Patent Grant
6974685
The present invention relates to a high production method of prenyl alcohol using microorganisms.
Geranylgeraniol and farnesol, typical members of prenyl alcohol, 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.
In the production of the geranylgeraniol derivatives stated above, an erg mutant of Saccharoinyces cerevisiae is known to produce and secrete farnesol [Curr. Genet., 18, 41-46 (1990)], but this mutant is not practical for use because it provides a low farnesol production (1 mg/L). Also, there has been developed a technique for producing an arachidonate-containing lipid by culturing a mutant derived from a microorganism having the ability to produce an arachidonate-containing lipid in a medium supplemented with a hydrocarbon, a fatty acid, and/or a fat or oil (Japanese Patent Application Laid-Open (kokai) No. 2000-69987). However, the above supplemental ingredients are only used for conversion into arachidonate (i.e., used as precursors for a final product) or consumed as nutrient sources, and therefore have no effect on extracting a substance produced in the mutant cells.
To produce an useful substance, it is often advantageous to use an elevated energy level for synthesis and an increased concentration of sugar as a starting material. However, when an oily substance is to be produced under an increased sugar concentration, since it has poor permeability through a cell membrane, the oily substance accumulates in cells, thereby inducing product inhibition. In such a case, the oily substance is not expected to be produced at a high yield.
Accordingly, the object of the present invention is to provide a method for effective secretory production of prenyl alcohol using a microorganism capable of producing prenyl alcohol, which involves allowing prenyl alcohol accumulated in the microorganism to be secreted into an extracellular environment.
Our research efforts were directed to achieving the above object, and we have found that when cells capable of producing prenyl alcohol, such as yeast cells (ascomycetes and deuteromycetes), bacterial cells, actinomycete cells and filamentous fungus cells, are cultured in a medium with an increased sugar content in the presence of at least one member selected from the group consisting of a surfactant, a fat or oil, and a terpene, prenyl alcohol accumulated in the cells can be actively secreted into an extracellular environment to reduce intracellular product concentration, thereby effecting an improvement in productivity per se. Surprisingly, we further have found that cells belonging to the above-mentioned microorganisms which do not produce prenyl alcohol under normal culture conditions also enable the production of the compound when cultured under the same conditions as stated above, thereby finally completing the invention.
Namely, the present invention provides a high production method of prenyl alcohol, which comprises culturing prenyl alcohol-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,
Bacillus,
Staphylococcus,
Pseudomonas,
Williopsis,
Nakazawaea,
Kluyveromyces,
Torulaspora,
Citeromyces,
Waltomyces,
Micrococcus,
Cryptococcus,
Exiguobacterium,
Nocardia,
Mucor,
Ambrosiozyma,
Cystofilobasidium,
Metschnikowia,
Trichosporon,
Xanthophyllomyces,
Bullera,
Fellomyces,
Filobasidium,
Holtermannia,
Phaffia,
Rhodotorula,
Sporidiobolus,
Sporobolomyces,
Willopsis,
Zygoascus,
Haloferax,
Brevibacterium,
Leucosporidium,
Myxozyma,
Trichosporiella, and
Alcaligenes
in a medium with an increased sugar content in the presence of at least one member selected from the group consisting of a surfactant, a fat or oil, and a terpene to produce and accumulate prenyl alcohol in the cells; allowing prenyl alcohol to be secreted from the cells; and then collecting prenyl alcohol.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application Nos.2000-401951 and No.2001-375842, which are priority documents of the present application.
The present invention will be described below in more detail.
In the present invention, a microorganism fermentation method is used to produce prenyl alcohol, typically geranylgeraniol and analogous compounds thereof. As used herein, an analogous compound of geranylgeraniol refers to geranylgeranyl pyrophosphate and compounds produced in association with the synthesis thereof, i.e., farnesyl pyrophosphate, farnesyl monophosphate, farnesol, geranyl pyrophosphate, geranyl monophosphate, geraniol, nerolidol, geranyllinanol, linalool and the like.
In the present invention, any microorganism may be used for geranylgeraniol production, so long as it has a potential 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, Torulaspora, Cryptococcus, Bullera, Rhodotorula, Sporobolomyces, Kloeckera and Willopsis; filamentous fungus strains belonging to Mucor; archaebacterial strains belonging to Haloferax; or bacterial strains belonging to Alcaligenes.
Specific examples of geranylgeraniol-producing cells will be presented below:
In the present invention, any microorganism may be used for farnesol production, so long as it has a potential ability to produce farnesol. Examples include yeast strains belonging to any one of Saccharomyces, Saccharomycodes, Schizosaccharomyces, Wickerharnia, 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; actinomycete strains belonging to Nocardia; 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, Willopsis, Zygoascus, Leucosporidium, Myxozyma, Trichosporiella, Haloferax and Brevibacterium.
Specific examples of farnesol-producing cells will be presented below:
In the present invention, any microorganism may be used for nerolidol production, so long as it has a potential ability to produce nerolidol. Examples include yeast strains belonging to Saccharomyces or Candida; filamentous fungus strains belonging to Mucor; or microbial strains belonging to any one of Cystofilobasidium, Rhodotorula, Willopsis, Zygoascus and Haloferax.
Specific examples of nerolidol-producing cells will be presented below:
In addition to the above-listed microorganisms, further examples of microorganisms capable of producing prenyl alcohol such as 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.
The microorganisms used in the present invention also encompass recombinant microorganisms modified by gene transfer into the naturally occurring microorganisms listed above.
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, actinomycete cells and filamentous fungus cells.
Any carbon compound may be used as a carbon source, so long as the microorganisms can assimilate it for growth.
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.
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.
However, it is naturally important to select culture conditions for maximum production of prenyl alcohol, according to the type of microorganism to be used and the composition of the medium.
In the present invention, the prenyl alcohol-producing cells are cultured in a medium with an increased sugar content in the presence of at least one member selected from the group consisting of a surfactant, a fat or oil, and a terpene in order to improve prenyl alcohol production and stimulate product secretion from the cells.
As used herein, a “medium with an increased sugar content” means a medium having a sugar content of 2% to 7%. Examples of sugars include glucose, sucrose and the like. As used herein, the percentage (%) used to express a sugar content etc. is based on w/v (%).
Examples of a fat or oil used in the present invention include soybean oil, fish oil, almond oil, olive oil and the like. For example, it may be added to a medium at a concentration of 0.01% or more, preferably 1% or more. Since a concentration exceeding 3% gives no further effect, it is desirable to add a fat or oil at a concentration of 0.01% to 3% in view of cost.
In the present invention, a preferred surfactant is a non-ionic surfactant including polyethylene glycol-type surfactants (e.g., an ethylene oxide adduct of a higher alcohol, an ethylene oxide adduct of an alkylphenol, an ethylene oxide adduct of a fatty acid, an ethylene oxide adduct of a polyhydric alcohol fatty acid ester, an ethylene oxide adduct of a higher alkylamine, an ethylene oxide adduct of a fatty amide, an ethylene oxide adduct of polypropylene ethylene glycol), polyhydric alcohol-type surfactants (e.g., a fatty acid ester of sucrose, an alkylether of a polyhydric alcohol), or a silicone oil (e.g., dimethyl silicone, a polyether-modified silicone oil).
More specifically, the following commercially available surfactants can be used in the present invention: Tergitol NP-40 (Nacalai), cholic acid (Nacalai), deoxycholic acid (Nacalai), N-lauryl sarcosine (Nacalai), sucrose monolaurate (Nacalai), Triton X-100 (Nacalai), Triton X-305 (Nacalai), Nonidet P-40 (Iwai Chemicals Company), Tween 20 (Nacalai), Tween 80 (Nacalai), Span 20 (Nacalai), Span 85 (Nacalai), CTAB (Nacalai), nonyl-β-D-glucose (Sigma), Adekapluronic L-61 (Asahi Denka Kogyo K.K.; PPG with attached ethylene oxide), Adekanol LG-109 (Asahi Denka Kogyo K.K.; polyether-type PPG), Adekanol LG-294 (Asahi Denka Kogyo K.K.; polyether-type PPG), Adekanol LG-295S (Asahi Denka Kogyo K.K.; polyether-type PPG), Adekanol LG-297 (Asahi Denka Kogyo K.K.; polyether-type PPG), Adekanol B3009A (Asahi Denka Kogyo K.K.; fat or oil/fatty acid ester), and silicone anti-foaming agents KS66 (Shin-Etsu Chemical Co., Ltd.), KS69 (Shin-Etsu Chemical Co., Ltd.), KS502 (Shin-Etsu Chemical Co., Ltd.), KM73 (Shin-Etsu Chemical Co., Ltd.) and KM82F (Shin-Etsu Chemical Co., Ltd.).
For example, a surfactant without anti-foaming activity may be added to a medium at a concentration of 0.005% to 1%, preferably 0.05% to 0.5%. A concentration exceeding 1% is not preferred because such a concentration causes foaming. In contrast, a surfactant with anti-foaming activity can be added at a concentration exceeding 1%.
In the present invention, examples of a terpene include squalene, tocopherol and the like. For example, it may be added to a medium at a concentration of 0.01% or more, preferably 1% or more. Since a concentration exceeding 3% gives no further effect, it is desirable to add a terpene at a concentration of 0.01% to 3% in view of cost.
Among these additives stated above, it is preferable to use a fat or oil, more preferably in combination with a surfactant (particularly, an anti-foaming surfactant), in order to improve prenyl alcohol production and stimulate product secretion from the cells.
In the present invention, prenyl alcohol may be produced in a batch manner or in a continuous manner using a bioreactor. Microorganism cells may be provided as such for prenyl alcohol 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 prenyl alcohol in the cells or culture supernatant, which is then collected.
To collect prenyl alcohol 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.
To collect prenyl alcohol 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.
The above solvent extraction step may be performed in combination with a known purification technique such as chromatography, as needed.
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.
In the production method of the present invention, prenyl alcohol is detected by gas chromatography/mass spectrometry (GC/MS) using a commercially available column and then quantified from the ratio of peak area between prenyl alcohol and 1-undecanol as an internal standard.
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.
(1) Preparation of Liquid Medium
In each of the following examples, yeast cells were cultured in YM medium (Difco) or KY medium prepared as presented below. In particular, squalene synthase-defective yeast cells were cultured in YM medium supplemented with ergosterol.
Bacterial cells or actinomycete cells were cultured in KB medium. A plate was prepared from the same medium by addition of Bactoagar (Difco) at a final concentration of 2%.
YM Medium Supplemented with Ergosterol
20 mg of ergosterol (Sigma) was suspended in an ethanol solution containing 50% Tergitol (Nacalai) and completely dissolved by heating in a boiling water bath. The resulting solution was added to 1 L of YM broth (Difco), followed by autoclaving.
KY Medium
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.
KB Medium
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.
(2) Extraction of Prenyl Alcohol from Supernatant Fraction
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 (φ18mm×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.
(3) Extraction of Prenyl Alcohol from Cell Fraction
(i) Extraction from Bacterial and Actinomycete Cells
10 ml of the liquid culture solution was transferred into a 50 ml Corning 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 (φ18 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 (2) above.
(ii) Extraction from Yeast Cells
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 washed φ=425-600 μm) was added to the tube and the cells were crushed using a Multi-Beads Shocker 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 (2) above.
(4) Analysis of Prenyl Alcohol
Analysis was performed using a Agilent HP6890/5973 GC/MS system under the following conditions:
(1) Strain
Colony selection was performed on squalene synthase-defective yeast strain ATCC#64031 (purchased from ATCC) to select a colony giving an increased farnesol production, which was used in this example.
(2) Preparation of Medium
The above YM medium supplemented with ergosterol was mixed individually with the following surfactants and then autoclaved.
Surfactants:
A loopful of the colony was inoculated from the slant into a 300 ml Erlenmeyer flask containing 50 ml of YM medium supplemented with ergosterol and then cultured at 26° C. while rotating at 150 rpm. After culturing for two days, 50 μl of the culture was added to 5 ml of the medium prepared in (2) above, followed by shaking culture in a test tube (φ18 mm×150 mm) at 26° C. for two days.
(4) Determination of Cell Counts
100 μl of the culture solution was diluted with physiological saline to determine its O.D. at 660 nm with a spectrophotometer.
(5) Extraction and Analysis of Prenyl Alcohol from Supernatant Fraction
Extraction and analysis were performed according to the procedures presented in the Reference Example.
(6) Results
A medium prepared by adding a fat or oil (almond oil, fish oil, soybean oil, or olive oil; each commercially available from Sigma), at a final concentration of 0.1%, to the YM medium supplemented with ergosterol stated above was used for culture of the squalene synthase-defective yeast strain (ATCC#64031) to examine an effect of the fat or oil on farnesol secretory production in the same manner as described in Example 1.
Each system containing the above fat or oil provides not only an increased farnesol secretion, but also an increased farnesol production as the concentration of fat or oil is increased.
Thus, the addition of a fat or oil was also shown to give a farnesol secretion-stimulating effect as in the case of a surfactant.
A medium prepared by adding a sugar (glucose or sucrose; each commercially available from Nacalai) to the YM medium supplemented with ergosterol stated above was used for culture of the squalene synthase-defective yeast strain (ATCC#64031) to examine an effect of the sugar on farnesol secretory production in the same manner as described in Example 1.
Since YM medium (Difco) originally contains 1% glucose, the final sugar concentration is expressed as 1% glucose plus additional glucose or sucrose (0% to 5%).
Farnesol secretory production per cell (O.D. at 660 nm) also shows a similar tendency, and therefore an increased sugar concentration in the medium results in an increased farnesol production per cell.
A medium prepared by adding 6% glucose as a sugar, 1% soybean oil as a fat or oil, and/or 0.1% Triton X-100 as a surfactant, alone or in combination, to the YM medium supplemented with ergosterol stated above was used for culture of the squalene synthase-defective yeast strain (ATCC#64031) to examine effects of these ingredients on geranylgeraniol and farnesol secretory production in the same manner as described in Example 1.
In contrast, geranylgeraniol is not detected in a cell fraction at all. Only the system containing glucose and soybean oil or Triton X-100 allows geranylgeraniol to be secreted from the cells. This indicates that the addition of these ingredients also stimulates geranylgeraniol secretion, thereby resulting in an increased production.
A medium prepared by adding tocopherol or squalene at a concentration shown in Tables 1 and 2 to the YM medium supplemented with ergosterol stated above was used for culture of Saccharomyces cerevisiae strains ATCC 64031 and IFO 0538 to examine an effect of tocopherol or squalene on prenyl alcohol secretory production. Tables 1 and 2 also show the results obtained. Saccharomyces cerevisiae strain IFO 0538 is found to produce no prenyl alcohol in YM medium commonly used for yeast culture. However, this strain enables prenyl alcohol production in the presence of squalene, and geranylgeraniol and farnesol are found in the supernatant at a higher concentration than that found in the cells (Table 2). Table 1 shows the results obtained using Saccharomyces cerevisiae strain ATCC 64031 which inherently produces farnesol. This strain produces more farnesol in the supernatant fraction as the concentration of tocopherol is increased.
Hanseniaspora valbyensis strain IFO 0115, Saccharomycodes ludwigii strain IFO 0339 and Candida glabrata strain IFO 0005, all of which inherently provide high production of farnesol and geranylgeraniol without addition of a specific ingredient to a medium, were separately cultured in an Erlenmeyer flask. The results, including days of culture, medium composition, and changes in production with or without phosphatase treatment, are shown in Table 3 (supernatant fraction) and Table 4 (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 cells. The addition of 1% soybean oil along with glucose stimulated Candida glabrata strain to secrete, farnesol and geranylgeraniol from the cells. In contrast, Hanseniaspora valbyensis strain IFO 0115 and Saccharomycodes ludwigii strain IFO 0339 showed no secretion-stimulating effect attributed to soybean oil in this example. This is because uniform sampling of farnesol and geranylgeraniol was difficult due to phase separation between the oil phase with a high product content and the aqueous phase with a low product content. However, each of these strains was cultured in a test tube for full volume analysis, indicating that these two strains showed the same secretion-stimulating effects (Tables 9 to 11).
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 both the cell fraction and the supernatant fraction (i.e., to be secreted from the cells).
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.
Candida
glabrata
Hanseniaspora
valbyensis
Saccharomycodes
ludwigii
Candida
glabrata
Hanseniaspora
valbyensis
Saccharomycodes
ludwigii
Some of the strains shown in Table 5 were test-tube cultured in YM medium alone and in YM medium supplemented with 5% glucose and 1% soybean oil to examine farnesol and geranylgeraniol production. Tables 5 and 6 show the results obtained. The medium supplemented with 5% glucose and 1% soybean oil effected a significantly increased production in both the cell fraction and the supernatant fraction (i.e., a significantly increased secretion from the cells).
Saccharomyces
cerevisiae
Saccharomyces rosei
Saccharomyces
cerevisiae
Williopsis saturnus
Ogataea polymorpha
Kluyveromyces lactis
Saccharomyces cerevisiae
Saccharomyces rosei
Saccharomyces cerevisiae
Williopsis saturnus var. saturnus
Ogataea polymorpha
Kluyveromyces lactis
Each of the strains shown in Tables 7 and 8 below was test-tube cultured in (i) YM medium supplemented with 4 mg/L ergosterol and 0-20 mg/L squalene synthesis inhibitor (SQAD: Japanese Patent Application No. 8-508245) or (ii) 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 7 and 8 also show the results obtained.
These tables indicate that the addition of soybean oil, glucose and a squalene synthesis inhibitor effects an increased production in both the cell fraction and the supernatant fraction (i.e., an increased secretion from the cells).
Saccharomyces unisporus
Saccharomyces cerevisiae
Candida glabrata
Yarrowia lopolytica
Komagataella pastoris
Kuraishia capsulata
Ogataea glucozyma
Saccharomyces kluyeri
Candida cariosilignicola
Candia glabrata
Saccharomyces
unisporus
Saccharomyces
cerevisiae
Candida
glabrata
Yarrowia
lopolytica
Komagataella
pastoris
Kuraishia
capsulata
Ogataea
glucozyma
Saccharomyces
kluyeri
Candida
cariosilignicola
Candia
glabrata
Each of the strains shown in Tables 9 and 10 below was cultured in YM, KB or KY 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 9 (supernatant fraction) and Table 10 (cell fraction).
As a control, each strain was cultured in YM medium without the above supplemental ingredients to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. Table 11 shows the results obtained.
These tables indicate that the addition of soybean oil, glucose and a squalene synthesis inhibitor effects an increased production in both the cell fraction and the supernatant fraction (i.e., an increased secretion from the cells).
Saccharomycopsis
fibuligera
Saccharomycopsis
fibuligera
Saccharomycopsis
fibuligera
Norcadia asteroides
Saccharomyces
cerevisiae
Saccharomyces
ellipsoideus
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Schizosaccharomyces
pombe
Ogataea polymorpha
Saccharomycodes
ludwigii
Kluyveromyces lactis
Candida glabrata
Candida solani
Cryptococcus humicolus
Wickerhamia fluorescens
Saccharomycopsis fibuligera
Saccharomycopsis fibuligera
Saccharomycopsis fibuligera
Norcadia asteroides
Saccharomyces cerevisiae
Saccharomyces ellipsoideus
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Ogataea polymorpha
Saccharomycodes ludwigii
Kluyveromyces lactis
Candida glabrata
Candida solani
Cryptococcus humicolus
Wickerhamia fluorescens
Norcadia asteroides
Mucor Javanicus
Saccharomyces
cerevisiae
Saccharomyces
ellipsoideus
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Schizosaccharomyces
pombe
Ogataea polymorpha
Saccharomycodes
ludwigii
Kluyveromyces lactis
Candida glabrata
Candida solani
Cryptococcus humicolus
Wickerhamia fluorescens
Each of the strains shown in Table 12 below was cultured in Medium A (YM medium supplemented with 0.1% Adekanol and 4 mg/L ergosterol) or Medium B (YM medium supplemented with 0.1% Adekanol, 4 mg/L ergosterol, 5% glucose and 3% soybean oil) at 30° C. for 4 or 10 days to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. Table 12 also shows the results obtained (supernatant and cell fractions).
Pichia pastorias
Schizosaccharomyces
pombe
Saccharomyces
cerevisiae
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.
As a control, each strain was cultured in KB medium without the above supplemental ingredients to examine the respective production of nerolidol, geranylgeraniol and farnesol in the same manner as stated above. Table 14 shows the results obtained.
These tables indicate that the addition of a squalene synthesis inhibitor effects an increased production in bacterial strains as in the case of yeast strains.
Baccilus
amyloliquefaciens
Baccilus pumilus
Staphylococcus
epidermidis
Micrococcus lutenus
Exiguobacterium
acetylicum
Baccilus amyloliquefaciens
Baccilus pumilus
Staphylococcus epidermidis
Micrococcus lutenus
Saccharomyces cerevisiae strain pRS435GGF/pRS434GAP-HMG1/YPH499#1 was prepared and cultured in a 5 L jar fermenter in the following manner. This strain allows high level expression of the HMG CoA reductase gene and a fusion gene comprising the geranylgeranyl pyrophosphate synthase gene and the farnesyl pyrophosphate synthase gene.
(1) Obtaining of GGPS and HMG-CoAR1 Genes Derived from Yeast Saccharomyces cerevisiae
Based on the GenBank information of S. cerevisiae-derived GGPS (A.N.U 31632) and HMG-CoAR1 (A.N.M 22002) genes, the following primers matching N- and C-termini of these genes were prepared and then used for PCR along with a yeast cDNA library (TOYOBO No. CL7220-1) as a template under the following conditions. PCR of the HMG-CoAR1 gene was performed using Perfect match (Stratagene).
PCR fragments were confirmed at positions of interest (about 1.0 kbp and 3.2 kbp, respectively). These gene fragments were then cloned into a TA-clonable pT7Blue-T vector to determine the full nucleotide sequence of GGPS and about 40% nucleotide sequence of HMG-CoAR1. The resulting sequences were completely matched with the GenBank sequences and therefore confirmed as genes derived from S. cerevisiae.
The resulting vector carrying the HMG-CoAR1 gene cloned into the pT7Blue-T vector was designated pT7HMG1.
(2) Construction of Vector pYES2 for Expression in Yeast Cells (Preparation of pYES-GGPS6)
pYES2 (Invitrogen) is a shuttle vector for extraction in yeast cells, which carries yeast 2 μm DNA ori as a replication origin and GAL1 promoter inducible with galactose. pT7Blue-T vector carrying the cloned GGPS gene was treated with BamHI and SalI to collect the GGPS gene, which was then introduced into a BamHI-XhoI site of pYES2 to give a vector designated pYES-GGPS6.
(3) Cloning of Farnesyl Pyrophosphate Synthase Gene (FPS) ERG20 Derived from Yeast Saccharomyces cerevisiae
Using the following primers, the FPP synthase gene, ERG20, was cloned from Saccharomyces cerevisiae YPH499 (Stratagene) cDNA by PCR:
An amplified fragment of approximately 0.9 kbp was purified by agarose gel electrophoresis and then T/A ligated into pT7Blue-T to prepare plasmid DNA designated pT7ERG20.
(4) Construction of Expression Vector
(4-1) Preparation of pRS405Tcyc and pRS404Tcyc (insertion of CYC1t fragments into pRS vectors)
CYC1 transcription terminator CYC1t fragments were prepared by PCR using the following primer sets.
The DNA fragments amplified with the above primer sets (i) and (ii) were cleaved with XhoI plus KpnI and XhoI plus ApaI, respectively, and then electrophoresed on agarose gels to give purified 260 bp DNA fragments designated CYC1t-XK and CYC1tXA. CYC1t-XK and CYC1tXA were inserted into a XhoI-KpnI site of pRS405 (Stratagene) and a XhoI-ApaI site of pRS404 (Stratagene), respectively, to give plasmids designated pRS405Tcyc and pRS404Tcyc.
(4-2) Preparation of TDH3p (Preparation of Transcription Promoter)
Saccharomyces cerevisiae YPH499 (Stratagene) genomic DNA was prepared using a yeast genomic DNA preparation kit “Gen TLE” (Takara Shuzo, Co., Ltd.), and then used as a PCR template to prepare a DNA fragment containing TDH3p (PGK) promoter.
Saccharomyces cerevisiae YPH499 (Stratagene)
The amplified DNA fragment was cleaved with SacI and SacII, and then electrophoresed on an agarose gel to give a purified 680 bp DNA fragment designated TDH3p.
(4-3) Preparation of 2μOriSN (Preparation of 2μDNA Replication Origin Region)
pYES2 (Invitrogen) was cleaved with SspI and NheI, and then electrophoresed on an agarose gel to give a purified 1.5 kbp fragment containing the 2μDNA replication origin (2μOri). The resulting fragment was blunt-ended with Klenow enzyme and designated 2μOriSN.
(4-4) Preparation of pRS434GAP and pRS435GAP (Preparation of YEp-type Expression Vector)
2μOriSN was inserted into a NaeI site of pRS404Tcyc or pRS405Tcyc treated with BAP (bacterial alkaline phosphatase, TaKaRa), and then transformed into E. coli SURE2 to prepare plasmid DNA. The resulting plasmid DNA was cleaved with DraIII plus EcoRI, HpaI, or PstI plus PvuII, and then electrophoresed on an agarose gel to confirm the insertion and orientation of 2μori. pRS404Tcyc and pRS405Tcyc carrying 2μori inserted in the same orientation as pYES2 were designated pRS434Tcyc2μOri and pRS435Tcyc2μOri, respectively. The fragment TDH3p containing the transcription promoter was inserted into SacI-SacII sites of these two plasmids pRS434Tcyc2μOri and pRS435Tcyc2μOri to give plasmids designated pRS434GAP and pRS435GAP, respectively.
(5) Preparation of pRS435GGF (FPS-GGPS Fusion Protein Gene)
pYES-GGPS6 carrying the inserted GGPS gene BTS1 and pT7-ERG20 carrying the inserted FPS gene ERG20 were used as templates to perform PCR under the following conditions.
wherein the sequence CCG CGG in primer 1 represents a SacII, XhoI or XbaI recognition site for vector ligation, and the sequence GGG ACC C in primer 2 represents a EcoO109I recognition site for fusion gene preparation.
wherein the sequence GGG TCC T in primer 3 represents a EcoO109I recognition site for fusion gene preparation.
The reaction products obtained by (i) and (ii) above were designated #9 and #11, respectively. After cleavage with restriction enzyme EcoO109I, #9 and #11 were ligated to each other and then used as a PCR template to perform a second round of PCR using the above SacII-BTS1 and −21 as primers under the same conditions, thereby obtaining DNA fragment #9-#11. The second PCR product #9-#11 was cleaved with SacII and BamHI, and then inserted into a SacII-BamHI site of pRS435GAP to obtain pRS435GGF.
As expression vectors for non-fusion genes BTS1 and ERG20, pRS435GAP-BTS1, pRS445GAP-BTS1, pRS435GAP-ERG20 and pRS445GAP-ERG20 were used. pRS434TEF-HMG1 and pRS434GAP-HMG1 were used for HMG1 expression.
(6) Preparation of pRS434GAP-HMG1
pT7HMG1 was cleaved with SmaI and SalI and then electrophoresed on an agarose gel to give a purified 3.2 kbp HMG1 gene fragment. This fragment was inserted into a SmaI-SalI site of pRS434GAP to obtain pRS434GAP-HMG 1.
(7) Creation of Transgenic Yeast (pRS435GGF/pRS434GAP-HMG1/YPH499#1)
pRS435GGF and pRS434GAP-HMG1 were introduced into Saccharomyces cerevisiae strain YPH499 (Stratagene) 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) or a Frozen-EZ Yeast Transformation II technique (Zymo Research, Orange, Calif.). The transformant was cultured on SD (-URA) medium to select a colony growing at 30° C. on a SD (-URA) agar plate (DOB+CSM (-URA), BIO 101, Vista, Calif.), thereby obtaining pRS435GGF/pRS434GAP-HMG1/YPH499#1.
(8) Culture of Transgenic Yeast (pRS435GGF/pRS434GAP-HMG1/YPH499#1)
A loopful of the colony was inoculated from the slant into a 500 ml baffled Erlenmeyer flask containing 200 ml of SD-Leu-Trp medium (BIO 101 Inc.) supplemented with 40 mg/L adenine (Sigma). After culturing at 130 rpm and at 30° C. for 2 days, centrifugation (1500×g, 5 minutes, 4° C.) and washing with sterilized physiological saline were repeated three times to completely remove glucose contained in the culture solution. An aliquot of the culture solution (50 ml; 1%) was then inoculated into a fermenter.
Fermentation Medium
5% glucose (including 1% glucose originally contained in YM broth)
YM broth (Difco)
0% or 3% soybean oil (Nacalai)
0.1% Adekanol LG-109 (Asahi Denka Kogyo K.K.)
Operation Conditions
Culture apparatus: MSJ-U 10 L culture apparatus (B. E. Marubishi Co., Ltd.)
Medium volume: 5 L
Medium temperature: 33° C.
Aeration rate: 1 vvm
Agitation: 300 rpm
pH: pH is proportionally controlled with 4N sodium hydroxide and 2N hydrochloric acid under the following parameter conditions, unless otherwise specified:
Analyses of extracellular and intracellular geranylgeraniol indicate that the addition of soybean oil allows geranylgeraniol to be secreted from the cells. The addition of soybean oil also resulted in an approximately 4-fold increase in production per se (FIG. 10).
Farnesol-producing recombinant yeast strain pYHMG044/AURGG101 was prepared and cultured in the following manner.
(1) Preparation of HMG1Δ Expression Vector pYHMG044
pT7HMG1 prepared in Example 12 was used as a template to prepare a fragment containing the vector backbone and a partially deleted HMG1 coding region by PCR. The resulting fragment was blunt-ended with Klenow enzyme, re-cyclized through self-ligation, and then transformed into E. coli JM109 to prepare plasmid DNA pYHMG044. The synthetic DNA sequences used as a primer set are presented below.
By using a 373A DNA sequencer (Perkin Elmer, Foster City, Calif.), the resulting DNA plasmid was confirmed to show no shift in reading frame for amino acids located upstream and downstream of HMG1 and to contain no PCR error-based amino acid replacement in the neighborhood of its binding site.
(2) Preparation of AURGG101
pAUR101 (TaKaRa, Japan) was linearized with EcoO65I and then introduced into Saccharomyces cerevisiae strain A451 (ATCC 200598) according to 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). The transformant was cultured on an YPD agar plate (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) containing 1 μg/ml Aureobasidin to select a colony growing thereon at 30° C.
(3) Creation of Transgenic Yeast (pYHMG044/AURGG 101)
pYHMG044 was introduced into AURGG101 according to 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). The transformant was cultured on SD (-URA) medium to select a colony growing at 30° C. on an SD (-URA) agar plate (DOB+CSM (-URA), BIO 101, Vista, Calif.), thereby obtaining pYHMG044/AURGG101.
(4) Culture Conditions
A loopful of the recombinant yeast strain pYHMG044/AURGG101 was inoculated from the slant into 200 ml of CSM-URA (BIO 101 Inc.) and DOB (BIO 101 Inc.) medium in a 500 ml baffled Erlenmeyer flask. After culturing at 130 rpm and at 30° C. for 2 days, centrifugation (1500×g, 5 minutes, 4° C.) and washing with sterilized physiological saline were repeated three times to completely remove glucose contained in the culture solution. An aliquot of the culture solution (50 ml; 1%) was then inoculated into a fermenter.
Fermentation Medium
5% glucose
YNB containing all amino acids (Difco)
1% soybean oil (Nacalai)
0.1% Adekanol LG-109 (Asahi Denka Kogyo K.K.)
Operation Conditions
Culture apparatus: MSJ-U 10 L culture apparatus (B. E. Marubishi Co., Ltd.)
Medium volume: 5 L
Medium temperature: 26° C.
Aeration rate: 1 vvm
Agitation: 300 rpm
pH: pH is proportionally controlled with 4N sodium hydroxide and 2N hydrochloric acid under the following parameter conditions, unless otherwise specified:
Cell counting was performed on 100 μl culture solution diluted 1- to 20-fold with physiological saline using a hemocytometer (supplier: HAYASHI RIKAGAKU, manufacturer: Sunlead Glass Co.). Cells found in a 0.06 mm square (corresponding to 9 minimum grids) were averaged over quadruplicate measurements to calculate cell counts per liter of culture medium from the following equation:
Cell counts (1×109/L broth)=0.444×(cell counts in 0.06 mm square)×dilution factor
Soybean oil stimulated the secretory production of farnesol in recombinant yeast cells. It allowed farnesol to be secreted from the cells in an amount of approximately 150 mg/L over 150 hours (FIG. 11).
Under the culture conditions shown in Table 15 (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 prenyl alcohol from both cell and supernatant fractions. Analysis was performed on these fractions according to the procedures presented in the Reference Example. Table 15 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.
LBO-SSI Medium
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.
YPDO-SSI Medium
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.
YMO-SSI Medium
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; Eisai Co., Ltd.) was added to the medium to give a final concentration of 1 to 20 mg/L.
YMOL-SSI Medium
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.
HVO-SSI Medium
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.
Alcaligenes faecalis
Brevibacterium divaricatum
Brevibacterium fuscum
Brevibacterium linens
Candida catenulata
Candida fragicola
Candida krusei
Candida lambica
Candida maltosa
Candida mycoderma
Candida parapsilosis
Candida rugosa
Candida succiphila
Candida tropicalis
Candida zeylanoides
Cryptococcus albidus
Cryptococcus glutinis
Dipodascus ovetensis
Haloferax volcanii
Hanseniaspora valbyensis
Issatchenkia orientalis
Kloeckera africana
Kloeckera apiculata
Kluyveromyces marxianus
Kuraishia capsulata
Mortierella ramanniana
Nakazawaea holstii
Pichia capsulata
Pichia henricii
Pichia holstii
Pichia naganishii
Pichia rhodanensis
Pichia saitoi
Rhodosporidium toruloides
Rhodotorula aurantinaca
Rhodotorula rubra
Saccharomycopsis fibuligera
Saccharomycopsis lipolytica
Schizosaccharomyces octosporus
Staphylococcus aureus
Torulaspora delbrueckii
Trichosporon cutaneum
Tsukamurella paurometabolum
Yamadazyma farinosa
Yerroiwa lipolytica
Zygosaccharomyces japonicus
Ambrosiozyma ambrosiae
Ambrosiozyma monospora
Ambrosiozyma philentoma
Ambrosiozyma platypodis
Bensingtonia intermedia
Botryozyma nematodophila
Brettanomyces anomalus
Brettanomyces bruxellensis
Brettanomyces custersianus
Bullera crocea
Bullera sinensis
Citeromyces matritensis
Clavispora lusitaniae
Cystofilobasidium infirmominiatum
Debaryomyces occidentalis
Dekkera bruxellensis
Dipodascus armillariae
Dipodascus tetrasperma
Eremascus albus
Eremascus fertilis
Eremothecium gossypii
Erythrobasidium hasegawianum
Hanseniaspora guilliermondii
Hanseniaspora uvarum
Kloeckeraspora vineae
Kockovaella imperatae
Kodamaea ohmeri
Kurtzmanomyces nectairei
Leucosporidium scottii
Lodderomyces elongisporus
Malassezia furfur
Metschnikowia hawaiiensis
Metschnikowia krissii
Metschnikowia lunata
Metschnikowia pulcherrima
Mrakia frigida
Myxozyma lipomycoides
Nadsonia commutata
Pachysolen tannophilus
Pichia burtonii
Pichia misumaiensis
Pichia ofunaensis
Pichia pijperi
Saccharomyces transvaalensis
Saccharomycodes sinensis
Saccharomycopsis fibuligera
Saccharomycopsis javaensis
Saccharomycopsis schoenii
Saccharomycopsis synnaedendra
Saccharomycopsis vini
Saturnispora zaruensis
Schizoblastosporion kobayasii
Schizoblastosporion starkeyi-henric
Sporopachydermia cereana
Stephanoascus ciferrii
Sterigmatomyces elviae
Sterigmatomyces halophilus
Sterigmatosporidium polymorphum
Sympodiomyces parvus
Sympodiomycopsis paphiopedili
Trichosporon brassicae
Trichosporon pullulans
Trigonopsis variabilis
Tsuchiyaea wingfieldii
Wickerhamilla domercqiae
Xanthophyllomyces dendrorhous
Zygozyma oligophaga
Aciculoconidium aculeatum
Bullera pseudoalba
Candida albicans
Candida glabrata
Candida guilliermondii
Candida intermedia
Candida kefyr
Candida krusei
Candida tenuis
Candida utilis
Cryptococcus humicola
Cryptococcus terreus
Debaryomyces castellii
Fellomyces penicillatus
Filobasidium capsuligenum
Filobasidium uniguttulatum
Kloeckera corticis
Holtermannia corniformis
Kluyveromyces marxianus
Phaffia rhodozyma
Pichia anomala
Pichia fabianii
Pichia farinosa
Pichia jadinii
Pichia polymorpha
Pichia silvicola
Rhodotorula glutinis
Rhodotorula minuta
Rhodotorula rubra
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomycodes ludwigii
Saccharomycopsis fermentans
Sporidiobolus samonicolar
Sporobolomyces salmonicolor
Trichosporiella flavificans
Trichosporon penicillatum
Williopsis californica
Willopsis saturnus
Yamadazyma farinosa
Zygoascus hellenicus
The present invention enables prenyl alcohol to be highly produced in and effectively secreted from prenyl alcohol-producing cells by culturing the cells in a medium with an increased sugar content in the presence of at least one member selected from the group consisting of a surfactant, a fat or oil, and a terpene.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2000-401951 | Dec 2000 | JP | national |
| 2001-375842 | Dec 2001 | JP | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6410755 | Millis et al. | Jun 2002 | B1 |
| Number | Date | Country |
|---|---|---|
| 2000-69987 | Mar 2000 | JP |
| WO 0001650 | Jan 2000 | WO |
| WO 0001685 | Jan 2000 | WO |
| WO 0001686 | Jan 2000 | WO |
| WO 00016949 | Jan 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20030096385 A1 | May 2003 | US |
