Microbial culture medium composition for production of retinol comprising antioxidant and use thereof

Abstract

The present application relates to: a method for producing retinol, the method comprising a step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant; a method for increasing the yield of retinol; a process for the production of a retinoid; a culture medium composition of a microorganism of the genus Yarrowia for producing retinol, the composition comprising an antioxidant; and a composition for producing retinol, the composition comprising the microorganism or a culture thereof and an antioxidant.

Description

Technical Field

[0001] The present disclosure relates to a method for producing retinol, the method comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant; a method for increasing retinol production; a method for producing a retinoid; a culture medium composition for a microorganism of the genus Yarrowia for producing retinol, the composition comprising an antioxidant; a composition for producing retinol, the composition comprising a microorganism or a culture thereof and an antioxidant; and uses thereof. Background Art

[0002] Retinol is a fat-soluble vitamin that is an essential vitamin associated with eye health, immunity enhancement, skin health, etc. to improve night blindness. However, since retinol is very unstable to heat, light, temperature, humidity, oxygen and extended time, it is easily oxidized when exposed to air or in aqueous solution. This leads to reduced potency of the raw material, major stability issues such as discoloration, odor, etc., and negative impacts on retinol production.

[0003] Therefore, many technologies have been developed to stabilize the retinol compound itself in a composition or product containing retinol (US Pat. No. 6858217). However, the development of methods for stably increasing retinol production remains insignificant. Summary of the invention

[0004] [Technical issues]

[0005] The problem to be solved by the present disclosure is to provide a microbial culture medium composition for producing retinol, the composition comprising an antioxidant, a method for producing retinoids using the composition, and uses thereof.

[0006] [Technical solution]

[0007] An object of the present disclosure is to provide a method for producing retinol using an antioxidant.

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

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

[0010] Another object of the present disclosure is to provide a microbial culture medium composition for producing retinol using an antioxidant.

[0011] Another object of the present disclosure is to provide a composition for producing retinol.

[0012] Another object of the present disclosure is to provide use of antioxidants in the production of retinoids.

[0013] [Beneficial Effects]

[0014] By including an antioxidant, the culture medium of the present disclosure can effectively increase the production of retinoids such as retinol. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The growth rate of microorganisms according to the culture time in the culture medium supplemented with antioxidants is shown.

[0016] Figure 2 It was shown that the production of retinol increased due to the addition of antioxidants. DETAILED DESCRIPTION

[0017] Hereinafter, the present disclosure will be described in detail as follows. Meanwhile, each embodiment and implementation scheme disclosed in the present disclosure may also be applied to other embodiments and implementation schemes. In addition, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the documents described in the present disclosure are incorporated herein by reference. In addition, the scope of the present disclosure is not limited by the specific implementation schemes described below.

[0018] One aspect of the present disclosure provides a method for producing retinol, the method comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant.

[0019] As used herein, the term "antioxidant" refers to substances that prevent oxidation. Antioxidation can be used interchangeably with preventing oxidative stress, preventing active oxygen and / or anti-aging, but antioxidant is not limited thereto as long as it can prevent oxidation and increase retinol production.

[0020] The antioxidant may be any one or more selected from the group consisting of 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), vitamin C (ascorbic acid), and glutathione (GSH), but is not limited thereto.

[0021] The concentration of the antioxidant relative to the total culture medium composition may be 0.0001% (w / v) to 10% (w / v), 0.0001% (w / v) to 5% (w / v), 0.0001% (w / v) to 1% (w / v), 0.001% (w / v) to 10% (w / v), 0.001% (w / v) to 5% (w / v), 0.001% (w / v) to 1% (w / v), 0.01% (w / v) to 10 % (w / v), 0.01% (w / v) to 5% (w / v), 0.01% (w / v) to 1% (w / v), 0.01% (w / v) to 0.09% (w / v), 0.01% (w / v) to 0.08% (w / v), 0.01% (w / v) to 0.07% (w / v), 0.01% (w / v) to 0.06% (w / v) or 0.01% (w / v) to 0.05% (w / v), but are not limited to these.

[0022] In one embodiment, by including the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant, retinol can be stably produced while minimizing consumption of time and human resources.

[0023] As used herein, the term "culture medium" refers to a mixture containing as main components nutrients required for culturing the Yarrowia microorganism of the present disclosure, wherein the culture medium provides nutrients including water, growth factors, etc. necessary for survival and growth.

[0024] In one embodiment, the culture medium of the present disclosure may be a culture medium for producing retinol, and may further include substances required for retinol production, but is not limited thereto.

[0025] As for the culture medium and other culture conditions for culturing the Yarrowia microorganism of the present disclosure, any common culture medium already containing an antioxidant or any culture medium used for conventional culture of microorganisms while also containing an antioxidant can be used without particular limitation.

[0026] The culture medium disclosed herein may be a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, while controlling temperature, pH and the like, but is not limited thereto.

[0027] In the present disclosure, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of other carbon sources may be used without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0028] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0029] The phosphate source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition, amino acids, vitamins and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0030] In addition, the pH of the culture medium can be adjusted by adding compounds (e.g., ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc.) to the culture medium in an appropriate manner during the cultivation of the Yarrowia microorganism of the present disclosure. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress the formation of bubbles during the cultivation process. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can be not injected or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain an anaerobic or anaerobic state, but is not limited thereto.

[0031] As used herein, the term "microorganism" or "strain" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it may also include microorganisms comprising genetic modifications for retinol production, which are microorganisms in which a specific mechanism is weakened or enhanced due to the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes.

[0032] In one embodiment, the microorganism of the present disclosure may be a microorganism of the genus Yarrowia (Yarrowia species), but is not limited thereto.

[0033] In one embodiment, the microorganism of the present disclosure may be Yarrowia lipolytica, but is not limited thereto.

[0034] In one embodiment, the microorganism of the present disclosure may be a microorganism for producing retinol. The microorganism or strain for producing retinol may be a microorganism naturally having the ability to produce retinol, or a microorganism that enhances or provides the ability to produce retinol due to natural or artificial genetic modification in a parent strain without the ability to produce retinol, but is not limited thereto. Specifically, the microorganism for producing retinol of the present disclosure may be a microorganism of the genus Yarrowia, which is modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI) and β-carotene 15,15'-oxygenase (BLH) proteins.

[0035] The microorganism of the present disclosure may be a microorganism modified to further comprise a polynucleotide encoding a lycopene cyclase / phytoene synthase (crtYB) and a phytoene desaturase (crtI) protein so as to exhibit 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 may be 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 comprise a nucleotide sequence based on GenBank: AY177204.1 or GenBank: AY177424.1, which are registered in the National Center for Biotechnology Information Search Database (NCBI), respectively. In one embodiment, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or comprise SEQ ID NO: 1 or 2, respectively. Taking into account codon degeneracy or preferred codons in microorganisms intended to express proteins, the polynucleotide may undergo various modifications in the coding region without changing the amino acid sequence. Specifically, the polynucleotide may have or include a nucleotide 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 with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or may consist of or be substantially composed of a nucleotide 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 with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

[0036] The microorganism of the present disclosure may be a microorganism modified to further include a polynucleotide encoding a geranylgeranyl pyrophosphate synthase (GGPPS) protein to exhibit the activity of the protein, or a microorganism in which the activity of the protein is enhanced, but is not limited thereto. The geranylgeranyl pyrophosphate synthase may be a protein from Haematococcus pluvialis, but is not limited thereto. In one embodiment, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have or include a nucleotide sequence based on GenBank: APX64485.1, which is registered in the National Center for Biotechnology Information Search Database (NCBI). In one embodiment, the polynucleotide encoding the geranylgeranyl pyrophosphate synthase may have or include the sequence of SEQ ID NO: 33. Taking into account codon degeneracy or preferred codons in a microorganism intended to express a protein, the polynucleotide may undergo various modifications in the coding region within the range of not changing the amino acid sequence. Specifically, the polynucleotide may have or comprise a nucleotide 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 with the sequence of SEQ ID NO:33, or may consist of or consist essentially of a nucleotide 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 with the sequence of SEQ ID NO:33, but is not limited thereto.

[0037] The microorganism of the present disclosure may be a microorganism modified to further include a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein to exhibit the activity of the protein, or a microorganism in which the activity of the protein is enhanced, but is not limited thereto. The β-carotene 15,15'-oxygenase may be a protein from uncultured marine bacterium 66A03, but is not limited thereto. In one embodiment, the β-carotene 15,15'-oxygenase polypeptide and the polynucleotide encoding it may have or include an amino acid sequence based on Q4PNI0 registered in the UniProt Knowledge Base (UniProtKB). In one embodiment, the β-carotene 15,15'-oxygenase polypeptide may have or include the sequence of SEQ ID NO:57. Taking into account codon degeneracy or preferred codons in a microorganism intended to express a protein, the polynucleotide may undergo various modifications in the coding region within the range of not changing the amino acid sequence. Specifically, the polynucleotide may have or comprise a nucleotide 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 with the sequence of SEQ ID NO:57, or may consist of or consist essentially of a nucleotide 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 with the sequence of SEQ ID NO:57, but is not limited thereto.

[0038] As used herein, the term "culture" refers to growing the Yarrowia microorganism of the present disclosure under appropriately adjusted environmental conditions. In the present disclosure, as long as a culture medium containing an antioxidant is used, the culture procedure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust this culture procedure according to the selected strain. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0039] The Yarrowia microorganism of the present disclosure can be cultured under aerobic conditions in a common culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., while controlling temperature, pH, etc.

[0040] In the culture of the present disclosure, the culture temperature can be maintained at 20°C to 35°C, specifically, at 25°C to 35°C, and the culture can be performed for about 10 hours to 160 hours, about 20 hours to 130 hours, about 24 hours to 120 hours, about 36 hours to 120 hours, about 48 hours to 120 hours, about 48 hours, about 72 hours or about 120 hours, but is not limited thereto.

[0041] Retinol produced by the culture of the present disclosure may be released into the culture medium, or may be retained in the microorganism.

[0042] As used herein, the term "retinol" is a substance called vitamin A and is a retinoid. Retinol can be used as it is, but can be converted into other retinoids (e.g., retinal, retinoic acid, retinyl esters, etc.) or carotenoid compounds by methods known in the art.

[0043] In the present disclosure, antioxidants may be added to a culture medium of a microorganism for producing retinol to significantly increase the retinol production capacity.

[0044] For example, when a microorganism is cultured in a medium to which an antioxidant is added, its retinol production capacity may be increased 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 a microorganism cultured in a medium to which an antioxidant is not added. However, as long as the microorganism has an increased retinol production capacity compared to before the addition of an antioxidant, it is not limited thereto.

[0045] As used herein, the term "about" is meant to include all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​equivalent or similar to the value after the term "about", but the range is not limited thereto.

[0046] The method for producing retinol of the present disclosure may further include a step of preparing the Yarrowia microorganism of the present disclosure, a step of preparing a culture medium for culturing the microorganism, or a combination of these steps (regardless of the order, in any order), for example, before or after the culturing step.

[0047] The method for producing retinol of the present disclosure may further include a step of recovering retinol from a culture medium produced by the culture (a culture medium in which the culture has been performed) or from the microorganism of the present disclosure. A recovery step may be further included after the culture step.

[0048] Recovery can be a method of culturing microorganisms according to the present disclosure, such as batch, continuous or fed-batch type culture, and collecting the desired retinol by using appropriate methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, cell disruption, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and combinations of these methods can be used, and retinol can be recovered from the culture medium or microorganism by using appropriate methods known in the art.

[0049] In addition, the method for producing retinol disclosed herein may further include a purification step. Purification may be performed using a suitable method known in the art. In an exemplary embodiment, when the method for producing retinol disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed discontinuously (or continuously) without regard to order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0050] Another aspect of the present disclosure provides a method for producing a retinoid, the method comprising the steps of: culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant; and converting retinol produced by the microorganism into a retinoid other than retinol.

[0051] The antioxidant, culture medium, microorganism, culture, retinol and retinoid are as described in other aspects, and the recovery and purification of retinol are also applicable to the recovery and purification of retinoids.

[0052] The method for producing retinoids disclosed herein may further include the step of converting the retinol produced by the microorganism disclosed herein into a retinoid other than retinol. In the method for producing retinoids disclosed herein, a conversion step may also be included after the culturing step or the recovery step. The conversion step may be performed using a suitable method known in the art. For example, retinol acyltransferase may be used for conversion, but is not limited thereto.

[0053] In one embodiment, the retinoid may be any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester, but is not limited thereto as long as it is contained in the retinoid.

[0054] Another aspect of the present disclosure provides a method for increasing retinol production, the method comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant.

[0055] The antioxidants, culture medium, microorganisms, culture and retinol are as described in other aspects.

[0056] Another aspect of the present disclosure provides a culture medium composition for producing retinol by a microorganism of the genus Yarrowia, the composition comprising an antioxidant.

[0057] In one embodiment, the culture medium composition can increase the retinol production of a Yarrowia microorganism, but is not limited thereto.

[0058] In one embodiment, the culture medium composition can increase the growth of microorganisms, but is not limited thereto.

[0059] The antioxidant, retinol, microorganism and culture medium are as described in other aspects.

[0060] Yet another aspect of the present disclosure provides a composition for producing retinol, the composition comprising a microorganism of the genus Yarrowia or a culture thereof, and an antioxidant.

[0061] The composition of the present disclosure may further include any suitable excipients commonly used in compositions for producing retinol, examples of which may include preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0062] The microorganism, antioxidant and retinol are as described in other aspects.

[0063] Another aspect of the present disclosure provides the use of an antioxidant in producing retinol; the use of a culture medium composition for a Yarrowia microorganism in producing retinol, the composition comprising an antioxidant; and the use of a composition comprising a Yarrowia microorganism or a culture thereof and an antioxidant in producing a retinoid. With regard to the use in producing a retinoid, the retinoid may be retinol or a retinoid other than retinol.

[0064] The antioxidant, microorganism, culture medium, retinoid and retinol are as described in other aspects.

[0065] Example

[0066] Hereinafter, the present disclosure will be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are only used to illustrate preferred embodiments of the present disclosure, and are therefore not intended to limit the scope of the present disclosure thereto. At the same time, technical matters not described in this specification can be fully understood and easily implemented by those skilled in the art of the present disclosure or similar technical fields.

[0067] Example 1. Preparation of a Yarrowia lipolytica platform strain for producing retinol

[0068] Example 1-1. Preparation of crtYB-crtI Insertion Strain from Phaffia rhodozyma

[0069] To prepare a Yarrowia platform strain for producing retinol, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes from Phaffia rhodozyma were inserted into the genome of the high-fat yeast KCCM12972P strain.

[0070] Based on the nucleotide sequence registered in the National Center for Biotechnology Information search database (NCBI) (GenBank: AY177204.1), the polynucleotide of SEQ ID NO: 1 of crtYB was obtained, and based on the nucleotide sequence registered in NCBI (GenBank: AY177424.1), the polynucleotide of SEQ ID NO: 2 of crtI was obtained. 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), respectively. The URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica was used as a selection marker to design a cassette for insertion into the MHY1 (YALI0B21582g) gene site.

[0071] Each PCR was performed using the synthetic crtYB and crtI genes and KCCM12972P genomic DNA as templates, and primers of SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, and SEQ ID NOs: 16 and 17 as shown in Table 1. PCR was performed by 35 cycles, including 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 a single cassette by overlap extension PCR.

[0072] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. Colonies in which the cassette was inserted into the genome using primers of SEQ ID NOs: 18 and 19 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0073] <5-Fluoroorotic acid (5-FOA)>

[0074] 20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 2 g / L yeast synthetic dropout medium supplement without uracil, 50 μg / mL uracil, 1 g / L 5-fluoroorotic acid (5-FOA), 15 g / L agar.

[0075] [Table 1]

[0076]

[0077] Example 1-2. Preparation of HMGR enhanced strains

[0078] A cassette for replacing the natural promoter (SEQ ID NO: 20) region of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene of the strain prepared by Example 1-1 with the TEFINt promoter was designed, and each PCR was performed using the genomic DNA of KCCM12972P as a template, and primers of SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, and SEQ ID NOs: 29 and 30 as shown in Table 2. PCR was performed by 35 cycles, including 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 prepared into a single cassette by overlap extension PCR.

[0079] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette insertion was confirmed using primers of SEQ ID NO: 31 and 32 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0080] [Table 2]

[0081]

[0082] Example 1-3. Preparation of strains introduced with GGPPS

[0083] The geranylgeranyl pyrophosphate synthase (GGPPS) gene derived from Haematococcus pluvialis was inserted into the genome of the strain prepared in Example 1-2.

[0084] Based on the nucleotide sequence registered in the National Center for Biotechnology Information search database (NCBI) (GenBank: APX64485.1), the polynucleotide of SEQ ID NO: 33 of GGPPS was obtained. The polynucleotide sequence of GGPPS was codon-optimized for Yarrowia lipolytica through http: / / atgme.org, and the gene was synthesized by Macrogen in the form of TEFINtp-GGPPS-CYC1t (SEQ ID NO: 34). Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, a cassette for insertion into the LIG4 (YALI0D21384g) gene site was designed.

[0085] Each PCR was performed using the synthetic GGPPS gene and the genomic DNA of KCCM12972P as templates, and primers of SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44 as shown in Table 3. PCR was performed by 35 cycles, including 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 prepared into a single cassette by overlap extension PCR.

[0086] The cassette thus prepared was introduced into the strain prepared in Example 1-2 by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette was inserted into the genome using primers of SEQ ID NO: 45 and 46 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0087] [Table 3]

[0088]

[0089] Example 1-4. Preparation of KU80 deletion strain

[0090] To facilitate future strain preparation, the KU80 (YALI0E02068g) gene was deleted. To this end, the KU80 gene deletion cassette was designed using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker. Each PCR was performed using the genomic DNA of KCCM12972P as a template and the primers of SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, and SEQ ID NOs: 53 and 54 as shown in Table 4. PCR was performed through 35 cycles, including 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 prepared into a single cassette by overlap extension PCR.

[0091] The cassette thus prepared was introduced into the strain prepared in Example 1-3 by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette was inserted into the genome using primers of SEQ ID NO: 55 and 56 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0092] [Table 4]

[0093]

[0094] Example 1-5. Preparation of BLH-introduced strains

[0095] The β-carotene 15,15′-oxygenase (BLH) gene derived from the uncultured marine bacterium 66A03 was inserted into the genome of the strain prepared by Example 1-4.

[0096] Based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledge Base), the polypeptide sequence of SEQ ID NO:57 of the BLH gene was obtained. It was codon-optimized for Yarrowia lipolytica by http: / / atgme.org and synthesized by Macrogen in the form of TEFINtp-BLH-CYC1t (SEQ ID NO:58). Using the URA3 gene (SEQ ID NO:5) of Yarrowia lipolytica as a selection marker, a cassette was designed to insert into the KU70 (yali co 08701g) gene locus. Each PCR was performed using the synthesized BLH gene and the genomic DNA of KCCM12972P as templates, and the primers of 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 as shown in Table 5. PCR was performed by 35 cycles, including 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 prepared into a single cassette by overlap extension PCR.

[0097] The cassette thus prepared was introduced into the strain prepared in Example 1-4 by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette was inserted into the genome using primers of SEQ ID NO: 69 and 70 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0098] [Table 5]

[0099]

[0100] Example 2. Evaluation of the Effect of Antioxidants on Retinol Production

[0101] Flask experiments were performed to compare the extent of retinol production of retinol-producing strains according to the presence and absence of antioxidants and the type. Each retinol-producing platform strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of Yarrowia lipolytica minimal medium 2 (YLMM2) with an initial OD of 4, and 4 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%, respectively. Cultivation was performed at 30°C and 200 rpm.

[0102] In order to evaluate the degree of growth according to the culture time, the OD value at a wavelength of 600 nm was measured using a spectrophotometer, and the method for measuring the retinol concentration was as follows: After the culture was completed, 1 ml of the culture medium was centrifuged to remove the supernatant, and then 0.5 ml of dimethyl sulfoxide (DMSO, Sigma) was added and the cells were disrupted by stirring at 55°C for 10 minutes (stirring; 2000 rpm). Next, 0.5 ml of acetone (Sigma) containing 4% BHT was added and stirred at 45°C (2000 rpm) for 15 minutes, and the retinoid extracted in this way was quantitatively analyzed using an HPLC device. The analyzed OD value is shown in Figure 1 and retinoid concentrations are shown in Figure 2 middle.

[0103] Considering that the consumption rate of sugars may vary depending on the type of antioxidant added to the culture medium, BHT, PG, vitamin C, and GSH were incubated for 48 hours each. After 48 hours of incubation, retinal and retinol were extracted and quantified.

[0104] As a result, when BHT, PG, vitamin C, and GSH were added, the retinol concentration increased up to 1.89-fold, 1.82-fold, 1.44-fold, and 1.21-fold, respectively, compared with the control group without antioxidants, and in some cases, the biomass (OD) was overall higher than that of the control group without antioxidants, indicating that antioxidants also promoted the growth of microorganisms ( Figure 2 ).

[0105] The above results confirmed that when culturing the retinol-producing strain, adding an antioxidant to the culture medium not only promoted the growth of the retinol-producing strain but also increased the retinol production concentration.

[0106] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic features. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all respects. The scope of the present disclosure is limited by the appended claims rather than by the description before them, so all changes and modifications falling within the boundaries and scope of the claims, or the equivalents of these boundaries and scopes, are included by the claims.

[0107] Each sequence of SEQ ID NO. according to the present disclosure is shown in Table 6 below.

[0108] [Table 6]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

Claims

1. A method for producing retinol, comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant.

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

3. The method of claim 1, wherein the microorganism is Yarrowia lipolytica. The method according to claim 1 , wherein the microorganism is used to produce retinol.

5. The method according to claim 1, wherein the concentration of the antioxidant is 0.001% (w / v) or more relative to the total medium composition.

6. The method according to claim 1, further comprising the step of recovering retinol from the culture medium or the microorganism.

7. A method for increasing retinol production, the method comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing an antioxidant.

8. A method for producing a retinoid, the method comprising the steps of: Cultivating a Yarrowia microorganism in a medium containing an antioxidant; and The retinol produced by the microorganism is converted into a retinoid other than retinol.

9. A culture medium composition for a microorganism of the genus Yarrowia for producing retinol, the composition comprising an antioxidant. 10 . The culture medium composition according to claim 9 , wherein the antioxidant is any one or more selected from the group consisting of 3,5-di-tert-4-butylhydroxytoluene (BHT), propyl gallate (PG), vitamin C (ascorbic acid) and glutathione (GSH).

11. The medium composition according to claim 10, wherein the concentration of the antioxidant is 0.001% (w / v) or more relative to the total medium composition.

12. The culture medium composition according to claim 9, wherein the microorganism is Yarrowia lipolytica.

13. The culture medium composition of claim 9, wherein the culture medium composition increases retinol production by the microorganism.

14. The culture medium composition of claim 9, wherein the culture medium composition promotes the growth of the microorganism.

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

16. An antioxidant; a culture medium composition of a Yarrowia microorganism comprising an antioxidant; or use of a composition comprising a Yarrowia microorganism or a culture thereof and an antioxidant in producing a retinoid.

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