Wickham yeasts with high efficiency in producing plant sphingosine derivatives

Abstract

This invention relates to a microorganism capable of producing plant sphingosine derivatives and resistant to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid, a composition comprising the microorganism, and a method for producing plant sphingosine derivatives using the microorganism.

Description

Technical Field

[0001] This invention relates to a microorganism with high production efficiency of phytosphingosine derivatives, such as tetraacetylphytosphingosine (TAPS) and / or triacetylphytosphingosine (TriAPS), its preparation method, and a method for producing tetraacetylphytosphingosine and triacetylphytosphingosine using the microorganism. Specifically, it relates to a microorganism resistant to cerulenine and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid (EGTA), a composition comprising the microorganism, and a method for producing phytosphingosine derivatives using the microorganism. Background Technology

[0002] Ceramides are sphingolipids with a structure linking fatty acids and sphingosine. They account for approximately 40% of the intercellular lipids that make up the stratum corneum of the skin and function as a lipid barrier to inhibit water evaporation and maintain the orderly structure of the stratum corneum. It has been reported that the stratum corneum is composed of keratinized cells in a brick-like, multi-layered structure. Because these keratinized cells are firmly bound together by ceramides, cholesterol, and free fatty acids, applying ceramide-containing preparations to the skin helps improve skin barrier function and hydration (Comparative Study on the Skin Moisturizing Effects and Barrier Function of Natural Ceramides and Ceramide-like Agents, Kwon Minsu, 2005).

[0003] Ceramides can be produced in yeast and mold, but not in the yeast *Wickham Severus* (*Ceramides*). Wickerhamomycesciferrii Also known as Severinia sifida ( Pichia ciferrii It has the property of synthesizing tetraacetyl phytosphingosine, an acetylated derivative of phytosphingosine, through palmitoyl-CoA and the amino acid L-serine, and then secreting it outside the cell.

[0004] Phytosphingosine secreted extracellularly by *Wickham Severus* is in an acetyl-substituted form. Depending on the number of acetyl groups substituted, tetraacetylphytosphingosine, triacetylphytosphingosine, or diacetylphytosphingosine have been reported. Tetraacetylphytosphingosine is converted to phytosphingosine through the removal of the acetyl group. Phytosphingosine can be used as a precursor for ceramide synthesis and therefore has industrial value.

[0005] In 1960, Wickerham, L.J. and Stodola, F.H., first discovered Hansenula yeast. Hansenula ciferrii NRRL14091 secretes sphingolipids extracellularly and forms crystals around the cells in solid culture medium. However, when cultured in liquid medium, it produces almost no sphingolipids (J. Bacteriol. 80: 484-491, 1960). In contrast, haploid F-60-10 (NRRL1301), isolated from the marker strain of diploid 14091, has been reported to secrete large amounts of sphingolipids in liquid medium.

[0006] Wickham's Severinia sericulture is a yeast that produces acetylated phytosphingosine, with the highest yield of tetraacetylated phytosphingosine. To improve the content and production efficiency of acetylated phytosphingosine in strains, studies have been conducted using genetic engineering and metabolic engineering to regulate its production. For example, Korean Patent No. 10-0287483 discloses a technique for improving tetraacetylated phytosphingosine production using a mutant strain of Severinia sericulture with an inserted GAPDH-enhancing promoter. Summary of the Invention

[0007] Technical issues This invention provides a microorganism with the ability to produce plant sphingosine derivatives and resistance to phytosphingosine.

[0008] The present invention provides a composition for producing plant sphingosine derivatives, comprising one or more selected from the microorganisms, the cells of the microorganisms, cell lysates of the microorganisms, cultures of the microorganisms, and extracts thereof.

[0009] The present invention provides a method for producing plant sphingosine derivatives, which includes the step of culturing the microorganisms.

[0010] Technical solution This invention relates to a Wickham yeast microorganism with the ability to produce plant sphingosine derivatives and resistance to cyanobacterium.

[0011] The microorganism may further possess resistance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

[0012] The microorganism may be Wickham Silver Saccharomyces cerevisiae (Wickham Silver Saccharomyces cerevisiae) Wickerhamomyces ciferrii ).

[0013] The present invention provides a composition for producing plant sphingosine derivatives, comprising one or more selected from the microorganisms, the cells of the microorganisms, cell lysates of the microorganisms, cultures of the microorganisms, and extracts thereof.

[0014] The composition may further contain one or more selected from cyanobacterium and ethylene glycol bis(3-aminoethyl ether)tetraacetic acid, but is not limited thereto.

[0015] The present invention provides a method for producing plant sphingosine derivatives, which includes the step of culturing the microorganisms.

[0016] This invention provides the use of a plant sphingosine derivative in the production of microorganisms with the ability to produce plant sphingosine derivatives and resistance to phytosphingosine.

[0017] The present invention provides the use of a microorganism in the preparation of a composition for producing plant sphingosine derivatives, said microorganism having the ability to produce plant sphingosine derivatives and resistance to phytosphingosine.

[0018] The microorganisms provided by this invention may have superior phytosphingosine production efficiency compared to wild-type microorganisms (e.g., wild-type Wickham Severus yeast).

[0019] The following describes this application in more detail.

[0020] This invention provides a Wickham yeast microorganism with the ability to produce plant sphingosine derivatives and resistance to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

[0021] The phytosphingosine derivative may be selected from one or more of monoacetyl phytosphingosine, diacetyl phytosphingosine, triacetyl phytosphingosine, and tetraacetyl phytosphingosine, specifically triacetyl phytosphingosine and / or tetraacetyl phytosphingosine.

[0022] The phytosphingosine derivative production capacity refers to having a high yield of tetraacetyl phytosphingosine and / or triacetyl phytosphingosine (e.g., yield per unit culture volume (L), mg / L) and / or the yield of tetraacetyl phytosphingosine and / or triacetyl phytosphingosine per unit cell dry weight (e.g., yield per unit cell dry weight, mg / g-cell)).

[0023] The yield of phytosphingosine derivatives of microorganisms provided by this invention, such as the yield of tetraacetyl phytosphingosine and / or triacetyl phytosphingosine (e.g., yield per unit culture volume (L), mg / L) and / or the yield of tetraacetyl phytosphingosine and / or triacetyl phytosphingosine per unit cell dry weight (e.g., yield per unit cell dry weight, mg / g-cell), can be measured after culturing at 30°C for 96 hours under aerobic conditions. Specifically, it can be measured by shaking culture in YMGL medium and extracting tetraacetyl phytosphingosine and / or triacetyl phytosphingosine, but is not limited thereto. The YMGL medium may contain glycerol, yeast extract, malt extract, and peptone.

[0024] The yields of different types of phytosphingosine derivatives from microorganisms provided by the present invention can be confirmed by HPLC analysis after culturing and / or fermenting the microorganisms and extracting the phytosphingosine derivatives, but are not limited thereto.

[0025] Based on a 100% yield of tetraacetyl phytosphingosine (mg / L) in wild-type Wickham Severinia, the yield of tetraacetyl phytosphingosine in the microorganisms can reach 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%, but not limited to this.

[0026] The microorganism's yield of tetraacetyl phytosphingosine per unit dry weight (mg / g-cell) can reach more than 15 mg, more than 20 mg, more than 25 mg, more than 30 mg, or more than 33 mg, such as 35 mg, but is not limited thereto.

[0027] Based on 100% of the unit cell dry weight tetraacetyl phytosphingosine yield (mg / g-cell) of wild-type Wickham Severinia yeast, the unit cell dry weight tetraacetyl phytosphingosine yield (mg / g-cell) of the aforementioned microorganism can reach 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%, but is not limited thereto. The unit of the unit cell dry weight tetraacetyl phytosphingosine yield can be mg / g-cell, but is not limited thereto.

[0028] Based on a total yield of 100% of tetraacetyl phytosphingosine and triacetyl phytosphingosine in wild-type Wickham Severus yeast, the total yield (mg / L) of tetraacetyl phytosphingosine and triacetyl phytosphingosine in the microorganism can reach more than 150%, more than 170%, more than 190%, more than 210%, more than 230%, more than 245%, or more than 249%, for example, more than 250%, but not limited thereto.

[0029] The total yield (mg / g-cell) of tetraacetyl phytosphingosine and triacetyl phytosphingosine per unit dry cell of the microorganism can reach 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.

[0030] Based on a baseline of 100% total yield of tetraacetyl phytosphingosine and triacetyl phytosphingosine per unit dry weight of wild-type Wickham Severinia, the total yield (mg / L) of tetraacetyl phytosphingosine and triacetyl phytosphingosine per unit dry weight of the microorganism can reach 150% or more, 180% or more, 200% or more, 210% or more, or 240% or more, for example, 244%, but is not limited thereto. The unit for the yield of tetraacetyl phytosphingosine per unit dry weight can be mg / g-cell, but is not limited thereto.

[0031] Among the phytosphingosine derivatives produced by the microorganisms, including monoacetyl-, diacetyl-, triacetyl-, and tetraacetyl phytosphingosine, the yields of tetraacetyl phytosphingosine and triacetyl phytosphingosine may be higher than the yields of the other types of phytosphingosine derivatives. For example, the yield of tetraacetyl phytosphingosine may be the highest, and the yield of triacetyl phytosphingosine may be the second highest.

[0032] In the phytosphingosine derivatives of the microorganisms containing monoacetyl, diacetyl, triacetyl, and tetraacetyl phytosphingosine, the yield of triacetyl phytosphingosine by weight may be more than 15%, more than 18%, more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, 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 not limited thereto.

[0033] In the phytosphingosine derivatives of the microorganisms containing monoacetyl, diacetyl, triacetyl and tetraacetyl phytosphingosine, the yield of tetraacetyl phytosphingosine by weight may be more than 65%, more than 68%, more than 70%, more than 71%, more than 72%, more than 73%, 65% to 76%, 68% to 76%, 70% to 76%, 71% to 76%, 72% to 76%, 73% to 76%, for example 73% or 75%, but not limited thereto.

[0034] The microorganism provided by the present invention is resistant to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid, which may have toxic effects on microbial growth and the production of phytosphingosine derivatives, and has excellent phytosphingosine derivative production capacity, specifically, excellent tetraacetyl phytosphingosine and / or triacetyl phytosphingosine production capacity.

[0035] The microorganisms according to the present invention are resistant to cyanobacterin.

[0036] Phytosphingomyelin is a fatty acid synthase inhibitor. When added to yeast growth medium, it inhibits yeast growth by blocking fatty acid synthesis. However, mutations in the fatty acid synthesis pathway can improve the production efficiency of tetraacetylphytosphingosine and triacetylphytosphingosine. Therefore, when using phytosphingomyelin to improve the production efficiency of tetraacetylphytosphingosine and triacetylphytosphingosine, the microorganisms used should have the ability to produce phytosphingosine derivatives and be resistant to phytosphingomyelin. This is more preferable for improving the production efficiency of tetraacetylphytosphingosine and triacetylphytosphingosine.

[0037] The resistance to phytosphingosine may refer to the ability to grow and produce phytosphingosine derivatives in a culture medium containing phytosphingosine at a concentration of 40 μg / ml or higher, but is not limited to this.

[0038] The microorganisms according to the present invention are resistant to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

[0039] "Ethylene glycol bis(3-aminoethyl ether)tetraacetic acid" can induce mutations in microorganisms through enzymatic chelation. These mutations can enhance the production efficiency of tetraacetyl phytosphingosine and triacetyl phytosphingosine in microorganisms. As a chelating agent, aminopolycarboxylic acid can be effectively used in buffer solutions that are similar to the cellular environment.

[0040] The resistance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid can refer to the ability to grow and produce phytosphingosine derivatives in a culture medium containing the microorganism at a concentration of ethylene glycol bis(3-aminoethyl ether)tetraacetic acid of 30 μg / ml or higher, but is not limited thereto.

[0041] The microorganism can be a strain of the genus *Wickhamia*, specifically *Wickhamia serratifolia*. Wickerhamomyces ciferrii ), Wickham Siam yeast ( W. siamensis Wickham alder yeast ( W.alni ), Wickham's dipyrtomyces ( W.bisporus Wickham Forest Yeast ( W.silvicola ) 、 Wickham Sildenafil yeast ( W.sydowiorum ), Wickham Chambard yeast ( W.chambardii ), abnormal Wickham yeast ( W.anomalus ), Wickham Arabian Praru yeast ( W.Arabprarous ) 、 Wickham Canadian Yeast ( W. canadensis ) 、 Wickham Pippoli yeast ( W.pijperi ) 、 Wickhamstrass yeast ( W. strasburgensis ) 、 Wickham Sildenafil yeast ( W.sydowiorum ), Wickham Bovine Yeast ( W.bovis ) 、 Wickham Shomie yeast ( W. chaumierensis ) 、 Wickham Soil Yeast ( W.edaphicus ), Wickham Hampshire yeast ( W. hampshirensis ) 、 Wickham Yutzman yeast ( W.jurtsmanii ) 、 Wickhamlineford yeast ( W.lynferdii ) 、 Wickham Mongola Yeast ( W. menglaensis ) 、 Wickham yeast ( W.mori ) 、 Wickham slime yeast ( W. mucosus Wickham Changcheng Yeast ( W.changensis ) 、 Wickham claw yeast ( W.onychis ) 、 Wickham Oriental Yeast W.orientalis ) 、 Wickham Patagonian yeast ( W. patagonicus ) 、 Wickham Quilloli yeast ( W.queroliae ) 、 Wickham Rabaul yeast ( W. rabaulensis ) 、 Wickham bark beetle yeast ( W.scolytoplatypi ), Wickham submembrane yeast ( W. subpelliculosus Wickham Darat yeast ( W.tratensis) or Wickham xylo-yeast ( W.xylosica (but not limited to this).

[0042] The microorganism may be Wickham Severin Yeast SYEC2-36, and may be the microorganism deposited at the Korean Microbial Collection Center on February 22, 2023, with accession number KCCM13333P.

[0043] The preserved strain has one or more characteristics selected from (1) to (4): (1) The yield of tetraacetyl phytosphingosine per unit dry weight of microorganisms with a cell weight of 15 mg or more (mg / g-cell). (2) Total yield of tetraacetyl phytosphingosine and triacetyl phytosphingosine per unit dry weight of microorganisms with a cell weight of 80 mg or more (mg / g-cell). (3) Resistance to cyanobacterium; and (4) Resistance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

[0044] The present invention may include the following steps: treating wild-type *Wickham Severus* with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and / or ultraviolet light (UV), and confirming that its tetraacetyl phytosphingosine and / or triacetyl phytosphingosine production capacity is higher than that of the parent strain *Wickham Severus* and screening it. Furthermore, the microorganism may further possess resistance to one or more compounds selected from ethylene glycol bis(3-aminoethyl ether)tetraacetic acid and cyanobacterin.

[0045] An example of the present invention provides a composition for producing phytosphingosine derivatives, the composition comprising a *Wickhamia* spp. microorganism having phytosphingosine derivative production capability and resistance to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid (EDTA). Wickerhamomyces.Sp The composition may contain one or more of the following: the microbial cell, the microbial cell lysate, the microbial culture, and its extract. The composition may further contain one or more selected from cyanobacterium and ethylene glycol bis(3-aminoethyl ether)tetraacetic acid, but is not limited thereto.

[0046] Another embodiment of the present invention relates to a method for producing plant sphingosine derivatives, the method comprising the steps of culturing (fermenting) one or more of the following: *Wickhamia spp.* microorganisms having the ability to produce plant sphingosine derivatives and resistance to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid; cells of said microorganisms; cell lysates of said microorganisms; cultures of said microorganisms; and extracts thereof.

[0047] Specifically, the step may be carried out by culturing *Wickhamia sphingosine* microorganisms capable of producing phytosphingosine derivatives and resistant to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid. The method for producing phytosphingosine derivatives may further include a step of recovering the phytosphingosine derivatives, based on the step of culturing *Wickhamia sphingosine* microorganisms.

[0048] The step of culturing the microorganism may include, but is not limited to, culturing the microorganism in a culture medium for producing plant sphingosine derivatives, or in a seed culture medium, and / or in a production culture medium for plant sphingosine derivatives.

[0049] When culturing in the seed culture medium, the culture temperature can 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.

[0050] When culturing in the seed culture medium, the culturing time can 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.

[0051] The culture in the seed culture medium can be carried out by shaking culture and can be carried out at various rpm conditions, such as 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, at 250 rpm, but is not limited thereto.

[0052] The seed culture medium may contain carbon sources, nitrogen sources, etc., and may specifically contain one or more selected from yeast extract, malt extract, peptone and glycerol, for example, YMGL medium, but is not limited thereto.

[0053] The carbon source can be one or more selected from glycerol, malt, dextrin, glucose, sucrose, acetic acid, ethanol, molasses and sulfite pulp waste liquor commonly used for microbial culture, specifically glycerol and / or malt, but not limited to these.

[0054] The nitrogen source may be one or more nitrogen-containing organic compounds selected from yeast, peptone, corn steep powder (CSP), corn steep liquor (CSL), urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, and casein, specifically yeast and / or peptone, but not limited to these.

[0055] The culture medium for producing the plant sphingosine derivative may include, but is not limited to, one or more of glycerol, yeast extract, corn steep liquor powder, ammonium sulfate, amino acids (e.g., serine, glycine, monosodium glutamate), sodium acetate, and calcium chloride.

[0056] When culturing in the production medium, the culture temperature may be the same as or different from that of the seed culture. 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.

[0057] Culture in the production medium can be carried out under various rpm conditions, such as 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, at 180 rpm, but is not limited thereto.

[0058] When culturing in the production medium, the pH conditions can 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 not limited thereto.

[0059] The cultivation in the production medium can be batch culture, fed-batch culture (e.g., fed-batch fermentation), or continuous culture, and can be carried out in more than one of the forms, but is not limited thereto. The fed-batch fermentation refers to the addition of culture medium during continuous culture or culture.

[0060] The culture in the production medium can be carried out by fed-batch fermentation, but is not limited to this.

[0061] The step of fed-batch fermentation in the production medium can involve inoculating and culturing microorganisms in the production medium while simultaneously supplying additional culture medium. Specifically, after all the glycerol in the production medium is depleted, the additional culture medium can be added to supply glycerol. The "additional culture medium" refers to a culture medium that is intermittently or continuously supplied to the culture medium after the start of microbial cultivation, in addition to the initial culture medium.

[0062] When the feed is used for batch fermentation, the production culture medium may contain one or more of the following, but is not limited to: glycerol, yeast extract, corn steep liquor powder, amino acids (e.g., serine, glycine, glutamic acid (monosodium glutamate)), ammonium sulfate, and calcium chloride (e.g., calcium chloride dihydrate).

[0063] The additional culture medium may contain one or more of the following, but is not limited to: 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.

[0064] The additional culture 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 per hour, for example 4 g / L, but not limited thereto.

[0065] When fermenting with the aforementioned feed in batches, the OD value of the production medium can be further supplied with corn steep liquor powder when it 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.

[0066] The OD value can be measured under light with 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 wavelength, but is not limited thereto.

[0067] The corn steep liquor powder can be supplied to the production culture medium at a concentration of 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 not limited thereto.

[0068] The corn steep liquor powder can be supplied dissolved in distilled water, for example, it can be supplied dissolved in primary distilled water, secondary distilled water and / or tertiary distilled water, but is not limited thereto.

[0069] When fermenting with the feed in batches and culturing in the production medium, if sodium acetate is added to the production medium, the pH of the production medium will increase, which may lead to difficulties in the initial culture. However, after the initial culture is completed and the glycerol is completely depleted, if sodium acetate is added to the additional medium, the growth of the cells and / or the yield of phytosphingosine (e.g., monoacetyl phytosphingosine, diacetyl phytosphingosine, triacetyl phytosphingosine and / or tetraacetyl phytosphingosine) can be increased.

[0070] When the culture is carried out in the production medium by fed-batch fermentation, the culture temperature can 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.

[0071] When cultured in the production medium by fed-batch fermentation, it can be carried out under various rpm conditions, such as 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 carried out at 900 rpm, but is not limited thereto.

[0072] When the culture is carried out in the production medium by fed-batch fermentation, the pH conditions can 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 not limited thereto.

[0073] When cultured in the production medium using fed-batch fermentation, the culture can be carried out at temperatures of 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 0.8 to 1.2 vvm, for example, 1 vvm, but is not limited thereto.

[0074] The recovery step of the phytosphingosine derivatives can use conventional extraction and recovery techniques to recover them from the microorganisms (cells) and / or fermentation broth (e.g., the culture medium of the microorganisms provided by the present invention), specifically from the culture cells and / or the fermentation supernatant. Once the desired product is obtained, it can be used directly or further processed according to the intended use. For example, acetylated derivatives of sphingosine, dihydrosphingosine, and / or phytosphingosine can be deacetylated by enzymatic or chemical methods.

[0075] The plant sphingosine derivatives obtained according to the present invention may include the use of the compound in compositions for use in food, cosmetics and skin, and specifically, may be used in the form of food ceramide in the form of glycoceramide.

[0076] Invention Effects The microorganisms resistant to cyanobacterium and / or ethylene glycol bis(3-aminoethyl ether)tetraacetic acid according to the present invention have excellent production capacity of phytosphingosine derivatives. When the microorganisms are used to produce phytosphingosine derivatives, phytosphingosine can be produced in an environmentally friendly and efficient manner compared with chemical synthesis methods. Detailed Implementation

[0077] The present invention will be described in more detail through the following embodiments, but is not intended to limit the scope of the claims through the following embodiments.

[0078] Example 1: Preparation of mutant microorganisms with improved tetraacetyl phytosphingosine production efficiency (1) Example 1-1: Cultivation and Recovery of Parental Strains In order to obtain Wickham Severin yeast ( Wickerhamomyces ciferrii A mutant microorganism with high tetraacetyl phytosphingosine production efficiency was obtained from the wild strain and then mutagenized. Specifically, strain ATCC14091 was obtained as the wild strain of *Wickham Severin*.

[0079] According to the composition in Table 1 below, 50 μl of stock glycerol solution was mixed into 3 ml of YMGL liquid medium with a pH of 5.5 and cultured with shaking at 30°C for 24 hours. The stock glycerol solution was prepared by mixing wild-type strain culture broth with 40 (w / w)% glycerol solution at a ratio of 1:1 and storing at -70°C.

[0080] Table 1

[0081] After shaking culture, 50 μl of the culture medium was inoculated into 3 ml of fresh YMGL liquid medium and incubated at 30 °C for 12 hours. Cell concentration was determined by measuring absorbance at 600 nm in the culture medium, and 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 cells were washed twice with 50 mM citrate buffer to remove culture medium components.

[0082] Examples 1-2: Strain Mutation Treatment The washed bacterial cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) at a concentration of 0.05 mg / ml for 30 minutes at room temperature 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 (EDTA) was added at a concentration ranging from 2 to 50 μg / ml to confirm the degree of growth inhibition. Inhibition was observed from a concentration of 30 μg / ml. Therefore, the microbial cells diluted with the sterile distilled water were plated onto YNB (amino acid-free) agar plates containing 30 μg / ml EDTA.

[0083] The mutant library was obtained by irradiating the agar plate containing the bacterial cells with ultraviolet light at a wavelength of 254 nm for 20 seconds at a distance of 15 cm from the plate and then culturing it at 30°C. The mutagenic microorganism was a microorganism with high tetraacetyl phytosphingosine production capacity and tolerance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid. Each microorganism was assigned an arbitrary number and named in the form of an E number (e.g., E284).

[0084] Examples 1-3: Primary screening and culture of bacterial colonies In order to screen out microorganisms with excellent tetraacetyl phytosphingosine production capacity from the mutagenic microorganisms of Examples 1-2, individual colonies with different shapes and sizes that did not overlap with other colonies were screened from the colonies grown in the plate culture medium of Examples 1-2.

[0085] The screened colonies and the parental strains from Example 1-1 before mutation treatment were inoculated separately into test tubes containing 3 ml of YMGL liquid medium as described in Table 1, and cultured with shaking at 30°C and 250 rpm for 96 hours. Nine times the volume of 100% methanol was added to the culture medium, and the mixture was vigorously stirred for 60 minutes at room temperature using a fine mixer to extract tetraacetyl phytosphingosine.

[0086] Subsequently, after centrifugation at 12,000 rpm for 10 minutes, the supernatant was filtered through a 0.2 μm filter, and the yield of tetraacetyl phytosphingosine was confirmed by HPLC analysis. The results are shown in Table 2.

[0087] Specific HPLC analysis was performed using HPLC-UV with a ZORBAX SB-C8 column (Agilent, 4.6 mm × 150 mm, 3.5 μm, USA). For the quantitative calculation of tetraacetyl phytosphingosine, tetraacetyl phytosphingosine was purchased from Sigma-Aldrich to ensure the standard curve, and the yield of tetraacetyl phytosphingosine (tetraacetyl phytosphingosine potency (mg / L)) and the yield of tetraacetyl phytosphingosine per unit cell weight (tetraacetyl phytosphingosine yield (mg / g-cell)) were measured.

[0088] Table 2

[0089] The production of tetraacetylphthysine was measured by HPLC analysis. The results showed that the tetraacetylphthysine production of the wild-type strain was 162 mg / mL. Six mutant microorganisms (E284, E218, E201, E125, E28, and E19) with higher tetraacetylphthysine production (393 to 528 mg / L) than the wild-type strain were screened. Among the six screened microorganisms, the E284 mutant strain showed the highest tetraacetylphthysine production (528 mg / L).

[0090] Example 2 Preparation of mutant microorganisms with improved tetraacetyl phytosphingosine production efficiency (2) Among the six strains that showed increased tetraacetyl phlosphingosine production compared to wild-type strains, strain E284, which exhibited significantly increased tetraacetyl phlosphingosine production and yield, underwent secondary mutation. The mutant screening method was largely the same as in Examples 1-1 and 1-2. For the mutagen-treated strains, cyanobacterium was used instead of ethylene glycol bis(3-aminoethyl ether)tetraacetic acid as a marker, with a concentration ranging from 0 to 50 μg / ml, to confirm the degree of growth inhibition. Inhibition was observed starting at a concentration of 40 μg / ml. Therefore, the mutagen-treated strains were plated on agar plates containing 40 μg / ml cyanobacterium to identify secondary mutant strains.

[0091] The additional mutagenic microorganism is a microorganism that is further screened by cyanobacterium from a primary screening of microorganisms that are resistant to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid, and each microorganism is assigned an arbitrary number and named in the form of EC number-number (e.g., EC2-34).

[0092] For the additional mutagenic microorganisms, the same methods as in Examples 1-3 were used to confirm the yield of tetraacetyl phytosphingosine and the yield per unit dry weight of tetraacetyl phytosphingosine (yield) through tetraacetyl phytosphingosine extraction and HPLC analysis. The extraction, yield, and yield of tetraacetyl phytosphingosine were also confirmed again for the E284 strain of Examples 1-3 and the wild-type strain of Example 1, and the results are shown in Table 3.

[0093] Table 3

[0094] The results of HPLC analysis on the yield of tetraacetyl phytosphingosine showed that the yield of tetraacetyl phytosphingosine in the wild plant was 162 mg / L, with a yield of 11 mg / g-cell. The yield of tetraacetyl phytosphingosine in the E284 plant selected in the first screening was 393 mg / L, with a yield of 31 mg / g-cell.

[0095] Among the mutant strains screened in a medium supplemented with phytosphingosine, five microorganisms (EC1-4, EC2-34, EC2-36, EC3-14, and EC4-43) were selected that showed increased tetraacetyl phytosphingosine production (528 to 705 mg / L) and tetraacetyl phytosphingosine yield (35 to 46 mg / g-cell) compared to the wild-type strain and E284.

[0096] Example 3: Confirmation of shake-flask yield of tetraacetylphytosphoprotein / acetylated phytosphoprotein Example 3-1: Strain Culture To compare the production of tetraacetyl phytosphingosine in wild-type and mutant strains of *Wickham Severus*, shake-flask cultures were performed. Five mutant strains tolerant to both p-ethylene glycol bis(3-aminoethyl ether)tetraacetic acid and cyanobacterium, along with the wild-type strain, were cultured in shake flasks to confirm the production of tetraacetyl phytosphingosine and triacetyl phytosphingosine.

[0097] Specifically, 3 ml of YMGL liquid culture medium from Table 1 was dispensed into 15 ml test tubes, and colonies of wild-type strains and five mutant strains resistant to both ethylene glycol bis(3-aminoethyl ether)tetraacetic acid and cyanobacterium obtained in Example 2 were inoculated respectively, and the cultures were shaken at 30°C and 250 rpm for 24 hours to obtain seed culture.

[0098] The seed culture was inoculated into a 250ml baffled flask containing 30ml of production culture medium with the composition shown in Table 4 below, until the OD value of the culture medium reached 0.3. The flask was then incubated at 30°C and 180rpm with shaking for 96 hours to obtain the production culture medium. The pH of the production culture medium was maintained at 5.2 by adding 50mM MES buffer.

[0099] Table 4

[0100] Example 3-2: Analysis of the yields of triacetylphytosphingosine and tetraacetylphytosphingosine The production culture medium obtained in Example 3-1 was subjected to a process that was substantially the same as in Examples 1-3, in which 100% methanol was added to the culture medium, and the supernatant obtained by centrifugation was separated and purified to obtain the analytical sample.

[0101] For the analyzed samples, the same method as in Examples 1-3 was used to confirm the yield of tetraacetyl phytosphingosine and triacetyl phytosphingosine (potency of triacetyl phytosphingosine + tetraacetyl phytosphingosine (mg / L)) and the yield per unit dry cell (yield) (yield of triacetyl phytosphingosine + tetraacetyl phytosphingosine (mg / g-cell)) by HPLC analysis.

[0102] Specifically, the lyophilized powder of the analytical sample was dissolved in CDCl3, and NMR analysis was performed using a Bruker Avance DPX400 (400MHz) to confirm the structure, thus confirming the presence of triacetylphytosphingosine in the sample. Furthermore, the analytical sample was separated, purified, and concentrated using a preparative HPLC system (manufacturer: Jai, Nippon Analytical Industries Co., Ltd. / resin: C18) to obtain a lyophilized powder. HPLC analysis was performed using methods substantially the same as in Examples 1-3 to measure the yield of triacetylphytosphingosine and the yield per unit cell weight of triacetylphytosphingosine. The yields of tetraacetylphytosphingosine and triacetylphytosphingosine obtained by the HPLC analysis and the yields per unit cell weight of tetraacetylphytosphingosine and triacetylphytosphingosine are shown in Table 5.

[0103] Table 5

[0104] The yields and rates of tetraacetylphthyrosine and triacetylphthyrosine were measured, ultimately identifying two strains: strain EC3-14, which showed the highest increase in the yields of both triacetylphthyrosine and tetraacetylphthyrosine compared to the wild-type strain; and strain EC2-36, which showed the highest yields of both tetraacetylphthyrosine and triacetylphthyrosine per cell. The yields of tetraacetylphthyrosine and acetylated phthyrosine were then confirmed by culturing these two selected strains, EC2-36 and EC3-14, in a fermenter.

[0105] Example 4: Production of acetylated phytosphoprotein in a fermenter According to Example 3-2, the two selected strains EC2-36 and EC3-14 were cultured in a fermenter and fed-batch fermentation was carried out to finally confirm the yield of tetraacetyl phytosphingosine and acetylated phytosphingosine.

[0106] Specifically, colonies of the microorganisms EC2-36 and EC3-14 were obtained in YMGL agar medium containing 15 g / L agar as described in Example 1. 3 ml of the YMGL liquid medium from Table 1 was dispensed into 15 ml test tubes, and colonies of strains EC2-36 and EC3-14 were inoculated separately. The cultures were then incubated with shaking at 30°C and 250 rpm for 24 hours to obtain seed culture solutions.

[0107] The seed culture solution was inoculated into a 500ml baffled shaker flask containing 100ml of YMGL liquid culture medium as described in Table 1, so that the OD value of the culture solution reached 0.3, and then cultured at 30℃ and 180rpm for 24 hours with shaking to obtain a preculture solution.

[0108] The pre-culture solution was inoculated into a 5L fermenter to bring the OD value of the fermentation culture to 0.3. The culture medium used for culturing was 2L of production culture medium containing the composition of Table 6 below. When the glycerol in the production culture medium was 20 to 30 g / L, glycerol was supplied at a rate of 4 g / L per hour using a feeding solution containing the composition of Table 7 below and culturing was carried out.

[0109] Table 6

[0110] Table 7

[0111] When the OD values ​​of the culture medium under 600 nm wavelength light were 130 and 260, a sterilized solution containing corn steep liquor powder dissolved in 30 mL of DDW was added to achieve a corn steep liquor powder concentration of 3 g / L. Filtered air was supplied to the production medium at a flow rate of 2 L / min, reaching an air flow rate of 1 vvm, and the mixture was stirred at 900 rpm at 30 °C. The pH of the culture medium was adjusted to 5.2 using 9% NH4OH, and antifoaming agent (SB2121, struktol) stock solution was added at 10 μl / 20 min, followed by fermentation for 151 hours.

[0112] Using a method largely the same as in Examples 3-2, 100% methanol was added to the obtained culture medium, and phytosphingosine and its derivatives were extracted by centrifugation. The yields of various types of acetylated phytosphingosine were analyzed. The product yields of strains EC2-36 and EC3-14 are shown in Table 8.

[0113] Table 8

[0114] Analysis of the products showed that the EC2-36 microorganism produced the highest yields of acetylated phytosphingosine, with triacetylphingosine and tetraacetylphingosine being the most abundant, specifically 4.19 g / L for triacetylphingosine and 13 g / L for tetraacetylphingosine. The EC3-14 strain also produced the highest yields of acetylated phytosphingosine, with triacetylphingosine being 2.76 g / L and tetraacetylphingosine being 8.1 g / L.

[0115] The EC2-36 strain, which was identified as having high production efficiency of triacetyl phytosphingosine and tetraacetyl phytosphingosine, was deposited at the Korea Center for Microbiology on February 22, 2023, with accession number KCCM13333P, and named SYEC2-36.

[0116] [Collection Number] Name of depository: Korea Center for Microbial Preservation Accession number: KCCM13333P Preservation date: 20230222

Claims

1. A type of microorganism belonging to the genus *Wickhamia* ( Wickerhamomyces.Sp It has the ability to produce plant sphingosine derivatives and resistance to phytosphingosine.

2. The *Wickhamomilla* microorganism according to claim 1, wherein, The phytosphingosine derivative is selected from one or more of the following groups: tetraacetyl phytosphingosine and triacetyl phytosphingosine.

3. The microorganism according to claim 2, wherein, The yield of tetraacetyl phytosphingosine per unit dry weight of the microorganism (mg / g-cell) reached more than 160% of the yield per unit dry weight of wild-type Wickham Severus yeast, based on 100% of the yield per unit dry weight.

4. The microorganism according to claim 2, wherein, The total yield (mg / g-cell) of tetraacetyl phytosphingosine and triacetyl phytosphingosine per unit dry weight of the microorganisms reached more than 150% of the yield per unit dry weight of wild-type Wickham Severus yeast, based on 100% of the yield per unit dry weight.

5. The microorganism according to claim 1, wherein, The resistance to phytosphingosine refers to the ability to grow and produce phytosphingosine derivatives in a culture medium containing phytosphingosine at a concentration of 40 μg / ml or higher.

6. The microorganism according to claim 1, wherein, The microorganism further exhibits resistance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

7. The microorganism according to claim 6, wherein, The resistance to ethylene glycol bis(3-aminoethyl ether)tetraacetic acid (EDTA) refers to the ability to grow and produce phytosphingosine derivatives in a culture medium containing the microorganism at a concentration of EDTA of 30 μg / ml or higher.

8. The microorganism according to claim 1, wherein, The microorganism is Wickham Severinia ( Wickerhamomyces ciferrii ).

9. The microorganism according to claim 1, wherein, The microorganism is Wickham Silver Saccharomyces cerevisiae with the preservation number KCCM13333P. Wickerhamomyces ciferrii ).

10. A composition for producing plant sphingosine derivatives, wherein, The composition comprises one or more selected from the group consisting of: cells of *Wickhamia spp.* microorganisms according to any one of claims 1-9, cell lysates of the microorganisms, cultures of the microorganisms, and extracts thereof.

11. The composition for producing phytosphingosine derivatives according to claim 10, wherein, The composition further comprises one or more selected from the group consisting of: cyanobacterium and ethylene glycol bis(3-aminoethyl ether)tetraacetic acid.

12. A method for producing a plant sphingosine derivative, wherein, The method includes the following steps: culturing *Wickhamia* microorganisms as described in any one of claims 1-9, and recovering phytosphingosine derivatives from the microbial culture medium.

13. The method for producing plant sphingosine derivatives according to claim 12, wherein, The microbial culture process is carried out using fed-batch fermentation.

14. The method for producing plant sphingosine derivatives according to claim 13, wherein, The step of culturing microorganisms involves inoculating and culturing microorganisms in a production culture medium while simultaneously supplying additional culture medium.

15. The method for producing plant sphingosine derivatives according to claim 14, wherein, The microbial culture process is carried out under conditions of pH 4 to 7.

16. The method for producing plant sphingosine derivatives according to claim 14, wherein, The production culture medium comprises one or more selected from the group consisting of glycerol, yeast extract, corn steep liquor powder, amino acids, ammonium sulfate, and calcium chloride.

17. The method for producing plant sphingosine derivatives according to claim 14, wherein, The additional culture medium is added at a concentration of 1 to 10 g / L per hour.

18. The method for producing plant sphingosine derivatives according to claim 14, wherein, The additional culture medium comprises one or more selected from the group consisting of glycerol, yeast extract, amino acids, ammonium sulfate, calcium chloride, and sodium acetate.

19. The method for producing plant sphingosine derivatives according to claim 14, wherein, When the OD value of the production culture medium is between 100 and 150 or between 230 and 300, corn steep liquor powder is further supplied.

20. The method for producing plant sphingosine derivatives according to claim 19, wherein, The corn steep liquor powder is supplied to achieve a concentration of 1 to 5 g / L in the production medium.

21. The method for producing plant sphingosine derivatives according to claim 14, wherein the method is carried out at a temperature of 10 to 50°C.

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