Microbial medium composition for retinol production containing antioxidants 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 an antioxidant, a method for increasing retinol production, a method for producing a retinoid, a medium composition for a Yarrowia microorganism for producing retinol containing an antioxidant, and a composition for producing retinol comprising the microorganism or a culture thereof and an antioxidant.

Description

[Technical Field]

[0001] The present application relates to a method for producing retinol, which comprises culturing a Yarrowia microorganism in a medium containing an antioxidant; a method for increasing retinol production; a method for producing a retinoid; a medium composition for a Yarrowia microorganism for producing retinol, which comprises an antioxidant; a composition for producing retinol, which comprises the microorganism or a culture thereof and an antioxidant; and uses thereof. [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 an antioxidant, 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 an antioxidant.

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

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

[0010] Another object of the present application is to provide a microbial medium composition for producing retinol that uses an antioxidant.

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

[0012] Furthermore, the present application aims to provide the use of antioxidants in the production of retinoids. [Effects of the Invention]

[0013] The medium of the present application contains an antioxidant, and therefore can efficiently increase the production of retinoids such as retinol. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the microbial growth rate at each culture time in a medium supplemented with an antioxidant. [Figure 2] FIG. 1 shows the increase in retinol with the addition of antioxidants. 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 microorganism in a medium containing an antioxidant.

[0017] In this application, the term "antioxidant" refers collectively to substances that prevent oxidation. The term "antioxidant" is used interchangeably with "antioxidant for oxidative stress," "antioxidant for active oxygen," and / or "antiaging," and may refer to any substance that can prevent oxidation and increase retinol production.

[0018] The antioxidant is at least one selected from the group consisting of 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), vitamin C (or ascorbic acid), and glutathione (GSH), but is not limited thereto.

[0019] The antioxidant is contained at a concentration of 0.0001 to 10%, 0.0001 to 5%, 0.0001 to 1%, 0.001 to 10%, 0.001 to 5%, 0.001 to 1%, 0.01 to 10%, 0.01 to 5%, 0.01 to 1%, 0.01 to 0.09%, 0.01 to 0.08%, 0.01 to 0.07%, 0.01 to 0.06%, or 0.01 to 0.05% (w / v) relative to the total medium composition, but is not limited to these contents.

[0020] In one embodiment, the method includes culturing a Yarrowia microorganism in a medium containing the antioxidant, thereby minimizing the consumption of time and labor resources and enabling stable production of retinol.

[0021] 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.

[0022] 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.

[0023] The medium and other culture conditions used for culturing the Yarrowia microorganism of the present application may be a conventional medium that already contains an antioxidant, or may further contain an antioxidant, and may be any medium used for culturing conventional microorganisms.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The term "microorganism" or "strain" in this application includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and includes microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and also includes microorganisms that have been genetically modified for retinol production.

[0030] In one example, the microorganism of the present application is a microorganism of the genus Yarrowia (Yarrowia sp.), but is not limited thereto.

[0031] In one example, but not limited to, the microorganism of the present application is Yarrowia lipolytica.

[0032] In one embodiment, the microorganism of the present application is a microorganism for producing retinol. The microorganism or strain for producing retinol 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 that does not have the ability to produce retinol has been genetically modified, either naturally or artificially, to enhance or confer retinol production ability. Specifically, the microorganism for producing retinol of the present application may be a Yarrowia microorganism that has been modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and beta-carotene 15,15'-oxygenase (BLH) proteins.

[0033] 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.

[0034] 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.

[0035] 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 polypeptide encoding it may have or contain the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). For example, the β-carotene 15,15'-oxygenase polypeptide may have or contain the sequence of SEQ ID NO: 57. 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% 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.

[0036] 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 an antioxidant 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.

[0037] 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.

[0038] 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, about 72 hours, or about 120 hours, but is not limited to these.

[0039] The retinol produced by the culture of the present application is either secreted into the medium or remains within the microorganism.

[0040] 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.

[0041] The present application may significantly improve retinol production capacity by adding an antioxidant to a microbial culture medium for retinol production.

[0042] For example, when a microorganism is cultured in a medium containing an antioxidant, the retinol production ability is improved by about 1% or more, specifically about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% or more, compared to when the microorganism is cultured in a medium without the antioxidant. However, any increase in the + value compared to before the antioxidant is added is acceptable.

[0043] 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.

[0044] 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.

[0045] The method for producing retinol of the present application may further include a step of recovering retinol from the culture medium (culture medium) or the microorganism of the present application. The recovery step may be further included after the culturing step.

[0046] 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.

[0047] 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.

[0048] 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 an antioxidant, and converting retinol produced by the microorganism into a retinoid other than retinol.

[0049] The antioxidant, medium, microorganism, culture, retinol, retinoid, etc. are as described above, and the above-described contents regarding the recovery and purification of retinol are equally applicable to the recovery and purification of retinoids.

[0050] 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.

[0051] 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.

[0052] Yet another aspect of the present application provides a method for increasing retinol production, comprising culturing a Yarrowia microorganism in a medium containing an antioxidant.

[0053] The antioxidant, medium, microorganism, culture, retinol, etc. are as described above.

[0054] Yet another aspect of the present application provides a Yarrowia microorganism medium composition for producing retinol, which comprises an antioxidant.

[0055] In one example, the medium composition increases retinol production in a microorganism of the genus Yarrowia, but is not limited thereto.

[0056] In one example, but not limited to, the medium composition promotes the growth of microorganisms.

[0057] The antioxidant, retinol, microorganisms and medium are as described above.

[0058] Yet another aspect of the present application provides a composition for producing retinol, comprising a Yarrowia microorganism or a culture thereof, and an antioxidant.

[0059] 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.

[0060] The microorganisms, antioxidants, retinol, etc. are as described above.

[0061] Further aspects of the present application provide use of an antioxidant for retinol production, use of a Yarrowia microorganism medium composition containing an antioxidant for retinol production, and use of a composition containing a Yarrowia microorganism or a culture thereof and an antioxidant for retinoid production. The retinoid used for retinoid production may be retinol or a retinoid other than retinol.

[0062] The antioxidant, microorganism, medium, retinoid, retinol, etc. are as described above. [Example]

[0063] 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]

[0064] Preparation of Yarrowia lipolytica platform strains for retinol production Example 1-1. Construction of a crtYB-crtI insertion strain derived from Xanthophyllomyces dendroas To construct a platform strain of Yarrowia for retinol production, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes from Xanthophyllomyces dendroas were inserted into the genome of the high-fat yeast strain KCCM12972P.

[0065] The polynucleotide sequence of crtYB (SEQ ID NO: 1) was obtained based on the nucleotide sequence (GenBank: AY177204.1) registered with the National Center for Biotechnology Information Search database (NCBI), and the polynucleotide sequence of crtI (SEQ ID NO: 2) was obtained based on the nucleotide sequence (GenBank: AY177424.1) registered with NCBI. 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.

[0066] Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers represented by 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. 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 3 minutes. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0067] 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 was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers represented by SEQ ID NOs: 18 and 19 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on the 5-FOA solid medium. 5-Fluoroorotic Acid (5-FOA) Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, uracil 50μg / mL, 5-Fluoroorotic acid (5-FOA) 1g / L, agar 15g / L

[0068] [Table 1]

[0069] Example 1-2. Construction of HMGR-enhanced strain A cassette was designed to replace the endogenous (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.

[0070] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by the heat shock method, and then colonies formed on uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette insertion was confirmed were smeared on 5-FOA solid medium using primers of SEQ ID NOs: 31 and 32 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium.

[0071] [Table 2]

[0072] Example 1-3. Preparation of GGPPS-introduced strain First, the geranylgeranyl pyrophosphate synthase (GGPPS) gene derived from Haematococcus pluvialis was inserted into the genome of the strain prepared in Example 1-2.

[0073] 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 codons of the GGPPS polynucleotide sequence were optimized for Yarrowia lipolytica according to Non-Patent Document 2, and the gene was synthesized by Macrogen in the form of TEFINtp-GGPPS-CYC1t (SEQ ID NO: 34). 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.

[0074] Using the synthesized GGPPS gene and KCCM12972P genomic DNA as templates, PCR was performed using primers represented by 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 converted into a single cassette by overlap extension PCR.

[0075] The cassette thus prepared was introduced into the strain prepared in Example 1-2 by the heat shock method, and then colonies formed on uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers of SEQ ID NOs: 45 and 46 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium.

[0076] [Table 3]

[0077] Example 1-4. Construction of KU80 deletion strain To facilitate future strain construction, the KU80 (YALI0E02068g) gene was deleted. To achieve this, a KU80 gene deletion cassette was designed using the Y. lipolytica URA3 gene (SEQ ID NO: 5) as a selectable marker. PCR was performed using the genomic DNA of KCCM12972P (listed in Table 4) 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.

[0078] 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 formed on uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers represented by SEQ ID NOs: 55 and 56 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium.

[0079] [Table 4]

[0080] 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 Yarrowia lipolytica URA3 gene (SEQ ID NO: 5) as a selection marker. Using the synthesized BLH gene and KCCM12972P genomic DNA as templates, PCR was performed using primers represented by SEQ ID NOs: 59 and 60, 61 and 62, 63 and 64, 65 and 66, and 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.

[0081] The cassette thus prepared was introduced into the strain prepared in Examples 1-4 by the heat shock method, and then colonies formed on uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on 5-FOA solid medium using primers represented by SEQ ID NOs: 69 and 70 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium.

[0082] [Table 5] [Example]

[0083] Evaluation of the effects of antioxidants on retinol production A flask assay was performed to compare the level of retinol production by retinol-producing strains with and without the addition of antioxidants and with different types of antioxidants. The retinol-producing platform strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of YLMM2 (Yarrowia lipolytica minimal media 2) medium to an initial OD of 4. Four antioxidants, 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), vitamin C, and glutathione (GSH), were added to the medium at concentrations ranging from 0.01 to 0.05%. Cultures were performed at 30°C and 200 rpm.

[0084] To assess the degree of growth over time, the OD value was measured at 600 nm using a spectrophotometer. The retinol concentration was measured as follows: After incubation, 1 ml of the culture medium was centrifuged to remove the supernatant. Then, 0.5 ml of DMSO (dimethyl sulfoxide, Sigma) was added and the cells were disrupted by shaking (agitation; 2,000 rpm) at 55°C for 10 minutes. Next, 0.5 ml of acetone (Sigma) containing 4% BHT was added and the cells were shaken (agitation; 2,000 rpm) at 45°C for 15 minutes. The extracted retinoids were quantitatively analyzed using an HPLC system. The analyzed OD values ​​are shown in Figure 1, and the retinoid concentrations are shown in Figure 2.

[0085] Because the sugar consumption rate differs depending on the type of antioxidant added to the medium, BHT, PG, vitamin C, and GSH were cultured for 48 hours. After 48 hours of culture, retinal and retinol were extracted and quantified.

[0086] As a result, when BHT, PG, vitamin C, and GSH were added, the retinol concentration increased by up to 1.89 times, 1.82 times, 1.44 times, and 1.21 times, respectively, compared to the control group, which did not contain any antioxidants. In some cases, the biomass (OD) was generally higher than in the control group, and it was confirmed that the antioxidants also promoted the growth of microorganisms (Figure 2).

[0087] These results confirmed that adding an antioxidant to the medium during the cultivation of a retinol-producing strain not only promotes the growth of the retinol-producing strain but also improves the concentration of retinol produced.

[0088] 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.

[0089] The sequences of the SEQ ID NOs of the present application are shown in Table 6.

[0090] [Table 6-1]

[0091] [Table 6-2]

[0092] [Table 6-3]

[0093] [Table 6-4]

[0094] [Table 6-5]

[0095] [Table 6-6]

[0096] [Table 6-7]

[0097] [Table 6-8]

[0098] [Table 6-9]

[0099] [Table 6-10]

[0100] Table 6-11

[0101] Table 6-12

[0102] Table 6-13

[0103] Table 6-14

[0104] Table 6-15

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Claims

1. A method for producing retinol, comprising culturing a retinol-producing Yarrowia lipolytica in a medium containing an antioxidant.

2. 2. The method for producing retinol according to claim 1, wherein the antioxidant is at least one selected from the group consisting of 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), ascorbic acid (vitamin C), and glutathione (GSH).

3. 2. The method for producing retinol according to claim 1, wherein the antioxidant is contained at a concentration of 0.001% (w / v) or more relative to the total medium composition.

4. 2. The method for producing retinol according to claim 1, further comprising recovering retinol from the medium or Yarrowia lipolytica.

5. A method for increasing retinol production, comprising culturing retinol-producing Yarrowia lipolytica in a medium containing an antioxidant.

6. Culturing retinol-producing Yarrowia lipolytica in a medium containing an antioxidant; and converting the retinol produced by the Yarrowia lipolytica into a retinoid other than retinol.

7. A Yarrowia lipolytica medium composition for retinol production containing an antioxidant.

8. 8. The medium composition according to claim 7, wherein the antioxidant is at least one selected from the group consisting of 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), ascorbic acid (vitamin C), and glutathione (GSH).

9. The medium composition according to claim 8 , wherein the antioxidant is contained at a concentration of 0.001% or more based on the total medium composition.

10. The medium composition of claim 7, wherein the medium composition increases retinol production in Yarrowia lipolytica.

11. The medium composition of claim 7 , wherein the medium composition promotes the growth of Yarrowia lipolytica.

12. A composition for producing retinol, comprising Yarrowia lipolytica for producing retinol or a culture thereof, and an antioxidant.

13. A use of a retinol-producing Yarrowia lipolytica medium composition containing an antioxidant, or a composition containing retinol-producing Yarrowia lipolytica or a culture thereof and an antioxidant, for retinoid production.

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