruki-kun” (manufactured by Takara Shuzo Co., Ltd.) yeast yeast DNA preparation kit. Prepared.
(Primer)
DNA primer 100 pmol
SacI-Ptdh3FW: 5'-CAC GGA GCT CCA GTT CGA GTT TAT CAT TAT CAA-3 '(SEQ ID NO: 11)
SacII-Ptdh3RV: 5 'CTC TCC GCG GTT TGT TTG TTT ATG TGT GTT TAT TC 3' (SEQ ID NO: 12)
(PCR conditions)
Template: Saccharomyces cerevisiae YPH499 (Stratagene)
Genomic DNA 0.46μg
Reaction solution: 1 x ExTaq buffer (TaKaRa), 20 nmol dNTP,
0.5 u ExTaq DNA polymerase (TaKaRa),
100 μl solution containing 1 μl perfect match polymerase enhancer
Reaction: 95 ° C for 2 minutes (95 ° C, 45 seconds, 60 ° C, 1 minute, 72 ° C, 2 minutes) x 30 cycles
72 ° C for 4 minutes, 4 ° C stock The amplified DNA was cleaved with SacI and SacII, and a 680 bp DNA fragment was purified by agarose gel electrophoresis to obtain TDH3p.

(4-3) Preparation of 2μOriSN (Preparation of 2μDNA replication initiation region)
After cleaving pYES2 (Invitrogen) with SspI and NheI, a 1.5 kbp fragment containing 2 μDNA replication origin (2 μori) was purified by agarose gel electrophoresis, blunted with Klenow enzyme, and this DNA fragment was designated as 2 μOriSN.

(4-4) Construction of pRS434GAP and pRS435GAP (production of YEp type expression vector)
2 μOriSN was inserted into the NaeI site of pRS404Tcyc and pRS405Tcyc treated with BAP (bacterial alkaline phosphatase, TaKaRa), and transformed into E. coli SURE2, and plasmid DNA was prepared. This was cleaved with DraIII and EcoRI, HpaI, or PstI and PvuII and then subjected to agarose gel electrophoresis to check the insertion of 2 μori and its orientation. The plasmids in which 2 μori was inserted into the prepared pRS404 Tcyc and pRS405Tcyc in the same direction as pYES2 were designated as pRS434Tcyc2μOripRS435Tcyc2μOri, respectively. A fragment TDH3p containing a transcription promoter was inserted into the SacI-SacII site of two plasmids, pRS434Tcyc2μOri and pRS435Tcyc2μOri, to obtain pRS434GAP and pRS435GAP, respectively.

(5) Preparation of pRS435GGF (FPS-GGPS fusion protein gene)
PCR was performed under the following conditions using pYES-GGPS6 incorporating the GGPS gene BTS1 and pT7-ERG20 incorporating the FPS gene ERG20 as templates.
1 PCR1
Mold: pYES-GGPS6
Primer 1: SacII-BTS1, 5'-TCC CCG CGG ATG GAG GCC AAG ATA GAT-3 '(SEQ ID NO: 13)
Primer 2: BTSI-109I, 5'-GCA GGG ACC CCA ATT CGG ATA AGT GGT C-3 '(SEQ ID NO: 14)
(In primer 1, CCG CGG is a SacII, XhoI or XbaI recognition site for ligation of vector, and in primer 2, GGG ACC C is an EcoO109I recognition site for preparing a fusion gene).
2 PCR2
Template: pT7-ERG20
Primer 3: 109I-ERG20, 5'-GTA GGG TCC TCA GAA AAA GAA ATT AGG AG-3 '(SEQ ID NO: 15)
Primer 4: -21, 5′-TGT AAA ACG ACG GCC AGT-3 ′ (SEQ ID NO: 16); T7,5′-TAA TAC GAC TCA CTA TAG GG-3 ′ (SEQ ID NO: 17)
In primer 3, GGG TCC T represents an EcoO109I recognition site for preparing a fusion gene).
Reaction solution: 1 x KOD-Plus buffer (Toyobo, Osaka, Japan),
0.2 mM dNTPs,
0.25 mM MgSO ₄ ,
15 pmol primer 1, 15 pmol primer 2, 0.01-0.1μg template DNA
1u KOD-Plus DNA polymerase (Toyobo) in 50 μl
reaction cocktail
KOD-Plus contains 1.6 μg / μl of KOD antibody.
Reaction conditions: 94 ° C for 2 minutes, then 94 ° C for 15 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute for 30 cycles.

  The reaction products obtained in 1 and 2 were designated as # 9 and # 11, respectively. After digesting # 9 and # 11 with the restriction enzyme EcoO109I and ligating this solution, using this solution as a template for PCR and further using the above SacII-BTS1 and -21 as primers, perform 2nd PCR under the same conditions to obtain DNA fragment # 9- # 11 Obtained. The 2nd PCR product # 9- # 11 was cleaved with SacII and BamHI and then inserted into the SacII-BamHI site of pRS435GAP to obtain pRS435GGF. PRS435GAP-BTS1, pRS445GAP-BTS1, pRS435GAP-ERG20, and pRS445GAP-ERG20 were used as expression vectors for BTS1 and ERG20 which are not fusion genes, and pRS434TEF-HMG1 and pRS434GAP-HMG1 were used as the plasmids used for HMG1 expression.

(6) Creation of pRS434GAP-HMG1
After cutting pT7HMG1 with SmaI and SalI, a 3.2 kbp HMG1 gene fragment was purified by agarose gel electrophoresis. This was inserted into the SmaI-SalI site of pRS434GAP to obtain pRS434GAP-HMG1.

(7) Preparation of transgenic yeast (pRS435GGF / pRS434GAP-HMG1 / YPH499 # 1)
pRS434GAP-HMG1 and pRS434GAP-HMG1 were converted into "Introduction of DNA into Yeast Cells" Current Protocols in Molecular Biology, John Wiley & Sons, Inc., pp.13.7.1-13.7.2 (contributed by Daniel M. Becher and Victoria Lundblad) The Saccharomyces cerevisiae YPH499 strain (Stratagene) was introduced using the lithium acetate method described in 1) or Frozen-EZ Yeast Transformation II (Zymo Research, Orange, CA). Colonies that grow at 30 ° C on SD (-URA) agar plates (DOB + CSM (-URA), BIO 101, Vista, CA) are selected with SD (-URA) medium and pRS435GGF / pRS434GAP-HMG1 / YPH499 # 1 was obtained.

(8) Preparation of transgenic yeast (pRS435GGF / pRS434GAP-HMG1 / YPH499 # 1)
An SD-Leu-Trp medium (manufactured by BIO 101 Inc.) containing 200 ml of 40 mg / L adenine (manufactured by Sigma) was prepared as a 500 ml-baffled Erlenmeyer flask, and one strain of the same strain was inoculated from a slant. After culturing at 30 ° C and 130rpm for 2 days, in order to completely remove glucose contained in the culture solution, centrifugation (1500g, 5 minutes, 4 ° C) and washing with sterilized physiological saline are repeated 3 times. Inoculated 50 ml (1%).

<Fermenter medium>
5% glucose (YM contains 1% glucose, resulting in a final concentration of 5%)
YM broth (Difco)
0 or 3% soybean oil (Nacalai)
0.1% Adecanol LG-109 (Asahi Denka)
<Operating conditions>
Culture device: MSJ-U 10L culture device (Maruhishi Bioengineering)
Medium volume: 5 L
Medium temperature: 33 ° C
Airflow: 1vvm
Agitation: 300rpm
pH: Unless otherwise specified, 4N sodium hydroxide solution and 2N hydrochloric acid solution are used, and the pH is proportionally controlled with the following parameters.
proportional Band 1.00
Non Sensitive Band 0.15
Control Period 16 sec
Full Stroke 1 sec
Minimum Stroke 0 sec
Analysis of geranylgeraniol in the culture solution or bacterial cells reveals that it is secreted into the culture solution by the addition of soybean oil. In addition, the production amount itself increased by about 4 times (FIG. 10).

Example 13 (mass production of farnesol using recombinant yeast)
Farnesol-producing recombinant yeast pYHMG044 / AURGG101 was prepared and cultured according to the following procedure.
(1) Preparation of HMG1 Δ expression vector pYHMG044 Using pT7HMG1 of Example 12 as a template, a fragment in which a part of the HMG1 coding region was deleted together with the vector portion by PCR was prepared, blunt-ended with Klenow enzyme, self- After recirculation by ligation and transformation into E. coli JM109, plasmid DNA pYHMG044 was prepared. The synthetic DNA sequences used as primers and their combinations are shown below.
(Primer)
5'-AGA AGA TAC GGA TTT CTT TTC TGC TTT-3 '(SEQ ID NO: 18)
5'-AAC TTT GGT GCA AAT TGG GTC AAT GAT-3 '(SEQ ID NO: 19)

  The 373A DNA sequencer (Perkin Elmer, Foster City, CA) shows that the reading frame of the amino acids upstream and downstream of HMG1 in the obtained plasmid DNA is not shifted and that no amino acid substitution due to PCR error has occurred in the vicinity of the binding site. )

(2) Fabrication of AURGG101
pAUR101 (TaKaRa, Japan) was linearized with EcoO65I, "Introduction of DNA into Yeast Cells" Current Protocols in Molecular Biology, John Wiley & Sons, Inc., pp. 13.7.1-13.7.2 (contributed by Daniel M. Becher and Victoria Lundblad) was introduced into the Saccharomyces cerevisiae A451 (ATCC 200598) strain by the lithium acetate method, and YPD agar plates containing 1 μg / ml aureobasidin (1% yeastextract, 2% peptone, 2% dextrose, 2 % agar) colonies growing at 30 ° C. were obtained.

(3) Preparation of transgenic yeast (pYHMG044 / AURGG101)
pYHMG044 was prepared using the lithium acetate method described in "Introduction of DNA into Yeast Cells" Current Protocols in Molecular Biology, John Wiley & Sons, Inc., pp. 13.7.1-13.7.2 (contributed by Daniel M. Becher and Victoria Lundblad). Used and introduced into AURGG101. Colonies growing at 30 ° C on SD (-URA) agar plates (DOB + CSM (-URA), BIO 101, Vista, CA) were selected with SD (-URA) medium to obtain pYHMG044 / AURGG101 .

(4) Culture conditions
Recombinant yeast pYHMG044 / AURGG101 was inoculated from a slant of CSM-URA (manufactured by BIO 101 Inc.) and D0B (manufactured by BIO 101 Inc.) medium (200 ml / 500 ml- Erlenmeyer flask with baffle). The cells were cultured at 30 ° C. and 130 rpm for 2 days. Next, in order to completely remove glucose contained in the culture solution, centrifugation (1500 g, 5 minutes, 4 ° C) and washing with sterilized physiological saline are repeated three times to inoculate 50 ml of fermenter (1%). did.

<Fermenter medium>
YNB with 5% glucose total amino acid (Difco)
1% soybean oil (Nacalai)
0.1% Adecanol LG-109 (Asahi Denka)
<Operating conditions>
Culture device: MSJ-U 10L culture device (Maruhishi Bioengineering)
Medium volume: 5 L
Medium temperature: 26 ℃
Airflow: 1vvm
Agitation: 300rpm
pH: Unless otherwise specified, 4N sodium hydroxide solution and 2N hydrochloric acid solution are used, and the pH is proportionally controlled with the following parameters.
proportional Band 1.00
Non Sensitive Band 0.15
Control Period 16 sec
Full Stroke 1 sec
Minimum Stroke 0 sec

  The number of cells is measured by diluting 100 μl of the culture solution 1 to 20 times with physiological saline, and counting the number of cells with a hemocytometer (distributor: Hayashi Chemical, manufacturer: Sunlead Glass Co.). went. The number of bacteria in 0.06 mm square (9 pieces of the smallest grid) was averaged, and the number of bacteria per liter of the culture was calculated from the following formula.

[Equation 1]
Number of bacteria (1 × 10 ⁹ /L-broth)=0.444×(0.06 mm square number of cells) × dilution rate

  During the production of farnesol using recombinant yeast, soybean oil promoted secretory production, and 150 mg / L of farnesol was secreted and produced outside the cells in 150 hours (FIG. 11).

Example 14
Various bacterial cells are cultured by the same culture procedure as in Example 1 according to the culture conditions such as the number of culture days, culture temperature, and medium shown in Table 15 below, and prenyl alcohol is extracted from the bacterial cell fraction and the supernatant fraction. The analysis was performed according to the method described in Reference Example. The results are shown in Table 15. The LBO-SSI medium, YPDO-SSI medium, YMO-SSI medium, YMOL-SSI medium, and HVO-SSI medium were prepared as follows.

The LBO-SSI medium and lower were dissolved in 1 L of deionized water and autoclaved. After sterilization, the squalene synthesis inhibitor SQAD aqueous solution (2.5 mg / ml) sterilized by filter after the medium was sufficiently cooled was added to 20 mg / L.
Yeast Extract (Difco) 5g
Bactopeptone (Difco) 10g
NaCl (Nacalai) 5g
Glucose (Nacalai) 50g
Soybean oil (made by Nacalai) 10ml
200 µl of ergosterol solution (ethanol solution containing 2% ergosterol and 50% tarditol)

The following YPDO-SSI medium was dissolved in 1 L of deionized water and sterilized by autoclave. After sterilization, the squalene synthesis inhibitor SQAD aqueous solution (2.5 mg / ml) sterilized by filter after the medium was sufficiently cooled was added to 20 mg / L.
Yeast Extract (Difco) 10g
Bactopeptone (Difco) 20g
Glucose (Nacalai) 50g
Soybean oil (made by Nacalai) 10ml
200 µl of ergosterol solution (ethanol solution containing 2% ergosterol and 50% tarditol)

YMO-SSI medium
The following was added to YM medium (Difco), made 1 L with deionized water, and autoclaved. After sterilization, filter sterilized squalene synthase inhibitor SQAD (manufactured by Eisai Co., Ltd., 2.5 mg / ml aqueous solution) was added to 20 mg / L.
Glucose (Nacalai) 50g
Soybean oil (Nacalai) 10ml
Ergosterol (manufactured by Nacalai) 4mg (20mg / 50% ethanol, 50% tarditol solution 200μl added)

YMOL-SSI medium 10 ml of olive oil (manufactured by Nacalai) was added to the YMO medium and prepared in the same manner as YMO-SSI.

The HVO-SSI medium and lower were dissolved in 1 L of deionized water and autoclaved. After sterilization, the squalene synthesis inhibitor SQAD aqueous solution (2.5 mg / ml) sterilized by filter after the medium was sufficiently cooled was added to 20 mg / L.
NaCl (Nacalai) 156g
MgCl ₂ · 6H ₂ O (Nacalai) 13 g
MgSO ₄・ 7H ₂ O (Nacalai) 20g
CaCl ₂・ 2H ₂ O (Nacalai) 1g
KCl (Nacalai) 4g
NaHCO ₃ (Nacalai) 0.2g
KBr (made by Nacalai) 0.5g
Yeast Extract (Difco) 5g
Glucose (Nacalai) 50g
Soybean oil (made by Nacalai) 10ml
200 µl of ergosterol solution (ethanol solution containing 2% ergosterol and 50% tarditol)

The effects of surfactants on the production of geranylgeraniol and farnesol are shown (A: per culture broth, B: per broth O.D.). The effect of various surfactants on the production of farnesol is shown (A: per culture broth, B: per broth O.D.). The effect of various surfactants on the production of farnesol is shown (A: per culture broth, B: per broth O.D.). The effect of various concentrations of surfactant (Adecanol LG109) on farnesol secretion production is shown (A: per culture broth, B: per broth O.D.). The effect of fats and oils on farnesol secretion production is shown (A: per culture broth, B: per culture broth O.D.). The effect of fats and oils concentration on farnesol secretion production is shown (A: per culture broth, B: per broth O.D.). The effect of saccharide concentration on farnesol secretion production is shown (A: per culture broth, B: per culture broth O.D.). The effects of sugars, fats and oils, surfactants, and combinations thereof on the production of geranylgeraniol and farnesol are shown. The effect of combined use of saccharides and fats and oils on farnesol secretion production is shown. The effect of fats and oils on geranylgeraniol secretion production in a recombinant yeast is shown. The effect of fats and oils addition on farnesol secretion production in a recombinant yeast is shown.

Claims (2)

  A prenyl alcohol-producing bacterium belonging to the genus Candida is cultured in a medium having a sugar concentration of 2 to 7% in the presence of oils and fats and / or terpenoids, and at least one selected from geranylgeraniol, farnesol and nerolidol Is produced and accumulated in the microbial cells, and is secreted and collected outside the microbial cells.   The method for high production of prenyl alcohol according to claim 1, wherein the medium further contains a surfactant.

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