JP6849183B2 - Monoacyl type MEL - Google Patents

Description

Techniques relating to biosurfactants (more specifically, monoacyl-type MELs) are disclosed.

Biosurfactant is a natural surfactant produced by microorganisms, which is highly biodegradable, has a low environmental load, and has various beneficial physiological functions. Therefore, if it is used in the food industry, cosmetics industry, pharmaceutical industry, chemical industry, environmental field, etc., it is meaningful in realizing an environment-friendly society.

Biosurfactants are classified into five types: glycolipid type, acylpeptide type, phospholipid type, fatty acid type and polymer type. Glycolipid-based biosurfactants include mannoseyl erythritol (hereinafter, also referred to as ME) in which erythritol is glycosidic bonded to mannose, mannosyl erythritol lipid (hereinafter, also referred to as MEL) in which fatty acid is ester-bonded, and ramnolipid. Justylazinic acid, trehalose lipid, sophorose lipid and the like are known.

MEL has various structures in which the fatty acid residues to be bound and the positions and numbers of acetyl groups are different. The general structural formula of MEL is shown in FIG. In MEL-A, in the structural formula of FIG. 1, R ₁ and R ₂ are aliphatic acyl groups, and R ₃ and R ₄ are acetyl groups. In MEL-B, R ₁ and R ₂ are aliphatic acyl groups, R ₃ is a hydrogen atom, and R ₄ is an acetyl group. In MEL-C, R ₁ and R ₂ are aliphatic acyl groups, R ₃ is an acetyl group, and R ₄ is a hydrogen atom. In MEL-D, R ₁ and R ₂ are aliphatic acyl groups, and R ₃ and R ₄ are hydrogen atoms. The optical isomers shown in FIGS. 2 (a) and 2 (b) are present in MEL depending on whether the hydroxymethyl group of erythritol bonded to mannose is derived from the carbon at the 1-position or the carbon at the 4-position. The MEL having the structure of FIG. 2 (a) is referred to as 4-O-β-MEL, and the MEL having the structure of FIG. 2 (b) is referred to as 1-O-β-MEL. Pseudozaima tsukubaensis is known to produce 1-O-β-MEL-B, which is more hydrated than 4-O-β-MEL-B. It also has a high vesicle forming ability.

By culturing MEL-producing bacteria using only glucose as the carbon source, fatty acids are bound only to _{R 2 in the MEL of FIG. 1, and R 1} , R ₃ and R ₄ are monoacyl-type MELs (single strands) which are hydrogen atoms. It has been reported that type MEL) is produced (Non-Patent Document 1). This monoacyl type MEL has improved hydrophilicity as compared with the conventional diacyl MEL (Non-Patent Document 1).

The MEL biosynthetic pathway has already been reported, and MEL is synthesized intracellularly by the reaction of glycosyltransferase that binds mannose and erythritol, acyltransferase that binds fatty acids, and acetyltransferase that binds acetyl groups ( Non-Patent Document 2).

Fukuoka et al., Appl Microbiol Biotechnol (2007) 76: 801-810 Hewald et al., Applied and Environmental Microbiology, Aug. 2006, p. 5469-5477

Under the current situation as described above, it is one of the issues to provide a new MEL.

Result of intensive studies to solve the such problem, by deleting the gene of acyltransferase of a microorganism having the ability to produce a biosurfactant, only aliphatic acyl R ₁ in the structural formula of Figure 1 We have found that a new monoacyl-type MEL with an attached group can be obtained. As a result of further research and examination based on these findings, inventions represented by the following are provided.

（式中、Ｒ_１は炭素数２〜２４の脂肪族アシル基を表す。Ｒ_２、Ｒ_３及びＲ_４は各々独立して水素原子又はアセチル基を表す。）
項Ａ２．
下記式（２）の構造を有するモノアシル型ＭＥＬ。

（式中、Ｒ_１は炭素数２〜２４の脂肪族アシル基を表す。）
項Ａ３．
下記式（３）の構造を有するモノアシル型ＭＥＬ。

（式中、Ｒ_１は炭素数２〜２４の脂肪族アシル基を表す。）
項Ａ４．
炭素数が４〜２４である、項Ａ１〜Ａ３のいずれかに記載のモノアシル型ＭＥＬ。 A. Monoacyl type MEL
Item A1.
A monoacyl type MEL having the structure of the following formula (1).

(In the formula, R ₁ represents an aliphatic acyl group having ₂ to 24 carbon atoms. R 2, R ₃ and R ₄ each independently represent a hydrogen atom or an acetyl group.)
Item A2.
A monoacyl type MEL having the structure of the following formula (2).

(In the formula, R ₁ represents an aliphatic acyl group having 2 to 24 carbon atoms.)
Item A3.
A monoacyl type MEL having the structure of the following formula (3).

(In the formula, R ₁ represents an aliphatic acyl group having 2 to 24 carbon atoms.)
Item A4.
Item 4. The monoacyl type MEL according to any one of Items A1 to A3, which has 4 to 24 carbon atoms.

B. Monoacyl-type MEL-producing microorganisms B1.
The microorganism that produces the monoacyl-type MEL according to any one of Items A1 to A4.
Item B2.
Item 2. The microorganism according to Item B1, wherein the gene encoding the mannose acyltransferase is deficient.
Item B3.
Item 2. The microorganism according to Item B1 or B2, wherein the microorganism belongs to the genus Pseudozaima.
Item B4.
Item 4. The microorganism according to any one of Items B1 to B3, wherein the microorganism belongs to Pseudozyma tsukubaensis.

C. Method for manufacturing monoacyl type MEL Item C1.
A method for producing a monoacyl type MEL using the microorganism according to any one of Items B1 to B4.
Item C2.
The method according to Item C1, wherein the monoacyl type MEL is the monoacyl type MEL according to the deviation of items A2 to A4.
Item C3.
Item 6. The method according to Item C1 or C2, which comprises the step of culturing the microorganism in a medium containing vegetable oil.

D. Method for producing monoacyl-type MEL-producing microorganism D1.
A method for producing a monoacyl-type MEL-producing microorganism, which comprises a step of disrupting a gene encoding a mannose acyltransferase of a microorganism capable of producing MEL.
D2.
Item 2. The method according to Item D1, wherein the microorganism capable of producing MEL is a microorganism belonging to the genus Pseudozaima.
D3.
Item 2. The method according to Item D1 or D2, wherein the microorganism capable of producing MEL is a microorganism belonging to Pseudozyma tsukubaensis.
D4.
The method according to any one of Items D1 to D3, wherein the monoacyl type MEL is the monoacyl type MEL according to any one of Items A1 to A4.

In one embodiment, a new monoacyl type MEL having excellent hydrophilicity is provided. In one embodiment, a means for producing a new monoacyl type MEL is provided.

The structure of MEL is shown. The structures of 4-O-β-D-MEL (a) and 1-O-β-D-MEL (b) are shown. The structure of the MAC2 gene disruption vector is shown. The result of thin layer chromatography which confirmed the pattern of MEL produced by the MAC2 gene gene disruption strain is shown. The result of structural analysis of MEL produced by the MAC2 gene disruption strain is shown. The structure of the monoacyl type MEL-D produced by the MAC2 gene disruption strain is shown. n is 4 to 24.

（式中、Ｒ_１は炭素数２〜２４の脂肪族アシル基を表す。Ｒ_２、Ｒ_３及びＲ_４は各々独立して水素原子又はアセチル基を表す。） A. Monoacyl type MEL
A monoacyl type MEL having the structure of the following formula (1) is provided.

(In the formula, R ₁ represents an aliphatic acyl group having ₂ to 24 carbon atoms. R 2, R ₃ and R ₄ each independently represent a hydrogen atom or an acetyl group.)

In one embodiment, the monoacyl type MEL of formula (1), it is preferable that the number of carbon atoms of the aliphatic acyl groups _{R 1} is 4 to 24, more preferably from 8 to 14. In one embodiment, _{the aliphatic acyl group of R 1} can have 3 or more carbon atoms, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 23 or less, 22 or less. , 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, or 13 or less. In one embodiment, in the monoacyl type MEL of the formula (1), it is preferable that _{R 2 is a hydrogen atom.} In one embodiment, in the monoacyl type MEL of the formula (1), _{it is preferable that R 3} is a hydrogen atom. In one embodiment, in the monoacyl type MEL of the formula (1), _{it is preferable that R 4} is a hydrogen atom.

（式中、Ｒ_１は炭素数２〜２４の脂肪族アシル基を表す。） In one preferred embodiment, the monoacyl type MEL has the structure of the following formula (2).

(In the formula, R ₁ represents an aliphatic acyl group having 2 to 24 carbon atoms.)

In the monoacyl type MEL of the formula (2), R ₁ is preferably an aliphatic acyl group having 4 to 24 carbon atoms, and preferably an aliphatic acyl group having 8 to 14 carbon atoms. In one embodiment, _{the aliphatic acyl group of R 1} can have 3 or more carbon atoms, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 23 or less, 22 or less. , 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, or 13 or less.

In one embodiment, the monoacyl type MEL is preferably a 1-O-β-monoacyl type MEL. In one preferred embodiment, the monoacyl type MEL preferably has the structure of the following formula (3).

In the monoacyl type MEL of the formula (3), R ₁ is preferably an aliphatic acyl group having 4 to 24 carbon atoms, and preferably an aliphatic acyl group having 8 to 14 carbon atoms. In one embodiment, _{the aliphatic acyl group of R 1} can have 3 or more carbon atoms, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and 23 or less, 22 or less. , 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, or 13 or less.

The monoacyl type MEL can be produced by any method. For example, in one embodiment, the monoacyl-type MEL can be produced by a microorganism. In other embodiments, the monoacyl type MEL can be produced by chemical synthesis. In one preferred embodiment, the monoacyl-type MEL is produced by subjecting a gene mutation to a microorganism that produces the MEL. A gene mutation is preferably a mutation that deletes an acyltransferase.

B. C. And D. Monoacyl-type MEL-producing microorganisms and production thereof The microorganisms that produce the monoacyl-type MEL according to A above are provided. The type of microorganism that produces monoacyl-type MEL is not particularly limited. In one embodiment, the monoacyl-type MEL-producing microorganism preferably belongs to the genus Pseudozyma, the genus Moijiomyces, the genus Ustilago, the genus Spolisolium, the genus Melanopsichium, or the genus Kurtumanomyces. Preferred Pseudozyma microorganisms are Pseudozyma antarctica, Pseudozyma parantarctica, Pseudozyma rugulosa, Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma aphidis, Pseudozyma hub. Preferred microorganisms of the genus Moesziomyces are Moesziomyces antarcticus, Moesziomyces aphidis. Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis. Preferred Sporisorium spp. are Sporisorium reilianum and Sporisorium scitamineum. A preferred Melanopsichium genus microorganism is Melanopsichium pennsylvanicum. A preferred microorganism of the genus Kurtzmanomyces is Kurtzmanomyces sp. I-11. In a preferred embodiment, the MEL-producing microorganism is a Pseudozyma genus microorganism, more preferably a microorganism belonging to Pseudozyma tsukubaensis, more specifically Pseudozyma tsukubaensis 1E5 (JCM16987 strain), NBRC1940 (ATCC24555, CBS422.96). , CBS6389, DBVPG6988, PYCC4855, JCM10324, MUCL29894, NCYC1510, NRRLY-7792). Microorganisms belonging to Pseudozyma tsukubaensis are known to selectively produce 1-O-β-MEL-B.

In one embodiment, the monoacyl-type MEL-producing microorganism can be obtained by mutating the conventional MEL-producing microorganism. Here, the conventional MEL is a diacyl type MEL. The type of mutation is not particularly limited, but it is preferably a mutation that disrupts the gene encoding the acyltransferase of the MEL-producing microorganism. Gene disruption means that the protein encoded by the gene (for example, acyltransferase) does not function, and the mode thereof is not particularly limited. In one embodiment, a microorganism that produces a monoacyl-type MEL can be obtained by disrupting a gene encoding an acyltransferase possessed by a MEL-producing microorganism. MEL-producing microorganisms generally have two types of mannose acyltransferases (MAC1 and MAC2). MAC1 and MAC2 are acyltransferases that catalyze the reaction of binding fatty acids to the hydroxyl groups at the 2- and 4-positions of mannose. In order to produce a monoacyl-type MEL-producing microorganism, either the gene encoding MAC1 or MAC2 may be disrupted, or both may be disrupted. In one preferred embodiment, it is preferred to disrupt the gene encoding MAC2.

Gene disruption can be performed by any method. For example, gene disruption is carried out by a method of introducing a mutation into the base sequence of the gene, a method of disrupting or deleting an expression control region (promoter, etc.) of the gene, or a method of inhibiting translation of a transcript of the gene. Can be carried out. These include, for example, homologous recombination method, transposon method, transgene method, posttranscription gene silencing method, RNAi method, nonsense mediated decay (NMD) method, ribozyme method, antisense method, miRNA (micro-RNA). ) Method, siRNA (small interfering RNA) method, etc. can be used.

In one embodiment, gene disruption is preferably carried out using a homologous recombination method. Techniques for disrupting genes by homologous recombination are known. For example, for disruption of a target gene by a homologous recombination method, a gene cassette in which a selectable marker gene such as a gene that complements drug resistance or nutritional requirement is inserted in the ORF of the target gene is prepared, and an appropriate vector (for example, for example) is used. , A plasmid), introduced into a host microorganism (for example, a conventional MEL-producing microorganism), and a marker gene is inserted into a target gene by homologous recombination. Microorganisms in which the target gene is disrupted can be selected by expressing the above marker gene.

As the marker gene used in the homologous recombination method, a selectable marker gene of a transformant usually used in genetic engineering can be used. Complementing nutritional requirements such as genes that confer resistance to drugs such as hyglomycin, zeosin, kanamycin, chloramphenicol, and G418, uracil synthase, leucine synthase, adenine synthase, and lysine synthase. Genes can be mentioned.

In one embodiment, the target gene is preferably the MAC2 gene. Examples of typical MAC2 genes are as follows. SEQ ID NO: 1 is a nucleotide sequence encoding an acyltransferase (PaMAC2) derived from the Pseudozyma antaractica T34 strain. SEQ ID NO: 2 is a nucleotide sequence encoding an acyltransferase (PaMAC2) derived from the Pseudozyma antaractica JCM10317 strain. SEQ ID NO: 3 is a nucleotide sequence encoding an acyltransferase (PhMAC2) derived from the Pseudozyma hubeiensis SY62 strain. SEQ ID NO: 4 is a nucleotide sequence encoding an acyltransferase (PtMAC2) derived from the Pseudozyma tsukubaensis NBRC1940 strain. SEQ ID NO: 5 is a nucleotide sequence encoding an acyltransferase (PtMAC2) derived from the Pseudozyma tsukubaensis 1E5 strain. Based on these sequence information, a vector for disrupting the acyltransferase gene can be constructed. The P. antarctica T-34 is also referred to as the "Moesziomyces antarcticus T-34". P. aphidis is also called "Moesziomyces aphidis".

Vectors for the host of the genus Pseudozaima include, for example, pUXV1 ATCC 77463, pUXV2 ATCC 77464, pUXV5 ATCC 77468, pUXV6 ATCC 77469, pUXV7 ATCC 77470, pUXV8 ATCC 77473, pUXV8 ATCC 77473, pUXV8 ATCC Examples thereof include pTA2, pUXV1-neo, pPAX1-neo, pPAA1-neo (Appl Microbiol Biotechnol (2016) 100: 3207-3217) and pUC_neo.

The vector can be introduced into the host cell by any method, and can be appropriately selected depending on the type of the host cell and the vector. The introduction of the vector can be carried out by, for example, electroporation, calcium phosphate co-precipitation method, lipofection, microinjection, lithium acetate method and the like.

The production of monoacyl-type MEL using a gene-disrupted microorganism can be carried out by any method. For example, a monoacyl-type MEL can be produced by culturing a gene-disrupted microorganism in a medium suitable for culturing a MEL-producing microorganism before gene disruption. The medium is not particularly limited, but for example, it is desirable to use sugars such as glucose, sucrose, and molasses as the carbon raw material. In addition to or in place of sugar, fats and oils can be used as a carbon source. The type of fats and oils is not particularly limited, and for example, it is preferable to add vegetable fats and oils, fatty acids or esters thereof. In one embodiment, it is preferable to add vegetable oil to the medium. The type of vegetable oil is not particularly limited, and can be appropriately selected depending on the type of target MEL and the like. For example, soybean oil, olive oil, rapeseed oil, safflower oil, sesame oil, palm oil, sunflower oil, coconut oil, cocoa butter, sunflower oil and the like can be mentioned. Examples of fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, behenic acid, and nervonic acid. In one embodiment, the preferred fatty acid is oleic acid. In one embodiment, a microorganism that produces monoacyl-type MEL can be cultured in a medium containing only glucose as a carbon source. As the nitrogen source, an organic nitrogen source and an inorganic nitrogen source can be used in combination. For example, as the organic nitrogen source, one or a combination of two or more selected from the group consisting of yeast extract, malt extract, peptone, polypeptone, corn steep liquor, casamino acid, and urea can be used. As the inorganic nitrogen source, one type or a combination of two or more types selected from the group consisting of sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, and ammonia can be used. In another embodiment, a method for producing mannosyl erythritol lipid is provided, which comprises culturing a microorganism capable of producing mannosyl erythritol lipid in a medium supplemented with fatty acid and glycerin.

The amounts of fatty acids and fats and oils are not particularly limited, but for example, they can be added so that the concentration in each medium is 0.1 to 20% by volume.

The culture conditions for microorganisms are not particularly limited. For example, when the microorganism belongs to the genus Pseudozaima, it can be cultured for 3 to 7 days under the conditions of pH 5 to 8, preferably pH 6, temperature 20 to 35 ° C., preferably 22 to 28 ° C. The monoacyl type MEL can be recovered from the culture medium according to a conventional method.

E. Monoacyl-type MEL-containing products Monoacyl-type MEL is considered to have higher hydrophilicity than conventional MEL. Therefore, it can be used in fields where conventional MEL is used (for example, cosmetics and pharmaceuticals), and is particularly suitable for use in fields where hydrophilicity is required. In one embodiment, a skin external preparation containing a monoacyl-type MEL is provided. The use of the external preparation for skin is not particularly limited, and for example, it can be used as a cosmetic, a quasi-drug, and / or a pharmaceutical product. Cosmetics containing monoacyl-type MEL include, for example, hair cosmetics such as shampoos, conditioners, and treatments, pigments, cleansing cosmetics, sunscreen cosmetics, packs, massage cosmetics, lotions, milky lotions, creams, and other skin. Examples thereof include make-up cosmetics such as cosmetics, eyeliners, mascara, lipsticks, and foundations, as well as bath cosmetics.

The monoacyl-type MEL-containing external preparation for skin may contain any component other than any monoacyl-type MEL, depending on the intended purpose. Such components include, for example: water; alcohols such as ethanol, fatty acids, coloring pigments such as iron oxide; preservatives such as parabens, phenoxyethanol; olive squalane, rice squalane, sharks. Squalane such as squalane; silicone oil such as dimethylpolysiloxane and cyclic silicone; hydrocarbons such as paraffin, liquid paraffin, vaseline, olefin oligomer, squalane; jojoba oil, olive oil, macadamia nut oil, myristic oil, red flower oil, sunflower oil, Vegetable oils such as avocado oil, canola oil, kyonin oil, rice germ oil, rice bran oil; glyceryl triacetylhydroxystearate, glyceryl triacetyllysinolate, glyceryl triisopalmitate, glyceryl triisostearate, glyceryl triundecanoate, trihydroxy Glyceryl stearate, glyceryl trioleate, tri (capril capric acid) glyceryl, tri (capril caprin myristic acid stearate) glyceryl, tri (capril caprin lauric acid) glyceryl, tri (capril caprin linoleic acid) ) Glyceryl, glyceryl tricaprate, glyceryl tri-beef fatty acid, glyceryl trimyristine, glyceryl tristearate, glyceryl tripalmitate, glyceryl tri2-heptylundecanoate, glyceryl tribehenate, glyceryl trimyristate, glyceryl trimyristic, trilaurin Triglycerides such as synthetic glycerides such as glyceryl acid, glyceryl fatty acid trilanolin, glyceryl 2-ethylhexanoate, glyceryl trilinolex; waxes such as mitsuro, mokuro, carnauba wax; octyldodecyl myristate, cetyl palmitate, isostearyl isostearate, Ester oils such as isopropyl myristic acid; higher alcohols such as cetanol, behenyl alcohol, isostearyl alcohol, jojoba alcohol, oleyl alcohol, stearyl alcohol, long-chain branched fatty acid alcohols; cholesterol, phytosterol, branched fatty acid cholesterol ester, macadamia nut fatty acid phytosteri Sterols such as ruesters and their derivatives; Processing oils such as hardened oils; Higher fatty acids such as stearic acid, myristic acid, iso-type long-chain fatty acids, anteiso-type long-chain fatty acids; Ethers such as dicapryl ether; Limonen, hydrogenated bi Terpenes such as saborol, anionic surfactants such as sodium cetyl sulfate, N-stearoyl-L-glutamate; polyhydric alcohol fatty acid esters (excluding polyoxyethylene hydrogenated castor oil), modified silicones, sugar esters, etc. Nonionic surfactants; Cationic surfactants such as tetraalkylammonium salts; Amphoteric surfactants such as betaine type, sulfobetaine type, sulfoamino acid type; natural systems such as lecithin, lysophosphatidylcholine, ceramide and celebroside. Surfactants; Pigments such as titanium oxide and zinc oxide; Antioxidants such as dibutylhydroxytoluene; Inorganic salts such as sodium chloride, magnesium chloride, sodium sulfate, potassium nitrate; sodium citrate, potassium acetate, sodium amber, aspartic acid Organic acid salts such as sodium, sodium lactate, gamma aminobutyric acid, lipoic acid; salts such as ethanolamine hydrochloride, ammonium nitrate, arginine hydrochloride, chelating agents such as edetic acid; among potassium hydroxide, diisopropanolamine, triethanolamine, etc. Japanese agent; biopolymers such as hyaluronic acid and collagen; placenta extract; ultraviolet absorbers such as hydroxymethoxybenzophenone sulfonate; vitamin A and its derivatives such as retinol, retinol acetate and retinol palmitate; α-tocopherol, γ-tocopherol , Δ tocopherol, tocopherol nicotinate, tocopherol acetate and other vitamin E and its derivatives; oil-soluble vitamin C derivatives such as ascorbyl palmitate, ascorbyl stearate, ascorbyl tetraisostearate; from xanthan gum, beta glucan, oats, white sardines, etc. Extracted polysaccharides, seaweed extracts such as carrageenan and alginic acid, agar, water-soluble polymers such as carboxyvinyl polymers, pectin, alkyl-modified carboxyvinyl polymers; dipropylene glycol, 1,3 butylene glycol, glycerin, propylene Examples thereof include, but are not limited to, polyhydric alcohols such as glycol, sorbitol, malbitol, diglycerin, raffinose and hexylene glycol.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

1. 1. Acquisition of uracil-requiring mutant strain
Pseudozyma tsukubaensis 1E5 strain was inoculated into 3 ml of 1 platinum loop YM medium (Yeast extract 0.3%, Malt extract 0.3%, Peptone 0.5%, Glucose 1%), and 24 in a 10 ml volume test tube at 25 ° C and 180 rpm. It was cultured with shaking for hours. The culture solution was spread on a petri dish, and the plate was placed 45 cm from the UV lamp (Panasonic GL15 germicidal lamp). After irradiation with UV, 0.2 ml of the culture solution was collected. Incubate the collected culture medium at 25 ° C for 3 hours and 5FOA plate (Yeast nitrogen base w / o AA 0.67%, -Ura DO supplement 0.078%, Uracil 0.05%, 5-FOA 0.2%, Glucose 2%, Agar 1.5%. ) Was coated and inoculated. Incubated plates were incubated at 25 ° C. for 6 days to grow colonies. The colonies grown on the 5FOA plate were subcultured in YNB + 5FOA + uracil medium with a toothpick, and some of the cells were subjected to colony PCR and sequence analysis. A PCR primer having the following base sequence was used.
PtURA3coloP_F: AATTAAGCTTCTGTGCGGGGCTCCTTCCTCCTT (SEQ ID NO: 6)
PtURA3coloP_R: CCATTGCTTACTTCAGTCTTGTCTGTGAGTGTGC (SEQ ID NO: 7)
PtURA5coloP_F: ACGTCAATTCTCGTCTCAGCCAAGCAAGAAGGCG (SEQ ID NO: 8)
PtURA5coloP_R: ATCATGACTCTTTCTCCCACTTGGCTCTTCTCAAG (SEQ ID NO: 9)
As a result of sequence analysis, it was confirmed that a mutation was inserted in the URA3 gene. The obtained uracil-requiring mutant was subjected to an auxotrophic test, and it was confirmed that it had uracil-requiring and 5FOA resistance. It was also confirmed that the MEL productivity was maintained.

2. Acquisition of a disrupted strain of the MAC2 gene 2-1. Construction of MAC2 vector
The MAC2 gene region was amplified by PCR using the genomic DNA of the Pseudozyma tsukubaensis 1E5 strain as a template. A PCR primer having the following base sequence was used.
PtMAC2_U2.0K_F: agtgtgggaccgcctcttccgggtgcttctgaagc (SEQ ID NO: 10)
PtMAC2_D2.0K_R: gatttgacgagaagaagtagagtgctcatgttttg (SEQ ID NO: 11)
The amplified DNA fragment was TA cloned using TArget Clone-Plus- (Toyobo Co., Ltd.) to prepare a pTA-MAC2 vector.

2-2. Construction of MAC2 destruction vector
The URA5 gene region was amplified by PCR using the genomic DNA of the Pseudozyma tsukubaensis 1E5 strain as a template, and a URA5 gene fragment was obtained. A PCR primer having the following base sequence was used.
PtURA5U1K_F: CCGAAGGTCATGGTGTTCCCGGTGGCTTGCCATAC (SEQ ID NO: 12)
PtURA5D05K_R: ACAAGCCAGATCAAGTTCGTCATGTCGGCGGTGAA (SEQ ID NO: 13)
Then, the linearized pTA-MAC2 vector was amplified by PCR using the pTA-MAC2 vector as a template, and a gene fragment was obtained. A PCR primer having the following base sequence was used.
PtMAC2_D2KU5_fF acgaacttgatctggcttgtgccgtctttgcttttgctcctgtgttccgc (SEQ ID NO: 14)
PtMAC2_U2KU5_fR gggaacaccatgaccttcggggttgcgatgtagattttccttgacagtgc (SEQ ID NO: 15)
The linearized pTA-MAC2 vector and URA5 gene fragment obtained above were subjected to Fusion (R) HD Cloning Kit (manufactured by TAKARA), transformed into JM109 strain (manufactured by Toyobo), and pTA-MAC2 disruption vector (manufactured by Toyobo). pTA-MAC2 del-ura5) was prepared. It was confirmed that the target fragment was inserted by pTA-MAC2 disruption vector and sequence analysis. The structure of the pTA-MAC2 disruption vector is shown in FIG.

2-3. Preparation of transformant 2-2. Using the pTA-MAC2 disruption vector obtained in 1) amplified by PCR, the electroporation method described above 1. It was transformed into the uracil-requiring mutant strain obtained in. Uracil demand loss was used to select transformants. It was confirmed by colony PCR and sequence analysis that the target DNA fragment was inserted into the target genome position by homologous recombination, and two mutants in which the MAC2 gene was disrupted were obtained.

2-4. Evaluation of MEL production ability of MAC2 gene-disrupted transformants Two strains of MAC2 gene-disrupted transformants were cultured in 60 mL (500 mL volume Sakaguchi flask) of YM medium containing 3% glycerol by shaking at 25 ° C. for 1 day, and pre-cultured. Obtained liquid. Next, 0.2 mL of the preculture solution was inoculated into 20 mL (500 mL volume Sakaguchi flask) of a medium in which 10% olive oil was added to the MEL medium, and the culture was shaken at 25 ° C. for 7 days. An equal amount of ethyl acetate was added to the obtained cell culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate layer was separated. The MEL contained in the ethyl acetate layer was confirmed by thin layer chromatography. As a result, as shown in FIG. 4, the production of MEL-B disappeared for all the mutant strains (# 2 and # 5), and the production of an unknown glycolipid having a lower Rf value than MEL-B was confirmed. It was.

3. 3. Structural analysis of glycolipids An unknown glycolipid fraction detected by thin layer chromatography was extracted with ethyl acetate, and an unknown glycolipid was obtained by heating and depressurizing. The obtained unknown glycolipid was structurally analyzed using the following devices and conditions.
(Analysis 1)
Device: Fourier transform nuclear magnetic resonance device (AVANCE500 manufactured by Bruker Biospin)
Measurement solution: 10 to 100 mg of the sample was dissolved in 0.6 ml of deuterated dimethyl sulfoxide.
1H resonance frequency: 500.13MHz
Flip angle of detection pulse: 45 °
Data acquisition time: 4.0 seconds Delay time: 1.0 seconds Number of integrations: 43 times Measurement temperature: 35 ° C.
(Analysis 2)
Device: Fourier transform nuclear magnetic resonance device (AVANCE500 manufactured by Bruker Biospin)
Measurement solution: 10 to 100 mg of the sample was dissolved in 0.6 ml of deuterated dimethyl sulfoxide.
13C resonance frequency: 125.76MHz
Flip angle of detection pulse: 45 °
Data acquisition time: 2.0 seconds Delay time: 0.5 seconds Proton decoupling: Full decouple.
Measurement temperature: Room temperature integration frequency: 4096 times

The results of the structural analysis are shown in 5A and 5B. From the result of 1H-NMR in FIG. 5A, the hydroxyl group at the 3-position of mannose is free, and the peak of the proton is observed around 3.5 ppm, whereas at the 2-position, the fatty acid is ester-bonded to the hydroxyl group. As a result, the peak of the proton was shifted to around 5.2 ppm by a low magnetic field, and it was found that the substance produced by any MAC2 gene-deficient strain was a monoacyl-type MEL-D having the structure shown in FIG. Similarly, from the results of 13C-NMR in FIG. 5B, the shift of the carbon peaks at the 2nd and 3rd positions of mannose was confirmed. From the above analysis results, it was found that the substances produced by any of the MAC2 gene-deficient strains were monoacyl-type MEL-D having the structure shown in FIG. As described above, it was confirmed that a new monoacyl-type MEL-D is produced by disrupting the MAC2 gene of the Pseudozyma tsukubaensis 1E5 strain that originally selectively produces MEL-B.

Claims (5)

A monoacyl type MEL having the structure of the following formula (1).

(In the formula, R ₁ represents an aliphatic acyl group having ₂ to 24 carbon atoms. R 2, R _{3 and} R ₄ each independently represent a hydrogen atom or an acetyl group.) A microorganism of the genus Pseudozaima in which the MAC2 gene is disrupted, which produces the monoacyl-type MEL according to claim 1. The microorganism according to claim 2, wherein the microorganism of the genus Pseudozyma is Pseudozyma tsukubaensis, Pseudozyma antarctica, Pseudozyma parantarctica, Pseudozyma aphidis, or Pseudozyma rugulosa. The microorganism according to claim 2 or 3, wherein the microorganism of the genus Pseudozyma is Pseudozyma tsukubaensis. A method for producing a monoacyl type MEL using the microorganism according to any one of claims 2 to 4.

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