KR101687726B1 - Novel carotinoid and use thereof - Google Patents

Abstract

The present invention relates to novel carotenoids and processes for their preparation. The present invention also relates to a composition for antioxidation comprising the carotenoid, and a natural coloring composition. The present invention also relates to an apo-carotenal wherein the novel carotenoid is an enzyme degradation product used as a substrate. The present invention also relates to a stem cell differentiation inducing composition comprising carotenoid as an active ingredient.
The novel carotene nodule of the present invention is a carotenoid having a novel structure using artificial molecular evolution and combinatorial biosynthesis which can not be produced in a conventional natural environment and can be usefully used as a novel antioxidant material.

Description

Novel carotinoids and uses thereof < RTI ID = 0.0 >

The present invention relates to novel carotenoids and processes for their preparation.

The present invention also relates to a composition for antioxidation comprising the carotenoid, and a natural coloring composition.

The present invention also relates to an apo-carotenal wherein the novel carotenoid is an enzyme degradation product used as a substrate.

The present invention also relates to a stem cell differentiation inducing composition comprising carotenoid as an active ingredient.

Isoprenoids are the most widely distributed class of secondary metabolites in nature, and are found in all species in a variety of forms including sterols, hormones, terpins, and quinones. They consist of a C5 material called isopentenyl diphosphate (IPP) as a unit element.

Carotenoids are a natural coloring matter expressed in nature, and are found in fruits, plants, fungi, microorganisms, etc., and its structure is found in various structures such as 700 kinds. Recently, they are attracting attention as a substance having high physiological activities such as anticancer and antioxidation, and they are attracting attention as a useful substance in various industries such as cosmetics and feed.

Microbial production of carotenoids, or metabolic engineering studies, has been around since the 1980s, focusing primarily on C40 carotenoids, and studies on C30 carotenoids are not yet active. Studies on the production of representative C40 carotenoids and C30 carotenoids via E. coli were carried out in 2003 by the team of Prof. Schmidt-dannert at the University of Minnesota. 5) Studies on the artificial molecular evolution of C30 carotenoids Schmidt-dannert and Professor Arnold of the California Institute of Technology. In these studies, however, the biosynthesis of the C30 carotenoid series, similar to the natural C40 carotenoids, was not achieved, and the production was also limited.

Carotenoids Cleavage Dioxygenase (CCD) is one of the carotenoid metabolizing enzymes found mainly in plants, cyanobacteria, and animals, and is sometimes found in microorganisms. The enzyme acts on natural carotene nodules (mainly beta carotene) to cleave carotenoids to produce retinal or other apo-carotenal. Previous studies on carotenoid cleavage enzymes have been conducted only on C40 carotenoids, and metabolism studies of C30 carotenoids, which are not common in nature, have not been conducted.

Therefore, the present inventors changed the activity of 4,4'-diapophytoene desaturase (CrtN) enzyme, which is important for the production of C30 carotenoid, through artificial molecular evolution, Here, an artificial molecular evolution of the C40 carotenoid activity enzyme lycopene cyclase (CrtY) is carried out, and 4,4'-diapotorulene (4,4'-diapotorulene) and 4,4'- The biosynthetic circuit of 4,4'-diapo-β-carotene was constructed and optimized. It is also possible to use it for various carotene modifying enzymes (lycopene elongase, CrtEb, beta-carotene ketolase, CrtO and CrtW, beta carotene hydroxylase-beta-carotene hydroxylase, CrtZ, Alpha-glucosyl transferase, CrtX, spheroidenone monooxygenase, CrtA, 4,4'-diaponurosporaldehyde dehydrogenase or 4,4'-diaponeurosporen-aldehyde dehydrogenase, AldH) Carotenoids of 14 new structures were biosynthesized in E. coli and identified by LC / MS analysis. In addition, these C30 carotenoids were reacted with one of the CCDs, NSC3 (an enzyme isolated from Nostoc sp. PCC 7120), to synthesize seven new apo-carotenal structures. In addition, the antioxidative activity of a novel carotenoid was confirmed, and the present invention was completed by confirming the ability of carotenoid to differentiate into neurons.

Application number: 1020057012813

One aspect of the present invention is to provide a novel carotenoid and a process for preparing the same.

Another aspect of the present invention is to provide a composition for antioxidation comprising the carotenoid, and a natural coloring composition.

In addition, an aspect of the present invention is to provide an apo-carotenal wherein the novel carotenoid is an enzyme degradation product which is used as a substrate.

Another aspect of the present invention is to provide a stem cell differentiation inducing composition comprising carotenoid as an active ingredient.

One aspect of the present invention provides novel carotenoids and processes for their preparation.

In the present invention, " carotenoid " is a representative coloring matter of nature, and is one of highly valuable substances such as an industrial food coloring additive, a medicinal material (anti-cancer, anti- inflammatory), a fragrance, and a fine chemical material.

The novel carotenoids provided in the present invention

4,4'-diaponeurosporen-6,6'-dione;

4,4'-diapolycopene-6,6'-dione;

7-hydroxy-8-keto-4,4'-diapotorulene;

4,4'-diapo-β-cryptoxanthin;

4,4'-diapozeaxanthin;

4,4'-diapoechinenone;

4,4'-diapo-beta-cryptoxanthin glucoside;

4,4'-diapotorulene-4'-al (4,4'-diapotorulene-4'-al); And

(4,4'-diapotorulene-4'-oic acid). The term " carotenoid "

The carotenoids according to the present invention can be produced by transforming E. coli with a vector comprising a polynucleotide encoding CrtM (diapophytoene synthase) and CrtN (diapophytoene desaturase).

The vector may further comprise a polynucleotide encoding preferably CrtY (lycopene cyclase).

In addition, the CrtN may be CrtN whose activity is changed through an artificial molecular evolution in addition to the wild type, and the CrtN can be preferably selected from the group consisting of SEQ ID NOS: 1-12.

Sequence information of CrtN of SEQ ID NOS: 1 to 12 is as follows.

The CrtY may be not only a wild-type but also CrtY whose activity has been changed through an artificial molecular evolution, and the CrtY may be preferably selected from the group consisting of SEQ ID NOS: 13 to 28.

The carotenoid may be selected from the group consisting of CrtZ, beta-carotene ketolase, CrtO, CrtX, CrtP, CrtP, further comprising the step of transforming E. coli into a vector comprising a polynucleotide encoding at least one enzyme selected from the group consisting of spheroidenone monooxygenase, CrtEb (Lycopene elongase), and AldH (diaponeurosporene-aldehyde dehydrogenase) .

In a preferred embodiment of the present invention, the 4,4'-diapophytoene desaturase (CrtN) enzyme, which is important for the production of C30 carotenoid, is activated through artificial molecular evolution , And an artificial molecular evolution of lycopene cyclase (CrtY), which is a C40 carotenoid enzyme, was carried out to produce 4,4'-diapotorulene, a C30 carotenoid, The biosynthetic circuit of 4,4'-diapo-β-carotene was constructed and optimized. It is also possible to use it for various carotene modifying enzymes (lycopene elongase, CrtEb, beta-carotene ketolase, CrtO and CrtW, beta carotene hydroxylase-beta-carotene hydroxylase, CrtZ, Alpha-glucosyl transferase, CrtX, spheroidenone monooxygenase, CrtA, 4,4'-diaponurosporaldehyde dehydrogenase or 4,4'-diaponeurosporen-aldehyde dehydrogenase, AldH) Carotenoids of 14 new structures were biosynthesized in E. coli and identified by LC / MS analysis.

Further, one aspect of the present invention provides a composition for antioxidation comprising the carotenoid.

The carotenoid as an active ingredient of the present invention exhibits a very high antioxidative effect and can be used as a pharmaceutical composition, a food composition, and a cosmetic composition.

The pharmaceutical composition may be used for cancer, inflammatory diseases, aging inhibition, fatigue, degenerative diseases, neurological diseases, cardiovascular diseases and the like.

Non-limiting examples of such cancers include, but are not limited to, ACTH-producing tumors, acute lymphocytic or lymphocytic leukemia, acute or chronic lymphocytic leukemia, acute non-lymphoid leukemia, bladder cancer, brain tumor, breast cancer, The present invention provides a method of treating a cancer selected from the group consisting of T-cell lymphoma, endometriosis, esophageal cancer, gall bladder cancer, Ewing's sarcoma, dandelion, stomach cancer, Hopkins lymphoma, A tumor of the stomach, a cancer of the thyroid gland, a tumor of the uterus, a testicular cancer, a Wilms' tumor, or a tropoblastoma tumor, .

The inflammatory disease refers to a disease caused by a common inflammatory reaction and specifically includes gastritis, gastric ulcer, bronchitis, asthma, edema, hepatitis, nephritis, arteriosclerosis, inflammatory bowel disease (IBD) Rheumatoid arthritis, osteoarthritis, joint inflammation related diseases, cancer, degenerative diseases, and the like.

May be a neurological disorder such as neurological disease neuralgia, myasthenia gravis, muscular atrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, Guillain-Barre syndrome, Huntington's disease, Alzheimer's disease, Parkinson's disease , ≪ / RTI >

The cardiovascular disease may be, but is not limited to, a cardiovascular disease such as atherosclerosis, angina pectoris, myocardial infarction, cardiac arrest or cardiovascular damage due to bypass surgery.

The content of carotenoid contained in the pharmaceutical composition of the present invention can be appropriately controlled depending on the method of using the therapeutic agent, the condition of the recipient, the kind of the disease, and the severity. In the pharmaceutical composition of the present invention, the carotenoid is preferably contained in an amount of 0.001 to 50% by weight, but is not limited thereto. When the content of carotenoid is less than 0.001% by weight, the antioxidative effect may be insignificant. When the carotenoid content is more than 50% by weight, it may be uneconomical because the rate of increase of effect is low.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The present invention relates to pharmaceutical compositions comprising an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, RTI ID = 0.0 > effective < / RTI > amount. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, or may be administered once a day, . ≪ / RTI >

The pharmaceutical compositions of the present invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In addition, the carotenoids can be used in cosmetic compositions for antioxidation.

The carotenoid is preferably contained in an amount of 0.001 to 50% by weight based on the total weight of the cosmetic composition, but is not limited thereto. If the content of carotenoid is less than 0.001% by weight, the antioxidative effect may be insignificant. If the carotenoid content is more than 50% by weight, it may be uneconomical because the rate of increase of effect is low.

The cosmetic composition of the present invention may further contain conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and the like which are conventionally used in cosmetic compositions, in addition to the above-mentioned effective ingredients. For example, the cosmetic composition may further contain an auxiliary component such as glycerin, butylene glycol, polyoxyethylene hardened castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin and the like .

Since the cosmetic composition of the present invention is basically applied to the skin, it can be prepared into any formulation conventionally produced by referring to the cosmetic composition of the related art. For example, they may be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritive cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, the carrier component may include an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, have.

When the formulation of the present invention is a powder or a spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like. Particularly in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane, dimethyl ether, and the like.

When the formulation of the present invention is a solution or an emulsion, the carrier component may include a solvent, a solubilizing agent, and an emulsifying agent. Specific examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol, propylene glycol or the like as a carrier component; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, trachant, and the like.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like.

The present invention also provides a food composition for antioxidation comprising carotenoid as an active ingredient. When the carotenoid of the present invention is used as a food additive, it can be suitably used according to a conventional method such as adding the carotenoid directly, mixing it with another food or food ingredient, and the like.

In addition, the mixing amount of the carotenoid as the active ingredient may be suitably changed according to the purpose of use (prevention, health or therapeutic treatment), and the carotenoid may be contained in an amount of 0.001 to 50% by weight based on the total weight of the food composition But is not limited thereto. When the content is less than 0.001% by weight, the antioxidant activity may be insignificant. When the content is more than 50% by weight, the rate of effect increase relative to the amount of use may be low, which may be uneconomical.

As a specific example, the carotenoid of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of the food to which the carotenoid of the present invention can be added include meat products, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, , A drink, an alcoholic beverage, a vitamin complex, and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Further, one aspect of the present invention provides a pigment composition comprising the novel carotenoid. When the novel carotenoid is used as a coloring matter, it can be used variously as a cosmetic, a dyeing, a food additive, a coloring agent, an industrial coloring agent and the like.

In addition, one aspect of the present invention provides an apo-carotenal wherein said novel caroteneoid is an enzyme degradation product that is used as a substrate. The apo-carotenal

C17 Apo-14 ' -diaponeurosporenal (C17 Apo-14 '-diaponeurosporenal);

C13 Apo-13'-diaponeurosporenone (C13 Apo-13'-diaponeurosporenone);

C22 Apo-10'-diaponeurosporenal (C22 Apo-10'-diaponeurosporenal);

C17 Apo-14 ' -diaponeurosporenal (C17 Apo-14 '-diaponeurosporenal);

C17 Apo-14'-diapotorulenal (C17 Apo-14'-diapotorulenal); or

C22 Apo-10'-diapotorulenal. ≪ / RTI >

Further, one aspect of the present invention provides a stem cell differentiation inducing composition comprising carotenoid as an active ingredient.

As used herein, the term " stem cells " is undifferentiated cells capable of differentiating into various body tissues, including totipotent stem cells, pluripotent stem cells, multipotential stem cells (multipotent stem cells).

Adult stem cells, embryonic stem cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, umbilical cord blood stem cells, and degenerated stem cells, and may be homogenous or autologous stem cells.

The stem cells may preferably be derived from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic membrane, decidual membrane, and placenta. The stem cells may also be derived from mammals other than humans, fetuses or humans. The mammal other than the human is more preferably a canine animal, a feline animal, a monkey animal, a cow, a sheep, a pig, a horse, a rat, a mouse or a guinea pig, and the origin thereof is not limited.

The differentiation may be preferably into neuronal differentiation.

The composition may further include a physiologically active substance to promote induction of differentiation during stem cell culture. The physiologically active substance may be selected from the group consisting of fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), bone morphogenic proteins (BMPs), lipopolysaccharide at least one selected from the group consisting of tumor necrosis factor-alpha, transforming growth factor-alpha, transforming growth factor-beta, ascorbic acid and dexamethasone. And may further include additives.

The stem cells can be cultured using Dulbecco's modified essential medium (DMEM) or Neural progenitor cell basal medium (Clonetics), which is a conventional medium used for cell culture.

There are no particular restrictions on the culturing conditions when the stem cells are cultured, and the stem cells can be cultured in the floating state or attached to the culture vessel. As the culture container, for example, a container widely used in the art such as a chamber glass, a non-coating dish and the like can be used.

The novel carotene nodule of the present invention is a carotenoid having a novel structure using artificial molecular evolution and combinatorial biosynthesis which can not be produced in a conventional natural environment and can be usefully used as a novel antioxidant material.

Fig. 1 is a diagram showing the results of expression of a module C ₃₀ carotenoid metabolism circuit.
Figure 2 shows the results of carotenoid production of new structures in E. coli via CrtA and CrtD.
Figure 3 shows the production of 4,4'-diapononaflavuxanthin, a novel structured C ₃₅ carotenoid, via Lycopene elongase (CrtEb).
FIG. 4 is a diagram showing screened CrtN mutant clones. FIG.
Figure 5 shows confirmation of changes in the HPLC profile of CrtN mutants.
6 is a graph showing changes in the production profile of carotenoids through HPLC analysis of clones obtained through site-directed evolution.
Figure 7 shows the carotenoid profile of pale yellowish clones screened.
FIG. 8 is a diagram showing the results of expression in E. coli by complementation of pACM-M _SA -Ny _SA , which is a diaponeurosporene production module, with CrtY.
FIG. 9 is a graph showing the results of HPLC analysis of the mutant CrtY clones screened by complementation with pACM-M _SA -N _SA .
Figure 10 shows a diapotorulene metabolism circuit optimized to produce a homogeneous product through a combination of directed evolution enzymes.
Figure 11 shows the functional complementation of the diapo-zeta-carotene biosynthesis gene module and CrtY. CrtY of P. agglomerans is most effective for biosynthesis of diapo-β-carotene.
12 shows the results of modification of diapotorulene using CrtZ, CrtO / CrtW and CrtX. Structures 3, 4, 5, and 6 are carotenoids of new structure that are not found in nature.
FIG. 13 shows the results of production of monocyclic C ₃₀ carotenoid having a structure similar to that of C ₂₀ retinal. The 8, 10 structure is a C ₃₀ carotenoid with a new structure that does not exist in nature.
14 is a diagram showing a novel bicyclic C ₃₀ carotenoid biosynthesized in E. coli. Structures 4, 5, 6, 7, and 8 are carotenoids of novel structure that have never been found in nature.
15 is a ¹ H NMR spectrum of diapotorulene and its assignments.
16 is a diagram showing the ¹ H- ¹ H COYY NMR spectrum of diapotorulene.
17 is a diagram showing the biosynthesis of the metabolic pathways of C ₃₀ carotenoids carotene novel structure obtained by the present invention.
FIG. 18 is a graph showing the results (HPLC-MS) of the activity of NSC3 against β-apo-8'-carotenal.
19 is a diagram showing the results (HPLC-MS) confirmed the activity in E. coli by the action of a C ₃₀ carotenoids carotene biosynthesis in the present invention as a substrate on NSC3.
Figure 20 is to separate the C _30-carotene, carotenoids novel structure in the biosynthesis in the present invention is a diagram showing a result of the reaction NSC3 on in vitro (HPLC).
21 is a graph showing the result 1 of the activity measurement of the NSC3 protein utilized in the present invention.
FIG. 22 is a graph showing the result 2 of the activity measurement of the NSC3 protein utilized in the present invention. FIG.
23 is a diagram showing apo-carotenals of a new structure biosynthesized in the present invention.
24 is a diagram showing the DPPH radical scavenging assay analysis of C ₃₀ carotenoids carotene analysis in the present invention.
25 is a diagram showing chemical structures of retinal, retinoic acid, diapotorulene, diapotorulene-al, and diapotorulenoic acid.
26 is a morphology observation of rBMSCs treated with diapotorulene at different concentrations. cell morphology is thinner and longer than normal cells.
FIG. 27 is a diagram showing a morphology observation of rBMSCs on the fourth day of diapotorulene treatment. FIG. Cells are thinner and longer, and they have similar morphology to neuron cells.
FIG. 28 is a diagram showing morphology observation of rBMSCs cell after 7 days of diapotorulene treatment. FIG. It is evident that the morphology of the cell is shaped like a neuron cell.
FIG. 29 shows cell cytotoxicity results of diapotorulene in rBMSCs cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Samples and methods

Experimental Example 1 - Artificial Molecular Evolution and Screening

An artificial molecular evolution of CrtN and CrtY was performed by error-prone PCR. For error-prone PCR reactions, MgCl2 was added to MgCl2-free buffer at a final concentration of 1.5 mM or 2.5 mM, and dNTP (ATP: TTP: CTP: GTP 1: 1: 1: 4) . The wild-type gene used as a template was pUCM-CrtN _SA and pUCM-CrtYcYd _BL cloned in the pUCM vector and PCR was performed to include a promoter and a terminator. The PCR product was reacted with cloned restriction enzyme sites (EcoRI, XbaI) and inserted into the pUCM vector to generate a library. The resulting libraries were pUCM-CrtN ^* and pUCM-CrtY ^* , respectively, and transformed into E. coli having pACM-M _SA and pACM-M _SA -N _SA , respectively, and plated on agar medium containing both ampicillin and chloramphenicol. Colonies were cultured at 37 ℃ for one day until they were well expressed at room temperature. The clones were inoculated into 4 ml of LB containing ampicillin and chloramphenicol by changing the color to obtain original clones through the plasmid preparation. From these clones, pUCM-CrtN ^* and pUCM-CrtY ^* were separated and purified through agarose gel, respectively. This plasmid was sequenced and again transformed into pACM-M _SA and pACM-M _SA -N _SA , The profile was confirmed.

Experimental Example 2 - Modulation of metabolic gene

Modulation of metabolic gene has been modularized by using previously established module or by using previously used method. The modules used or built are listed in the following table.

Experimental Example 3 - Escherichia coli culture and carotenoid production

Escherichia coli strain SURE was used for cloning except for the pBBR1MCS vector and for producing carotenoid. E. coli XL1-Blue strain was used for cloning into pBBR1MCS and expressing the gene module containing pBBR1MCS vector. For the production of carotenoids, recombinant E. coli was cultured in TB 50ml or 200ml at 30 ° C for 36 hours at 250 rpm and appropriate antibiotics were added according to the transformed plasmid (chloramphenicol 50 μg / ml, ampicillin 100 μg / ml, kanamycin 50 g / ml).

Experimental Example 4 - Isolation of Carotenoid

Cells cultured for the separation of carotenoids were centrifuged and 5 ml or 10 ml of acetone was repeatedly extracted until the color completely disappeared. The extracted solution was concentrated using a vacuum centrifugal dryer (EZ2-Plus, Genevac). 5 ml of ethyl acetate was added to the concentrated solution, and 5 N NaCl solution was added to separate the solution layer. After separating the upper layer containing the color, it was washed twice with the third order water to remove the remaining water, and was completely dried using a vacuum centrifugal dryer. 100-200 μl of ethyl acetate was added to the completely dried sample to dissolve and used for further analysis. For the LC / MS analysis, the sample was loaded on the silica column and eluted with hexane: ethyl acetate (9: 1). The eluted samples were used for LC / MS analysis after confirmation of purity by TLC and HPLC analysis.

Experimental Example 5 - Analysis of carotenoids

TLC analysis of carotenoids was performed using hexane: ethyl acetate: acetone (9: 1: 1). A 9: 1: 3 solution was used for the analysis of the glucosylated material. HPLC was performed using 10-20 μl of the prepared sample. The HPLC was performed using a Zorbax eclipse XDB-C18 column (4.6 × 150 mm or 250 mm, 5 μm; Agilent Technology) was analyzed at a flow rate of 1 ml / min in a stationary phase. For structural analysis, HPLC retention time, absorption spectrum and mass spectrum were compared. The mass spectra were monitored for both positive and negative modes using a Varian 1200L LC / MS system and the atmosphere pressure chemical ionization (APCI) module was used for ionization.

Experimental Example 6- C ₃₀  Identification of antioxidant activity of carotenoid (DPPH radical scavenging assay)

As a running condition, DPPH was dissolved in methanol at 200 mM concentration and used as a stock solution. Buffered methanol was used in 40 ml of 0.1 M acetic acid buffer (pH 5.5) mixed with 60 ml of methanol. For the reaction conditions, 250 μl of 200 mM DPPH, 200 μl of buffered methanol and 50 μl of carotenoid were added to a total volume of 500 μl volume reaction. Carotenoids were reacted at final concentrations of 10 μM, 5 μM, 2.5 μM, 0 μM Concentration. The reactants were reacted at room temperature for one hour in the absence of light and proceeded three times to calculate the IC ₅₀ value with mean ± SD. To compensate for the natural degradation of carotenoids, a DPPH-free reaction batch was reacted together and used as a blank reaction at 517 nm.

Experimental Example 7 - Determination of the ability of 4,4'-diapotorulene to differentiate into mesenchymal stem cells (rBMSCs) into neurons

The stem cells used in the experiment were isolated from the femoral region of the rat with rat bone marrow mesenchymal stem cells (rBMSCs). Passages 4 to 6 were used. Cells were seeded at 3 × 10 ³ cells / well in a 48-well plate, cultured in culture media for one day, and cultured in DMEM medium containing 20% FBS and 10 ng / ml basic fibroblast growth factor (bFGF) media) for 12 hours. Twelve hours later, Diapotorulene dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM was diluted to prepare 2 μM, 10 μM, 20 μM and 50 μM neuron differentiation inducing medium, and the amount of DMSO was the same . The control group was the same group as the control group, but the control group was the same group with the same amount of DMSO.

result

Example 1 - Preparation of C ₃₀  Expression of carotenoids and the combination of new diatomic C ₃₀  Carotenoid expression

In the staphyloxanthin metabolism pathway, one enzyme (4,4'-diaponeurosporen-aldehyde dehydrogenase, AldH) was excluded and expressed in E. coli. Carotenoids similar in structure to staphyloxanthin were biosynthesized in E. coli. As a result of expression in E. coli through the completed C ₃₀ carotenoid gene module, a part of 4,4'-diaponeurosporene and 4,4'-diapolycopene were mixed and expressed ( FIG. 1 ). Expression of wildtype diapophytoene synthase (CrtM) and diapophytoene desaturase (CrtN) gene modules in Escherichia coli revealed that diapolycopene and diaponeurosporene coexisted, and when CrtN was expressed in high copy pUCM, the proportion of diapolycopene increased ( Figure 1 ) . The completed diaponeurosporene-diapolycopene-producing module was modularized with Rhodobacter capsulatus Spheroidene monooxygenase (CrtA) and combined with C3 ', 4'-desaturase (CrtD) to extend the linear C ₃₀ carotenoid metabolism circuit. Carotenoids were produced in E. coli ( Fig. 2 ). In addition, lycopene elongase (CrtEb) of Corynebacterium glutamicum was combined with elongation of IPP in diaponeurosporene to produce a new C ₃₅ structure linear 4,4'-diapononaflavuxanthin ( FIG. 3 ).

Example 2- An artificial molecular evolution of 4,4'-diapophytoene desaturase (CrtN)

In order to expand the metabolism cycle of C ₃₀ carotenoid, lycopene cyclase (CrtY) can not function if there is a double bond conjugated to both ends of the carotenoid backbone. Therefore, CrtY enzyme is added to 4,4'-diapolycopene It can not work. Therefore, in order to change the activity of CrtN, we changed the activity of the CrtN enzyme through an artificial molecular evolution technique, and selected mutant clones with altered activity. CrtNr, CrtNy, CrtNy, and CrtNz clones were selected, and the profile and sequence analysis of the selected clones were performed. Sequenced clones Each mutated site was identified by site-directed mutagenesis

The results are shown in Figs. 4, 5, 6 and 7 and Tables 1 and 2.

FIG. 4 is a diagram showing screened CrtN mutant clones. FIG.

Figure 5 shows confirmation of changes in the HPLC profile of CrtN mutants.

6 is a graph showing changes in the production profile of carotenoids through HPLC analysis of clones obtained through site-directed evolution.

Figure 7 shows the carotenoid profile of pale yellowish clones screened.

Characterization of CrtN mutant clones and mutant sites. nucleotide Codon usage Amino acid CrtNwt CrtNrO4
C401T (GCA? GTA)
A737G (GAG? GGG) 0.22? 0.17
0.3? 0.13 A134V
E246G CrtNr07

A75G (GAA → GAG)
A626G (GAA → GGA)
A695G (AAT → AGT) 0.7? 0.3
0.7? 0.09
0.39 - > 0.13 -
E209G
N232S CrtNyO2

T635C (TTT to TCT)
T840C (AGT → AGC)
A1049G (GAT to GGT) 0.51? 0.19
0.13? 0.27
0.59 - > 0.38 F212S
-
D350G CrtNy08



A185G (TAT → TGT)
T367C (TTT? CTT)
T578G (ATT → AGT)
A627G (GAA → GAG)
A1046G (CAT? CGT) 0.53 - > 0.43
0.51? 0.1
0.47 - > 0.13
0.7? 0.3
0.52 - > 0.42 Y62C
F123L
I193S
-
H348R CrtNy11

T173C (ATG? ACG)
T1327C (TTC → CTC)
T1458C (AGT → AGC) 1.0 - > 0.23
0.49 - > 0.1
0.13? 0.27 M58T
F443L
-

Profile comparison of CrtNz screen clones for Diapo-ζ-carotene production (peak area). CrtN J-1 CrtNz01 CrtNz02 CrtNz03 CrtNz12 CrtNz14 CrtNz17 diapo-ζ-carotene 2311.2 1755.1 8714.7 10119.9 10033.1 2186.8 8978.7 diaponeurosporene 1662.6 225.8 9478.5 7246.1 14133.7 325.5 20045.6 diapo-ζ-carotene /
diaponeurosporene 1.4 7.8 0.9 1.4 0.7 6.7 0.4 CrtN J-1 CrtNz18 CrtNz20 CrtNz24 CrtNz28 CrtNz31 CrtNz32 CrtNz34 CrtNz37 diapo-ζ-carotene 2311.2 7927.1 5932.3 4436.5 4368.1 994.2 943.4 727.8 1461.1 diaponeurosporene 1662.6 11150.7 4396.2 3655.6 3304.1 692.7 945.1 541.1 2267.5 diapo-ζ-carotene /
diaponeurosporene 1.4 0.7 1.3 1.2 1.3 1.4 One 1.3 0.6 CrtN J-1 CrtNz 2-3 CrtNz 2-6 CrtNz 2-9 CrtNz 2-12 CrtNz 2-13 CrtNz 2-15 CrtN z2-18 CrtNz 2-20 diapo-ζ-carotene 2311.2 2841.8 4212.4 2134.6 5526.5 5073.6 3933.2 3162.8 4600.9 diaponeurosporene 1662.6 1605.9 2538.6 917.7 3034.3 3243.1 1568.9 1580.7 2680.8 diapo-ζ-carotene /
diaponeurosporene 1.4 1.8 1.7 2.3 1.8 1.6 2.5 2.0 1.7 CrtNz2-24 CrtNz2-25 CrtNz2-26 CrtNz2-28 CrtNz2-30 CrtNz2-31 CrtNz2-33 CrtNz2-36 CrtNz2-39 diapo-ζ-carotene 8546.6 3200.1 4796 5785.9 5810.5 1866.6 1655.4 1492.2 5478.7 diaponeurosporene 4608.9 1847.8 2794.4 4165.4 5209.7 544.7 802.9 1077 3449.6 diapo-ζ-carotene /
diaponeurosporene 1.85 1.64 1.72 1.4 1.1 3.4 2.1 1.4 1.6

The CrtNr clones were highly expressed in 4,4'-diapolycopene. The CrtNy clones were 4,4'-diaponeurosporene and the CrtNz clones were 4,4'-diapo-ζ-carotene. The amino acid sequence of each clone analyzed is as follows.

In addition, one of the mutant clone CrtNy11 proceeds CrtM module and granulation to build pACM-M _SA -N _ySA was used in the subsequent extension pathway again, CrtN _z 2-24 clone is added to the study as pACM-M _SA -N _zSA Respectively.

Example 3 - Artificial Molecular Evolution of Lycopene cyclase (CrtY)

The synthesis of cyclic C ₃₀ carotenoids was confirmed by combining pacM-M _SA -Ny _SA from above with CrtY from various sources. The use CrtY analyzed the Pantoea agglomerans, Pantoea ananatis, Brevibacterium linens , Corynebacterium glutamicum, was derived Salinibacter ruber, pACM-M _SA -Ny then proceeds co-transformation with a culture _SA, carotene, carotenoids profile in E. coli by HPLC (Fig. 8 ). As a result, the production of diapotorulene was greatly increased in the complementation of CrtY of Brevibacterium linens . Surprisingly, the production of diapo-β-carotene by CrtY of P. agglomerans was also confirmed. First, the directed evolution of CrtY in B. linens enhanced the substrate specificity for diaponeurosporene and produced a more homogeneous diapotorulene. E. coli comp in progress method was the same as CrtN, has a pACM-M _SA -N _SA. The mutant CrtY library was constructed in the cell and screened for the clones that were light in color. As a result of the experiment, it was possible to screen various clones having weak color, and their profiles were analyzed by their HPLC analysis ( FIG. 9 ).

Sequence analysis of the CrtY mutant clones screened above identified mutated sites.

The results are as follows.

Among the clones, CrtYt01 clone was used to construct a pACM-M _SA- Ny _SA -Yt _BL module and a system was constructed to homogenize the production of diapotorulene. This result is a result of homogenization of heterogeneous production using an artificial molecular evolution and combination method, thereby enhancing the selectivity of the metabolic pathway and thereby further elongating the metabolic pathway ( FIG. 10 ).

On the other hand, for the production of diapo-β-carotene, a bicyclic C ₃₀ carotenoid, CrtY of B. linens was applied to pACM-M _SA -Nz _SA to proceed with directed evolution. In this case, . Alternatively, a module for biosynthesis of diapo-β-carotene was obtained by modifying CrtY of P. agglomerans ( FIG. 11 ). In this case, the production of diapo-β-carotene appears to be significant, but since the pACM-M _SA -Nz _SA module does not produce only diapo-ζ-carotene as a major, diaponeurosporene also biosynthesizes, The biosynthesis of carotene was not homogeneous.

Example 4 - Diapotorulene, diapo-β-carotene A series of novel structures through the functional extension of the biosynthetic circuit C ₃₀  Carotenoid biosynthesis.

The diapotorulene and diapo-β-carotene derivative carotenoids were synthesized by combining with various C ₄₀ carotenoid biosynthesis-related genes using the biosynthesis gene module of diapotorulene and diapo-β-carotene obtained above. First, a mono-cyclic C ₃₀ carotenoid metabolism circuit was constructed. Diapotorulene was modified with β-carotene hydroxylase (CrtZ), β-carotene ketolase (CrtW / CrtO) and glycosyl transferase (CrtX) New monocyclic C _30s such as 4,4'-diapotorulene, 8-keto-4,4'-diapotorulene, 7-hydroxy-8-keto-4,4'-diapotorulene and 7-hydroxy- Carotenoids were biosynthesized in E. coli ( Fig. 12 ). Also diaponeurosporene oxidase (crtP), diaponeurosporen- aldehyde dehydrogenase (aldH) mutant using the CrtY having an aldehyde group or a carboxyl group C ₃₀ monocyclic carotene Carcinoid of 4,4'-diapotorulen-4'-al, diapotorulen 4,4'- -4'-oic acid was produced in E. coli ( FIG. 13 ). Next, diapo-β-carotene was modified by using CrtZ, CrtO, and CrtX enzymes to extend diapo-β-carotene metabolism. As a result, 4,4'-diapo-β-cryptoxanthin, 4,4 ' -diapozeaxanthin, 4,4'-diapoechinenone, 4,4'- diapo-β-cryptoxanthin in the production of the novel bicyclic cannabinoids carotene C ₃₀ was confirmed in the glucoside (Fig. 14).

Example 5 - Structural analysis of newly synthesized carotenoids

Secured on the carotene structure of the solenoid is HPLC retention time, UV / Vis spectrum , it was confirmed through the three results of the MS spectrum, of these, diapotorulene was isolated and purified through further ^{silica fractionation, 1 H NMR, 1} H- The structure was confirmed by ¹ H COZY NMR. ( Figs. 15 and 16 ). The MS spectra of the C ₃₀ carotenoids identified in the present invention are as shown in Table 3 below, and the C ₃₀ carotenoid metabolism circuit of novel structure biosynthesized in E. coli is shown in FIG .

The MS spectrum of the C ₃₀ carotenoid identified in the present invention number structure name MS spectrum One 4,4'-diapo-ζ-carotene

2 4,4'-diaponeurosporene

3 4,4'-diapolycopene

4 4,4'-diaponeurosporene-6,6'-dione

5 4,4'-diapolycopene-6,6'-dione

6 4,4'-diapononaflavuxanthin

7 4,4'-diapotorulene

8 4,4'-diapo-β-carotene

9 7-hydroxy-4,4'-diapothorulene

10 8-keto-4,4'-diapotorulene,

11 7-hydroxy-8-keto-4,4'-diapotorulene

12 7-hydroxy-4,4'-diapotorulene glucoside

13 4,4 ' -diaphthorulene-4 ' -al

14 4,4 ' -diaphthorulene-4 ' -oic acid

15 4,4'-diapo-β-cryptoxanthin

16 4,4'-diapozeaxanthin

17 4,4'-diapoechinenone

18 4,4'-diapo-β-cryptoxanthin glucoside

Example 6 - Cloning and activity confirmation of NSC3 enzyme

As other carotenoid cleavage enzymes, cyanobacteria Nostoc sp. The NSC3 enzyme was cloned from PCC 7120 and expressed in E. coli. Carotenoid cleaving enzymes are found in plant and animal species, including cyanobacteria, and work to produce apo-carotenal, including retinal, by cleaving carotene nodules such as beta carotene . The produced retinoids or apo-carotene eggs are used as vitamin precursors and hormones, and the metabolic circuits have not yet been fully understood. In addition, since interest in carotenoids including antioxidant and antioxidant effects of carotenoids has increased, research on the physiological activity of carotenoids of various structures is also becoming more and more active. However, existing carotenoid cleavage enzymes have been studied only on C ₄₀ carotenoid substrates, and studies using C ₃₀ carotenoids as substrates have been made in the present invention for the first time. In the present invention, the NSC3 enzyme was cloned and expressed in Escherichia coli to confirm the enzymatic characteristics, and a new structure of apo-carotenal was biosynthesized in E. coli using C30 carotenoid as a substrate.

The activity of NSC3 obtained earlier was confirmed in vitro using β-apo-8'-carotenal as a substrate ( FIG. 18 ). As a result, NSC3 was active at C13-C14, C15-C15 'and C14'-C13' bonds, resulting in retinoids, β-apo-14'-carotenal and β-apo-13- This is because Nostoc sp. Showed distinctive activity from NSC1 and NSC2 identified from PCC 7120. Additionally, use of a C _30-carotene, carotenoids biosynthetic modules obtained in the present invention, saw out the in vivo activity in the NSC3 of Escherichia coli (Figure 19). NSC3 was well used for the C ₃₀ carotenoid substrate, and it was confirmed that several new structures of apo-carotenal were produced. However, the C ₄₀ carotenoid substrate did not act on NSC3 in E. coli, and only truncated products of 3,4,3 ', 4'-tetradehydrolycopene and torulene were identified. , 3 ', 4'-tetradehydrolycopene did not accurately analyze the product. Were analyzed additionally, various properties of the accurate C _30-carotene, carotenoids NSC3 enzyme the C _30-carotene using a solenoid, NSC3 separation to make the temperament over has confirmed that the activity (Fig. 20), in vitro on the in vitro (FIG. 21 , 22 ). Finally, the apo-carotenals of the structure identified in the present invention are shown in FIG .

Example 7 - Measurement of antioxidative effect of various / novel carotene nodal structures

As antioxidant capacity of natural substances such as carotenoids has increased, antioxidative effects of various representative carotenoids have been measured and studied in various ways. Most of these studies are concentrated on zeaxanthin, lycopene, β-carotene, and astaxanthin, which are commonly found in the natural world, and their antioxidant capacity is measured in comparison with representative antioxidants such as DL-α-tocopherol. However, studies on the antioxidant capacity of C ₃₀ carotenoids have been limited, and studies on the antioxidant activity of staphyloxanthin have been conducted to confirm the viability of Staphylococcus aureus in the host.

In order to confirm the antioxidant ability of the carotenoids obtained in the present invention, the extraction and separation / purification of carotenoids biosynthesized from E. coli must be preliminarily performed. Therefore, a plasmid in which a carotenoid of each structure is dorminantly expressed is transformed, Escherichia coli cells were cultured. After cell culture, the cells were recovered and extracted with acetone. Separation / purification was carried out through silica fractionation. All procedures were carried out in glass tubes and vials to avoid plastic contamination. The final samples were dissolved in methanol and quantified. The samples were stored at -80 ° C until they were filled with nitrogen until they were used for measurement.

The antioxidative effect of C ₃₀ carotenoid was confirmed by DPPH radical scavenging assay. This is a radical scavenging assay based on a single electron transfer reaction (SET), which is widely used to measure the antioxidant activity of carotenoids. However, in the DPPH assay, the IC ₅₀ value varies greatly depending on the method and condition to be used. In the present study, the method of the 2009 Bhat study group was modified to compensate for the lack of a formalized method suitable for carotenoids Respectively. As a running condition, DPPH was dissolved in methanol at 200 mM concentration and used as a stock solution. Buffered methanol was used in 40 ml of 0.1 M acetic acid buffer (pH 5.5) mixed with 60 ml of methanol. For the reaction conditions, 250 μl of 200 mM DPPH, 200 μl of buffered methanol and 50 μl of carotenoid were added to a total volume of 500 μl volume reaction. Carotenoids were reacted at final concentrations of 10 μM, 5 μM, 2.5 μM, 0 μM Concentration. Reactants were reacted at room temperature for one hour in the absence of light, and the IC ₅₀ values were calculated by the mean ± standard deviation ( FIG. 24, Table 4 ).

DPPH IC ₅₀ value of C ₃₀ carotenoid DPPH IC ₅₀ Diapolycopene 8.68 μM Diaponeurosporene 11.58 [mu] M Diaponeurosporene-al 10.17 [mu] M Diapolycopen-dial 7.5 μM Diaponeurosporenoic acid 9.67 [mu] M Diapotorulene 70.25 μM Diapo-β-carotene 77.78 [mu] M DL-α-Tocopherol 33.22 [mu] M

As a result of this experiment, the reaction occurred well at 100 mM DPPH and 10-20 μM carotenoid ratio, but the radical scavenging reaction did not occur at a lower rate. To compensate for the natural breakdown of carotenoids, a reaction batch without DPPH was reacted together and used as a blank reaction at 517 nm. As a result, the radical scavenging ability of the reacted C ₃₀ carotenoids was confirmed in the order of diapolycopene-dial>diapolycopene> diaponeurosporenoic acid>diaponeurosporen-al>diaponeurosporene>diapotorulene> diapo-β-carotene, The number of conjugated double bonds is the number of conjugated double bonds. In case of the same number of oxygen, the radical substitution is more favorable when oxygen is substituted, and a linear carotenoid rather than a β-ring carotenoid (β-carotene, torulene etc.) It can be seen that it is more effective for radical removal. Furthermore, except for carotenoids with β-rings, linear C ₃₀ carotenoids showed a higher degree of radical elimination than DL-α-tocopherol. The results of this experiment will be helpful for the selection of candidate carotenoids with high antioxidant ability and it will be a good model test method for the antioxidant ability of carotenoid.

Example 8 - Measurement of stem cell differentiation ability of carotenoid according to various / new structure

Studies on the ability of carotenoids to differentiate into stem cells are still in its infancy and little research has been done. However, the ability of stem cells to differentiate into neurons has been studied for retinal and retinoic acid, and retinoic acid has been known to be very effective. As interest in stem cell research has increased in recent years, there has been a growing interest in the study of the ability of natural biologically active substances to increase stem cell differentiation. In addition, studies on the ability of carotenoids to differentiate into stem cells have been carried out gradually. The C ₃₀ carotenoids biosynthesized in the present invention are shorter than conventional C ₄₀ carotenoids and smaller in size than small (C ₂₀ ) carotenoids such as retinal or retinoic acid. Carotene cannabinoid used in the experiments is a C _30-carotene cannabinoid structure, such as 4,4'-diapotorulene as, retinal or retinoic acid and the structure is similar to 4,4'-diapotorulen-4-al, or 4,4'-diapotorulenoic acid However, 4,4'-diapotorulene was used as a model because of difficulties in separation / purification of the above substances ( Fig. 25) . 4,4'-diapotorulene was transformed into pACM-M _SA- Ny _SA -Yt _BL plasmid into E. coli and cultured in TB medium for 36 hours at 30 ° C. Cells were extracted from the cell with acetone, column. The purity was analyzed by HPLC. The stem cells used in the experiment were isolated from the femoral region of the rat with rat bone marrow mesenchymal stem cells (rBMSCs). Passages 4 to 6 were used. Cells were seeded at 3 × 10 ³ cells / well in a 48-well plate, cultured in culture media for one day, and cultured in DMEM medium containing 20% FBS and 10 ng / ml basic fibroblast growth factor (bFGF) media) for 12 hours. Twelve hours later, diapotorulene dissolved at 5 mM concentration in dimethyl sulfoxide (DMSO) was diluted to prepare 2 μM, 10 μM, 20 μM and 50 μM concentrations of neurogenic differentiation medium, and the amount of DMSO was the same The normal group was treated with no treatment, while the control group was treated with the same amount of DMSO. After the treatment with diapotorulene, morphology changes of cells were observed for one week and cytotoxicity was confirmed by MTT assay ( FIGS. 26, 27, 28, 29 ). The results suggest that 4,4'-diapotorulene significantly differentiates into the neural cells of the stem cells and has the ability to differentiate into neurons.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Novel carotinoid and use thereof <130> 1-28p <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNrO4 amino acids <400> 1 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Val Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Gly Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 2 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNr08 amino acids <400> 2 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Ile Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 3 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> crtN_wt amino acids <400> 3 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 4 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNr05 amino acids <400> 4 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Gly Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Gly Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 5 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNr07 amino acids <400> 5 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Gly Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Ser Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 6 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNr06 amino acids <400> 6 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Gly Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 7 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN Reddish clone crtNrO2 amino acids <400> 7 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Ser His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Gly Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Ala Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Arg Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 8 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN yellowish clone crtNy06 amino acids <400> 8 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Gly Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 9 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN yellowish clone crtNy11 amino acids <400> 9 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Thr Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Leu Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 10 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN yellowish clone crtN_wt amino acids <400> 10 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 11 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN yellowish clone crtNy02 amino acids <400> 11 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Tyr Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Phe Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ile Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Ser Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser His Gly Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 12 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> CrtN yellowish clone crtNy08 amino acids <400> 12 Met Lys Ile Ala Val Ile Gly Ala Gly Val Thr Gly Leu Ala Ala Ala   1 5 10 15 Ala Arg Ile Ala Ser Gln Gly His Glu Val Thr Ile Phe Glu Lys Asn              20 25 30 Asn Asn Val Gly Gly Arg Met Asn Gln Leu Lys Lys Asp Gly Phe Thr          35 40 45 Phe Asp Met Gly Pro Thr Ile Val Met Met Pro Asp Val Cys Lys Asp      50 55 60 Val Phe Thr Met Cys Gly Lys Asn Tyr Glu Asp Tyr Ile Glu Leu Arg  65 70 75 80 Gln Leu Arg Tyr Ile Tyr Asp Val Tyr Phe Asp Arg Asp Asp Cys Ile                  85 90 95 Thr Val Pro Thr Asp Leu Ala Glu Leu Gln His Met Leu Glu Ser Ile             100 105 110 Glu Pro Gly Ser Thr His Gly Phe Met Ser Leu Leu Thr Asp Val Tyr         115 120 125 Lys Lys Tyr Glu Ile Ala Arg Arg Tyr Phe Leu Glu Arg Thr Tyr Arg     130 135 140 Lys Pro Ser Asp Phe Tyr Asn Met Thr Ser Leu Val Gln Gly Ala Lys 145 150 155 160 Leu Lys Thr Leu Asn His Ala Asp Gln Leu Ile Glu His Tyr Ile Asp                 165 170 175 Asn Glu Lys Ile Gln Lys Leu Leu Ala Phe Gln Thr Leu Tyr Ile Gly             180 185 190 Ser Asp Pro Lys Arg Gly Pro Ser Leu Tyr Ser Ile Ile Pro Met Ile         195 200 205 Glu Met Met Phe Gly Val His Phe Ile Lys Gly Gly Met Tyr Gly Met     210 215 220 Ala Gln Gly Leu Ala Gln Leu Asn Lys Asp Leu Gly Val Asn Ile Glu 225 230 235 240 Leu Asn Ala Glu Ile Glu Gln Ile Ile Ile Asp Pro Lys Phe Lys Arg                 245 250 255 Ala Asp Ala Ile Lys Val Asn Gly Asp Ile Arg Lys Phe Asp Lys Ile             260 265 270 Leu Cys Thr Ala Asp Phe Pro Ser Val Ala Glu Ser Leu Met Pro Asp         275 280 285 Phe Ala Pro Ile Lys Lys Tyr Pro Pro His Lys Ile Ala Asp Leu Asp     290 295 300 Tyr Ser Cys Ser Ala Phe Leu Met Tyr Ile Gly Ile Asp Ile Asp Val 305 310 315 320 Thr Asp Gln Val Arg Leu His Asn Val Ile Phe Ala Asp Asp Phe Arg                 325 330 335 Gly Asn Ile Glu Glu Ile Phe Glu Gly Arg Leu Ser Arg Asp Pro Ser             340 345 350 Ile Tyr Val Tyr Val Pro Ala Val Ala Asp Lys Ser Leu Ala Pro Gln         355 360 365 Gly Gln Thr Gly Ile Tyr Val Leu Met Pro Thr Pro Glu Leu Lys Thr     370 375 380 Gly Ser Gly Ile Asp Trp Ser Asp Glu Ala Leu Thr Asp Gln Ile Lys 385 390 395 400 Asp Val Ile Tyr Arg Lys Leu Ala Thr Ile Glu Val Phe Glu Asp Ile                 405 410 415 Lys Ser His Ile Val Ser Glu Thr Ile Phe Thr Pro Asn Asp Phe Glu             420 425 430 Gln Thr Tyr His Ala Lys Phe Gly Ser Ala Phe Gly Leu Met Pro Thr         435 440 445 Leu Ala Gln Ser Asn Tyr Tyr Arg Pro Gln Asn Val Ser Ser Asp Tyr     450 455 460 Lys Asp Leu Tyr Phe Ala Gly Ala Ser Thr His Pro Gly Ala Gly Val 465 470 475 480 Pro Ile Val Leu Thr Ser Ala Lys Ile Thr Val Asp Glu Met Ile Lys                 485 490 495 Asp Ile Glu Gln Gly Val             500 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT1_Yc amino acids <400> 13 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Val Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 14 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT9_Yc amino acids <400> 14 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Val Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 15 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT11_Yc amino acids <400> 15 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Val Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 16 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT6_Yc amino acids <400> 16 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Ile Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 17 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> crtY_Yc_wild amino acids <400> 17 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Ile Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 18 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT10_Yc amino acids <400> 18 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Val Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Ile Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 19 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT5_Yc amino acids <400> 19 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Ile Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Thr His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Ile Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 20 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> crtY_Yc mutation clone Consensus amino acids <400> 20 Met Ser Ala Phe Glu Tyr Leu Leu Leu Met Gly Ala Cys Leu Leu Ile   1 5 10 15 Thr Leu Pro Leu Glu Leu Leu Phe Ser Ala Arg Val Tyr Arg Arg Pro              20 25 30 Lys Leu Leu Ile Gly Ser Leu Ile Pro Ile Leu Val Phe Ser Leu          35 40 45 Trp Asp Ile Ile Ala Xaa Asp Arg Asp His Trp Thr Tyr Asn Gln Gln      50 55 60 Phe Val Thr Gly Ile His Ile Gly Asn Leu Pro Leu Glu Glu Leu Val  65 70 75 80 Phe Phe Ile Val Xaa Pro Ile Cys Ala Leu Leu Ser Tyr Glu Ala Val                  85 90 95 Gly Thr Val Leu Lys Phe Val Ala Lys Lys Ser Gly Thr Arg Ala Gly             100 105 110 Arg Lys Ser Gly Asn Arg Lys Asp Gly Gly Asp Val Ala         115 120 125 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT1_Yd amino acids <400> 21 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Arg Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT5_Yd amino acids <400> 22 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT9_Yd amino acids <400> 23 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> crtY_Yd_wild amino acids <400> 24 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 25 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT11_Yd amino acids <400> 25 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT10_Yd amino acids <400> 26 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Cys Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY mutation clone YT6_Yd amino acids <400> 27 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Tyr Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Gln Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CrtY_Yd mutation clone Consensus amino acids <400> 28 Met Leu Pro Glu Tyr Thr Val Met Thr Val Val Gly Ile Val Ile Val   1 5 10 15 Ile Gly Leu Glu Ile Phe Val Phe Arg Ser Gly Ile Phe Arg Arg Gly              20 25 30 Gln Tyr Trp Ala Ala Leu Ala Ile Xaa Leu Ala Phe Gln Cys Leu Val          35 40 45 Asp Gly Trp Leu Thr Lys Leu Ser Asp Pro Ile Val Arg Tyr Asn Pro      50 55 60 Ser Gln Phe Leu Asn Val Arg Phe Pro Trp Asp Ile Pro Ile Glu Asp  65 70 75 80 Phe Gly Phe Gly Phe Ala Met Ile Thr Ser Val Leu Met Leu Trp Gln                  85 90 95 Trp Xaa Leu Asn Arg Ser Ser Lys Asp Thr Gln             100 105

Claims (15)

4,4'-diaponeurosporen-6,6'-dione;
4,4'-diapolycopene-6,6'-dione;
7-hydroxy-8-keto-4,4'-diapotorulene;
4,4'-diapo-β-cryptoxanthin;
4,4'-diapozeaxanthin;
4,4'-diapoechinenone;
4,4'-diapo-beta-cryptoxanthin glucoside; And
At least one novel carotene nodine selected from the group consisting of 4,4'-diapotorulene-4'-oic acid. The method according to claim 1,
The carotenoid
A method for producing a carotenoid comprising the step of transforming E. coli with a vector comprising a polynucleotide encoding CrtM (diapophytoene synthase) and CrtN (diapophytoene desaturase). 3. The carotenoid of claim 2, wherein said vector further comprises a polynucleotide encoding CrtY (lycopene cyclase). The method according to claim 2, wherein the CrtN comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 12, 4. The carotenoid according to claim 3, wherein the CrtY comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 13 to 28. The method of claim 2,
The method for producing carotenoids
CrtZ (beta-carotene ketolase), CrtO (beta-carotene ketolase), CrtX (glycosyl transferase), CrtP (diaponeurosporene oxidase), CrtA (spheroidenone monooxygenase), CrtEb elongase), and AldH (diaponeurosporene-aldehyde dehydrogenase), which comprises the step of transforming E. coli with a vector comprising a polynucleotide encoding at least one enzyme selected from the group consisting of carotenoids . A method for producing a carotenoid of the novel structure according to claim 1, comprising the step of transforming E. coli with a vector comprising a polynucleotide encoding CrtM (diapophytoene synthase) and CrtN (diapophytoene desaturase). 8. The method of claim 7, wherein the vector further comprises a polynucleotide encoding CrtY (lycopene cyclase). The method of claim 7,
CrtZ (beta-carotene ketolase), CrtO (beta-carotene ketolase), CrtX (glycosyl transferase), CrtP (diaponeurosporene oxidase), CrtA (spheroidenone monooxygenase), CrtEb elongase), and AldH (diaponeurosporene-aldehyde dehydrogenase), which comprises the step of transforming E. coli with a vector comprising a polynucleotide encoding at least one enzyme selected from the group consisting of carotenoids Gt; A composition for antioxidation comprising the carotenoid of claim 1. A colorant composition comprising the carotenoid of claim 1. delete delete 1. A stem cell differentiation inducing composition comprising carotenoid as an active ingredient, wherein said carotenoid is
4,4'-diaponeurosporen-6,6'-dione;
4,4'-diapolycopene-6,6'-dione;
7-hydroxy-8-keto-4,4'-diapotorulene;
4,4'-diapo-β-cryptoxanthin;
4,4'-diapozeaxanthin;
4,4'-diapoechinenone;
4,4'-diapo-beta-cryptoxanthin glucoside; And
And 4,4'-diapotorulene-4'-oic acid. 4. The composition for inducing differentiation of stem cells according to claim 1, wherein the composition is at least one selected from the group consisting of 4,4'-diapotorulene-4'-oic acid.
delete

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