KR102335035B1 - Process for preparing crocin-4 using enzyme reaction - Google Patents

Abstract

The present invention provides a composition for producing crosin-4 comprising a CaUGT3 enzyme derived from a Catharanthus roseus strain, and a transformant into which a gene encoding the CaUGT3 enzyme is introduced. Composition, method for producing crosin-4 from crosin- 2 using the composition, NtUGT enzyme derived from Nicotiana tabacum strain or GjUGT1 derived from Gardenia jasminoides strain Crosin-4 production composition comprising an enzyme and CaUGT3 enzyme derived from Catharanthus roseus strain, Crosin-4 production composition comprising a transformant into which the gene encoding each enzyme is introduced and to a method for producing crosine-4 from crocetin using the composition. Using the method for producing crosin-4 provided in the present invention, crosin-4 can be efficiently produced using a simple enzymatic reaction or bioconversion process, so it can be widely used for economical production of saffron compounds. will be.

Description

Method for producing crocin-4 using an enzyme reaction {Process for preparing crocin-4 using enzyme reaction}

The present invention relates to a method for producing crosin- 4 using an enzymatic reaction, and more particularly, the present invention relates to a composition for producing crosin-4 comprising a CaUGT3 enzyme derived from a Catharanthus roseus strain; A composition for producing crosin-4 comprising a transformant into which a gene encoding the CaUGT3 enzyme is introduced, a method for producing crosin-4 from crosin-2 using the composition, Nicotiana tabacum ) NtUGT enzyme derived from the strain or Gardenia jasminoides ( Gardenia jasminoides ) GjUGT1 enzyme derived from the strain Catharanthus roseus ( Catharanthus roseus ) Crosin-4 production composition comprising a CaUGT3 enzyme derived from the strain, the It relates to a composition for producing crosin-4 including a transformant into which a gene encoding each enzyme is introduced, and a method for producing crocetin-4 from crocetin using the composition. Using the method for producing crosin-4 provided in the present invention, it is possible to efficiently produce crosin-4 using a simple enzymatic reaction or bioconversion process, so it can be widely used for economical production of saffron compounds. will be.

Saffron is a dried spice prepared by extraction from the stigma of the flower Crocus sativus L., which is thought to have been used for over 3500 years. This spice has been used historically for a number of medicinal purposes, but more recently it is mainly used for its colorant properties. Crocetin, one of the main components of saffron, has antioxidant properties similar to the related carotenoid-type molecules, and is also a colorant. The main pigment in saffron is crosine, a mixture of glycosides that gives it its yellow-red color. The main component of crosine is α-crosine, which is yellow in color. Safranal is believed to be a product of a drying process, and also has olfactory properties that can be used in food preparation. Safranal is the aglycone form of the bitter part of picrocrosin, a colorless extract of saffron. Thus, saffron extract is used for many purposes as a colorant or flavorant or for its olfactory-stimulant properties.

The saffron plant is grown commercially in many countries including Italy, France, India, Spain, Greece, Morocco, Turkey, Switzerland, Israel, Pakistan, Azerbaijan, China, Egypt, United Arab Emirates, Japan, Australia, and Iran. Iran produces about 80% of the world's annual saffron production (estimated just above 200 tonnes). It has been reported that more than 150,000 flowers are required to produce 1 kg. Efforts to improve plant breeding to increase yield are complicated by the triplet in the genome of plants that give rise to non-bearing plants. In addition, the plant exists in a flowering state only for about 15 days starting in mid or late October. Typically, production also involves the manual removal of the stigma from the flower, an inefficient process. Selling prices in excess of $1000 per kg of saffron are typical.

As a means to replace such expensive production costs, research to produce saffron components through a bioconversion process is being actively conducted. For example, U.S. Patent Application Publication No. 2017-0306376 discloses a method using a recombinant microorganism engineered to express zeaxanthin-degrading dioxygenase alone or in combination with a recombinant gene encoding UDP-glycosyltransferase (UGT). Methods for producing compounds from saffron, such as cetin, crocetin dialdehyde, crosine, or picrocrosine, are disclosed. However, the production yield of the saffron compound by the used UDP-glycosyltransferase was low, so it was necessary to improve the yield thereof.

Under this background, the present inventors made intensive research efforts to develop a method for increasing the production yield of saffron compounds, and as a result, UDP-glycosyltrans derived from Nicotiana tabacum strain, Gardenia jasminoides strain and Catalanthus roseus strain. When ferase was used, it was confirmed that crosin-4 could be produced in high yield, and the present invention was completed.

The main object of the present invention is to provide a composition for producing crosin-4 comprising UDP-glycosyltransferase derived from a Catalanthus roseus strain.

Another object of the present invention is to provide a method for producing crosin-4 from crosin-2 using UDP-glycosyltransferase derived from Cat. roseus strain.

Another object of the present invention is to provide a composition for producing crosin-4 comprising a transformant into which a gene encoding UDP-glycosyltransferase derived from a Catalanthus roseus strain is introduced.

Another object of the present invention is to provide a method for producing crosin-4 from crosin-2 using a transformant into which a gene encoding UDP-glycosyltransferase derived from a Cat. roseus strain is introduced. will do

Another object of the present invention is a crosin containing UDP-glycosyltransferase derived from a Nicotiana tabacum strain or a Gardenia jasminoides strain and a UDP-glycosyltransferase derived from a Catalanthus roseus strain. -4 to provide a composition for production.

Another object of the present invention is Crocetin using UDP-glycosyltransferase derived from Nicotiana tabacum strain or Gardenia jasminoides strain and UDP-glycosyltransferase derived from Catalanthus roseus strain. To provide a method for producing crosine-4 from

Another object of the present invention is a gene encoding a UDP-glycosyltransferase derived from a Nicotiana tabacum strain or a Gardenia jasminoides strain and a UDP-glycosyltransferase derived from a Catalanthus roseus strain. To provide a composition for producing crosin-4 comprising a transformant into which a coding gene is introduced.

Another object of the present invention is to provide a method for producing crosin-4 from crocetin using the transformant.

One embodiment of the present invention for achieving the above object provides a composition for producing crosin-4 comprising UDP-glycosyltransferase derived from Catalanthus roseus strain.

As used herein, the term "UDP-glycosyltransferase (UDP-glucosyltransferase, UGT)" refers to an enzyme that catalyzes the transfer of the glucuronic acid component of various UDP-glucuronic acids, such as UDP-glucose, to small hydrophobic molecules. means

In the present invention, the UGT can be interpreted as an enzyme directly involved in a reaction for producing crosin-2 from crocetin or crosin-4 from crocetin-2.

As clarified in the present invention, all types of UGT are not directly involved in the reaction of producing crosin-2 from crocetin or crosin-4 from crocetin, and are derived from a specific strain. UGT may be involved in a reaction that produces crosin-2 from crocetin or crosin-4 from crosin-2.

As an example, GjUGT1 enzyme (SEQ ID NO: 1), which is a UGT derived from a Gardenia jasminoides strain, or a NtUGT enzyme (SEQ ID NO: 9) that is a UGT derived from a Nicotiana tabacum strain It may be directly involved in the reaction that produces crosine-2 from cetin.

As another example, CaUGT3 enzyme (SEQ ID NO: 21), which is a UGT derived from a Catharanthus roseus strain, may be directly involved in a reaction for producing crosin-4 from crosin-2.

As described above, the CaUGT3 enzyme (SEQ ID NO: 21), which is a UGT derived from a Catharanthus roseus strain, can be directly involved in the reaction for producing crosin-4 from crosin-2, so that the CaUGT3 The composition comprising the enzyme (SEQ ID NO: 21) may be used as a composition for producing Crosin-4.

The composition for crosin-4 production may further include crosin-2 and UGT-glucose required for crosin-4 production in addition to CaUGT3 enzyme (SEQ ID NO: 21).

Another embodiment of the present invention provides a method for producing crosin-4 from crosin-2 using UDP-glycosyltransferase derived from a Cat. roseus strain.

The enzymatic reaction may be performed in a reactant containing the substrates crocetin, crosine-2 and UGT-glucose.

The temperature conditions for carrying out the enzymatic reaction are not particularly limited thereto, but as an example, may be 20 to 40 ℃, as another example, may be 25 to 35 ℃, as another example, 30 ℃ can be

The time condition for performing the enzymatic reaction is not particularly limited thereto, but as an example, it may be 10 to 240 minutes, as another example, it may be 60 to 180 minutes, and as another example, it may be 120 minutes. .

Another embodiment of the present invention is a transformant into which a gene encoding a UDP-glycosyltransferase derived from a Cat. roseus strain is introduced or Crosin-4 production comprising a culture product of the transformant A composition is provided.

As described above, since crosin-4 can be produced from crosin-2 by using UGT derived from a specific strain, crosin- Crocin-4 can be produced from 2.

In this case, the transformant may be one in which the CaUGT3 gene (SEQ ID NO: 22) directly involved in the reaction for producing crosin-4 from crosin-2 is also introduced.

The culture product of the transformant is not particularly limited as long as it can be used to produce crosin-4 from crosin-2 in high yield, but as an example, the transformant culture, culture supernatant, and lysate , their fractions, and the like, and as another example, a culture supernatant obtained by centrifuging the transformant culture, a lysate obtained by physically or sonicating the transformant, the culture, the culture supernatant, It may be a fraction obtained by subjecting the lysate to a method such as centrifugation or chromatography.

Another embodiment of the present invention provides a method for producing crosin-4 from crosin-2 using a transformant into which a gene encoding UDP-glycosyltransferase derived from a Cat. roseus strain is introduced. to provide.

Specifically, the method for producing crosin-4 from crosin-2 includes culturing the transformant into which the CaUGT3 gene (SEQ ID NO: 22) is introduced in a medium containing crosin-2.

As described above, when using the CaUGT3 enzyme (SEQ ID NO: 21), crosin-4 can be produced from crosin-2, so when using the transformant into which the gene encoding the CaUGT3 enzyme (SEQ ID NO: 22) is introduced , Crosin-4 can be produced from Crosin-2 by the bioconversion process.

In addition, it may further comprise the step of recovering crosin-4 from the culture obtained through the culture.

As used herein, the term "cultivation" refers to a method of growing microorganisms in an appropriately artificially controlled environmental condition.

In the present invention, the method for culturing the transformant can be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as it can produce crosin-4 from the transformant, but it can be continuously cultured in a batch process or injection batch or repeated fed batch process. can

The medium used for culture should meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acids, vitamins, and the like. Carbon sources that can be used include sugars and carbohydrates such as glucose, xylose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitate, etc. Acids, fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, and the like may additionally be used, and these substances may be used individually or as a mixture. Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products, etc. can These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate and the corresponding sodium-containing salt as phosphorus. The phosphorus that may be used includes potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used as the inorganic compound. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials may be added in a batch, fed-batch or continuous manner by an appropriate method to the culture during the culturing process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture.

In addition, an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles. Oxygen or oxygen-containing gas (eg air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. Cultivation is continued until the maximum amount of the peptide is obtained. This is usually achieved in 10 to 100 hours for this purpose.

In addition, the step of recovering crosin-4 from the reaction solution may be performed by methods known in the art, such as dialysis, centrifugation, filtration, solvent extraction, chromatography, and crystallization. For example, a method of recovering the desired crosin-4 by centrifuging the reaction solution to remove the transformant and applying the obtained supernatant to a solvent extraction method may be used. If it is a method capable of recovering the crossin-4 by combining known experimental methods according to the characteristics, it may be used without particular limitation.

Another embodiment of the present invention is a croissant comprising UDP-glycosyltransferase derived from a Nicotiana tabacum strain or a Gardenia jasminoides strain and a UDP-glycosyltransferase derived from a Catalanthus roseus strain. A composition for producing Shin-4 is provided.

As described above, GjUGT1 enzyme (SEQ ID NO: 1), which is a UGT derived from a Gardenia jasminoides strain, or NtUGT enzyme (SEQ ID NO: 9), which is a UGT derived from a Nicotiana tabacum strain, is CaUGT3 enzyme (SEQ ID NO: 21), which can be directly involved in the reaction to produce crosin-2 from crocetin, and is a UGT derived from a Catharanthus roseus strain, is crosin-4 from crosin-2 Since it can be directly involved in the reaction producing

Therefore, the composition including each UGT can be used as a composition for producing Crosin-4.

The composition for crosin-4 production further includes crocetin and UGT-glucose required for crosin-4 production in addition to GjUGT1 enzyme (SEQ ID NO: 1) or NtUGT enzyme (SEQ ID NO: 9) and CaUGT3 enzyme (SEQ ID NO: 21) can do.

According to one embodiment of the present invention, as a result of testing the combination of GjUGT1 enzyme or NtUGT enzyme and CaUGT3 enzyme, SpUGT enzyme or NsUGT enzyme in vitro, crosine produced by GjUGT1 enzyme or NtUGT enzyme- 2, it was confirmed that the CaUGT3 enzyme can convert crosine-4 (FIG. 4).

Another embodiment of the present invention uses a UDP-glycosyltransferase derived from a Nicotiana tabacum strain or a Gardenia jasminoides strain and a UDP-glycosyltransferase derived from a Cataranthus roseus strain to cross A method for producing crosine-4 from cetin is provided.

Specifically, the method for producing crosine-4 from crocetin includes (a) the presence of NtUGT enzyme derived from Nicotiana tabacum strain or GjUGT1 enzyme derived from Gardenia jasminoides strain. a step of reacting crocetin with UDP-glucose to obtain a reactant containing crosine-2; and (b) adding and reacting CaUGT3 enzyme derived from Catharanthus roseus strain to the reaction to obtain crosin-4.

The enzymatic reaction may be carried out in a reactant containing the substrates Crocetin, UGT and UGT-glucose.

The temperature conditions for carrying out the enzymatic reaction are not particularly limited thereto, but as an example, may be 20 to 40 ℃, as another example, may be 25 to 35 ℃, as another example, 30 ℃ can be

The time condition for performing the enzymatic reaction is not particularly limited thereto, but as an example, it may be 10 to 240 minutes, as another example, it may be 60 to 180 minutes, and as another example, it may be 120 minutes. .

Meanwhile, in performing the reaction of step (b), UGT-glucose may be added in addition to the CaUGT3 enzyme.

Another embodiment of the present invention provides a composition for producing crosin-4 comprising a transformant or a culture product of the transformant into which the gene encoding each enzyme is introduced.

As described above, when UGT derived from a specific strain is used, crocetin or crosin-4 can be produced from crocetin or crosin-2. The composition containing the culture product may be used as a composition for producing crosin-4.

At this time, in the transformant, the GjUGT1 gene (SEQ ID NO: 2) or NtUGT gene (SEQ ID NO: 10) directly involved in the reaction for producing crosin-2 from crocetin must be introduced, as well as from crosin-2 The CaUGT3 gene (SEQ ID NO: 22) directly involved in the reaction for producing crosin-4 must also be introduced together.

The GjUGT1 gene (SEQ ID NO: 2) or NtUGT gene (SEQ ID NO: 10) and CaUGT3 gene (SEQ ID NO: 22) may be introduced together into a transformant using one vector, or transformants using each vector may be introduced individually.

In an embodiment of the present invention, it was confirmed that crosin-4 could be produced by a bioconversion process using a transformant into which the NtUGT gene (SEQ ID NO: 10) and the CaUGT3 gene (SEQ ID NO: 22) were introduced (FIG. 6) )

In addition, the transformant may be in a form in which the biosynthesis gene is additionally introduced. As such, the transformant in the form in which the gene biosynthesis is introduced can be used in the bioconversion process without separately using the substrate, crocetin. Crosin-4 can be produced by

In this case, the used crocetin biosynthesis gene is not particularly limited thereto, but may be a CsCCD2 gene or aldH gene.

In addition, the composition for producing crosin-4 may further include crocetin and UGT-glucose necessary for production of crosin-4 in addition to the transformant.

Another embodiment of the present invention provides a method for producing crosine-4 from crocetin using a transformant into which a gene encoding each of the above enzymes has been introduced.

Specifically, the method for producing crosin-4 from crocetin includes culturing a transformant into which the gene encoding each enzyme is introduced.

At this time, even without separately adding crocetin and UDP-glucose to the medium used for the culture, crosin-4 can be produced by the metabolic process of the transformant performed during the culture. However, in order to improve the productivity of crosin-4, the medium may additionally contain crocetin and UDP-glucose.

In addition, it may further comprise the step of recovering crosin-4 from the culture obtained through the culture.

According to one embodiment of the present invention, in one embodiment of the present invention, crosin-4 is converted into It was confirmed that it can be produced (FIG. 6)

Using the method for producing crosin-4 provided in the present invention, crosin-4 can be efficiently produced using a simple enzymatic reaction or bioconversion process, so it can be widely used for economical production of saffron compounds. will be.

1 is an electrophoretic photograph showing the result of expressing a candidate gene of a sugar transferase capable of converting crocetin to crosin-2.
2 is a graph showing the results of converting crocetin into crosin-2 using GjUGT1 enzyme, GT1-316 enzyme, or NtUGT enzyme under laboratory conditions ( in vitro).
3 is an electrophoretic photograph showing the result of expressing a candidate gene of a sugar transferase capable of converting crosin-2 to crosin-4.
4 is a graph showing the result of converting crocetin to crosine-4 using a combination of GjUGT1 enzyme or NtUGT enzyme and CaUGT3 enzyme, SpUGT enzyme or NsUGT enzyme under laboratory conditions ( in vitro).
5 is a schematic diagram showing the composition of a transformed microorganism and a graph showing a growth curve.
6 is a graph showing the results of the production of crosin-2 and crosin-4 using the transformed microorganism.

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

Example 1: Identification of primary sugar transferase

To discover a sugar transferase (UDP-glucosyltransferase-1, UGT-1) that can convert crocetin into crocetin-2 (Crocetin diglucosyl-ester), UGT75L6, a sugar transferase derived from Gardenia jasminoides strain by performing a BLAST search on the basis of the gene, the gene derived from Gardenia jasminoides GjUGT1 strain (SEQ ID NO: 2), the GT1-316 gene derived from Populus sp (Populus fremontii X Populus angustifolia) (SEQ ID NO: 6), Nicotiana tabacum The NtUGT gene (SEQ ID NO: 10) derived from the strain, the FaGT2 gene (SEQ ID NO: 14) derived from the Fragaria ananassa strain and the StUGT gene (SEQ ID NO: 18) derived from the Solanum tuberosum strain were discovered, and their nucleotide sequences were codon-optimized After that, it was synthesized.

At this time, the base sequence of each primer used is as follows:

GjUGT1_F: 5'-CGGAATTCATGGTTCAGCAGCGTCAC-3' (SEQ ID NO: 3)

GjUGT1_R: 5'-CCGCTCGAGGTTGCTCTCCGCTTGATAG-3' (SEQ ID NO: 4)

GT1-316_F: 5'-CGGGATCCATGGTCTCCGAGAGTCTAG-3' (SEQ ID NO: 7)

GT1-316_R: 5'-ACGCGTCGACTGCGGATGATCCGACTAAC-3' (SEQ ID NO: 8)

NtUGT_F: 5'-CGGGATCCATGGTTCAACCTCATGTCCT-3' (SEQ ID NO: 11)

NtUGT_R: 5'-CCCAAGCTTACAACCTTTTCCTACTTCTTGC-3' (SEQ ID NO: 12)

FaGT2_F: 5'-CGGGATCCATGGGTAGTGAGTCTT-3' (SEQ ID NO: 15)

FaGT2_R: 5'-CCCAAGCTTTGACTCTACTAACTCGACTT-3' (SEQ ID NO: 16)

StUGT_F: 5'-CCGGAATTCATGGTTCAACCTCATGTGCTT-3' (SEQ ID NO: 19)

StUGT_R: 5'-CCGCTCGAGACAAGATTTTCCCACTTCCTG-3' (SEQ ID NO: 20)

Each of the synthesized pET21α(+) vectors cloned together with the empty vector pET21α(+) was introduced into the BL21 strain, respectively, and then cultured in 50 mL of LB (Luria bertani) medium at 250 rpm under aerobic conditions. In the case of incubation temperature, culture was performed at 37°C, and when the OD reached 0.6, 1 mM IPTG was inducted, followed by incubation at 30°C for 4 hours. Thereafter, the cells were recovered by centrifugation at 4° C. and 3800 rpm for 20 minutes and washed with 50 mL of 50 mM Tris buffer (pH 7.0). Then, suspended in 10 mL of Tris buffer, treated with 0.2 mg/mL lysozyme and 0.1 mM PMSF, pulsed at 25% amp. for 10 seconds and disrupted by sonication with rest for 10 seconds. Thereafter, centrifugation was performed at 4° C. 13200 rpm for 10 minutes to separate a supernatant (soluble protein) and a precipitate (insoluble pellet), and the precipitate was suspended in a small amount of Tris buffer. Using the Bradford protein quantification method, the same amount of protein was obtained, and protein expression was confirmed through SDS-PAGE analysis (FIG. 1).

1 is an electrophoretic photograph showing the result of expressing a candidate gene of a sugar transferase capable of converting crocetin to crosin-2.

As shown in FIG. 1, various sugar transferases having a size of 52030 to 61389Da were expressed, GjUGT1 gene (SEQ ID NO: 2), GT1-316 gene (SEQ ID NO: 6) and NtUGT gene (SEQ ID NO: 10) were normally expressed, but , FaGT2 gene (SEQ ID NO: 14) and StUGT gene (SEQ ID NO: 18) were confirmed not to be expressed normally.

Then, it was evaluated in vitro whether the three normally expressed sugar transferases (GjUGT1, GT1-316 and NtUGT) could convert crocetin to crosin-2.

Roughly, the reaction volume for confirming glucose transferase activity is 100 μl, and the composition is 50 mM Tris-HCl buffer (pH 7.0), 5 mM UDP-glucose, 0.1 mM encapsulated crocetin, and 25 ㎍ cell lysate (crude protein extract) was prepared

After adding all final concentration 50 mM Tris-HCl buffer (pH 7.0), 5 mM UDP-glucose corresponding to the substrate, and 0.1 mM encapsulated crocetin, finally, 25 μg cell lysate (GjUGT1 enzyme, GT1-316 enzyme or NtUGT enzymes) were added and reacted at 30°C for 2 hours. Then, 200 μl of cold methanol (-20°C chilled MeOH) was added to terminate the reaction. Each reaction sample was centrifuged at 4° C., 13200 rpm for 10 minutes, and it was confirmed how much Crocetin was converted to Croxin-2 through HPLC analysis of the supernatant ( FIG. 2 ). At this time, crocetin and crosin-2 were confirmed through HPLC retention time, ultraviolet-visible light (UV-Vis) spectrum. Roughly, A: 100% MeOH (2% formic acid) and B: 100% DDW (2% formic acid) were analyzed as mobile phases. For gradient conditions, start with solvent A at 50%, with solvent A at 80% for 60 minutes, then with solvent A at 100% for 80 minutes, then again with solvent A at 50% for 90 minutes, then with solvent A at 50 % was continued until 100 minutes and a total of 100 minutes were analyzed. A Zorbax eclipse XDB-C18 column (4.6150 mm, 5 ; Agilent Technology) was analyzed as a stationary phase at a flow rate of 0.8 mL/min.

2 is a graph showing the results of converting crocetin into crosin-2 using GjUGT1 enzyme, GT1-316 enzyme, or NtUGT enzyme under laboratory conditions ( in vitro).

As shown in FIG. 2 , GT1-316 enzyme failed to convert crossetin to crossin-2, and GjUGT1 enzyme converted most of crossetin to crossin-1 and only a small amount of crossetin to crossin-2. However, it was confirmed that the NtUGT enzyme converts crocetin to the equivalent level of crosin-1 and crosin-2.

Therefore, it was confirmed that the enzymes capable of converting crocetin to crosin-2 are the NtUGT enzyme and the GjUGT1 enzyme.

Example 2: Identification of secondary sugar transferase

Crocin -2 to -4 to crocin (Crocetin digentiobiosyl-ester) to identify transferase (UDP-glucosyltransferase-2, UGT -2) each capable of converting, a CaUGT3 gene derived from Catharanthus roseus isolates (SEQ ID NO: 22), the NsUGT gene (SEQ ID NO: 26) derived from the Nicotiana sylvestris strain and the SpUGT gene (SEQ ID NO: 30) derived from the Solanum pennellii strain were discovered, and their nucleotide sequences were codon-optimized, and then synthesized, in the above Example It was expressed by the method of 3 (FIG. 3). At this time, the base sequence of each primer used in the synthesis of each gene is as follows:

CaUGT3_F: 5'-CGGGATCCATGGCCACCGAACAGCA-3' (SEQ ID NO: 23)

CaUGT3_R: 5'-ACGCGTCGACTACGCACAATTGCTTCAGC-3' (SEQ ID NO: 24)

NsUGT_F: 5'-CGGGATCCATGGACACAGAACACAGCA-3' (SEQ ID NO: 27)

NsUGT_R: 5'-ACGCGTCGACCAACATCATGTTATTACTTTTGC-3' (SEQ ID NO: 28)

SpUGT_F: 5'-CGGGATCCATGGGAACTCAAGTTACAGAA-3' (SEQ ID NO: 31)

SpUGT_R: 5'-ACGCGTCGACATTGGTGTTGATCTTACAGAAC-3' (SEQ ID NO: 32)

3 is an electrophoretic photograph showing the result of expressing a candidate gene of a sugar transferase capable of converting crosin-2 to crosin-4.

As shown in FIG. 3 , it was confirmed that various sugar transferases having a size of 50 to 52 kDa were expressed.

Then, whether each of the expressed sugar transferases can convert crosin-2 to crosin-4 was evaluated under laboratory conditions (in vitro ).

Roughly, 16 μg of GjUGT1 or NtUGT expressed in Example 3 was added to a mixture of 50 mM Tris-HCl (pH 7.0), 5 mM UDP-glucose, and 0.1 mM encapsulated crocetin, and reacted at 30° C. for 30 minutes. . Then, 50 mM Tris-HCl (pH 7.0) and 7.5 mM UDP-glucose were added, and then, 55 μg of CaUGT3, SpUGT or NsUGT was combined and treated, followed by reaction at 30° C. for 2 hours. Finally, 200 μl of cold methanol (-20°C chilled MeOH) was added to terminate the reaction. Each reaction sample was centrifuged at 4° C. and 13200 rpm for 10 minutes to determine how much crossetin was converted to crossin-4 through HPLC analysis of the supernatant ( FIG. 4 ).

Unlike the analysis method for confirming the conversion to crosin-2, the analysis was conducted using different mobile phase and gradient conditions to prevent the retention time of substances from being concentrated in the front time. A: 100% ACN and B: 100% DDW were analyzed as mobile phases. For gradient conditions, start with solvent A at 20%, with solvent A at 40% for 20 minutes, with solvent A at 60% for 25 minutes, with solvent A at 80% for 30 minutes, and with solvent A 100% The analysis was conducted for a total of 45 minutes by setting the solvent A to 35 minutes, 100% solvent A for 40 minutes, and 20% solvent A to 45 minutes. A Zorbax eclipse XDB-C18 column (4.6150 nm, 5 μm; Agilent Technology) was analyzed as a stationary phase at a flow rate of 1 mL/min.

4 is a graph showing the results of converting crocetin to crosine-4 using a combination of GjUGT1 enzyme or NtUGT enzyme and CaUGT3 enzyme, SpUGT enzyme or NsUGT enzyme under laboratory conditions ( in vitro).

As shown in FIG. 4 , it was confirmed that only the combination of the GjUGT1 enzyme or the NtUGT enzyme and the CaUGT3 enzyme could produce crosine-4 from crocetin.

Example 3: Production of Crosin-4 using a transgenic microorganism

Crocetin-4 was produced from crocetin by using the transgenic microorganism into which the genes of each enzyme identified in Examples 1 and 2 were introduced.

Example 3-1: Advancement and modularization of metabolic circuit genes

First, in the chromosome of E. coli MG1655 strain, E. coli-derived ispA (geranyl diphosphate/farnesyl diphosphate synthase), idi (isopentenyl-diphosphate Δ-isomerase), dxs (1-deoxy-D-xylulose-5-phosphate synthase) and dxr ( 1-deoxy-D-xylulose 5-phosphate reductoisomerase) genes were introduced, and these introduced genes were transformed to be expressed by the lac promoter, thereby preparing an E. coli strain (primary mutant) with enhanced MEP metabolic circuit.

Next, the CrtE, CrtB, CrtI, CrtY and CrtZ genes, which are zeaxanthin synthesis genes derived from Pantoea agglomerans, are introduced into the prepared primary mutant strain, and these introduced genes are transformed to be expressed by the trc promoter, and the MEP metabolic cycle is strengthened and produced a ZEA-1 strain (secondary mutant) capable of producing zeaxanthin.

Example 3-2: Construction of expression vector containing crocetin biosynthesis gene

First, the gene of CsCCD2, a carotenoid cleaving enzyme used for biosynthesis of crocetin dialdehyde, was synthesized and amplified, and the amplified product was cloned into EcoRI and HindIII sites of pKK223-3 vector. Then, the expression vector pSTVM_CsCCD2 was constructed by subcloning in the BglII and NotI sites of the pSTVM vector.

The base sequences of the primers used for cloning and subcloning of the CsCCD2 gene are as follows:

CsCCD2_F ; 5'-CGGAATTCATGGCGAACAAAGAAGAGG-3' (SEQ ID NO: 33)

CsCCD2_R ; 5'-CCCAAGCTTTTAGGTCTCCGCTTGATGC-3' (SEQ ID NO: 34)

sub_CCD2_F ; 5'-GGAAGATCTGCTGTGCAGGTCGTAAA-3' (SEQ ID NO: 37)

sub_CCD2_R ; 5'-ATAAGAATGCGGCCGCGAAACGCAAAAAGGCCA-3' (SEQ ID NO: 38)

Next, the gene of aldH used for biosynthesis of crocetin was obtained and amplified from Synechococcus elongatus PCC 7942 strain, and the amplified product was cloned into the XbaI and EcoRI sites of the pUCM vector. Then, it was subcloned into the prepared pSTVM_CsCCD2 vector to construct pSTVM_CsCCD2_aldH7942.

The nucleotide sequences of the primers used for cloning and subcloning of the aldH gene are as follows:

aldH_F ; 5'-GCTCTAGAAGGAGGATTACAAAATGACTGCTGTCGTTCTCC-3' (SEQ ID NO: 35)

aldH_R ; 5'-CGGAATTCCTAGAGCTTGCGGAAG-3' (SEQ ID NO: 36)

sub_aldH_F ; 5'-AAGAGCGUCTAGAAGGAGGATTACAAAA-3' (SEQ ID NO: 39)

sub_aldH_R ; 5'-AGCTGATUGTACTGAGAGTGCACCAT-3' (SEQ ID NO: 40)

Example 3-3: Construction of expression vector containing glycotransferase gene

First, the NtUGT gene confirmed to be suitable for the production of crosin-2 from crocetin through Example 1 was cloned into the pUCrop vector to construct the pUCrop_NtUGT vector. In this case, the base sequence of the primer used in the cloning is as follows:

pUCrop_NtUGT_F: 5'-GCTCTAGAAGAGGATTACAAAATGGTTCAACCTCATGTCC-3' (SEQ ID NO: 41)

pUCrop_NtUGT_R: 5'-TCCCCCCGGGTTAACAACCTTTTCCTACTTCTTG-3' (SEQ ID NO: 42)

Next, the CaUGT3 gene confirmed to be suitable for the production of crosin-4 from crosin-2 through Example 2 was cloned into the prepared pUCrop_NtUGT vector, and an expression vector containing a glycosyltransferase gene (pUCrop_NtUGT_CaUGT3) was produced. In this case, the base sequence of the primer used in the cloning is as follows:

pUCrop_NtUGT_CaUGT_F: 5'-TCCCCCCGGGAGGAGGACAGCTAAATGGCCACCGAACAGC-3' (SEQ ID NO: 43)

pUCrop_NtUGT_CaUGT_R: 5'-AAGGAAAAAAGCGGCCGCTTATACGCACAATTGCTTCAGC-3' (SEQ ID NO: 44)

Example 3-4: Production of transformed microorganisms

To the ZEA-1 strain prepared in Example 3-1, the expression vector containing the crocetin biosynthesis gene prepared in Example 3-2 and the expression vector containing the glycosyltransferase gene prepared in Example 3-3 Introduced and spread on a plate medium to which ampicillin and chloramphenicol were added, the colonies were secured by stationary culture at 37° C. for 12 hours. The obtained colonies were inoculated in 150ml TB (terrific broth) medium in a 500ml flask and cultured with shaking at 37°C and 250rpm for 12 hours.

Non-continuous fermentation is performed using the culture medium obtained through shaking culture for 12 hours. At this time, the medium composition of the discontinuous fermentation was 1.5L TB (terrific broth) medium containing 20 g/L of glycerol as a carbon source, and 50 μg/ml chloramphenicol and 100 μg/ml ampicillin were all added. In the case of incubation temperature, culture was carried out at 30°C, and when the OD600 reached between 5 and 6, the temperature was changed to 20°C, and the culture was continued. During incubation, the oxygen concentration was 30%, and air supply was performed at 1vvm (FIG. 5).

5 is a schematic diagram showing the composition of a transformed microorganism and a graph showing a growth curve.

After the fermentation was completed, the crosine compound contained in the culture was confirmed through HPLC analysis (FIG. 6).

At this time, HPLC was analyzed using A: 100% MeOH (25 mM formic acid) and B: 100% DDW (25 mM formic acid) as mobile phases. The concentration gradient conditions were: 50% of solvent A for 50 minutes, 80% of solvent A for 60 minutes, and 100% of solvent A for 80 minutes. A Zorbax eclipse XDB-C18 column (4.6×150 mm or 250 mm, 5 μm; Agilent Technology) was analyzed as a stationary phase at a flow rate of 0.8 ml/min. For structural analysis, HPLC retention time, absorption spectrum, and mass spectrum were compared and analyzed. For mass spectrum, both positive and negative modes were monitored using Varian 1200L LC/MS system, and APCI (atmosphere pressure chemical ionization) module was used for ionization.

6 is a graph showing the results of the production of crosin-2 and crosin-4 using the transformed microorganism.

As shown in FIG. 6 , it was confirmed that crosin-2 and crosin-4 were produced.

As can be seen from the above results, crosin-2 produced from the transformed microorganism is converted from crocetin produced by the crocetin biosynthesis gene introduced into the microorganism, and crosin-2 from the transformed crosin-2 -4 was analyzed to be generated.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Process for preparing crocin-4 using enzyme reaction <130> KPA190916-KR <160> 44 <170> KopatentIn 2.0 <210> 1 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> recombinant GjUGT1 <400> 1 Met Val Gln Gln Arg His Val Leu Leu Ile Thr Tyr Pro Ala Gln Gly 1 5 10 15 His Ile Asn Pro Ala Leu Gln Phe Ala Gln Arg Leu Leu Arg Met Gly 20 25 30 Ile Gln Val Thr Leu Ala Thr Ser Val Tyr Ala Leu Ser Arg Met Lys 35 40 45 Lys Ser Ser Gly Ser Thr Pro Lys Gly Leu Thr Phe Ala Thr Phe Ser 50 55 60 Asp Gly Tyr Asp Asp Gly Phe Arg Pro Lys Gly Val Asp His Thr Glu 65 70 75 80 Tyr Met Ser Ser Leu Ala Lys Gin Gly Ser Asn Thr Leu Arg Asn Val 85 90 95 Ile Asn Thr Ser Ala Asp Gin Gly Cys Pro Val Thr Cys Leu Val Tyr 100 105 110 Thr Leu Leu Leu Pro Trp Ala Ala Thr Val Ala Arg Glu Cys His Ile 115 120 125 Pro Ser Ala Leu Leu Trp Ile Gln Pro Val Ala Val Met Asp Ile Tyr 130 135 140 Tyr Tyr Tyr Phe Arg Gly Tyr Glu Asp Asp Val Lys Asn Asn Ser Asn 145 150 155 160 Asp Pro Thr Trp Ser Ile Gln Phe Pro Gly Leu Pro Ser Met Lys Ala 165 170 175 Lys Asp Leu Pro Ser Phe Ile Leu Pro Ser Ser Asp Asn Ile Tyr Ser 180 185 190 Phe Ala Leu Pro Thr Phe Lys Lys Gln Leu Glu Thr Leu Asp Glu Glu 195 200 205 Glu Arg Pro Lys Val Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Gln 210 215 220 Ala Leu Lys Ala Ile Glu Ser Tyr Asn Leu Ile Ala Ile Gly Pro Leu 225 230 235 240 Thr Pro Ser Ala Phe Leu Asp Gly Lys Asp Pro Ser Glu Thr Ser Phe 245 250 255 Ser Gly Asp Leu Phe Gln Lys Ser Lys Asp Tyr Lys Glu Trp Leu Asn 260 265 270 Ser Arg Pro Ala Gly Ser Val Val Tyr Val Ser Phe Gly Ser Leu Leu 275 280 285 Thr Leu Pro Lys Gln Gln Met Glu Glu Ile Ala Arg Gly Leu Leu Lys 290 295 300 Ser Gly Arg Pro Phe Leu Trp Val Ile Arg Ala Lys Glu Asn Gly Glu 305 310 315 320 Glu Glu Lys Glu Glu Asp Arg Leu Ile Cys Met Glu Glu Leu Glu Glu 325 330 335 Gln Gly Met Ile Val Pro Trp Cys Ser Gln Ile Glu Val Leu Thr His 340 345 350 Pro Ser Leu Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Leu 355 360 365 Glu Thr Leu Val Cys Gly Val Pro Val Val Ala Phe Pro His Trp Thr 370 375 380 Asp Gln Gly Thr Asn Ala Lys Leu Ile Glu Asp Val Trp Glu Thr Gly 385 390 395 400 Val Arg Val Val Pro Asn Glu Asp Gly Thr Val Glu Ser Asp Glu Ile 405 410 415 Lys Arg Cys Ile Glu Thr Val Met Asp Asp Gly Glu Lys Gly Val Glu 420 425 430 Leu Lys Arg Asn Ala Lys Lys Trp Lys Glu Leu Ala Arg Glu Ala Met 435 440 445 Gln Glu Asp Gly Ser Ser Asp Lys Asn Leu Lys Ala Phe Val Glu Asp 450 455 460 Ala Gly Lys Gly Tyr Gln Ala Glu Ser Asn 465 470 <210> 2 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> recombinant GjUGT1 <400> 2 atggttcagc agcgtcacgt tttgttgatt acctatccag cacagggtca cattaaccca 60 gcgttgcagt tcgcacagag attgttgcgt atgggtattc aagttaccct ggcgaccagc 120 gtgtacgctc tgagccgtat gaaaaagagc agcggtagca ccccgaaagg tctgaccttt 180 gcaaccttca gcgatggtta cgatgacggt tttcgtccga aaggtgttga ccataccgaa 240 tatatgagca gcctggcgaa gcaaggtagc aataccctgc gtaatgtgat caacaccagc 300 gctgatcagg gttgcccggt tacctgtttg gtgtatacct tgttgctgcc atgggctgct 360 accgttgcac gtgaatgcca cattccgagc gcgctgctgt ggatccaacc ggttgctgtg 420 atggatatct actattacta tttccgtggt tatgaagatg acgttaagaa taacagcaat 480 gacccgacct ggagcatcca gtttccgggt ctgccgagca tgaaagctaa ggatctgccg 540 agcttcattc tgccgagcag cgacaacatc tacagctttg cactgccgac cttcaaaaag 600 caactggaaa ccctggatga agaagaacgt ccgaaagttc tggtgaatac ctttgacgcg 660 ctggaaccgc aggcactgaa ggcgattgaa agctataacc tgattgcaat tggtccactg 720 accccgagcg cgttcctgga tggtaaagac ccgagcgaaa ccagctttag cggtgacctg 780 tttcagaaaa gcaaggacta caaggaatgg ctgaatagcc gtccggctgg tagcgttgtg 840 tatgttagct ttggtagcct gctgaccctg ccgaaacaac agatggaaga aattgcacgt 900 ggtctgctga agagcggtcg tccgttcctg tgggttattc gtgcgaaaga aaacggtgaa 960 gaagaaaagg aagaagatcg tctgatttgc atggaagaac tggaagaaca gggtatgatt 1020 gttccgtggt gtagccagat cgaagtgctg acccatccga gcctgggttg ctttgttacc 1080 cactgtggtt ggaatagcac cctggaaacc ctggtttgcg gtgtgccggt tgtggctttc 1140 ccgcattgga ccgatcaagg taccaatgca aaactgatcg aagacgtttg ggaaaccggt 1200 gtgcgtgttg tgccgaacga agatggtacc gttgaaagcg acgaaattaa acgttgtatc 1260 gaaaccgtta tggatgacgg tgaaaaaggt gtggaactga agcgtaacgc gaaaaagtgg 1320 aaggaactgg ctcgtgaagc aatgcaggaa gatggtagca gcgacaaaaa cctgaaagcg 1380 ttcgtggagg atgcgggcaa gggctatcaa gcggagagca actaa 1425 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggaattcat ggttcagcag cgtcac 26 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccgctcgagg ttgctctccg cttgatag 28 <210> 5 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> recombinant GT1-316 <400> 5 Met Val Ser Glu Ser Leu Gly His Leu Phe Leu Val Ser Phe Pro Gly 1 5 10 15 Gln Gly His Val Asn Pro Leu Leu Arg Leu Gly Lys Ile Leu Ala Ser 20 25 30 Lys Gly Phe Leu Val Thr Phe Ser Thr Thr Glu Thr Thr Gly Glu Gln 35 40 45 Met Arg Lys Ala Ser Asp Ile Ile Asp Lys Leu Thr Pro Phe Gly Asp 50 55 60 Gly Phe Ile Arg Phe Glu Phe Ile Ala Asp Gly Trp Glu Glu Asp Glu 65 70 75 80 Pro Arg Arg Gln Asp Leu Asp Gln Tyr Leu Leu Gln Leu Glu Leu Val 85 90 95 Gly Lys Gln Val Ile Pro Gln Met Ile Lys Lys Asn Ala Glu Gln Gly 100 105 110 Arg Pro Val Ser Cys Leu Ile Asn Asn Pro Phe Ile Pro Trp Val Thr 115 120 125 Asp Val Ala Thr Thr Leu Gly Leu Pro Ser Ala Met Leu Trp Val Gln 130 135 140 Ser Cys Ala Cys Phe Ala Ser Tyr Tyr His Tyr Tyr His Gly Thr Val 145 150 155 160 Pro Phe Pro Asp Glu Glu His Pro Glu Ile Asp Val Gln Leu Pro Trp 165 170 175 Met Pro Leu Leu Lys Tyr Asp Glu Val Pro Ser Tyr Leu Tyr Pro Thr 180 185 190 Thr Pro Tyr Pro Phe Leu Arg Arg Ala Ile Leu Gly Gln Tyr Lys Asn 195 200 205 Leu Asp Lys Pro Phe Cys Ile Leu Met Glu Thr Phe Glu Glu Leu Glu 210 215 220 Pro Glu Leu Ile Lys His Met Ser Glu Ile Phe Pro Ile Lys Ala Val 225 230 235 240 Gly Pro Leu Phe Arg Asn Thr Lys Ala Pro Lys Thr Thr Val His Gly 245 250 255 Asp Phe Leu Lys Ala Asp Asp Cys Ile Glu Trp Leu Asp Thr Lys Pro 260 265 270 Pro Ser Ser Val Val Tyr Val Ser Phe Gly Ser Val Val Gln Leu Lys 275 280 285 Gln Asp Gln Trp Asn Glu Ile Ala Tyr Gly Leu Leu Asn Ser Gly Val 290 295 300 Ser Phe Leu Leu Val Met Lys Pro Ala His Lys Asp Ala Gly His Asp 305 310 315 320 Leu Leu Val Leu Pro Asp Gly Phe Leu Glu Lys Ala Gly Asp Arg Gly 325 330 335 Lys Val Val Gln Trp Ser Pro Gln Glu Lys Val Leu Gly His Pro Ser 340 345 350 Val Ala Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Met Glu Ala 355 360 365 Leu Thr Ser Gly Met Pro Val Val Ala Phe Pro Gln Trp Gly Asp Gln 370 375 380 Val Thr Asn Ala Lys Tyr Leu Val Asp Ile Leu Lys Val Gly Val Arg 385 390 395 400 Met Cys Arg Gly Glu Ala Glu Asn Lys Leu Ile Thr Arg Asp Glu Ile 405 410 415 Glu Lys Cys Leu Leu Glu Ala Thr Val Gly Pro Lys Ala Val Glu Met 420 425 430 Lys Gln Asn Ala Met Lys Trp Lys Glu Ala Ala Glu Ala Ala Val Ala 435 440 445 Glu Gly Gly Ser Ser Asp Gln Asn Ile Arg Tyr Phe Thr Asp Asp Ile 450 455 460 Val Lys Ala Asn Glu Ser Glu Ile Ala Arg Lys Cys Ile Gly Ser Asn 465 470 475 480 Glu Phe Pro Val Ser Val Val Val Lys Ser Asn Glu Lys Val Asp Glu 485 490 495 Leu Val Gly Ser Ser Ala 500 <210> 6 <211> 1506 <212> DNA <213> Artificial Sequence <220> <223> recombinant GT1-316 <400> 6 atggtctccg agagtctagg tcacctattc ctagtatctt ttccggggca ggggcatgta 60 aaccctttat taagattagg aaaaatcctt gctagtaaag gtttcctagt taccttttcc 120 accaccgaga caacagggga gcagatgcgt aaggcaagtg acatcataga taaacttaca 180 ccattcggtg acggcttcat tcgttttgaa ttcattgccg acggttggga ggaggatgaa 240 ccccgtaggc aagaccttga tcaatatcta ctacaacttg aattagtggg caagcaggta 300 ataccgcaaa tgattaagaa gaatgccgaa cagggtagac ccgtgtcctg cctgatcaac 360 aaccccttca tcccatgggt cacagatgtc gctacgactc taggccttcc ttcagctatg 420 ctgtgggtac aaagttgtgc ttgttttgcg agctactatc actactacca tggcacagtc 480 ccttttcccg atgaggagca ccccgaaatt gatgtacagc taccgtggat gcctttgctt 540 aagtatgatg aggtccctag ttatctttac cctacgacgc cgtacccctt tttaaggcgt 600 gcaatattgg gtcagtataa aaacctggac aagccgtttt gcatcttaat ggagactttt 660 gaagagcttg aaccggaact tatcaagcat atgagtgaaa tttttccaat taaggcggtt 720 ggaccgctat tccgtaacac aaaagcgccc aaaacaactg tgcacggcga cttcttaaaa 780 gcagatgatt gtatagaatg gttggatacc aaacctccat cttctgtcgt ttacgtatca 840 ttcggctctg ttgtgcagct aaaacaggac caatggaacg agatagctta cggtttactt 900 aactctgggg tttccttttt attggtgatg aagcccgctc ataaagacgc tgggcatgac 960 cttctagtac ttcctgacgg cttcttagag aaagctggcg acagagggaa ggtggtccag 1020 tggtcacctc aagaaaaggt actaggtcac ccgagcgtag cctgctttgt aactcactgt 1080 ggatggaaca gcacgatgga agctctgacc tctggaatgc ccgtggtcgc ttttccccag 1140 tggggtgatc aggttacaaa tgcgaaatac ttggtagaca tactaaaggt aggagtcagg 1200 atgtgtaggg gagaggccga aaataagctt attacaagag atgaaattga aaaatgccta 1260 ttagaagcta cggtaggacc taaagctgtt gagatgaagc aaaacgccat gaaatggaag 1320 gaagctgctg aagccgcggt cgccgaaggt ggctcaagtg accaaaacat acgttacttt 1380 actgatgaca tcgtgaaagc aaatgagtcc gagattgcta gaaagtgtat cggctctaac 1440 gaattcccgg tgtcagtcgt tgttaaatct aatgagaaag tggatgagtt agtcggatca 1500 tccgca 1506 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cgggatccat ggtctccgag agtctag 27 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 acgcgtcgac tgcggatgat ccgactaac 29 <210> 9 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> recombinant NtUGT <400> 9 Met Val Gln Pro His Val Leu Leu Val Thr Phe Pro Ala Gln Gly His 1 5 10 15 Ile Asn Pro Cys Leu Gln Phe Ala Met Arg Leu Ile Arg Met Gly Ile 20 25 30 Glu Val Thr Phe Ala Thr Ser Val Phe Ala His Arg Arg Met Ala Lys 35 40 45 Thr Ala Thr Ser Thr Leu Pro Lys Gly Leu Asn Phe Ala Ala Phe Ser 50 55 60 Asp Gly Tyr Asp Asp Gly Phe Lys Ala Asp Glu His Asp Ser Gln His 65 70 75 80 Tyr Met Ser Glu Ile Lys Ser Arg Gly Ser Glu Thr Leu Lys Asp Ile 85 90 95 Ile Leu Lys Ser Ser Asp Glu Gly Arg Pro Val Thr Ser Leu Val Tyr 100 105 110 Ser Leu Leu Leu Pro Trp Ala Ala Asn Val Ala Arg Glu Phe His Ile 115 120 125 Pro Cys Ala Leu Leu Trp Ile Gln Pro Ala Thr Val Leu Asp Ile Tyr 130 135 140 Tyr Tyr Tyr Phe Asn Gly Ser Glu Asp Ala Ile Lys Gly Ser Thr Asn 145 150 155 160 Asp Pro Asn Trp Cys Ile Gln Leu Pro Asn Leu Pro Leu Leu Lys Ser 165 170 175 Gln Asp Leu Pro Ser Phe Leu Leu Ser Ser Asn Asn Asp Glu Lys Tyr 180 185 190 Ser Phe Ala Leu Pro Thr Phe Lys Glu Gln Leu Asp Thr Leu Asp Val 195 200 205 Glu Glu Asn Pro Lys Val Leu Val Asn Thr Phe Asp Ala Leu Glu Gln 210 215 220 Glu Glu Leu Lys Ala Ile Glu Arg Tyr Asn Leu Ile Gly Ile Gly Pro 225 230 235 240 Leu Ile Pro Ser Ser Phe Leu Asp Gly Lys Asp Pro Leu Asp Ser Ser 245 250 255 Phe Gly Gly Asp Leu Phe Gln Lys Ser Asn Asp Tyr Ile Glu Trp Leu 260 265 270 Asn Ser Lys Asp Asn Ser Ser Val Ile Tyr Ile Ser Phe Gly Ser Leu 275 280 285 Leu Asn Leu Ser Lys Asn Gln Lys Glu Glu Ile Ala Lys Gly Leu Ile 290 295 300 Glu Ile Lys Arg Pro Phe Leu Trp Val Ile Arg Asp Gln Glu Asn Gly 305 310 315 320 Lys Gly Asp Glu Lys Glu Glu Glu Lys Leu Ser Cys Met Met Glu Leu 325 330 335 Glu Lys Gln Gly Lys Ile Val Pro Trp Cys Ser Gln Leu Glu Val Leu 340 345 350 Thr His Pro Ser Leu Gly Cys Phe Val Ser His Cys Gly Trp Asn Ser 355 360 365 Thr Leu Glu Ser Leu Ser Thr Gly Val Pro Val Val Ala Phe Pro His 370 375 380 Trp Thr Asp Gln Gly Thr Asn Ala Lys Leu Ile Glu Asp Val Trp Lys 385 390 395 400 Ile Gly Val Arg Leu Lys Lys Asn Glu Asp Gly Val Val Glu Ser Glu 405 410 415 Glu Ile Lys Arg Cys Ile Asp Met Val Met Asn Gly Gly Glu Lys Gly 420 425 430 Glu Glu Met Arg Arg Asn Ala Gln Lys Trp Lys Glu Leu Ala Arg Glu 435 440 445 Ala Val Lys Glu Gly Gly Ser Ser Tyr Met Asn Leu Lys Ala Phe Val 450 455 460 Gln Glu Val Gly Lys Gly Cys 465 470 <210> 10 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> recombinant NtUGT <400> 10 atggttcaac ctcatgtcct acttgttaca tttccagcac aaggacatat taacccctgc 60 ttgcagtttg ccatgaggct gatccgtatg ggaatcgaag taacgtttgc tacctcagtg 120 tttgctcacc gtagaatggc taagaccgca acaagcaccc tgcccaaggg cctaaacttt 180 gccgcgttca gcgacggata cgacgatggg tttaaggcag acgagcatga ttcccagcac 240 tacatgtctg aaatcaagtc tagggggagc gagacgctga aggatataat tctgaagagt 300 tctgatgaag ggagacccgt cacgtccctt gtgtatagcc tgcttttacc ctgggctgca 360 aatgttgcga gggaatttca cataccctgt gcgttgctat ggatacaacc ggcaactgtt 420 ttggatattt attactacta ctttaatggg agtgaggacg ccataaaagg atctaccaac 480 gatccgaatt ggtgcatcca gttgccaaat ctacctctat tgaaatctca agacttacct 540 tccttcctac tgtcctcaaa taacgatgag aaatacagtt ttgctttgcc aacgttcaaa 600 gaacagctgg acacattgga tgtggaagaa aaccccaagg ttttggtcaa tacattcgat 660 gcgctagagc aggaagagct gaaggcgatt gagagatata atttaatcgg gatcggacct 720 ttgataccga gcagtttctt agatggaaag gacccgctgg attcaagttt tggtggcgac 780 ttattcaga agtccaacga ttatattgaa tggctaaact ccaaagataa cagtagcgta 840 atatatatct cattcggttc actgttgaac ctatccaaaa accagaagga ggaaatagcg 900 aaagggctaa tcgagataaa aaggccattc ttgtgggtca tcagagacca ggaaaatggc 960 aaaggcgatg aaaaagaaga agaaaagttg agttgtatga tggaactaga gaagcaaggt 1020 aaaatcgtac catggtgctc acaactagaa gtcttgactc acccctccct tggttgcttc 1080 gtctctcact gcggttggaa ttcaacactt gagagtttgt ctacaggcgt gccagtggtt 1140 gcgttccctc attggaccga tcagggaact aacgcgaaac ttatcgaaga cgtatggaag 1200 atcggtgtgc gtttgaagaa aaatgaagat ggagtagttg agagcgagga gataaaaagg 1260 tgtatagaca tggttatgaa cggaggtgaa aagggagaag agatgcgtcg taacgcacag 1320 aagtggaaag aactagctcg tgaagccgtt aaagagggag gaagttctta tatgaactta 1380 aaagccttcg tgcaagaagt aggaaaaggt tgt 1413 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cgggatccat ggttcaacct catgtcct 28 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cccaagctta caaccttttc ctacttcttg c 31 <210> 13 <211> 555 <212> PRT <213> Artificial Sequence <220> <223> recombinant FaGT2 <400> 13 Met Gly Ser Glu Ser Leu Val His Val Phe Leu Val Ser Phe Ile Gly 1 5 10 15 Gln Gly His Val Asn Pro Leu Leu Arg Leu Gly Lys Arg Leu Ala Ala 20 25 30 Lys Gly Leu Leu Val Thr Phe Cys Thr Ala Glu Cys Val Gly Lys Glu 35 40 45 Met Arg Lys Ser Asn Gly Ile Thr Asp Glu Pro Lys Pro Val Gly Asp 50 55 60 Gly Phe Ile Arg Phe Glu Phe Phe Lys Asp Arg Trp Ala Glu Asp Glu 65 70 75 80 Pro Met Arg Gln Asp Leu Asp Leu Tyr Leu Pro Gln Leu Glu Leu Val 85 90 95 Gly Lys Glu Val Ile Pro Glu Met Ile Lys Lys Asn Ala Glu Gln Gly 100 105 110 Arg Pro Val Ser Cys Leu Ile Asn Asn Pro Phe Ile Pro Trp Val Cys 115 120 125 Asp Val Ala Glu Ser Leu Gly Leu Pro Ser Ala Met Leu Trp Val Gln 130 135 140 Ser Ala Ala Cys Leu Ala Ala Tyr Tyr His Tyr Tyr His Gly Leu Val 145 150 155 160 Pro Phe Pro Ser Glu Ser Asp Met Phe Cys Asp Val Gln Ile Pro Ser 165 170 175 Met Pro Leu Leu Lys Tyr Asp Glu Val Pro Ser Phe Leu Tyr Pro Thr 180 185 190 Ser Pro Tyr Pro Phe Leu Arg Arg Ala Ile Leu Gly Gln Tyr Gly Asn 195 200 205 Leu Glu Lys Pro Phe Cys Ile Leu Met Asp Thr Phe Gln Glu Leu Glu 210 215 220 Ser Glu Ile Ile Glu Tyr Met Ala Arg Leu Cys Pro Ile Lys Ala Val 225 230 235 240 Gly Pro Leu Phe Lys Asn Pro Lys Ala Gln Asn Ala Val Arg Gly Asp 245 250 255 Phe Met Glu Ala Asp Asp Ser Ile Ile Gly Trp Leu Asp Thr Lys Pro 260 265 270 Lys Ser Ser Val Val Tyr Ile Ser Phe Gly Ser Val Val Tyr Leu Lys 275 280 285 Gln Glu Gln Val Asp Glu Ile Ala His Gly Leu Leu Ser Ser Gly Val 290 295 300 Ser Phe Ile Trp Val Met Lys Pro His Pro Asp Ser Gly Phe Glu 305 310 315 320 Leu Leu Val Leu Pro Glu Gly Phe Leu Glu Lys Ala Gly Asp Arg Gly 325 330 335 Lys Val Val Gln Trp Ser Pro Gln Glu Lys Ile Leu Glu His Pro Ser 340 345 350 Thr Ala Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Met Glu Ser 355 360 365 Leu Thr Ser Gly Met Pro Val Val Ala Phe Pro Gln Trp Gly Asp Gln 370 375 380 Val Thr Asp Ala Lys Tyr Leu Val Asp Glu Phe Lys Val Gly Val Arg 385 390 395 400 Met Cys Arg Gly Glu Ala Glu Asp Arg Val Ile Pro Arg Asp Glu Val 405 410 415 Glu Lys Cys Leu Leu Glu Ala Thr Ser Gly Ser Lys Ala Ala Glu Met 420 425 430 Lys Gln Asn Ala Leu Lys Trp Lys Ala Ala Ala Glu Ala Ala Phe Ser 435 440 445 Glu Gly Gly Ser Ser Asp Arg Asn Leu Gln Ala Phe Val Asp Glu Val 450 455 460 Arg Arg Ile Ser Ala Ser Leu Asn Ser Lys Ser Ser Ala Val Gly Tyr 465 470 475 480 Val Lys Ser Lys Ile Asn Gly Val Val Glu Tyr Val Asp Ser Lys Leu 485 490 495 Asn Gly Lys Ala Ala Pro Val Glu Glu Ala Asn Thr Arg Thr Asn Gly 500 505 510 Ile Ala Lys Val Glu Gln Pro Lys Ala Ala Asn Gly Lys Val Glu Ile 515 520 525 Ala Glu Leu Thr Pro Ile Asn Gly Lys Val Glu Ile Ala Glu Leu Lys 530 535 540 Pro Ile Asn Gly Lys Val Glu Leu Val Glu Ser 545 550 555 <210> 14 <211> 1665 <212> DNA <213> Artificial Sequence <220> <223> recombinant FaGT2 <400> 14 atgggtagtg agtctttggt tcacgtcttt cttgtgtcct ttattggcca aggacacgtc 60 aacccgcttc tgaggctggg gaaaagactg gcggctaaag ggctgcttgt aaccttttgc 120 acggcggagt gtgtcgggaa agaaatgagg aaatctaatg ggataaccga tgagcctaaa 180 ccagtaggag acggatttat acgttttgaa ttcttcaaag ataggtgggc agaagacgaa 240 cccatgcgtc aggacctgga tctatattta ccccagttgg agctagttgg aaaagaagtg 300 atacctgaga tgataaagaa gaacgcagaa caagggagac cagtatcatg cctaatcaat 360 aatccgttta ttccgtgggt ctgtgacgtc gccgaatctc ttggtttacc atccgcaatg 420 ctgtgggttc aatctgccgc ctgtctagct gcttactatc actactacca tggactagtc 480 ccattcccat ctgagtctga catgttttgt gatgtccaaa tacccagcat gccattgtta 540 aaatatgacg aggttcccag ctttttgtat ccaacgagcc catatccatt tttgagacgt 600 gcgatcttgg gtcaatacgg gaacttagag aagccgtttt gtatccttat ggatacgttt 660 caagaactag agtctgaaat aattgaatat atggccagat tatgtcccat aaaggccgtc 720 gggccattgt tcaagaatcc caaggctcaa aacgcggtta gaggcgactt tatggaagcc 780 gatgactcaa tcatcggatg gttggacaca aaacccaaga gttcagttgt ttacattagc 840 ttcgggagcg ttgtatactt aaagcaggag caagtggatg aaattgctca cggactttta 900 tcatccggcg tgtcttttat atgggtgatg aaacctcctc atccggatag cggatttgaa 960 ttgttggtat taccagaagg tttcctagaa aaggcgggag accgtggaaa agttgtgcaa 1020 tggagtccac aagaaaagat attagaacac ccttcaactg catgttttgt gacacactgc 1080 gggtggaata gtacgatgga aagtttaacc agcgggatgc ccgtggtcgc gtttccacag 1140 tggggggatc aggtaactga cgcgaagtac ctagtggatg aatttaaggt aggagttagg 1200 atgtgcagag gcgaggccga ggacagggtg attcctagag acgaggttga aaagtgtctt 1260 ttggaagcta cgtctggttc aaaagctgca gagatgaaac aaaatgcgct taagtggaaa 1320 gctgctgcag aggctgcgtt ctccgaagga ggatcttcag ataggaatct gcaagccttc 1380 gtcgacgaag tcagacgtat tagcgccagc ttaaattcta agtcctctgc tgtcggctat 1440 gtcaagagta agatcaacgg agtggtggaa tatgtcgata gtaagttaaa cgggaaagcc 1500 gccccggtgg aggaggcgaa cactcgtaca aatggcatcg caaaggtaga acaacctaag 1560 gcagcaaacg gcaaggtgga aatagctgaa ttaactccga ttaatggcaa ggttgagatt 1620 gccgaactga agccaataaa cgggaaagtc gagttagtag agtca 1665 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgggatccat gggtagtgag tctt 24 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cccaagcttt gactctacta actcgactt 29 <210> 17 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> recombinant StUGT <400> 17 Met Val Gln Pro His Val Leu Leu Val Thr Phe Pro Ala Gln Gly His 1 5 10 15 Ile Asn Pro Ser Leu Gln Phe Ala Lys Arg Leu Ile Lys Met Gly Ile 20 25 30 Glu Val Thr Phe Thr Thr Ser Val Phe Ala His Arg Arg Met Ala Lys 35 40 45 Thr Ala Ala Ser Asn Ala Pro Lys Gly Leu Asn Leu Ala Ala Phe Ser 50 55 60 Asp Gly Phe Asp Asp Gly Phe Lys Ser Asn Val Asp Asp Ser Lys Arg 65 70 75 80 Tyr Met Ser Glu Ile Arg Ser Arg Gly Ser Gln Thr Leu Arg Asp Ile 85 90 95 Ile Leu Lys Ser Ser Asp Glu Gly Arg Pro Val Thr Ser Leu Val Tyr 100 105 110 Thr Leu Leu Leu Pro Trp Ala Ala Glu Val Ala Arg Glu Leu His Ile 115 120 125 Pro Ser Ala Leu Leu Trp Ile Gln Pro Ala Thr Val Leu Asp Ile Tyr 130 135 140 Tyr Tyr Tyr Phe Asn Gly Tyr Glu Asp Glu Met Lys Cys Ser Ser Asn 145 150 155 160 Asp Pro Asn Trp Ser Ile Gln Leu Pro Arg Leu Pro Leu Leu Lys Ser 165 170 175 Gln Asp Leu Pro Ser Phe Leu Val Ser Ser Ser Ser Lys Asp Asp Lys 180 185 190 Tyr Ser Phe Ala Leu Pro Thr Phe Lys Glu Gln Leu Asp Thr Leu Asp 195 200 205 Gly Glu Glu Asn Pro Lys Val Leu Val Asn Thr Phe Asp Ala Leu Glu 210 215 220 Leu Glu Pro Leu Lys Ala Ile Glu Lys Tyr Asn Leu Ile Gly Ile Gly 225 230 235 240 Pro Leu Ile Pro Ser Ser Phe Leu Gly Gly Lys Asp Ser Leu Glu Ser 245 250 255 Ser Phe Gly Gly Asp Leu Phe Gln Lys Ser Asp Asp Asp Tyr Met Glu 260 265 270 Trp Leu Asn Thr Lys Pro Lys Ser Ser Ile Val Tyr Ile Ser Phe Gly 275 280 285 Ser Leu Leu Asn Leu Ser Arg Asn Gln Lys Glu Glu Ile Ala Lys Gly 290 295 300 Leu Ile Glu Ile Lys Arg Pro Phe Leu Trp Val Ile Arg Asp Gln Glu 305 310 315 320 Asn Ile Lys Glu Val Glu Lys Glu Glu Glu Lys Leu Ser Cys Met Met 325 330 335 Glu Leu Glu Lys Gln Gly Lys Ile Val Pro Trp Cys Ser Gln Leu Glu 340 345 350 Val Leu Thr His Pro Ser Leu Gly Cys Phe Val Ser His Cys Gly Trp 355 360 365 Asn Ser Thr Leu Glu Ser Leu Ser Ser Gly Val Pro Val Val Ala Phe 370 375 380 Pro His Trp Thr Asp Gin Gly Thr Asn Ala Lys Leu Ile Glu Asp Val 385 390 395 400 Trp Lys Thr Gly Val Arg Met Arg Val Ser Glu Asp Gly Val Val Glu 405 410 415 Ser Glu Glu Ile Lys Arg Cys Ile Glu Ile Val Met Asp Gly Gly Glu 420 425 430 Lys Gly Glu Glu Met Arg Lys Asn Ala Gln Lys Trp Lys Glu Leu Ala 435 440 445 Arg Glu Ala Val Lys Glu Gly Gly Ser Ser Glu Val Asn Leu Lys Ala 450 455 460 Phe Val Gln Glu Val Gly Lys Ser Cys 465 470 <210> 18 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> recombinant StUGT <400> 18 atggttcaac ctcatgtgct tcttgtaacg ttcccggcac aaggtcacat caacccctca 60 cttcagtttg ccaagagatt aattaagatg ggcatcgagg ttacctttac tacaagcgtc 120 ttcgcccaca gacgtatggc gaagacggct gcttccaacg ctcccaaggg ccttaatctg 180 gccgccttct cagatggttt tgatgatggc ttcaaatcta atgtcgatga ttcaaagcgt 240 tatatgtctg agattagatc taggggcagc cagaccctgc gtgacattat cttaaagagt 300 agcgacgaag gaaggcccgt aacaagtcta gtatataccc tactattgcc ttgggctgct 360 gaagtagcaa gggagctgca tattccttca gctttgttat ggattcagcc agcgacagtc 420 ctagacatat attattatta cttcaatggc tacgaagatg agatgaaatg tagctcaaac 480 gatcctaatt ggtcaataca attacctcgt ttgcccttac ttaagtccca agatttaccc 540 tctttccttg tgtcctctag ctccaaggat gataaataca gctttgctct accaacgttc 600 aaagaacaac ttgacactct tgacggagag gagaatccca aggtcttagt gaatacgttt 660 gacgccctag aattagagcc gttaaaagca atcgagaagt ataatcttat cgggattgga 720 cctctaattc cgtcttcatt cctgggcggg aaagattcac tagaaagtag cttcggaggc 780 gatttatttc agaaaagcga cgatgattac atggaatggc taaacactaa acccaaatct 840 tcaattgtgt acatttcctt cggttcacta cttaacctat ccagaaatca aaaagaggaa 900 atcgcaaaag ggttaattga aatcaagcgt ccttttctgt gggtcattag ggatcaggaa 960 aatataaagg aggtggagaa ggaggaagag aagttaagct gcatgatgga gctggaaaaa 1020 caaggtaaaa ttgttccttg gtgtagccaa ctagaggtat taactcatcc ctccctggga 1080 tgcttcgtaa gccattgtgg atggaacagt acacttgaaa gtttatcttc aggcgtgccc 1140 gtggtcgcct tccctcactg gacagaccaa ggtactaacg cgaaattaat agaagatgtt 1200 tggaagacag gtgtacgtat gcgtgttagc gaggatggcg tcgtcgaatc agaggagatt 1260 aaaagatgta tagagatagt gatggatggg ggcgagaaag gcgaggaaat gagaaaaaac 1320 gctcagaaat ggaaagaact agccagggaa gcggtgaagg agggggggag cagtgaggtc 1380 aacctaaagg cttttgtgca ggaagtggga aaatcttgt 1419 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ccggaattca tggttcaacc tcatgtgctt 30 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ccgctcgaga caagattttc ccacttcctg 30 <210> 21 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> recombinant CaUGT3 <400> 21 Met Ala Thr Glu Gln Gln Gln Ala Ser Ile Ser Cys Lys Ile Leu Met 1 5 10 15 Phe Pro Trp Leu Ala Phe Gly His Ile Ser Phe Leu Gln Leu Ala 20 25 30 Lys Lys Leu Ser Asp Arg Gly Phe Tyr Phe Tyr Ile Cys Ser Thr Pro 35 40 45 Ile Asn Leu Asp Ser Ile Lys Asn Lys Ile Asn Gln Asn Tyr Ser Ser 50 55 60 Ser Ile Gln Leu Val Asp Leu His Leu Pro Asn Ser Pro Gln Leu Pro 65 70 75 80 Pro Ser Leu His Thr Thr Asn Gly Leu Pro His Leu Met Ser Thr 85 90 95 Leu Lys Asn Ala Leu Ile Asp Ala Asn Pro Asp Leu Cys Lys Ile Ile 100 105 110 Ala Ser Ile Lys Pro Asp Leu Ile Ile Tyr Asp Leu His Gln Pro Trp 115 120 125 Thr Glu Ala Leu Ala Ser Arg His Asn Ile Pro Ala Val Ser Phe Ser 130 135 140 Thr Met Asn Ala Val Ser Phe Ala Tyr Val Met His Met Phe Met Asn 145 150 155 160 Pro Gly Ile Glu Phe Pro Phe Lys Ala Ile His Leu Ser Asp Phe Glu 165 170 175 Gln Ala Arg Phe Leu Glu Gln Leu Glu Ser Ala Lys Asn Asp Ala Ser 180 185 190 Ala Lys Asp Pro Glu Leu Gln Gly Ser Lys Gly Phe Phe Asn Ser Thr 195 200 205 Phe Ile Val Arg Ser Ser Arg Glu Ile Glu Gly Lys Tyr Val Asp Tyr 210 215 220 Leu Ser Glu Ile Leu Lys Ser Lys Val Ile Pro Val Cys Pro Val Ile 225 230 235 240 Ser Leu Asn Asn Asn Asn Asp Gln Gly Gln Gly Asn Lys Asp Glu Asp Glu 245 250 255 Ile Ile Gln Trp Leu Asp Lys Lys Ser His Arg Ser Ser Val Phe Val 260 265 270 Ser Phe Gly Ser Glu Tyr Phe Leu Asn Met Gln Glu Ile Glu Glu Ile 275 280 285 Ala Ile Gly Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Leu Arg 290 295 300 Phe Pro Lys Gly Glu Asp Thr Lys Ile Glu Glu Val Leu Pro Glu Gly 305 310 315 320 Phe Leu Asp Arg Val Lys Thr Lys Gly Arg Ile Val His Gly Trp Ala 325 330 335 Pro Gln Ala Arg Ile Leu Gly His Pro Ser Ile Gly Gly Phe Val Ser 340 345 350 His Cys Gly Trp Asn Ser Val Met Glu Ser Ile Gln Ile Gly Val Pro 355 360 365 Ile Ile Ala Met Pro Met Asn Leu Asp Gln Pro Phe Asn Ala Arg Leu 370 375 380 Val Val Glu Ile Gly Val Gly Ile Glu Val Gly Arg Asp Glu Asn Gly 385 390 395 400 Lys Leu Lys Arg Glu Arg Ile Gly Glu Val Ile Lys Glu Val Ala Ile 405 410 415 Gly Lys Lys Gly Glu Lys Leu Arg Lys Thr Ala Lys Asp Leu Gly Gln 420 425 430 Lys Leu Arg Asp Arg Glu Lys Gln Asp Phe Asp Glu Leu Ala Ala Thr 435 440 445 Leu Lys Gln Leu Cys Val 450 <210> 22 <211> 1362 <212> DNA <213> Artificial Sequence <220> <223> recombinant CaUGT3 <400> 22 atggccaccg aacagcaaca ggcatccatc tcttgtaaaa ttttgatgtt cccttggcta 60 gctttcggac atatctccag cttcttgcaa ttagcgaaga agcttagcga cagggggttc 120 tatttttata tctgctccac ccctataaat cttgactcca taaaaaacaa aataaatcag 180 aattattcat cctccattca gctagtcgat ctgcacctac ccaatagccc tcagttgccc 240 ccgagtttac acacaaccaa cggactaccg ccgcatttga tgtcaactct gaagaatgcg 300 cttattgacg ctaatcccga tctgtgtaaa ataatagcaa gcattaagcc ggatctgatc 360 atatatgatc tacatcaacc gtggacagag gcgctagcct cacgtcacaa cattcctgcg 420 gtcagtttct ctaccatgaa tgctgtatcc tttgcctacg ttatgcatat gtttatgaat 480 ccgggcatag aatttccatt caaagctata catctgtctg atttcgagca agcgcgtttc 540 ttggagcagc tggagtcagc aaaaaacgat gcctcagcaa aagaccccga actacaggga 600 agtaaagggt tttttaactc cacctttata gttagatcca gtcgtgaaat cgaggggaaa 660 tacgtcgatt acttgagtga gatcttaaaa agtaaggtta tacctgtttg ccccgtcatt 720 agccttaaca ataatgatca gggacaaggg aacaaagacg aggacgaaat cattcaatgg 780 ttggacaaaa aaagtcatag aagctccgtg tttgtttcat tcggtagtga atacttcctt 840 aacatgcaag agattgaaga gatagccatt ggtcttgagc tgtctaatgt caacttcatc 900 tgggttttga ggtttcccaa aggtgaagat acgaaaatag aagaggtgct accggaagga 960 tttctagata gggtgaagac caaagggaga atagtgcatg gctgggcccc acaggctcgt 1020 attctgggtc atccatctat aggtgggttt gtatcccatt gtggctggaa cagtgtgatg 1080 gaaagtattc aaatcggtgt gcccatcatt gcgatgccga tgaatttaga ccaacctttc 1140 aatgcaaggc ttgtcgtgga aattggtgtg ggtattgagg tcggcaggga cgagaacggg 1200 aaacttaaaa gggaacgtat cggtgaggtc attaaggaag ttgcaatcgg aaagaagggg 1260 gagaagctta gaaaaaccgc gaaagacctg ggccagaagc taagggaccg tgagaaacag 1320 gattttgatg agcttgcagc gacgctgaag caattgtgcg ta 1362 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 cgggatccat ggccaccgaa cagca 25 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 acgcgtcgac tacgcacaat tgcttcagc 29 <210> 25 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> recombinant NsUGT <400> 25 Met Asp Thr Glu His Ser Thr Leu Arg Val Leu Met Phe Pro Trp Leu 1 5 10 15 Ala His Gly His Ile Ser Pro Tyr Leu Thr Val Ala Lys Lys Leu Ala 20 25 30 Cys Arg Gly Phe Tyr Val Tyr Leu Cys Ser Thr Pro Val Asn Leu Asn 35 40 45 Phe Ile Lys Lys Lys Ile Pro Gln Lys Tyr Ser Ile Ser Ile Gln Leu 50 55 60 Val Glu Phe His Leu Pro Asp Leu Pro Glu Leu Pro Leu Ser Tyr His 65 70 75 80 Thr Thr Asn Gly Ile Pro His Leu Val Ser Thr Leu Lys Lys Ala 85 90 95 Val Lys Met Ser Lys Pro Asn Phe Tyr Lys Ile Ile Glu Asn Leu Lys 100 105 110 Pro Asn Met Leu Ile Tyr Asp Ile Leu Gln Pro Trp Ala Lys Glu Val 115 120 125 Ala Asn Ser Tyr Asn Ile Pro Ala Val Met Leu Leu Thr Phe Cys Ala 130 135 140 Ala Met Leu Ser Tyr Arg Leu His Pro Val Lys Lys Pro Gly Thr Glu 145 150 155 160 Phe Pro Phe Pro Ala Leu Tyr Leu Arg Lys Ile Glu Arg Gln Gln Arg 165 170 175 Glu Glu Met Leu Glu Lys Ala Ala Lys Glu Lys Asp Pro Asp Asp Lys 180 185 190 Asp Pro Phe Ala Glu Glu Glu Thr Met Asn Lys Ile Ile Leu Met Ser 195 200 205 Thr Ser Arg Ala Thr Glu Ala Lys Tyr Ile Asp Tyr Phe Thr Glu Leu 210 215 220 Ile Gln Trp Lys Ile Ile Pro Val Gly Pro Pro Val Gln Glu Thr Thr 225 230 235 240 Asn Glu Tyr Asp Gly Asp Val Asp Asp Leu Ile Asp Trp Leu Gly Asn 245 250 255 Lys Tyr Glu Asn Ser Thr Val Phe Val Ser Phe Gly Ser Gln Tyr Phe 260 265 270 Leu Ser Lys Glu Asp Leu Glu Glu Ile Ala Leu Gly Leu Glu Leu Ser 275 280 285 Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro Lys Gly Glu Glu Val 290 295 300 Arg Val Glu Glu Ala Leu Pro Glu Gly Phe Leu Glu Arg Ile Gly Asp 305 310 315 320 Arg Gly Arg Val Val Asp Gly Trp Ala Pro Gln Leu Arg Ile Leu Ser 325 330 335 His Pro Ser Thr Gly Gly Phe Val Ser His Cys Gly Trp Asn Ser Val 340 345 350 Met Glu Ser Ile Asp Phe Arg Val Pro Ile Ile Ala Leu Pro Met His 355 360 365 Leu Asp Gln Pro Ile Asn Ala Arg Leu Ile Val Glu Leu Gly Val Ala 370 375 380 Val Glu Ile Val Arg Asp Asp Glu Gly Lys Val His Arg Gly Glu Val 385 390 395 400 Ala Glu Ile Val Lys Ser Ile Ile Cys Glu Lys Thr Gly Glu Asn Leu 405 410 415 Arg Asn Lys Val Arg Glu Ile Ser Glu Asn Leu Lys Lys Glu Arg Glu 420 425 430 Glu Glu Met Asp Ala Ala Ile Gly Glu Leu Val Gln Leu Cys Lys Thr 435 440 445 Ser Lys Ser Asn Asn Met Met Leu 450 455 <210> 26 <211> 1368 <212> DNA <213> Artificial Sequence <220> <223> recombinant NsUGT <400> 26 atggacacag aacacagcac gctacgtgtg ctaatgttcc cgtggctagc gcacggacac 60 atatctccct atttaacagt tgcaaagaag ctagcatgta gaggatttta cgtctatctt 120 tgttccacgc cggtcaatct aaacttcatc aagaagaaga taccccagaa gtattcaatt 180 agtatacagt tggtcgaatt ccacctacct gacctacctg agcttccatt aagttaccat 240 acgacaaacg gcattcctcc ccacctagtg tcaacgttaa aaaaagccgt aaagatgagc 300 aaaccgaatt tttataaaat aattgagaat ttgaaaccaa atatgttgat atatgacatt 360 cttcagccat gggcgaagga agtcgccaac tcctacaata tccccgctgt tatgttattg 420 acgttttgcg ctgccatgct ttcttaccgt ttgcacccgg tcaaaaagcc gggcacagag 480 ttcccattcc cggcgctata tttgaggaaa attgaacgtc agcaaaggga ggagatgctt 540 gaaaaagccg ccaaggagaa ggacccggat gataaagacc cgtttgcgga ggaagaaact 600 atgaacaaga taatcctgat gtctacaagc agggcgacgg aggccaagta catcgactat 660 ttcactgaac taattcagtg gaagataatt cctgtgggcc cgcccgtcca ggaaacgact 720 aacgagtacg acggagatgt agatgattta attgattggt tgggaaacaa atatgaaaac 780 tccactgtat ttgtcagctt tgggagccaa tatttcttga gcaaagaaga cttagaggag 840 attgctctag gattggaact atccaatgtg aacttcatat gggtcgtgag attccccaag 900 ggggaggaag ttagagtaga agaagcgttg ccagaaggtt tccttgagag gattggggac 960 agagggaggg ttgttgacgg ttgggcacct caattgcgta ttctgagtca cccttcaacc 1020 ggaggatttg tcagtcactg tgggtggaac tctgtgatgg agtccatcga ttttcgtgtc 1080 cctatcatcg ccctgccaat gcacctggat cagccaatca atgcccgtct aattgtcgaa 1140 ttaggagttg ccgtcgaaat agttcgtgac gacgagggca aagttcacag gggggaggta 1200 gcagagatcg ttaaaagcat tatatgtgag aagacggggg aaaatttgcg taataaagtc 1260 cgtgaaatct ctgaaaatct taagaaagaa agggaagagg agatggacgc agcgatagga 1320 gaactggtgc aattatgcaa gactagcaaa agtaataaca tgatgttg 1368 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 cgggatccat ggacacagaa cacagca 27 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 acgcgtcgac caacatcatg ttattacttt tgc 33 <210> 29 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> recombinant SpUGT <400> 29 Met Gly Thr Gln Val Thr Glu His Gly Thr Ser Asn Leu Arg Val Val 1 5 10 15 Met Phe Pro Trp Leu Ala Tyr Gly His Ile Ser Pro Phe Leu Tyr Val 20 25 30 Ala Lys Lys Leu Ala Asp Arg Gly Phe Leu Ile Tyr Leu Cys Ser Thr 35 40 45 Pro Ile Asn Leu Lys Ser Thr Ile Lys Lys Ile Pro Glu Lys Tyr Ala 50 55 60 Asp Ser Ile His Leu Ile Glu Leu His Ile Pro Glu Leu Pro Glu Leu 65 70 75 80 Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro His Leu Asn His 85 90 95 Thr Leu Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Leu Ser Lys Ile 100 105 110 Leu Lys Asn Leu Lys Pro Asp Leu Met Ile Tyr Asp Val Leu Gln Gln 115 120 125 Trp Ala Glu Arg Val Ala Asn Glu Gln Ser Ile Pro Ala Val Arg Leu 130 135 140 Leu Thr Phe Gly Ala Ala Val Phe Ser Tyr Phe Cys Asn Leu Val Lys 145 150 155 160 Lys Pro Gly Val Glu Phe Pro Phe Pro Asp Ile Tyr Leu Arg Lys Ile 165 170 175 Glu Gln Val Lys Leu Gly Glu Met Leu Glu Lys Ser Ala Lys Asp Gln 180 185 190 Asp Pro Asp Asp Glu Glu Arg Leu Val Asp Glu Tyr Lys Gln Ile Ala 195 200 205 Leu Ile Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile Asp Phe Leu 210 215 220 Leu Glu Leu Ser Asn Leu Lys Val Val Pro Val Gly Pro Pro Val Gln 225 230 235 240 Asp Leu Ile Thr Asn Asp Ala Asp Asp Met Glu Leu Ile Asp Trp Leu 245 250 255 Gly Ser Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly Ser Glu 260 265 270 Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Leu Gly Leu Glu 275 280 285 Leu Ser Asn Val Asn Phe Val Trp Val Ala Arg Phe Pro Lys Gly Glu 290 295 300 Glu Gln Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu Arg Ile 305 310 315 320 Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Leu Arg Ile 325 330 335 Leu Asn His Thr Ser Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn 340 345 350 Ser Val Met Glu Ser Ile His Phe Gly Val Pro Ile Val Ala Met Pro 355 360 365 Met His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val Glu Leu Gly 370 375 380 Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile His Arg Glu 385 390 395 400 Glu Ile Ala Lys Thr Leu Lys Asp Val Ile Thr Glu Arg Ile Gly Glu 405 410 415 Asn Leu Arg Ala Lys Met Arg Asp Ile Ser Met Asn Leu Asn Ser Ile 420 425 430 Ser Gly Glu Glu Met Asp Ala Ala Ala His Glu Leu Ile Gln Phe Cys 435 440 445 Lys Ile Asn Thr Asn 450 <210> 30 <211> 1359 <212> DNA <213> Artificial Sequence <220> <223> recombinant SpUGT <400> 30 atgggaactc aagttacaga acatggaaca tctaatctaa gggtagtcat gttcccttgg 60 ctggcatacg gtcacatctc accattcctt tatgtcgcaa aaaaactggc agacaggggt 120 ttcctgattt acttatgtag tactcccatt aatcttaagt ccacaataaa gaaaatacca 180 gaaaaatacg ccgactccat ccatttaatc gagcttcata ttccagaatt gccagagttg 240 cctcctcatt atcataccac gaacggcctt ccacctcatc taaatcacac actacagaag 300 gccttgaaga tgtcaaagcc caatctatcc aaaatactaa aaaaccttaa gccagattta 360 atgatctacg acgtactaca acagtgggct gagagggtag cgaacgaaca gtccattccg 420 gctgtaaggt tattaacctt cggtgccgca gttttttcat atttttgcaa tttggttaag 480 aaacccggag tcgagtttcc attccccgac atttatttga gaaaaattga gcaggtcaaa 540 ctaggtgaaa tgttggagaa atctgccaaa gaccaagatc ctgacgatga agaaaggtta 600 gtagatgagt acaaacaaat tgctttaatt tgtaccagta gaactattga agccaaatac 660 atcgacttcc tattggagct gagtaaccta aaggtcgttc cagtcggtcc tcccgtccag 720 gacttgatca ccaatgatgc cgatgatatg gaattaatcg actggcttgg ctcaaaggat 780 gaaaattcca ccgttttcgt aagtttcggg agtgagtatt ttttaagcaa agaagatatg 840 gaagaggtcg cgctaggcct tgaactgagc aacgttaact ttgtatgggt cgctaggttc 900 cccaagggag aagaacaaaa tctagaagat gccttaccga aggggttcct ggagagaata 960 ggggaacgtg ggagagtcct ggataagttt gcaccgcagc tgaggatatt aaatcacact 1020 tccactggtg gatttatatc acattgtggt tggaacagcg tgatggaatc tatacacttc 1080 ggagttccga ttgtggcgat gccaatgcat ttggaccagc caatgaacgc gaggcttatc 1140 gttgagcttg gagtagcggt ggagattgtt agggacgatg atggcaaaat tcacagggaa 1200 gaaattgcga agactcttaa ggacgtgata acagagcgta ttggagagaa tctaagggct 1260 aagatgcgtg atatttcaat gaacctaaac agtattagcg gagaggagat ggacgcagct 1320 gctcatgaac ttatacagtt ctgtaagatc aacaccaat 1359 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 cgggatccat gggaactcaa gttacagaa 29 <210> 32 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 acgcgtcgac attggtgttg atcttacaga ac 32 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 cggaattcat ggcgaacaaa gaagagg 27 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 cccaagcttt taggtctccg cttgatgc 28 <210> 35 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gctctagaag gaggattaca aaatgactgc tgtcgttctc c 41 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 cggaattcct agagcttgcg gaagag 26 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 ggaagatctg ctgtgcaggt cgtaaa 26 <210> 38 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 ataagaatgc ggccgcgaaa cgcaaaaagg cca 33 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 aagagcguct agaaggagga ttacaaaa 28 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 agctgatugt actgagagtg caccat 26 <210> 41 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 gctctagaag aggattacaa aatggttcaa cctcatgtcc 40 <210> 42 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 tccccccggg ttaacaacct tttcctactt cttg 34 <210> 43 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 tccccccggg aggaggacag ctaaatggcc accgaacagc 40 <210> 44 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 aaggaaaaaa gcggccgctt atacgcacaa ttgcttcagc 40

Claims (21)

Catharanthus roseus ( Catharanthus roseus ) consisting of the amino acid sequence of SEQ ID NO: 21 comprising a CaUGT3 enzyme derived from the strain, Crosin-4 production composition.
delete According to claim 1,
The composition further comprises crosin-2 and UDP-glucose, crosin-4 production composition.
Crosin-4 from Crosin-2, comprising the step of reacting Crosin-2 with UDP-glucose in the presence of CaUGT3 enzyme consisting of the amino acid sequence of SEQ ID NO: 21 to obtain a reaction product containing Crosin-4 how to produce it.
5. The method of claim 4,
The method of producing crosin-4 from crosin-2, wherein the reaction is carried out under a temperature condition of 20 to 40 ℃ and a time condition of 10 to 240 minutes.
A composition for producing crosin-4 comprising a transformant into which CaUGT3 gene derived from a Catharanthus roseus strain consisting of the nucleotide sequence of SEQ ID NO: 22 or a culture product thereof is introduced.
delete A method for producing crosin-4 from crosin-2, comprising culturing a transformant into which the CaUGT3 gene consisting of the nucleotide sequence of SEQ ID NO: 22 is introduced in a medium containing crosin-2 and UDP-glucose .
9. The method of claim 8,
The method for producing crosin-4 from crosin-2, further comprising the step of recovering crosin-4 from the culture obtained through the culture.
(a) GjUGT1 enzyme derived from the Gardenia jasminoides strain consisting of the amino acid sequence of SEQ ID NO: 1 or Nicotiana tabacum consisting of the amino acid sequence of SEQ ID NO: 9 NtUGT enzyme derived from the strain; And (b) Catharanthus roseus ( Catharanthus roseus ) consisting of the amino acid sequence of SEQ ID NO: 21 Containing a CaUGT3 enzyme derived from the strain, Crosin-4 production composition.
delete 11. The method of claim 10,
The composition further comprises crocetin and UDP-glucose, crosin-4 production composition.
(a) reacting crocetin with UDP-glucose in the presence of a GjUGT1 enzyme consisting of the amino acid sequence of SEQ ID NO: 1 or an NtUGT enzyme consisting of the amino acid sequence of SEQ ID NO: 9 to obtain a reaction product containing crosine-2; and
(b) adding and reacting CaUGT3 enzyme consisting of the amino acid sequence of SEQ ID NO: 21 to the reaction to obtain crosin-4.
14. The method of claim 13,
The reaction of steps (a) and (b) is a method for producing crosine-4 from crocetin, which is performed under a temperature condition of 20 to 40°C and a time condition of 10 to 240 minutes.
14. The method of claim 13,
In the step (b), the method for producing crosine-4 from crocetin further comprising the step of adding UDP-glucose.
(A) Gardenia jasminoides consisting of the nucleotide sequence of SEQ ID NO: 2 GjUGT1 gene derived from the strain or Nicotiana tabacum consisting of the nucleotide sequence of SEQ ID NO: 10 NtUGT gene derived from the strain; And (b) the nucleotide sequence of SEQ ID NO: 22 Catharanthus roseus ( Catharanthus roseus ) Crosin-4 production composition comprising a transformant or a culture product of the CaUGT3 gene derived from the strain.
delete 17. The method of claim 16,
The transformant is a composition for producing crocetin-4, wherein the crocetin biosynthesis gene is additionally introduced.
19. The method of claim 18,
The crocetin biosynthesis gene is a gene of CsCCD2 and aldH gene, a composition for crosine-4 production.
(a) the GjUGT1 gene consisting of the nucleotide sequence of SEQ ID NO: 2 or the NtUGT gene consisting of the nucleotide sequence of SEQ ID NO: 10; and (b) culturing the transformant into which the CaUGT3 gene consisting of the nucleotide sequence of SEQ ID NO: 22 is introduced in a medium containing crocetin and UDP-glucose, a method for producing crosine-4 from crocetin .
21. The method of claim 20,
The method for producing crosine-4 from crocetin further comprising the step of recovering crosin-4 from the culture obtained through the culture.

Images