Marine microorganism cellulophaga omnivescoria MSK1 strain producing zeaxanthin and method for producing zeaxanthin from biomass using same

Abstract

The present invention provides the Cellulophaga omnivescoria MSK1 strain, which biosynthesizes the carotenoid zeaxanthin, and relates to a method for producing zeaxanthin from agarose, which is a substance extractable from biomass, by culturing the strain and utilizing same. The zeaxanthin produced by the novel MSK1 strain provided in the present invention can be sustainably produced, and thus can be widely applied as a natural antioxidant material for health supplements, functional cosmetics, and pharmaceuticals. In addition, the MSK1 strain can serve as a fundamental bio-industrial material for the continuous supply and commercialization of zeaxanthin.

Description

Cellulophaga Omnibus Korea MSK1 (CELLULOPHAGA OMNIVESCORIA MSK1) strain, a marine microorganism producing zeaxanthin, and a method for producing zeaxanthin from biomass using the strain.

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to Patent Application No. 10-2023-0110698, filed August 23, 2023, and Patent Application No. 10-2024-0112810, filed August 22, 2023, the entire contents of each of which are incorporated herein by reference.

[0003]

[0004] Sequence list information

[0005] The sequence listing associated with this application is provided in XML format instead of a paper copy and is incorporated herein by reference. The XML file containing the sequence listing is named CELLULOPHAGA OMNIVESCORIA MSK1 0819.xml. The XML file is 9,733 bytes in size, was created on August 19, 2023, and was submitted electronically via PatentCenter.

[0006]

[0007] Technology field

[0008] The present invention relates to a Cellulophaga omnivescoriaMSK1 strain having zeaxanthin production ability and a method for producing zeaxanthin from biomass using the strain.

[0009]

[0010] Carotenoids are natural pigments synthesized in plants, fungi, algae, and bacteria, and are also antioxidants. Carotenoids are classified by the number of skeletal carbon atoms in their structure. 30 , C 40 , C 50Carotenoids are classified into a family, and about 700 types of carotenoids have been discovered to date. Specifically, carotenoids are classified into carotene and xanthophyll carotenoids depending on the presence or absence of an oxygenated functional group. Representative xanthophyll carotenoids currently used commercially include astaxanthin, lutein, and zeaxanthin, and they are known to have effective functions in human diseases and health promotion, such as antioxidant, anticancer, and anti-inflammation. Among these, zeaxanthin, which is mainly synthesized in plants, plays an important role in the xanthophyll circulation and is known to be effective in promoting eye health, such as preventing and improving cataracts and macular degeneration, and its demand as a health food is gradually increasing. However, as consumer concerns about secondary pollution, side effects, and harmfulness of chemically synthesized zeaxanthin increase, interest in zeaxanthin biosynthesis is further increasing.

[0011] For this reason, active research has recently been conducted on plants that synthesize zeaxanthin, zeaxanthin-producing microorganisms, or through genetic engineering. While zeaxanthin can be obtained through plant synthesis from paprika and marigold, it suffers from slow growth rates and low zeaxanthin yields. In particular, plant extracts exhibit significant variation in the amount of pigment extracted, depending on the variety, climate, cultivation conditions, and harvesting method.

[0012] In addition, in the case of zeaxanthin production by microorganisms into which recombinant plasmids have been inserted as engineering, there are problems that make industrial application difficult due to reasons such as the use of antibiotics for screening recombinant insertions, decreased yield due to plasmid detachment, and increased costs due to additional processes.

[0013] Therefore, there have been attempts to find microorganisms that are relatively less affected by the environment, grow quickly, and have the activity to produce zeaxanthin at a stable and constant yield, and to develop a method for producing zeaxanthin using such microorganisms (Public Patent No. 10-2023-0091552). Specific examples of microorganisms with zeaxanthin-producing activity include Zeaxanthinibacter enoshimensis TD-ZE3, Hyunsoonleella jejuensis, Kordia aquimaris CC-AMZ-301, Aquibacter zeaxanthinifaciens CC-AMZ-304, Gramella oceani CC-AMSZ-T, all within the Flavobacteriaceae genus, and Nubsella zeaxanthinifaciens TDMA-5, Sphingomonas jaspsi TDMA-16, all within the Sphingomonadaceae genus, which have been reported to produce zeaxanthin. In addition, some microorganisms within the Flavobacteriaceae genus have the ability to decompose and utilize agarose, which can be extracted from sustainably produced biomass.

[0014] Biomass is an alternative energy source that can alleviate concerns about environmental pollution caused by excessive dependence on fossil fuels. It refers to biological organisms that include plants and microorganisms that receive solar energy and produce photosynthesis, as well as bacteria and animals that eat them. Agar (seaweed) and agarose, the main component of agar, also fall into this category.

[0015] The discovery of microorganisms capable of producing zeaxanthin and decomposing substances extractable from biomass is expected to be very advantageous in terms of stable production and utilization of zeaxanthin, which has useful applications in various industrial fields such as food and medicine, as it enables biological conversion of sustainable substances into zeaxanthin without environmental pollution concerns.

[0016] Accordingly, the inventors of the present invention have made efforts to produce high value-added zeaxanthin from biomass, and as a result, they have found that a novel marine microorganism, Cellulophaga omnivescoriaMSK1 strain, has excellent agarose decomposition ability, and thus, when the strain is cultured in an agarose culture medium, it secretes an agarose-decomposing protein to convert agarose into a monosaccharide, and using this monosaccharide as a carbon source, zeaxanthin can be produced in a high yield within a short period of time, thereby completing the present invention.

[0017]

[0018] The main object of the present invention is to provide a novel microorganism that produces zeaxanthin and decomposes agarose extractable from biomass, and specifically, to provide a strain of Cellulophaga omnivescoria MSK1 that produces zeaxanthin, a carotenoid. Another object of the present invention is to provide a method for sustainably producing zeaxanthin using a culture of the strain, a lysate of the strain, a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof.

[0019]

[0020] The present invention provides a novel Cellulophaga omnivescoriaMSK1 strain.

[0021] In addition, the present invention provides a method for producing zeaxanthin using a culture of Cellulophaga omnivescoriaMSK1 strain, a lysate of the strain, a dried product of the culture and / or lysate of the strain, or an extract thereof.

[0022] In addition, the present invention provides a composition comprising, as an active ingredient, a culture of Cellulophaga omnivescoriaMSK1 strain, a lysate of the strain, a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof.

[0023]

[0024] The novel zeaxanthin-producing strain MSK1 of the present invention possesses excellent zeaxanthin production capacity and a highly active agarase protein secreted by the strain outside the cell. This allows for growth using marine biomass, such as red algae and agar, as a carbon source, thereby enabling sustainable zeaxanthin production, leading to stable zeaxanthin production and reduced production costs. It is expected to be utilized in high value-added industries such as food, cosmetics, and pharmaceuticals. Furthermore, it can realize biorefinery and carbon-neutral values.

[0025]

[0026] Figure 1 is a strain of Cellulophaga omnivescoriaMSK1 cultured purely on a marine agar medium.

[0027] Figure 2 is a diagram showing a taxonomic phylogenetic tree of the Cellulophaga omnivescoriaMSK1 strain.

[0028] Figure 3a is a graph showing the results of UV-Visible spectrum analysis of zeaxanthin extracted from Cellulophaga omnivescoriaMSK1 strain.

[0029] Figure 3b is a graph showing the HPLC analysis results of an extract extracted using methanol from the Cellulophaga omnivescoriaMSK1 strain and a zeaxanthin standard solution.

[0030] Figure 3c is a graph showing the results of LC-MS analysis of zeaxanthin extracted from Cellulophaga omnivescoriaMSK1 strain.

[0031] Figure 4 is a graph showing the growth curve of Cellulophaga omnivescoriaMSK1 when agarose was used as the sole carbon source.

[0032] Figure 5a is a graph showing the phylogenetic tree of agarose decomposition protein (Aga3) isolated from Cellulophaga omnivescoriaMSK1 strain.

[0033] Figure 5b is a diagram comparing the amino acid sequences of agarose decomposition protein (Aga3) isolated from Cellulophaga omnivescoriaMSK1 strain and agarases of other microorganisms with high homology to Aga3.

[0034] Figure 6a is a picture showing the purified agarose-degrading protein (Aga3) isolated from Cellulophaga omnivescoriaMSK1 strain and compared with a protein standard solution.

[0035] Figure 6b is a graph showing the agarose-degrading activity of an agarose-degrading protein (Aga3) isolated from the strain Cellulophaga omnivescoriaMSK1 over time. An enzyme unit (U) was defined as the release of 1 μmol of D-galactose per minute.

[0036] Figure 6c shows the results of thin-layer chromatography (TLC) that confirmed the final degradation product of the agarose degradation protein (Aga3) isolated from the strain Cellulophaga omnivescoriaMSK1. The reaction products of the agarose degradation protein (Aga3) and 0.2% agarose with the standards of neoagarobios (NA2), neoagarotetetrose (NA4), and neoagarohexaose (NA6) are shown on TLC.

[0037]

[0038] Hereinafter, the present invention will be described in detail.

[0039] The present invention provides a strain of Cellulophaga omnivescoriaMSK1 (hereinafter referred to as MSK1 strain) having zeaxanthin production ability.

[0040] The MSK1 strain of the present invention was confirmed to have approximately 99% homology with the Cellulophaga omnivescoria strain as a result of 16S rRNA base sequence analysis and phylogenetic tree analysis, and is a novel microorganism belonging to the genus Cellulophaga. The MSK1 strain of the present invention has a 16S rRNA base sequence described as SEQ ID NO: 3.

[0041] The MSK1 strain of the present invention is a strain deposited with the Microbial Bank of the National Institute of Agricultural Sciences, Rural Development Administration, on August 1, 2023, under the accession number KACC 81269BP.

[0042] The MSK1 strain of the present invention is a strain that exhibits the activity of biosynthesizing xanthophylls zeaxanthin.

[0043] The MSK1 strain of the present invention can provide a novel Aga3 agarase, i.e., a protein having agarose-decomposing activity, which decomposes agarose into neoagarotetrose and neoagarobiose. The Aga3 agarase has a DNA base sequence represented by SEQ ID NO: 4 and an amino acid sequence represented by SEQ ID NO: 5.

[0044] The MSK1 strain of the present invention can grow using agarose as the sole carbon source and simultaneously produce zeaxanthin.

[0045] The MSK1 strain of the present invention has excellent zeaxanthin production ability.

[0046] As used herein, the term 'zeaxanthin' is named 4-[18-(4-hydroxy-2,6,6-trimethyl-1-cyclohexenyl)-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethyl-cyclohex-3-en-1-ol (IUPAC nomenclature), C 40 H 56 Refers to a compound represented by the chemical formula O2.

[0047] Zeaxanthin is an oxycarotenoid pigment produced by the oxygenation of carotenoids and belongs to the xanthophylls. While plants and some microorganisms synthesize zeaxanthin, animals cannot produce it directly and must obtain it through food.

[0048] Zeaxanthin is a component of the yellow substance (macular pigment) found in animals, including the macula, fat, skin, and egg yolk. Regular intake of zeaxanthin and macular pigment can help prevent or alleviate the symptoms of macular degeneration, a disease characterized by degeneration of the macula, the central nerve tissue of the retina, causing vision impairment.

[0049] The MSK1 strain of the present invention is a strain that can be cultured in a medium containing agarose. During the culture process, agarose can be the sole carbon source.

[0050] As used herein, the term 'agarose' is a polysaccharide contained in large quantities in marine algae, and is a linear polysaccharide composed of a β-galactopyranose residue bonded at the 1,3 position and a 3,6-anhydro-α-L-galactopyranose residue bonded at the 1,4 position.

[0051] Agarose is one of the main components of agar, and is purified from agar by removing agaropectin, another main component of agar.

[0052] Agarose is mainly used as a carbon source in cell culture media, and can be used in the form of a hydrogel because the hardness of the structure can be easily controlled.

[0053] In the present invention, agarose may be obtained from Lonza Company located in Rockland, USA (Catalog No.: 50004), but is not limited thereto.

[0054] The present invention provides a method for producing zeaxanthin, comprising: a step of culturing an MSK1 strain isolated from seawater in a medium; a step of extracting zeaxanthin from the MSK1 strain obtained by the culturing, a culture of the strain, a lysate of the strain, or any one selected from the strain, the culture, and the lysate, or an extract thereof.

[0055] For example, the MSK1 strain of the present invention is inoculated into Marine broth or Minimal Marine broth containing 50 ml of 0.2% agarose, stirred for 48 hours under conditions of 30°C and 200 rpm to recover cells, and 5 ml of methanol is added to the obtained cells after lyophilization and zeaxanthin can be completely extracted from the cells by sonication.

[0056] The present invention provides a culture of the MSK1 strain containing zeaxanthin biosynthesized within the strain, a lysate of the strain, or a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof.

[0057] The culture of the strain of the present invention refers to a material obtained by culturing the novel strain of the present invention in a known medium, and is a concept that includes the Cellulophaga Omnibus Korea MSK1 strain.

[0058] The lysate of the strain of the present invention may be a product obtained by lysing the strain itself by chemical or physical force.

[0059] The extract of the strain of the present invention refers to a product containing zeaxanthin as an indicator component obtained by extracting the strain itself, a dead strain, a lysate of the strain, a culture of the strain, or a dried product of each or a mixture of the strain, the dead strain, the lysate, and / or the culture using an appropriate extraction solvent, temperature, and number of times, and also includes a material that can be obtained by processing or treating it by other methods such as fractionation or concentration after extraction.

[0060] The composition of the present invention can be formulated as a pharmaceutical composition, and can be prepared using a pharmaceutically acceptable carrier according to a method easily performed by a person of ordinary skill in the art. The pharmaceutically acceptable carrier includes, but is not limited to, water, cellulose, phosphoric acid, calcium, gelatin, calcium silicate, flavinylpyrrolidone, sorbitol, mannitol, propylhydroxybenzoate, and mineral oil.

[0061] The above pharmaceutical composition can be applied to the prevention or treatment of ophthalmic diseases such as glaucoma, cataract, macular degeneration, dry eye, and low vision.

[0062] The above pharmaceutical composition may be formulated as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs for oral administration, but is not limited thereto.

[0063] In addition, the above composition may be formulated into a liquid dosage form suitable for parenteral administration such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc., but is not limited thereto.

[0064] The dosage of the above pharmaceutical composition may be prescribed in various ways depending on factors such as the type of disease of the patient, severity of the disease, type of formulation, age, sex, weight, health condition, diet, administration time and method of the pharmaceutical therapeutic composition, administration route, excretion rate, and response sensitivity of the patient, and is 0.001 to 100 mg / kg per day for adults.

[0065] The composition of the present invention can be manufactured as a health functional food composition by including conventional food additives. The additives include, but are not limited to, glycine, nicotinic acid, potassium citrate, cinnamic acid, and the like.

[0066] The food composition of the present invention may contain 0.1 to 99.9 wt% of the extract of the present invention as an effective ingredient based on the total weight of the composition. In addition, the composition of the present invention may additionally contain one or more effective ingredients exhibiting the same or similar function in addition to the above effective ingredients.

[0067] The above food composition may be formulated in the form of tablets, capsules, powders, granules, liquids, pills, etc., but is not limited thereto.

[0068]

[0069] According to specific examples and experimental examples of the present invention, the inventors first identified a single species of microorganism having a yellow pigment (carotenoid pigment) from marine microorganisms, and then analyzed the 16s rRNA base sequence to confirm that Cellulophaga is a novel strain belonging to Omnibus Korea (Figs. 1 and 2), and named Cellulophaga Omnibus Korea MSK1 strain.

[0070] A single colony of the selected MSK1 strain was inoculated into Marine broth and cultured, and the pigment was completely extracted from the obtained strain. The UV spectrum of the pigment extract obtained first was examined, and it was confirmed that the highest absorption was at a wavelength of 450 nm, which was the same as that of the general yellow zeaxanthin standard substance (Fig. 3a). HPLC analysis of the mobile phase of the secondarily obtained pigment extract was performed, and it was confirmed that the retention time and peak were consistent with those of the zeaxanthin mobile phase (Fig. 3b). In addition, the molecular weight of the extract was determined by LC-MS analysis of the obtained pigment extract, and C 40 H 56 It was found that it was zeaxanthin with the chemical formula O2 (Fig. 3c).

[0071] Then, the MSK1 strain was inoculated and cultured in a minimal medium (Minimal marine broth) containing agarose as the sole carbon source, and the growth pattern and zeaxanthin production amount were observed. As a result of the observation, it was confirmed that the MSK1 strain can be cultured in a minimal medium containing only agarose as a sole carbon source, and that it produces zeaxanthin from agarose as a sole carbon source (Fig. 4).

[0072] In order to more clearly elucidate the agarose decomposition activity exhibited by the MSK1 strain, the present inventors analyzed the amino acid sequence of the agarose decomposition protein (referred to as Aga3 agarase) secreted by the MSK1 strain, and as a result, the Aga3 agarase has a sequence similar to that of Cellulophaga lytica beta-agarase and an amino acid sequence (Glu) commonly possessed by Glycoside hydrolase family 16 (GH16). 175 , Asp 177 , Glu 180 ) was confirmed to belong to the GH16 family (Fig. 5a).

[0073] Additionally, through the Aga3 purification and activity experiments conducted, it was confirmed that Aga3 agarase can decompose agarose into polysaccharide forms, namely neoagarohexaose, neoagarotetraose, and neoagarobiose (Fig. 6c).

[0074]

[0075] Hereinafter, the process leading to the present invention will be described in detail. However, the following examples are intended only to illustrate the present invention and are not intended to limit the present invention.

[0076]

[0077] <Example 1> Isolation and identification of microorganisms with zeaxanthin biosynthetic ability from seawater samples

[0078] 1-1. Pure culture and base sequence analysis of pigment-producing marine microorganisms

[0079] First, 37.35 g of Marine broth (Kisan Bio, MB-M1512) and 15 g of Bacto agar (BD, France) were dissolved in 1 ℓ of purified water to prepare a Marine agar medium, which was then autoclaved at 121℃ for 15 minutes. 100 ㎕ of the diluted solution was spread on the prepared medium, diluted with sterilized saline solution, and cultured at 30℃ for 3 days. After culture, only yellow colonies were selected from among the colonies that grew, and passaged 2 to 3 times on new Marine agar medium to isolate only a single species of microorganism with yellow pigment (Fig. 1).

[0080] Afterwards, in order to identify the microorganism obtained in the above process, the DNA of the purely cultured microorganism was extracted and PCR was performed using 16s universal primers (SEQ ID NO: 1 - forward; and SEQ ID NO: 2 - reverse), and the 16s rRNA base sequence of approximately 1.5 kb obtained was analyzed (SEQ ID NO: 3).

[0081] The 16s rRNA sequence obtained from the above process was registered in NCBI GenBank (GenBank accession number: GCA_027915275.1). The species of the microorganism was identified using the NCBI BLAST program, and through phylogenetic analysis using the Mega 7.0 program (Fig. 2), the yellow-pigmented microorganism isolated from the seawater off the coast of Jeju was named Cellulophaga omnivescoriaMSK1.

[0082] The present inventors deposited the Cellulophaga omnivescoriaMSK1 strain producing the above-mentioned zeaxanthin to the Microbial Bank of the National Institute of Agricultural Sciences, Rural Development Administration, and were assigned the accession number KACC 81269BP.

[0083]

[0084] 1-2. Extraction of zeaxanthin biosynthesized by MSK1 strain

[0085] A single colony of the MSK1 strain obtained in Example 1-1 was cultured in 3 ml of Marine broth with stirring at 200 rpm for 24 hours. After culture, 1% of the culture solution of the MSK1 strain was reinoculated into a fresh 50 ml of Marine broth and cultured with stirring at 30°C and 200 rpm for 24 hours.

[0086] After culturing, the culture medium was centrifuged for 10 minutes at 4°C and 10,000 rpm to recover the cells, and 5 ml of methanol was added to the strain obtained after lyophilization, and the pigment was completely extracted from the strain through sonication. The obtained extract was filtered using a filter paper with a pore size of 0.25 μm to obtain a pure pigment extract from which cell debris was finally removed.

[0087]

[0088] 1-3. Analysis of zeaxanthin in MSK1 using UV spectrum, high-performance liquid chromatography (HPLC), and LC-MS analysis.

[0089] First, the UV spectrum was investigated for the pigment extract obtained in Example 1-2.

[0090] Specifically, 200 μL of the extract was placed in a 96-well clear plate and scanned using a microplate reader (Biotek, Winooski, VA, USA) at wavelengths between 300 nm and 700 nm. As a result, the extract was confirmed to have the highest absorption at a wavelength of 450 nm, similar to that of a typical yellow zeaxanthin standard substance (Fig. 3a).

[0091] Afterwards, the column for the separation of the extract was Zorbax Eclipse XDB-C18 5 ㎛ (4.6 x 150 mm), and the column temperature was set to 35℃, the flow rate to 1 ㎖ / min, and the UV wavelength to 450 nm, and the analysis was conducted for 5 minutes. 5 ㎕ of the prepared extract was injected. The mobile phase used was a mixture of acetonitrile, methanol, and isopropanol in a ratio of 40:50:10.

[0092] As a result, as shown in Fig. 3b, it was confirmed that the pigment extract of MSK1 detected peaks at retention times of approximately 1.5 min and 2 min, respectively. In particular, it was confirmed that the highest peak was detected at a retention time of 1.5 min, and when compared to the zeaxanthin standard substance, it was confirmed that it was zeaxanthin as the retention time and peak shape were the same.

[0093] Afterwards, the molecular weight of the extract was determined by LC-MS analysis of the pigment extract of the MSK1 strain. The ion spectrum analysis results confirmed that it was detected at 568.4269 m / z [M] and 569.4327 m / z [M+H], and the result was C 40 H 56 It was found that it was zeaxanthin with the chemical formula O2 (Fig. 3c).

[0094] The above results confirmed that Cellulophaga omnivescoriaMSK1 can produce zeaxanthin in a short period of time, thus having high economic feasibility.

[0095]

[0096] <Example 2> Production of Cellulophaga omnivescoriaMSK1 strain using agarose and production of zeaxanthin

[0097] In order to obtain zeaxanthin from agarose using the purely isolated MSK1 strain in Example 1, 200 mg / L agarose was added as the sole carbon source to Minimal Marine broth (23 g / L Instant Ocean Sea salt, 1 g / L Yeast extract, 50 mM Tris-HCl buffer, 0.5 g / L NaCl2) without a carbon source, and then the MSK1 strain solution cultured for 24 hours was inoculated to 1% (v / v) and the growth pattern of the MSK1 strain was observed at an absorbance of 600 nm while stirring and culturing for 24 hours under the conditions of 30°C and 200 rpm.

[0098] As a result of observation, the maximum OD value in Minimal Marine broth containing agarose was 0.48 after 18 hours of incubation, and the maximum zeaxanthin production was 2.22 mg / L after 42 hours of incubation. Therefore, it was confirmed that zeaxanthin could be produced from agarose extractable from biomass using the MSK1 strain that can be cultured in a minimal medium containing only agarose as a single carbon source (Fig. 4).

[0099]

[0100] <Example 3> Identification and purification of agarose-degrading protein derived from MSK1 strain

[0101] The protein exhibiting agarose decomposition activity secreted by the MSK1 strain Cellulophaga omnivescoria was identified and purified through expression in Escherichia coli [Escherichia coliBL21(DE3)]. The amino acid sequence of the purified protein was analyzed and confirmed to have the amino acid sequence of SEQ ID NO: 5. This sequence was confirmed to be a novel sequence and similar to Cellulophaga lyticabeta-agarase (Fig. 5a), and also has an amino acid sequence common to Glycoside hydrolase family 16 (Glu 175 , Asp177 , Glu 180 ) was confirmed to contain it (Fig. 5b). Accordingly, the protein with agarose decomposition activity isolated from the MSK1 strain was identified as Aga3 agarase, and it was also confirmed that the agarase belongs to the GH16 family.

[0102]

[0103] 3-2. Purification of agarose-degraded proteins derived from MSK1 strain

[0104] To purify Aga3 agarase from MSK1 strain, the base sequence region of Aga3 was denatured at 95°C for 5 minutes using primers (SEQ ID NO: 6-forward; and SEQ ID NO: 7-reverse), followed by 30 cycles of binding at 60°C for 30 seconds and extension at 72°C for 2 minutes, and preservation reaction at 72°C for 5 minutes, to obtain 936 bp Aga3 base sequence (SEQ ID NO: 4).

[0105] To introduce and express the amplified Aga3 DNA into E. coli, the Aga3 DNA fragment and the protein expression plasmid pET21a were reacted with restriction enzymes BamHI and XhoI at 37°C for 3 h to obtain the Aga3 DNA fragment and the pET21a fragment. The Aga3 DNA fragment and the pET21a fragment were ligated with T4 ligase (Enzynomics, South Korea) at room temperature for 1 h to produce the recombinant plasmid.

[0106] To construct a transformed E. coli strain expressing Aga3 protein, competent E. coli BL21(DE3) cells treated with CaCl2 were constructed. 1 μg of the ligated recombinant plasmid was added to 100 μl of BL21(DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 1 min, and then cultured at 37°C for 1 h. To obtain transformed E. coli, the cells were plated on LB medium (Luria-Bertani broth media) containing ampicillin antibiotics and cultured at 37°C for 1 day. Among the transformed E. coli strains generated in LB medium containing ampicillin antibiotics, only the strain containing Aga3 DNA was cultured in pure form again.

[0107] After seed culture of transformed E. coli expressing Aga3 protein in liquid medium containing Ampicillin antibiotic for one day, the transformed E. coli seed culture was added to 500 ml of LB liquid medium containing Ampicillin antibiotic at 1% of the total volume, and when the absorbance (optical density 600 nm) reached 0.4, 1 mM IPTG was treated and the transformed E. coli was cultured overnight at 16℃ to express the protein. After overnight, the culture was centrifuged at 13,000g for 10 min at 4℃ and washed with distilled water. The centrifuged culture was then resuspended in 5 ml of lysis buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 500 mM NaCl, pH 7.4) and the cells were disrupted using an ultrasonic analyzer with the conditions of power 30 s and pulse 30 s for 5 min. The disrupted cell fluid was centrifuged at 13,000 g for 10 min at 4°C to obtain only the supernatant. The obtained supernatant was purified using a Ni-NTA-agarose column (Qiagen, Germany) and imidazole. The purified protein was size-confirmed by SDS-PAGE, and it was confirmed that the actual size of the Aga3 protein matched the predicted size (Fig. 6a).

[0108] Purified Aga3 was dialyzed through Amicon Ultra Centrifugal filters (Millipore, Ireland) to remove imidazole and concentrate Aga3. The optimal activity condition of the concentrated Aga3 was determined by reacting with 0.2% agarose (v / v) in Na2HPO4 / citric acid buffer, pH 5, 50°C, and the activity of Aga3 over time was confirmed by a reducing sugar assay (3,5-dinitrosalic acid assay). Optimal activity appeared to be observed when reacted for 15 minutes (Fig. 6b). Finally, to confirm the decomposition products, 10 ㎕ of the reaction solution in which Aga3 and 0.2% agarose (v / v) were reacted in 50 ㎕ of Na2HPO4 / Citric acid buffer at 50℃ for 15 minutes was dispensed onto a thin-layer chromatography (TLC) plate, and then developed with a developing solution (n-butanol: ethanol: H2O = 3:1:1, v / v) and developed with 10% (v / v) sulfuric acid and ethanol, and heated at 121℃ for 5 minutes to visualize the spots on the TLC plate. In conclusion, it was confirmed that the standard substances neoagarohexaose, neoagarotetraose, and neoagarobiose appeared as the final decomposition products (Fig. 6c).

[0109] Aga3 purification and activity experiments confirmed that Cellulophaga omnivescoriaMSK1 can decompose the polysaccharide agarose into the polysaccharide forms neoagarohexaose, neoagarotetraose, and neoagarobiose, which can be easily decomposed into monosaccharides.

[0110] In conclusion, it was found that the Aga3 agarase of Cellulophaga omnivescoriaMSK1 plays a key role in efficiently decomposing agarose, enabling the microorganism to grow using agarose-derived monosaccharides as a carbon source and produce zeaxanthin.

[0111] Hereinafter, the present invention will be described in detail.

[0112] The present invention provides a strain of Cellulophaga omnivescoriaMSK1 (hereinafter referred to as MSK1 strain) having zeaxanthin production ability.

[0113] The MSK1 strain of the present invention was confirmed to have approximately 99% homology with the Cellulophaga omnivescoria strain as a result of 16S rRNA base sequence analysis and phylogenetic tree analysis, and is a novel microorganism belonging to the genus Cellulophaga. The MSK1 strain of the present invention has a 16S rRNA base sequence described as SEQ ID NO: 3.

[0114] The MSK1 strain of the present invention is a strain deposited with the Microbial Bank of the National Institute of Agricultural Sciences, Rural Development Administration, on August 1, 2023, under the accession number KACC 81269BP.

[0115] The MSK1 strain of the present invention is a strain that exhibits the activity of biosynthesizing xanthophylls zeaxanthin.

[0116] The MSK1 strain of the present invention can provide a novel Aga3 agarase, i.e., a protein having agarose-decomposing activity, which decomposes agarose into neoagarotetrose and neoagarobiose. The Aga3 agarase has a DNA base sequence represented by SEQ ID NO: 4 and an amino acid sequence represented by SEQ ID NO: 5.

[0117] The MSK1 strain of the present invention can grow using agarose as the sole carbon source and simultaneously produce zeaxanthin.

[0118] The MSK1 strain of the present invention has excellent zeaxanthin production ability.

[0119] As used herein, the term 'zeaxanthin' is named 4-[18-(4-hydroxy-2,6,6-trimethyl-1-cyclohexenyl)-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethyl-cyclohex-3-en-1-ol (IUPAC nomenclature), C 40 H 56 Refers to a compound represented by the chemical formula O2.

[0120] Zeaxanthin is an oxycarotenoid pigment produced by the oxygenation of carotenoids and belongs to the xanthophylls. While plants and some microorganisms synthesize zeaxanthin, animals cannot produce it directly and must obtain it through food.

[0121] Zeaxanthin is a component of the yellow substance (macular pigment) found in animals, including the macula, fat, skin, and egg yolk. Regular intake of zeaxanthin and macular pigment can help prevent or alleviate the symptoms of macular degeneration, a disease characterized by degeneration of the macula, the central nerve tissue of the retina, causing vision impairment.

[0122] The MSK1 strain of the present invention is a strain that can be cultured in a medium containing agarose. During the culture process, agarose can be the sole carbon source.

[0123] As used herein, the term 'agarose' is a polysaccharide contained in large quantities in marine algae, and is a linear polysaccharide composed of a β-galactopyranose residue bonded at the 1,3 position and a 3,6-anhydro-α-L-galactopyranose residue bonded at the 1,4 position.

[0124] Agarose is one of the main components of agar, and is purified from agar by removing agaropectin, another main component of agar.

[0125] Agarose is mainly used as a carbon source in cell culture media, and can be used in the form of a hydrogel because the hardness of the structure can be easily controlled.

[0126] In the present invention, agarose may be obtained from Lonza Company located in Rockland, USA (Catalog No.: 50004), but is not limited thereto.

[0127] The present invention provides a method for producing zeaxanthin, comprising: a step of culturing an MSK1 strain isolated from seawater in a medium; a step of extracting zeaxanthin from the MSK1 strain obtained by the culturing, a culture of the strain, a lysate of the strain, or any one selected from the strain, the culture, and the lysate, or an extract thereof.

[0128] For example, the MSK1 strain of the present invention is inoculated into Marine broth or Minimal Marine broth containing 50 ml of 0.2% agarose, stirred for 48 hours under conditions of 30°C and 200 rpm to recover cells, and 5 ml of methanol is added to the obtained cells after lyophilization and zeaxanthin can be completely extracted from the cells by sonication.

[0129] The present invention provides a culture of the MSK1 strain containing zeaxanthin biosynthesized within the strain, a lysate of the strain, or a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof.

[0130] The culture of the strain of the present invention refers to a material obtained by culturing the novel strain of the present invention in a known medium, and is a concept that includes the Cellulophaga Omnibus Korea MSK1 strain.

[0131] The lysate of the strain of the present invention may be a product obtained by lysing the strain itself by chemical or physical force.

[0132] The extract of the strain of the present invention refers to a product containing zeaxanthin as an indicator component obtained by extracting the strain itself, a dead strain, a lysate of the strain, a culture of the strain, or a dried product of each or a mixture of the strain, the dead strain, the lysate, and / or the culture using an appropriate extraction solvent, temperature, and number of times, and also includes a material that can be obtained by processing or treating it by other methods such as fractionation or concentration after extraction.

[0133] The composition of the present invention can be formulated as a pharmaceutical composition, and can be prepared using a pharmaceutically acceptable carrier according to a method easily performed by a person of ordinary skill in the art. The pharmaceutically acceptable carrier includes, but is not limited to, water, cellulose, phosphoric acid, calcium, gelatin, calcium silicate, flavinylpyrrolidone, sorbitol, mannitol, propylhydroxybenzoate, and mineral oil.

[0134] The above pharmaceutical composition can be applied to the prevention or treatment of ophthalmic diseases such as glaucoma, cataract, macular degeneration, dry eye, and low vision.

[0135] The above pharmaceutical composition may be formulated as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs for oral administration, but is not limited thereto.

[0136] In addition, the above composition may be formulated into a liquid dosage form suitable for parenteral administration such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc., but is not limited thereto.

[0137] The dosage of the above pharmaceutical composition may be prescribed in various ways depending on factors such as the type of disease of the patient, severity of the disease, type of formulation, age, sex, weight, health condition, diet, administration time and method of the pharmaceutical therapeutic composition, administration route, excretion rate, and response sensitivity of the patient, and is 0.001 to 100 mg / kg per day for adults.

[0138] The composition of the present invention can be manufactured as a health functional food composition by including conventional food additives. The additives include, but are not limited to, glycine, nicotinic acid, potassium citrate, cinnamic acid, and the like.

[0139] The food composition of the present invention may contain 0.1 to 99.9 wt% of the extract of the present invention as an effective ingredient based on the total weight of the composition. In addition, the composition of the present invention may additionally contain one or more effective ingredients exhibiting the same or similar function in addition to the above effective ingredients.

[0140] The above food composition may be formulated in the form of tablets, capsules, powders, granules, liquids, pills, etc., but is not limited thereto.

[0141]

[0142] According to specific examples and experimental examples of the present invention, the inventors first identified a single species of microorganism having a yellow pigment (carotenoid pigment) from marine microorganisms, and then analyzed the 16s rRNA base sequence to confirm that Cellulophaga is a novel strain belonging to Omnibus Korea (Figs. 1 and 2), and named Cellulophaga Omnibus Korea MSK1 strain.

[0143] A single colony of the selected MSK1 strain was inoculated into Marine broth and cultured, and the pigment was completely extracted from the obtained strain. The UV spectrum of the pigment extract obtained first was examined, and it was confirmed that the highest absorption was at a wavelength of 450 nm, which was the same as that of the general yellow zeaxanthin standard substance (Fig. 3a). HPLC analysis of the mobile phase of the secondarily obtained pigment extract was performed, and it was confirmed that the retention time and peak were consistent with those of the zeaxanthin mobile phase (Fig. 3b). In addition, the molecular weight of the extract was determined by LC-MS analysis of the obtained pigment extract, and C 40 H 56 It was found that it was zeaxanthin with the chemical formula O2 (Fig. 3c).

[0144] Then, the MSK1 strain was inoculated and cultured in a minimal medium (Minimal marine broth) containing agarose as the sole carbon source, and the growth pattern and zeaxanthin production amount were observed. As a result of the observation, it was confirmed that the MSK1 strain can be cultured in a minimal medium containing only agarose as a sole carbon source, and that it produces zeaxanthin from agarose as a sole carbon source (Fig. 4).

[0145] In order to more clearly elucidate the agarose decomposition activity exhibited by the MSK1 strain, the present inventors analyzed the amino acid sequence of the agarose decomposition protein (referred to as Aga3 agarase) secreted by the MSK1 strain, and as a result, the Aga3 agarase has a sequence similar to that of Cellulophaga lytica beta-agarase and an amino acid sequence (Glu) common to Glycoside hydrolase family 16 (GH16). 175 , Asp 177 , Glu 180 ) was confirmed to belong to the GH16 family (Fig. 5a).

[0146] Additionally, through the Aga3 purification and activity experiments conducted, it was confirmed that Aga3 agarase can decompose agarose into polysaccharide forms, namely neoagarohexaose, neoagarotetraose, and neoagarobiose (Fig. 6c).

[0147]

[0148] Hereinafter, the process leading to the present invention will be described in detail. However, the following examples are intended only to illustrate the present invention and are not intended to limit the present invention.

[0149]

[0150] <Example 1> Isolation and identification of microorganisms with zeaxanthin biosynthetic ability from seawater samples

[0151] 1-1. Pure culture and base sequence analysis of pigment-producing marine microorganisms

[0152] First, 37.35 g of Marine broth (Kisan Bio, MB-M1512) and 15 g of Bacto agar (BD, France) were dissolved in 1 ℓ of purified water to prepare a Marine agar medium, which was then autoclaved at 121℃ for 15 minutes. 100 ㎕ of the diluted solution was spread on the prepared medium, diluted with sterilized saline solution, and cultured at 30℃ for 3 days. After culture, only yellow colonies were selected from among the colonies that grew, and passaged 2 to 3 times on new Marine agar medium to isolate only a single species of microorganism with yellow pigment (Fig. 1).

[0153] Afterwards, in order to identify the microorganism obtained in the above process, the DNA of the purely cultured microorganism was extracted and PCR was performed using 16s universal primers (SEQ ID NO: 1 - forward; and SEQ ID NO: 2 - reverse), and the 16s rRNA base sequence of approximately 1.5 kb obtained was analyzed (SEQ ID NO: 3).

[0154] The 16s rRNA sequence obtained from the above process was registered in NCBI GenBank (GenBank accession number: GCA_027915275.1). The species of the microorganism was identified using the NCBI BLAST program, and through phylogenetic analysis using the Mega 7.0 program (Fig. 2), the yellow-pigmented microorganism isolated from the seawater off the coast of Jeju was named Cellulophaga omnivescoriaMSK1.

[0155] The present inventors deposited the Cellulophaga omnivescoriaMSK1 strain producing the above-mentioned zeaxanthin to the Microbial Bank of the National Institute of Agricultural Sciences, Rural Development Administration, and were assigned the accession number KACC 81269BP.

[0156]

[0157] 1-2. Extraction of zeaxanthin biosynthesized by MSK1 strain

[0158] A single colony of the MSK1 strain obtained in Example 1-1 was cultured in 3 ml of Marine broth with stirring at 200 rpm for 24 hours. After culture, 1% of the culture solution of the MSK1 strain was reinoculated into a fresh 50 ml of Marine broth and cultured with stirring at 30°C and 200 rpm for 24 hours.

[0159] After culturing, the culture medium was centrifuged for 10 minutes at 4°C and 10,000 rpm to recover the cells, and 5 ml of methanol was added to the strain obtained after lyophilization, and the pigment was completely extracted from the strain through sonication. The obtained extract was filtered using a filter paper with a pore size of 0.25 μm to obtain a pure pigment extract from which cell debris was finally removed.

[0160]

[0161] 1-3. Analysis of zeaxanthin in MSK1 using UV spectrum, high-performance liquid chromatography (HPLC), and LC-MS analysis.

[0162] First, the UV spectrum was investigated for the pigment extract obtained in Example 1-2.

[0163] Specifically, 200 μL of the extract was placed in a 96-well clear plate and scanned using a microplate reader (Biotek, Winooski, VA, USA) at wavelengths between 300 nm and 700 nm. As a result, the extract was confirmed to have the highest absorption at a wavelength of 450 nm, similar to that of a typical yellow zeaxanthin standard substance (Fig. 3a).

[0164] Afterwards, the column for the separation of the extract was Zorbax Eclipse XDB-C18 5 ㎛ (4.6 x 150 mm), and the column temperature was set to 35℃, the flow rate to 1 ㎖ / min, and the UV wavelength to 450 nm, and the analysis was conducted for 5 minutes. 5 ㎕ of the prepared extract was injected. The mobile phase used was a mixture of acetonitrile, methanol, and isopropanol in a ratio of 40:50:10.

[0165] As a result, as shown in Fig. 3b, it was confirmed that the pigment extract of MSK1 detected peaks at retention times of approximately 1.5 min and 2 min, respectively. In particular, it was confirmed that the highest peak was detected at a retention time of 1.5 min, and when compared to the zeaxanthin standard substance, it was confirmed that it was zeaxanthin as the retention time and peak shape were the same.

[0166] Afterwards, the molecular weight of the extract was determined by LC-MS analysis of the pigment extract of the MSK1 strain. The ion spectrum analysis results confirmed that it was detected at 568.4269 m / z [M] and 569.4327 m / z [M+H], and the result was C 40 H 56 It was found that it was zeaxanthin with the chemical formula O2 (Fig. 3c).

[0167] The above results confirmed that Cellulophaga omnivescoriaMSK1 can produce zeaxanthin in a short period of time, thus having high economic feasibility.

[0168]

[0169] <Example 2> Production of Cellulophaga omnivescoriaMSK1 strain using agarose and production of zeaxanthin

[0170] In order to obtain zeaxanthin from agarose using the purely isolated MSK1 strain in Example 1, 200 mg / L agarose was added as the sole carbon source to Minimal Marine broth (23 g / L Instant Ocean Sea salt, 1 g / L Yeast extract, 50 mM Tris-HCl buffer, 0.5 g / L NaCl2) without a carbon source, and then the MSK1 strain solution cultured for 24 hours was inoculated to 1% (v / v) and the growth pattern of the MSK1 strain was observed at an absorbance of 600 nm while stirring and culturing for 24 hours under the conditions of 30°C and 200 rpm.

[0171] As a result of observation, the maximum OD value in Minimal Marine broth containing agarose was 0.48 after 18 hours of incubation, and the maximum zeaxanthin production was 2.22 mg / L after 42 hours of incubation. Therefore, it was confirmed that zeaxanthin could be produced from agarose extractable from biomass using the MSK1 strain that can be cultured in a minimal medium containing only agarose as a single carbon source (Fig. 4).

[0172]

[0173] <Example 3> Identification and purification of agarose-degrading protein derived from MSK1 strain

[0174] The protein exhibiting agarose decomposition activity secreted by the MSK1 strain Cellulophaga omnivescoria was identified and purified through expression in Escherichia coli [Escherichia coliBL21(DE3)]. The amino acid sequence of the purified protein was analyzed and confirmed to have the amino acid sequence of SEQ ID NO: 5. This sequence was confirmed to be a novel sequence and similar to Cellulophaga lyticabeta-agarase (Fig. 5a), and also has an amino acid sequence common to Glycoside hydrolase family 16 (Glu 175 , Asp177 , Glu 180 ) was confirmed to contain it (Fig. 5b). Accordingly, the protein with agarose decomposition activity isolated from the MSK1 strain was identified as Aga3 agarase, and it was also confirmed that the agarase belongs to the GH16 family.

[0175]

[0176] 3-2. Purification of agarose-degraded proteins derived from MSK1 strain

[0177] To purify Aga3 agarase from MSK1 strain, the base sequence region of Aga3 was denatured at 95°C for 5 minutes using primers (SEQ ID NO: 6-forward; and SEQ ID NO: 7-reverse), followed by 30 cycles of binding at 60°C for 30 seconds and extension at 72°C for 2 minutes, and preservation reaction at 72°C for 5 minutes, to obtain 936 bp Aga3 base sequence (SEQ ID NO: 4).

[0178] To introduce and express the amplified Aga3 DNA into E. coli, the Aga3 DNA fragment and the protein expression plasmid pET21a were reacted with restriction enzymes BamHI and XhoI at 37°C for 3 h to obtain the Aga3 DNA fragment and the pET21a fragment. The Aga3 DNA fragment and the pET21a fragment were ligated with T4 ligase (Enzynomics, South Korea) at room temperature for 1 h to produce the recombinant plasmid.

[0179] To construct a transformed E. coli strain expressing Aga3 protein, competent E. coli BL21(DE3) cells treated with CaCl2 were constructed. 1 μg of the ligated recombinant plasmid was added to 100 μl of BL21(DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 1 min, and then cultured at 37°C for 1 h. To obtain transformed E. coli, the cells were plated on LB medium (Luria-Bertani broth media) containing ampicillin antibiotics and cultured at 37°C for 1 day. Among the transformed E. coli strains generated in LB medium containing ampicillin antibiotics, only the strain containing Aga3 DNA was cultured in pure form again.

[0180] After seed culture of transformed E. coli expressing Aga3 protein in liquid medium containing Ampicillin antibiotic for one day, the transformed E. coli seed culture was added to 500 ml of LB liquid medium containing Ampicillin antibiotic at 1% of the total volume, and when the absorbance (optical density 600 nm) reached 0.4, 1 mM IPTG was treated and the transformed E. coli was cultured overnight at 16℃ to express the protein. After overnight, the culture was centrifuged at 13,000g for 10 min at 4℃ and washed with distilled water. The centrifuged culture was then resuspended in 5 ml of lysis buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 500 mM NaCl, pH 7.4) and the cells were disrupted using an ultrasonic analyzer with the conditions of power 30 s and pulse 30 s for 5 min. The disrupted cell fluid was centrifuged at 13,000 g for 10 min at 4°C to obtain only the supernatant. The obtained supernatant was purified using a Ni-NTA-agarose column (Qiagen, Germany) and imidazole. The purified protein was size-confirmed by SDS-PAGE, and it was confirmed that the actual size of the Aga3 protein matched the predicted size (Fig. 6a).

[0181] Purified Aga3 was dialyzed through Amicon Ultra Centrifugal filters (Millipore, Ireland) to remove imidazole and concentrate Aga3. The optimal activity condition of the concentrated Aga3 was determined by reacting with 0.2% agarose (v / v) in Na2HPO4 / citric acid buffer, pH 5, 50°C, and the activity of Aga3 over time was confirmed by a reducing sugar assay (3,5-dinitrosalic acid assay). Optimal activity appeared to be observed when reacted for 15 minutes (Fig. 6b). Finally, to confirm the decomposition products, 10 ㎕ of the reaction solution in which Aga3 and 0.2% agarose (v / v) were reacted in 50 ㎕ of Na2HPO4 / Citric acid buffer at 50℃ for 15 minutes was dispensed onto a thin-layer chromatography (TLC) plate, and then developed with a developing solution (n-butanol: ethanol: H2O = 3:1:1, v / v) and developed with 10% (v / v) sulfuric acid and ethanol, and heated at 121℃ for 5 minutes to visualize the spots on the TLC plate. In conclusion, it was confirmed that the standard substances neoagarohexaose, neoagarotetraose, and neoagarobiose appeared as the final decomposition products (Fig. 6c).

[0182] Aga3 purification and activity experiments confirmed that Cellulophaga omnivescoriaMSK1 can decompose the polysaccharide agarose into the polysaccharide forms neoagarohexaose, neoagarotetraose, and neoagarobiose, which can be easily decomposed into monosaccharides.

[0183] In conclusion, it was found that the Aga3 agarase of Cellulophaga omnivescoriaMSK1 plays a key role in efficiently decomposing agarose, enabling the microorganism to grow using agarose-derived monosaccharides as a carbon source and produce zeaxanthin.

[0184]

[0185]

[0186] Statement on Deposit

[0187] The inventor of the present invention deposited the strain Cellulophaga omnivescoriaMSK1 with the Microbial Bank (KACC) of the National Institute of Agricultural Sciences, Rural Development Administration on August 1, 2023, and was assigned the accession number KACC81269BP.

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[0200] Translation text

[0201] Budapest Convention on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (International Form)

[0202] Deposit certificate for original deposit

[0203] TO: Seoul Metropolitan University Industry-Academic Cooperation Foundation

[0204] 163 Seoulsiripdaero, Dongdaemun-gu, South Korea, 02504

[0205] I. Characteristics of the microorganism Specific designation given by the depositor Cellulophaga omnivescoria MSK1 Accession number given by the international depositary authority KACC 81269BP II. Scientific properties and / or taxonomic positions Microorganisms identified as I are described by 〔 〕 scientific properties 〔 〕 taxonomic positions (indicate where applicable). III. The international depositary authority deposited the microorganism identified as I on August 1, 2023. IV. Request for conversion of deposit The microorganism identified as I was deposited with the Microbial Bank of the National Institute of Agricultural Sciences, Rural Development Administration, an international depositary authority, and a request for conversion of the original deposit for the microorganism to a deposit under the Budapest Treaty has been received. V. Name of the international depository: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Address: 166 Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, Agricultural Microbiology Division, National Institute of Agricultural Sciences, Agricultural Biology Department, 55365 Signature of the person authorized to represent the international depository Date: August 16, 2023

Claims

1. A novel Cellulophaga omnivescoriaMSK1 strain having zeaxanthin production ability and comprising a 16S rRNA base sequence represented by sequence number 3.

2. In paragraph 1, The above strain is a strain deposited under the deposit number KACC 81269BP.

3. In paragraph 1, The above strain is a strain capable of producing zeaxanthin using agarose as a carbon source.

4. In paragraph 1, The strain is capable of being cultured and grown in a medium containing agarose as a single carbon source.

5. In paragraph 1, The strain comprises an Aga3 agarase exhibiting agarose decomposition activity having an amino acid sequence represented by sequence number 5.

6. In paragraph 5, A strain wherein the above agarose decomposition protein is encoded by a base sequence represented by sequence number 4.

7. A method for producing zeaxanthin, comprising: a step of extracting zeaxanthin from a culture of the Cellulophaga omnivescoriaMSK1 strain of paragraph 1, a lysate of the strain, or a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof.

8. A method for producing zeaxanthin, wherein the strain comprises an agarose decomposition protein having an amino acid sequence represented by sequence number 5.

9. A method for producing zeaxanthin, wherein the agarose decomposition protein in paragraph 8 is encoded by a base sequence represented by sequence number 4.

10. A composition comprising a culture of the Cellulophaga omnivescoriaMSK1 strain of paragraph 1, a lysate of the strain, a dried product of one or a mixture selected from the culture and lysate of the strain, or an extract thereof as an active ingredient.

11. A composition containing zeaxanthin obtained by the method of Article 7 as an active ingredient.

12. Aga3 agarase exhibiting agarose decomposition activity having an amino acid sequence represented by sequence number 5.

13. Aga3 agarase, which is encoded by a base sequence represented by sequence number 4 in the 12th paragraph.

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