JP6514867B2 - Process for producing acetyl sphingoid containing unsaturation - Google Patents

Description

  The present invention relates to a method for producing acetyl sphingoid containing unsaturation using a microorganism.

  The sphingolipids are synthesized by biosynthesis starting from the condensation reaction of L-serine and an acyl-CoA such as palmitoyl-CoA. As sphingoid bases that are basic structures, sphingosine, phytosphingosine, dihydrosphingosine (sphinginin), 6-hydroxysphingosine, etc. are known, and various types of ceramides are formed by the amide bond of these sphingoid bases with various fatty acids. It is synthesized. Ceramides I, II, V, IV, VII, and VIII have an alkenyl group in the side chain of sphingoid.

  Sphingolipids have many physiological functions. In particular, ceramide and sphingoid bases are involved in the moisturizing function and barrier function of the skin, prevent water evaporation from the skin, and play a role in protecting the human body from various external stimuli. Sphingosine, phytosphingosine, sphinganine, and 6-hydroxysphingosine exist as sphingoid bases in human skin, but sphingolipids containing sphingoid bases of various structures are detected from other organisms such as animals, plants and insects. (Non-patent document 1), their function and physiological activity analysis is in progress. However, many of them have not yet constructed efficient production methods.

Although many methods for producing sphingolipids have been studied, since it is difficult to separate and purify animal and plant-derived sphingolipids, in recent years, development of methods for producing sphingolipids using fermentation with yeast has been advanced. As candidate yeast strain Pichia ciferrii (Pichia ciferrii; now Wicker conger Mrs. ciferrii (Wickerhamomyces ciferrii)), although Candida utilis (Candida utilis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) are mentioned, tetraacetyl phytosphingosine It is useful to obtain tetraacetylphytosphingosine by utilizing Wicker Hacomyces siferli which secretes sphingosine out of the cell (Patent Document 1).

  The rate-limiting step of tetraacetylphytosphingosine synthesis in such Wicker Hacomyces siferliin is revealed from the in vitro analysis of the biosynthetic pathway to be the condensation reaction of serine and palmitoyl-CoA and the acetylation reaction of phytosphingosine ( Non-Patent Document 2), two acetylation enzymes, SLI1 and ATF2, have been found. Among these, since SLI1 produces triacetylphytosphingosine when it is expressed in Saccharomyces cerevisiae (Non-patent document 3), any one of three hydroxyl groups and one amino group of phytosphingosine It is believed to be involved in the acetylation of three sites.

  However, in this production method, the production of phytosphingosine, sphinganine and sphingosine (Non-patent Document 4) has been confirmed, but the production of acetylsphingoids having other structures is not known at all.

Japanese Patent Publication No. 9-504434

Sarah T. P. et. Al., J. Lipid Res., 49, 1621, (2008) Barenholz, Y. et. Al., Biochem. Biophys. Acta, 306, 341, (1973) Veld, F. et. Al., Appl. Microbiol, Biotechnol., 97, 19, 8537 (2013) Borgel, D. et. Al., Metab. Eng., 14, 412, (2012)

  The present invention relates to providing a method for producing an acetyl sphingoid having an alkenyl group in a side chain using yeast.

The inventors of the present invention have found that when yeasts of the genus Wickerhamomyces ( Wickerhamomyces ) are cultured in a medium containing an unsaturated fatty acid ester represented by the following formula (1), acetylsphingines represented by the following formula (2) I found that I could produce an id.

That is, the present invention relates to the following method.
The following formula (1):

[Wherein, X represents an alkyl group having 1 to 4 carbon atoms, and n represents an integer of 6 or 7]. ]
And a step of culturing in a medium containing an unsaturated fatty acid ester represented by the following formula (2):

Wherein, Y ¹ to Y ³ represents a hydroxyl group or an acetyl group independently, Y ⁴ represents a hydrogen atom or an acetyl radical, n represents the same number as said. However, at least three or more of Y ^{1 to} Y ⁴ represent an acetyl group. ]
A process for producing acetyl sphingoid represented by

  According to the method of the present invention, an acetyl sphingoid having an alkenyl group in a side chain can be produced by a simple method of adding a desired unsaturated fatty acid ester to a culture medium. The said acetyl sphingoid is useful as a synthetic raw material of the ceramide containing the sphingoid which has an alkenyl group in a side chain.

The yeast of the genus Wickerhamomyces (Wickerhamomyces) used in the present invention may be any one that produces a sphingoid from serine and an unsaturated fatty acid ester and has the ability to acetylate the sphingoid, and is isolated from nature. It may be a wild strain or a mutant derived from the wild strain. The Wicker conger Mrs. genus yeasts, e.g., Wicker conger Mrs. ciferrii (formerly known as Pichia ciferrii (Pichia ciferrii)) are preferred. In addition, specifically, Wicker hydroxylase / Siferiella strains can be used, for example, Wicker hydroxylase / Siferiella strain ATCC 14091, ATCC 32292 strain, Wicker hydroxylase siferliei NBRC 0793 strain, NBRC 0905 strain, and the like.
The use amount of such Wickerhamomyces genus yeast is appropriately determined in consideration of the reaction time and the activity per cell, but it is 1 × 10 ⁶ CFU / mL or more, preferably 1 × 10 ⁷ CFU / mL or more. And 1 × 10 ¹⁰ CFU / mL or less, preferably 1 × 10 ⁹ CFU / mL or less. CFU / mL is an abbreviation of Colony Forming Unit, and indicates the number of cells present in 1 mL.

  In the unsaturated fatty acid ester represented by the formula (1) contained in the medium, the alkyl group having 1 to 4 carbon atoms represented by X may be either linear or branched. For example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group etc. are mentioned, Preferably it is a methyl group and an ethyl group.

In the present invention, as the unsaturated fatty acid ester, as shown by the formula (1), one is used in which the stereoisomerism at the double bond is trans (E). By using the trans form as shown in the examples described later, the corresponding acetyl sphingoid can be produced.
Suitable unsaturated fatty acid esters include methyl trans-9-hexadecenoate where n is 6 and methyl trans-10-heptadecenoate where n is 7.

  The concentration of such unsaturated fatty acid ester in the reaction system is preferably 1 mM or more, more preferably 5 mM or more, still more preferably 20 mM or more, preferably 200 mM or less, more preferably 100 mM or less from the viewpoint of reaction efficiency. More preferably, it is used to be 50 mM or less. In addition, it is preferably used so as to be 1 to 200 mM, more preferably 5 to 100 mM, and still more preferably 20 to 50 mM.

  The culture medium used for the culture can be a conventional medium containing carbon sources, nitrogen sources, inorganic salts, and other organic trace nutrients such as amino acids and vitamins as required, and either synthetic or natural media can be used. It is. Commercial culture media may be used.

  The carbon source and nitrogen source used for the culture medium may be any type as long as a microorganism strain to be cultured can be used. As a carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate, molasses can be used, and in addition, organic acids such as acetic acid and citric acid and alcohols such as ethanol can be used alone. Alternatively, it can be used in combination with other carbon sources. As the nitrogen source, ammonium salts such as ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate or nitrates can be used. As the organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, peptones containing these, casamino acids, yeast extract, soybean protein degradation products etc. can be used, and auxotrophic mutations requiring amino acids etc for growth When using a strain, it is preferable to supplement the required nutrients. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.

  Moreover, as a culture medium, for example, YPD medium containing 1% Yeast Extract, 2% Peptone, 2% Glucose, 0.3% Yeast Extract, 0.3% Malt Extract, 0.5% Peptone, 1% Glucose YM medium containing 0.3 to 0.5% Yeast Extract, 0.3 to 0.5% Malt Extract, 0.5 to 0.7% Peptone, YMgl medium containing 1 to 10% Glycerol, etc. may be mentioned.

  In addition, L-serine, calcium chloride and the like can be appropriately added to such a medium. Thereby, the generation efficiency of acetylsphingoid can be improved.

The culture temperature is, for example, 20 ° C. or more, preferably 25 ° C. or more, 35 ° C. or less, preferably 33 ° C. or less, and 20 to 35 ° C., preferably 25 to 33 ° C.
The culture time is, for example, 24 hours or more, preferably 48 hours or more, preferably 72 hours or more, and includes 120 hours or less, preferably 96 hours or less. Moreover, 24 to 120 hours, preferably 48 to 96 hours can be mentioned.

By such culture, an acetyl sphingoid represented by the formula (2) is accumulated in the culture solution. Here, the acetyl sphingoid obtained is a triacetylated compound in which any three of Y ¹ to Y ⁴ in the formula (2) are acetyl groups, and tetraacetyl compounds in which all Y ^{1 to} Y ⁴ are acetyl groups. A mixture of

  The method for recovering acetyl sphingoid from the culture solution after completion of the culture is not particularly limited, and may be performed according to a known recovery method. For example, after removing the cells from the culture solution as necessary, it is possible to extract acetylsphingoid by solvent extraction with an organic solvent.

As an organic solvent used for the said solvent extraction, chloroform, hexane, butanol, methanol, an ethyl acetate etc. can be mentioned, for example. Among them, one or two or more selected from the group consisting of chloroform, methanol, ethyl acetate and hexane is preferable from the viewpoint of improvement in extraction efficiency of acetylsphingoid and reduction in production cost, and more preferably a chloroform-methanol mixed solution It is.
The weight ratio of the culture to the organic solvent at the time of extraction (culture / organic solvent) is preferably 1/1 to 1/20, more preferably 1/20, from the viewpoint of improving the extraction efficiency of acetyl sphingoid and reducing the production cost. It is 1 to 1/10, more preferably 1/1 to 1/5.

Regarding the embodiment described above, the following aspects are further disclosed in the present invention.
<1> Wicker Hammomyces genus yeast, the following formula (1):

[Wherein, X represents an alkyl group having 1 to 4 carbon atoms, and n represents an integer of 6 or 7]. ]
And a step of culturing in a medium containing an unsaturated fatty acid ester represented by the following formula (2):

Wherein, Y ¹ to Y ³ represents a hydroxyl group or an acetyl group independently, Y ⁴ represents a hydrogen atom or an acetyl radical, n represents the same number as said. However, at least three or more of Y ^{1 to} Y ⁴ represent an acetyl group. ]
A process for producing acetyl sphingoid represented by
<2> A method for producing <1>, wherein the Wicker Hammomyces genus yeast is Wicker Hacomyces siferli.
The method of <1> or <2> whose <3> unsaturated fatty acid ester is methyl trans-9-hexadecenoate or methyl trans-10-heptadecenoate.
<4> The concentration of unsaturated fatty acid ester in the reaction system is preferably 1 mM or more, more preferably 5 mM or more, still more preferably 20 mM or more, preferably 200 mM or less, more preferably 100 mM or less, still more preferably 50 mM or less The method according to any one of <1> to <3>, which is preferably 1 to 200 mM, more preferably 5 to 100 mM, and still more preferably 20 to 50 mM.

Example 1-2 Production of Acetyl Sphingoid (1) Wicker hydroxylase siferli strain ATCC 14091 is applied to YPD agar medium and cultured, and grown colonies are cultured in 5 mL of YPD medium (2% glucose, 2% Bacto Peptone, 1) After inoculating a platinum loop tube into a 100 mL test tube containing 1% Yeast Extract, the cells were cultured at 25 ° C. and 250 rpm for 24 hours.
The obtained culture broth was mixed with 5 mL of YMgl medium (10% Glycerol, 0.5% Bacto Peptone, 0.4% Malt Extract, 0.4% Yeast Extract, 50 mM calcium chloride dihydrate and 25 mM L-serine) Inoculate a 100 mL test tube containing the solution, add unsaturated fatty acid ester (methyl trans-9-hexadecenoate, trans-10-heptadecenoate) shown in Table 2 to a specified concentration, 25 ° C, 250 rpm Culture for 3 days.
After completion of the reaction, add 4 mL of chloroform: methanol = 2: 1 mixed solution to 1 mL of culture solution, mix well by vortexing, allow to stand for about 15 minutes, and centrifuge at 3000 rpm for 15 minutes. The product was recovered from (chloroform layer). The recovered chloroform layer was dried with nitrogen, re-suspended in methanol and appropriately diluted, filtered, and analyzed by LC-MS / MS under the following conditions.

LC conditions: Capcell core C18 2.7 umφ2.1 × 50 mm (Shiseido), Oven Temp. 40 ° C., Sol. A: 0.1% HCO2H in water, Sol. B: MECN, (A 60%, B 40%) 0.5 min → [(A 60%, B 40%) → (A 0%, B 100%) 5.5 min] → B 100% 2 min, [(A 0%, B 100%) → (( A 60%, B 40%) 0.01 min → (A 60%, B 40%) 2 min, Flow rate 0.6 ml / min. , Inject 5μl, MS / MS device: API 3200 QTrap (AB SCIEX)
The obtained analytical value was used as a quantitative value by calculating the concentration using a calibration curve of triacetylphytosphingosine for triacetylated products and tetraacetylphytosphingosine for tetraacetylated products.

(2) Results As shown in Table 2, when methyl trans-9-hexadecenoate (Example 1) and methyl trans-10-heptadecenoate (Example 2) are added, the corresponding acetyl sphingoid is formed It was done.

Comparative Examples 1 to 3
In the above example, the unsaturated fatty acid ester to be added is changed to methyl cis-9-hexadecenoate or methyl cis-10-heptadecenoate shown in Table 3 and methyl trans-9-octadecenoate shown in Table 4; The product was collected by culture and quantified. The results are shown together in Tables 3 and 4.

  As shown in Tables 3 and 4, when methyl cis-9-hexadecenoate (comparative example 1) and cis-10-heptadecenoic acid methyl (comparative example 2) are added, the corresponding acetyl sphingoid is hardly produced. The In addition, even when methyl trans-9-octadecenoate (Comparative Example 3) having a large number of carbon atoms in the side chain was added, the corresponding acetyl sphingoid was not produced.

Claims (3)

The following formula (1):

[Wherein, X represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 6 or 7]. ]
And a step of culturing in a medium containing an unsaturated fatty acid ester represented by the following formula (2):

[Wherein, Y ^{1 to} Y ³ independently represent a hydroxyl group or an acetyl group, Y 4 represents a hydrogen atom or an acetyl group, and n represents the same number as that described above. However, at least three or more of Y ^{1 to} Y ⁴ represent an acetyl group. ]
A process for producing acetyl sphingoid represented by   The method according to claim 1, wherein the Wicker Hacomyces genus yeast is Wicker Hacomyces siferli. The method according to claim 1 or 2, wherein the X turtles methyl group or an ethyl group.