Preparation of monoacyl-MEL

Abstract

The present invention provides a means for efficiently producing a monoacyl MEL. This means comprises culturing a monoacyl MEL-producing microorganism in the presence of a surfactant.

Description

[Technical field]

[0001] A technique for producing monoacyl-MEL using microorganisms is disclosed. [Background technology]

[0002] Biosurfactants are natural surfactants produced by microorganisms, which are highly biodegradable, have low environmental impact, and have various beneficial physiological functions. Therefore, their use in the food, cosmetics, pharmaceutical, chemical, and environmental industries is meaningful in realizing an environmentally harmonious society.

[0003] Biosurfactants are classified into five types: glycolipids, acyl peptides, phospholipids, fatty acids, and polymers. Among these, glycolipid surfactants have been most extensively studied. Known examples of such glycolipid biosurfactants include mannosylerythritol (hereinafter also referred to as ME), in which erythritol is glycoside-bonded to mannose, and mannosylerythritol lipid (hereinafter also referred to as MEL), in which fatty acids are further ester-bonded to mannosylerythritol lipid (hereinafter also referred to as MEL), as well as rhamnolipid, ustyladic acid, trehalose lipid, and sophorose lipid.

[0004] MEL has various structures that differ in the position and number of fatty acid residues and acetyl groups bound to it. FIG. 1 shows the structural formula of MEL, in which hydrogen atoms, acetyl groups, and fatty acid residues with 3 to 18 carbon atoms are indicated by R1 to R5. A structure in which R1 and R2 are fatty acid residues and R3 and R4 are acetyl groups is defined as MEL-A, a structure in which R3 is a hydrogen atom and R4 is an acetyl group is defined as MEL-B, a structure in which R3 is an acetyl group and R4 is a hydrogen atom is defined as MEL-C, and a structure in which R3 and R4 are hydrogen atoms is defined as MEL-D. Depending on whether the hydroxymethyl group of erythritol bound to mannose is derived from the 1st carbon or the 4th carbon, the structure of the obtained ME differs as shown in FIG. 2(a) and (b). MEL having 4-O-β-D-mannopyranosyl-erythritol as the sugar skeleton shown in FIG. 2(a) is called 4-O-β-D-MEL. Pseudozyma tsukubaensis is known to produce 1-O-β-D-MEL-B, which has 1-O-β-D-mannopyranosyl-erythritol as the sugar skeleton, as shown in Figure 2(b). 1-O-β-MEL-B has improved hydration and vesicle-forming ability compared to 4-O-β-MEL-B, making it a promising biomaterial for use in skin care products.

[0005] It has been reported that by culturing MEL-producing bacteria using glucose as the only carbon source, monoacyl-type MEL (single-chain MEL) is produced, in which a fatty acid is bound only to R2 in MEL in Figure 1, and R1, R3, and R4 are hydrogen atoms (Non-Patent Document 1). This monoacyl-type MEL has improved hydrophilicity compared to conventional diacyl-type MEL (Non-Patent Document 1).

[0006] The MEL biosynthetic pathway has already been reported, in which MEL is synthesized intracellularly through the reactions of glycosyltransferases that link mannose and erythritol, acyltransferases that link fatty acids, and acetyltransferases that link acetyl groups (Non-Patent Document 2).

[0007] The present inventors discovered that by deleting the gene for acyltransferase in a microorganism capable of producing a biosurfactant, it is possible to obtain monoacyl-type MEL in which an aliphatic acyl group is bound only to R1 in the structural formula shown in Figure 1 (Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent application 2016-191438 [Non-patent literature]

[0009] [Non-Patent Document 1] Fukuoka et al., Appl. Microbiol. Biotechnol. (2007) 76: 801-810. [Non-Patent Document 2] Hewald et al., Appl. Environ. Microbiol. (2006) 72: 5469-5477 Summary of the Invention [Problem to be solved by the invention]

[0010] One challenge is to efficiently produce monoacyl-MEL. [Means for solving the problem]

[0011] As a result of intensive research aimed at solving such problems, it was found that the production efficiency of monoacyl-MEL can be improved by adding a surfactant to the medium during the cultivation of a monoacyl-MEL-producing bacterium. Based on this knowledge, further research and studies were carried out, and the following representative inventions are provided.

[0012] Item 1 A method for producing monoacyl-MEL, comprising culturing a microorganism capable of producing monoacyl-MEL in the presence of a surfactant. Section 2 Item 3. The method according to Item 1, wherein the surfactant is a nonionic surfactant. Section 3 Item 3. The method according to Item 1 or 2, wherein the microorganism is a microorganism belonging to the genus Pseudozyma. Section 4 Item 4. The method according to any one of Items 1 to 3, wherein the microorganism is deficient in a gene encoding mannose acyltransferase. Section 5 Item 5. The method according to any one of Items 1 to 4, further comprising a step of extracting monoacyl-MEL with one or more selected from the group consisting of methanol, ethanol, acetone, and a mixture thereof. Section 6 A monoacyl-type MEL production ability enhancer for a microorganism having monoacyl-type MEL production ability, comprising a surfactant. Section 7 Use of a surfactant for improving the ability of a microorganism having an ability to produce monoacyl-MEL to produce monoacyl-MEL. Effect of the Invention

[0013] The production efficiency of monoacyl-MEL can be improved. [Brief description of the drawings]

[0014] [Figure 1] The structure of MEL is shown below. [Diagram 2] The structures of 4-O-β-D-mannopyranosyl-erythritol (a) and 1-O-β-D-mannopyranosyl-erythritol (b) are shown. [Diagram 3] The results of thin-layer chromatography of monoacyl-type MEL extracted with ethyl acetate and methanol are shown. [Figure 4] The results of evaluation of monoacyl-type MEL extracted with methanol, ethanol, and acetone by thin-layer chromatography are shown. [Diagram 5] The results of evaluating the amount of monoacyl-MEL produced by a monoacyl-MEL-producing bacterium when it was cultured in a medium containing no surfactant, using thin-layer chromatography, are shown. [Figure 6] The results of thin-layer chromatography evaluation of the amount of bacterial cell growth (a) and the amount of monoacyl-MEL produced (b) when a monoacyl-MEL-producing bacterium was cultured in a medium supplemented with MEL-B are shown. [Figure 7] The results of thin-layer chromatography evaluation of the amount of bacterial cell growth (a) and the amount of monoacyl-MEL produced (b) when a monoacyl-MEL producing bacterium was cultured in a medium supplemented with Tween 20 are shown. [Figure 8] The results of thin-layer chromatography evaluation of the amount of monoacyl-MEL produced when a monoacyl-MEL-producing bacterium was cultured in a medium supplemented with Triton X-100 are shown. [Figure 9] The results of the evaluation of the amount of monoacyl-MEL produced by monoacyl-MEL-producing bacteria cultured in a medium supplemented with BRIJ35 using thin-layer chromatography are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] When culturing a microorganism that produces monoacyl-type MEL, it is preferable to culture in the presence of a surfactant. The type of surfactant is not particularly limited and can be any. For example, the surfactant can be a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant. In one embodiment, the surfactant is preferably a nonionic surfactant. Examples of the nonionic surfactant include ether type, ester type, and ester-ether type. In one embodiment, the nonionic surfactant is preferably an ether type. Examples of the ether-type nonionic surfactant include polyoxyethylene-based surfactants (e.g., polyoxyethylene octylphenyl ether (TritonX-100), polyoxyethylene sorbitan monolaurate (Tween20), and polyoxyethylene lauryl ether (BRIJ35)), block polymer-based surfactants (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene alkyl ether), MEL, sophorolipid, rhamnolipid, and trehalose lipid. Any one or more of these surfactants can be used in combination. In one embodiment, the surfactant is preferably an anionic surfactant. Examples of anionic surfactants include carboxylic acid surfactants (e.g., sodium octanoate, sodium decanoate, sodium laurate, sodium myristate, sodium palmitate, sodium stearate, etc.), sulfonic acid surfactants (e.g., sodium 1-hexanesulfonate, sodium 1-octanesulfonate, sodium 1-decanesulfonate, sodium 1-dodecanesulfonate, perfluorobutanesulfonic acid, sodium toluenesulfonate, etc.), and phosphate surfactants (e.g., lauryl phosphoric acid, sodium lauryl phosphate, potassium lauryl phosphate, etc.). Any one or more of these surfactants can be used in combination.

[0016] The manner of culturing the monoacyl-type MEL-producing microorganism in the presence of a surfactant is not particularly limited. For example, a surfactant can be added to a liquid medium, and the monoacyl-type MEL-producing microorganism can be cultured in the medium. The amount of surfactant added to the medium is not particularly limited, and can be appropriately set in consideration of the type of surfactant, the type of microorganism, the type of medium, and the like. For example, the surfactant can be set so that the concentration in the medium is 0.0001% by mass to 1% by mass, 0.001% by mass to 1% by mass, 0.0001% by mass to 0.1% by mass, 0.001% by mass to 0.1% by mass, 0.01% by mass to 0.1% by mass, 0.0001% by mass to 0.05% by mass, 0.001% by mass to 0.05% by mass, 0.0001% by mass to 0.01% by mass, or 0.001% by mass to 0.01% by mass.

[0017] Conventional ethyl acetate is not suitable as a solvent for extracting monoacyl-MEL from the culture medium. In one embodiment, monoacyl-MEL can be extracted with methanol, ethanol, or acetone after freeze-drying the culture medium.

[0018] The type of microorganism that produces monoacyl-type MEL is not particularly limited. For example, the microorganism that produces monoacyl-type MEL described in Patent Document 1 can be used. The patent document is incorporated herein in its entirety by reference. In one embodiment, the microorganism that produces monoacyl-type MEL preferably belongs to the genus Pseudozyma, Moidiomyces, Ustilago, Sporisorium, Melanopsichium, or Kurtzmannomyces. Preferred Pseudozyma microorganisms are Pseudozyma antarctica (Moesziomyces antarcticus), Pseudozyma parantarctica, Pseudozyma rugulosa, Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma aphidis (Moesziomyces aphidis), Pseudozyma hubeiensis, and Pseudozyma tsukubaensis. Preferred Moesziomyces microorganisms are Moesziomyces antarcticus and Moesziomyces aphidis. Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis. Preferred Sporisorium microorganisms are Sporisorium reilianum and Sporisorium scitamineum. A preferred microorganism of the genus Melanopsichium 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 microorganism of the genus Pseudozyma, more preferably a microorganism belonging to Pseudozyma tsukubaensis, and more specifically, Pseudozyma tsukubaensis 1E5 (JCM16987 strain) or 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.

[0019] In one embodiment, the monoacyl MEL-producing microorganism can be obtained by mutating a microorganism that produces conventional MEL. Here, conventional MEL is diacyl MEL. The type of mutation is not particularly limited, but preferably, it is a mutation that disrupts a gene encoding an acyltransferase contained in the MEL-producing microorganism. Disruption of a gene means that a protein (e.g., acyltransferase) encoded by the gene becomes non-functional, and the manner is not particularly limited. In one embodiment, a microorganism that produces monoacyl MEL can be obtained by disrupting a gene encoding an acyltransferase contained in the MEL-producing microorganism. MEL-producing microorganisms generally have two types of mannose acyltransferase (MAC1 and MAC2). MAC1 and MAC2 are acyltransferases that catalyze the reaction of binding a fatty acid to the hydroxyl groups at the 2- and 3-positions of mannose. To produce a microorganism that produces monoacyl MEL, either the gene encoding MAC1 or MAC2 may be disrupted, or both may be disrupted. In a preferred embodiment, it is preferable to disrupt the gene encoding MAC2.

[0020] Gene disruption can be carried out by any method. For example, gene disruption can be carried out by a method of introducing a mutation into the base sequence of the gene, a method of disrupting or deleting the expression control region (promoter, etc.) of the gene, or a method of inhibiting the translation of the transcription product of the gene. These can be carried out using, for example, the homologous recombination method, the transposon method, the transgene method, the post-transcriptional gene silencing method, the RNAi method, the nonsense mediated decay (NMD) method, the ribozyme method, the antisense method, the miRNA (micro-RNA) method, the siRNA (small interfering RNA) method, etc.

[0021] In one embodiment, gene disruption is preferably performed using a homologous recombination method. Techniques for disrupting genes by homologous recombination are known. For example, target gene disruption by homologous recombination is a method in which a gene cassette is created by inserting a selection marker gene, such as a gene that complements drug resistance or auxotrophy, into the ORF of the target gene, and the gene cassette is incorporated into an appropriate vector (e.g., a plasmid), which is then introduced into a host microorganism (e.g., a conventional MEL-producing microorganism), and the marker gene is inserted into the target gene by homologous recombination. A microorganism in which the target gene has been disrupted can be selected by the expression of the marker gene.

[0022] The marker gene used in the homologous recombination method may be a selectable marker gene for transformants that is commonly used in genetic engineering, such as genes that confer resistance to drugs such as hygromycin, zeocin, kanamycin, chloramphenicol, and G418, and genes that complement auxotrophy such as uracil synthase, leucine synthase, adenine synthase, and lysine synthase.

[0023] In one embodiment, the target gene is preferably a MAC2 gene. Representative examples of MAC2 genes are as follows: SEQ ID NO: 1 is a nucleotide sequence encoding an acyltransferase (PaMAC2) derived from Pseudozyma antaractica T34 strain; SEQ ID NO: 2 is a nucleotide sequence encoding an acyltransferase (PaMAC2) derived from Pseudozyma antaractica JCM10317 strain; SEQ ID NO: 3 is a nucleotide sequence encoding an acyltransferase (PhMAC2) derived from Pseudozyma hubeiensis SY62 strain; SEQ ID NO: 4 is a nucleotide sequence encoding an acyltransferase (PtMAC2) derived from Pseudozyma tsukubaensis NBRC1940 strain; SEQ ID NO: 5 is a nucleotide sequence encoding an acyltransferase (PtMAC2) derived from Pseudozyma tsukubaensis 1E5 strain; SEQ ID NO: 6 is a nucleotide sequence encoding an acyltransferase (MAC2) derived from Pseudozyma aphidis DSM70725 strain. Based on this sequence information, a vector for disrupting the acyltransferase gene can be constructed. P. antarctica T-34 is also called "Moesziomyces antarcticus T-34". P. aphidis is also called "Moesziomyces aphidis".

[0024] Examples of vectors for use with the genus Pseudozyma as a host include pUXV1 ATCC 77463, pUXV2 ATCC 77464, pUXV5 ATCC 77468, pUXV6 ATCC 77469, pUXV7 ATCC 77470, pUXV8 ATCC 77471, pUXV3 ATCC 77465, pU2X1 ATCC 77466, pU2X2 ATCC 77467, pTA2, pUXV1-neo, pPAX1-neo, pPAA1-neo (Appl Microbiol Biotechnol (2016) 100:3207-3217, pUC_neo and pUC T Examples include _neo.

[0025] The vector can be introduced into the host cell by any method, which can be appropriately selected depending on the type of the host cell and the vector, etc. The vector can be introduced by, for example, electroporation, calcium phosphate coprecipitation method, lipofection, microinjection, lithium acetate method, etc.

[0026] The production of monoacyl-MEL using a microorganism that produces monoacyl-MEL can be carried out by any method. For example, monoacyl-MEL can be produced by culturing the MEL-producing microorganism in a medium suitable for culturing. Such a medium is not particularly limited, but for example, it is desirable to use carbohydrates such as glucose, sucrose, and blackstrap molasses as the carbon source. In addition to or instead of carbohydrates, fats and oils can also be used as carbon sources. The type of fats and oils is not particularly limited, and it is preferable to add, for example, vegetable fats and oils, or fatty acids or their esters.

[0027] 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 MEL of interest. Examples of the vegetable oil include soybean oil, olive oil, rapeseed oil, safflower oil, sesame oil, palm oil, sunflower oil, coconut oil, cocoa butter, and castor oil. 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.

[0028] In one embodiment, a microorganism producing monoacyl-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 more selected from the group consisting of yeast extract, malt extract, peptone, polypeptone, corn steep liquor, casamino acid, and urea can be used in combination.

[0029] The inorganic nitrogen source may be one or a combination of two or more selected from the group consisting of sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, and ammonia. In another embodiment, there is provided a method for producing monoacyl-MEL, comprising culturing a microorganism capable of producing monoacyl-MEL in a medium supplemented with fatty acid and glycerin.

[0030] The amounts of fatty acids and oils and fats are not particularly limited, but for example, each can be added so that the concentration in the medium is 0.1 to 40% by volume.

[0031] The culture conditions for the microorganism are not particularly limited. For example, the microorganism can be cultured at pH 5 to 8, preferably pH 6, at a temperature of 20 to 35° C., preferably 22 to 28° C., for 3 to 7 days.

[0032] The monoacyl-MEL produced by a microorganism capable of producing monoacyl-MEL can be extracted by any method. For example, the culture solution or cell lysate is centrifuged, the supernatant is collected, an appropriate extraction solvent is added to the supernatant, the extraction solvent layer is collected, and further purification is performed as necessary to obtain monoacyl-MEL. In one embodiment, the extraction solvent used for extracting monoacyl-MEL is preferably one or more selected from the group consisting of methanol, ethanol, acetone, and mixtures thereof, from the viewpoint of extraction efficiency. Acetone is preferable from the viewpoint of efficiently separating hydrophilic impurities from monoacyl-MEL to obtain monoacyl-MEL.

[0033] As described above, by using a surfactant, the production efficiency (capacity) of monoacyl-type MEL by microorganisms can be improved, and monoacyl-type MEL can be obtained more stably. Thus, in one embodiment, a surfactant can be used as a promoter of monoacyl-type MEL production by monoacyl-type MEL-producing microorganisms. In another embodiment, a surfactant can be used to promote the production of monoacyl-type MEL by monoacyl-type MEL-producing microorganisms and stabilize monoacyl-type MEL production. EXAMPLES

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] 1.Material ·Bacteria used Monoacyl-type MEL producing strain: Pseudozyma tsukubaensis 1E5 strain (JCM16987) acyltransferase (PtMAC2) disruptant Surfactants MEL-B (Pseudozyma tsukubaensis culture purified product) Tween20 (SIGMA) TritonX-100 (MP Biomedicals) BRIJ35 (MP Biomedicals) Sodium laurate (Fujifilm Wako Pure Chemical Industries, Ltd.) Culture medium Glycerol-added YM medium: prepared by dissolving 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and 50 g of glycerol in 1 L of deionized water. MEL production medium: Prepared by dissolving 5 g of yeast extract, 3 g of sodium nitrate, 0.3 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate heptahydrate, 20 g of glycerol, and a surfactant (described below) in 1 L of deionized water.

[0036] 2. Study of extraction solvents for monoacyl-MEL 2-1. Cultivation of monoacyl-MEL producing strains The monoacyl-type MEL producing strain was cultured in 2 mL of glycerol-added YM medium at 25°C for 2 days with shaking to obtain a preculture solution. Next, 1 mL of the preculture solution was inoculated into 20 mL of MEL production medium (containing 0.005% TritonX-100) supplemented with 3% olive oil, and cultured at 25°C for 7 days with shaking. On the third day of culture, 2% olive oil was added (total amount of added oil was 5%).

[0037] 2-2. Extraction of monoacyl-MEL Extraction of monoacyl-MEL from the culture medium was tested using various solvents (ethyl acetate, methanol, ethanol, acetone). The culture medium obtained in 2-1 above was centrifuged at 3,000 rpm to collect the culture supernatant. Ethyl acetate was added directly to the collected supernatant, and the ethyl acetate layer containing monoacyl-MEL was collected and used as the ethyl acetate extract. In the extraction with methanol, the first collected supernatant was frozen at -20°C and then freeze-dried. Methanol was added to the dried supernatant, which was stirred with a vortex mixer and then allowed to stand overnight at room temperature. After standing, the methanol layer was collected and filtered through a 0.45 μm filter to obtain the methanol extract. Extraction with ethanol or acetone was also performed in the same manner as for methanol.

[0038] 2-3. Evaluation of monoacyl-MEL content in extracts The amount of monoacyl-MEL contained in each solvent extract was analyzed by thin-layer chromatography (TLC). The developing phase was chloroform:methanol:12% ammonia water = 55:25:2. After development, the TLC plate was sprayed with 2% anthrone sulfate reagent and heated at 95°C for 5 minutes to detect the spots of monoacyl-MEL.

[0039] As shown in Figure 3, it was confirmed that the amount of monoacyl-MEL extracted by ethyl acetate was smaller than that by methanol extraction. The fact that the spot of monoacyl-MEL in the methanol extract was separated into two is thought to be due to the influence of water in the solvent.

[0040] Similarly, the results of extraction with ethanol and acetone are shown in Figure 4. As with methanol, it was confirmed that ethanol and acetone can extract monoacyl MEL more efficiently than ethyl acetate. In particular, with acetone extraction, the highly hydrophilic impurities detected at the origin with methanol and ethanol extraction were hardly detected, and it was confirmed that only monoacyl MEL can be extracted with higher purity. Also, unlike methanol and ethanol, it was confirmed that a single spot of monoacyl MEL was detected.

[0041] 3. Evaluation of monoacyl-MEL production 3-1. Cultivation of monoacyl-MEL producing strains The monoacyl-type MEL producing strain was cultured in 2 mL of glycerol-supplemented YM medium at 25°C for 2 days with shaking to obtain a preculture solution. Next, 1 mL of the preculture solution was inoculated into 20 mL of MEL production medium supplemented with 3% olive oil, and cultured at 25°C for 7 days with shaking. On the third day of culture, 2% olive oil was added (total amount of added oil: 5%). The MEL production medium used was one that did not contain a surfactant, and one that contained a surfactant (MEL-B, Tween20, TritonX-100, or BRIJ35) at a concentration of 0.01% or 0.1%.

[0042] 3-2. Extraction of monoacyl-MEL The culture medium obtained in 3-1 above was centrifuged at 3,000 rpm to recover the culture supernatant. The recovered supernatant was frozen at -20°C and then freeze-dried. Acetone was added to the dried supernatant, which was then stirred with a vortex mixer and allowed to stand overnight at room temperature. After standing, the acetone layer was recovered and filtered through a 0.45 μm filter to obtain the acetone extract.

[0043] 3-3. Evaluation of monoacyl-MEL content in extracts The amount of monoacyl-MEL contained in each acetone extract was analyzed by thin-layer chromatography (TLC). The developing phase was chloroform:methanol:12% ammonia water = 55:25:2. After development, the TLC plate was sprayed with 2% anthrone sulfate reagent and heated at 95°C for 5 minutes to detect the spots of monoacyl-MEL.

[0044] 3-4. Production of monoacyl-MEL in the absence of surfactant The results of culturing the monoacyl-MEL producing strain three times in a MEL production medium without the addition of surfactant are shown in Figure 5 (n=3 for each culture). The TLC plates on the left, center, and right in Figure 5 represent the results of the first, second, and third cultures, respectively. As shown in Figure 5, it was confirmed that under surfactant-free conditions, monoacyl-MEL was not produced (third culture) or was produced in very small amounts (first culture). Even when culture was performed with n=3, there were cases where monoacyl-MEL was detected only in one sample (second culture), confirming that production was unstable. The average amount of bacterial cell growth was 21.9±2.8g / L for the three cultures.

[0045] 3-5. Production of monoacyl-MEL under MEL-B addition conditions The results of culturing a monoacyl-MEL producing strain in a MEL production medium supplemented with MEL-B are shown in Figure 6. In Figure 6, (a) shows the amount of bacterial cell growth, and (b) shows the results of TLC analysis. As shown in Figure 6, the addition of 0.01% and 0.1% MEL-B significantly improved the amount of monoacyl-MEL produced compared to the non-supplemented condition, and it was confirmed that monoacyl-MEL was stably produced in all cultures. The amount of bacterial cell growth was also higher than that in the non-supplemented condition.

[0046] 3-6. Production of monoacyl-MEL under Tween 20 conditions The results of culturing the monoacyl-MEL producing strain in MEL production medium supplemented with Tween 20 are shown in Figure 7. In Figure 7, (a) shows the amount of bacterial cell growth, and (b) shows the results of TLC analysis. As shown in Figure 7, the addition of Tween 20 improved the amount of monoacyl-MEL produced compared to the non-supplemented condition, and it was confirmed that monoacyl-MEL was stably produced in all cultures. The amount of bacterial cell growth was also higher than that in the non-supplemented condition.

[0047] 3-7. Production of monoacyl-MEL under TritonX-100 supplementation conditions The results of culturing a monoacyl-MEL producing strain in a MEL production medium supplemented with TritonX-100 are shown in Figure 8. As shown in Figure 8, the production amount of monoacyl-MEL was significantly improved by adding 0.01% TritonX-100 compared to the non-supplemented condition, and it was confirmed that monoacyl-MEL was stably produced in all cultures. In addition, the bacterial cell growth rate was 25.0 ± 0.4 g / L, which was higher than the non-supplemented condition.

[0048] 3-8. Monoacyl-MEL production under BRIJ35 addition conditions The results of culturing the monoacyl-MEL producing strain in the MEL production medium supplemented with BRIJ35 are shown in Figure 9. As shown in Figure 9, the production amount of monoacyl-MEL was improved by adding 0.01% BRIJ35 compared to the non-supplemented condition, and it was confirmed that monoacyl-MEL was stably produced in all cultures. The bacterial cell growth amount was 20.6 ± 0.4 g / L.

[0049] 3-9. Production of monoacyl-MEL under sodium laurate added conditions When the monoacyl MEL-producing strain was cultured in a MEL production medium containing 0.01% sodium laurate, an anionic surfactant, in the same manner as in the above test, it was confirmed that the amount of monoacyl MEL produced increased and the production of monoacyl MEL was stabilized compared to when no surfactant was added.

[0050] It is presumed that the addition of MEL-B to the medium promoted the emulsification and dispersion of the raw oil, thereby promoting the production of monoacyl-type MEL.

Claims

1. A method for producing monoacyl-MEL, comprising culturing a microorganism capable of producing monoacyl-MEL in the presence of one or more surfactants selected from the group consisting of polyoxyethylene-based surfactants and carboxylic acid-based surfactants.

2. The method according to claim 1 , wherein the microorganism is a microorganism belonging to the genus Pseudozyma.

3. The method according to claim 1 or 2, wherein the microorganism is deficient in a gene encoding mannose acyltransferase.

4. The method according to any one of claims 1 to 3, further comprising the step of extracting the monoacyl-MEL with one or more selected from the group consisting of methanol, ethanol and acetone, and mixtures thereof.

5. An agent for improving the production ability of monoacyl-type MEL in a microorganism having an ability to produce monoacyl-type MEL, comprising one or more surfactants selected from the group consisting of polyoxyethylene-based surfactants and carboxylic acid-type surfactants.

6. 2. Use of one or more surfactants selected from the group consisting of polyoxyethylene surfactants and carboxylic acid surfactants for improving the ability of a microorganism having an ability to produce monoacyl-type MEL.

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