Microbial medium composition for retinol production containing surfactant and use thereof

Abstract

The present application relates to a method for producing retinol comprising a step of culturing a Yarrowia microorganism in a medium containing a nonionic surfactant, a method for increasing retinol production, a method for producing a retinoid, a Yarrowia microorganism medium composition for producing retinol comprising a nonionic surfactant, and a composition for producing retinol comprising the microorganism or a culture thereof and a nonionic surfactant.

Description

[Technical Field]

[0001] The present application relates to a method for producing retinol, which includes a step of culturing a Yarrowia microorganism in a medium containing a nonionic surfactant; a method for increasing retinol production; a method for producing a retinoid; a medium composition for a Yarrowia microorganism for producing retinol, which contains a nonionic surfactant; a composition for producing retinol, which contains the microorganism or a culture thereof and a nonionic surfactant; and use for producing a retinoid. [Background technology]

[0002] Retinol, a fat-soluble vitamin, is an essential vitamin involved in eye health (for improving night blindness), strengthening the immune system, and healthy skin. However, retinol is highly unstable against heat, light, temperature, moisture, oxygen, and the passage of time, and is easily oxidized when exposed to the air or in aqueous solutions. This poses a major stability problem, causing discoloration and odor due to a decrease in the potency of the raw material, and negatively impacting retinol production.

[0003] Therefore, although many techniques have been developed to stabilize the retinol compound itself in compositions or products containing retinol (Patent Document 1), there are currently few methods developed to stably increase retinol production. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,858,217 [Non-patent literature]

[0005] [Non-Patent Document 1] D.-C. Chen et al., Appl Microbiol Biotechnol, 1997 [Non-patent document 2] http: / / atgme.org Summary of the Invention [Problem to be solved by the invention]

[0006] The problem to be solved by the present application is to provide a microbial medium composition for retinol production containing a surfactant, a method for producing retinoids using the same, and uses thereof. [Means for solving the problem]

[0007] The present application aims to provide a method for producing retinol using a nonionic surfactant.

[0008] Another object of the present application is to provide a method for increasing retinol production using a nonionic surfactant.

[0009] A further object of the present application is to provide a method for producing retinoids other than retinol using a nonionic surfactant.

[0010] Another object of the present application is to provide a microbial medium composition for retinol production that uses a nonionic surfactant.

[0011] A further object of the present application is to provide a composition for producing retinol using a nonionic surfactant.

[0012] A further object of the present application is to provide use of a nonionic surfactant in the production of retinoids. [Effects of the Invention]

[0013] The medium of the present application contains a non-ionic surfactant, and therefore can efficiently increase the production of retinoids such as retinol. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1A shows OD values ​​as a result of flask culture evaluation of a β-carotene-producing strain in the presence or absence and concentration of a surfactant, and FIG. 1B shows OD values ​​as a result of flask culture evaluation of a retinol-producing strain in the presence or absence and concentration of a surfactant. [Figure 2] FIG. 1A is a graph showing the β-carotene concentration as a result of flask culture evaluation of a β-carotene-producing strain in the presence or absence and concentration of a surfactant, and FIG. 1B is a graph showing the β-carotene, retinal, and retinol concentrations as a result of flask culture evaluation of a retinol-producing strain in the presence or absence and concentration of a surfactant. [Figure 3] FIG. 1A is a graph showing OD values ​​as a result of flask culture evaluation of a β-carotene-producing strain depending on the presence, type, and concentration of a Tween surfactant; and FIG. 1B is a graph showing OD values ​​as a result of flask culture evaluation of a retinol-producing strain depending on the presence, type, and concentration of a Tween surfactant. [Figure 4] FIG. 1A is a graph showing β-carotene concentration as a result of flask culture evaluation of a β-carotene-producing strain depending on the presence, type, and concentration of a Tween surfactant; and FIG. 1B is a graph showing β-carotene, retinal, and retinol concentrations as a result of flask culture evaluation of a retinol-producing strain depending on the presence, type, and concentration of a Tween surfactant. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present application will be described in detail below. Note that the description and embodiment of one aspect disclosed in this application also apply to the description and embodiment of other aspects with respect to common matters. Furthermore, all combinations of various elements disclosed in this application are included in this application. Furthermore, documents cited in this application are incorporated by reference into this application. Furthermore, the present application is not limited to the following specific description.

[0016] One aspect of the present application provides a method for producing retinol, comprising culturing a Yarrowia sp. microorganism in a medium comprising a non-ionic surfactant.

[0017] In this application, the term "surfactant" refers to a compound having both a hydrophilic and a hydrophobic portion. Surfactants are classified into anionic, cationic, nonionic (polar), zwitterionic, and special surfactants depending on the charge of the hydrophilic portion when dissociated in water. The surfactants in this application are nonionic surfactants, and are classified into sorbitan fatty acid salts (e.g., span), polyoxyethyl sorbitan fatty acid salts (e.g., tween), polyethyl ether fatty acid salts (e.g., Triton), etc. depending on their chemical structure.

[0018] In one embodiment, the surfactant is at least one selected from the group consisting of Tween and Triton, but is not limited thereto.

[0019] In one embodiment, the twin is at least one selected from the group consisting of twin 20, twin 40, twin 60 and twin 80, but is not limited thereto.

[0020] In one embodiment, the Triton is Triton X-100, but is not limited thereto.

[0021] The nonionic surfactant may be used in an amount of 0.001% or more, 0.001 to 30%, 0.01% or more, 0.01 to 30%, 0.01 to 25%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 2%, 0.01 to 1%, 0.05 to 30%, 0.05 to 25%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.01 to 2%, 0.01 to 1%, 0.1 to 30%, 0.1 to 25%, 0.1 to 20%, 0.1 to 15%, 0.1 to 10%, 0.1 to 5%, 0.1 These may be present at concentrations of, but are not limited to, 0.2%, 0.1% to 1%, 0.5% to 30%, 0.5% to 25%, 0.5% to 20%, 0.5% to 15%, 0.5% to 10%, 0.5% to 5%, 0.5% to 2%, 0.5% to 1%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 2%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 15%, 2% to 10%, 2% to 5%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 30%, 10% to 25%, 10% to 20%, and 10% to 15% (w / v).

[0022] In one example, but not limited to, the nonionic surfactant increases the extracellular excretion of retinol.

[0023] In one embodiment, the method includes culturing a Yarrowia microorganism in a medium containing the nonionic surfactant, thereby minimizing the consumption of time and labor resources and enabling stable production of retinol.

[0024] The term "culture medium" as used herein refers to a substance containing a mixture of nutrients, as its main components, necessary for culturing the Yarrowia microorganism of the present application, and supplies nutrients, growth factors, and the like, including water, which are essential for survival and growth.

[0025] As an example, the medium of the present application may be a medium for producing retinol, and may further contain substances necessary for retinol production, but is not limited thereto.

[0026] The culture medium and other culture conditions used for culturing the Yarrowia microorganism of the present application may be a conventional culture medium that already contains a nonionic surfactant, or may further contain a nonionic surfactant, and may be any medium used for culturing conventional microorganisms.

[0027] The medium of the present application is a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., with temperature, pH, etc. adjusted, but is not limited thereto.

[0028] Examples of carbon sources used in the present application include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0029] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0030] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0031] During cultivation of the Yarrowia microorganism of the present application, the pH of the medium may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid to the medium in a suitable manner. Furthermore, foam formation may be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic or microaerobic states, but these are not limiting examples.

[0032] In the present application, "Yarrowia microorganism" or "Yarrowia strain" includes all wild-type Yarrowia microorganisms and Yarrowia microorganisms that have been genetically modified, either naturally or artificially, and includes Yarrowia microorganisms that have been genetically modified for retinol production, which are microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by strengthening or inactivating the activity of an endogenous gene.

[0033] In one example, the Yarrowia microorganism of the present application is Yarrowia lipolytica, but is not limited thereto.

[0034] In one embodiment, the Yarrowia microorganism of the present application is a microorganism for producing retinol. The retinol-producing microorganism or strain may be, but is not limited to, a microorganism that naturally has the ability to produce retinol, or a microorganism in which a parent strain lacking retinol production has been genetically modified, either naturally or artificially, to enhance or confer retinol production ability. Specifically, the retinol-producing Yarrowia microorganism of the present application may be a microorganism modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and beta-carotene 15,15'-oxygenase (BLH) proteins.

[0035] The microorganism of the present application may be a microorganism that has been modified to further contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, and thus exhibits or enhances the activity of these proteins. The lycopene cyclase / phytoene synthase or phytoene desaturase may be a protein derived from, but not limited to, Xanthophyllomyces dendrorhous. In one example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include the nucleotide sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1 or GenBank: AY177424.1), respectively. In one example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. The coding region of the polynucleotide may be modified in various ways, taking into account codon degeneracy or codons preferred in the microorganism in which the protein is to be expressed, as long as the amino acid sequence is not changed.Specifically, the polynucleotide has a base sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or contains the base sequence; or consists of a base sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or is essentially composed of the base sequence, but is not limited to these.

[0036] The microorganism of the present application may be, but is not limited to, a microorganism that has been modified to further contain a polynucleotide encoding a geranylgeranyl pyrophosphate synthase (GGPPS) protein and exhibits or enhances the activity of the protein. The geranylgeranyl pyrophosphate synthase is a protein derived from Haematococcus pluvialis, but is not limited thereto. For example, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have or include the nucleotide sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: APX64485.1). For example, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have or include the sequence set forth in SEQ ID NO: 33. The coding region of the polynucleotide can be modified in various ways without changing the amino acid sequence, taking into account codon degeneracy or codons preferred in the microorganism in which the protein is to be expressed. Specifically, the polynucleotide has or contains a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 33, or consists of or essentially consists of a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 33, but is not limited thereto.

[0037] The microorganisms of the present application include, but are not limited to, microorganisms that have been modified to further contain a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein and exhibit the activity of the protein, or microorganisms with enhanced activity of the protein. β-Carotene 15,15'-oxygenase is a protein derived from uncultured marine bacterium 66A03, but is not limited to this. For example, the β-carotene 15,15'-oxygenase polypeptide and the polynucleotide encoding it may have or include the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). For example, the β-carotene 15,15'-oxygenase polypeptide may have or include the sequence of SEQ ID NO: 57. The polynucleotide encoding the polypeptide can be modified in various ways within the coding region, without altering the amino acid sequence, taking into account codon degeneracy or the codons preferred in the microorganism to be expressed in. Specifically, the polypeptide has or contains a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% identical to the sequence of SEQ ID NO: 57, or consists of or essentially consists of a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% identical to the sequence of SEQ ID NO: 57, but is not limited thereto.

[0038] In the present application, "culturing" refers to growing a Yarrowia microorganism of the present application under appropriately adjusted environmental conditions. In the present application, the culturing process can be carried out using a suitable medium and culture conditions known in the art, as long as a medium containing a nonionic surfactant is used. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0039] The Yarrowia microorganism of the present application is cultured under aerobic conditions in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, with temperature, pH, and other parameters controlled.

[0040] In the culture of the present application, the culture temperature is maintained at 20 to 35°C, specifically 25 to 35°C, and the culture is carried out for about 10 to 160 hours, about 20 to 130 hours, about 24 to 120 hours, about 36 to 120 hours, about 48 to 120 hours, about 48 hours or more, about 48 hours, about 72 hours, or about 120 hours, but is not limited to these.

[0041] The retinol produced by the culture of the present application is either secreted into the medium or remains within the microorganism.

[0042] In this application, "retinol" refers to a substance known as vitamin A and is a type of retinoid. Retinol may be used as it is, or may be converted into other retinoids (e.g., retinal, retinoic acid, retinyl esters, etc.) or carotenoid compounds by methods known in the art.

[0043] The present application may significantly improve retinol production ability by adding a nonionic surfactant to a microbial culture medium for retinol production.

[0044] In one embodiment, the nonionic surfactant increases the extracellular secretion of retinol, but is not limited thereto. In one embodiment, when a microorganism is cultured in a medium containing the nonionic surfactant, the retinol production ability is increased by about 1% or more, specifically about 3%, about 5%, about 10%, about 50%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 380%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, or about 680% or more, compared to when the microorganism is cultured in a medium without the nonionic surfactant. However, any nonionic surfactant may be used as long as it has an increase in the + value compared to the microorganism before the addition of the nonionic surfactant.

[0045] The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0046] The retinol production method of the present application may further include a step of preparing the Yarrowia microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0047] The medium may be supplemented with a non-ionic surfactant to increase retinol production.

[0048] The retinol production method of the present application may further include a step of recovering retinol from the culture medium (the medium in which the culture was carried out) or the microorganism of the present application. The recovery step may be further included after the culturing step.

[0049] The recovery may involve collecting the target retinol using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used, and the target retinol can be collected from the medium or the microorganism using a suitable method known in the art.

[0050] The retinol production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the retinol production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed chronologically (or consecutively) regardless of the order, simultaneously, or integrated into a single step, but are not limited thereto.

[0051] Another aspect of the present application provides a method for producing a retinoid, comprising the steps of culturing a Yarrowia microorganism in a medium containing a nonionic surfactant, and converting retinol produced by the microorganism into a retinoid other than retinol.

[0052] The nonionic surfactant, medium, microorganism, culture, retinol, retinoid, etc. are as described above, and the recovery and purification of retinol described above are similarly applicable to the recovery and purification of retinoid.

[0053] The retinoid production method of the present application may further include a step of converting the retinol produced by the microorganism of the present application into a retinoid other than retinol. In the retinoid production method of the present application, the conversion step may be further included after the culturing step or the recovery step. The conversion step can be carried out by a suitable method known in the art. For example, the conversion can be carried out using retinol acyltransferase, but is not limited thereto.

[0054] In one embodiment, the retinoid is any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester, but may be any one included in the retinoid.

[0055] Yet another aspect of the present application provides a method for increasing retinol production, comprising culturing a Yarrowia microorganism in a medium comprising a non-ionic surfactant.

[0056] The nonionic surfactant, medium, microorganism, culture, retinol, etc. are as described above.

[0057] Yet another aspect of the present application provides a Yarrowia microorganism medium composition for producing retinol, comprising a non-ionic surfactant.

[0058] In one example, the medium composition increases retinol production in a microorganism of the genus Yarrowia, but is not limited to this example.

[0059] In one example, but not limited to, the medium composition promotes the growth of microorganisms.

[0060] The nonionic surfactant, retinol, microorganisms, and medium are as described above.

[0061] Yet another aspect of the present application provides a composition for producing retinol, comprising a Yarrowia microorganism or a culture thereof, and a nonionic surfactant.

[0062] The compositions of the present application may further comprise any suitable excipients commonly used in compositions for producing retinol, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0063] The microorganisms, nonionic surfactants, retinol, etc. are as described above.

[0064] Still other aspects of the present application provide the use of a nonionic surfactant for retinol production, the use of a Yarrowia microorganism medium composition comprising a nonionic surfactant for retinol production, and the use of a composition comprising a Yarrowia microorganism or a culture thereof and a nonionic surfactant for retinoid production.

[0065] The retinoid used in the production of the retinoid may be retinol or a retinoid other than retinol. The nonionic surfactant, microorganism, medium, retinoid, retinol, etc. are as described above. [Example]

[0066] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0067] Construction of platform strains for retinol production Example 1-1. Construction of a crtYB-crtI insertion strain derived from Xanthophyllomyces dendroas To construct a platform Yarrowia strain for retinol production, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes from Xanthophyllomyces dendroas were inserted into the genome of Yarrowia liplytica KCCM12972P.

[0068] The polynucleotide sequence of crtYB (SEQ ID NO: 1) was obtained based on the nucleotide sequence registered with the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1), and the polynucleotide sequence of crtI (SEQ ID NO: 2) was obtained based on the nucleotide sequence registered with NCBI (GenBank: AY177424.1). The polynucleotide sequences of crtYB and crtI were synthesized by Macrogen in the form of TEFINtp-crtYB-CYC1t (SEQ ID NO: 3) and TEFINtp-crtI-CYC1t (SEQ ID NO: 4). A cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene locus using the Yarrowia lipolytica URA3 gene (SEQ ID NO: 5) as a selectable marker.

[0069] Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers SEQ ID NOs: 6 and 7, 8 and 9, 10 and 11, 12 and 13, 14 and 15, and 16 and 17 shown in Table 1. PCR conditions included 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 3 minutes. The resulting DNA fragments were combined into a single cassette by overlap extension PCR.

[0070] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (Non-Patent Document 1), and colonies were then grown on a uracil-free solid medium (YLMM1). Colonies in which the cassette had been confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers set forth in SEQ ID NOs: 18 and 19, and cultured at 30°C for 3 days. The URA3 marker was removed by obtaining colonies grown on the 5-FOA solid medium.

[0071] [Table 1]

[0072] The compositions of the uracil-free solid medium (YLMM1) and the 5-FOA medium are as follows: <Yarrowia lipolytica minimal media 1(YLMM1)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Agar 15g / L 5-Fluoroorotic Acid (5-FOA) Glucose 20 g / L, Yeast nitrogen base without amino acids 6.7 g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2 g / L, Uracil 50 μg / mL, 5-fluoroorotic acid (5-FOA) 1 g / L, and agar 15 g / L

[0073] Example 1-2. Construction of HMGR-enhanced strain A cassette was designed to replace the native promoter (SEQ ID NO: 20) of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain prepared in Example 1-1 with the TEFINt promoter. PCR was performed using the genomic DNA of KCCM12972P as a template and primers SEQ ID NOs: 21 and 22, 23 and 24, 25 and 26, 27 and 28, and 29 and 30 shown in Table 2. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute and 30 seconds. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0074] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted were smeared on 5-FOA solid medium using primers of SEQ ID NO: 31 and SEQ ID NO: 32 and cultured at 30°C for 3 days. The URA3 marker was removed by obtaining colonies formed on the 5-FOA solid medium.

[0075] [Table 2]

[0076] Example 1-3. Preparation of GGPPS-introduced strain As described above, the geranylgeranyl pyrophosphate synthase (GGPPS) gene derived from Haematococcus pluvialis was inserted into the genome of the strain prepared in Example 1-2.

[0077] The polynucleotide sequence of GGPPS (SEQ ID NO: 33) was obtained based on the nucleotide sequence (GenBank: APX64485.1) registered with the National Center for Biotechnology Information Search database (NCBI). The GGPPS polynucleotide sequence was codon-optimized for Yarrowia lipolytica according to Non-Patent Document 2, and the gene was synthesized in the form of TEFINtp-GGPPS-CYC1t (SEQ ID NO: 34) by requesting Macrogen. A cassette was designed to be inserted into the LIG4 (YALI0D21384g) gene position using the Yarrowia lipolytica URA3 gene (SEQ ID NO: 5) as a selection marker. PCR was performed using the synthesized GGPPS gene and KCCM12972P genomic DNA as templates and primers (SEQ ID NOs: 35 and 36, 37 and 38, 39 and 40, 41 and 42, and 43 and 44) ​​shown in Table 3. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes. The resulting DNA fragments were then synthesized into a single cassette by overlap extension PCR.

[0078] The cassette thus prepared was introduced into the strain prepared in Example 1-2 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers set forth in SEQ ID NOs: 45 and 46, and cultured at 30°C for 3 days. The URA3 marker was removed by obtaining colonies formed on the 5-FOA solid medium.

[0079] [Table 3]

[0080] Example 1-4. Construction of KU80 deletion strain To facilitate future strain construction, the KU80 (YALI0E02068g) gene was deleted from the strain constructed in Examples 1-3 as described above. To this end, a KU80 gene deletion cassette was designed using the Y. lipolytica URA3 gene (SEQ ID NO: 5) as a selectable marker. PCR was then performed using the genomic DNA of KCCM12972P as a template and primers SEQ ID NOs: 47 and 48, 49 and 50, 51 and 52, and 53 and 54. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute and 30 seconds. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0081] The cassette thus prepared was introduced into the strain prepared in Examples 1-3 by the heat shock method as described above, and then colonies were grown on uracil-free solid medium (YLMM1). Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers set forth in SEQ ID NOs: 55 and 56 and cultured at 30°C for 3 days. The URA3 marker was removed by culturing the colonies grown on 5-FOA solid medium, and the resulting strain was designated CC08-1043.

[0082] [Table 4]

[0083] Example 1-5. Construction of BLH-introduced strains The β-carotene 15,15'-oxygenase (BLH) gene from uncultured marine bacterium 66A03 was inserted into the genome of the strain prepared in Examples 1-4. The polypeptide sequence of the BLH gene, SEQ ID NO: 57, was obtained based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). The codons were then optimized for Yarrowia lipolytica according to Non-Patent Document 2, and the gene was synthesized by Macrogen in the form of TEFINtp-BLH-CYC1t (SEQ ID NO: 58). A cassette was designed to be inserted at the KU70 (YALI0C08701g) gene position using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker. Using the synthesized BLH gene and KCCM12972P genomic DNA as templates, PCR was performed using primers SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, and SEQ ID NOs: 67 and 68 shown in Table 5. PCR conditions included 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes. The resulting DNA fragments were combined into a single cassette by overlap extension PCR.

[0084] The cassette thus prepared was introduced into the strain prepared in Examples 1-4 by the heat shock method, and colonies were then grown on a uracil-free solid medium (YLMM1). Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers set forth in SEQ ID NOs: 69 and 70 and cultured at 30°C for 3 days. The URA3 marker was removed by culturing the colonies grown on the 5-FOA solid medium, and the resulting strain was designated CC08-2163.

[0085] [Table 5] [Example]

[0086] Comparative evaluation of retinol production capacity by type of surfactant Example 2-1. Cultivation of Microorganisms To compare the levels of β-carotene and retinol production depending on the type of surfactant and whether or not it was added, flask evaluation was performed on strains CC08-1043 and CC08-2163. The CC08-1043 or CC08-2163 strain was inoculated into 20 ml of YLMM2 (Yarrowia lipolytica minimal media 2) medium in a 250 ml corner-baffled flask to an initial OD of 2. Five surfactants, Tween 20 (TW20, Sigma, CAS Number 9005-64-5), Tween 40 (TW40, Sigma, CAS Number 9005-66-7), Tween 60 (TW60, Sigma, CAS Number 9005-67-8), Tween 80 (TW80, Sigma, CAS Number 9005-65-6), and Span 80 (SP80, Sigma, CAS Number 1338-43-8), were added at 2% concentration, and two surfactants, sodium dodecyl sulfate (SDS, Sigma, CAS Number 151-21-3) and Triton Xanthan Gum, were added. X-100 (TX, Sigma, CAS Number 9036-19-5) was added to the medium at a concentration of 0.05 or 1%. Cultivation was carried out at 30°C and 200 rpm. Because the sugar consumption rate differed depending on the type of surfactant added, the culture was continued for 48 hours until all residual sugar was consumed.

[0087] Example 2-2: Evaluation of microbial growth To check the degree of growth over time, the OD value was measured at a wavelength of 600 nm using a spectrophotometer.

[0088] The OD values ​​of the two strains are shown in Table 6, and the OD values ​​of the β-carotene-producing strain (CC08-1043) and the retinol-producing strain (CC08-2163) are shown in Figure 1a and Figure 1b, respectively.

[0089] [Table 6]

[0090] As a result, growth was not achieved under any concentration conditions when SDS, an anionic surfactant, was added among the surfactants used in this experiment. Under all surfactant addition conditions except SDS, biomass (OD) generally tended to be higher than under no surfactant addition conditions.

[0091] After 48 hours of culture, in which all sugar had been consumed, retinal, retinol, and β-carotene were extracted and their concentrations analyzed in the surfactant-added groups (Tween 20, Tween 40, Tween 60, Tween 80, Span 80, and Triton X-100-added groups).

[0092] Example 2-3: Evaluation of β-carotene, retinal, and retinol concentrations The intracellular and extracellular concentrations of β-carotene, retinol, and retinal were measured as follows.

[0093] To measure intracellular β-carotene, retinol, and retinal, 0.05 ml of culture medium was centrifuged and the supernatant removed. 0.5 ml of DMSO (dimethyl sulfoxide, Sigma, Cas Number 67-68-5) was added and the cells were disrupted by shaking at 55°C for 10 minutes at 2,000 rpm. Next, 0.5 ml of acetone (Sigma, Cas Number 67-64-1) containing 4% BHT (Sigma, Cas Number 128-37-0) was added and the cells were shaken at 45°C for 15 minutes at 2,000 rpm. The extracted β-carotene, retinol, and retinal were quantitatively analyzed using HPLC.

[0094] To measure extracellular (Ext.) β-carotene, retinol, and retinal, 0.95 ml of acetone (Sigma) containing 4% BHT was added to 0.05 ml of the supernatant after removing the cells from the culture, and the mixture was shaken (2,000 rpm) at 45°C for 15 minutes. The extracted β-carotene, retinal, and retinol were quantitatively analyzed using an HPLC system.

[0095] The β-carotene, retinol, and retinal concentrations (mg / L) in the two strains are shown in Table 7. Among these analytical results, the β-carotene concentration measured in the culture of the β-carotene-producing strain (CC08-1043) is shown in Figure 2(a), and the β-carotene, retinol, and retinal concentrations measured in the culture of the retinol-producing strain (CC08-2163) are shown in Figure 2(b).

[0096] [Table 7]

[0097] As a result, in an evaluation of the β-carotene-producing CC08-1043 strain, it was confirmed that the β-carotene concentration increased by up to 1.4 times in the group containing Tween surfactants (Tween 20, Tween 40, Tween 60, and Tween 80), known as nonionic surfactants, compared to the untreated group, while the β-carotene concentration decreased by more than 10% in the group containing Span 80 and 0.05% Triton X-100 (Figure 2a).

[0098] In contrast to this result, Tween-based surfactants (TW20, 40, 60, and 80) and Triton-based surfactants (Triton X-100) all increased retinol production. In the control group without surfactant, no extracellular retinol was detected, and only intracellular retinol was measured.

[0099] These results confirmed that the addition of nonionic surfactants increased the retinol production concentration by up to 6.8 times or more compared to the no-addition condition. [Example]

[0100] Comparative evaluation of retinol production capacity at different concentrations of Tween surfactants Example 3-1. Cultivation of Microorganisms To compare the effects of Tween-based nonionic surfactants on β-carotene and retinol production, strains CC08-1043 and CC08-2163 were subjected to flask assays. 20 ml of YLMM2 (Yarrowia lipolytica minimal media 2) medium was added to a 250 ml corner-baffled flask containing strains CC08-1043 and CC08-2163 at an initial OD of 2. Four surfactants, Tween 20 (TW20), Tween 40 (TW40), Tween 60 (TW60), and Tween 80 (TW80), were added to the medium at concentrations of 5, 10, or 15%, respectively. Cultures were performed at 30°C and 200 rpm. Because the sugar consumption rate varied depending on the surfactant concentration, the cultures were allowed to continue for 48 hours, at which time all sugars were consumed.

[0101] Example 3-2: Evaluation of microbial growth Microbial growth was evaluated in the same manner as in Example 2-2. The OD values ​​of the two strains are shown in Table 8, and the analyzed OD values ​​of the β-carotene-producing strain (CC08-1043) and the retinol-producing strain (CC08-2163) are shown in Figure 3a and Figure 3b, respectively.

[0102] [Table 8]

[0103] As a result, regardless of the surfactant concentration, biomass (OD) tended to be generally higher in all Tween surfactant-added groups compared to the unadded group, and biomass (OD) tended to be even higher in groups added with Tween 40, Tween 60, or Tween 80. However, it was confirmed that within the same type of Tween-added groups, the difference in biomass (OD) due to concentration was not significant (Figure 3).

[0104] Example 3-3: Evaluation of β-carotene, retinal, and retinol concentrations The concentrations of β-carotene, retinal and retinol were evaluated in the same manner as in Example 2-3.

[0105] The β-carotene, retinol, and retinal concentrations (mg / L) in the two strains are shown in Table 9. Among these analytical results, the β-carotene concentration measured in the culture of the β-carotene-producing strain (CC08-1043) is shown in Figure 4(a), and the β-carotene, retinol, and retinal concentrations measured in the culture of the retinol-producing strain (CC08-2163) are shown in Figure 4(b).

[0106] [Table 9]

[0107] As a result, in the evaluation of the β-carotene-producing CC08-1043 strain, regardless of the surfactant concentration or type, the intracellular β-carotene production concentration tended to be higher overall in all Tween surfactant-added groups compared to the non-added group (Figure 4a). As a result, in the Tween surfactant-added group, the β-carotene concentration increased by up to 1.5 times compared to the non-added group.

[0108] Evaluation of the retinol-producing strain CC08-2163 confirmed that extracellular retinol was measured only in the group containing Tween-based surfactants (Figure 4b). In contrast, in the group without surfactants, only relatively small amounts of intracellular retinol were measured. The optimal concentration for retinol extracellular secretion varied depending on the type of Tween. For example, while there was no difference in retinol production depending on the concentration of Tween 20, the highest extracellular retinol concentrations were measured at 10% Tween 40 and 5% Tween 60 and 80.

[0109] To summarize these results, the degree of retinol production and extracellular excretion differs depending on the type of Tween, but the retinol production concentration increases by up to 3.8 times or more under certain Tween-added conditions compared to the unadded group (Figure 4b).

[0110] These results confirmed that the addition of a nonionic surfactant to the medium not only promoted the growth of the retinol-producing strain but also increased the concentration of retinol produced.

[0111] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

[0112] The sequences of the SEQ ID NOs of this application are shown in Table 10.

[0113] [Table 10-1]

[0114] [Table 10-2]

[0115] Table 10-3

[0116] Table 10-4

[0117] Table 10-5

[0118] Table 10-6

[0119] Table 10-7

[0120] Table 10-8

[0121] Table 10-9

[0122] Table 10-10

[0123] Table 10-11

[0124] Table 10-12

[0125] Table 10-13

[0126] Table 10-14

[0127] Table 10-15

[0128] JPEG0007819351000025.jpg208145

Claims

1. 1. A method for producing retinol, comprising: culturing a Yarrowia microorganism in a medium containing a nonionic surfactant, the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and The method for producing retinol, wherein the nonionic surfactant increases the extracellular excretion of retinol.

2. 2. The method for producing retinol according to claim 1, wherein the Tween® is at least one selected from the group consisting of Tween 20, Tween 40, Tween 60, and Tween 80.

3. The method for producing retinol according to claim 1 , wherein the microorganism is for producing retinol.

4. 2. The method for producing retinol according to claim 1, wherein the nonionic surfactant is contained at a concentration of 0.01% (w / v) or more relative to the total medium composition.

5. 2. The method for producing retinol according to claim 1, further comprising recovering retinol from the medium or microorganism.

6. 1. A method for increasing retinol production, comprising culturing a Yarrowia microorganism in a medium containing a non-ionic surfactant, the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and A method for increasing retinol production, wherein the nonionic surfactant increases the extracellular excretion of retinol.

7. Cultivating a Yarrowia microorganism in a medium containing a nonionic surfactant; and converting the retinol produced by the microorganism into a retinoid other than retinol, the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and The method for producing a retinoid, wherein the nonionic surfactant increases the extracellular excretion of retinol.

8. A medium composition for a Yarrowia microorganism for producing retinol, comprising a nonionic surfactant, the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and A medium composition, wherein the nonionic surfactant increases extracellular excretion of retinol.

9. The medium composition according to claim 8 , wherein the nonionic surfactant is contained at a concentration of 0.01% or more based on the total medium composition.

10. The medium composition according to claim 8, wherein the medium composition increases retinol production by a microorganism.

11. A composition for producing retinol, comprising a Yarrowia microorganism or a culture thereof and a nonionic surfactant, the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and A composition for producing retinol, wherein the nonionic surfactant increases the extracellular excretion of retinol.

12. The composition for producing retinol according to claim 11 , wherein the microorganism is for producing retinol.

13. Use of a Yarrowia microorganism medium composition containing a nonionic surfactant, or a composition containing a Yarrowia microorganism or a culture thereof and a nonionic surfactant, for producing a retinoid, comprising: the non-ionic surfactant is at least one selected from the group consisting of Tween® and Triton®; and The nonionic surfactant increases the extracellular excretion of retinol.

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