Microbial culture medium composition for production of retinol comprising surfactant 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 comprising a nonionic surfactant; a method for increasing the yield of retinol; a process for the production of a retinoid; a culture medium composition for microorganisms of the genus Yarrowia for the production of retinol, the composition comprising a nonionic surfactant; and a composition for producing retinol, the composition comprising the microorganism or a culture thereof and a nonionic surfactant.

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 a nonionic surfactant; 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 a nonionic surfactant; a composition for producing retinol, the composition comprising a microorganism or a culture thereof and a nonionic surfactant; and use thereof in producing a retinoid. 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 a surfactant, 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 a nonionic surfactant.

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

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

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

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

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

[0013] [Beneficial Effects]

[0014] By including a nonionic surfactant, 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 A shows the OD values ​​as a result of the flask culture test of the β-carotene-producing strain according to the presence or absence of the surfactant and its concentration;

[0016] Figure 1 B shows the OD values ​​as a result of the flask culture test of the retinol-producing strain according to the presence or absence of the surfactant and its concentration;

[0017] Figure 2 A shows the β-carotene concentration as a result of a flask culture test of a β-carotene-producing strain according to the presence or absence and concentration of a surfactant;

[0018] Figure 2 B shows the concentrations of β-carotene, retinal, and retinol as a result of flask culture tests of the retinol-producing strain according to the presence or absence of surfactant and its concentration;

[0019] Figure 3 A shows the OD values ​​as a result of the flask culture test of the β-carotene-producing strain according to the presence or absence and type and concentration of Tween series surfactants;

[0020] Figure 3 B shows the OD values ​​as a result of the flask culture test of the retinol-producing strain according to the presence or absence and type and concentration of Tween series surfactants;

[0021] Figure 4 A shows the β-carotene concentration as a result of a flask culture test of a β-carotene-producing strain according to the presence or absence and type of Tween series surfactants and their concentrations; and

[0022] Figure 4 B shows the concentrations of β-carotene, retinal and retinol as a result of flask culture test of the retinol-producing strain according to the presence or absence and type of Tween series surfactants and their concentrations. DETAILED DESCRIPTION

[0023] 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.

[0024] 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 a nonionic surfactant.

[0025] As used herein, the term "surfactant" refers to a compound having both a hydrophilic part and a hydrophobic part. According to the charge of the hydrophilic part when dissociated in water, surfactants can be divided into anionic, cationic, nonionic (polar), amphoteric and special surfactants. The surfactant of the present disclosure is a nonionic surfactant, and according to its chemical structure, it can be divided into a sorbitan fatty acid salt series (such as span), a polyoxyethyl sorbitan fatty acid salt series (such as Tween) and a polyethyl ether fatty acid salt series (such as Triton), etc.

[0026] In one embodiment, the surfactant may be any one or more selected from the group consisting of Tween and Triton, but is not limited thereto.

[0027] In one embodiment, the Tween may be any one or more selected from the group consisting of Tween 20, Tween 40, Tween 60 and Tween 80, but is not limited thereto.

[0028] In one embodiment, the Triton may be Triton X-100, but is not limited thereto.

[0029] Relative to the total medium composition, 0.001% (w / v) or more, 0.001% (w / v) to 30% (w / v), 0.01% (w / v) or more, 0.01% (w / v) to 30% (w / v), 0.01% (w / v) to 25% (w / v), 0.01% (w / v) to 20% (w / v), 0.01% (w / v) to 15% (w / v), 0.01% (w / v) to 10% (w / v), 0.01% (w / v) to 5% (w / v), 0.01% (w / v) to 2% (w / v), 0.01% (w / v) to 1% (w / v), 0.05% (w / v) to 30% (w / v), 0.05% (w / v) to 25% (w / v), 0.05% (w / v) to 20% (w / v), 0.05% (w / v) to 15% (w / v), 0.05% (w / v) to 10% (w / v), 0.05% (w / v) to 15% (w / v), 0.05% (w / v) to 10% (w / v), 0.05% (w / v) to 20% (w / v), 0.05% (w / v) to 25% (w / v), 0.05% (w / v) to 2 ...25% (w / v), 0.05% (w / v v) to 5% (w / v), 0.01% (w / v) to 2% (w / v), 0.01% (w / v) to 1% (w / v), 0.1% (w / v) to 30% (w / v), 0.1% (w / v) to 25% (w / v), 0.1% (w / v) to 20% (w / v), 0.1% (w / v) to 15% (w / v), 0.1% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), 0.1 % (w / v) to 2% (w / v), 0.1% (w / v) to 1% (w / v), 0.5% (w / v) to 30% (w / v), 0.5% (w / v) to 25% (w / v), 0.5% (w / v) to 20% (w / v), 0.5% (w / v) to 15% (w / v), 0.5% (w / v) to 10% (w / v), 0.5% (w / v) to 5% (w / v), 0.5% (w / v) to 2% (w / v), 0.5% (w / v) to 1% (w / v), 1% (w / v) to 30% (w / v), 1% (w / v) to 25% (w / v), 1% (w / v) to 20% (w / v), 1% (w / v) to 15% (w / v), 1% (w / v) to 10% (w / v), 1% (w / v) to 5% (w / v), 1% (w / v) to 2% (w / v), 2% (w / v) to 30% (w / v), 2% (w / v) to 25% (w / v), 2% (w / v) to 20% (w / v), 2% (w / v) to 15% (w / v), 2% (w / v) to The nonionic surfactant is contained in a concentration of 5% (w / v) to 10% (w / v), 2% (w / v) to 5% (w / v), 5% (w / v) to 30% (w / v), 5% (w / v) to 25% (w / v), 5% (w / v) to 20% (w / v), 5% (w / v) to 15% (w / v), 5% (w / v) to 10% (w / v), 10% (w / v) to 30% (w / v), 10% (w / v) to 25% (w / v), 10% (w / v) to 20% (w / v), 10% (w / v) to 15% (w / v), but is not limited thereto. .

[0030] In one embodiment, the nonionic surfactant can increase the extracellular secretion of retinol, but is not limited thereto.

[0031] In one embodiment, by including the step of culturing a microorganism of the genus Yarrowia in a culture medium containing a nonionic surfactant, retinol can be stably produced while minimizing consumption of time and human resources.

[0032] 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.

[0033] 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.

[0034] As for the culture medium and other culture conditions for culturing the Yarrowia microorganism of the present disclosure, any common culture medium already containing a nonionic surfactant or any culture medium used for conventional culture of microorganisms while also containing a nonionic surfactant can be used without particular limitation.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] As used herein, the term "microorganism of the genus Yarrowia" or "strain of the genus Yarrowia" includes all wild-type microorganisms of the genus Yarrowia or naturally or artificially genetically modified microorganisms of the genus Yarrowia, and it may also include genetically modified microorganisms of the genus Yarrowia 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.

[0041] In one embodiment, the Yarrowia microorganism of the present disclosure may be Yarrowia lipolytica, but is not limited thereto.

[0042] In one embodiment, the Yarrowia microorganism of the present disclosure may be a microorganism for producing retinol. The microorganism or strain for producing retinol may be a microorganism that naturally has 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 that does not have the ability to produce retinol, but is not limited thereto. Specifically, the Yarrowia microorganism for producing retinol of the present disclosure may be a microorganism modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and β-carotene 15,15'-oxygenase (BLH) proteins.

[0043] 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.

[0044] 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.

[0045] 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 encoding the polypeptide may undergo various modifications in the coding region within the range of not changing the amino acid sequence. Specifically, the polypeptide 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.

[0046] 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 a nonionic surfactant 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.

[0047] The Yarrowia microorganism of the present disclosure can be cultured under aerobic conditions in a common medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc., while controlling temperature, pH and the like.

[0048] 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 or longer, or about 48 hours, about 72 hours, or about 120 hours, but is not limited thereto.

[0049] Retinol produced by the culture of the present disclosure may be released into the culture medium, or may be retained in the microorganism.

[0050] 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.

[0051] In the present disclosure, a nonionic surfactant may be added to a microbial culture medium for producing retinol to significantly increase the retinol production capacity.

[0052] In one embodiment, nonionic surfactants can increase the extracellular secretion of retinol, but are not limited thereto. In one embodiment, when the microorganism is cultured in a culture medium to which a nonionic surfactant has been added, its retinol production capacity can be increased by about 1% or more, specifically, about 3%, about 5% or more, about 10% or more, about 50% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 380% or more, about 400% or more, about 450% or more, about 500% or more, about 550% or more, about 600% or more, about 650% or more, about 680% or more, compared to before the addition of the nonionic surfactant. However, as long as the microorganism has an increased + value capacity, it is not limited thereto.

[0053] 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.

[0054] 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.

[0055] The culture medium may be one to which a nonionic surfactant is added to increase the production of retinol.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 a nonionic surfactant; and converting retinol produced by the microorganism into a retinoid other than retinol.

[0060] The nonionic surfactant, 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.

[0061] The method for producing retinoids disclosed herein may further include the step of converting the retinol expressed 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.

[0062] 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.

[0063] 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 comprising a nonionic surfactant.

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

[0065] Another aspect of the present disclosure provides a culture medium composition for producing retinol by a microorganism of the genus Yarrowia, the composition comprising a nonionic surfactant.

[0066] In one embodiment, the culture medium composition can increase the retinol production of a Yarrowia microorganism, but is not limited thereto.

[0067] In one embodiment, the culture medium composition can increase the growth of microorganisms, but is not limited thereto.

[0068] The nonionic surfactant, retinol, microorganism and culture medium are as described in other aspects.

[0069] 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 a nonionic surfactant.

[0070] 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.

[0071] The microorganism, nonionic surfactant and retinol are as described in other aspects.

[0072] Another aspect of the present disclosure provides use of a nonionic surfactant in producing retinol; use of a culture medium composition for a Yarrowia microorganism in producing retinol, the composition comprising a nonionic surfactant; and use of a composition comprising a Yarrowia microorganism or a culture thereof and a nonionic surfactant in producing a retinoid.

[0073] Regarding the use in producing retinoid, the retinoid may be retinol or a retinoid other than retinol. The nonionic surfactant, microorganism, culture medium, retinoid and retinol are as described in other aspects.

[0074] Example

[0075] 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.

[0076] Example 1. Preparation of platform strain for retinol production

[0077] Example 1-1. Preparation of crtYB-crtI Insertion Strain from Phaffia rhodozyma

[0078] 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 Yarrowia lipolytica KCCM12972P strain.

[0079] 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.

[0080] Each PCR was performed using the synthetic crtYB and crtI genes and KCCM12972P genomic DNA as templates, and primers of SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and SEQ ID NO: 16 and SEQ ID NO: 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.

[0081] 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 whose cassette insertion into the genome was confirmed using primers of SEQ ID NO: 18 and SEQ ID NO: 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 remove the URA3 marker.

[0082] [Table 1]

[0083]

[0084] In addition, the compositions of the uracil-free solid medium (YLMM1) and the 5-FOA medium are as follows.

[0085] <Yarrowia lipolytica minimal medium 1 (YLMM1)>

[0086] 20g / L glucose, 6.7g / L yeast nitrogen base without amino acids, 2g / L yeast synthetic dropout medium supplement without uracil, 15g / L agar

[0087] <5-Fluoroorotic acid (5-FOA)>

[0088] 20g / L glucose, 6.7g / L yeast nitrogen base without amino acids, 2g / L yeast synthetic dropout medium supplement without uracil, 50μg / mL uracil, 1g / L 5-fluoroorotic acid (5-FOA), 15g / L agar

[0089] Example 1-2. Preparation of HMGR enhanced strains

[0090] 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 NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, and SEQ ID NO: 29 and SEQ ID NO: 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.

[0091] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by a 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 SEQ ID NO: 32 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies formed on a 5-FOA solid medium to remove the URA3 marker.

[0092] [Table 2]

[0093]

[0094] Example 1-3. Preparation of strains introduced with GGPPS

[0095] The geranylgeranyl pyrophosphate synthase (GGPPS) gene derived from Haematococcus pluvialis was inserted into the genome of the strain prepared in Example 1-2.

[0096] 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. Each PCR was performed using the synthetic GGPPS gene and the genomic DNA of KCCM12972P as templates, and primers of SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, and SEQ ID NO: 43 and SEQ ID NO: 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.

[0097] 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 SEQ ID NO: 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 remove the URA3 marker.

[0098] [Table 3]

[0099]

[0100] Example 1-4. Preparation of KU80 deletion strain

[0101] To facilitate future strain preparation, the KU80 (YALI0E02068g) gene of the strain prepared in Example 1-3 was deleted. To this end, the URA3 gene (SEQ ID NO:5) of Yarrowia lipolytica was used as a selection marker to design a KU80 gene deletion cassette. Each PCR was performed using the genomic DNA of KCCM12972P as a template, and primers of SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, and SEQ ID NO:53 and SEQ ID NO:54. 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.

[0102] The cassette thus prepared was introduced into the strain prepared in Example 1-3 by the heat shock method, and then a colony was obtained, which was formed on a solid medium (YLMM1) without uracil. The colony whose cassette was inserted into the genome using primers of SEQ ID NO: 55 and SEQ ID NO: 56 was plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain a colony formed on a 5-FOA solid medium to remove the URA3 marker. The corresponding strain was named CC08-1043.

[0103] [Table 4]

[0104]

[0105] Example 1-5. Preparation of BLH-introduced strains

[0106] The β-carotene 15,15'-oxygenase (BLH) gene from the uncultured marine bacterium 66A03 was inserted into the genome of the strain prepared by Example 1-4. 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 through http: / / atgme.org, and the gene was synthesized by Macrogen in the form of TEFINtp-BLH-CYC1t (SEQID NO:58). Using the URA3 gene (SEQ ID NO:5) of Yarrowia lipolytica as a selection marker, a cassette was designed to be inserted into the KU70 (YALI0C08701g) gene site. Each PCR was performed using the synthetic BLH gene and the genomic DNA of KCCM12972P as templates, and primers of SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 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.

[0107] The cassette thus prepared was introduced into the strain prepared in Example 1-4 by the heat shock method, and then a colony was obtained, which was formed on a solid medium (YLMM1) without uracil. The colony whose cassette was inserted into the genome using primers of SEQ ID NO: 69 and SEQ ID NO: 70 was plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain a colony formed on a 5-FOA solid medium to remove the URA3 marker. The corresponding strain was named CC08-2163.

[0108] [Table 5]

[0109]

[0110]

[0111] Example 2. Comparative evaluation of the effect of surfactant type on retinol production capacity

[0112] Example 2-1. Microbial culture

[0113] In order to compare the effects of the type and amount of surfactant added on the production of β-carotene and retinol, a flask experiment was conducted on CC08-1043 and CC08-2163 strains. CC08-1043 or CC08-2163 strains were inoculated into a 250 ml angle plate flask containing 20 ml of Yarrowia lipolytica minimal medium 2 (YLMM2) with an initial OD = 2, and five types of surfactants were added at a concentration of 2%, namely Tween 20 (TW20, Sigma, CAS No. 9005-64-5), Tween 40 (TW40, Sigma, CAS No. 9005-66-7), Tween 60 (TW60, Sigma, C 9005-67-8), Tween 80 (TW80, Sigma, CAS No. 9005-65-6) and Span 80 (SP80, Sigma, CAS No. 1338-43-8), and two types of surfactants sodium dodecyl sulfate (SDS, Sigma, CAS No. 151-21-3) and Triton X-100 (TX, Sigma, CAS No. 9036-19-5) were added to the culture medium at a concentration of 0.05% or 1%, respectively. The culture was carried out at 30°C and 200 rpm. Since the consumption rate of sugars may vary depending on the type of surfactant added, the culture was continued to 48 hours, at which time all residual sugars were consumed.

[0114] Example 2-2: Evaluation of microbial growth

[0115] To examine the growth status over the culture time, the OD value at a wavelength of 600 nm was measured using a spectrophotometer.

[0116] The OD values ​​of the two strains are shown in Table 6. The OD values ​​of the β-carotene producing strain (CC08-1043) and the retinol producing strain (CC08-2163) are shown in Figure 1 A and 1B.

[0117] [Table 6]

[0118]

[0119]

[0120] As a result, when the anionic surfactant SDS among the surfactants used in this experiment was added, no growth was observed under all concentration conditions. Compared with the condition without addition, under the condition of adding each surfactant except SDS, the biomass (OD) tended to be higher overall.

[0121] The extraction and concentration analysis of retinal, retinol and β-carotene were performed in the groups with added surfactant (groups with added Tween 20, Tween 40, Tween 60, Tween 80, Span 80 and Triton X-100), in which all sugars were consumed in 48 hr of culture.

[0122] Example 2-3: Evaluation of β-carotene, retinal and retinol concentrations

[0123] The method for measuring intracellular and extracellular β-carotene, retinol, and retinal concentrations is as follows.

[0124] In order to measure intracellular (Int.) β-carotene, retinol and retinal, 0.05 ml of the cultured medium was centrifuged to remove the supernatant, and then 0.5 ml of dimethyl sulfoxide (DMSO, Sigma, Cas No. 67-68-5) was added, and the cells were disrupted by stirring (2,000 rpm) at 55° C. for 10 minutes. Then, 0.5 ml of acetone (Sigma, Cas No. 67-64-1) containing 4% BHT (Sigma, Cas No. 128-37-0) was added, and shaken (2,000 rpm) at 45° C. for 15 minutes, and the β-carotene, retinol and retinal extracted in this way were quantitatively analyzed using an HPLC device.

[0125] In order to measure extracellular (Ext.) β-carotene, retinol and retinal, 0.95 ml of acetone (Sigma) containing 4% BHT was added to 0.05 ml of supernatant prepared by removing cells after completion of culture, followed by shaking (2,000 rpm) at 45°C for 15 minutes, and β-carotene, retinal and retinol extracted in this manner were quantitatively analyzed, respectively, using an HPLC apparatus.

[0126] The concentrations of β-carotene, retinol and retinal (mg / L) in the two strains are shown in Table 7. Regarding the analytical results, the β-carotene concentration measured when the β-carotene producing strain (CC08-1043) was cultured is shown in Figure 2 A, β-carotene, retinol, and retinal concentrations measured when the retinol-producing strain (CC08-2163) was cultured are shown in Figure 2 B.

[0127] [Table 7]

[0128]

[0129] As a result, in the evaluation of the β-carotene-producing strain CC08-1043, the group to which Tween series surfactants (Tween 20, Tween 40, Tween 60 and Tween 80) known as nonionic surfactants were added showed an increase in β-carotene concentration up to 1.4 times, while the group to which Span 80 or 0.05% Triton X-100 was added showed a decrease in β-carotene concentration of 10% or more ( Figure 2 A).

[0130] Different from the above results, Tween series (TW20, 40, 60 and 80) and Triton series (Triton X-100) surfactants all increased the production of retinol. In the group without surfactant (control group), no extracellular retinol was observed, and only intracellular retinol was measured.

[0131] These results demonstrate that when a nonionic surfactant was added, the retinol production concentration increased by up to 6.8 times or more compared to the condition without addition.

[0132] Example 3. Comparative evaluation of the effect of Tween series surfactant concentration on retinol production capacity Example 3-1. Microbial culture

[0133] In order to compare the effect of the added concentration of Tween series surfactants as nonionic surfactants on the yield of β-carotene and retinol, flask experiments were carried out on CC08-1043 and CC08-2163 strains. Each CC08-1043 and CC08-2163 strain was inoculated into a 250ml angle plate flask containing 20ml of Yarrowia lipolytica minimal medium 2 (YLMM2) with an initial OD=2, and 4 types of surfactants, Tween 20 (TW20), Tween 40 (TW40), Tween 60 (TW60) and Tween 80 (TW80) were added to the culture medium at a concentration of 5%, 10% or 15%, respectively. Cultivation was carried out under the conditions of 30°C and 200rpm. Since the consumption rate of sugar may vary with the concentration of surfactant, the cultivation lasted for 48 hours, at which time all sugars were consumed.

[0134] Example 3-2: Evaluation of microbial growth

[0135] Microbial growth was evaluated in the same manner as in Example 2-2. The OD values ​​of the two strains are shown in Table 8 below. The analytical OD values ​​of the β-carotene producing strain (CC08-1043) and the retinol producing strain (CC08-2163) are shown in Table 8, respectively. Figure 3 A and 3B.

[0136] [Table 8]

[0137]

[0138]

[0139] As a result, regardless of the surfactant concentration, the biomass (OD) in all groups to which the Tween series surfactant was added tended to be higher overall compared to the group to which it was not added, and the biomass (OD) in the group to which Tween 40, Tween 60 or Tween 80 was added tended to be higher. However, it was confirmed that there was no significant difference in biomass (OD) according to concentration between groups to which the same type of Tween was added ( Figure 3 ).

[0140] Example 3-3: Evaluation of β-carotene, retinal and retinol concentrations

[0141] The concentrations of β-carotene, retinal, and retinol were evaluated in the same manner as in Example 2-3.

[0142] The concentrations of β-carotene, retinol and retinal (mg / L) in the two strains are shown in Table 9. Regarding the analytical results, the β-carotene concentration measured when the β-carotene producing strain (CC08-1043) was cultured is shown in Figure 4 A, β-carotene, retinol, and retinal concentrations measured when the retinol-producing strain (CC08-2163) was cultured are shown in Figure 4 B.

[0143] [Table 9]

[0144]

[0145]

[0146] As a result, in the evaluation of β-carotene-producing strain CC08-1043, regardless of the concentration and type of surfactant, the intracellular β-carotene production concentration tended to be generally higher in all groups with all concentrations of Tween series surfactants added compared to the group without addition ( Figure 4 A). Thus, the group supplemented with Tween series surfactants showed an increase of up to 1.5 times in β-carotene concentration compared to the group without addition.

[0147] In the evaluation of retinol-producing strain CC08-2163, extracellular retinol was measured only in the group supplemented with Tween series surfactants ( Figure 4B). In contrast, a relatively small amount of intracellular retinol was measured in the group without the addition of surfactant. With regard to the extracellular secretion capacity, the optimal concentration of each Tween was different. For example, there was no difference in the amount of retinol produced according to the concentration of Tween 20, but the highest extracellular retinol concentration was measured under the conditions of adding 10% Tween 40, and 5% Tween 60 and Tween 80.

[0148] In general, although the production and extracellular secretion of retinol varied depending on the type of Tween, the concentration of retinol produced under specific Tween addition conditions increased by up to 3.8 times or more compared with the group without addition ( Figure 4 B).

[0149] The above results confirmed that when a nonionic surfactant was added to the culture medium, the growth of the retinol-producing strain could be promoted and the retinol production concentration could also be increased.

[0150] 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.

[0151] Each sequence of SEQ ID NO. according to the present disclosure is shown in Table 10 below.

[0152] [Table 10]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

Claims

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

2. The method according to claim 1, wherein the nonionic surfactant is any one or more selected from the group consisting of Tween and Triton.

3. The method according to claim 2, wherein the Tween is any one or more selected from the group consisting of Tween 20, Tween 40, Tween 60 and Tween 80.

4. The method of claim 1, wherein the nonionic surfactant increases the extracellular secretion of retinol. The method according to claim 1 , wherein the microorganism is used to produce retinol.

6. The method according to claim 1, wherein the concentration of the nonionic surfactant is 0.01% (w / v) or more relative to the total medium composition.

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

8. A method for increasing retinol production, the method comprising the step of culturing a microorganism of the genus Yarrowia in a culture medium containing a nonionic surfactant.

9. A method for producing a retinoid, the method comprising the steps of: culturing a microorganism of the genus Yarrowia in a culture medium comprising a nonionic surfactant; and The retinol expressed by the microorganism is converted into a retinoid other than retinol.

10. A culture medium composition for a microorganism of the genus Yarrowia for producing retinol, the composition comprising a nonionic surfactant.

11. The culture medium composition according to claim 10, wherein the nonionic surfactant is any one or more selected from the group consisting of Tween and Triton. 12 . The medium composition according to claim 10 , wherein the concentration of the nonionic surfactant is 0.01% (w / v) or more relative to the total medium composition.

13. The culture medium composition according to claim 10, wherein the culture medium composition increases the retinol production of the microorganism.

14. A composition for producing retinol, comprising a microorganism of the genus Yarrowia or a culture thereof and a nonionic surfactant.

15. The composition of claim 14, wherein the microorganism is used to produce retinol.

16. A nonionic surfactant; a culture medium composition of a microorganism of the genus Yarrowia comprising a nonionic surfactant; or use of a composition comprising a microorganism of the genus Yarrowia or a culture thereof and a nonionic surfactant in producing a retinoid.

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