JP2018033433A - Strain belonging to mortierella, and oils and fats production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a strain belonging to Mortierella which is excellent in nervonic acid productivity, and to provide oils and fats production methods using the strain.SOLUTION: A strain belonging to Mortierella according to the invention is capable of producing 400 mg or more of nervonic acid per 1 L of culture medium as a constituent fatty acids of oils and fats, when 0.4 mg/ml of the strain is inoculated into the culture medium represented below and cultured at the conditions of 28°C and 215 rpm for 7 days: glucose 10% (w/v), yeast extract 0.4% (w/v), potato peptone 0.5% (w/v), Magnesium sulfate heptahydrate 0.00135% (w/v), and calcium chloride 0.00475% (w/v).SELECTED DRAWING: Figure 1

Description

  The present invention relates to a strain belonging to the genus Mortierella and a method for producing fats and oils.

  Nervonic acid accounts for 30 to 45% of the fatty acids constituting the myelin of vertebrates, and when the concentration of nervonic acid in the myelin is insufficient, a neurological disease called demyelinating disease is induced. For this reason, it is known that administration of nervonic acid and its derivatives is effective for the treatment of demyelinating diseases. In addition, nervonic acid has a high nutritional value. Therefore, it is preferred to use nervonic acid in the food and drink and pharmaceutical fields. Furthermore, since it is a constituent fatty acid of ceramide, application to cosmetic raw materials is also expected.

  Patent Document 1 discloses a method for producing an oil having nervonic acid in a constituent fatty acid by a bacterium belonging to the genus Mortierella. In particular, as a strain having a large production amount of nervonic acid, a strain having a deposit number of NITE P-1508 (storage number RD000969 which can be sold by NITE) is disclosed at the Patent Microorganism Depositary Center.

JP 2014-168418 A

  However, the strain with the accession number NITE P-1508 cannot be said to produce sufficient nervonic acid from the viewpoint of industrialization, and a strain with higher nervonic acid-producing ability is demanded.

  This invention is made | formed in view of the above situation, and it aims at providing the manufacturing method of the fats and oils which used the strain which belongs to the genus Mortierella excellent in the nervonic acid production ability, and this strain.

  The present inventors have found that a strain belonging to the genus Mortierella having high nervonic acid-producing ability can be obtained by a predetermined method, and have completed the present invention. More specifically, the present invention aims to provide the following.

(1) Medium shown below:
Glucose 10% (w / v)
Yeast extract 0.4% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
A strain belonging to the genus Mortierella capable of producing 400 mg or more of nervonic acid as a constituent fatty acid in fats and oils when inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 215 rpm for 7 days.

  (2) The strain according to (1), which can produce 750 mg or more of nervonic acid as a constituent fatty acid of fats and oils per liter of medium.

(3) Medium shown below:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
A strain belonging to the genus Mortierella capable of producing 1000 mg or more of nervonic acid as a constituent fatty acid in fats and oils when inoculated with 0.4 mg / ml of strain and cultured for 14 days at 28 ° C. and 215 rpm.

(4) Medium shown below:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
A strain belonging to the genus Mortierella capable of producing 1000 mg or more per liter of nervonic acid as a constituent fatty acid when inoculated with 0.4 mg / ml of strain and cultured for 14 days at 28 ° C. and 180 rpm.

  (5) Fatty acid elongation activity is 700% or more with respect to Mortierella capitata RD000969 strain deposited at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center under the accession number NITE P-1508 The strain according to any one of (1) to (4).

  (6) Mortierella capitata RD000969 strain in which the desaturation activity at the Δ9 position of the fatty acid component of fats and oils has been deposited under the deposit number NITE P-1508 at the National Institute of Technology and Technology Patent Microorganisms The strain according to any one of (1) to (5), which is 200% or more.

  (7) The strain according to any one of (1) to (6), which is a strain having cerulenin resistance.

  (8) A strain of Mortierella capitata deposited with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, NITE P-02299, NITE P-02300, or NITE P-02301, The strain according to any one of (1) to (7).

(9) A method for producing fats and oils having nervonic acid as a constituent fatty acid,
Culturing the strain according to any one of (1) to (8);
And a step of recovering fats and oils from the cultured cells.

  According to the present invention, a strain belonging to the genus Mortierella having high nervonic acid-producing ability can be obtained.

It is a flow of elongation and desaturation of fatty acids constituting the oils and fats of Mortierella capitata.

  Hereinafter, although embodiment of this invention is described, this invention is not specifically limited to this.

<Strain belonging to the genus Mortierella>
The strain belonging to the genus Mortierella of the present invention produces a large amount of nervonic acid as a constituent fatty acid of fats and oils. Although the strain described in Patent Document 1 did not have sufficient ability to produce fats and oils in the cells, the strain belonging to the genus Mortierella of the present invention can produce many fats and oils in the cells. The fats and oils that can be produced by the strain of the present invention refers to fats and oils that are produced in the cells. Hereinafter, the strain of the present invention will be described.

The strain belonging to the genus Mortierella of the present invention is a medium shown below:
Glucose 10% (w / v)
Yeast extract 0.4% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
Inoculated with 0.4 mg / ml of strain and cultured for 7 days at 28 ° C. and 215 rpm (hereinafter, this culture condition may be referred to as “culture condition (a)” in this specification). Is a strain capable of producing 400 mg or more of nervonic acid as a constituent fatty acid per liter of medium.

  The culture medium in the culture condition (a) was glucose 10% (w / v), yeast extract 0.4% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0 .00135% (w / v) and calcium chloride 0.00475% (w / v) dissolved in water (hereinafter sometimes referred to as “medium (A)”). In the present specification, “% (w / v)” means a component contained in 100 mL of the medium (in the medium (A), glucose, yeast extract, potato peptone, magnesium sulfate heptahydrate, calcium chloride. ) Mass (g).

  In the culture condition (a), the amount (mg / ml) of the strain inoculated on the medium (A) is a numerical value converted to the dry cell weight and is a weight relative to the volume (ml) of the medium (A). The strain to be inoculated into the medium (A) can be obtained by a known method. For example, the strain can be prepared by pre-culturing or further pre-culturing in the following procedure.

  First, a frozen stock (an agar medium containing bacterial cells) is transplanted into a thinly spread potato dextrose medium (commercial product, solid medium containing agar), and cultured at 28 ° C. in advance. After the pre-culture, bacteria that spread in a flower shape are collected with a straw having a diameter of 8 mm, and a medium for pre-culture (potato peptone: 0.5% (w / v), glucose: 3% (w / v), yeast Extract: 0.1% (w / v)) Inoculated into 200 ml (conical flask with 500 ml capacity baffle), pre-cultured at 140 rpm, 28 ° C. for 2 days, and inoculated into the medium (A). .

  In the culture condition (a), 200 ml of the medium (A) is used. In the culture medium (A), the strain may be inoculated to an amount such that the dry cell weight is 0.4 mg / ml and finally adjusted to 200 ml. As the culture vessel, a conical flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple constant temperature shaker TAL-RS310, manufactured by Thomas Scientific Instruments Co., Ltd.) at 28 ° C. and 215 rpm.

  The fats and oils produced by the strain of the present invention are those in which nervonic acid as a constituent fatty acid of fats and oils is produced under the culture conditions (a). The fats and oils produced under the culture condition (a) are not particularly limited as long as nervonic acid as a constituent fatty acid has an amount of 400 mg or more per liter of the medium. For example, other constituent fatty acids As, it may have eicosenoic acid, hexacosenoic acid and the like.

  The strain of the present invention is not particularly limited as long as it can produce 400 mg or more of nervonic acid as a constituent fatty acid of fat / oil in culture conditions (a), but 500 mg of nervonic acid as a constituent fatty acid of oil / fat per liter of medium. More preferably, it can be produced more preferably 550 mg or more, more preferably 600 mg or more can be produced, still more preferably 700 mg or more can be produced, and 750 mg or more can be produced. It is particularly preferred. On the other hand, the strain of the present invention may be a bacterium capable of producing 1000 mg or less of nervonic acid as a constituent fatty acid of fats and oils per liter of culture medium under culture conditions (a).

  In the culturing condition (a) of the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is preferably 20.0 mg or more per 1 g of microbial cells (dry microbial cell weight). Preferably, it is 30.0 mg or more, and particularly preferably 35.0 mg or more. In addition, in the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is 100.0 mg or less (90.0 mg or less, 80 or less per 1 g (dry cell weight)) in the culture condition (a). 0.0 mg or less, 70.0 mg or less, 60.0 mg or less, 50.0 mg or less, etc.).

  The strain of the present invention is particularly useful in that respect because the proportion of nervonic acid in the constituent fatty acid in the fat increases. In this respect, in the culture condition (a), the proportion of nervonic acid in the constituent fatty acids of the fat and oil is preferably 5.0% by mass or more with respect to the total constituent fatty acids of the fat and oil, and 5.5% by mass or more. It is preferable that it is 6.0 mass% or more, and it is especially preferable that it is 7.0 mass% or more. In addition, in the culture condition (a), the strain of the present invention is such that the ratio of nervonic acid in the fatty acid constituent fatty acid is 20.0 mass% or less (18.0 mass% or less, 16.0% by mass or less, 14.0% by mass or less, 13.0% by mass or less, 12.0% by mass or less).

The strain of the present invention includes the following media:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
Inoculated with 0.4 mg / ml of the strain and cultured for 14 days at 28 ° C. and 215 rpm (hereinafter, this culture condition may be referred to as “culture condition (b)” in this specification). A strain belonging to the genus Mortierella capable of producing 1000 mg or more of nervonic acid as a constituent fatty acid per liter of the medium may be used.

  The culture medium in the culture condition (b) was glucose 12% (w / v), yeast extract 0.8% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0 .00135% (w / v) and calcium chloride 0.00475% (w / v) dissolved in water (hereinafter sometimes referred to as “medium (B)”). Moreover, under culture conditions (b), 70 ml of a 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have elapsed since the start of the culture. This “culture start” refers to the time when 0.4 mg / ml of the strain was inoculated into the medium (B).

  In the culture condition (b), the amount (mg / ml) of the strain inoculated on the medium (B) is a numerical value converted to the dry cell weight and is a weight relative to the volume (ml) of the medium (B). The strain inoculated into the medium (B) can be obtained by the same method as in the culture condition (a).

  In the culture condition (b), 200 ml of the medium (B) is used. In the culture medium (B), the strain may be inoculated to an amount such that the dry cell weight is 0.4 mg / ml, and finally 200 ml may be prepared. As the culture vessel, a conical flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple constant temperature shaker TAL-RS310, manufactured by Thomas Scientific Instruments Co., Ltd.) at 28 ° C. and 215 rpm.

  The fats and oils produced by the strain of the present invention may be those that produce nervonic acid as a constituent fatty acid of the fats and oils under the culture conditions (b). The fats and oils produced under the culture conditions (b) are not particularly limited as long as nervonic acid as a constituent fatty acid has an amount of 1000 mg or more per 1 L of the medium. For example, other constituent fatty acids As, it may have eicosenoic acid, hexacosenoic acid and the like.

  The strain of the present invention is not particularly limited as long as it can produce 1000 mg or more of nervonic acid as a constituent fatty acid of fat / oil in culture conditions (b), but 1050 mg of nervonic acid as a constituent fatty acid of oil / fat per liter of culture medium. More preferably, it is possible to produce 1100 mg or more, more preferably 1200 mg or more can be produced, still more preferably 1300 mg or more can be produced, and 1400 mg or more can be produced. It is particularly preferred. On the other hand, the strain of the present invention is a bacterium capable of producing 2000 mg or less (1900 mg or less, 1800 mg or less, 1700 mg or less, 1600 mg or less, etc.) of nervonic acid as a constituent fatty acid in fats and oils in culture conditions (b). Also good.

  In the culture condition (b) of the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is preferably 25.0 mg or more per 1 g of microbial cells (dry cell weight), and 28.0 mg The above is preferable, and 33.0 mg or more is particularly preferable. In addition, in the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is 100.0 mg or less (90.0 mg or less, 80 or less per 1 g of cells (dry cell weight) under the culture conditions (b). 0.0 mg or less, 70.0 mg or less, 60.0 mg or less, 50.0 mg or less, etc.).

  The strain of the present invention is particularly useful in that respect because the proportion of nervonic acid in the constituent fatty acid in the fat increases. In this respect, in the culture condition (b), the proportion of nervonic acid in the constituent fatty acids of the fats and oils is preferably 5.5% by mass or more based on the total constituent fatty acids of the fats and oils, and is 5.6% by mass or more. It is preferable that it is 5.8 mass% or more, and it is especially preferable that it is 6.0 mass% or more. Moreover, the strain of this invention WHEREIN: In culture conditions (b), the ratio of the nervonic acid in the fatty-acid constituent fatty acid is 15.0 mass% or less (14.0 mass% or less, 13.0% by mass or less, 12.0% by mass or less, 11.0% by mass or less, 10.0% by mass or less, and the like.

The strain of the present invention includes the following media:
Medium shown below:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
0.4 mg / ml of the strain was inoculated and cultured at 28 ° C. and 180 rpm for 14 days (hereinafter, this culture condition may be referred to as “culture condition (c)” in this specification). The strain which belongs to the genus Mortierella which can produce 1000 mg or more per liter of nervonic acid as a constituent fatty acid of fats and oils may be used.

  The culture medium in the culture condition (c) is glucose 12% (w / v), yeast extract 0.8% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0 .00135% (w / v) and calcium chloride 0.00475% (w / v) dissolved in water (hereinafter referred to as “medium (C)” in this specification). Moreover, in culture condition (c), 70 ml of 60% (w / v) aqueous glucose solution is added per 1 L of the medium when 10 days have elapsed since the start of the culture. This “culture start” refers to the time when 0.4 mg / ml of the strain was inoculated into the medium (C).

  In the culture condition (c), the amount (mg / ml) of the strain inoculated on the medium (C) is a numerical value converted to the dry cell weight and is a weight relative to the volume (ml) of the medium (C). The strain inoculated into the medium (C) can be obtained by the same method as in the culture condition (a).

  In the culture condition (c), 200 ml of the medium (C) is used. In the medium (C), the strain may be inoculated to an amount such that the dry cell weight is 0.4 mg / ml, and finally 200 ml may be prepared. As the culture vessel, a conical flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple constant-temperature shake incubator TAL-RS310, manufactured by Thomas Scientific Instruments Co., Ltd.) under the conditions of 28 ° C. and 180 rpm.

  The fats and oils produced by the strain of the present invention may be those that produce nervonic acid as a constituent fatty acid of the fats and oils under the culture conditions (c). The fats and oils produced under the culture condition (c) are not particularly limited as long as nervonic acid as a constituent fatty acid has an amount of 1000 mg or more per liter of the medium. For example, other constituent fatty acids As, it may have eicosenoic acid, hexacosenoic acid and the like.

  The strain of the present invention is not particularly limited as long as it can produce 1000 mg or more of nervonic acid as a constituent fatty acid of fat / oil in culture conditions (c), but 1100 mg of nervonic acid as a constituent fatty acid of oil / fat per liter of medium. More preferably, it can be produced, more preferably 1200 mg or more can be produced, still more preferably 1300 mg or more can be produced, still more preferably 1400 mg or more can be produced, even more preferably 1500 mg or more can be produced. It is particularly preferred. On the other hand, the strain of the present invention may be a bacterium capable of producing 2000 mg or less (1900 mg or less, 1800 mg or less, 1700 mg or less, etc.) of nervonic acid as a constituent fatty acid of fats and oils per liter of culture medium under culture conditions (c).

  In the culturing condition (c) of the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is preferably 20.0 mg or more per 1 g of microbial cells (dry microbial cell weight). Preferably, it is 35.0 mg or more, and particularly preferably 39.0 mg or more. In addition, in the strain of the present invention, the production amount of nervonic acid as a constituent fatty acid of fats and oils is 100.0 mg or less (90.0 mg or less, 80 or less per 1 g (dry cell weight)) in the culture condition (c). 0.0 mg or less, 70.0 mg or less, 60.0 mg or less, 50.0 mg or less, etc.).

  The strain of the present invention is particularly useful in that respect because the proportion of nervonic acid in the constituent fatty acid in the fat increases. In this respect, in the culture condition (c), the proportion of nervonic acid in the constituent fatty acids of the fat and oil is preferably 5.0% by mass or more with respect to the total constituent fatty acids of the fat and oil, and 5.5% by mass or more. It is preferable that it is 6.0 mass% or more, and it is especially preferable that it is 6.6 mass% or more. Moreover, the strain of this invention WHEREIN: In culture conditions (c), the ratio of the nervonic acid in the fatty-acid constituent fatty acid is 15.0 mass% or less (14.0 mass% or less, 13.0% by mass or less, 12.0% by mass or less, 11.0% by mass or less, 10.0% by mass or less, and the like.

  Since the strain of the present invention has high fatty acid elongation activity, it is considered to have high nervonic acid production ability. Therefore, it is preferable that the strain has a high fatty acid elongation activity. In the present invention, “fatty acid elongation activity” specifically means that the expression level of a gene that extends the carbon chain of a constituent fatty acid of fats and oils is large, and more specifically, a constituent fatty acid having 16 carbon atoms (palmitin). This means that the expression level of a gene encoding an enzyme that extends an acid to a constituent fatty acid having 18 carbon atoms (stearic acid) is high. In this respect, the strain of the present invention is higher in fatty acid elongation than the fatty acid elongation activity of Mortierella capitata RD000969 strain deposited at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center under the accession number NITE P-1508. More specifically, the expression level of the gene (SEQ ID NO: 1) encoding the enzyme having the fatty acid elongation activity in the RD000969 strain (the fatty acid elongation activity of the RD000969 strain) is 100%. Sometimes, the expression level of the corresponding gene (for example, SEQ ID NO: 1) in the strain of the present invention (fatty acid elongation activity of the strain of the present invention) has a fatty acid elongation activity of more than 100% relative to the fatty acid elongation activity of the RD000969 strain. Preferably 300% It is more preferable to have the above fatty acid elongation activity, more preferably 500% or more fatty acid elongation activity, still more preferably 700% or more fatty acid elongation activity, It is even more preferable that the fatty acid elongation activity is 900% or more, and it is particularly preferable that the fatty acid elongation activity is 1200% or more. In addition, the strain of the present invention is 1700% or less (1600% or less, 1500% or less) with respect to the fatty acid elongation activity of the RD000969 strain when the expression level of the gene of SEQ ID NO: 1 of the RD000969 strain is 100%. 1400% or less) or the like.

  Further, the fatty acid elongation activity of the strain of the present invention is (1) in addition to the activity for the expression level of SEQ ID NO: 1 described above under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 1. DNA having a base sequence capable of hybridizing, (2) having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence of SEQ ID NO: 2, and DNA having fatty acid elongation activity, (3) amino acid sequence having 85% or more (90% or more, 94% or more, 95% or more, 97% or more, 99% or more) homology with the amino acid sequence of SEQ ID NO: 2 It is preferable that the activity is related to the expression level of any one of DNAs having a fatty acid elongation activity.

  Since the strain of the present invention also has high desaturation activity at the Δ9 position of the constituent fatty acids of fats and oils, it is considered that the nervonic acid producing ability is high. Therefore, it is preferable that the strain has a high desaturation activity at the Δ9 position. In the present invention, “the desaturation activity at the Δ9 position” specifically refers to a gene that converts a saturated fatty acid (stearic acid) among the constituent fatty acids of fats and oils to an unsaturated fatty acid (oleic acid) that is unsaturated. It means that there is much expression level. In this respect, the strain of the present invention is higher than the fatty acid desaturation activity of Mortierella capitata RD000969 strain deposited at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center under the accession number NITE P-1508. It is preferable that it has a desaturation activity at the Δ9 position. More specifically, the expression level of the gene (SEQ ID NO: 3) encoding the enzyme having the desaturation activity at the Δ9 position in the RD000969 strain (RD000969 strain) When the expression level of the corresponding gene (for example, SEQ ID NO: 3) in the strain of the present invention (the desaturation activity at the Δ9 position of the strain of the present invention) is 100%, It has a desaturation activity at the Δ9 position exceeding 100% with respect to the desaturation activity at the Δ9 position. More preferably 120% or more of Δ9-position desaturation activity, more preferably 150% or more of Δ9-position desaturation activity, more preferably 200% It is more preferable to have the above-mentioned desaturation activity at the Δ9 position, even more preferable to have a desaturation activity at the Δ9 position of 300% or more, and a Δ9 position of 350% or more. It is particularly preferred that it has a saturation activity. In addition, the strain of the present invention is 1000% or less with respect to the desaturation activity at the Δ9 position of the RD000969 strain when the expression level of the gene of SEQ ID NO: 3 of the RD000969 strain is defined as the desaturation activity at the Δ9 position of 100%. It may have fatty acid desaturation activity (900% or less, 800% or less, 700% or less, 600% or less, 500% or less, etc.).

  In addition, the desaturation activity at the Δ9 position of the strain of the present invention includes (1) a base sequence complementary to the base sequence described in SEQ ID NO: 3 and a stringent in addition to the activity regarding the expression level of SEQ ID NO: 3 above. DNA having a base sequence capable of hybridizing under various conditions, (2) having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 4 And (3) the amino acid sequence set forth in SEQ ID NO: 4 and 85% or more (90% or more, 94% or more, 95% or more, 97% or more, 99% or more) It is preferable that the activity is related to the expression level of any one of the DNAs having a base sequence encoding an amino acid sequence having homology (2) and having desaturation activity at the Δ9 position.

  In the present invention, as "stringent conditions", for example, the reaction is performed in a normal hybridization buffer at 40 to 70 ° C (preferably 50 to 67 ° C, more preferably 60 to 65 ° C), Examples include conditions for washing in a washing solution having a salt concentration of 15 to 300 mM (preferably 15 to 150 mM, more preferably 15 to 60 mM, and still more preferably 30 to 50 mM).

  In the present invention, “one or more” is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 1 amino acid). . When maintaining the fatty acid elongation activity, the amino acid residue to be mutated is desirably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, as the properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acids and amide-containing side chains (D, N, E, Q), amino groups having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses represent single letter amino acids).

  In the present invention, homology between amino acid sequences and base sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When a base sequence is analyzed by BLASTN based on BLAST, parameters are set to score = 100 and wordlength = 12, for example. When an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

  Since the strain of the present invention tends to have a high ability to produce nervonic acid, it is preferably a strain having cerulenin resistance.

  Examples of the strain belonging to the genus Mortierella of the present invention include, for example, Mortierella capitata, Mortierella elongata, Mortierella exigua, Mortierella higroll, Mortierella alpina, Mortierella parvispora, Mortierella beljakova, Mortierella globa, Mortierella globa rtierella epigama), Mortierella kuhlmanii, Mortierella acrotona, Mortierella zychae, Mortierella zychae, Mortierella zychae, Mortierella zychae Mention may be made of Rella Schmuckeri (Mortierella schmuckeri) and the like. Of these, Mortierella capitata is particularly preferable because of its high ability to produce nervonic acid. Among Mortierella capitata, in particular, an independent administrative agency, Product Evaluation Technology Base Organization, Patent Microorganism Depositary Center has accession number NITE P-02299, The strain of Mortierella capitata deposited as NITE P-02300 or NITE P-02301 is preferred.

  The amount of nervonic acid in the present invention means the amount of nervonic acid produced in the cells. The amount of nervonic acid (the amount of nervonic acid as a constituent fatty acid in the fat or oil) is measured by a gas chromatograph mass spectrometer.

  In the present invention, a strain belonging to the genus Mortierella capable of producing 400 mg or more of nervonic acid per liter of the medium under the above-described culture condition (a), and Mortierella genus capable of producing 1000 mg or more of nervonic acid per liter under the culture conditions (b). A strain belonging to the genus Mortierella capable of producing 1000 mg or more of nervonic acid per liter according to the strain and culture conditions (c) can be produced by the following method.

  Mortierella capitata RD000969 strain is inoculated into 5 ml of potato dextrose liquid medium and cultured with shaking at 28 ° C. for 2 days, for example. Bacteria are collected from this culture solution by filtration, a buffer solution is added, and ultrasound (for example, frequency 20 kHz, amplitude 40 μm) is irradiated for 2 minutes to prepare a mycelium suspension. For example, after adding 1 ml of NTG solution to the suspension and allowing to stand at 28 ° C. for 15 minutes, the cells are collected by centrifugation, washed with distilled water, and suspended in 1 ml of distilled water. This suspension may be applied to a potato dextrose medium supplemented with cerulenin to isolate cerulenin resistant bacteria, which may be used as the strain of the present invention.

  Furthermore, it is preferable to perform a mutation treatment by irradiating ultraviolet rays (253 nm) with respect to the separated cerulenin-resistant bacteria. Specifically, the isolated cerulenin-resistant bacterium is inoculated into 15 ml of potato dextrose liquid medium and cultured with shaking at 28 ° C. for 2 days, for example. Bacteria are collected from the culture solution by filtration, washed, added with a buffer solution, and irradiated with ultrasonic waves (for example, frequency 20 kHz, amplitude 40 μm) to prepare a mycelium suspension. After the suspension is transferred to a petri dish and irradiated with an ultraviolet lamp, 50 μl is applied to a potato dextrose medium supplemented with 0.04% cerulenin to isolate cerulenin-resistant bacteria. Thus, by selecting a cerulenin resistant strain, the strain of the present invention having a high ability to produce nervonic acid can be obtained.

  In addition, it can be confirmed by cultivating by the said culture condition (a) and measuring the quantity of nervonic acid whether the obtained strain is the above-mentioned strain.

<Oil and fat manufacturing method>
The method for producing fats and oils of the present invention is a method for producing fats and oils having nervonic acid as a constituent fatty acid, the step of culturing the strain belonging to the genus Mortierella, and the step of recovering fats and oils from the cultured cells And having.

  As the strain belonging to the genus Mortierella, the above-described strain of the present invention can be used.

  The method for culturing a strain belonging to the genus Mortierella is not particularly limited. For example, a preculture solution obtained by culturing in advance may be inoculated into a liquid medium and cultured. Alternatively, strains (spores, mycelia, etc.) that have been cultured in a solid medium may be inoculated into a liquid medium and cultured.

  The medium used in the medium of the present invention is not particularly limited as long as it can grow a strain belonging to the genus Mortierella. Specific carbon sources include, for example, glucose, sucrose, fructose, xylose, mannose, soluble Starch, glycerol, mannitol and the like can be used. Of these, glucose is preferred. Moreover, it is preferable that the density | concentration of a carbon source is 5-15% (w / v), and it is preferable that glucose is contained at 5-15% (w / v).

  The medium preferably further contains a nitrogen source. The nitrogen source is not particularly limited, and for example, yeast extract, malt extract, meat extract, peptone, casamino acid, corn steep liquor and the like can be used. Especially, since the effect which raises the density | concentration of the microbial cell per culture solution is large, it is preferable to use a yeast extract and peptone, and it is preferable to use both a yeast extract and peptone. In particular, the concentration of nitrogen source is preferably 0.1 to 1.0% (w / v), in particular, yeast extract 0.1 to 1.0% (w / v), potato peptone 0.1 to It is preferably 1.0% (w / v).

  Other components include magnesium salts (magnesium sulfate, heptahydrate, etc.), calcium salts (calcium chloride, etc.), phosphates (potassium phosphate, etc.), iron salts (iron sulfate, etc.), copper salts It is preferable to include an isotonic agent such as an inorganic salt such as (copper sulfate) or sodium chloride. Regarding inorganic salts (especially magnesium sulfate and calcium chloride), magnesium sulfate is preferably 0.00001 to 0.01000% (w / v), and calcium chloride is 0.00001 to 0.02000% (w / v). It is preferable that

  In the medium, fatty acids or fats and oils containing fatty acids may be used as a substrate. The fatty acid is not particularly limited, and examples thereof include palmitic acid, stearic acid, oleic acid, arachidic acid, eicosenoic acid, behenic acid, erucic acid, and lignoceric acid. Among them, erucic acid is preferable because it has a large effect of increasing the concentration of nervonic acid in the oil and fat composition. Examples of the oils and fats include olive oil, palm oil, and rapeseed oil. Of these, rapeseed oil is preferred because of its high erucic acid content.

  Moreover, the well-known culture medium used for the strain which belongs to the conventional Mortierella genus may be used, Czapek culture medium, Czapek-dox culture medium, PD (potato dextrose) culture medium, etc. can be used.

  The culture temperature is not particularly limited as long as it is a temperature at which a strain belonging to the genus Mortierella can grow. For example, it may be 5 ° C or higher, 10 ° C or higher, 20 ° C or higher, 50 ° C or lower, 40 ° C or lower, It may be 30 ° C. or lower.

  The pH of the medium is not particularly limited as long as it is a pH at which a strain belonging to the genus Mortierella can grow. For example, it may be 3 or more, 4 or more, 5 or more, 12 or less, 11 or less, 10 or less. It may be.

  The culture method is not particularly limited as long as a strain belonging to the genus Mortierella can grow. For example, the culture can be performed under stirring conditions, shaking culture, stationary culture, etc. . In the case of shaking culture, the number of rotations of shaking is not particularly limited as long as it is a method capable of growing a strain belonging to the genus Mortierella, but it is preferably performed at 150 to 250 rpm.

  The culture time of the strain belonging to the genus Mortierella may be determined according to the desired amount of nervonic acid, for example, may be within the range of 10 hours to 30 days, preferably 7 days or more, It is preferable that it is 10 days or more. In particular, when culturing for 7 days or more, it is preferable to add an aqueous glucose solution during the cultivation. Further, since the production amount of nervonic acid starts to decrease, the culture time is preferably within 30 days, and more preferably within 25 days.

  The method for recovering fats and oils from the cultured cells can be performed by a conventionally known method. For example, the cells may be first separated and collected from the culture solution by centrifugation, filtration, etc., and the fats and oils having nervonic acid as a constituent fatty acid are extracted from the cells with normal hexane.

  The production method of the present invention may further include a step of separating nervonic acid from fats and oils. Separation of nervonic acid from fats and oils can be performed by known methods such as solvent fractionation and preparative HPLC.

  In addition, as for manufacture of fats and oils, you may use a well-known method as it described in Unexamined-Japanese-Patent No. 2014-168418.

  Nervonic acid can be separated from the fats and oils produced by the strain of the present invention and used in foods and drinks and pharmaceuticals.

<Production of mutant strain>
According to NTG (N-methyl-N'-nitro-N-nitrosoguanidine) against Mortierella capitata RD000969 strain deposited with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation of the National Institute for Product Evaluation as NITE P-1508 Mutation treatment was performed.

  First, the cells were inoculated into 5 ml of potato dextrose liquid medium and cultured with shaking at 28 ° C. for 2 days. Bacteria were collected from this culture by filtration, washed with 50 mM trismaleic acid buffer (pH 7.5), 1 ml of 100 mM trismaleic acid buffer (pH 7.5) was added, and ultrasonic waves (frequency 20 kHz, (Amplitude 40 μm) was irradiated for 2 minutes to prepare a mycelial suspension. After adding 1 ml of NTG solution (40 μg / ml) to the suspension and allowing to stand at 28 ° C. for 15 minutes, the cells were collected by centrifugation, washed with distilled water, and suspended in 1 ml of distilled water. 50 μl of this suspension was applied to a potato dextrose medium supplemented with 0.04% cerulenin to isolate two strains (M209 strain, M324 strain) of cerulenin-resistant bacteria. The M209 strain was used as Example 1, and the M324 strain was used as the strain according to Example 2. The accession number NITE P-02299 for the M209 strain and the accession number for the M324 strain are NITE P-02300.

  The strain according to Example 2 was further subjected to mutation treatment by ultraviolet irradiation (253 nm). The cerulenin-resistant bacterium was inoculated into 15 ml of potato dextrose liquid medium and cultured with shaking at 28 ° C. for 2 days. Bacteria were collected from this culture by filtration, washed with 50 mM trismaleic acid buffer (pH 7.5), 1 ml of 100 mM trismaleic acid buffer (pH 7.5) was added, and ultrasonic waves (frequency 20 kHz, (Amplitude 40 μm) was irradiated for 2 minutes to prepare a mycelial suspension. The suspension was transferred to a petri dish, and an ultraviolet lamp was irradiated for 22 minutes from a location 30 cm away, and then 50 μl was applied to a potato dextrose medium supplemented with 0.04% cerulenin to obtain a cerulenin resistant strain (M463 strain). separated. The M463 strain was used as the strain according to Example 3. The accession number of the M463 strain is NITE P-02301.

<Manufacture of fats and oils>
(Pre-culture, pre-culture)
As Comparative Example 1, the RD000969 strain was prepared, and as the Comparative Example 2, the NBRC102984 strain (Pareichytrium sp.) Was prepared. The cells of Comparative Examples 1 and 2 and the cells of Examples 1 to 3 were pre-cultured and pre-cultured.

  First, frozen stocks of each strain (an agar medium containing bacterial cells) were transplanted, and cultured in advance on a potato dextrose medium (commercially available agar) at 28 ° C. while monitoring the growth status. After pre-culture, bacteria that spread in a flower shape were collected with a straw having a diameter of 8 mm, and a liquid medium for preculture (potato peptone: 0.5% (w / v), glucose: 3% (w / v), Yeast extract: 0.1% (w / v)) In addition to 200 ml (flask with 500 ml capacity baffle), pre-culture was performed at 140 rpm and 28 ° C. for 2 days.

(Main culture)
After pre-culture, 10 ml of the bacterial cells according to Examples 1 to 3 and Comparative Examples 1 and 2 were collected (including the culture solution), and 10 ml of the bacterial cells were added to 190 ml of the medium to make a total of 200 ml. Inoculated to 4 mg / ml and main culture was performed under the following culture conditions (a).

[Culture conditions (a)]
Medium: glucose 10% (w / v), yeast extract 0.4% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0.00135% (w / v ),
Calcium chloride 0.00475% (w / v)
Capacity: 200ml (500ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 215 rpm (rotary shaker (vertical triple constant temperature shaking incubator TAL-RS310, manufactured by Thomas Scientific Instruments))
Culture period: 7 days

  For the strains according to Examples 2 and 3 and Comparative Example 1, after preculture, 10 ml of the bacterial cells were collected (including the culture solution), and 10 ml of the bacterial cells were added to 190 ml of the medium to make a total of 200 ml, and the dry cell weight Was inoculated to 0.4 mg / ml and main culture was performed under the following culture conditions (b).

[Culture conditions (b)]
Medium: glucose 12% (w / v), yeast extract 0.8% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0.00135% (w / v )
Calcium chloride 0.00475% (w / v)
(When 10 days have elapsed from the start of the culture, a 60% (w / v) aqueous glucose solution added at 70 ml per 1 L of the medium)
Capacity: 200ml (500ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 215 rpm (rotary shaker (vertical triple constant temperature shaking incubator TAL-RS310, manufactured by Thomas Scientific Instruments))
Culture period: 14 days

  For the strain according to Example 2, after pre-culture, 10 ml of the microbial cells are collected (including the culture solution), and 10 ml of the microbial cells are added to a 190 ml medium to make a total of 200 ml, and the dry microbial weight is 0.4 mg / ml. The main culture was performed under the following culture conditions (c).

[Culture conditions (c)]
Medium: glucose 12% (w / v), yeast extract 0.8% (w / v), potato peptone 0.5% (w / v), magnesium sulfate heptahydrate 0.00135% (w / v ),
Calcium chloride 0.00475% (w / v)
(When 10 days have passed since the start of the culture, 60% (w / v) aqueous glucose solution was added to 1 L of the medium.
70ml added)
Capacity: 200ml (500ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 180 rpm (rotary shaker (vertical triple constant temperature shake incubator TAL-RS310, manufactured by Thomas Scientific Instruments))
Culture period: 14 days

<Measurement of the amount of nervonic acid>
The amount of nervonic acid was measured by the following method.

  First, after the main culture, the cells were collected by filtration and lyophilized. 3 ml of a 2M hydrochloric acid methanol solution was added to 30 mg of dried cells and heated at 100 ° C. for 2 hours to methylate the cell fatty acids. After washing the reaction solution with 1 ml of distilled water, fatty acid methyl ester was extracted with 6 ml of normal hexane, and the amount of nervonic acid was measured with a gas chromatograph mass spectrometer (TSQ8000, Thermo Fisher Scientific).

<Measurement results>
The culture results are shown in Tables 1 to 3 below. The nervonic acid production amount in Tables 1 to 3 is the amount of nervonic acid (the amount of nervonic acid as a constituent fatty acid in fats and oils) contained in the cells after the main culture. Further, “ratio (%) of nervonic acid in lipid” means the ratio (% by mass) of nervonic acid as a constituent fatty acid in fats and oils recovered from the cells. Table 1 shows the results of the main culture performed under the culture conditions (a), Table 2 shows the results of the main culture performed under the culture conditions (b), and Table 3 shows the results of the main culture performed under the culture conditions (c). It is the result of having gone.

  As shown in Tables 1 to 3, it was found that the strains according to Examples 1 to 3 had higher nervonic acid production than the RD000969 strain of Comparative Example 1, and also increased the proportion of nervonic acid in the lipid. .

<Analysis of gene expression level>
RNA extraction was performed for the strain according to Example 1 and the strain according to Comparative Example 1, and the amino acid sequence encoded by the expressed gene was searched for homology with known fatty acid synthases shown in Table 4 below. .

  As a result of the homology search, the amino acid sequence (SEQ ID NO: 2) encoded by the TR3595 gene (SEQ ID NO: 1) of Example 1 and Comparative Example 1 had 89% homology with the amino acid sequence of MALCE1p in Table 4. Moreover, as a result of analysis, it was found that it belonged to the same phylogenetic branch as MALCE1p and had a transmembrane mode similar to MALCE1p. Further, the amino acid sequence (SEQ ID NO: 4) encoded by the TR13919 gene (SEQ ID NO: 3) of Example 1 and Comparative Example 1 has 85% homology with the amino acid sequence of Ole1p in Table 4, and is Δ9 desaturated It was found to belong to a family of enzyme families. Regarding the TR3595 gene and TR13919 gene of Example 1 and Comparative Example 1, the gene expression levels were analyzed by RNA-sequencing (Illumina HiSeq sequencing system). The expression level results are shown in Table 5 below. In addition, the unit of the expression level shown in Table 5 is FPKM (Fragments Per Kilobiase of transcript per Migration mapped reads).

From the results of the above homology search, the TR3595 gene is a fatty acid elongation activity gene (a gene encoding an enzyme that expands a constituent fatty acid having 16 carbon atoms (palmitic acid) to a constituent fatty acid having 18 carbon atoms (stearic acid)). It was suggested that the TR13919 gene is a desaturation activity gene at the Δ9 position (a gene that converts the 9th position of the saturated fatty acid (stearic acid) to an unsaturated fatty acid (oleic acid) that is unsaturated). In Mortierella capitata, it is considered that the elongation and desaturation of the fatty acids constituting the fats and oils are progressing through the route shown in FIG. In FIG. 1, C _{24: 1} is nervonic acid. In Example 1, the expression level of TR3595 gene was about 13.6 times higher than that of Comparative Example 1, and the expression level of TR13919 gene was about 3.9 times higher than that of Comparative Example 1. From these results, the production amount of nervonic acid in Examples 1 to 3 was high because the expression level of the fatty acid elongation activity gene (TR3595) and the desaturation activity gene (TR13919) at the Δ9 position in the flow shown in FIG. However, it is estimated that this is due to the increase.

Claims (9)

Medium shown below:
Glucose 10% (w / v)
Yeast extract 0.4% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
A strain belonging to the genus Mortierella capable of producing 400 mg or more of nervonic acid as a constituent fatty acid in fats and oils when inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 215 rpm for 7 days.   The strain according to claim 1, which can produce 750 mg or more of nervonic acid as a constituent fatty acid of fats and oils per liter of medium. Medium shown below:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
A strain belonging to the genus Mortierella capable of producing 1000 mg or more of nervonic acid as a constituent fatty acid in fats and oils when inoculated with 0.4 mg / ml of strain and cultured for 14 days at 28 ° C. and 215 rpm. Medium shown below:
Glucose 12% (w / v)
Yeast extract 0.8% (w / v)
Potato peptone 0.5% (w / v)
Magnesium sulfate heptahydrate 0.00135% (w / v)
Calcium chloride 0.00475% (w / v)
(However, 70 ml of 60% (w / v) aqueous glucose solution is added per liter of the medium when 10 days have passed since the start of the culture.)
A strain belonging to the genus Mortierella capable of producing 1000 mg or more per liter of nervonic acid as a constituent fatty acid when inoculated with 0.4 mg / ml of strain and cultured for 14 days at 28 ° C. and 180 rpm.   The fatty acid elongation activity is 700% or more with respect to Mortierella capitata RD000969 strain deposited as an accession number NITE P-1508 at the Patent Evaluation Microorganism Depositary of the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation. The strain according to any one of 1 to 4.   Desaturation activity at the Δ9 position of the fatty acid constituent fatty acid is against Mortierella capitata RD000969 strain deposited with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation as Deposit Number NITE P-1508 The strain according to any one of claims 1 to 5, which is 200% or more.   The strain according to any one of claims 1 to 6, which is a strain having cerulenin resistance.   Claim 1. The strain of Mortierella capitata deposited with the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center, NITE P-02299, NITE P-02300, or NITE P-02301. The strain according to any one of 7 to 7. A method for producing fats and oils having nervonic acid as a constituent fatty acid,
Culturing the strain according to any one of claims 1 to 8,
And a step of recovering fats and oils from the cultured cells.

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