JP2017023091A - Process for producing hydroxy unsaturated fatty acid or ester thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel process for producing a hydroxy unsaturated fatty acid or an ester thereof from an unsaturated fatty acid or an ester thereof using a microorganism, the process being capable of further increasing production efficiency of the hydroxy unsaturated fatty acid or the ester thereof.SOLUTION: The present invention provides a process for producing a hydroxy unsaturated fatty acid or an ester thereof. The process comprises culturing a microorganism belonging to the genus Candida or a microorganism belonging to the genus Yarrowia in a medium containing glucose and an unsaturated fatty acid or an ester thereof. The medium keeps the glucose concentration at 1 to 20 g/L and/or contains an alcohol dehydrogenase inhibitor.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a hydroxy unsaturated fatty acid or an ester thereof. More specifically, the present invention relates to a method for producing a hydroxy unsaturated fatty acid or ester thereof from an unsaturated fatty acid or ester thereof using a microorganism.

  Royal jelly of bees contains proteins, medium chain fatty acids, hydroxy fatty acids and the like, and among them, royal jelly-derived hydroxy fatty acids are known to have various physiological activities. For example, 10-hydroxy-trans-2-decenoic acid is also referred to as royal jelly acid because it is a hydroxy unsaturated fatty acid that is naturally contained only in royal jelly, and transforming growth factor (TGF) -β1 in a dose-dependent manner. Has the effect of inducing the production of dermal fibroblasts to enhance the collagen production ability of human dermal fibroblasts (Non-patent Document 1), promoting the generation of neural stem cells to neurons (Non-patent Document 2), estrogen receptor It has been reported so far that it binds to β (ERβ) and acts as an agonist (Non-patent Document 3).

  However, the amount of hydroxy unsaturated fatty acids such as 10-hydroxy-trans-2-decenoic acid is very small in royal jelly, and further, royal jelly itself is rare. It is difficult to produce and supply stably. As a method for producing a hydroxy unsaturated fatty acid, for example, a method of chemically synthesizing is known (Patent Document 1). However, the synthesis method described in Patent Document 1 is a method of synthesizing a hydroxy unsaturated fatty acid using a diol compound as a starting material. Since this method requires many steps, it is complicated and expensive, and the production efficiency is also high. There is a problem that it is low.

  In order to solve such problems, the present inventors have achieved an epoch-making method capable of producing a hydroxy unsaturated fatty acid or an ester thereof by a simple operation at a low cost by using a specific microorganism. Developed. In the method developed by the present inventors, a hydroxy unsaturated fatty acid is obtained by a simple operation of culturing a microorganism belonging to the genus Yarrowia or Candida in a medium containing an unsaturated fatty acid or an ester thereof. Or it is possible to manufacture the ester (patent document 2). Furthermore, the present inventors have been developing a method capable of producing a hydroxy unsaturated fatty acid or an ester thereof more efficiently, for example, a microorganism having a higher ability to produce a hydroxy unsaturated fatty acid or an ester thereof. A mutant has been successfully produced (Patent Document 3).

JP 2005-502692 A JP 2008-136488 A JP 2011-155908 A

Biosci. Biotechnol. Biochem. 68 (4), 767-773 (2004) Biomedical Research, 28 (5), 261-266 (2007) American Naturalist, 128, 13-34 (1986)

  The present invention has been made in view of the above-described problems of the prior art and the current situation. In a method for producing a hydroxy unsaturated fatty acid or an ester thereof from an unsaturated fatty acid or an ester thereof using a microorganism, the hydroxy unsaturated fatty acid is produced. Or it aims at providing the new method which can raise the production efficiency of the ester more.

  The inventors of the present invention belong to the genus Yarrowia or Candida, while intensively studying to develop a new method capable of more efficiently producing a hydroxy unsaturated fatty acid or an ester thereof. It has been confirmed that when the microorganism is continuously cultured in a medium containing an unsaturated fatty acid or an ester thereof, the amount of the product hydroxy unsaturated fatty acid or an ester thereof decreases over time ( See the reference example below).

  Specifically, when 10-hydroxy-trans-2-decenoic acid, which is a hydroxy unsaturated fatty acid, is produced from a trans-2-decenoic acid, which is an unsaturated fatty acid, in a medium for a while after the start of culture. It was confirmed that the amount of 10-hydroxy-trans-2-decenoic acid contained increased with time, but decreased rapidly after a certain point in time.

  As a result of proceeding with the analysis of such a phenomenon, the present inventors have detected trans-2-decendioic acid, which is an unsaturated dicarboxylic acid, as the amount of 10-hydroxy-trans-2-decenoic acid decreases. I found out that Although this phenomenon is not necessarily clear, hydroxy unsaturated fatty acids are metabolized to dicarboxylic acids by the ω oxidation system inherent in microorganisms. More specifically, alcohol dehydrogenase and aldehyde dehydration contained in microorganisms are used. The present inventors presume that the hydroxyl group of a hydroxy unsaturated fatty acid is oxidized by the action of an enzyme to be converted into an unsaturated dicarboxylic acid or the like.

  Further, when 10-hydroxy-trans-2-decenoic acid is purified from the medium, if the medium contains trans-2-decenedioic acid, trans-2-decenedioic acid and 10-hydroxy-trans It was very difficult to separate and purify from 2-decenoic acid, and it was confirmed that there was a problem that high-purity 10-hydroxy-trans-2-decenoic acid could not be obtained.

  As a result of further earnest studies to solve the above-mentioned problems, the present inventors have surprisingly been able to produce the hydroxy unsaturated fatty acid produced by maintaining the glucose concentration in the medium within a specific concentration range. Or it discovered that the fall of the quantity of the ester could be suppressed. Surprisingly, it has been found that a decrease in the amount of the hydroxy unsaturated fatty acid or ester produced can also be suppressed by adding an alcohol dehydrogenase inhibitor to the culture solution. The inventors of the present invention have completed the present invention through further research based on such knowledge.

  That is, the present invention typically includes the subject matters described in the following sections.

Item 1.
A process for producing a hydroxy unsaturated fatty acid or ester thereof,
Culturing a microorganism belonging to the genus Candida or a microorganism belonging to the genus Yarrowia in a medium containing glucose and an unsaturated fatty acid or an ester thereof,
A method comprising maintaining a glucose concentration in the medium at 1 to 20 g / L, and / or containing an alcohol dehydrogenase inhibitor in the medium.

Item 2.
The hydroxy unsaturated fatty acid or ester thereof is
Formula (I):

[In formula, R shows a hydrogen atom, an alkali metal, or a C1-C4 alkyl group, and n shows the integer of 1-14. ]
The method according to Item 1, wherein the compound is represented by the formula:

Item 3.
The unsaturated fatty acid or ester thereof,
General formula (II):

[Wherein, R and n are the same as defined above. ]
Item 3. The method according to Item 1 or 2, which is a compound represented by:

Item 4.
The microorganism belonging to the genus Candida is at least selected from the group consisting of Candida maltosa, Candida succiphila, Candida pignalae, and at least one species selected from the group Candida nitratotra. The method according to any one of Items 1 to 3, wherein

Item 5.
Item 4. The method according to any one of Items 1 to 3, wherein the microorganism belonging to the genus Candida is a wild strain of Candida maltosa (Candida maltosa) or a mutant thereof.

Item 6.
Item 4. The method according to any one of Items 1 to 3, wherein the microorganism belonging to the genus Yarrowia is Yarrowia lypolytica.

Item 7.
Item 7. The method according to any one of Items 1 to 6, wherein the alcohol dehydrogenase inhibitor is 4-methylpyrazole and / or N-ethylmaleimide.

  The method of the present invention can inhibit metabolism of a hydroxy unsaturated fatty acid or ester thereof by a microorganism in a method for producing a hydroxy unsaturated fatty acid or ester thereof from an unsaturated fatty acid or ester thereof using a microorganism. Therefore, according to the method of the present invention, a hydroxy unsaturated fatty acid or an ester thereof that is rare in nature can be produced efficiently.

Measurement results of the turbidity _{(OD 660)} and glucose concentration in the culture solution in reference example and illustrates 2DAEt, quantitative results of 10HDA and 2DDA by HPLC analysis. In FIG. 1, (A) shows the measurement result of the turbidity (OD ₆₆₀ ) of the culture solution, (B) shows the measurement result of glucose concentration, (C) shows the peak area by HPLC analysis of 2DAEt, (D) shows The peak area by HPLC analysis of 10HDA, (E) shows the peak area by HPLC analysis of 2DDA, respectively. Moreover, "↓" in (A), (C), and (E) of FIG. 1 indicates that 10 mL of 2DAEt was additionally added. Implementation turbidity of the culture solution in Example 1 _{(OD 660)} and glucose concentration in the measurement results, and illustrates 2DAEt, quantitative results of 10HDA and 2DDA by HPLC analysis. In FIG. 2, (A) shows the measurement result of the turbidity (OD ₆₆₀ ) of the culture solution, (B) shows the measurement result of glucose concentration, (C) shows the peak area by HPLC analysis of 2DAEt, (D) shows The peak area by HPLC analysis of 10HDA, (E) shows the peak area by HPLC analysis of 2DDA, respectively. In addition, “↓” in FIGS. 2A and 2B indicates that glucose was additionally added, and “↓” in (C) and (E) indicates that 10 mL of 2DAEt was additionally added. Example turbidity of the culture solution in 2 _{(OD 660)} and glucose concentration in the measurement results, and illustrates 2DAEt, quantitative results of 10HDA and 2DDA by HPLC analysis. In FIG. 3, (A) shows the measurement result of the turbidity (OD ₆₆₀ ) of the culture solution, (B) shows the measurement result of glucose concentration, (C) shows the peak area by HPLC analysis of 2DAEt, and (D) shows The peak area by HPLC analysis of 10HDA, (E) shows the peak area by HPLC analysis of 2DDA, respectively. In addition, “↓” in FIGS. 3A, 3C, and 3D indicates that 10 mL of 2DAEt was additionally added.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a method for producing a hydroxy unsaturated fatty acid or an ester thereof. In the following, the method for producing a hydroxy unsaturated fatty acid or ester thereof of the present invention may be simply referred to as “the method of the present invention”.

  The hydroxy unsaturated fatty acid or ester thereof produced by the method of the present invention is a compound having a hydroxy group at the end of the hydrocarbon chain of a monovalent lower to intermediate unsaturated fatty acid (that is, the end not on the carboxyl group side) or its Ester. In the present specification, “lower” means 1 to 7 carbon atoms, and “intermediate” means 8 to 17 carbon atoms. More specifically, the hydroxy unsaturated fatty acid or ester thereof produced by the method of the present invention is represented by the following formula (I).

  R in the above formula (I) represents a hydrogen atom, an alkali metal, or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include linear or branched alkyl groups such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, and tert-butyl group. Groups. Moreover, the case where R is an alkali metal means that it is an alkali metal salt of a hydroxy unsaturated fatty acid. Examples of the alkali metal salt of the hydroxy unsaturated fatty acid include sodium salt and potassium salt.

  N in the above formula (I) represents an integer of 1 to 14, preferably an integer of 4 to 14, more preferably an integer of 8 to 12.

  Among the compounds represented by the above formula (I), specific examples of the hydroxy unsaturated fatty acid include 8-hydroxy-trans-2-octenoic acid, 10-hydroxy-trans-2-decenoic acid, 12-hydroxy-trans- Specific examples of hydroxy unsaturated fatty acid esters include methyl 10-hydroxy-trans-2-decenoate, ethyl 10-hydroxy-trans-2-decenoate, 10-hydroxy-trans-2. -Propyl decenoate, 10-hydroxy-trans-2-butyl decenoate and the like.

  In the method of this invention, the process of culture | cultivating the microorganisms mentioned later in the culture medium containing unsaturated fatty acid or its ester, and glucose is included.

  The unsaturated fatty acid or ester thereof contained in the medium used in the method of the present invention is converted into a hydroxy unsaturated fatty acid or ester thereof by a microorganism described later, and is a monovalent lower to intermediate unsaturated fatty acid or its Ester. More specifically, the unsaturated fatty acid or ester thereof used in the method of the present invention is represented by the following formula (II).

  R in the above formula (II) is the same as R in the above formula (I). Specifically, R represents a hydrogen atom, an alkali metal, or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include linear or branched alkyl groups such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, and tert-butyl group. Groups. Moreover, the case where R is an alkali metal indicates that it is an alkali metal salt of an unsaturated fatty acid. Examples of the alkali metal salt of unsaturated fatty acid include sodium salt and potassium salt.

  N in the formula (II) is the same as n in the formula (I). Specifically, n is an integer of 1 to 14, preferably an integer of 4 to 14, more preferably 8 to 12. Indicates an integer.

  Among the compounds represented by the above formula (II), specific examples of unsaturated fatty acids include trans-2-octenoic acid, trans-2-decenoic acid, trans-2-dodecenoic acid, and the like. Specific examples of the ester include methyl trans-2-decenoate, ethyl trans-2-decenoate, propyl trans-2-decenoate, and butyl trans-2-decenoate.

  The microorganism used in the method of the present invention is a microorganism belonging to the genus Candida or a microorganism belonging to the genus Yarrowia. The microorganism has an ability to convert unsaturated fatty acid or ester thereof into hydroxy unsaturated fatty acid or ester thereof. In the following, microorganisms belonging to the genus Candida and microorganisms belonging to the genus Yarrowia are sometimes simply referred to as “microorganisms”.

  Examples of microorganisms belonging to the genus Candida include Candida maltosa, Candida succiphila, Candida pignariae, and Candida nitratofil. Among these, Candida maltosa is preferable because of its high ability to convert an unsaturated fatty acid or an ester thereof to a hydroxy unsaturated fatty acid or an ester thereof. These microorganisms belonging to the genus Candida may be wild strains or mutant strains.

  Among Candida maltosa, Candida maltosa KU83 strain or a mutant thereof has a higher conversion ability from unsaturated fatty acid or ester thereof to hydroxy unsaturated fatty acid or ester thereof. In addition, even when the amount of the hydroxy unsaturated fatty acid or its ester in the system is increased, it is more preferable because the growth is good. Further, among the KU83 strains, the Candida maltosa KU83-26 strain or a mutant thereof is more preferable.

  Candida maltosa KU83 strain is named and displayed as Candida maltosa KU83, date of acceptance: October 4, 2006, receipt number: NITE P-266, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Postal code: 292-0818, Kisarazu City, Chiba Prefecture Kazuka 2-5-8).

  Candida maltosa KU83-26 strain is named and displayed as Candida maltosa KU83-26. Date of entrustment: December 24, 2009, Receipt number: NITE AP-855 It is deposited at the Patent Microorganism Deposit Center (Zip code: 292-0818, Kisarazu City, Kazuka, Kamaka 2-5-8).

  Examples of microorganisms belonging to the genus Yarrowia include Yarrowia lypolytica. These microorganisms belonging to the genus Yarrowia may be wild strains or mutant strains.

  The medium used in the method of the present invention is not particularly limited as long as it contains an unsaturated fatty acid or an ester thereof and glucose.

  The concentration of the unsaturated fatty acid or ester thereof in the medium is not particularly limited as long as it is converted into the hydroxy unsaturated fatty acid or ester thereof by the action of the microorganism, and is, for example, about 0.1 to 100 g / L, preferably 0. About 5 to 50 g / L, more preferably about 0.5 to 10 g / L.

  As will be described later, the method of the present invention is characterized in that the glucose concentration in the medium is maintained in a specific concentration range. Therefore, the glucose concentration at the start of culture (glucose start concentration) is not particularly limited as long as the microorganism grows well, and is, for example, about 1 to 200 g / L, preferably about 40 to 120 g / L, more preferably 40 to It is about 80 g / L.

  In addition, the medium may contain other components as necessary from the viewpoint of improving the growth of microorganisms or increasing the conversion efficiency of unsaturated fatty acids or esters thereof to hydroxy unsaturated fatty acids or esters thereof. May be included.

Examples of other components include yeast extract, polypeptone, urea, manganese (II) chloride tetrahydrate (MnCl ₂ .4H ₂ O), magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O), iron sulfate (II) Heptahydrate (FeSO ₄ · 7H ₂ O), zinc sulfate heptahydrate (ZnSO ₄ · 7H ₂ O) and the like.

  Although it does not restrict | limit especially as a composition of a specific culture medium, For example, the yeast and mold culture medium (YM culture medium) is used as a basic culture medium, Furthermore, glucose is 30-200 g / L, and potassium dihydrogen phosphate is 0.5-50 g /. L, disodium hydrogen phosphate 0.5-50 g / L, magnesium sulfate heptahydrate 0.1-10 g / L, yeast extract 0.1-50 g / L, polypeptone 0.1-50 g / L L, urea 0.3 to 30 g / L, manganese (II) chloride tetrahydrate 0.001 to 0.1 g / L, iron (II) sulfate heptahydrate 0.001 to 0.1 g / L Examples thereof include a medium containing L and zinc sulfate heptahydrate at 0.001 to 0.1 g / L.

The pH of the medium is not particularly limited as long as the microorganism can grow well, and may be, for example, about 5 to 8, preferably about 6.5 to 7.5. Moreover, pH can be adjusted using potassium dihydrogen phosphate (KH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), or the like.

  The method for culturing the microorganism in the above medium is not particularly limited, and examples thereof include a method in which the above medium is used as a liquid medium and cultured by stirring, shaking, or the like.

  Moreover, culture conditions such as culture temperature and culture time are not particularly limited, and known culture conditions can be applied. For example, the culture may be performed at a culture temperature of about 20 to 37 ° C., preferably about 24 to 30 ° C., more preferably about 27 to 29 ° C., for about 12 to 120 hours, preferably about 24 to 60 hours.

  Furthermore, the method of the present invention is characterized in that the glucose concentration in the medium is maintained within a specific concentration range, and / or the medium contains an alcohol dehydrogenase inhibitor.

  The glucose concentration in the medium is maintained at about 1 to 20 g / L, preferably about 1 to 10 g / L, more preferably about 2 to 10 g / L, and particularly preferably about 2 to 6 g / L.

  Here, “maintaining the glucose concentration at X to Yg / L” means that the glucose concentration in the medium is adjusted to X to Yg / L from the start of the culture to the end of the culture. Therefore, during the culture, the glucose concentration in the medium may be temporarily less than X% or may exceed Y%.

  As the culture of microorganisms continues, the glucose concentration in the medium decreases, and a specific method for maintaining the glucose concentration in the above-described range includes, for example, a method of adding glucose to the medium. In this case, it may be added to the medium in a powder state, or a solution containing glucose may be prepared, and the solution may be added to the medium.

  In addition, the method for maintaining the glucose concentration is not particularly limited, but to give some specific examples, the glucose concentration in the medium is measured at regular intervals, or the glucose concentration is monitored over time, When the glucose concentration is less than the above-described concentration range, the method of adding glucose so as to be in the above-described concentration range, or when the glucose consumption rate of the microorganism is empirically known, from the consumption rate A method of estimating the time when the glucose concentration is less than the above-described concentration range and adding glucose every time is included. More specifically, when it is known that glucose is consumed in 12 hours, a method of adding glucose every 12 hours may be mentioned.

  The alcohol dehydrogenase inhibitor contained in the medium is not particularly limited as long as it is a substance having an inhibitory action on alcohol dehydrogenase. However, from the viewpoint of availability, 4-methylpyrazole, N-ethylmaleimide is used. It is preferable to use it.

  There is no particular limitation on the timing at which the alcohol dehydrogenase inhibitor is contained in the medium, and it may be added to the medium before the start of the culture or may be added to the medium during the culture. Especially, it is preferable to add to a culture medium before the culture | cultivation start. Further, the method for adding the alcohol dehydrogenase inhibitor is not particularly limited, and the alcohol dehydrogenase inhibitor may be added to the medium in a powder state, or a solution containing the alcohol dehydrogenase inhibitor is prepared, and the solution is added to the medium. It may be added.

  The concentration of the alcohol dehydrogenase inhibitor contained in the medium is not particularly limited as long as the alcohol dehydrogenase inhibitory action can be exerted, and is, for example, about 0.1 to 2 g / L, preferably 0.1 to 1 g / L. About L.

  In the method of the present invention, any one of maintaining the glucose concentration in the medium in a specific concentration range and containing the alcohol dehydrogenase inhibitor in the medium may be achieved. May be achieved.

  In this way, by maintaining the glucose concentration in the medium within a specific concentration range and / or by including an alcohol dehydrogenase inhibitor in the medium, the unsaturated dicarboxylic acid derived from the hydroxy unsaturated fatty acid or ester thereof by the microorganism can be obtained. Metabolism to acid can be suppressed.

  In the above-described method of the present invention, hydroxy unsaturated fatty acid or ester thereof is produced from unsaturated fatty acid or ester thereof by the action of microorganisms, and hydroxy unsaturated fatty acid or ester thereof accumulates in the medium. A hydroxy unsaturated fatty acid or ester thereof can be obtained by isolating or purifying the hydroxy unsaturated fatty acid or ester thereof from the medium.

  It does not restrict | limit especially as a method to obtain a hydroxy unsaturated fatty acid or its ester from a culture medium, What is necessary is just to carry out according to a conventional method. For example, subjecting it to a fractionation means such as HPLC using an appropriate eluent, monitoring the absorbance at a wavelength of 215 nm, and fractionating a fraction showing the same retention time as a standard of a hydroxy unsaturated fatty acid or its ester. Can isolate the hydroxy unsaturated fatty acid or its ester.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using a reference example and an Example, this invention is not limited to the following example. In addition, the culture medium used in the following reference examples and Examples 1 and 2 was previously sterilized at 120 ° C. for 15 minutes in an autoclave.

Reference Example YM broth (manufactured by BD Biosciences) 21 mL of YM medium dissolved in 1 L of distilled water was dispensed into a flask with a 300 mL baffle, and Candida maltosa KU83-26-2-2-106 (hereinafter referred to as “Candida maltosa”) And 0.25 mL of a cryopreserved bacterial cell suspension of KU-106) is inoculated on the YM medium and shaken at 28 ° C. under a stirring speed of 140 rpm for 24 hours. went.

  Next, 10 mL of trans-2-decenoic acid ethyl (2DAEt) previously autoclaved and 2500 mL of the LM modified medium described in Table 1 below were placed in a 5 L jar fermenter, and 120 mL of the preculture solution obtained above was added. After adjusting the pH to 7.5, the cells were cultured for 96 hours under the conditions of 28 ° C., a stirring speed of 350 rpm, and an aeration rate of 3.5 mL / min (1.44 vvm). An antifoaming agent was added when the culture solution foamed during the continuation of the culture. During the continuation of the culture, 10 mL of 2DAEt was added when it was confirmed that 2DAEt in the culture was almost consumed in the determination of ethyl trans-2-decenoate (2DAEt) described later.

After collecting 50 mL of the culture solution at each time point of 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours from the start of the culture and measuring the turbidity (OD ₆₆₀ ) of the culture solution, 14400 × g Centrifugation was performed for 5 minutes under the above conditions, and the supernatant was recovered. About the collect | recovered supernatant, the measurement of glucose concentration was performed using glucose CII-Test Wako (made by Wako Pure Chemical Industries Ltd.). Further, for the collected supernatant, according to the following method, ethyl trans-2-decenoate (2DAEt), 10-hydroxy-trans-2-decenoic acid (10HDA), and trans-2-decenedioic acid (2DDA) Quantification was performed.

<Quantification of 2DAEt, 10HDA and 2DDA>
After 5 mL of the supernatant collected above was adjusted to pH 2.0 with 6N HCl, 1 mL of acetophenone (5000 rpm) was added as an internal standard, and an equal amount of ethyl acetate was added and stirred for 1 minute for extraction. After the extraction operation was repeated twice, the extract was dried under reduced pressure, dissolved in 5 mL of methanol, filtered, and subjected to HPLC analysis. The conditions for HPLC analysis are shown in Table 2 below.

The measurement results of the turbidity (OD ₆₆₀ ) and glucose concentration of the culture solution at each time point described above, and the quantification results of 2DAEt, 10HDA and 2DDA are shown in FIG.

  As apparent from FIG. 1 (A), it was confirmed that the turbidity of the culture broth increased up to 72 hours. Although it is not necessarily clear, from FIG. 1 (B), since the glucose concentration was almost consumed at the time of 33 hours, the KU-106 strain continued to grow by consuming 2DAEt added additionally. Inferred.

  Further, as is clear from FIGS. 1C to 1E, it was confirmed that the peak area of 10HDA increased with a decrease in the peak area of 2DAEt from the start of the culture to 33 hours. Note that almost no increase in the peak area of 2DDA was observed until 33 hours after the start of culture. On the other hand, it was confirmed that the peak area of 10HDA decreased from around 33 hours when glucose was almost consumed, and the peak area was almost zero at 96 hours. Furthermore, it was confirmed that the peak area of 2DDA increases as the peak area of 10HDA decreases. From the results, it was suggested that 2DAEt was converted to 10HDA and the conversion from 10HDA to 2DDA was suppressed while glucose was present, but the conversion from 10HDA to 2DDA was promoted when glucose was depleted.

Example 1
In the same manner as in the above reference example, the glucose concentration in the culture solution at each time point of 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours from the start of the culture was measured, and the glucose concentration was less than 10 g / L. Except that glucose was added to the culture solution when it was confirmed that the turbidity (OD ₆₆₀ ) and glucose concentration of the culture solution were measured, and 2DAET, 10HDA and Quantification of 2DDA was performed. Glucose was added in an amount of 40 g after 24 hours, 120 g after 35 hours, 80 g after 48 hours, and 80 g after 74 hours, respectively. It maintained so that it might be in the range of about -20 g / L. The results are shown in FIG.

  As apparent from FIG. 2 (A), it was confirmed that the turbidity of the culture broth increased up to 48 hours and then gradually decreased.

  Further, as is clear from FIGS. 2C to 2E, it was confirmed that the peak area of 10HDA continuously increased from the start of the culture to 96 hours. Although a continuous increase in the peak area of 2DDA was confirmed from the start of culture to 96 hours, the degree of increase in the peak area of 2DDA was higher than that in the above Reference Example in which no additional glucose was added. It was confirmed to be much lower. From the above results, it was found that by maintaining the glucose concentration in the culture solution, the decrease in 10HDA can be suppressed, and further, the conversion from 10HDA to 2DDA can be suppressed.

Example 2
Cultivation was carried out in the same manner as in the above Reference Example except that an LM improved medium supplemented with 1 g / L of 4-methylpyrazole, an alcohol dehydrogenase inhibitor, was used, and the turbidity (OD ₆₆₀ ) and glucose concentration of the culture solution And 2DAEt, 10HDA and 2DDA were quantified. The results are shown in FIG.

  As is clear from FIG. 3 (A), it was confirmed that the turbidity of the culture broth increased up to 24 hours and then gradually decreased. Furthermore, when FIG. 1 (B) and FIG. 3 (B) were compared, it was confirmed that the consumption rate of glucose decreased by addition of 4-methylpyrazole.

  Further, as is clear from FIGS. 3C to 3D, it was confirmed that the peak area of 10HDA continuously increased from the start of the culture to 96 hours. Although a gradual increase in the peak area of 2DDA was confirmed from the start of the culture to 96 hours, the degree of increase in the peak area of 2DDA was much lower than that in the above Reference Example, and Example 1 It was confirmed that it was lower than the case of. From the above results, it was found that by adding an alcohol dehydrogenase inhibitor such as 4-methylpyrazole to the culture solution, the decrease of 10HDA can be suppressed, and further, the conversion from 10HDA to 2DDA can be suppressed.

  From the results of Examples 1 and 2 above, by maintaining the concentration of glucose in the culture solution within the range of 1 to 20 g / L, and / or by adding an alcohol dehydrogenase inhibitor to the culture solution, It has been found that hydroxy unsaturated fatty acids or esters thereof can be produced efficiently.

Claims (7)

A process for producing a hydroxy unsaturated fatty acid or ester thereof,
Culturing a microorganism belonging to the genus Candida or a microorganism belonging to the genus Yarrowia in a medium containing glucose and an unsaturated fatty acid or an ester thereof,
A method comprising maintaining a glucose concentration in the medium at 1 to 20 g / L, and / or containing an alcohol dehydrogenase inhibitor in the medium. The hydroxy unsaturated fatty acid or ester thereof is
Formula (I):
[In formula, R shows a hydrogen atom, an alkali metal, or a C1-C4 alkyl group, and n shows the integer of 1-14. ]
The method of Claim 1 which is a compound represented by these. The unsaturated fatty acid or ester thereof,
General formula (II):
[Wherein, R and n are the same as defined above. ]
The method of Claim 1 or 2 which is a compound represented by these. The microorganism belonging to the genus Candida is Candida maltosa, Candida succiphila, Candida pignariae, and at least one species selected from the group Candida nitratolato. The method according to claim 1, wherein The method according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Candida is a wild strain of Candida maltosa or a mutant thereof. The method according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Yarrowia is Yarrowia lypolytica. The method according to any one of claims 1 to 6, wherein the alcohol dehydrogenase inhibitor is 4-methylpyrazole and / or N-ethylmaleimide.