Chlamydomonas reinhardtii mutant strain with improved ability to produce keto-carotenoids

Abstract

The present invention relates to a Chlamydomonas reinhardtii mutant strain having an improved ability to produce keto-carotenoid pigments, particularly astaxanthin and canthaxanthin. When used, the strain of the present invention exhibits excellent lipid production ability compared to conventional strains, and can produce keto-carotenoid pigments, specifically astaxanthin and canthaxanthin, and thus can efficiently produce pigments on an industrial scale. In addition, the strain can be applied as a raw material for foods, health functional foods and medicines comprising pigments.

Description

A mutant strain of Chlamydomonas reinhardtii with enhanced keto-carotenoid production

[0001] The present invention relates to a mutant strain of Chlamydomonas reinhardtii having improved keto-carotenoid production ability and its use, and more particularly, to a novel strain having keto-carotenoid production ability, a composition comprising the same, and a method for producing food, food raw material, or pigment using the same.

[0002]

[0003] Microalgae are unicellular organisms known for their high photosynthetic efficiency and growth rate, along with their potential to produce bioactive compounds. Research on microalgae has primarily focused on the production of bio-oils, fatty acid nutrients (e.g., docosahexaenoic acid and eicosapentaenoic acid (EPA)), and carotenoid pigments. More recently, microalgae producing high-value carotenoids, such as astaxanthin, β-carotene, lutein, and zeaxanthin, have attracted significant attention.

[0004] Carotenoids (keto-carotenoids) are isoprenoids that are considered natural pigments, and their characteristic colors range from yellow to red and are found in many flowers, fruits, and vegetables.

[0005] Keto-carotenoids, a type of carotenoid, are carotenoids containing a ketone (carbonyl) group. They belong to a group of compounds called terpenoids, and are metabolic products of zeaxanthin, canthaxanthin, and astaxanthin, containing both a hydroxyl group and a ketone.

[0006] Astaxanthin is a powerful antioxidant, 550 times more potent than vitamin E. It is primarily used as a coloring agent in aquaculture feeds for salmon, trout, and shrimp. It is also used as a health functional food ingredient to prevent age-related macular degeneration, Alzheimer's and Parkinson's diseases, heart disease, and skin damage caused by UV rays. Specifically, astaxanthin is a keto-carotenoid naturally produced by the freshwater microalgae Haematococcus lacustris and the yeast fungus Xanthophyllomyces dendrorhous (or Phaffia rhodozyma). It helps protect cells from free radicals. Studies have also shown that astaxanthin intake can improve skin and muscle health, strengthen eye health and immunity, and support healthy aging. Astaxanthin can help reduce exercise-induced oxidative stress, and may have a positive effect on alleviating exercise fatigue and improving endurance.

[0007] The global astaxanthin market size was 200 tons as of last year. Demand for salmon and trout is increasing due to population growth, and demand for natural products rather than synthetic ones is also increasing, so steady growth is expected. Furthermore, rapid aging and rising consumer health awareness are supporting market expansion.

[0008] Canthaxanthin is a keto-carotenoid pigment widely distributed in nature. It is produced by plants, algae, some bacteria, archaea, and fungi, and was first isolated from edible mushrooms. The chemical formula of canthaxanthin is C 40 H 52 It's O2

[0009] Canthaxanthin is known as a potent lipid-soluble antioxidant and is associated with biological functions such as free radical scavenging and vitamin E conservation in animal tissues. Furthermore, canthaxanthin is approved for use as a food colorant in over 70 countries in Europe, the United States, and Asia. It is also permitted for addition to mackerel, salmon, and poultry feed. The health benefits of dietary carotenoids are encouraging a shift in commercial production of canthaxanthin from chemical synthesis to consumption or extraction from natural sources. Therefore, exports may also be possible to meet international demand.

[0010] Astaxanthin and / or canthaxanthin are recognized as safe as food or feed additives, and demand is rapidly increasing in various industries such as health supplements, cosmetics, food and beverage, pharmaceuticals, aquaculture, and animal feed.

[0011] Currently, commercially widely used astaxanthin is mainly produced from Haematococcus lacustris, formerly known as Haematococcus pluvialis. The reported astaxanthin production rate of Haematococcus lacustris to date is approximately 5% on a dry weight basis, which is the highest among natural products. However, Haematococcus lacustris has a slow growth rate, the risk of contamination due to intermediate processes in the production process, and difficulties in extracting the pigment, resulting in an inefficient extraction method. This necessitates the development of novel microalgal strains that accumulate astaxanthin and / or canthaxanthin even under basic conditions.

[0012] Meanwhile, Chlamydomonas reinhardtii, a freshwater microalga approved as a food ingredient, can enhance the oxidative stability of extracts due to its rapid growth and high lipid content when used as a host. However, it rarely produces keto-carotenoids such as astaxanthin and / or canthaxanthin.

[0013] Moreover, antioxidant pigment components such as astaxanthin and canthaxanthin have the problem of being unstable when extracted from microalgae (as lipids).

[0014] Accordingly, it is necessary to develop a strain that is stable when extracted together with lipids and is effective in terms of storage after extraction, while producing raw materials for food, health functional foods, or pharmaceuticals.

[0015] [Prior Art Literature]

[0016] [Patent Document]

[0017] (Patent Document 1) Korean Patent Registration No. 10-2358538 (January 27, 2022)

[0018]

[0019] The purpose of the present invention is to provide microalgae capable of extracting antioxidant pigments used as raw materials for food, medicine, feed, etc. together with lipids while improving keto-carotenoid production ability, a composition including the same, and a method for producing pigments using the same.

[0020]

[0021] In order to achieve the above purpose, the inventors of the present invention have made efforts to utilize the strain as a keto-carotenoid production strain, and as a result, by introducing the BKT gene into a mutant in which the AGP gene is knocked out of the microalgae Chlamydomonas reinhardtii, a mutant strain of Chlamydomonas reinhardtii having an improved keto-carotenoid production ability, i.e., astaxanthin and / or canthaxanthin, utilizing microalgal oil rich in keto-carotenoids, including astaxanthin and canthaxanthin, compared to a conventional Chlamydomonas reinhardtii strain, thereby completing the present invention.

[0022] Accordingly, the present invention aims to provide a Chlamydomonas reinhardtiia agp / BKT mutant 1(a / B_1) strain (Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP) having keto-carotenoid production ability and a culture thereof.

[0023] In addition, the present invention provides a pigment composition comprising at least one selected from the group consisting of Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

[0024] The above composition may be for oral administration, feed, feed additive, food or food additive.

[0025] In addition, the present invention provides a method for producing a pigment comprising culturing Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP.

[0026] The above pigment production method may further include extracting the pigment from Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP or a culture thereof.

[0027] The pigment may include one or more antioxidant pigments selected from the group consisting of astaxanthin and cantaxanthin.

[0028] In addition, the present invention provides a method for producing a food or feed raw material comprising culturing Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP.

[0029]

[0030] The present invention relates to a microalgae oil-producing Chlamydomonas reinhardtii strain agp / BKT mutant 1(a / B_1) in which the AGP gene is knocked out in the wild type Chlamydomonas reinhardtii to increase lipids, and the BKT gene is overexpressed to enhance the production of astaxanthin and canthaxanthin.

[0031] When Chlamydomonas reinhardtii, which grows rapidly, is used as a host, the oxidative stability of the extract can be increased due to rapid cultivation and high lipid content, and productivity can be increased by using a lipid-accumulating strain (AGP knockout strain) compared to the actual wild type under nitrogen-depleted conditions.

[0032]

[0033] Figures 1a and 1b show the AGP gene sequence of the wild type Climidomonas.

[0034] Figure 2 shows the results of analyzing the sgRNA target site through PCR.

[0035] Figure 3 is a schematic diagram showing the process of producing a Chlamydomonas reinhardtii mutant strain (hereinafter, agp / BKT_1) of the present invention.

[0036] FIG. 4 shows the pOpt3_PsaD_CrBKT_aadA vector used in the present invention corresponding to the first drawing of FIG. 3. The BKT_aadA vector was provided through joint research with a German research team (Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany).

[0037] Figure 5 shows new pigments (astaxanthin, canthaxanthin) produced when BKT is expressed in the carotenoid biosynthetic pathway of the Chlamydomonas reinhardtii strain. [Carotenoid synthesis in Chlamydomonas reinhardtii begins with the process of generating C5 isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through 2-C-methyl-D-erythritol 4-phosphate (MEP) from 3-phosphate (G3P) and pyruvate. In the case of C5 building blocks, the intermediate step is C20 geranylgeranyl pyrophosphate (GGPP) to generate C40 carotenoid phytoene. After that, phytoene undergoes desaturation to become lycopene, which is then divided into α- and β-carotene branches. In the α-branch, α-carotene produces lutein and loroxanthin (in the case of chlamydomonas), and in the β-branch, β-carotene produces the xanthophylls zeaxanthin, antheraxanthin, and violaxanthin. Later, violaxanthin produces neoxanthin. In addition, astaxanthin is rarely produced in Chlamydomonas reinhardtii, but it can be produced from β-carotene and zeaxanthin through overexpression of the BKT gene inherent in Chlamydomonas reinhardtii. The pathway starting from β-carotene produces astaxanthin through echinenone, cantaxanthin, and adonirubin, and the pathway starting from zeaxanthin produces astaxanthin through adonixanthin.

[0038] Figures 6a and 6b show the carotenoid profiles of the wild type (WT) and mutant strains (AGP, WT / BKT, agp / BKT_1) of Chlamydomonas reinhardtii, showing pigment profiles different from those of the wild type (cc4349) and the AGP mutant strain, and indicating astaxanthin and canthaxanthin present within parallel lines [Neo: neoxanthin, Vio: violaxanthin, Trans-ast: trans-astaxanthin, Lut: lutein, Can: cantaxanthin, Chl b: chlorophyll b, chl a: chlorophyll a].

[0039] Figure 7a shows the growth curves (cell count per volume, cells / ml) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp_BKT_1) of Chlamydomonas reinhardtii.

[0040] Figure 7b shows the biomass (g / L, 3 days) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp_BKT_1) of Chlamydomonas reinhardtii.

[0041] It was confirmed that the growth of the mutant strain agp_BKT of the present invention did not differ significantly compared to the control group and the host.

[0042] Figure 8a shows the content of astaxanthin (mg / g) per biomass of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day.

[0043] Figure 8b shows the content of canthaxanthin (mg / g) per biomass of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day.

[0044] Figure 9a shows the total carotenoid content (mg / L) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day.

[0045] Figure 9b shows the content of astaxanthin (mg / L) per total carotenoid content of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day.

[0046] Figure 9c shows the content of canthaxanthin (mg / L) per total carotenoid content of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day.

[0047] Figure 10a shows the growth curves (cell count per volume, cells / ml) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions.

[0048] Figure 10b shows the biomass (g / L, 4 days) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions.

[0049] Figure 11a shows the content of astaxanthin (mg / g) per biomass of wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions on the 4th day.

[0050] Figure 11b shows the content of canthaxanthin (mg / g) per biomass of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions on the 4th day.

[0051] Figure 12a shows the total carotenoid content (mg / L) of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions on the 4th day.

[0052] Figure 12b shows the content of astaxanthin (mg / L) per total carotenoid content of wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under primary nitrogen deficiency conditions.

[0053] Figure 12c shows the content of canthaxanthin (mg / L) per total carotenoid content of the wild type (WT) and mutant strains (AGP, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions on the 4th day.

[0054] Figure 13 shows the lipid content per weight (g / g) of the wild type (WT) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen deficiency conditions on the 4th day.

[0055]

[0056] Hereinafter, the present invention will be described in more detail.

[0057] The present invention relates to Chlamydomonas reinhardtiia agp / BKT mutant 1(a / B_1) (Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1), KCTC 15839BP) [hereinafter referred to as Chlamydomonas reinhardti mutant].

[0058] Chlamydomonas reinhardtii, a unicellular green alga (Chlorophyta), is a eukaryotic organism found in diverse environments, including freshwater and marine waters, with a doubling time of 6 to 8 hours. Furthermore, it is one of the most widely used microalgal model systems and can be produced in bioreactors.

[0059] The above mutant was produced by knocking out the AGP (ADP-glucose pyrophosphorylase) gene using CRISPR gene scissors technology without the introduction of external DNA, i.e., using RGNE RNPs rather than general mutagenesis.

[0060] The above mutant strain was knocked out by Cas9 protein targeting the ADP-glucose pyrophosphorylase (AGP) gene (Cre03.g188250.t1.2) [Fig. 10] of Chlamydomonas reinhardtii cc4349WT represented by SEQ ID NO: 1, and was performed in the same manner as the paper by the present inventors (Song et al., Microbial Cell Factories 2020; 19: 220. The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii) and Korean Patent Application No. 10-2019-0002969.

[0061] In one embodiment of the present invention, Chlamydomonas reinhardtii cc4349 cw15 mt+ [Goodenough 330A] was obtained from the Chlamydomonas Resource Center (www.chlamycollection.org) [http: / www.chlamycollection.org / product / cc-4349-cw15-mt-goodenough-330a / ]. Using this as the original strain, the BKT gene was expressed using a strain in which the AGP gene was knocked out according to the existing gene editing method (Baek, Kwangryul, et al. "DNA-free two-gene knockout in Chlamydomonas reinhardtiivia CRISPR-Cas9 ribonucleoproteins." Scientific reports 6.1 (2016): 30620.), and the selected mutant was named Chlamydomonas reinhardtiiviagp / BKT mutant 1(a / B_1) and deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC) on February 28, 2024, and assigned the accession number KCTC 15839BP.

[0062] The Chlamydomonas reinhardtii mutant strain of the present invention has a significantly higher keto-carotenoid production ability than the wild type, and it can be confirmed that astaxanthin, which was previously hardly produced, accumulates 1 to 10 parts by weight or 5 to 7 parts by weight of astaxanthin relative to 100 parts by weight of total carotenoid pigments, and cantaxanthin accumulates 5 to 15 parts by weight or 7 to 10 parts by weight of cantaxanthin relative to 100 parts by weight of total carotenoid pigments [Figure 5].

[0063] The above mutant strain can be cultured in an environment in which general Chlamydomonas rainhardtii algae can be cultured, and specifically, a culture medium capable of culturing algae under low light conditions can be used. In order to cultivate a specific microorganism, it may be a mixture containing nutrients required by the microorganism to be cultured, that is, the cultured body, and additional substances for special purposes may be added. The above medium is also called a culture medium or culture solution, and is a concept that includes all of a natural medium, a synthetic medium, or a selective medium. The Chlamydomonas rainhardtii mutant strain can be cultured according to a conventional culture method. For example, it can be cultured in a photosynthetic medium such as HS medium or TAP medium, and a carbon source can be added.

[0064] The pH of the above culture medium is not particularly limited as long as it is within the range where Chlamydomonas reinhardtii can survive and grow. For example, it can survive at pH 6 or higher, specifically at pH 6 to pH 9, and can have an optimal growth rate at pH 7.0 or higher and less than pH 8.0.

[0065] The Chlamydomonas reinhardtii mutant of the present invention can accumulate a high content of lipids within cells and can contain a higher content of keto-carotenoids, so that the algae can be effectively used as a raw material for food, feed, medicine, etc. by culturing them.

[0066] In this respect, the present invention relates to a culture of the above Chlamydomonas reinhardtii mutant.

[0067] In the present invention, "culture" refers to a medium in which a specific microorganism is cultured, i.e., a cultured medium, and the culture includes the Chlamydomonas reinhardtii mutant. In addition, the culture includes both a concentrate of the culture obtained by concentrating, drying, or processing the culture medium after culture, and a dried product of the culture. The culture may include its byproducts, and its formulation is not limited, and may be, for example, a liquid or a solid.

[0068] The above medium contains nutrients required by the microorganisms to be cultured, i.e. the cultured organism, for culturing specific microorganisms, and may be a mixture in which substances for special purposes are additionally added. The above medium is also called a culture medium or culture solution, and is a concept that includes natural media, synthetic media, or selective media. The pH of the above medium may be a range in which the Chlamydomonas reinhardtii mutant can grow, and for example, may be pH 6 or higher, preferably pH 6 to pH 9.

[0069] In addition, the present invention relates to a composition comprising at least one selected from the group consisting of a mutant strain of Chlamydomonas reinhardtii of the present invention, a culture of the algae, a dried product thereof, and an extract thereof.

[0070] The above composition can be used for promoting human and animal health.

[0071] The mutant strain of the present invention has the property of producing and accumulating in the body an antioxidant pigment including astaxanthin and / or canthaxanthin, and in this respect, the composition may be a pigment composition.

[0072] The above pigment composition may contain astaxanthin in an amount of 1 to 10 parts by weight or 5 to 7 parts by weight based on 100 parts by weight of the total carotenoid pigment, and / or cantaxanthin in an amount of 5 to 15 parts by weight or 7 to 10 parts by weight based on 100 parts by weight of the total carotenoid pigment.

[0073] According to one embodiment of the present invention, as a result of measuring the content of astaxanthin in the total pigment per cell of each Chlamydomonas reinhardtii wild type algae and Chlamydomonas reinhardtii mutant, it was confirmed that the content of astaxanthin in the pigment was significantly higher in the Chlamydomonas reinhardtii mutant than in the wild type (Fig. 5a).

[0074] The above pigment composition can be used as a raw material for food or feed, and can be used as a preparation for oral administration.

[0075] Accordingly, the pigment composition comprising the above composition or extract may be a composition for oral administration in that it can be supplied orally as a food, medicine, feed, etc.

[0076] In the case of compositions for oral administration, the active ingredient may be included in oral preparations formulated using methods known in the art, such as powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture to obtain a tablet or a sugar tablet. Examples of suitable excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; fillers such as gelatin and polyvinylpyrrolidone. Additionally, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrating agent.

[0077] In addition, since the composition can be added to food or feed to achieve a special purpose, in this respect, it can be a food composition, a composition for food additives, a feed composition, or a composition for feed additives. When the composition is used in feed or food, it can maintain or enhance body health by the antioxidant pigments, particularly astaxanthin and canthaxanthin, produced by the Chlamydomonas reinhardtii mutant and accumulated in cells. Specifically, since the astaxanthin and canthaxanthin have antioxidant, immune system strengthening, athletic performance enhancement, eye health strengthening or maintenance, and eye function deterioration prevention or improvement effects, the feed or food composition can be used for the prevention or improvement of the symptoms, or for the purposes for the effects.

[0078] In the present invention, “for additives” includes all components added to food or feed other than the main raw material, and specific examples thereof include effective active substances having functionality in food or feed, or food additives defined by the Ministry of Food and Drug Safety that are added for coloring, preservation, etc. in processed foods.

[0079] The above food may be a health functional food. More specifically, it may be a health functional food for antioxidant properties, immune system enhancement, athletic performance enhancement, eye health enhancement or maintenance, or prevention or improvement of eye function decline.

[0080] The above food, food additive, feed, or feed additive composition may further include other effective ingredients as long as they do not impair the activity of the Chlamydomonas reinhardtii mutant strain of the present invention, the culture of the mutant strain, the dried product thereof, and the extract thereof. In addition, the composition may further include additional ingredients such as a carrier.

[0081] In the present invention, the feed composition can be manufactured in the form of fermented feed, compound feed, pellet form, silage, etc. The fermented feed includes the Chlamydomonas reinhardtii mutant of the present invention, dried cells of the mutant, culture of the mutant, and extract thereof, and can be manufactured by additionally including various microbial strains or enzymes. The compound feed can be manufactured by mixing various types of general feed with the mutant of the present invention, dried cells of the mutant, culture of the algae, and extract thereof. Feed in pellet form can be manufactured by formulating the fermented feed or compound feed in a pellet machine. Silage can be manufactured by mixing green feed with the Chlamydomonas reinhardtii mutant, dried cells of the mutant, culture of the mutant, and / or extract thereof, but the use of the composition of the present invention is not limited thereto.

[0082] The above composition can be manufactured and administered in the form of tablets, troches, capsules, elixirs, syrups, powders, suspensions, or granules by mixing with carriers and flavorings commonly used in the food or pharmaceutical fields. Carriers that can be used include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, and suspending agents. The administration method can be oral, parenteral, or topical, but oral administration is preferred. In addition, the administration dosage can be appropriately selected depending on the absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, and the age, sex, condition, etc. of the recipient. The pH of the composition can be easily changed depending on the manufacturing conditions of the drug, food, etc. in which the composition is used.

[0083] The above composition may contain 0.001 to 99.99 wt%, preferably 0.1 to 99 wt%, of any one selected from the group consisting of a Chlamydomonas reinhardtii mutant, a culture of the mutant, a dried product thereof, and an extract thereof, based on the total weight of the composition, and the content of the active ingredient may be appropriately adjusted depending on the method of use and purpose of use of the composition.

[0084] The above Chlamydomonas reinhardtii mutant strain may be included in the composition as is or in a dried form, and the algal culture may be included in the composition in a concentrated or dried form. In addition, the dried product refers to the dried form of the algae or its culture, and may be in the form of a powder manufactured by freeze-drying or the like.

[0085] In addition, the above extract refers to an extract obtained by extracting from the Chlamydomonas reinhardtii mutant of the present invention, a culture medium thereof, or a dried product thereof, and includes an extract using a solvent, etc., and an extract obtained by crushing the Chlamydomonas reinhardtii mutant of the present invention. Specifically, it may be an extract obtained by extracting and separating a pigment accumulated in the cells of the Chlamydomonas reinhardtii mutant of the present invention by a physical or chemical method.

[0086] The above extraction process can be performed by a conventional method. For example, an extraction solvent can be added, homogenized, and then the cells can be crushed to extract the target pigment. After extraction, the algae fragments can be removed by centrifugation, and the extraction solvent can be removed by a method such as reduced pressure distillation. In addition, a conventional purification process can be further included. Since the pigment is insoluble in water, it can be more easily extracted from the algae of the present invention.

[0087] Since the Chlamydomonas reinhardtii mutant of the present invention has an excellent ability to produce keto-carotenoids, particularly astaxanthin and canthaxanthin, a composition comprising the mutant and its by-products has the effect of improving physical activity, maintaining physical function, and preventing its decline. Specifically, since the keto-carotenoid pigment is known to have antioxidant, immune-enhancing, and eye-health-enhancing properties, the composition of the present invention can be used as a raw material included in foods, health functional foods, medicines, or feeds for the purpose of maintaining physical health, specifically, maintaining, preventing decline, or improving physical functions in which the xanthophyll pigment is involved.

[0088] In addition, another object of the present invention is to provide a method for producing a pigment using the Chlamydomonas reinhardtii mutant of the present invention.

[0089] In addition, another object of the present invention is to provide a method for producing food or feed raw materials, which comprises a step of culturing a Chlamydomonas reinhardtii mutant of the present invention.

[0090] When the Chlamydomonas reinhardtii mutant of the present invention is used, the accumulation of keto-carotenoid in the cultured algae can be increased, thereby efficiently supplying raw materials used industrially, etc.

[0091] The above production method may include a step of culturing the Chlamydomonas reinhardtii mutant strain of the present invention.

[0092] Additionally, the above production method may further include, after the above culturing step, a step of isolating the Chlamydomonas reinhardtii mutant strain of the present invention from the culture. The isolated algae may undergo further processing steps, including drying.

[0093] In addition, the above production method may further include a step of extracting a pigment from the Chlamydomonas reinhardtii mutant of the present invention, a culture of the mutant, a concentrate of the culture, or a dried product of the culture.

[0094] The above culture can be performed in a medium with pH 6.0 to 8.0. In addition, it can be performed under weak light conditions, specifically 10 to 2,000 μmol photons / m 2 It can be performed under light intensity conditions of the s range. In the case of the Chlamydomonas reinhardtii mutant of the present invention, pigment production ability is excellent even at low light intensity, and thus the content of keto-carotenoids in the body can be increased. Therefore, excellent keto-carotenoid accumulation can be achieved without administering high light energy, and thus, it can be effectively used industrially.

[0095] The above extraction can be performed by a conventional method for extracting pigments from microorganisms, including but not limited to an enzymatic method, an ultrasonic extraction method, and a mechanical extraction method.

[0096] In addition to the cultivation step, the above production method may further include a concentration step to increase the algae content after cultivation, and a drying step to further reduce the moisture content of the algae after the concentration step and thereby dry them. However, the concentration step or the drying step is not necessarily required, and can be performed using any concentration and drying method or machine commonly used in the field to which the present invention pertains.

[0097] The above production method may be performed by further including a purification step after the extraction step, and this may be performed by a conventional purification method in the technical field to which the present invention belongs.

[0098] Keto-carotenoids manufactured through the above concentration or drying steps can be used as raw materials for foods, health functional foods, cosmetics, or pharmaceuticals.

[0099] The above keto-carotenoid production method can be performed by employing other methods within a range that does not impair the effects of the present invention.

[0100] The above-mentioned mutant strain and composition can also be applied to the production method of the present invention.

[0101]

[0102] [Example]

[0103] Hereinafter, the present invention will be described in detail through manufacturing examples and experimental examples. The following examples and experimental examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0104]

[0105] Example 1. Production of AGP gene knockout strain

[0106] 1-1. Wild-type AGP gene sequence of Chlamydomonas

[0107] SEQ ID NO: 1 and the attached drawings are shown in FIG. 1a and FIG. 1b.

[0108]

[0109] 1-2. Selection of gRNA target site and sgRNA synthesis

[0110] The experiment was conducted in the same manner as in the paper (Song et al., Microbial Cell Factories (2020)) and patent (domestic patent registration no. 2286087).

[0111] Gene knockout was performed targeting the ADP-glucose pyrophosphorylase (AGP) gene (Cre03.g188250.t1.2). The experiment was conducted with the sgRNA target sequence AGP4_sg1 5'-TAGCATGGCCCTGAAGATGCGGG-3' (SEQ ID NO: 2).

[0112]

[0113] 1-3 Generation of AGP gene knockout Chlamydomonas mutant strain using CRISPR gene scissors technology (CRISPR-Cas9 RNP) in Chlamydomonas

[0114] To knockout the AGP gene, transformation was performed using the RNP method. The cc4349 strain was washed three times with TAP sucrose (40 mM), and 100 μg of Cas9 protein (ToolGen, Seoul, South Korea) and 70 μg of in vitro transcribed sgRNA (GeneArt Tm Precision gRNA Synthesis Kit, Invitrogen, CA, USA) at room temperature for 10 minutes, and 5 x 10 5Cells were incubated on ice for 5 minutes. Transferred to a 0.4 cm electroporation cuvette, the cells and RNP complex mixture were transformed using a Gene Pulser Xcell Electroporation System (Bio-Rad, CA, USA) under conditions of (600 V, 50 uF, 200 ohm). After electroporation, cells were plated on TAP-agar plates.

[0115]

[0116] 1-4 Confirmation of transformants using the CRISPR-Cas9 RNP method

[0117] After waiting for colonies to grow, the liquid-cultured cells in N-TAP were confirmed through iodine staining. Under nitrogen-deficient conditions, more starch and lipids are synthesized, but when the agp gene is knocked out, starch is not synthesized and therefore not stained by iodine. Colonies were transferred to 96-well culture plates, and the transferred cells were cultured in nitrogen-deficient TAP medium for 3 days. After that, starch accumulation was confirmed through 1 μg of iodine. Normal strains show dark green or dark gray, but the agp mutants did not show any color change (green, yellow-green). Those that did not show any color change were selected as AGP knockout mutants, and the sgRNA target site was analyzed through PCR. AGP (F: 5'-TGGGCACGACTTGCATTGTGT-3': SEQ ID NO: 3, R: 5'-AATGGGCCAGCGCGAGCATA-3': SEQ ID NO: 4). As a result, the WT / BKT strain was confirmed to have a PCR product of approximately 500 bp, which is the same as the WT strain when PCR was performed, and the agp / BKT_1 strain was confirmed to have a PCR product of approximately 1300 bp, confirming that a mutation occurred due to insertion at the target site [Figure 2].

[0118]

[0119] Example 2. Production of BKT overexpressing strain

[0120] 2-1 Obtaining expression vector for BKT overexpression

[0121] The BKT_aadA vector was provided through joint research with a German research team (Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany) [Fig. 2].

[0122]

[0123] 2-2 Production of mutant strains for BKT gene expression in Chlamydomonas

[0124] For the BKT overexpression strain, CC4349 (WT) and the agp mutant strain were used. The strain was inoculated into N+ TAP at 100 x 10^4 cells / ml two days before transformation, and then inoculated again into N- TAP at 100 x 10^4 cells / ml one day before. Afterwards, the BKT vector was cleaved using Xba1 and kpn1, and the DNA was recovered using gel electrophoresis and gel elution, which was then used for transformation. On the same day, in order to change the medium of the microalgae strain to TAP sucrose (40 mM), the process of washing with TAP sucrose (40 mM) was performed three times, and the cells were concentrated to 1 x 10^7 cells and then dissolved in 250 ul of TAP sucrose (40 mM). Afterwards, cc4349 cells and the agp knock out strain were cultured at 40 ℃ for 30 minutes with shaking to induce heat shock. Afterwards, the previously eluted DNA (860 ng) was added to the cells, transferred to an electroporation cuvette, and reacted for 5 minutes. Transformation was performed using an electroporation system (Biorad) at 600 V, 50 uF, and 200 ohm [2nd picture in Fig. 3]. After reacting for 15 minutes at room temperature, 750 μl of 40 mM TAP sucrose was added to the cuvette, and transferred to 6 wells containing 4 ml of TAP sucrose (40 mM), followed by incubation for 1 day (25 ℃, 5 uE). After concentrating the cells (2,500 rpm, 10 min, RT), the medium was discarded, and lysed using 250 μl of TAP-40 mM sucrose. It was plated on a medium containing TAP (Spectinomycin 200ug / ml) [3rd picture in Fig. 3].

[0125]

[0126] 2-3 Confirmation of transformants

[0127] After that, we waited for about 10 days at 25℃, 50uE for the colonies to come up. The colonies whose color changed from green to red due to the expression of the BKT gene [3rd picture in Fig. 1] were transferred to a 96-well plate and grown for about 3 days. After that, colony PCR was performed using primers for the transformation confirmation part, promoter part, and terminator part [4th picture in Fig. 1]. For primers to confirm the completion of transformation, Trafo confirmation for and Trafo confirmation rev were used, and to confirm the insertion of the promoter part, P7_pPSAD_promoter_primer_F and Trafo confirmation rev were used. Then, to confirm the insertion of the terminator part, primers of P9_aada_F and P8_FDX_terminator_R were used [4th picture in Fig. 3]. Afterwards, the astaxanthin content of the selected strains was measured using HPLC, and the values ​​were divided by the cell number to select two strains each with the highest astaxanthin content per cell from WT and agp [5th figure in Fig. 3].

[0128] [Table 1]

[0129]

[0130] For pigments, extraction was performed with 90% acetone, and the extract was analyzed at a wavelength of 445 nm using a Shimadzu Prominence HPLC model LC-20AD (Shimadzu, Kyoto, Japan), a Spherisorb 5.0uM ODS1 4.6 x 250 mm cartridge column (Waters, Milford, USA), and a photodiode array detector. The purified pigments were purchased and compared with their retention time values ​​to confirm the pigments contained in the mutant strains [Fig. 5].

[0131]

[0132] Example 3: Characterization of selected mutants in TAP medium

[0133] 3-1. Cultivation of mutant strains

[0134] Cultivation of the Chlamydomonas mutant of the present invention was performed according to the following medium and culture conditions.

[0135] In the case of mixotrophic culture, which is cultured by supplying photosynthesis and a carbon source, the cells were cultured in TAP medium supplemented with acetic acid. After making a medium with the composition shown in Table 1 below, it was sterilized at high pressure and high temperature using an autoclave. After adding the medium to the cells and allowing them to go through a growth phase, they were grown for about 3 days and then incubated for 10 6 cells / ml (nitrogen-sufficient conditions) or 10 7 Cells were seeded into 50 ml of TAP medium to initiate growth at a concentration of 10 cells / ml (nitrogen-deficient conditions). For 50 ml cultures, a 250 ml glass flask was used, and for 5 L cultures, a 5 L JAR was used. Illumination was provided using a fluorescent lamp at an intensity of 100 uE.

[0136] [Table 2]

[0137]

[0138]

[0139] 3-2. Pigment profile

[0140] When analyzing the pigment profiles as shown in Figures 6a and 6b, it can be seen that the strain has additional pigments different from the existing wild type (cc4349) and AGP mutant strain. After expressing BKT, it can be seen that it contains additional pigments (parallel lines), which represent astaxanthin and canthaxanthin, respectively.

[0141]

[0142] 3-3. Growth curve

[0143] When comparing the growth curves (cells / ml) of the wild type (CC4349) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii as shown in Fig. 7a, it can be seen that there is no significant difference in the BKT overexpressing strains compared to the WT or agp strains.

[0144]

[0145] 3-4 Biomass production

[0146] When comparing the biomass of the wild type (WT) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii, it can be seen that agp has a reduced biomass compared to WT. When BKT was expressed, it can be seen that each BKT gene overexpressor has a phenotype with a reduced biomass compared to WT and agp [Fig. 7b].

[0147]

[0148] 3-5 Analysis of keto-carotenoid production

[0149] When comparing the contents of astaxanthin and canthaxanthin of the wild type (WT) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in TAP medium on the third day, the strain that did not express BKT did not produce astaxanthin. In the case of the strain that expressed BKT, astaxanthin had 2.25 mg / L (WT / BKT) and 2.45 mg / L (agp / BKT_1), respectively. Canthaxanthin had 3.15–3.5 mg / L (WT / BKT) and 3.26 mg / L (agp / BKT_1), respectively.

[0150] When these were converted to content per biomass, astaxanthin had 3.6 mg / g (WT / BKT) and 4 mg / g (agp / BKT_1), respectively [Fig. 8a]. Cantaxanthin had 5.1–5.7 mg / g (WT / BKT) and 5.4 mg / g (agp / BKT_1), respectively [Fig. 8b]. When the content of astaxanthin and cantaxanthin per total carotenoid was confirmed, the total carotenoid had values ​​of 5.7 mg / L (WT / BKT_1), 6.2 mg / L (WT / BKT_2), and 6 mg / L (WT / BKT), respectively [Fig. 9a]. Among these, astaxanthin accounts for 0.30~0.4 mg / L (WT / BKT) and 0.33~0.42 mg / L (agp / BKT_1) among the total carotenoids [Fig. 9b], and cantaxanthin accounts for 0.46~0.61 mg / L (WT / BKT) and 0.52~0.56 mg / L (agp / BKT_1) [Fig. 9c].

[0151]

[0152] Example 4: Characterization of selected mutants under nitrogen deficiency conditions.

[0153] In the case of oil production in C. reinhardtii, it is produced in a two-step manner.

[0154] The first step is to grow the cells, and the second step is to induce TAG by stressing them by growing them in a nitrogen-free medium.

[0155] It is known that nutrients are accumulated as lipids and starch under nitrogen starvation conditions. In the case of agp mutants, starch cannot be synthesized, and the energy accumulated under nitrogen starvation is known to be used to synthesize lipids and accumulate high lipid contents. Therefore, in order to utilize the increased lipid content under nitrogen starvation conditions, the lipid content and pigment content under nitrogen starvation conditions were confirmed [Jang, S., Kong, F., Lee, J., Choi, BY, Wang, P., Gao, P. & Lee, Y. (2020). CrABCA2 facilitates triacylglycerol accumulation in Chlamydomonas reinhardtii under nitrogen starvation. Molecules and cells, 43(1), 48-57. (Song, I et al., (2022)., Microbial cell factories,)).

[0156]

[0157] 4-1 Cultivation of mutants in nitrogen-depleted medium

[0158] [Table 3]

[0159]

[0160] To compare the differences between the mutant strains of the present invention (agp, WT / BKT1, WT / BKT2, agp / BKT_1) and the wild type (WT) of Chlamydomonas reinhardtii, a culture experiment was conducted using TAP medium under nitrogen-deficient conditions. In the case of nitrogen-deficient conditions, 15 mM KCl was used instead of 15 mM NH4Cl in the conventional TAP medium.

[0161]

[0162] 4-2. Growth curve

[0163] When the growth curves (cells / ml) of the wild type (CC4349) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii were compared under nitrogen-deficient conditions, it was seen that the BKT overexpressing strains did not show a significant difference compared to the WT or agp strains [Fig. 10a].

[0164]

[0165] 4-3 Biomass production

[0166] When comparing the biomass of the wild type (WT) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii, the agp mutant has a reduced biomass compared to the WT. While the biomass of WT / BKT is reduced compared to the WT, the biomass of agp / BKT_1 is almost the same as that of the agp mutant [Fig. 10b].

[0167]

[0168] 4-4 Keto-carotenoid production analysis

[0169] When the contents of astaxanthin and canthaxanthin of wild type (WT) and mutant strains (agp, WT / BKT1, WT / BKT2, agp / BKT_1) of Chlamydomonas reinhardtii in nitrogen-deficient TAP medium on the 4th day were compared, the strain that did not express BKT did not produce astaxanthin. In the case of the strain that expressed BKT, astaxanthin had 0.59~0.61 mg / L (WT / BKT) and 0.56 mg / L (agp / BKT_1), respectively. Canthaxanthin had 1.93~2.00 mg / L (WT / BKT) and 2.32 mg / L (agp / BKT_1), respectively.

[0170] When these were converted to content per biomass, astaxanthin had 0.66–0.68 mg / g (WT / BKT) and 0.83 mg / g (agp / BKT_1), respectively [Fig. 11a]. Cantaxanthin had 2.16–2.24 mg / g (WT / BKT) and 3.42 mg / g (agp / BKT_1), respectively [Fig. 11b]. When the content of astaxanthin and cantaxanthin per total carotenoid was confirmed, the total carotenoid had values ​​of 3 mg / L (WT / BKT_1), 3.4 mg / L (WT / BKT_2), and 3.6 mg / L (WT / BKT), respectively [Fig. 12a]. Among these, astaxanthin accounts for 0.17~0.22 mg / L (WT / BKT) and 0.15~0.17 mg / L (agp / BKT_1) among all carotenoids [Fig. 12b], and cantaxanthin accounts for 0.48~0.75 mg / L (WT / BKT) and 0.53~0.95 mg / L (agp / BKT_1) [Fig. 12c].

[0171]

[0172] 4-5 Lipid content analysis

[0173] To compare the differences in lipid content between the mutant strains of the present invention and the wild-type strain, total lipid content was determined using the Bligh and dyer method. Cells were grown under nitrogen starvation for 4 days to induce lipid induction. The supernatant was removed from the precipitated cells using a centrifuge and resuspended in 4 ml of deionized water (DW). 10 ml of methanol and 5 ml of chloroform were added to obtain a water:methanol:chloroform ratio of 4:10:5. After reacting for approximately 5 minutes, 5 ml of deionized water and 5 ml of chloroform were added to obtain a water:methanol:chloroform volume ratio of 9:10:10. The extract was centrifuged at 3200 rpm for 10 minutes to separate into two layers. The chloroform layer containing the lipid was transferred to a pre-weighed aluminum foil dish and evaporated using a hot plate. The lipid content was then determined using the difference in weight before and after evaporation.

[0174] Figure 13 shows the lipid content per weight of the wild type (WT) and mutant strains (agp, WT / BKT_1, WT / BKT_2, agp / BKT_1) of Chlamydomonas reinhardtii under nitrogen starvation conditions on the 4th day. WT has a lipid content of 0.33 g / g, while the agp mutant has a lipid content of 0.66 g / g. The strains expressing BKT have 0.16–0.26 g / g (WT / BKT) and 0.40 g / g (agp / BKT_1), respectively. agp / BKT_1 has about twice as much lipid as WT / BKT, but has a lower lipid content than the existing agp mutant [Figure 13].

[0175]

[0176]

[0177] [Accession number]

[0178] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0179] Accession number: KCTC15839BP

[0180] [Correction pursuant to Article 91 of the Rules 11.06.2025] Date of acceptance: 20240228

Claims

1. Chlamydomonas reinhardtiiagp / BKT mutant 1(a / B_1) strain with improved keto-carotenoid production ability, accession number KCTC 15839BP.

2. In paragraph 1, Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain, wherein the above keto-carotenoid is at least one selected from the group consisting of astaxanthin and cantaxanthin.

3. Culture of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain with accession number KCTC 15839BP.

4. A pigment composition comprising at least one selected from the group consisting of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having accession number KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

5. In paragraph 4, A pigment composition comprising 1 to 10 parts by weight of astaxanthin per 100 parts by weight of total carotenoid pigment.

6. In paragraph 4, A pigment composition comprising 5 to 15 parts by weight of canthaxanthin per 100 parts by weight of total carotenoid pigment.

7. A composition for oral administration comprising at least one selected from the group consisting of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) of accession number KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

8. A composition for food or food additive comprising at least one selected from the group consisting of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having accession number KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

9. A composition for feed or feed additive comprising at least one selected from the group consisting of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having accession number KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

10. A health functional food for antioxidant activity, strengthening immunity, improving exercise capacity, strengthening or maintaining eye health, and preventing or improving eye function decline, comprising at least one selected from the group consisting of Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain with accession number KCTC 15839BP, a culture thereof, a dried product thereof, and an extract thereof.

11. A pigment production method comprising culturing a Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having the accession number KCTC 15839BP.

12. In paragraph 11, A method for producing a pigment, comprising extracting a pigment from the Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain or a culture thereof.

13. In paragraph 11 or 12, A method for producing a pigment, wherein the pigment comprises at least one selected from the group consisting of astaxanthin and cantaxanthin.

14. A method for producing food raw materials, comprising culturing a Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having the accession number KCTC 15839BP.

15. A method for producing feed raw materials, comprising culturing a Chlamydomonas reinhardtii agp / BKT_1 mutant 1 (a / B_1) strain having the accession number KCTC 15839BP.

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