JP6823298B2 - Strains belonging to the genus Mortierella and methods for producing fats and oils - Google Patents

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 that make up the myelin sheath of spinal animals, and insufficient concentration of nervonic acid in the myelin sheath induces a nervous system disease called demyelinating disease. Therefore, it is known that administration of nervonic acid or a derivative thereof is effective for the treatment of demyelinating disease. 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, it is expected to be applied to cosmetic raw materials.

Patent Document 1 discloses a method for producing an oil or fat containing nervonic acid as a constituent fatty acid by a bacterium belonging to the genus Mortierra. In particular, as a strain that produces a large amount of nervonic acid, a strain of accession number NITE P-1508 (storage number RD000969 that can be sold by NITE) is disclosed at the Patent Microorganisms Depositary Center.

Japanese Unexamined Patent Publication No. 2014-168418

However, the strain of Accession No. NITE P-1508 cannot be said to produce sufficient nervonic acid from the viewpoint of industrialization, and a strain having even higher nervonic acid-producing ability is required.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a strain belonging to the genus Mortierella, which has excellent nervonic acid-producing ability, and a method for producing fats and oils using this strain.

The present inventors have found that a strain belonging to the genus Mortierella having a high ability to produce nervonic acid can be obtained by a predetermined method, and have completed the present invention. More specifically, it is an object of the present invention 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 of fats and oils per 1 L of a medium 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 1 L of a 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 1 L of the medium 10 days after 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 of fats and oils per 1 L of a medium when inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 215 rpm for 14 days.

(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 1 L of the medium 10 days after 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 of fats and oils when inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 180 rpm for 14 days.

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

(6) Mortierella capitata RD000969 strain whose unsaturated activity at the Δ9 position of the constituent fatty acids of fats and oils was deposited with the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-1508. The strain according to any one of (1) to (5), which is 200% or more of the strain.

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

(8) A strain of Mortierella capitata deposited under the accession numbers NITE P-02299, NITE P-02300, or NITE P-02301 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center. The strain according to any one of (1) to (7).

(9) A method for producing fats and oils containing nervonic acid as a constituent fatty acid.
The step of culturing the strain according to any one of (1) to (8) and
A production method comprising a step of recovering fats and oils from bacterial cells after culturing.

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

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

Hereinafter, embodiments of the present invention will be described, but the present invention is not particularly limited thereto.

<Strains 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. The strain described in Patent Document 1 did not have sufficient ability to produce fats and oils in the cells, but the strain belonging to the genus Mortierella of the present invention can produce a large amount of fats and oils in the cells. The fats and oils produced by the strain of the present invention refer to fats and oils 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 the 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)
Was inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 215 rpm for 7 days (hereinafter, this culture condition may be referred to as “culture condition (a)” in the present specification). It is a strain capable of producing 400 mg or more of nervonic acid as a constituent fatty acid per 1 L of a medium.

The medium under 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. It is a liquid medium in which 0.0015% (w / v) and 0.00475% (w / v) of calcium chloride are dissolved in water (hereinafter, may be referred to as "medium (A)" in the present specification). In the present specification, "% (w / v)" is a component contained in 100 mL of the medium (in the medium (A), glucose, yeast extract, potato peptone, magnesium sulfate / heptahydrate, calcium chloride. ) Means the mass (g).

In the culture condition (a), the amount (mg / ml) of the strain inoculated into the medium (A) is a numerical value converted into the dry cell weight, and is the weight with respect 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 according to the following procedure.

First, the frozen stock (agar medium containing bacterial cells) is transplanted to a thinly spread potato dextrose medium (commercially available, solid medium containing agar), and cultured at 28 ° C. before the previous stage. After pre-culturing, the flower-shaped bacteria are collected with a straw having a diameter of 8 mm, and the medium for pre-culturing (potato peptone: 0.5% (w / v), glucose: 3% (w / v), yeast. Extract: 0.1% (w / v)) 200 ml (500 ml volume Erlenmeyer flask with baffle) can be inoculated and pre-cultured at 140 rpm and 28 ° C. for 2 days to obtain cells to be inoculated into medium (A). ..

Under the culture condition (a), 200 ml of the medium (A) is used. In the 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, an Erlenmeyer flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.) at 28 ° C. and 215 rpm.

The fats and oils produced by the strain of the present invention produce nervonic acid as a constituent fatty acid of the fats and oils under the culture condition (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 is contained in an amount of 400 mg or more per 1 L of the medium, but other constituent fatty acids are not particularly limited. 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 nerbonic acid as a constituent fatty acid of fats and oils per 1 L of medium under the culture condition (a), but 500 mg of nerbonic acid as a constituent fatty acid of fats and oils per 1 L of medium. It is more preferable to be able to produce the above, more preferably 550 mg or more, further preferably 600 mg or more, further preferably 700 mg or more, and 750 mg or more can be produced. It is particularly preferable to have. 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 1 L of the medium under the culture condition (a).

Under the culture condition (a), the strain of the present invention preferably produces 20.0 mg or more of nervonic acid as a constituent fatty acid of fats and oils per 1 g (dry cell weight) of 25.0 mg. It is preferably 30.0 mg or more, and particularly preferably 35.0 mg or more. Further, in the strain of the present invention, under the culture condition (a), the production amount of nervonic acid as a constituent fatty acid of fats and oils was 100.0 mg or less (90.0 mg or less, 80) per 1 g (dry cell weight) of the cells. It may be 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 acids in fats and oils is high. From this viewpoint, in the culture condition (a), the ratio 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% by mass or more, and it is particularly preferable that it is 7.0% by mass or more. Further, in the strain of the present invention, under the culture condition (a), the ratio of nervonic acid in the constituent fatty acids of the fat and oil was 20.0% by mass or less (18.0% by mass or less) with respect to the total constituent fatty acids of the fat and oil. It may be 16.0% by mass or less, 14.0% by mass or less, 13.0% by mass or less, 12.0% by mass or less, etc.).

The strain of the present invention is a 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 1 L of the medium 10 days after the start of the culture.)
Was inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 215 rpm for 14 days (hereinafter, this culture condition may be referred to as “culture condition (b)” in the present specification). A strain belonging to the genus Mortierra, which can produce 1000 mg or more of nervonic acid as a constituent fatty acid of the above, may be used.

The medium under 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. A liquid medium in which 0.0015% (w / v) and 0.00475% (w / v) calcium chloride are dissolved in water (hereinafter, may be referred to as "medium (B)" in the present specification). Further, under the culture condition (b), 70 ml of a 60% (w / v) glucose aqueous solution is added per 1 L of the medium 10 days after the start of the culture. The “culture start” refers to the time when the strain 0.4 mg / ml is inoculated into the medium (B).

In the culture condition (b), the amount (mg / ml) of the strain inoculated into the medium (B) is a numerical value converted to the weight of dried cells, and is the weight with respect to the volume (ml) of the medium (B). The strain to be inoculated into the medium (B) can be obtained by the same method as the method under the culture condition (a).

Under the culture condition (b), 200 ml of the medium (B) is used. In the 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 adjusted. As the culture vessel, an Erlenmeyer flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.) at 28 ° C. and 215 rpm.

The fat and oil produced by the strain of the present invention may produce nervonic acid as a constituent fatty acid of the fat and oil under the culture condition (b). The fats and oils produced under the culture condition (b) are not particularly limited as long as nervonic acid as a constituent fatty acid is contained in an amount of 1000 mg or more per 1 L of the medium, but other constituent fatty acids are not particularly limited. 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 nerbonic acid as a constituent fatty acid of fats and oils per 1 L of medium under the culture condition (b), but 1050 mg of nerbonic acid as a constituent fatty acid of fats and oils is produced per 1 L of medium. It is more preferable that the above-mentioned production is possible, further preferably 1100 mg or more, further preferably 1200 mg or more, further preferably 1300 mg or more, and 1400 mg or more. It is particularly preferable to have. 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 of fats and oils per 1 L of medium under the culture condition (b). May be good.

Under the culture condition (b), the strain of the present invention preferably produces 25.0 mg or more of nervonic acid as a constituent fatty acid of fats and oils, preferably 25.0 mg or more, per 1 g (dry cell weight). It is preferably 33.0 mg or more, and particularly preferably 33.0 mg or more. Further, in the strain of the present invention, under the culture condition (b), the amount of nervonic acid produced as a constituent fatty acid of fats and oils was 100.0 mg or less (90.0 mg or less, 80) per 1 g (dry cell weight) of the cells. It may be 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 acids in fats and oils is high. From this point of view, in the culture condition (b), the ratio of nervonic acid in the constituent fatty acids of the fat is preferably 5.5% by mass or more with respect to the total constituent fatty acids of the fat and oil, and is preferably 5.6% by mass or more. It is preferable that it is 5.8% by mass or more, and it is particularly preferable that it is 6.0% by mass or more. Further, in the strain of the present invention, under the culture condition (b), the ratio of nervonic acid in the constituent fatty acids of the fat and oil was 15.0% by mass or less (14.0% by mass or less) with respect to the total constituent fatty acids of the fat and oil. It may be 13.0% by mass or less, 12.0% by mass or less, 11.0% by mass or less, 10.0% by mass or less, etc.).

The strain of the present invention is a medium shown below:
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 1 L of the medium 10 days after the start of the culture.)
Was inoculated with 0.4 mg / ml of the strain and cultured at 28 ° C. and 180 rpm for 14 days (hereinafter, this culture condition may be referred to as “culture condition (c)” in the present specification). It may be a strain belonging to the genus Mortierra capable of producing 1000 mg or more of nerbonic acid as a constituent fatty acid of fats and oils per liter.

The medium under the culture condition (c) was glucose 12% (w / v), yeast extract 0.8% (w / v), potato peptone 0.5% (w / v), magnesium sulfate / heptahydrate 0. A liquid medium in which 0.0015% (w / v) and 0.00475% (w / v) calcium chloride are dissolved in water (hereinafter, may be referred to as "medium (C)" in the present specification). Further, under the culture condition (c), 70 ml of a 60% (w / v) glucose aqueous solution is added per 1 L of the medium 10 days after the start of the culture. This "culture start" refers to the time when the strain 0.4 mg / ml is inoculated into the medium (C).

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

Under 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 adjusted. As the culture vessel, an Erlenmeyer flask with a 500 ml capacity baffle is used. The medium is shaken using a rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.) at 28 ° C. and 180 rpm.

The fat and oil produced by the strain of the present invention may produce nervonic acid as a constituent fatty acid of the fat and oil under the culture condition (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 is contained in an amount of 1000 mg or more per 1 L of the medium, but other constituent fatty acids are not particularly limited. 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 nerbonic acid as a constituent fatty acid of fats and oils per 1 L of medium under the culture condition (c), but 1100 mg of nerbonic acid as a constituent fatty acid of fats and oils per 1 L of medium. It is more preferable to be able to produce the above, more preferably 1200 mg or more, further preferably 1300 mg or more, further preferably 1400 mg or more, and 1500 mg or more. It is particularly preferable to have. 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 1 L of the medium under the culture condition (c).

Under the culture condition (c), the strain of the present invention preferably produces 20.0 mg or more of nervonic acid as a constituent fatty acid of fats and oils per 1 g (dry cell weight) of 25.0 mg. It is preferably 35.0 mg or more, and particularly preferably 39.0 mg or more. Further, in the strain of the present invention, under the culture condition (c), the amount of nervonic acid produced as a constituent fatty acid of fats and oils was 100.0 mg or less (90.0 mg or less, 80) per 1 g (dry cell weight) of the cells. It may be 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 acids in fats and oils is high. From this viewpoint, in the culture condition (c), the ratio 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% by mass or more, and it is particularly preferable that it is 6.6% by mass or more. Further, in the strain of the present invention, under the culture condition (c), the ratio of nervonic acid in the constituent fatty acids of the fat and oil was 15.0% by mass or less (14.0% by mass or less) with respect to the total constituent fatty acids of the fat and oil. It may be 13.0% by mass or less, 12.0% by mass or less, 11.0% by mass or less, 10.0% by mass or less, etc.).

Since the strain of the present invention has a high fatty acid elongation activity, it is considered that the strain has a high ability to produce nervonic acid. Therefore, it is preferable that the strain has a high fatty acid elongation activity. In the present invention, the "fatty acid elongation activity" specifically means that the expression level of the gene that extends the carbon chain of the constituent fatty acids of fats and oils is high, and more specifically, the constituent fatty acids having 16 carbon atoms (palmitin). This means that the expression level of the gene encoding the enzyme that extends the acid) to the constituent fatty acid (stearic acid) having 18 carbon atoms is high. From this point of view, the strain of the present invention has a higher fatty acid elongation activity than the fatty acid elongation activity of the Mortierella capitata RD000969 strain deposited under the Patent Microorganisms Depositary Center of the Japan Product Evaluation Technology Infrastructure Organization under the accession number NITE P-1508. It is preferable that the substance has activity, and more specifically, the expression level of the gene (SEQ ID NO: 1) encoding the enzyme having fatty acid extension activity in the RD000969 strain is set to 100% (fatty acid extension activity of the RD000969 strain). Occasionally, the expression level of the corresponding gene (eg, SEQ ID NO: 1) in the strain of the present invention (fatty acid elongation activity of the strain of the present invention) is more than 100% of the fatty acid elongation activity of the RD000969 strain. It is preferably possessed, more preferably 300% or more fatty acid elongation activity, further preferably 500% or more fatty acid elongation activity, and 700% or more fatty acid elongation activity. It is even more preferable to have a fatty acid elongation activity of 900% or more, and it is particularly preferable that the fatty acid has a fatty acid elongation activity of 1200% or more. Further, 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%. It may have a fatty acid elongation activity (1400% or less, etc.).

In addition to the activity for the expression level of SEQ ID NO: 1 described above, the amino acid elongation activity of the strain of the present invention is (1) under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1. A DNA having a base sequence capable of hybridizing, (2) 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 shown in 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 shown in SEQ ID NO: 2. It is preferable that the activity is related to the expression level of any of the DNAs having the base sequence encoding the above and having the amino acid elongation activity.

Since the strain of the present invention also has a high unsaturated activity at the Δ9 position of the constituent fatty acids of fats and oils, it is considered that the strain has a high ability to produce nervonic acid. Therefore, it is preferable that the strain has a high unsaturated activity at the Δ9 position. In the present invention, the "desaturation activity at the Δ9 position" is specifically a gene that converts the 9th position of a saturated fatty acid (stearic acid) among the constituent fatty acids of fats and oils into an unsaturated fatty acid (oleic acid). It means that the expression level of is high. From this point of view, the strain of the present invention is higher than the fatty acid unsaturated activity of the Mortierella capitata RD000969 strain deposited under the Patent Microorganisms Depositary Center, Product Evaluation Technology Infrastructure Organization, under the accession number NITE P-1508. It is preferably one having an unsaturated activity at the Δ9 position, and more specifically, an expression level (RD000969 strain) of a gene (SEQ ID NO: 3) encoding an enzyme having an unsaturated activity at the Δ9 position in the RD000969 strain. The expression level of the corresponding gene (for example, SEQ ID NO: 3) in the strain of the present invention (for example, the unsaturated activity at position Δ9 of the strain of the present invention) is that of the RD000969 strain, when the fatty acid elongation activity of the strain of the present invention is 100%. It is preferable that the unsaturated activity at the Δ9 position is more than 100% with respect to the unsaturated activity at the Δ9 position, and more preferably it has an unsaturated activity at the Δ9 position of 120% or more. , It is more preferable that it has an unsaturated activity at the Δ9 position of 150% or more, it is even more preferable that it has an unsaturated activity at the Δ9 position of 200% or more, and it is more preferable that it has a Δ9 position of 300% or more. It is even more preferable that it has an unsaturated activity of 350% or more, and it is particularly preferable that it has an unsaturated activity at the Δ9 position. Further, the strain of the present invention is 1000% or less with respect to the unsaturated activity at position Δ9 of the RD000969 strain when the expression level of the gene of SEQ ID NO: 3 of the RD000969 strain is 100% of the unsaturated activity at position Δ9. It may have a fatty acid unsaturated activity (900% or less, 800% or less, 700% or less, 600% or less, 500% or less, etc.).

In addition to the activity for the expression level of SEQ ID NO: 3, the desaturation activity at position Δ9 of the strain of the present invention is (1) a base sequence complementary to the base sequence shown in SEQ ID NO: 3 and a stringent. 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 shown in SEQ ID NO: 4. However, DNA having unsaturated activity at position Δ9, (3) 85% or more (90% or more, 94% or more, 95% or more, 97% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO: 4. It is preferable that the activity is related to the expression level of any of the DNAs having the nucleotide sequence encoding the amino acid sequence having the homology of) and having the unsaturated activity at the Δ9 position.

In the present invention, as "stringent conditions", for example, the reaction is carried out in a normal hybridization buffer at 40 to 70 ° C. (preferably 50 to 67 ° C., more preferably 60 to 65 ° C.). Conditions 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, still more preferably 30 to 50 mM).

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

In the present invention, the homology of 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 analyzing the base sequence by BLASTN based on BLAST, the parameters are, for example, score = 100 and worldgth = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and worldgth = 3. 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 cellrenin resistance.

Examples of the strains belonging to the genus Mortierella of the present invention include Mortierella capitata, Mortierella elongata, Mortierella exigua, Mortierella exigua, Mortierella exigua, and Mortierella glo. Mortierella alpina, Mortierella parvispora, Mortierella beljakovae, Mortierella grobalpina Mortierella Mortierella kuhlmanii), Mortierella Akurotona (Mortierella acrotona), Mortierella Zaicha (Mortierella zychae), Mortierella Minuchishima (Mortierella minutissima), Mortierella Bainieri (Mortierella bainieri), the Mortierella sect (Mortierella schmuckeri), etc. Can be mentioned. Of these, Mortierla capitata is particularly preferable because of its high nervonic acid production capacity, and among Mortierra capitata, in particular, the NITE P-02299, which has been referred to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, Incorporated Administrative Agency, Strains of Mortierla capitata deposited as NITE P-02300 or NITE P-02301 are 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 fats and oils) 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 medium under the above-mentioned culture condition (a), and a genus Mortierella capable of producing 1000 mg or more of nervonic acid per liter under the culture condition (b). A strain belonging to the genus Mortierella, which can produce 1000 mg or more of nervonic acid per liter depending on the strain to which it belongs and the culture condition (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, for example, 28 ° C. for 2 days. Bacterial cells are collected from this culture solution by filter filtration, a buffer solution is added, and ultrasonic waves (for example, frequency 20 kHz, amplitude 40 μm) are irradiated for 2 minutes to prepare a hyphal suspension. 1 ml of NTG solution is added to the suspension, and the suspension is allowed to stand at 28 ° C. for 15 minutes, and then 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.

Further, it is preferable to mutate the isolated cerulenin-resistant bacteria by irradiating them with ultraviolet rays (253 nm). Specifically, the above-mentioned isolated cellrenin-resistant bacteria are inoculated into 15 ml of potato dextrose liquid medium and cultured at 28 ° C. for 2 days with shaking. Bacterial cells are collected from this culture solution by filter filtration, washed, a buffer solution is added, and ultrasonic waves (for example, frequency 20 kHz, amplitude 40 μm) are irradiated to prepare a hyphal suspension. After transferring the suspension to a petri dish and irradiating with an ultraviolet lamp, 50 μl is applied to a potato dextrose medium supplemented with 0.04% cerulenin to isolate cerulenin-resistant bacteria. By selecting the cellrenin-resistant strain in this way, the strain of the present invention having a high nervonic acid-producing ability can be obtained.

Whether or not the obtained strain is the above-mentioned strain can be confirmed by culturing under the above-mentioned culture condition (a) and measuring the amount of nervonic acid.

<Manufacturing method of fats and oils>
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, and is a step of culturing the above-mentioned strain belonging to the genus Mortierra and a step of recovering fats and oils from the cells after culturing. And have.

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

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

The medium used for the medium of the present invention is not particularly limited as long as it is a medium in which a strain belonging to the genus Mortierra can grow, but specific carbon sources include, for example, glucose, sucrose, fructose, xylose, mannose, and soluble. Starch, glycerol, mannitol and the like can be used. Of these, glucose is preferred. The concentration of the carbon source is preferably 5 to 15% (w / v), and glucose is preferably contained in 5 to 15% (w / v).

The medium preferably further contains a nitrogen source. The nitrogen source is not particularly limited, but for example, yeast extract, malt extract, meat extract, peptone, casamino acid, corn stippricer and the like can be used. Above all, since the effect of increasing the concentration of bacterial cells per culture solution is large, it is preferable to use yeast extract and peptone, and it is preferable to use both yeast extract and peptone. In particular, the concentration of the nitrogen source is preferably 0.1 to 1.0% (w / v), and in particular, yeast extract 0.1 to 1.0% (w / v) and potato peptone 0.1 to 0.1. 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.), and copper salts. It is preferable to contain an inorganic salt such as (copper sulfate or the like) and an isotonic agent such as sodium chloride. Regarding inorganic salts (particularly 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). Is preferable.

As the medium, fatty acids or fats and oils containing fatty acids may be used as substrates. 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, lignoceric acid and the like. Of these, erucic acid is preferable because it has a large effect of increasing the concentration of nervonic acid in the fat and oil composition. Examples of fats and oils include olive oil, palm oil, and rapeseed oil. Of these, rapeseed oil is preferable because it has a high content of erucic acid.

In addition, a known medium used for a conventional strain belonging to the genus Mortierra may be used, and a Czapek medium, a Czapek-dox medium, a PD (potato dextrose) medium, or the like can be used.

The culture temperature may be any temperature as long as the strain belonging to the genus Mortierra can grow, and is not particularly limited, but may be, for example, 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 may be any pH as long as the strain belonging to the genus Mortierra can grow, and is not particularly limited, but may be, for example, 3 or more, 4 or more, 5 or more, and 12 or less, 11 or less, 10 or less. It may be there.

The method of culturing is not particularly limited as long as it is a method in which a strain belonging to the genus Mortierra can grow, but for example, it can be carried out under the above-mentioned culture conditions by stirring culture, shaking culture, static culture and the like. .. In the case of shaking culture, the number of rotations of shaking is not particularly limited as long as it is a method in which a strain belonging to the genus Mortierra can grow, but it is preferably carried out at 150 to 250 rpm.

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

The method of recovering fats and oils from the cells after culturing can be carried out by a conventionally known method. For example, it may be carried out by first separating and recovering the bacterial cells from the culture solution by centrifugation, filtration or the like, and extracting the fat or oil containing nervonic acid as a constituent fatty acid from the bacterial cells with normal hexane.

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

For the production of fats and oils, a known method may be used as described in Japanese Patent Application Laid-Open 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.

<Preparation of mutant strain>
NTG (N-methyl-N'-nitro-N-nitrosoguanidine) was applied to the Mortierra capitata RD000969 strain deposited under NITE P-1508 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary. 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. The cells were collected from this culture solution by filter filtration, washed with 50 mM Trismaleic acid buffer (pH 7.5), and then 1 ml of 100 mM Trismaleic acid buffer (pH 7.5) was added to ultrasonic waves (frequency 20 kHz, A mycelial suspension was prepared by irradiating with an amplitude of 40 μm) for 2 minutes. After adding 1 ml of NTG solution (40 μg / ml) to the suspension and allowing it to stand at 28 ° C. for 15 minutes, the cells were recovered 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, and two cerulenin-resistant strains (M209 strain and M324 strain) were isolated. The M209 strain was designated as Example 1 and the M324 strain was designated as the strain according to Example 2. The trust number of the M209 strain is NITE P-02299, and the trust number of the M324 strain is NITE P-02300.

The strain according to Example 2 was further subjected to ultraviolet irradiation (253 nm) for mutation treatment. Cerlenin-resistant bacteria were inoculated into 15 ml of potato dextrose liquid medium and cultured with shaking at 28 ° C. for 2 days. The cells were collected from this culture solution by filter filtration, washed with 50 mM Trismaleic acid buffer (pH 7.5), and then 1 ml of 100 mM Trismaleic acid buffer (pH 7.5) was added to ultrasonic waves (frequency 20 kHz, A mycelial suspension was prepared by irradiating with an amplitude of 40 μm) for 2 minutes. The suspension was transferred to a petri dish, irradiated with an ultraviolet lamp from a distance of 30 cm for 22 minutes, 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 for the M463 strain is NITE P-02301.

<Manufacturing of fats and oils>
(Pre-culture, pre-culture)
As Comparative Example 1, the RD000969 strain was prepared, and as Comparative Example 2, the NBRC102984 strain (Parietichytrium 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, a frozen stock of each strain (agar medium containing bacterial cells) was transplanted, and the cells were cultured in a potato dextrose medium (commercially available with agar) at 28 ° C. while observing the growth condition. After pre-culturing, the flower-shaped bacteria were collected with a straw having a diameter of 8 mm, and the liquid medium for pre-culture (potato peptone: 0.5% (w / v), glucose: 3% (w / v), Yeast extract: 0.1% (w / v)) 200 ml (500 ml volume flask with baffle) was added, and preculture was performed at 140 rpm and 28 ° C. for 2 days.

(Main culture)
After pre-culture, 10 ml of the cells according to Examples 1 to 3 and Comparative Examples 1 and 2 were collected (including the culture solution), and 10 ml of the cells was added to 190 ml of the medium to make a total of 200 ml, and the dry cell weight was 0. The cells were inoculated to a concentration of 4 mg / ml, and the main culture was carried out 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: 200 ml (500 ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 215 rpm (Rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.))
Culture period: 7 days

For the strains according to Examples 2, 3 and Comparative Example 1, after pre-culture, 10 ml of the cells were collected (including the culture solution), and 10 ml of the cells was added to 190 ml of the medium to make a total of 200 ml, and the dry cell weight was adjusted to 200 ml. 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)
(Add 70 ml of 60% (w / v) glucose aqueous solution to 1 L of medium 10 days after the start of culture)
Capacity: 200 ml (500 ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 215 rpm (Rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.))
Culture period: 14 days

For the strain according to Example 2, after pre-culture, 10 ml of the cells were collected (including the culture solution), and 10 ml of the cells was added to 190 ml of the medium to make a total of 200 ml, and the dry cell weight was 0.4 mg / ml. The main culture was carried out 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)
(1 L of medium 1 L of 60% (w / v) glucose aqueous solution 10 days after the start of culture
70 ml per unit)
Capacity: 200 ml (500 ml Erlenmeyer flask with baffle)
Culture temperature: 28 ° C
Shaking speed: 180 rpm (Rotary shaker (vertical triple-barrel constant temperature shaking incubator TAL-RS310, manufactured by Thomas Kagaku Kikai Co., Ltd.))
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 filter filtration and freeze-dried. 3 ml of a 2M hydrochloric acid methanol solution was added to 30 mg of dried bacterial cells, and the cells were heated at 100 ° C. for 2 hours to methyl esterify the bacterial cell fatty acids. After washing the reaction solution with 1 ml of distilled water, the 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 result>
The culture results are shown in Tables 1 to 3 below. The amount of nervonic acid produced in Tables 1 to 3 is the amount of nervonic acid contained in the cells after the main culture (the amount of nervonic acid as a constituent fatty acid in fats and oils). In addition, "ratio (%) of nervonic acid in lipid" means the ratio (mass%) of nervonic acid as a constituent fatty acid in fats and oils recovered from cells. Table 1 shows the results of the main culture under the culture condition (a), Table 2 shows the results of the main culture under the culture condition (b), and Table 3 shows the results of the main culture under the culture condition (c). It is the result of doing.

As shown in Tables 1 to 3, it was found that the strains according to Examples 1 to 3 produced a higher amount of nervonic acid 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 on 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 the known fatty acid synthase 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 the analysis, it was found that it belonged to the same phylogenetic branch as MALCE1p and had a transmembrane mode similar to MALCE1p. In addition, 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 the lineage of the enzyme family. For the TR3595 gene and TR13919 gene of Example 1 and Comparative Example 1, the expression levels of the genes were analyzed by RNA-sequencing (HiSeq sequencing system of Illumina). The results of the expression level are shown in Table 5 below. The unit of the expression level shown in Table 5 is FPKM (Fragments Per Kilobase of transcript per Million mapped reads).

From the results of the above homology search, the TR3595 gene is a fatty acid elongation active gene (a gene encoding an enzyme that extends a 16-carbon constituent fatty acid (palmitic acid) to an 18-carbon constituent fatty acid (stearic acid)). , TR13919 gene was suggested to be an unsaturated fatty acid active gene at position Δ9 (a gene that converts the 9th position of saturated fatty acid (stearic acid) into unsaturated fatty acid (oleic acid)). It is considered that Mortierla capitata has been elongated and unsaturated in the constituent fatty acids of fats and oils by the route shown in FIG. In FIG. 1, C _{24: 1} is nervonic acid. In Example 1, the expression level of the TR3595 gene was about 13.6 times higher than that of Comparative Example 1, and the expression level of the TR13919 gene was about 3.9 times higher than that of Comparative Example 1. From this result, the amount of nervonic acid produced was high in Examples 1 to 3 because of the expression levels of the fatty acid elongation activity gene (TR3595) and the unsaturated activity gene at the Δ9 position (TR13919) in the flow shown in FIG. However, it is presumed that this is due to the increase.

Claims (2)

Ru strains der of the National Institute of Technology and Evaluation accession number in the Patent Microorganisms Depositary Center NITE P-02299, NITE P- 02300, or NITE P-02301 Mortierella Kapitata deposited as (Mortierella capitata), Strain .. A method for producing fats and oils containing nervonic acid as a constituent fatty acid.
The step of culturing the strain according to claim 1 and
A production method comprising a step of recovering fats and oils from bacterial cells after culturing.

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