Microbial medium composition for producing retinol containing a surfactant and use thereof

Abstract

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

Description

[Technical field]

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

[0002] Retinol, a fat-soluble vitamin, is an essential vitamin involved in eye health for improving night blindness, strengthening the immune system, and healthy skin. However, retinol is 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, which contains a surfactant, a method for producing a retinoid using the same, and use of the same. [Means for solving the problem]

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

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

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

[0010] Furthermore, the present application has an object to provide a microbial medium composition for producing retinol, which uses a nonionic surfactant.

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

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

[0013] Since the medium of the present application contains a non-ionic surfactant, it can efficiently increase the production of retinoids such as retinol. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1A shows OD values ​​as a result of flask culture evaluation of a β-carotene-producing strain in the presence or absence and at the same concentration of a surfactant, and FIG. 1B shows OD values ​​as a result of flask culture evaluation of a retinol-producing strain in the presence or absence and at the same concentration of a surfactant. [Diagram 2] FIG. 1A shows the β-carotene concentration as a result of flask culture evaluation of a β-carotene-producing strain in the presence or absence and its concentration of a surfactant, and FIG. 1B shows the β-carotene, retinal and retinol concentrations as a result of flask culture evaluation of a retinol-producing strain in the presence or absence and its concentration of a surfactant. [Diagram 3] FIG. 1A is a graph showing OD values ​​as a result of flask culture evaluation of a β-carotene-producing strain depending on the presence, type, and concentration of a Tween surfactant, and FIG. 1B is a graph showing OD values ​​as a result of flask culture evaluation of a retinol-producing strain depending on the presence, type, and concentration of a Tween surfactant. [Figure 4] FIG. 1A shows the β-carotene concentration as a result of flask culture evaluation of a β-carotene-producing strain depending on the presence, type, and concentration of a Tween-based surfactant, and FIG. 1B shows the β-carotene, retinal, and retinol concentrations as a result of flask culture evaluation of a retinol-producing strain depending on the presence, type, and concentration of a Tween-based surfactant. 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 sp. microorganism in a medium comprising a non-ionic surfactant.

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

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

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

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

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

[0022] As a non-limiting example, the non-ionic surfactant increases the extracellular excretion of retinol.

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

[0024] 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.

[0025] 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.

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

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

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

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

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

[0034] As an example, the Yarrowia microorganism of the present application is a microorganism for producing retinol. The retinol-producing microorganism or strain is a microorganism that naturally has retinol-producing ability, or a parent strain that does not have retinol-producing ability has been genetically modified naturally or artificially to enhance or impart retinol-producing ability, but is not limited thereto. Specifically, the retinol-producing Yarrowia microorganism of the present application may be a microorganism modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and beta-carotene 15,15'-oxygenase (BLH) proteins.

[0035] The microorganism of the present application may be a microorganism that has been modified to further contain 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 essentially composed of the base sequence, but is not limited to these.

[0036] 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.

[0037] 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 activity of the protein, or a microorganism in which the activity of the protein is enhanced, 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 polynucleotide encoding it may have an amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase), or may include the 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 the sequence. The polynucleotide encoding the polypeptide may be modified in various ways in the coding region, without altering the amino acid sequence, by taking into consideration the codon degeneracy or the codons preferred in the microorganism in which the protein is to be expressed. Specifically, the polypeptide has 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, contains the base sequence, or consists 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 is substantially composed of the base sequence, but is not limited thereto.

[0038] In the present application, "culturing" means growing the Yarrowia microorganism of the present application under appropriately adjusted environmental conditions. In the present application, the culturing process can be performed in a suitable medium and under suitable culture conditions known in the art, as long as a medium containing a nonionic surfactant is used. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing is batch, continuous and / or fed-batch culture, but is not limited thereto.

[0039] 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.

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

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

[0042] 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.

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

[0044] In one embodiment, the nonionic surfactant increases the extracellular excretion of retinol, but is not limited thereto. In one embodiment, when a microorganism is cultured in a medium containing a nonionic surfactant, the retinol production ability is 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, but any nonionic surfactant may be used as long as it has an increase in the + value compared to the one before the addition of the nonionic surfactant.

[0045] 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."

[0046] 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.

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

[0048] 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.

[0049] The recovery may involve collecting the target retinol using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, various 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.

[0050] 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.

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

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

[0053] The retinoid production method of the present application may further include a step of converting the retinol produced by the microorganism of the present application into a retinoid other than retinol. In the retinoid production method of the present application, the 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.

[0054] 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.

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

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

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

[0058] As an example, the medium composition increases retinol production in a Yarrowia microorganism, but is not limited thereto.

[0059] In one non-limiting example, the medium composition may be one that promotes the growth of microorganisms.

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

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

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

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

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

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

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

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

[0068] The polynucleotide sequence of crtYB 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.

[0069] Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using the primers 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 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 reaction at 72°C for 3 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0070] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (Non-Patent Document 1), and 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 NO: 18 and SEQ ID NO: 19, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0071] [Table 1]

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

[0073] Example 1-2. Preparation of HMGR-enhanced strain As described above, a cassette was designed to replace the 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 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 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.

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

[0075] [Table 2]

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

[0077] 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 GGPPS polynucleotide sequence 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) at the request of Macrogen. A cassette was designed to be inserted into the LIG4 (YALI0D21384g) gene position using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker. PCR was performed using the synthesized GGPPS gene and the genomic DNA of KCCM12972P as templates and the 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 shown in Table 3. The PCR conditions were 35 cycles of denaturation at 95° C. for 1 min, annealing at 55° C. for 1 min, and polymerization at 72° C. for 2 min. The resulting DNA fragments were generated into one cassette by overlap extension PCR.

[0078] The cassette thus prepared was introduced into the strain prepared in Example 1-2 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers of SEQ ID NO: 45 and SEQ ID NO: 46, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0079] [Table 3]

[0080] Example 1-4. Construction of KU80 deletion strain For ease of future strain construction, the KU80 (YALI0E02068g) gene was deleted in the strain constructed in Example 1-3 as described above. 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. In addition, PCR was performed using the genomic DNA of KCCM12972P as a template and primers 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. 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 constructed into one cassette by overlap extension PCR.

[0081] 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 NO: 55 and SEQ ID NO: 56 and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker, and the strain was named CC08-1043.

[0082] [Table 4]

[0083] 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 as a 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. 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 the primers 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, and SEQ ID NO:67 and SEQ ID NO: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.

[0084] 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 NO: 69 and SEQ ID NO: 70 and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker, and the strain was named CC08-2163.

[0085] [Table 5] EXAMPLES

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

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

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

[0089] [Table 6]

[0090] As a result, when SDS, an anionic surfactant, was added, the bacteria did not grow under any concentration conditions. Biomass (OD) generally tended to be higher under all surfactant addition conditions, except for SDS, compared to the no-addition condition.

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

[0092] Example 2-3: Evaluation of β-carotene, retinal and retinol concentrations The method for measuring the intracellular and extracellular concentrations of β-carotene, retinol and retinal is as follows.

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

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

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

[0096] [Table 7]

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

[0098] In contrast to the results, Tween-based (TW20, 40, 60, and 80) and Triton-based (Triton X-100) surfactants all increased retinol production. In the control group where no surfactant was added, no extracellular retinol was detected, and only intracellular retinol was measured.

[0099] These results confirmed that the addition of a nonionic surfactant increased the retinol production concentration by up to 6.8 times or more compared to when no nonionic surfactant was added. EXAMPLES

[0100] Comparative evaluation of retinol production by different concentrations of Tween surfactants Example 3-1. Cultivation of Microorganisms In order to compare the degree of β-carotene and retinol production depending on the concentration of Tween surfactants, which are nonionic surfactants, flask evaluation was performed on the CC08-1043 and CC08-2163 strains. The CC08-1043 and CC08-2163 strains were inoculated into 250 ml corner baffle flasks containing 20 ml of YLMM2 (Yarrowia lipolytica minimal media 2) medium to an initial OD of 2, and four types of surfactants, Tween 20 (TW20), Tween 40 (TW40), Tween 60 (TW60), and Tween 80 (TW80), were added to the medium at concentrations of 5, 10, or 15%, respectively. The culture was performed at 30°C and 200 rpm. Since the sugar consumption rate differs depending on the concentration of the surfactant, the culture was continued for 48 hours until all the sugar was consumed.

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

[0102] [Table 8]

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

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

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

[0106] [Table 9]

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

[0108] In the evaluation of the retinol-producing CC08-2163 strain, it was confirmed that extracellular retinol was measured only in the group to which a Tween-based surfactant was added (Figure 4b). In contrast, in the group to which no surfactant was added, it was confirmed that only a relatively small amount of intracellular retinol was measured. The optimal concentration for the extracellular excretion of retinol differed depending on the type of Tween. For example, there was no difference in the amount of retinol produced depending on the concentration of Tween 20, but the highest extracellular retinol concentration was measured when Tween 40 was added at 10%, and when Tween 60 and Tween 80 were added at 5%.

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

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

[0111] From the above description, 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.

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

[0113] [Table 10-1]

[0114] [Table 10-2]

[0115]

Table 10-3

[0116]

Table 10-4

[0117]

Table 10-5

[0118]

Table 10-6

[0119]

Table 10-7

[0120]

Table 10-8

[0121]

Table 10-9

[0122]

Table 10-10

[0123]

Table 10-11

[0124]

Table 10-12

[0125]

Table 10-13

[0126]

Table 10-14

[0127]

Table 10-15

[0128] JPEG2025510208000026.jpg208145

Claims

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

2. 2. The method for producing retinol according to claim 1, wherein the non-ionic surfactant is at least one selected from the group consisting of tween and Triton.

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

4. The method for producing retinol according to claim 1 , wherein the nonionic surfactant increases extracellular excretion of retinol.

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

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

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

8. 1. A method for increasing retinol production, comprising culturing a Yarrowia microorganism in a medium containing a non-ionic surfactant.

9. Cultivating a Yarrowia microorganism in a medium containing a nonionic surfactant; and converting the retinol produced by the microorganism into a retinoid other than retinol.

10. A medium composition for producing retinol from a Yarrowia microorganism, comprising a nonionic surfactant.

11. The medium composition according to claim 10, wherein the non-ionic surfactant is at least one selected from the group consisting of Tween and Triton.

12. The medium composition according to claim 10, wherein the nonionic surfactant is contained at a concentration of 0.01% or more based on the total medium composition.

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

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

15. The composition for producing retinol according to claim 14 , wherein the microorganism is for producing retinol.

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

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