Microbial medium composition for producing retinol containing an antioxidant 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 comprising the 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 Yarrowia microorganism medium composition for producing retinol containing an antioxidant, a composition for producing retinol comprising 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 very unstable to heat, light, temperature, moisture, oxygen, and the passage of time, and is easily oxidized when exposed to the air or in an aqueous solution. This causes discoloration and odor due to a decrease in the potency of the raw material, which is a major stability problem and has a negative impact on 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] US Patent No. 6858217 [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 producing retinol containing an antioxidant, a method for producing retinoid using the same, and use thereof. [Means for solving the problem]

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

[0008] It is also an object of the present application to provide a method for increasing retinol production using an antioxidant.

[0009] It is a further object of the present application to provide a method for producing a retinoid other than retinol using an antioxidant.

[0010] Furthermore, the present application has an object to provide a microbial medium composition for producing retinol using an antioxidant.

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

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

[0013] Since the medium of the present application contains an antioxidant, it can efficiently increase the production of retinoids such as retinol. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the microbial growth rate at each incubation time in a medium supplemented with an antioxidant. [Diagram 2] FIG. 1 shows the increase in retinol with the addition of antioxidants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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. In addition, 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" is a general term for a substance that prevents 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 0.0001-10%, 0.0001-5%, 0.0001-1%, 0.001-10%, 0.001-5%, 0.001-1%, 0.01-10%, 0.01-5%, 0.01-1%, 0.01-0.09%, 0.01-0.08%, 0.01-0.07%, 0.01-0.06%, or 0.01-0.05% (w / v) relative to the total medium composition, but is not limited to these contents.

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

[0021] The term "culture medium" in the present application refers to a substance that is a mixture of nutrients necessary for culturing the Yarrowia microorganism of the present application as the main components, and supplies nutrients such as water essential for survival and growth, as well as growth factors.

[0022] As an example, the medium of the present application may be a medium for producing retinol, and may further contain substances necessary for producing retinol, 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] The carbon source in the present application may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0026] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0027] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, such as amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0028] During the cultivation of the Yarrowia microorganism of the present application, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid may be added to the medium in a suitable manner to adjust the pH of the medium. Also, during the cultivation, an antifoaming agent such as a fatty acid polyglycol ester may be used to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0029] In the present application, the term "microorganism" or "strain" 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 enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and includes microorganisms that have been genetically modified for retinol production.

[0030] In one embodiment, but not limited to, the microorganism of the present application is a Yarrowia sp. microorganism.

[0031] In one embodiment, 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 retinol-producing ability, or a parent strain that does not have retinol-producing ability that has been genetically modified naturally or artificially to enhance or impart retinol-producing ability. Specifically, the microorganism for producing retinol of the present application may be a Yarrowia microorganism 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 a polynucleotide encoding a lycopene cyclase / phytoene synthase (crtYB) or a phytoene desaturase (crtI) protein and exhibits the activity of the protein, or a microorganism in which the activity of the protein is enhanced. The lycopene cyclase / phytoene synthase or phytoene desaturase is a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto. In one embodiment, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include a base sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1 or GenBank: AY177424.1), respectively. In one embodiment, 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 polynucleotide may have various modifications in the coding region, without altering the amino acid sequence, due to codon degeneracy or taking into account the codons preferred in the microorganism in which the protein is to be expressed.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 substantially composed of the base sequence, but is not limited to these.

[0034] The microorganism of the present application is a microorganism that has been modified to further contain a polynucleotide encoding a geranylgeranyl pyrophosphate synthase (GGPPS) protein and exhibits the protein activity or has enhanced protein activity, but is not limited thereto. The geranylgeranyl pyrophosphate synthase is a protein derived from Haematococcus pluvialis, but is not limited thereto. As an example, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have a base sequence (GenBank: APX64485.1) registered in the NCBI (National Center for Biotechnology Information Search database), or may include the base sequence. As an example, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have a sequence of SEQ ID NO: 33, or may include the sequence. The polynucleotide may be modified in various ways in the coding region without changing the amino acid sequence, taking into consideration the codon degeneracy or the codons preferred in the microorganism in which the protein is to be expressed. Specifically, the polynucleotide may have a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 33, or may contain the base sequence, or may consist of a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 33, or may essentially consist of the base sequence, but is not limited thereto.

[0035] The microorganism of the present application is a microorganism that has been modified to further contain a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein and exhibits the protein activity or has enhanced protein activity, but is not limited thereto. β-carotene 15,15'-oxygenase is a protein derived from uncultured marine bacterium 66A03, but is not limited thereto. As an example, the β-carotene 15,15'-oxygenase polypeptide and the polypeptide encoding it may have an amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase) or may include said amino acid sequence. As an example, the β-carotene 15,15'-oxygenase polypeptide may have a sequence of SEQ ID NO: 57 or may include said sequence. The polynucleotide may be modified in various ways in the coding region without changing the amino acid sequence, taking into consideration the codon degeneracy or the codons preferred in the microorganism in which the protein is to be expressed. Specifically, the polynucleotide may have a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 57, may contain the base sequence, may consist of a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 57, or may essentially consist of the base sequence, but is not limited thereto.

[0036] In the present application, "culturing" means growing the Yarrowia microorganism of the present application under appropriately adjusted environmental conditions. In the present application, as long as a medium containing an antioxidant is used, the culture process can be performed in a suitable medium and under suitable culture conditions known in the art. Such a culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0037] The Yarrowia microorganism of the present application is cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions with controlled temperature, pH, etc.

[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 performed 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 cultures of the present application is either secreted into the medium or remains within the microorganisms.

[0040] In this application, "retinol" refers to a substance known as vitamin A, which is a type of retinoid. The retinol may be used as it is, or may be converted to 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 medium for producing retinol.

[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% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, or about 80% or more, compared to when the microorganism is cultured in a medium without the addition of an antioxidant. However, any increase in the + value compared to before the addition of the antioxidant is acceptable.

[0043] The term "about" refers to a range including, but not limited to, ±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."

[0044] The retinol production method of the present application may further include a step of preparing a Yarrowia microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), e.g., prior to the culturing step.

[0045] The method for producing retinol of the present application may further include a step of recovering retinol from the medium used in the culture (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.

[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 chromatographies 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] In addition, 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 at different times (or consecutively) regardless of the order, or may be performed simultaneously or integrated into one step, but is 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 antioxidants, medium, microorganisms, culture, retinol, retinoids, etc. are as described above, and the contents regarding the recovery and purification of retinol are similarly applied 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 converting step may further be included after the culturing step or the recovering step. The converting step can be carried out by any 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 esters, but may be any one included in the retinoids.

[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 that includes an antioxidant.

[0055] As an example, the medium composition increases retinol production in a Yarrowia microorganism, but is not limited thereto.

[0056] In one non-limiting example, the medium composition may be one that promotes the growth of microorganisms.

[0057] The antioxidant, retinol, microorganism 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 the retinoid production may be retinol or a retinoid other than retinol.

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

[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 fully understood and easily implemented by a skilled engineer in the technical field of the present application or a similar technical field. EXAMPLES

[0064] Preparation of Yarrowia lipolytica platform strains for retinol production Example 1-1. Construction of a strain with crtYB-crtI insertion derived from Xanthophyllomyces dendroas To generate 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 KCCM12972P.

[0065] The polynucleotide sequence of crtYB was obtained as SEQ ID NO:1 based on the base sequence (GenBank: AY177204.1) registered in the National Center for Biotechnology Information Search database (NCBI), and the polynucleotide sequence of crtI was obtained as SEQ ID NO:2 based on the base sequence (GenBank: AY177424.1) registered in the 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 position using the URA3 gene (SEQ ID NO:5) of Yarrowia lipolytica as a selection marker.

[0066] Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers of 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 prepared into one 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 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 of SEQ ID NOs: 18 and 19, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker. <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. Preparation of HMGR-enhanced strain A cassette was designed to replace the endogenous (native) promoter (SEQ ID NO: 20) site of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain prepared in Example 1-1 with the TEFINt promoter, and PCR was performed using the genomic DNA of KCCM12972P as a template and the primers SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, and SEQ ID NO: 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 reaction at 72 ° C for 1 minute and 30 seconds. The resulting DNA fragment was prepared into one 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 a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette insertion was confirmed were smeared on a 5-FOA solid medium using primers of SEQ ID NOs: 31 and 32, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[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] GGPPS was obtained as a polynucleotide of SEQ ID NO: 33 based on the base sequence (GenBank: APX64485.1) registered in the National Center for Biotechnology Information Search database (NCBI). The polynucleotide sequence of GGPPS was codon-optimized to match 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 to be inserted into the LIG4 (YALI0D21384g) gene position was designed using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker.

[0074] Using the synthesized GGPPS gene and the genomic DNA of KCCM12972P as templates, PCR was performed using primers of 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 reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one 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 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 of SEQ ID NOs: 45 and 46, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[0076] [Table 3]

[0077] Example 1-4. Construction of KU80 deletion strain For ease of future strain construction, the KU80 (YALI0E02068g) gene was deleted. To this end, a KU80 gene deletion cassette was designed using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker. PCR was performed using the genomic DNA of KCCM12972P shown in Table 4 as a template and primers SEQ ID NO: 47 and 48, SEQ ID NO: 49 and 50, SEQ ID NO: 51 and 52, and SEQ ID NO: 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 made into one cassette by overlap extension PCR.

[0078] The cassette thus prepared was introduced into the strain prepared in Example 1-3 by the heat shock method as described above, 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 of SEQ ID NOs: 55 and 56, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[0079] [Table 4]

[0080] Example 1-5. Preparation of BLH-introduced strain The beta-carotene 15,15'-oxygenase (BLH) gene derived from the marine bacterium 66A03 was inserted into the genome of the strain prepared in Example 1-4. The BLH gene was obtained by obtaining the polypeptide sequence of SEQ ID NO: 57 based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). Next, the codons were optimized according to Yarrowia lipolytica according to Non-Patent Document 2, and the gene was then synthesized in the form of TEFINtp-BLH-CYC1t (SEQ ID NO: 58) by requesting Macrogen Corporation. A cassette to be inserted at the KU70 (YALI0C08701g) gene position was designed using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker. Using the synthesized BLH gene and the genomic DNA of KCCM12972P as templates, PCR was performed using primers of SEQ ID NOs: 59 and 60, 61 and 62, 63 and 64, 65 and 66, and 67 and 68 shown in Table 5. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0081] The cassette thus prepared was introduced into the strain prepared in Example 1-4 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 of SEQ ID NOs: 69 and 70, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[0082] [Table 5] EXAMPLES

[0083] Assessment of the effects of antioxidants on retinol production A flask evaluation was performed to compare the degree of retinol production by retinol-producing strains with and without the addition of antioxidants and their types. Retinol-producing platform strains were inoculated into 250-mL corner baffle flasks containing 25 mL of YLMM2 (Yarrowia lipolytica minimal media 2) medium to an initial OD of 4, and four types of antioxidants, 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), vitamin C, and glutathione (GSH), were added to the medium at concentrations of 0.01 to 0.05%. The culture was performed at 30°C and 200 rpm.

[0084] To evaluate the degree of growth depending on the culture time, the OD value was measured at a wavelength of 600 nm using a spectrophotometer. The retinol concentration was measured as follows. After the culture was completed, 1 ml of the culture solution was centrifuged to remove the supernatant, and 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 (2,000 rpm) at 45°C for 15 minutes. The retinoids extracted in this way were quantitatively analyzed using an HPLC device. The analyzed OD values ​​are shown in Figure 1, and the retinoid concentrations are shown in Figure 2.

[0085] Since 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 each. After the 48-hour 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 a maximum of 1.89 times, 1.82 times, 1.44 times, and 1.21 times, respectively, compared to the control group where no antioxidants were added, and in some cases, the biomass (OD) was generally higher than in the control group where no antioxidants were added, confirming that the antioxidants also promote the growth of microorganisms (Figure 2).

[0087] These results confirmed that the addition of 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, a person 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 its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[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

[0105] JPEG2025510211000022.jpg204145

Claims

1. A method for producing retinol, comprising the step of culturing a Yarrowia microorganism 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 microorganism is Yarrowia lipolytica.

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

5. 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 based on the total medium composition.

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

7. 1. A method for increasing retinol production, comprising culturing a Yarrowia microorganism in a medium containing an antioxidant.

8. Cultivating a Yarrowia microorganism in a medium containing an antioxidant; and converting the retinol produced by the microorganism into a retinoid other than retinol.

9. A medium composition for producing retinol from a Yarrowia microorganism comprising an antioxidant.

10. 10. The medium composition according to claim 9, 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, and glutathione (GSH).

11. The medium composition according to claim 10, wherein the antioxidant is contained at a concentration of 0.001% or more based on the total medium composition.

12. 10. The medium composition of claim 9, wherein the microorganism is Yarrowia lipolytica.

13. The medium composition according to claim 9 , which increases retinol production in a microorganism.

14. The medium composition according to claim 9 , which promotes the growth of a microorganism.

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

16. Use of an antioxidant, a medium composition for a Yarrowia microorganism containing an antioxidant, or a composition containing a Yarrowia microorganism or a culture thereof and an antioxidant in the production of a retinoid.

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