Wickerhamomyces spp. microorganisms with high productivity of phytosphingosine derivatives

Abstract

The present invention relates to a microorganism having the ability to produce phytosphingosine derivatives and having resistance to cerulenin and / or EGTA, a composition containing the same, and a method for producing phytosphingosine derivatives using the same.

Description

Technology Field

[0001] The present invention relates to a microorganism having high productivity for phytosphingosine derivatives, such as tetraacetylphytosphingosine (TAPS) and / or triacetylphytosphingosine (TriAPS), a method for producing the same, and a method for producing TAPS and TriAPS using the same, and more specifically, to a genus of Wicker Harmomyses (which is resistant to cerulenine and / or ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA) Wickerhamomyces The invention relates to a microorganism (spp), a composition containing the same, and a method for producing a phytosphingosine derivative using the same. Background Technology

[0002] Ceramide is a type of sphingolipid with a structure in which fatty acids are linked to sphingosine. It accounts for approximately 40% of the intercellular lipids of keratinocytes that make up the stratum corneum of the skin and functions as a lipid barrier that inhibits water evaporation and maintains the orderly structure of the stratum corneum. Since the stratum corneum consists of keratinized cells arranged in a brick-like multilayer structure and these cells are tightly bound by ceramide, cholesterol, and free fatty acids, it has been reported that applying preparations containing ceramide to the skin helps improve skin barrier function and skin hydration (Comparative Study on Skin Hydrating Effects and Barrier Function of Natural Ceramide and Ceramide-like Supposites, Kwon Minsu, 2005).

[0003] Ceramide can be produced from yeast and fungi, but among these, the yeast Wickerharmonyces ciferi ( Wickerhamomyces ciferrii ; Pikia Siperi ( Pichia ciferrii(also named) has the characteristic of synthesizing tetraacetylphytosphingosine (TAPS), an acetylated derivative of phytosphingosine, from palmiotyl-CoA and the amino acid L-serine and secreting it outside the cell.

[0004] Phytosphingosine secreted outside the cell by Wicker Harmomyces ciferi is in a form substituted with acetyl groups, and depending on the number of substituted acetyl groups, tetraacetylphytosphingosine, triacetylphytosphingosine, or diacetylphytosphingosine have been reported. TAPS is converted into phytosphingosine through the process of removing acetyl groups, and phytosphingosine has industrial value as it can be used as a precursor required for ceramide synthesis.

[0005] In 1960, Wickerham and Stodola (LJ Wickerham and FH Stodola) [published] the yeast Hansenula ciferi ( Hansenula ciferrii It was first discovered that NRRL 14091 secretes sphingolipids out of the cell and forms crystals around solid media. However, it produced almost no sphingolipids when cultured in liquid media (J. Bacteriol. 80: 484-491, 1960). On the other hand, the diploid F-60-10 (NRRL 1301), isolated from the spores of the diploid 14091, was reported to secrete and produce a significant amount of sphingolipids even in liquid media.

[0006] Wicker Harmomyces ciferi is a yeast that produces acetylated phytosphingosine, and among them, it produces the most TAPS. Research has been attempted to increase the content and productivity of acetylated phytosphingosine within the strain through regulation via genetic engineering and metabolic engineering. For example, Korean Patent No. 10-0287483 discloses a technology for improving the production capacity of TAPS using a Pichia ciferi mutant strain into which a GAPDH-enhanced promoter is inserted. The problem to be solved

[0007] The present invention provides a microorganism having the ability to produce phytosphingosine derivatives and resistance to cerulenin.

[0008] The present invention provides a composition for producing a phytosphingosine derivative comprising one or more selected from the group consisting of the microorganism, the cell of the microorganism, the lysate of the cell of the microorganism, the culture of the microorganism, and extracts thereof.

[0009] The present invention provides a method for producing a phytosphingosine derivative, comprising the step of culturing the microorganism. means of solving the problem

[0010] The present invention relates to a microorganism of the genus Wickerhamomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin.

[0011] The above microorganisms may additionally have resistance to ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA).

[0012] The above microorganism is Wicker Harmomyces ciferi ( Wickerhamomyces ciferrii It can be.

[0013] The present invention provides a composition for producing a phytosphingosine derivative comprising one or more selected from the group consisting of the microorganism, the cell of the microorganism, the lysate of the cell of the microorganism, the culture of the microorganism, and extracts thereof.

[0014] The above composition may additionally include one or more selected from the group consisting of cerulenin and EGTA, but is not limited thereto.

[0015] The present invention provides a method for producing a phytosphingosine derivative, comprising the step of culturing the microorganism.

[0016] The microorganism provided by the present invention may have superior phytosphingosine productivity compared to wild-type microorganisms (e.g., wild-type Wicker Harmomyces ciferi).

[0018] The present application will be described in more detail below.

[0020] The present invention relates to a microorganism of the genus Wicker Harmomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin and / or EGTA ( Wickerhamomyces. Sp It provides ).

[0021] The above phytosphingosine derivative may be one or more selected from the group consisting of monoacetylphytosphingosine, diacetylphytosphingosine, triacetylphytosphingosine (TriAPS), and tetraacetylphytosphingosine (TAPS), and specifically may be triacetylphytosphingosine (TriAPS) and / or tetraacetylphytosphingosine (TAPS).

[0022] The above phytosphingosine derivative production capacity is one that has high production capacity in terms of TAPS and / or TriAPS production (e.g., production per unit culture volume (L), mg / L) and / or TAPS and / or TriAPS production per unit cell dry weight (e.g., production per unit cell dry weight, mg / g-cell).

[0023] The phytosphingosine derivative production of the microorganism provided by the present invention, such as the production of TAPS and / or TriAPS (e.g., production per unit culture volume (L), mg / L) and / or the production of TAPS and / or TriAPS per unit cell dry weight (e.g., production per unit cell dry weight, mg / g-cell), may be the result of measuring after culturing at 30°C for 96 hours under aerobic conditions, and specifically, may be measured by shaking culture in YMGL medium and extracting TAPS and / or TriAPS, but is not limited thereto. The YMGL medium may include glycerol, yeast extract, malt extract, and peptone.

[0024] The production yield of each type of phytosphingosine derivative of the microorganism provided by the present invention can be confirmed by culturing and / or fermenting the microorganism and extracting the phytosphingosine derivative through HPLC analysis, but is not limited thereto.

[0025] The TAPS production of the above microorganism may be 180% or more, 200% or more, 220% or more, 240% or more, 260% or more, 280% or more, 300% or more, 320% or more, e.g., 326%, based on 100% of the TAPS production (mg / L) of wild-type Wickerharmonyces ciferi, but is not limited thereto.

[0026] The TAPS production per unit cell dry weight (mg / g-cell) of the above microorganism may be 15 mg or more, 20 mg or more, 25 mg or more, 30 mg or more, or 33 mg or more, e.g., 35 mg, but is not limited thereto.

[0027] The TAPS production per unit cell dry weight (mg / g-cell) of the above microorganism may be 160% or more, 180% or more, 200% or more, 220% or more, 240% or more, 260% or more, 280% or more, 300% or more, 310% or more, 315% or more, or 318% or more, e.g., 318%, based on 100% of the TAPS production per unit cell dry weight (mg / g-cell) of wild-type Wicker Harmomyces ciferi, but is not limited thereto. The unit of the TAPS production per unit cell dry weight may be mg / g-cell, but is not limited thereto.

[0028] The total production of TAPS and TriAPS (mg / L) of the above microorganism may be 150% or more, 170% or more, 190% or more, 210% or more, 230% or more, 245% or more, or 249% or more, e.g., 250%, based on 100% of the total production of TAPS and TriAPS of wild-type Wicker Harmomyces ciferi, but is not limited thereto.

[0029] The total production of TAPS and TriAPS per unit cell dry weight of the above microorganism (mg / g-cell) may be 80 mg or more, 90 mg or more, 95 mg or more, 100 mg or more, 105 mg or more, 110 mg or more, 115 mg or more, 120 mg or more, or 125 mg or more, e.g., 127.1 mg, but is not limited thereto.

[0030] The total production of TAPS and TriAPS per unit cell dry weight of the above microorganism (mg / g-cell) may be 150% or more, 180% or more, 200% or more, 210% or more, or 240% or more, e.g., 244%, based on 100% of the total production of TAPS and TriAPS per unit cell dry weight of wild-type Wicker Harmomyces ciferi (mg / g-cell), but is not limited thereto. The unit of the TAPS production per unit cell dry weight may be mg / g-cell, but is not limited thereto.

[0031] Among the phytosphingosine derivatives produced by the above microorganism, including monoacetyl-, diacetyl-, triacetyl-, and tetraacetylphytosphingosine, the production of TAPS and TriAPS may be higher than the production of each of the other types of phytosphingosine derivatives, for example, the production of TAPS may be the highest and the production of TriAPS may be the second highest.

[0032] Among the phytosphingosine derivatives including monoacetyl-, diacetyl-, triacetyl-, and tetraacetylphytosphingosine of the above microorganism, the TriAPS production ratio may be 15% or more, 18% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 15% to 26%, 18% to 26%, 20% to 26%, 21% to 26%, 22% to 26%, 23% to 26%, or 24% to 26%, for example, 24% or 25%, based on weight.

[0033] Among the phytosphingosine derivatives including monoacetyl-, diacetyl-, triacetyl-, and tetraacetylphytosphingosine of the above microorganism, the TAPS production ratio may be 65% or more, 68% or more, 70% or more, 71% or more, 72% or more, 73% or more, 65% to 76%, 68% to 76%, 70% to 76%, 71% to 76%, 72% to 76%, 73% to 76%, for example, 73% or 75% by weight.

[0034] The microorganism provided by the present invention has resistance to cerulenin and / or EGTA, which can act as toxicity in the growth of the microorganism and the production of phytosphingosine derivatives, and has excellent phytosphingosine derivative production ability, specifically having excellent TAPS and / or TriAPS production ability.

[0036] The microorganism according to the present invention has resistance to cerulenin.

[0037] Cerulenin is a fatty acid synthase inhibitor; when added to a culture medium for yeast growth, it inhibits yeast growth by suppressing fatty acid synthesis, but it can increase TAPS and TriAPS productivity through mutations in the aforementioned fatty acid synthesis pathway. Therefore, when cerulenin is used to increase TAPS and TriAPS productivity, the microorganisms used acquire resistance to cerulenin along with the ability to produce phytosphingosine derivatives, which is more desirable for improving TAPS and TriAPS productivity.

[0038] The resistance to the above-mentioned cerulenin may be the ability to grow and produce phytosphingosine derivatives at a cerulenin concentration of 40 μg / ml or higher in the microbial supplement medium, but is not limited thereto.

[0039] The microorganism according to the present invention has resistance to EGTA.

[0040] "Ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA)" can induce mutations in microorganisms through enzyme chelating, and the TAPS and TriAPS productivity of microorganisms can be increased by such mutations, and as an aminopolycarboxylic acid chelating agent, it can be usefully used in buffers similar to the cell environment.

[0041] The resistance to the above-mentioned EGTA may be the ability to grow and produce phytosphingosine derivatives at an EGTA concentration of 30 μg / ml or higher in the microbial supplement medium, but is not limited thereto.

[0042] The above microorganism may be a strain of the genus Wickerhamomyces, specifically Wickerhamomyces ciferi ( Wickerhamomyces ciferrii ), Wicker Harmomyces siamensis ( W. siamensis ) , Wicker Harmomyces alni ( W. alni ) , Wickerhamomyces bisporus ( W.bisporus ) , Wicker Harmomyces Silvicola ( W. Silvicola ) , Wicker harmonies sidowiorum ( W. sydowiorum ) , Wicker Harmomyces Chamberdi ( W. chambardii ) , Wickerhamomyces anomalus ( W. anomalus ) , Wicker Harmies Arab Praus ( W. arabprarous ) , Wickerhamomyces canadensis ( W. canadensis ) , Wickerhamomyces piperie ( W. pijperi ) , Wickerhamyces strasburgensis ( W. strasburgensis ) , Wicker harmonies sidowiorum ( W. sydowiorum ) , Wicker Harmomyces bobis ( W. Bovis ) , Wicker harmonies chaumierensis ( W. chaumierensis ) ,Wickerhamyses edapicus ( W. edaphicus ) , Wickerhamomyces hemsilensis ( W. hampshirensis ) , Wickerhamomises jurtsmani ( W. jurtsmanii ) , Wicker Harmies Linferdi ( W. lynferdii ) , Wickerhamomyces menglaensis ( W. menglaensis ) , Wicker Harmonise Mori ( W. mori ) , Wicker Harmomyces mucosus ( W. mucosus ) , Wicker harmonies changgensis ( W. changensis ) , Wicker Harmomyces Onikis ( W. onychis ) , Wickerhamomyces orientalis ( W. orientalis ) , Wicker Harmomyces Patagonicus ( W. patagonicus ) , Wickerhamysses querroli ( W. queroliae ) , Wickerhamomyces labaulensis ( W. rabaulensis ) , Wicker harmonies scolitoplaty ( W. scolytoplatypi ) , Wickerhamomyces subpeliculosus ( W. subpelliculosus ) , Wicker Harmomyces tratensis ( W. tratensis ) or Wicker harmonies xylousica ( W. xylosica It may be, but is not limited to.

[0043] The above microorganism may be Wickerhamyces ciferi SYEC2-36, and may be a microorganism that was deposited with the Korean Culture Collection on February 22, 2023, and assigned KCCM13333P.

[0044] The deposited strain above has one or more characteristics selected from the group consisting of (1) to (4) below:

[0045] (1) TAPS production per unit cell dry weight of 15 mg or more of microorganisms (mg / g-cell);

[0046] (2) Total production of TAPS and TriAPS per unit cell dry weight of 80 mg or more of microorganisms (mg / g-cell);

[0047] (3) Resistance to cerulenin; and

[0048] (4) Resistance to EGTA

[0050] The present invention may include the steps of treating wild-type Wicker Harmomyces ciferi with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and / or ultraviolet rays (UV), and selecting a strain that has a higher TAPS and / or TriAPS production capacity than the parent strain Wicker Harmomyces ciferi. Additionally, the microorganism may have resistance to one or more compounds selected from the group consisting of EGTA and cerulenin.

[0051] One example of the present invention is a microorganism of the genus Wicker Harmomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin and / or EGTA ( Wickerhamomyces. Sp The present invention provides a composition for producing a phytosphingosine derivative comprising one or more selected from the group consisting of the cell of the microorganism, lysate of the cell of the microorganism, culture of the microorganism, and extracts thereof. The composition may additionally include one or more selected from the group consisting of cerulenin and EGTA, but is not limited thereto.

[0052] A further example of the present invention is a microorganism of the genus Wicker Harmomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin and / or EGTA ( Wickerhamomyces. SpThe present invention relates to a method for producing a phytosphingosine derivative, comprising the step of culturing (fermenting) one or more selected from the group consisting of the cell of the microorganism, the lysate of the microorganism, the culture of the microorganism, and extracts thereof.

[0053] Specifically, the above step is a microorganism of the genus Wicker Harmomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin and / or EGTA ( Wickerhamomyces. Sp It may be performed by culturing ). The above method for producing phytosphingosine derivatives is a microorganism of the genus Wicker Harmomyces ( Wickerhamomyces. Sp In addition to the step of culturing ), a step of recovering the phytosphingosine derivative may be further included.

[0054] The step of culturing the microorganism may include, but is not limited to, culturing the microorganism in a medium for the production of a phytosphingosine derivative, or culturing it in a seed culture medium and / or culturing it in a medium for the production of a phytosphingosine derivative.

[0055] When culturing in the above seed culture medium, the culture temperature may be 10 to 50°C, 10 to 40°C, 10 to 35°C, 20 to 50°C, 20 to 40°C, 20 to 35°C, 25 to 50°C, 25 to 40°C, or 25 to 35°C, for example, 30°C, but is not limited thereto.

[0056] When culturing in the above seed culture medium, the culture time may be 50 to 150 hours, 50 to 120 hours, 50 to 100 hours, 70 to 150 hours, 70 to 120 hours, 70 to 100 hours, 90 to 150 hours, 90 to 120 hours, 90 to 100 hours, 95 to 150 hours, 95 to 120 hours, or 95 to 100 hours, for example, 96 hours, but is not limited thereto.

[0057] Culture in the above-mentioned seed culture medium can be performed by shaking culture and can be performed under various rpm conditions, for example, 100 to 500 rpm, 100 to 400 rpm, 100 to 300 rpm, 150 to 500 rpm, 150 to 400 rpm, 150 to 300 rpm, 200 to 500 rpm, 200 to 400 rpm, or 200 to 300 rpm, e.g., 250 rpm, but is not limited thereto.

[0058] The above-mentioned seed culture medium may include a carbon source, a nitrogen source, etc., and specifically may include one or more selected from the group consisting of yeast extract, malt extract, peptone, and glycerol, and may be, for example, a YMGL medium, but is not limited thereto.

[0059] The above carbon source may be one or more selected from the group consisting of glycerol, malt, dextrin, glucose, sucrose, acetic acid, ethanol, molasses, and sulfite pulp waste liquid used for the cultivation of ordinary microorganisms, and specifically may be glycerol and / or malt, but is not limited thereto.

[0060] The above nitrogen source may be one or more selected from the group consisting of yeast, peptone, corn steep powder (CSP), corn steep liquor (CSL), urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and nitrogen-containing organic substances such as casein, and specifically may be yeast and / or peptone, but is not limited thereto.

[0061] The production medium for the above-mentioned phytosphingosine derivative may include one or more selected from the group consisting of glycerol, yeast extract, corn soaking powder (CSP), ammonium sulfate, amino acids (e.g., serine, glycine, glutamic acid (monosodium glutamate)), sodium acetate, and calcium chloride, but is not limited thereto.

[0062] When culturing in the above production medium, the culture temperature may be the same as or different from the above seed culture, and specifically, it may be 10 to 50°C, 10 to 40°C, 10 to 35°C, 20 to 50°C, 20 to 40°C, 20 to 35°C, 25 to 50°C, 25 to 40°C, or 25 to 35°C, for example, 30°C, but is not limited thereto.

[0063] Culture in the above production medium can be performed under various rpm conditions, for example, 100 to 500 rpm, 100 to 400 rpm, 100 to 300 rpm, 100 to 200 rpm, 150 to 500 rpm, 150 to 400 rpm, 150 to 300 rpm, or 150 to 200 rpm, e.g., 180 rpm, but is not limited thereto.

[0064] When culturing in the above production medium, the pH conditions may be 4 to 7, 4 to 6, 4 to 5.5, 4.5 to 7, 4.5 to 6, 4.5 to 5.5, 5 to 7, 5 to 6, 5 to 5.5, for example, 5.2, but are not limited thereto.

[0065] Culture in the above-mentioned production medium may be batch culture, fed-batch culture (e.g., fed-batch continuous fermentation), or continuous culture, and culture may be carried out in one or more of the above forms, but is not limited thereto. The above-mentioned fed-batch continuous fermentation means performing continuous culture or adding a medium during the culture process.

[0066] Culture in the above production medium may be performed by fed-batch continuous fermentation, but is not limited thereto.

[0067] The step of culturing in the production medium of the above-mentioned fed-batch continuous fermentation process may involve supplying additional medium while inoculating and culturing microorganisms in the production medium; specifically, the additional medium may be added after all the glycerol in the production medium has been consumed, thereby supplying glycerol. The "additional medium" refers to a medium that is additionally supplied to the culture medium intermittently or continuously after the cultivation of microorganisms has begun, in addition to the initial culture medium.

[0068] When culturing with the above-mentioned fed-batch continuous fermentation, the production medium may include one or more selected from the group consisting of glycerol, yeast extract, corn soaking powder (CSP), amino acids (e.g., serine, glycine, glutamic acid (monosodium glutamate)), ammonium sulfate, and calcium chloride (e.g., calcium chloride dihydrate), but is not limited thereto.

[0069] The above additional medium may include one or more selected from the group consisting of glycerol, yeast extract, amino acids (e.g., serine, glycine and / or glutamic acid (monosodium glutamate)), ammonium sulfate, calcium chloride (e.g., calcium chloride dihydrate) and sodium acetate, but is not limited thereto.

[0070] The above additional medium may be added at a concentration of 1 to 10 g / L, 1 to 8 g / L, 1 to 6 g / L, 1 to 5 g / L, 3 to 10 g / L, 3 to 8 g / L, 3 to 6 g / L, or 3 to 5 g / L, for example, 4 g / L, but is not limited thereto.

[0071] When culturing with the above-mentioned fed-batch continuous fermentation, when the OD value of the production medium is 100 to 150, 100 to 140, 110 to 150, 110 to 140, 120 to 150, or 120 to 140, e.g., 130, and / or 230 to 300, 230 to 280, 230 to 270, 240 to 300, 240 to 280, 240 to 270, 250 to 300, 250 to 280, or 250 to 270, e.g., 260, corn soaking powder (CSP) may be additionally supplied.

[0072] The above OD value may be measured for light of a wavelength of 400 to 800 nm, 400 to 700 nm, 400 to 650 nm, 400 to 620 nm, 500 to 800 nm, 500 to 700 nm, 500 to 650 nm, 500 to 620 nm, 550 to 800 nm, 550 to 700 nm, 550 to 650 nm, 550 to 620 nm, 580 to 800 nm, 580 to 700 nm, 580 to 650 nm, or 580 to 620 nm, for example, 600 nm, but is not limited thereto.

[0073] The above corn soaking powder may be supplied such that the concentration of corn soaking powder in the production medium is 1 to 5 g / L, 1 to 4 g / L, 2 to 5 g / L, or 2 to 4 g / L, for example, 3 g / L, but is not limited thereto.

[0074] The above corn soaking powder may be supplied by dissolving it in distilled water, for example, by dissolving it in primary distilled water, secondary distilled water and / or tertiary distilled water, but is not limited thereto.

[0075] When culturing in the production medium of the above-mentioned fed-batch continuous fermentation, adding sodium acetate to the production medium may increase the pH in the production medium, making initial cultivation difficult; however, if sodium acetate is added to the additional medium after all the glycerol has been consumed following the initial cultivation, it can increase cell growth and / or the production of phytosphingosine (e.g., monoacetylphytosphingosine, diacetylphytosphingosine, triacetylphytosphingosine (TriAPS) and / or tetraacetylphytosphingosine (TAPS)) of the cells.

[0076] When culturing in the above production medium by fed-batch continuous fermentation, the culture temperature may be 10 to 50°C, 10 to 40°C, 10 to 35°C, 20 to 50°C, 20 to 40°C, 20 to 35°C, 25 to 50°C, 25 to 40°C, or 25 to 35°C, for example, 30°C, but is not limited thereto.

[0077] When culturing in the above production medium via fed-batch continuous fermentation, it may be performed under various rpm conditions, for example, 500 to 1,500 rpm, 700 to 1,500 rpm, 800 to 1,500 rpm, 850 to 1,500 rpm, 500 to 1,200 rpm, 700 to 1,200 rpm, 800 to 1,200 rpm, 850 to 1,200 rpm, 500 to 1,000 rpm, 700 to 1,000 rpm, 800 to 1,000 rpm, 850 to 1,000 rpm, 500 to 950 rpm, 700 to 950 rpm, 800 to 950 rpm, or 850 to 950 rpm, e.g., 900 It can be performed under rpm conditions, but is not limited thereto.

[0078] When culturing by fed-batch continuous fermentation in the above production medium, the pH conditions may be 4 to 7, 4 to 6, 4 to 5.5, 4.5 to 7, 4.5 to 6, 4.5 to 5.5, 5 to 7, 5 to 6, 5 to 5.5, for example, 5.2, but are not limited thereto.

[0079] When culturing by fed-batch continuous fermentation in the above production medium, the culture is 0.1 to 10 vvm, 0.1 to 8 vvm, 0.1 to 6 vvm, 0.1 to 4 vvm, 0.1 to 2 vvm, 0.1 to 1.5 vvm, 0.1 to 1.2 vvm, 0.5 to 10 vvm, 0.5 to 8 vvm, 0.5 to 6 vvm, 0.5 to 4 vvm, 0.5 to 2 vvm, 0.5 to 1.5 vvm, 0.5 to 1.2 vvm, 0.8 to 10 vvm, 0.8 to 8 vvm, 0.8 to 6 vvm, 0.8 to 4 vvm, 0.8 to 2 vvm, 0.8 to 1.5 vvm Or it may be performed under conditions of 0.8 to 1.2 vvm, for example, 1 vvm, but is not limited thereto.

[0080] The recovery step of the above phytosphingosine derivative may be performed using conventional extraction and recovery techniques from the microorganism (cell) and / or fermentation liquid (e.g., culture medium of the microorganism provided by the present invention), specifically, from the cultured cell and / or fermentation supernatant. Once the desired product is obtained, it may be used directly or subjected to another process depending on the intended use. For example, acetylated derivatives of sphingosine, dihydrosphingosine, and / or phytosphingosine may be deacetylated enzymatically or chemically.

[0081] The phytosphingosine derivative obtained according to the present invention includes the use of the compound in food, cosmetic, and skin compositions, and specifically, can be used in the form of a food-grade ceramide in the form of a glycoceramide. Effects of the invention

[0082] The present invention provides a microorganism having resistance to cerulenin and / or EGTA, a composition including the same, and a method for producing a phytosphingosine derivative using the same, wherein the microorganism has excellent ability to produce a phytosphingosine derivative. Specific details for implementing the invention

[0083] The present invention will be explained in more detail with reference to the following examples, but the scope of the rights is not intended to be limited to the following examples.

[0085] Example 1. Preparation of a variant microorganism with improved TAPS productivity (1)

[0086] Example 1-1: Culture and Recovery of Mother Strain

[0087] Mutations were induced from the wild strain of Wickerhamomyces ciferrii to obtain a mutant microorganism with high TAPS productivity. Specifically, the ATCC 14091 strain was obtained as the wild strain of Wickerhamomyces ciferrii.

[0088] 50 μl of glycerol stock was mixed into YMGL liquid medium with a pH of 5.5 having the composition of Table 1 below, and cultured with shaking at 30°C for 24 hours. The glycerol stock was prepared by mixing wild strain culture medium and a 40 (w / w)% glycerol solution in a 1:1 ratio and storing it at -70°C.

[0089] Components Concentration (g / L) Yeast extract 3 Malt extract 3 Peptone 5 Glycerol 30

[0090] After shaking culture, 50 μl of the culture medium was taken, inoculated into 3 ml of fresh YMGL liquid medium, and cultured at 30°C for 12 hours. The cell concentration was determined by measuring the absorbance at 600 nm in the culture medium, and the cells were recovered when the OD value reached 2. Specifically, the culture medium was centrifuged (12,000 rpm, 10 min) to recover only the cells, and the medium components were removed by washing twice with 50 mM citrate buffer.

[0092] Examples 1-2: Strain Mutation Treatment

[0093] The washed bacterial cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) at a concentration of 0.05 mg / ml and reacted at room temperature for 30 minutes to prepare mutagenic microbial cells. The mutagenic microbial cells were diluted with sterile distilled water, and the degree of growth inhibition was confirmed by adding ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA), a mutation marker, in a concentration range of 2 to 50 μg / ml. Inhibition was confirmed starting from a concentration of 30 μg / ml, so the microbial cells diluted with sterile distilled water were plated onto a YNB (w / o amino acid) agar plate containing 30 μg / ml.

[0094] A mutation library was obtained by irradiating UV light with a wavelength of 254 nm for 20 seconds at a distance of 15 cm from the plate inoculated with the cells, and then incubating at 30°C. The above-mentioned mutagenic microorganisms were microorganisms with high TAPS production capacity and EGTA resistance, and each microorganism was assigned an arbitrary number and named in the form of an E number (e.g., E284).

[0096] Examples 1-3: Primary Colony Selection and Culture

[0097] In order to select a microorganism with excellent TAPS production ability among the mutagenic microorganisms of Example 1-2, a single colony was selected from the colonies grown on the agar plates of Example 1-2 that differed in shape and size and did not grow overlapping with other colonies.

[0098] The selected colonies and the parent strain of Example 1-1 before mutation treatment were each inoculated into test tubes containing 3 ml of the YMGL liquid medium shown in Table 1, and cultured with shaking at 250 rpm for 96 hours at 30°C. 100% methanol, 9 times the volume of the culture medium, was added, and TAPS was extracted by vigorously stirring with a finemixer at room temperature for 60 minutes. Subsequently, after centrifugation at 12,000 rpm for 10 minutes, the supernatant was filtered through a 0.2 μm filter, and the TAPS production was confirmed by HPLC analysis. The results are shown in Table 2.

[0099] Specific HPLC analysis was performed using an HPLC-UV equipped with a ZORBAX SB-C8 column (Agilent, 4.6mm x 150mm, 3.5μm, USA), and to quantify TAPS, TAPS was purchased from Sigma-Aldrich to obtain a quantification curve, and TAPS production (TAPS titer (mg / L)) and TAPS production yield per unit cell weight (TAPS yield (mg / g-cell)) were measured.

[0100] microorganism TAPS content (mg / L) TAPS yield (mg / g-cell) Wild liquor 162 11.1 E19 393 31.0 E28 398 22.1 E125 315 19.9 E201 388 38.3 E218 406 26.8 E284 528 36.2

[0101] As a result of measuring TAPS production through the above HPLC analysis, the TAPS production of the wild strain was measured to be 162 mg / mL, and six mutant microorganisms (E284, E218, E201, E125, E28, and E19) were selected that showed higher TAPS production (393–528 mg / L) compared to the wild-type strain. Among the six selected microorganisms, the E284 mutant strain showed the highest TAPS production (528 mg / L).

[0103] Example 2. Preparation of a variant microorganism with improved TAPS productivity (2)

[0104] Among the six strains that showed increased TAPS production compared to the wild strain, a secondary mutation was performed using strain E284, which showed a significant increase in TAPS production and yield. The method for selecting mutant strains was carried out substantially the same as the method of Examples 1-1 and 1-2. For the strains treated with the mutagen, cerulenin was added as a marker in the concentration range of 0 to 50 μg / ml instead of EGTA to check the degree of growth inhibition. Since inhibition was confirmed starting from a concentration of 40 μg / ml, the strains treated with the mutagen were plated on a medium containing 40 μg / ml to discover secondary mutant strains.

[0105] The above additional mutagenic microorganisms are microorganisms that were additionally selected for cerulenin among the primary selected microorganisms with EGTA resistance, and were named in the form of EC number-number (e.g., EC2-34) by assigning a random number to each microorganism.

[0106] For the additional mutagenic microorganisms mentioned above, the TAPS production volume and TAPS production volume per unit cell dry weight (production yield) were determined through TAPS extraction and HPLC analysis in substantially the same manner as in Examples 1-3. For the E284 of Examples 1-3 and the wild-type strain of Example 1, TAPS extraction, TAPS production volume, and TAPS production yield were re-determined in the same manner, and the results are shown in Table 3.

[0107] microorganism TAPS content (mg / L) TAPS yield (mg / g-cell) Wild liquor 162 11 E284 393 31 EC1-4 654 45 EC2-34 705 46 EC2-36 528.2 35 EC3-14 540 37 EC4-43 649 44

[0108] As a result of measuring the TAPS production through the above HPLC analysis, the wild strain was measured to have a TAPS production of 162 mg / L and a TAPS production yield of 11 mg / g-cell, while the first selected E284 was measured to have a TAPS production of 393 mg / L and a TAPS production yield of 31 mg / g-cell.

[0109] Among the mutant strains selected from a medium supplemented with cerulenin, five types of microorganisms (EC1-4, EC2-34, EC2-36, EC3-14, and EC4-43) were selected that showed increased TAPS production (528 ~ 705 mg / L) and TAPS production yield (35 ~ 46 mg / g-cell) compared to the wild strain and E284.

[0111] Example 3: Verification of TAPS / acetylated phytosphingosine flask yield

[0112] Example 3-1: Strain Culture

[0113] Flask cultures were performed to compare the TAPS production of wild and mutant W. ciferrii strains. Five mutant strains resistant to both EGTA and cerulenin and the wild strain were cultured in flasks to determine the production of TAPS and TriAPS.

[0114] Specifically, 3 ml of the YMGL liquid medium of Table 1 above was dispensed into a 15 ml test tube, and colonies of five mutant strains resistant to both EGTA and cerulenin obtained in Example 2 were inoculated respectively, and the cultures were shaken at 250 rpm at 30°C for 24 hours to obtain a seed culture.

[0115] The above seed culture was inoculated into a 250 ml baffled flask containing 30 ml of production medium having the composition of Table 4 below so that the OD value of the culture medium became 0.3, and the culture was shaken for 96 hours at 30°C and 180 rpm to obtain the production culture medium. The pH of the production medium was maintained at pH 5.2 by adding 50 mM MES buffer.

[0116] furtherance Concentration (g / L) Glycerol 100 Yeast extract 0.7 Corn steep powder 3 (NH4)2SO4 1 Sodium acetate 2 CaCl2.2H2O 1 Serine 1 pH 5.2

[0117] Example 3-2: Analysis of TriAPS and TAPS Production Volume

[0118] For the production culture obtained in Example 3-1, 100% methanol was added to the culture in substantially the same manner as in Example 1-3, and the supernatant obtained by centrifugation was separated and purified to obtain an analysis sample.

[0119] For the above analysis samples, the production of TAPS and TriAPS (Tri + TAPS titer (mg / L)) and the production yield per unit cell dry weight (production yield) (Tri + TAPS yield (mg / g-cell)) were determined by HPLC analysis in substantially the same manner as in Examples 1-3.

[0120] Specifically, the freeze-dried powder of the above analysis sample was dissolved in CDCl3, and the structure was finally confirmed by NMR analysis using a Bruker Avance DPX 400 (400 MHz) to confirm the presence of TriAPS in the sample. Separately, the above analysis sample was separated, purified, and concentrated using a recycling preparative HPLC (Manufacturer: Jai, Japan Analytical Industry Co., LTD / Resin: C18) to obtain freeze-dried powder, and HPLC was performed in substantially the same manner as in Examples 1-3 to measure the production volume of TriAPS and the production yield of TriAPS per unit cell weight. The production volume of TAPS and TriAPS and the production yield of TAPS and TriAPS per unit cell weight obtained through the above HPLC analysis are shown in Table 5.

[0121] microorganism TAPS + TriAPS content (mg / L) TAPS Yield + TriAPS (mg / g-cell) Wild liquor 1257.0 52.0 E284 2759.7 97.6 EC1-4 1926.5 82.6 EC2-34 3079.3 97.7 EC2-36 3152.4 127.1 EC3-14 3234.7 112.0 EC4-43 2387.7 100.2

[0122] As a result of measuring the production volume and yield of TAPS and TriAPS, two strains were finally identified: strain EC3-14, which showed the highest increase in TriAPS and TAPS production compared to the wild strain, and strain EC2-36, which showed the highest yield of TAPS and TriAPS per cell unit. The two selected strains, EC2-36 and EC3-14, were cultured in a fermentation tank to finally confirm the production volume of TAPS and acetylated phytosphingosine.

[0124] Example 4: Production of acetylated phytosphingosine in a fermenter

[0125] According to Example 3-2, the two strains selected above, EC2-36 and EC3-14, were cultured in a fermentation tank, and fed-batch continuous fermentation was carried out to finally confirm the production of TAPS and acetylated phytosphingosine.

[0126] Specifically, colonies of each of the microorganisms EC2-36 and EC3-14 were obtained in a YMGL agar medium to which 15 g / L of agar was added to the YMGL liquid medium of Example 1. 3 ml of the YMGL liquid medium of Table 1 was dispensed into a 15 ml test tube, colonies of the EC2-36 and EC3-14 strains were inoculated, and incubated with shaking at 250 rpm at 30°C for 24 hours to obtain a seed culture.

[0127] The above seed culture was inoculated into a 500ml baffled flask containing 100ml of the YMGL liquid medium of Table 1 so that the OD value of the culture medium became 0.3, and a preculture was obtained by shaking culture at 30℃ and 180rpm for 24 hours.

[0128] The above pre-culture medium was inoculated into a 5L jar fermenter so that the OD value of the fermentation medium became 0.3, and 2L of production medium containing the composition of Table 6 below was used as the culture medium, and when 20 to 30 g / L of glycerol remained in the production medium, culture was carried out by supplying 4 g / L of glycerol per hour using a feeding solution containing the composition of Table 7 below.

[0129] furtherance Concentration (g / L) glycerol 100 yeast extract 0.7 corn steep powder 3 serine 1 glycine 1 monosodium glutamate 1.27 ammonium sulfate 1 calcium chloride dihydrate 1

[0130] furtherance Concentration (g / L) glycerol 1000 yeast extract 7 serine 5 glycine 5 monosodium glutamate 6.3 ammonium sulfate 5 calcium chloride dihydrate 5 sodium acetate 20

[0131] A sterilized solution prepared by mixing CSP (corn steep powder) with 30 mL of DDW so that the concentration of CSP in the culture medium was 3 g / L was added when the OD value of the culture medium under light of 600 nm was 130 and 260, respectively. Filtered air was supplied to the production medium at a rate of 2 L / min to achieve an air flow rate of 1 vvm, and the process was carried out with stirring at 900 rpm under a temperature of 30℃. The pH of the medium was adjusted to 5.2 using 9% NH4OH, and 10 μl / 20 min of stock antifoam (SB2121, struktol) was added, and fermentation was carried out for 151 hours.

[0132] Phytosphingosine was extracted by adding 100% methanol to the obtained culture medium and centrifuging it in substantially the same manner as in Example 3-2, and the production volume of each type of phytosphingosine was analyzed, and the results of the product production analysis of EC2-36 and EC3-14 strains are shown in Table 8.

[0133] Types of phytosphingosine EC2-36 Production volume (g / L) EC3-14 Production volume (g / L) Monoacetylphytosphingosine 0.01 0.02 diacetylphytosphingosine 0.11 0.14 Triacetylphytosphingosine (TriAPS) 4.19 2.76 Tetraacetylphytosphingosine (TAPS) 13 8.1 Total 17.31 11.02

[0134] As a result of the product analysis above, the acetylated phytosphingosine production of the EC2-36 microorganism was highest in TriAPS and TAPS, specifically 4.19 g / L for TriAPS and 13 g / L for TAPS. The acetylated phytosphingosine production of the EC3-14 strain was highest in TriAPS and TAPS, specifically 2.76 g / L for TriAPS and 8.1 g / L for TAPS.

[0135] The EC2-36 strain, which was confirmed to have high productivity in TriAPS and TAPS, was deposited with the Korean Culture Collection of Microorganisms on February 22, 2023, received the accession number KCCM13333P, and was named SYEC2-36.

[0137] Name of depositing institution: Korean Culture Collection of Microorganisms Trustee Number: KCCM13333P Date of Deposit: 2023-02-22

Claims

Claim 1 Microorganisms of the genus Wicker Harmomyces that have the ability to produce phytosphingosine derivatives and resistance to cerulenin ( Wickerhamomyces. Sp As ), the above microorganism is Wicker Harmomyces ciferi having accession number KCCM13333P ( Wickerhamomyces ciferrii Microorganisms that are ) Claim 2 In claim 1, the microorganism of the genus Wicker Harmomyces, wherein the phytosphingosine derivative is one or more selected from the group consisting of tetraacetylphytosphingosine (TAPS) and triacetylphytosphingosine (TriAPS). Claim 3 In paragraph 2, the microorganism is one in which the TAPS production per unit cell dry weight (mg / g-cell) of the microorganism is 160% or more based on 100% of the production per unit cell dry weight of wild-type Wickerharmonyces ciferi. Claim 4 In paragraph 2, the microorganism is one in which the total production of TAPS and TriAPS per unit cell dry weight (mg / g-cell) of the microorganism is 150% or more based on 100% of the production per unit cell dry weight of wild-type Wicker Harmomyces ciferi. Claim 5 In claim 1, the microorganism having resistance to cerulenin is capable of growing and producing phytosphingosine derivatives at a cerulenin concentration of 40 μg / ml or higher in the microbial-added medium. Claim 6 The microorganism of claim 1, wherein the microorganism further has resistance to ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA). Claim 7 In claim 6, the microorganism having resistance to EGTA is capable of growing and producing phytosphingosine derivatives at an EGTA concentration of 30 μg / ml or higher in the microbial-added medium. Claim 8 delete Claim 9 delete Claim 10 A composition for producing a phytosphingosine derivative, comprising one or more selected from the group consisting of the cell of a microorganism of the genus Wickerhamomyces according to any one of claims 1 to 7, a lysate of the cell of said microorganism, a culture of said microorganism, and extracts thereof. Claim 11 A composition for producing phytosphingosine derivatives according to claim 10, further comprising one or more selected from the group consisting of cerulenin and EGTA. Claim 12 A method for producing a phytosphingosine derivative comprising the steps of culturing a microorganism of the genus Wickerhamomyces according to any one of claims 1 to 7 and recovering a phytosphingosine derivative from the microorganism culture solution. Claim 13 A method for producing a phytosphingosine derivative according to claim 12, wherein the step of culturing the microorganism is performed by fed-batch continuous fermentation. Claim 14 A method for producing a phytosphingosine derivative according to claim 13, wherein the step of culturing the microorganism is to inoculate and culture the microorganism in a production medium while supplying an additional medium. Claim 15 A method for producing a phytosphingosine derivative according to claim 14, wherein the step of culturing the microorganism is performed under pH conditions of 4 to 7. Claim 16 A method for producing a phytosphingosine derivative according to claim 14, wherein the production medium comprises one or more selected from the group consisting of glycerol, yeast extract, corn soaking powder, amino acids, ammonium sulfate, and calcium chloride. Claim 17 A method for producing a phytosphingosine derivative according to claim 14, wherein the additional medium is added at a concentration of 1 to 10 g / L per hour. Claim 18 A method for producing a phytosphingosine derivative according to claim 14, wherein the additional medium comprises one or more selected from the group consisting of glycerol, yeast extract, amino acids, ammonium sulfate, calcium chloride, and sodium acetate. Claim 19 A method for producing a phytosphingosine derivative according to claim 14, wherein corn soaking powder is additionally supplied when the OD value of the production medium is 100 to 150 or 230 to 300. Claim 20 A method for producing a phytosphingosine derivative according to claim 19, wherein the corn soaking powder is supplied such that the concentration of the corn soaking powder in the production medium is 1 to 5 g / L. Claim 21 A method for producing a phytosphingosine derivative according to claim 14, wherein the derivative is cultured under temperature conditions of 10 to 50°C.

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