Wickerhamomyces sp. microorganism having high productivity of phytosphingosine derivative

Abstract

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

Description

Microorganisms of the genus Wickerhamomyces with high productivity of phytosphingosine derivatives

[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. Specifically, the present invention relates to a microorganism of the genus Wickerhamomyces having resistance to cerulenine and / or ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA), a composition containing the same, and a method for producing phytosphingosine derivatives using the same.

[0002] Ceramide is a type of sphingolipid with a structure in which a fatty acid is linked to sphingosine. It accounts for about 40% of the interstitial lipids of keratinocytes that make up the stratum corneum of the skin, and it has the function of acting as a lipid barrier to prevent moisture evaporation and maintaining the orderly structure of the stratum corneum. The stratum corneum of the skin is composed of keratinized cells in a brick-like multilayer structure, and keratinocytes are firmly bound together by ceramides, cholesterol, and free fatty acids. Therefore, it has been reported that applying a product containing ceramide to the skin helps with the skin barrier function and skin moisturizing (Comparative study on the skin moisturizing effect and barrier function of natural ceramide and pseudoceramide, Kwon Minsu, 2005).

[0003] Ceramides can be produced by yeast and molds, but among them, the yeast Wickerhamomyces ciferrii (also known as Pichia ciferrii) has the property 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 by Wickerhamomyces schiperi is in an acetyl-substituted form, and depending on the number of substituted acetyl groups, it is reported to be tetraacetylphytosphingosine, triacetylphytosphingosine, or diacetylphytosphingosine. TAPS is converted to phytosphingosine through the process of removing the acetyl group, and phytosphingosine can be used as a precursor required for ceramide synthesis, so it has industrial value.

[0005] In 1960, Wickerham (LJ) and Stodola (FH) first discovered that the yeast Hansenula ciferrii NRRL 14091 secretes sphingolipids extracellularly and forms crystals on the periphery of solid media. However, when cultured in liquid media, it produced almost no sphingolipids (J. Bacteriol. 80: 484-491, 1960). On the other hand, it was reported that the haploid strain F-60-10 (NRRL 1301), isolated from the diploid 14091 marker, secretes and produces significant amounts of sphingolipids even in liquid media.

[0006] Wickerhamomyces siferi is a yeast that produces acetylated phytosphingosine, of which TAPS is the most abundant. Research has attempted to increase the content and productivity of acetylated phytosphingosine within the strain, including through genetic engineering and metabolic engineering. For example, Korean Patent No. 10-0287483 discloses a technique for enhancing TAPS production using a Pichia siferi mutant containing a GAPDH-enhanced promoter.

[0007]

[0008] The present invention provides a microorganism having the ability to produce phytosphingosine derivatives and resistance to cerulenin.

[0009] The present invention provides a composition for producing a phytosphingosine derivative, comprising at least one selected from the group consisting of the microorganism, a cell of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof.

[0010] The present invention provides a method for producing a phytosphingosine derivative, comprising a step of culturing the above microorganism.

[0011] The present invention relates to a microorganism of the genus Wickerhamomyces having the ability to produce phytosphingosine derivatives and resistance to cerulenin.

[0012] The above microorganism may additionally have resistance to ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA).

[0013] The above microorganism may be Wickerhamomyces ciferrii.

[0014] The present invention provides a composition for producing a phytosphingosine derivative, comprising at least one selected from the group consisting of the microorganism, a cell of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof.

[0015] The above composition may additionally include, but is not limited to, one or more selected from the group consisting of cerulein and EGTA.

[0016] The present invention provides a method for producing a phytosphingosine derivative, comprising a step of culturing the above microorganism.

[0017] The present invention provides a use of a microorganism having the ability to produce phytosphingosine derivatives and resistance to cerulenin for producing phytosphingosine derivatives.

[0018] The present invention provides a use of a microorganism having the ability to produce phytosphingosine derivatives and resistance to cerulenin for preparing a composition for producing phytosphingosine derivatives.

[0019] The microorganism provided by the present invention may have superior phytosphingosine productivity compared to a wild type microorganism (e.g., wild type Wickerhamomyces siferi).

[0020]

[0021] Hereinafter, the present application will be described in more detail.

[0022]

[0023] The present invention provides a microorganism of the genus Wickerhamomyces (Wickerhamomyces.Sp) having the ability to produce phytosphingosine derivatives and resistance to cerulenin and / or EGTA.

[0024] The above phytosphingosine derivative may be at least one selected from the group consisting of monoacetylphytosphingosine, diacetylphytosphingosine, triacetylphytosphingosine (TriAPS), and tetraacetylphytosphingosine (TAPS), and specifically may be triacetylphytosphingosine (TriAPS) and / or tetraacetylphytosphingosine (TAPS).

[0025] The above phytosphingosine derivative production ability is a production ability having a high 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).

[0026] The production amount of phytosphingosine derivatives of the microorganism provided by the present invention, such as 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), may be measured after culturing for 96 hours at 30°C under aerobic conditions, and may be specifically 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.

[0027] The production amount of each type of phytosphingosine derivative provided by the microorganism of the present invention can be confirmed by culturing and / or fermenting the microorganism, extracting the phytosphingosine derivative, and analyzing it through HPLC, but is not limited thereto.

[0028] 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, for example, 326%, based on 100% of the TAPS production (mg / L) of the wild type Wickerhamomyces siferi, but is not limited thereto.

[0029] 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, for example, 35 mg, but is not limited thereto.

[0030] 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, for example, 318%, based on 100% of the TAPS production per unit cell dry weight (mg / g-cell) of the wild type Wickerhamomyces siferi, 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] The total TAPS and TriAPS production (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, for example, 250%, based on 100% of the total TAPS and TriAPS production of the wild type Wickerhamomyces siferi, but is not limited thereto.

[0032] The combined production of TAPS and TriAPS per unit cell dry weight (mg / g-cell) of the above microorganism 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, for example, 127.1 mg, but is not limited thereto.

[0033] The sum of TAPS and TriAPS production per unit cell dry weight (mg / g-cell) of the above microorganism may be 150% or more, 180% or more, 200% or more, 210% or more, or 240% or more, for example, 244%, based on 100% of the sum of TAPS and TriAPS production per unit cell dry weight (mg / g-cell) of the wild type Wickerhamomyces siferi, 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.

[0034] Among the phytosphingosine derivatives including monoacetyl-, diacetyl-, triacetyl-, and tetraacetylphytosphingosine produced by the above microorganism, the production of TAPS and TriAPS may be higher than the production of each of the remaining types of phytosphingosine derivatives, for example, the production of TAPS may be the highest and the production of TriAPS may be the second highest.

[0035] 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%, but is not limited thereto.

[0036] 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%, but is not limited thereto.

[0037] The microorganism provided by the present invention has resistance to cerulenin and / or EGTA, which may act as toxic agents in the growth of microorganisms and the production of phytosphingosine derivatives, and has excellent phytosphingosine derivative production ability, and specifically, excellent TAPS and / or TriAPS production ability.

[0038]

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

[0040] Cerulenin is a fatty acid synthase inhibitor. When added to a yeast growth medium, it inhibits fatty acid synthesis and thus yeast growth. However, by altering the fatty acid synthesis pathway, TAPS and TriAPS productivity can be increased. 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 further desirable for enhancing the productivity of TAPS and TriAPS.

[0041] The resistance to the above cerulenin may be, but is not limited to, growth and phytosphingosine derivative production ability at a cerulenin concentration of 40 μg / ml or more in the medium added to the above microorganism.

[0042] The microorganism according to the present invention has resistance to EGTA.

[0043] "Ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA)" can induce mutations in microorganisms through enzyme chelating, and the TAPS and TriAPS productivity of the microorganisms can be increased by the mutations, and as an aminopolycarboxylic acid, a chelating agent, it can be usefully used in a buffer solution similar to the cell environment.

[0044] The resistance to the above EGTA may be, but is not limited to, growth and phytosphingosine derivative production ability at an EGTA concentration of 30 μg / ml or more in the medium added to the above microorganism.

[0045] The above microorganism may be a strain of the genus Wickerhamomyces, and specifically, Wickerhamomyces ciferrii, W. siamensis, W. alni, W. bisporus, W. silvicola, W. sydowiorum, W. chambardii, W. anomalus, W. arabprarous, W. canadensis, W. pijperi, W. strasburgensis, W. sydowiorum), W. bovis, W. chaumierensis, W. edaphicus, W. hampshirensis, W. jurtsmanii, W. lynferdii, W. menglaensis, W. mori, W. mucosus, W. changensis, W. onychis, W. orientalis, W. patagonicus. patagonicus), W. queroliae, W. rabaulensis, W. scolytoplatypi, W. subpelliculosus, W. tratensis or W. xylosica.xylosica) but are not limited thereto.

[0046] The above microorganism may be Wickerhamomyces siferi SYEC2-36, and may be a microorganism deposited with the Korea Microbiological Conservation Center on February 22, 2023 and assigned the accession number KCCM13333P.

[0047] The above-deposited strain has at least one characteristic selected from the group consisting of (1) to (4):

[0048] (1) TAPS production per unit cell dry weight of 15 mg or more of microorganism (mg / g-cell);

[0049] (2) Total production of TAPS and TriAPS per unit dry weight of microbial cells of 80 mg or more (mg / g-cell);

[0050] (3) Resistance to cerulein; and

[0051] (4) Resistance to EGTA

[0052]

[0053] The present invention may include a step of treating a wild type Wickerhamomyces siferae with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and / or ultraviolet ray (UV), and a step of selecting a microorganism having a higher TAPS and / or TriAPS production ability than the parent strain Wickerhamomyces siferae. In addition, the microorganism may have resistance to one or more compounds selected from the group consisting of EGTA and cerulein.

[0054] One example of the present invention provides a composition for producing a phytosphingosine derivative, comprising at least one selected from the group consisting of a microorganism of the genus Wickerhamomyces (Sp) having the ability to produce a phytosphingosine derivative and resistance to cerulenin and / or EGTA, a cell of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof. The composition may additionally comprise at least one selected from the group consisting of cerulenin and EGTA, but is not limited thereto.

[0055] Another example of the present invention relates to a method for producing a phytosphingosine derivative, comprising a step of culturing (fermenting) at least one selected from the group consisting of a microorganism of the genus Wickerhamomyces (Sp), having a phytosphingosine derivative production ability and resistance to cerulenin and / or EGTA, a cell of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof.

[0056] Specifically, the above step may be performed by culturing a microorganism of the genus Wickerhamomyces (Wickerhamomyces. Sp) having the ability to produce a phytosphingosine derivative and resistance to cerulenin and / or EGTA. The method for producing a phytosphingosine derivative may further include a step of recovering a phytosphingosine derivative in addition to the step of culturing a microorganism of the genus Wickerhamomyces (Wickerhamomyces. Sp).

[0057] The step of culturing the above microorganism may include, but is not limited to, culturing the microorganism in a medium for producing a phytosphingosine derivative, or a step of culturing in a seed culture medium and / or a step of culturing in a medium for producing a phytosphingosine derivative.

[0058] 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.

[0059] When culturing in the above-mentioned seed culture medium, the culturing 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.

[0060] In the above seed culture medium, the culture 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, for example, 250 rpm, but is not limited thereto.

[0061] The above-mentioned seed culture medium may include a carbon source, a nitrogen source, etc., and specifically may include at least one 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.

[0062] The above carbon source may be at least one selected from the group consisting of glycerol, malt, dextrin, glucose, sucrose, acetic acid, ethanol, molasses, and sulfite pulp waste liquid, which are commonly used for culturing microorganisms, and may specifically be glycerol and / or malt, but is not limited thereto.

[0063] The above nitrogen source may be at least one selected from the group consisting of nitrogen-containing organic substances such as yeast, peptone, corn steep powder (CSP), corn steep liquor (CSL), urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and casein, and may specifically be yeast and / or peptone, but is not limited thereto.

[0064] The production medium of the above phytosphingosine derivative may include, but is not limited to, one or more selected from the group consisting of glycerol, yeast extract, corn steep liquor (CSP), ammonium sulfate, amino acids (e.g., serine, glycine, glutamic acid (monosodium glutamate)), sodium acetate, and calcium chloride.

[0065] The culture temperature during culture in the above production medium may be the same as or different from the above seed culture, and in detail, 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.

[0066] Cultivation 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, for example, 180 rpm, but is not limited thereto.

[0067] The pH conditions during cultivation in the above production medium may be, but are not limited to, 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.

[0068] In the above production medium, the cultivation may be batch culture, fed-batch culture (e.g., fed-batch continuous fermentation), or continuous culture, and the cultivation may be performed in one or more of the above formats, but is not limited thereto. The fed-batch continuous fermentation refers to continuous culture or culture performed by adding a medium during the cultivation process.

[0069] Cultivation in the above production medium may be performed by fed-batch continuous fermentation, but is not limited thereto.

[0070] The step of culturing in a production medium using the above-mentioned fed-batch continuous fermentation may be to inoculate microorganisms in the production medium and to culture them while supplying additional medium. Specifically, the additional medium may be added after all glycerol in the production medium is exhausted, thereby supplying glycerol. The above-mentioned "additional medium" refers to a medium that is additionally supplied intermittently or continuously to the culture medium after the initiation of microbial culture, other than the initial culture medium.

[0071] When culturing in the above fed-batch continuous fermentation, the production medium may include, but is not limited to, one or more selected from the group consisting of glycerol, yeast extract, corn steep liquor (CSP), amino acids (e.g., serine, glycine, glutamic acid (monosodium glutamate)), ammonium sulfate, and calcium chloride (e.g., calcium chloride dihydrate).

[0072] The above additional medium may include, but is not limited to, 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.

[0073] The above additional medium may be added at a concentration of, but is not limited to, 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, e.g., 4 g / L, per hour.

[0074] When the OD value of the production medium during the continuous fermentation in the fed-batch mode is 100 to 150, 100 to 140, 110 to 150, 110 to 140, 120 to 150, or 120 to 140, for example, 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, for example, 260, corn steep powder (CSP) may be additionally supplied.

[0075] The above OD value may be measured for light having 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.

[0076] The corn steeping powder may be supplied such that the corn steeping powder concentration 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.

[0077] The above corn steeping powder may be supplied by dissolving in distilled water, and may be supplied by dissolving in, for example, primary distilled water, secondary distilled water, and / or tertiary distilled water, but is not limited thereto.

[0078] When sodium acetate is added to the production medium during cultivation in the above-mentioned fed-batch continuous fermentation, the pH in the production medium increases, which may make initial cultivation difficult. However, after the initial cultivation, when sodium acetate is included in the additional medium and added after all glycerol is consumed, cell growth and / or cell phytosphingosine (e.g., monoacetylphytosphingosine, diacetylphytosphingosine, triacetylphytosphingosine (TriAPS) and / or tetraacetylphytosphingosine (TAPS)) production can be increased.

[0079] When culturing in a fed-batch continuous fermentation in the above production medium, the culturing 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.

[0080] The above production medium can be cultured under various rpm conditions in fed-batch continuous fermentation, 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, for example, It can be performed under conditions of 900 rpm, but is not limited thereto.

[0081] When culturing in a 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.

[0082] When cultured in a 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 It can be performed under vvm or 0.8 to 1.2 vvm, for example, 1 vvm conditions, but is not limited thereto.

[0083] The step of recovering the above phytosphingosine derivative can be recovered from the microorganism (cells) and / or fermentation broth (e.g., culture broth of the microorganism provided by the present invention), specifically, the cultured cells and / or fermentation supernatant, provided by the present invention, using conventional extraction and recovery techniques. Once the desired product is obtained, it can be used directly or subjected to another process depending on the intended use. For example, acetylated derivatives of sphingosine, dihydrosphingosine, and / or phytosphingosine can be deacetylated enzymatically or chemically.

[0084] 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 ceramide in the form of a sugar ceramide.

[0085] The microorganism having resistance to cerulein and / or EGTA according to the present invention has an excellent ability to produce phytosphingosine derivatives. When producing phytosphingosine derivatives using the microorganism, phytosphingosine can be produced with high efficiency and in an environmentally friendly manner compared to chemical synthesis methods.

[0086] The present invention will be described in more detail with reference to the following examples, but the scope of the invention is not intended to be limited to the following examples.

[0087]

[0088] Example 1. Production of mutant microorganisms with improved TAPS productivity (1)

[0089] Example 1-1: Cultivation and recovery of parent strain

[0090] To obtain a mutant microorganism with high TAPS productivity from a wild strain of Wickerhamomyces ciferrii, mutations were performed. Specifically, ATCC 14091 strain was obtained as a wild strain of Wickerhamomyces ciferrii.

[0091] 50 μl of glycerol stock was mixed with 3 ml of YMGL liquid medium with a pH of 5.5 and the composition shown in Table 1 below, and cultured with shaking at 30°C for 24 hours. The glycerol stock was prepared by mixing the wild strain culture and a 40 (w / w)% glycerol solution in a 1:1 ratio and storing at -70°C.

[0092] Constituent concentration (g / L) Yeast extract 3 Malt extract 3 Peptone 5 Glycerol 30

[0093] After shaking culture, 50 μl of the culture was taken and inoculated into 3 ml of new YMGL liquid medium and cultured at 30°C for 12 hours. The cell concentration was confirmed by measuring the absorbance at 600 nm in the culture medium, and the cells were harvested when the OD value reached 2. Specifically, the culture medium was centrifuged (12,000 rpm, 10 min) to harvest only the cells, and the medium components were removed by washing twice with 50 mM citrate buffer.

[0094]

[0095] Example 1-2: Strain mutation treatment

[0096] The washed 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 mutation marker ethylene glycol-bis(3-aminoethyl ether) tetraacetic acid (EGTA) was added at a concentration range of 2 to 50 μg / ml to confirm the degree of growth inhibition. Inhibition was confirmed starting from a concentration of 30 μg / ml, so the microbial cells diluted with sterile distilled water were plated on YNB (w / o amino acid) plate medium containing EGTA at a concentration of 30 μg / ml.

[0097] A mutant library was secured by culturing at 30°C after irradiating the plate with UV light at a wavelength of 254 nm for 20 seconds at a distance of 15 cm from the plate on which the fungi were spread. The mutagenic microorganisms were microorganisms with high TAPS production ability and EGTA resistance, and each microorganism was assigned a random number and named in the form of the number E (e.g., E284).

[0098]

[0099] Example 1-3: Primary colony selection and culture

[0100] In order to select a microorganism with excellent TAPS production ability among the mutagenic microorganisms of Example 1-2, a single colony that was different in shape and size and did not grow overlapping with other colonies was selected from among the colonies grown on the flat plate of Example 1-2.

[0101] The selected colonies and the parent strain before mutation treatment of Example 1-1 were each inoculated into a test tube containing 3 ml of YMGL liquid medium of Table 1, and cultured under shaking conditions of 250 rpm at 30°C for 96 hours. 100% methanol in an amount 9 times the volume of the culture medium was added, and TAPS was extracted by vigorously stirring using a fine mixer at room temperature for 60 minutes.

[0102] Afterwards, the supernatant was centrifuged for 10 minutes at 12,000 rpm, filtered through a 0.2 μm filter, and the TAPS production amount was confirmed through HPLC analysis, and the results are shown in Table 2.

[0103] Specific HPLC analysis was performed using HPLC-UV equipped with a ZORBAX SB-C8 column (Agilent, 4.6 mm x 150 mm, 3.5 μm, USA). For TAPS quantitative calculation, TAPS was purchased from Sigma-Aldrich to obtain a quantitative curve, and TAPS production (TAPS titer (mg / L)) and TAPS production yield per unit cell weight (TAPS yield (mg / g-cell)) were measured.

[0104] Microbial TAPS content (mg / L) TAPS yield (mg / g-cell) Wild strain 16211.1 E1939331.0 E2839822.1 E12531519.9 E20138838.3 E21840626.8 E28452836.2

[0105] 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) with higher TAPS production (393 to 528 mg / L) than the wild-type strain were selected. Among the six selected microorganisms, the E284 mutant showed the highest TAPS production (528 mg / L).

[0106]

[0107] Example 2. Production of mutant microorganisms with improved TAPS productivity (2)

[0108] Among the six strains with increased TAPS production compared to the wild type, the E284 strain with significantly increased TAPS production and yield was used to perform secondary mutations. The method for selecting mutants was substantially the same as the methods of Examples 1-1 and 1-2 above, and the strains treated with the mutagen were confirmed to have growth inhibition by adding cerulenin as a marker in the concentration range of 0 to 50 μg / ml instead of EGTA, and inhibition was confirmed starting from a concentration of 40 μg / ml. Therefore, the strains treated with the mutagen were plated on a plate containing 40 μg / ml of cerulenin to identify secondary mutant strains.

[0109] The above additional mutagenic microorganisms are microorganisms that were additionally selected for cerulein among the first-selected microorganisms with EGTA resistance, and each microorganism was assigned a random number and named in the form of EC number-number (e.g., EC2-34).

[0110] For the additional mutagenic microorganisms described above, TAPS production and TAPS production per unit cell dry weight (production yield) were confirmed through TAPS extraction and HPLC analysis using substantially the same method as in Examples 1-3. TAPS extraction, TAPS production, and TAPS production yield were also confirmed again for E284 of Examples 1-3 and the wild-type strain of Example 1, and the results are shown in Table 3.

[0111] Microbial TAPS content (mg / L) TAPS yield (mg / g-cell) Wild strain 16211E28439331EC1-465445EC2-3470546EC2-36528.235EC3-1454037EC4-4364944

[0112] As a result of measuring 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.

[0113] Among the mutant strains selected in the medium supplemented with cerulein, five microorganisms (EC1-4, EC2-34, EC2-36, EC3-14, and EC4-43) were selected that had increased TAPS production (528 to 705 mg / L) and TAPS production yield (35 to 46 mg / g-cell) compared to the wild type and E284.

[0114]

[0115] Example 3: Confirmation of TAPS / acetylated phytosphingosine flask production

[0116] Example 3-1: Strain cultivation

[0117] To compare the TAPS production of wild and mutant W. ciferrii strains, flask cultures were conducted. Five mutant strains resistant to both EGTA and cerulein, as well as the wild strain, were cultured in flasks to determine the production of TAPS and TriAPS.

[0118] Specifically, 3 ml of the YMGL liquid medium in Table 1 was dispensed into a 15 ml test tube, and five colonies of wild strains and mutant strains resistant to both EGTA and cerulein obtained in Example 2 were inoculated, respectively, and cultured by shaking at 250 rpm at 30°C for 24 hours to obtain a seed culture solution.

[0119] The above seed culture solution was inoculated into a 250 ml baffled flask containing 30 ml of a production medium having the composition shown in Table 4 below, so that the OD value of the culture solution became 0.3, and the culture was shaken for 96 hours at 30°C and 180 rpm to obtain a production culture solution. The pH of the production medium was maintained at pH 5.2 by adding 50 mM MES buffer.

[0120] Composition concentration (g / L)Glycerol100Yeast extract0.7Corn steep powder3(NH4)2SO41Sodium acetate2CaCl2.2H2O1Serine1pH 5.2

[0121]

[0122] Example 3-2: Analysis of TriAPS and TAPS production

[0123] For the production culture solution obtained in Example 3-1, 100% methanol was added to the culture solution 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.

[0124] For the above analysis samples, the TAPS and TriAPS production (Tri + TAPS titer (mg / L)) and the production per unit cell dry weight (production yield) (Tri + TAPS yield (mg / g-cell)) were confirmed through HPLC analysis in substantially the same manner as in Examples 1-3.

[0125] Specifically, the freeze-dried powder of the above-mentioned analysis sample was dissolved in CDCl3 and the structure was finally confirmed through NMR analysis using Bruker Avance DPX 400 (400MHz) to confirm the presence of TriAPS in the sample, and separately, the above-mentioned analysis sample was separated, purified, and concentrated using recycling preparative HPLC (manufacturer: Jai, Japan Analytical Industry Co., LTD, / Resin: C18) to obtain a freeze-dried powder, and HPLC analysis was performed in substantially the same manner as in Examples 1-3 to measure the production amount of TriAPS and the production yield of TriAPS per unit cell weight. The production amounts of TAPS and TriAPS and the production yields of TAPS and TriAPS per unit cell weight obtained through the HPLC analysis are shown in Table 5.

[0126] Microorganism TAPS + TriAPS content (mg / L) TAPS yield + TriAPS (mg / g-cell) Wild strain 1257.052.0 E2842759.797.6 EC1-41926.582.6 EC2-343079.397.7 EC2-363152.4127.1 EC3-143234.7112.0 EC4-432387.7100.2

[0127] As a result of measuring the production and production 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 type, and strain EC2-36, which showed the highest yield of TAPS and TriAPS per unit cell. The two selected strains, EC2-36 and EC3-14, were cultured in a fermenter to finally confirm the production of TAPS and acetylated phytosphingosine.

[0128]

[0129] Example 4: Production of acetylated phytosphingosine in a fermenter

[0130] In order to finally confirm the production amount of TAPS and acetylated phytosphingosine, fed-batch continuous fermentation was performed by culturing the two strains selected according to Example 3-2, EC2-36 and EC3-14, in a fermenter.

[0131] Specifically, colonies of each of the EC2-36 and EC3-14 microorganisms were obtained from YMGL agar medium in 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 each of the EC2-36 and EC3-14 strains were inoculated, and the culture was shaken at 250 rpm at 30°C for 24 hours to obtain a seed culture solution.

[0132] The above seed culture solution was inoculated into a 500 ml baffled flask containing 100 ml of YMGL liquid medium in Table 1 so that the OD value of the culture solution became 0.3, and the preculture solution was obtained by shaking and culturing for 24 hours under conditions of 30°C and 180 rpm.

[0133] The above pre-culture solution was inoculated into a 5 L jar fermenter so that the OD value of the fermentation culture solution became 0.3, and 2 L of a 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, 4 g / L of glycerol was supplied per hour using a feeding solution containing the composition of Table 7 below, and culture was conducted.

[0134] Composition concentration (g / L)glycerol100yeast extract0.7corn steep powder3serine1glycine1monosodium glutamate1.27ammonium sulfate1calcium chloride dihydrate1

[0135] Composition concentration (g / L)glycerol1000yeast extract7serine5glycine5monosodium glutamate6.3ammonium sulfate5calcium chloride dihydrate5sodium acetate20

[0136] A sterilized solution of CSP (corn steep powder) mixed with 30 mL of DDW so that the CSP concentration of the culture solution was 3 g / L was added when the OD values ​​of the culture solution under light with a wavelength of 600 nm were 130 and 260, respectively.

[0137] Filtered air was supplied to the above production medium at 2 L / min to ensure an air flow rate of 1 vvm, and the fermentation was conducted at 900 rpm under temperature conditions of 30°C. The medium pH was adjusted to 5.2 using 9% NH4OH, and antifoam (SB2121, struktol) was added at 10 μl / 20 min of the original solution, and fermentation was conducted for 151 hours.

[0138] In a method substantially the same as Example 3-2, 100% methanol was added to the culture solution obtained above, centrifuged to extract phytosphingosine and derivatives, and the production amount of each type of acetylated phytosphingosine was analyzed, and the results of the product production analysis of the EC2-36 and EC3-14 strains are shown in Table 8.

[0139] Phytosphingosine type EC2-36 production (g / L) EC3-14 production (g / L) Monoacetylphytosphingosine 0.01 0.02 Diacetylphytosphingosine 0.11 0.14 Triacetylphytosphingosine (TriAPS) 4.19 2.76 Tetraacetylphytosphingosine (TAPS) 138.1 Total 17.3 111.02

[0140] As a result of the above product analysis, the acetylated phytosphingosine production of the EC2-36 microorganism was the highest in TriAPS and TAPS, specifically, TriAPS was 4.19 g / L and TAPS was 13 g / L. The acetylated phytosphingosine production of the EC3-14 strain was the highest in TriAPS and TAPS, specifically, TriAPS was 2.76 g / L and TAPS was 8.1 g / L.

[0141] The strain EC2-36, which was confirmed to have high TriAPS and TAPS productivity, was deposited with the Korea Microbiological Conservation Center on February 22, 2023, and was given the accession number KCCM13333P and named SYEC2-36.

[0142]

[0143] [Accession number]

[0144] Name of depositor: Korea Microbiological Conservation Center

[0145] Accession number: KCCM13333P

[0146] Date of acceptance: 20230222

[0147]

[0148]

[0149]

Claims

1. A microorganism of the genus Wickerhamomyces (Wickerhamomyces. Sp) that has the ability to produce phytosphingosine derivatives and has resistance to cerulenin.

2. A microorganism of the genus Wickerhamomyces, wherein the phytosphingosine derivative in the first paragraph is at least one selected from the group consisting of tetraacetylphytosphingosine (TAPS) and triacetylphytosphingosine (TriAPS).

3. In the second paragraph, the microorganism has a TAPS production (mg / g-cell) per unit cell dry weight of the microorganism that is 160% or more of the production per unit cell dry weight of the wild type Wickerhamomyces siferi, which is 100% or more.

4. In the second paragraph, a microorganism, wherein the total production of TAPS and TriAPS per unit cell dry weight (mg / g-cell) of the microorganism is 150% or more of the production per unit cell dry weight of the wild type Wickerhamomyces siferi, which is 100% or more.

5. In the first paragraph, a microorganism having resistance to cerulenin has the ability to grow and produce phytosphingosine derivatives at a cerulenin concentration of 40 μg / ml or more in the medium to which the microorganism is added.

6. A microorganism according to claim 1, wherein the microorganism further has resistance to ethylene glycol-bis (3-aminoethyl ether) tetraacetic acid (EGTA).

7. In the 6th paragraph, a microorganism having resistance to EGTA has the ability to grow and produce phytosphingosine derivatives at an EGTA concentration of 30 μg / ml or more in the medium added to the microorganism.

8. A microorganism according to claim 1, wherein the microorganism is Wickerhamomyces ciferrii.

9. A microorganism according to claim 1, wherein the microorganism is Wickerhamomyces ciferrii having the accession number KCCM13333P.

10. A composition for producing a phytosphingosine derivative, comprising at least one selected from the group consisting of a cell of a microorganism of the genus Wickerhamomyces of any one of claims 1 to 9, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof.

11. A composition for producing a phytosphingosine derivative, further comprising at least one selected from the group consisting of cerulein and EGTA, in claim 10.

12. A method for producing a phytosphingosine derivative, comprising the step of culturing a microorganism of the genus Wickerhamomyces according to any one of claims 1 to 9 and the step of recovering a phytosphingosine derivative from the microorganism culture solution.

13. A method for producing a phytosphingosine derivative, wherein the step of culturing the microorganism in paragraph 12 is performed by fed-batch continuous fermentation.

14. A method for producing a phytosphingosine derivative, wherein the step of culturing the microorganism in the 13th paragraph is to inoculate the microorganism in a production medium and supply an additional medium while culturing the microorganism.

15. A method for producing a phytosphingosine derivative, wherein the step of culturing the microorganism in claim 14 is performed under pH conditions of 4 to 7.

16. A method for producing a phytosphingosine derivative, wherein the production medium comprises at least one selected from the group consisting of glycerol, yeast extract, corn steep liquor, amino acid, ammonium sulfate, and calcium chloride.

17. A method for producing a phytosphingosine derivative, wherein the additional medium is added at a concentration of 1 to 10 g / L per hour in the 14th paragraph.

18. A method for producing a phytosphingosine derivative, wherein the additional medium comprises at least one selected from the group consisting of glycerol, yeast extract, amino acid, ammonium sulfate, calcium chloride, and sodium acetate.

19. A method for producing a phytosphingosine derivative, wherein, in paragraph 14, when the OD value of the production medium is 100 to 150 or 230 to 300, corn steep powder is additionally supplied.

20. A method for producing a phytosphingosine derivative, wherein, in claim 19, the corn steep powder is supplied so that the concentration of the corn steep powder in the production medium is 1 to 5 g / L.

21. A method for producing a phytosphingosine derivative, wherein the method is cultured under temperature conditions of 10 to 50°C in accordance with claim 14.

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