KR20210157193A - Senna-tora derived gene having anthoraquinone biosynthesis function and use thereof - Google Patents

Abstract

The present invention relates to Senna tora-derived CHS-L protein having polyketide synthase activity and a gene thereof. The Senna tora-derived CHS-L of the present invention has activity in biosynthesis of atrochrysome carboxylic acid, which is an emodin precursor, or/and endocrosine anthrone and can be usefully used in developing a crop with an increased anthraquinone (emodin) content or in synthesizing the emodin precursor and emodin.

Description

Senna-tora derived gene having anthoraquinone biosynthesis function and use thereof

The present invention relates to anthraquinone biosynthesis-derived CHS-L protein, a gene thereof, and uses thereof.

Kyeomyeongja is an annual herb belonging to the legume family, and two varieties are known : Senna obtusifolia and Senna tora. Its mature seeds are known as Kyeomyeongja in the sense of clearing the eyes. Pharmacological ingredients include carotene, a precursor of vitamin A, alloin, Rhein, emodin, chrysophanol, and aloe-emodin. -emodin), Physcion, Obtusin, Aurantio-obtusin, Rubrofusrin, Torachryson, Tora lactone, etc. have. Among these components, Emodin is an anthraquinone-based compound and has a C ₆ -C ₂ -C ₆ basic skeleton of 14 carbon atoms and 1,3,8-trihydroxy-6-methyl- It has the chemical name 9,10-anthracenedione (1,3,8-trihydroxy-6-methyl-9,10-anthracenedione). Various biological activities such as anticancer, anti-inflammatory, antibacterial, diuretic and immunosuppressive have been reported.

Many studies have been reported on structural analysis or physiological effects of anthraquinone-based physiologically active substances from the extract of kaleidoscope. However, biosynthesis studies in microorganisms for anthraquinone-based physiologically active substances among the extracts of Kaleidoscope are scarce except for chrysophanol. A study on the biosynthesis of chrysophanol, which is effective in arteriosclerosis, was initiated in 2008 in The Journal of Antibiotics , in the study on the synthesis of chrysophanol intermediates in actinomycetes, and in phytochemistry in 2009, in phytochemistry. However, studies on the biosynthesis of emodin are quite incomplete.

Republic of Korea Patent Registration No. 10-2072017

The problem to be solved by the present invention is to provide a CHS-L protein derived from C. C. C. synthase having polyketide synthase activity, a CHS-L gene encoding the CHS-L protein, and a recombinant vector including the gene will do

In order to solve the above problems, the present invention provides a CHS-L protein derived from C. C., represented by the amino acid sequence of SEQ ID NO: 2 having polyketide synthase activity.

The polyketide synthase may be to synthesize an emodin precursor using a malonyl-CoA substrate.

The emodin precursor may be atrochrysome carboxylic acid or endocrocin anthrone.

The present invention also provides a CHS-L gene represented by SEQ ID NO: 1 encoding the CHS-L protein.

The present invention also provides a recombinant vector comprising the CHS-L gene.

The present invention also provides a transformant transformed with the recombinant vector.

The transformant may be an E. coli BL21 (DE3) strain.

In addition, the present invention provides a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 capable of specifically amplifying the CHS-L gene represented by SEQ ID NO: 1.

The present invention also provides a method for producing an emodin precursor comprising the step of reacting CHS-L protein with malonyl-CoA.

The production method may further comprise adding a NADPH cofactor.

The emodin precursor may be atrochrysome carboxylic acid or endocrocin anthrone.

According to the present invention, it is possible to produce an emodin precursor by utilizing the CHS-L derived from Kyomyungja, and it is possible to develop a crop with an improved anthraquinone (emodin) content.

1 is an anthraquinone predicted biosynthesis pathway by polyketide synthetase.
2 is a result of confirming the phylogenetic specificity of the CHS-L gene line in the genomes of four other legumes ( C. fasciculata , A. hypogaea , M. truncatula , G. max).
Figure 3 shows the results of confirming the expression of the recombinant protein CHS-L (Sto07g228250) or CHS (Sto03g054970) by SDS-PAGE. (a) Sto07g228250 SDS-PAGE analysis result, lane 1: standard protein marker; lane 2: soluble protein; lane 3: insoluble protein; Lanes 4 and 5: Purified protein. (b) Sto03g054970 SDS-PAGE analysis, lane 1: soluble protein; lane 2: insoluble protein; lanes 3 and 4: purified protein; Lane 5: standard protein marker.
4 is a result of TOF ESI-MS analysis of the result of the enzymatic reaction in CHS-L of Kyomyungja.

The present invention provides a polyketide synthase (polyketide synthase)-derived CHS-L protein represented by the amino acid sequence of SEQ ID NO: 2 having activity.

In addition, functional equivalents of proteins constituting the amino acid sequence of SEQ ID NO: 2 may be included within the scope of the present invention. The "functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution or deletion of amino acids. It refers to a protein having substantially the same physiological activity as the protein represented by SEQ ID NO: 2 as having 95% or more sequence homology.

In the present invention, Kyeomyeongja is an annual herb belonging to the legume family, and as a plant of the genus Gyeomyeong ( Senna ), two varieties are known: Senna obtusifolia and Kyeomyeongja ( Senna tora). In the past, Chogyeolmyeongja and Kyeomyeongja were classified as Cassia , but now they are classified as Senna. Gyeolmyeongja is also called Chogyeolmyeong or Gingangnamcha. Chogyeomyeongja is native to Central America and is cultivated in Japan and other places in tropical Asia, such as China and Korea.

The shape of the fruit of Kyeolmyungja is irregular hexagonal columnar shape and one side is pointed. The color of the outer surface is yellowish brown to blackish brown, and the length is 3 to 6 mm, and the diameter is 2 to 3.5 mm. The seed coat is firm and glossy, and has a single narrow line of glossy patterns on the left and right sides, and has a slightly peculiar smell. Chogyeomyeongja is the opposite of seeds, and Kyolmyungja is small.

In an embodiment of the present invention, the CHS-L (Sto07g228250) gene of SEQ ID NO: 1 was isolated from the coleoptera, and the isolated CHS-L protein has polyketide synthase activity and is involved in the biosynthetic pathway of emodin, Specifically, it was confirmed that emodin precursor atrochrysome carboxylic acid and endocrocin anthrone were synthesized using a malonyl-CoA substrate.

Therefore, the gyeolmyeongja of the present invention may be a gyeolmyeongja (Senna tora) variety.

In an embodiment of the present invention, the CHS-L protein derived from Kyomyungja has polyketide synthase activity, and atrochrysome carboxylic acid (atrochrysome carboxylic acid), a precursor of emodin, using a malonyl-CoA substrate acid) or endocrocin anthrone was confirmed to be synthesized, the CHS-L protein of the present invention has polyketide synthase activity.

In the present invention, polyketide synthase is an enzyme for synthesizing an emodin precursor or emodin using a malonyl-CoA substrate.

In the present invention, emodin is an anthraquinone-based compound, has a C6-C2-C6 basic skeleton of 14 carbon atoms, and 1,3,8-trihydroxy-6-methyl-9,10-anthracenedione It has the chemical name (1,3,8-trihydroxy-6-methyl-9,10-anthracenedione).

In the present invention, a precursor is a substance in a stage before becoming a specific substance in any metabolism or reaction, and is produced in the pathway in which malonyl-CoA is biosynthesized into emodin by polyketide synthase. means middle-class products.

In the present invention, the emodin precursor may be atrochrysome carboxylic acid or endocrocin anthrone.

The present invention also provides a CHS-L gene represented by SEQ ID NO: 1 encoding the CHS-L protein.

The CHS-L gene of the present invention is a gene encoding the CHS-L protein of SEQ ID NO: 2 and is represented by SEQ ID NO: 1.

The CHS-L gene is not limited to the nucleotide sequence of SEQ ID NO: 1, and includes a functionally equivalent codon or a nucleotide sequence including a codon encoding the same amino acid, or a codon encoding a biologically equivalent amino acid. In consideration of mutations having biologically equivalent activity, the nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity to the sequence described in the sequence listing. The substantial identity is at least 60% when the sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence that exhibits homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

In the example of the present invention, as a result of comparative analysis of the CHS-L gene derived from C. C. plant species with the genes of 15 plants (14 species of legumes, 1 type of grape family), there were no 11 kinds of CHS-L subfamily genes. On the other hand, it was confirmed that 16 genes of Senna tora , 5 of C. fasciculata , 2 of A. hypogaea , 1 of M. truncatula , and 1 of G. max contained CHS-L subfamily genes. .

In other words, compared to the genomes of 15 other plant species, Kyushu Myeongja confirmed that there are especially many CHS-L series genes. Therefore, the CHS-L gene of the present invention may be a gene derived from Kyomyungja.

The present invention also provides a recombinant vector comprising the CHS-L gene.

The recombinant vector of the present invention includes a CHS-L gene represented by SEQ ID NO: 1 encoding a CHS-L protein.

As it has been confirmed that the CHS-L protein derived from K. C. C. in the above-described embodiment is involved in the synthesis of the emodin precursor, the recombinant vector of the present invention may be a recombinant vector that promotes or enhances the biosynthesis of the emodin precursor.

As used herein, the term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form. Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.

In the present invention, the term "recombinant vector for transformation" refers to a gene construct including essential regulatory elements operably linked to express a gene insert, and includes all common vectors including plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. include

The "recombinant vector for transformation" of the present invention is functionally linked to an expression control sequence so that the CHS-L gene can be expressed. For example, the vector includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, operator, initiation codon, stop codon, polyadenylation signal, and enhancer, and may be prepared in various ways depending on the purpose. . In addition, the vector may include a selectable marker, and the vector may be self-replicating or integrated into host DNA. The vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation are performed using enzymes generally known in the art.

In an embodiment of the present invention, a recombinant vector for E. coli expression was prepared by recombination of the CHS-L gene in the pET28a(+) E. coli expression vector.

The present invention also provides a transformant transformed with the recombinant vector.

As used herein, the term "transformation" refers to a phenomenon in which DNA enters the cell and changes the genetic trait when DNA, which is a genetic material, is injected into a living cell of another lineage. Or also called type conversion.

In the present invention, "transformation" of a microorganism or plant with the vector can be performed by transformation techniques known to those skilled in the art.

Specifically, transformation method using Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated method ( liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, or Agrobacterium spraying method can be used. have.

In the present invention, the term "plant" is meant to include not only mature plants, but also plant cells, plant tissues, and seeds of plants that can develop into mature plants.

In the present invention, the plant is not particularly limited, and as an example, food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat or sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion or carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots or bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies or tulips; And it may be any one selected from the group consisting of forage crops including ryegrass, red clover, orchard grass, alpha alpha, tall fescue or perennial ryegrass.

Since the transformed plant in the present invention expresses the CHS-L gene or a protein thereof, the expression of the CHS-L gene present in the plant can be enhanced by introducing a recombinant vector containing the CHS-L gene. Accordingly, it is possible to promote the biosynthesis of the emodin precursor, and also promote or enhance the biosynthesis of the emodin precursor to promote the production of emodin.

In an embodiment of the present invention, the CHS-L recombinant vector was introduced into the E. coli BL21 (DE3) strain, to promote the expression of CHS-L, and these expressed proteins were isolated, and malonyl- using the isolated CHS-L protein CoA substrate was synthesized with atrochrysome carboxylic acid or endocrocin anthrone.

Therefore, the transformant of the present invention may be a microorganism, specifically E. coli BL21 (DE3).

In addition, the present invention provides a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 capable of specifically amplifying the CHS-L gene represented by SEQ ID NO: 1.

In an embodiment of the present invention, the Sto07g228250 (1,173 bp, SEQ ID NO: 1) gene encoding CHS-L was amplified using a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6.

Accordingly, the primer set of the present invention includes the primers represented by SEQ ID NO: 5 and SEQ ID NO: 6. The primer set specifically amplifies the CHS-L gene, and may specifically amplify the CHS-L gene of SEQ ID NO: 1.

In the present invention, the primer set includes all combinations of primer sets consisting of forward and reverse primers recognizing a target gene sequence, but preferably, a primer set that provides analysis results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, high specificity can be conferred when the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .

The present invention also provides a method for producing an emodin precursor comprising the step of reacting the CHS-L protein with malonyl-CoA.

The method for producing an emodin precursor of the present invention includes the step of reacting the CHS-L protein derived from K. seigenji represented by the amino acid sequence of SEQ ID NO: 2 with malonyl-CoA.

The step of reacting is to mix CHS-L protein and malonyl CoA and perform an enzymatic reaction at 20 to 40 ° C. Specifically, mix CHS-L protein and malonyl CoA and 3 to 12 hours at 25 to 35 ° C. It may be to proceed with the enzymatic reaction.

As a result of enzymatic reaction of malonyl-CoA substrate at 30 ° C. for 6 hours using CHS-L recombinant protein isolated from E. coli transformants in an example of the present invention, atrochrysome carboxylic acid was synthesized. It was confirmed, and it was confirmed that atrochrysome carboxylic acid or endocrocin anthrone was synthesized from the enzymatic reaction malonyl-CoA substrate to which 1 mM NADPH cofactor was added.

Accordingly, the method for producing an emodin precursor of the present invention may further include adding a NADPH cofactor.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

1. Analysis of the anthraquinone biosynthetic pathway of Kyeongmyungjae

Anthraquinone, which is biosynthesized by polyketide synthase, undergoes a condensation reaction of eight malonyl-CoA molecules to produce octaketide, and octaketide is a ring It is expected that anthranoid-type substances such as atrochrysome carboxylic acid will be biosynthesized from polyketide synthase through sequential reactions such as cyclization and enolization. After that, emodin, one of anthraquinones, is produced through decarboxylation and oxidation processes, and another biosynthetic process undergoes dehydration and enolization to produce chrysophanol and Islandi. Anthraquinones such as islandicin are expected to be biosynthesized ( FIG. 1 ).

2. Evolutionary analysis of CHS-L gene derived from K.

2.1. Comparative analysis of chalcone synthase (CHS) and chalcone synthase-like, CHS-L

Chalcone synthase (CHS) and chalcone synthase-like (CHS-L) genes are involved in the biosynthesis of various natural products in plant biological processes. Accordingly, subfamily genes of chalcone synthase (CHS) or chalcone synthase-like (CHS-L) were compared and analyzed in the genomes of 14 types of kaleidoscope, 14 types of legumes, and 1 type of grape (16 types in total).

Table 1 shows the results of analysis of CHS-L and CHS gene subfamily in the 16 plant genomes. Referring to Table 1, mimosa ( Mimosa pudica , MIMPU ), winter thorn acacia tree ( Faidherbia albida , FAIAL), chickpea ( Cicer arientinum , CICAR) , cicer reticulatum ( Cicer reticulatum , CICRE), pea ( Pisum sativum , PISSA ) ), pigeon beans ( Cajanus cajan , CAJCA), kidney beans ( Phaseolus vulgaris , PHAVU), mung beans ( Vigna radiata , VIGRA), red beans ( Vigna angularis , VIGAN), dong beans ( Vigna unguiculata , VIGUN), grapevines ( Vitis vinifera , VITVI), the CHS-L family gene did not appear in the genome. On the other hand, Senna tora (SETOT) is 16, Partridge pea ( Chamaecrista fasciculata , CHAFA) is 5, peanut ( Arachis hypogaea , ARHYP) is 2, Medicago truncatula (MEDTR) It was confirmed that one, soybean (Glycine max, GLYMA) contains one CHS-L family gene.

In other words, compared to the genomes of 15 other plant species, Kyushu Myeongja confirmed that there are especially many CHS-L series genes.

Twelve CHS genes were present in the genus of Kyomyungja, not in peas ( Pisum sativum , PISSA), and it was confirmed that 6 to 48 were present in the genomes of 14 other plant species.

＊E/C represents expansion/contraction.

† Rapid_E and Rapid_C indicate rapid expansion and rapid contraction.

‡ Species name: Senna tora ( SETOT ), Partridge pea ( Chamaecrista fasciculata , CHAFA ), Mimosa ( Mimosa pudica , MIMPU ), Winter thorn acacia ( Faidherbia albida , FAIAL), Peanut ( Arachis hypogaea , ARHYP) , Medicago truncatula ( MEDTR ), chickpea ( Cicer arientinum , CICAR ) , cicer reticulatum , CICRE , pea ( Pisum sativum , PISSA ), soybean ( Glycine max , GLYMA ), pigeon Beans ( Cajanus cajan , CAJCA ), kidney beans ( Phaseolus vulgaris , PHAVU ), mung beans ( Vigna radiata , VIGRA ), red beans ( Vigna angularis , VIGAN ), dong beans ( Vigna unguiculata , VIGUN ), grapevines ( Vitis vinifera , VITVI ).

Next, the phylogenetic specificity of each CHS-L family gene included in K. senna tora and four legumes ( Chamaecrista fasciculata , Arachis hypogaea , Medicago truncatula , Glycine max ) was confirmed. As a result, the CHS- One of 16 L genes was located on chromosome 2, and it was confirmed that the remaining 15 genes were distributed on chromosome 7 (FIG. 2).

2.2. Securing of the CHS-L and CHS gene sequences derived from Kyomyungja

The cDNA sequence of the type III polyketide synthetase derived from Kyomyungja was obtained through the Kyomyungja transcript database. ORFs of Sto07g228250 (SEQ ID NO: 1) encoding the CHS-L gene and Sto03g054970 (SEQ ID NO: 3) encoding the CHS gene were selected and their functions were analyzed.

base/amino acid sequence CHS-L gene
(SEQ ID NO: 1) ATGGAGAGTGCTGCAGAAAAGAAGGGATTCGCCACAGTGCTTGCTATTGGCACTGCAAATCCCCCAAACATTTATCTTCAATCTGAGTTTCCTGATTTCTTCTTCAGAGTCACCAATAGTGAGCATCATGTCCAACTCAAGCAGAAGTTCAAGCGCATGTGTGACAACTCCAATATCAGGAAGCGCCATTTTCTTGTTGATGAAGATATTCTTAAAGAGCATCCAAACATCAGCACCTATGGAGCTCCTTCGTTGGATACACGCAGAGAAATAGTAACTAAGTACATTCCTAAGCTTGGGAAGGAAGCAGCCTTGAAATGTATTAAAGAATGGGGCCAACCTTTATCTAAGATCACACATCTCATCTTCTGCACATCTTCATGCATCAACAGCATTCCTGGACCTGATTTCTACCTTGCCAGAGAAATTGGCCTCCCAGCCACTTGTAACCGCCTTGTGATTTATGATCATGGTTGTCACGCTGGTGGTTCAGTCATTCGTGTAGCCAAGGCTCTTGCTGAGAGCATCCCTGGCTCACGTGTGCTCACTGTGTGTGCTGAGACCATGCTTACTTCCTTCCAAGCCCCAAGTCCGTCACATATGGACATTGTGGTTGGGCACGCCTTGTTTGGAGATGGTGCTGCTGCCATGATTGTCGGTACAGATCCTATCCCCAATGTTGAACGTCCACTGTTTGAGTTTGTGTTGGCTGCACAACAGACAGTGGGAGGATCTGAGGAGGCAATTCATGGACATGCAACTGAAAGAGGTCAAACTTACTTTTTAGGAAAAGAGATTCCAAATGTTGTTGCTGGTAATGTGAAGAAGTGTATGCATGATGCATTTGGGACGCTTGGTATGACAATCAATGAAGGAGAGTGGAATTCGTTGTTCTACGTGGTGCATCCTGGTGGGAAGGCAGTACTGAATGGGATGGAAGAGGTGCTTGAGTTGAAGGAAGAGAAGCTTGCTGCGAGTAGGACTATTTTGAGAGAGTACG GAAACATGTGGAGTCCATGTGTGTTCTTCGTGTTGGATGAAATGAGAAAGAAATCTTCCAACGAAGGGAAGTCCACAACCGGAGAAGGACATGACTGGGGTGCTTTGATGACCTTTGGACCAGGTTTGACGGTTGAAACAATTGTTTTGCATTCCATTTCCCTGAAGGACTAG CHS-L protein
(SEQ ID NO:2) MESAAEKKGFATVLAIGTANPPNIYLQSEFPDFFFRVTNSEHHVQLKQKFKRMCDNSNIRKRHFLVDEDILKEHPNISTYGAPSLDTRREIVTKYIPKLGKEAALKCIKEWGQPLSKITHLIFCTSSCINSIPGPDFYLAREIGLPATCNRLVIYDHGCHAGGSVIRVAKALAESIPGSRVLTVCAETMLTSFQAPSPSHMDIVVGHALFGDGAAAMIVGTDPIPNVERPLFEFVLAAQQTVGGSEEAIHGHATERGQTYFLGKEIPNVVAGNVKKCMHDAFGTLGMTINEGEWNSLFYVVHPGGKAVLNGMEEVLELKEEKLAASRTILREYGNMWSPCVFFVLDEMRKKSSNEGKSTTGEGHDWGALMTFGPGLTVETIVLHSISLKD * CHS gene
(SEQ ID NO:3) ATGGTGAATGTGGAAGAGATCCGTAAGGCACAACGAGCGGAAGGTGCTGCTACAGTAATGGCTATCGGTACAGCGACCCCAACAAACTGTATAGAACAAAGTACCTATCCAGATTACTATTTTCGTATTACAAACAGTGAGCACATGACAGAGTTGAAAGAGAAATTCCAGCGCATGTGTGATAAGTCAATGATAAAAAAGAGATATATGCACTTGACAGAGGAGATCTTAAAGGAGAACCCTAATATGTGTGCTTACATGGCACCTTCTATTGATGCAAGGCAAGATATAGTGGTTTTGGAAGTACCAAAGCTTGGAAAAGAGGCTGCAACAAAGGCCATTAAGGAATGGGGCCAACCCAAATCCAAAATTACGCACTTGATCTTTTGCACTACAAGTGGTGTGGACATGCCAGGAGCTGACTACCAACTCACAAAGCTCTTAGGTCTTCGCCCATCTGTGAAGCGATACATGATGTACCAACAAGGTTGCTTTGCAGGCGGTACGGTGCTCCGTTTAGCCAAAGACTTGGCTGAGAATAACAAAGGAGCACGTGTACTTGTGGTTTGTTCTGAGATTACTGCAGTTACATTCCGTGGGCCTACTGATACCCATCTTGACAGCCTTGTGGGCCAAGCATTGTTTGGCGATGGGGCAGCTGCTGTTATTGTTGGATCTGACCCAATCCCACAAGTTGAGAAGCCTTTATTTGAACTTGTATGGACAGCACAAACTATTCTTCCCGATAGTGAAGGGGCTATTGATGGGCATCTTCGTGAAGTTGGGCTCACATTCCATCTTCTGAAAGATGTTCCTGGGCTCATCTCAAAGAACATTGAGAAAGCCTTGGTTGAAGCCTTCAAACCATTGGGAATTTCTGATTATAACTCAATTTTCTGGATTGCTCACCCAGGTGGGCCTGCAATTTTGGACCAAGTTGAGGCCAAATTAGAGTTGAAGCCCGAAAAGATGCAGGCCACTAGACACGTGCTTAGTGAGT ATGGAAACATGTCCAGTGCATGCGTGTTATTCATTTTGGATGAAATGAGGAGAAAATCAGCAAAAGATGGACTTGGCACAACAGGTGAAGGACTTGAGTGGGGTGTTCTATTTGGATTTGGGCCTGGCCTTACTGTTGAGACCGTTGTGCTCCACAGTATTGCTATTTAA CHS protein
(SEQ ID NO: 4) MVNVEEIRKAQRAEGAATVMAIGTATPTNCIEQSTYPDYYFRITNSEHMTELKEKFQRMCDKSMIKKRYMHLTEEILKENPNMCAYMAPSIDARQDIVVLEVPKLGKEAATKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPSVKRYMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPTDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLFELVWTAQTILPDSEGAIDGHLREVGLTFHLLKDVPGLISKNIEKALVEAFKPLGISDYNSIFWIAHPGGPAILDQVEAKLELKPEKMQATRHVLSEYGNMSSACVLFILDEMRRKSAKDGLGTTGEGLEWGVLFGFGPGLTVETVVLHSIAI *

3. Cloning and expression of C. C. CHS-L and CHS genes

3.1. CHS-L and CHS gene cloning

Primers were designed as shown in Table 3 to clone Sto07g228250 (1,173 bp, SEQ ID NO: 1) encoding CHS-L and Sto03g054970 (1,170 bp, SEQ ID NO: 3) encoding CHS.

gene name forward reverse CHS-L
Sto07g228250 AAGGATCCATGGAGAGTGCTGGAG
(SEQ ID NO: 5) AACTCGAGCTAGTCTCTCAGAGGG
(SEQ ID NO: 6) CHS
Sto03g054970 GGATCCATGGTGAATGTGGAAGAGATC
(SEQ ID NO: 7) GAATTCTTAGACAGCCACACTATGGAG
(SEQ ID NO:8)

After RT-PCR using the primers under the following conditions, CHS-L was recombined into pET28a(+) E. coli expression vector using BamHI and XhoI restriction enzyme sites, and CHS was pET28a ( +) was recombined into an E. coli expression vector. The plasmids recombined in the expression vector were named pET28a(+)_Sto07g228250 and pET28a(+)_Sto03g054970, respectively.

RT-PCR conditions:

94℃ 7 minutes 1 cycle,

94℃ 1 min, 58-62℃ 1 min 30 cycles,

72℃ 1 min, 72℃ 7 min 1 cycle

3.2. CHS-L and CHS gene expression

E. coli BL21(DE3) strain was transduced with the pET28a(+)_Sto07g228250 or pET28a(+)_Sto03g054970 plasmid to prepare a recombinant strain, and CHS-L and CHS proteins were expressed in these strains.

Specifically, the recombinant protein (CHS-L or CHS) was expressed by inoculating the recombinant strain in the LB medium and incubating for 20 to 24 hours at 20° C. after inducing expression with 0.4 mM IPTG. To isolate the recombinant protein, the culture medium of the recombinant strain was centrifuged to collect only the cells, washed twice with 100 mM phosphate buffer (pH 7.5) containing 10% glycerol, and then sonicated to destroy the cell wall. Thereafter, the precipitate (insoluble cell debris) was removed by centrifugation at 4° C., 12,000 rpm (13,475 × g) for 30 minutes to obtain a supernatant.

Next, the expressed protein from the supernatant was purified. Put the obtained supernatant into the purification column, 1 ml of His6 Ni-Superflow Resin (Takara, Japan) and 100 mM phosphate buffer (pH 7.5, 500 mM NaCl, 5 mM imidazole, 1 mM dithiothreitol, and 10% In the presence of glycerol), the protein was bound to His-tag resin for at least 1 hour. The recombinant protein bound to 6X His-tag was flowed through a phosphate buffer containing 8 times the column volume of 50 mM and 250 mM imidazole to obtain a purified recombinant protein.

The purified protein was concentrated using Amicon Ultra 15 (Millipore, 30 K NMWL centrifugal filters). Protein concentration was analyzed by Bradford assay (Bradford assay, Protein Assay Dc, Bio-Rad, Hercules, CA, USA), and albumin was used as a standard protein.

Purity and molecular mass of the isolated recombinant protein were confirmed using 12% SDS-PAGE. As a result of confirming the expression of the His-tag-modified recombinant protein from CHS-L (Sto07g228250) and CHS (Sto03g054970), it was confirmed that it was ~48 kDa (FIG. 3).

4. CHS-L and CHS Enzyme Reaction and Assay

4.1. Polyketide synthase activity of CHS-L and CHS

Next, to analyze the role of CHS-L (Sto07g228250) or CHS (Sto03g054970) protein in the anthraquinone biosynthesis process, the polyketide synthase activity of purified CHS-L or CHS recombinant protein was analyzed. .

To analyze the polyketide synthase activity of CHS-L protein, 5 mM malonyl-CoA, 10 mM MgCl ₂ , 100 mM phosphate buffer (pH 7.5), and 10 μg/ml purified protein were used. The reaction was carried out in ml volume. In addition, as a control reaction, the reaction was carried out using a recombinant protein inactivated by heat under the same conditions as above. Separate reactions were performed with similar reaction components with an additional 1 mM NADPH cofactor to the reaction mixture. In addition, the reaction was carried out with 13C3-malonyl-CoA labeled with a radioisotope instead of malonyl-CoA using the above reaction conditions.

The reaction time was 30° C. for 6 hours, and after the reaction, the reaction was terminated by heat treatment at 85° C. for 3 minutes.

In the case of the CHS (Sto03g054970) enzyme reaction, p-coumaroyl-CoA was used as the reaction starting material (substrate), and malonyl-CoA and 13C3-malonyl-CoA were used as the substrate to extend the carbon skeleton. , the reaction was repeated 3 times each.

4.2. CHS-L (Sto07g228250) and CHS (Sto03g054970) enzyme assays

The result of the enzymatic reaction of CHS-L or CHS protein was analyzed by UPLC coupled with TOF ESI-MS. As a result of analyzing the reaction product with the CHS-L (Sto07g228250) enzyme through ion chromatogram (EIC), molecular masses of 319.08 Da and 301.07 Da were obtained ( FIGS. 4a (i), b (i)). However, reaction products having the same mass were not detected in the CHS (Sto03g054970) and heat-denatured CHS-L (Sto07g228250 control) enzymes.

^{Specifically, the result value of m/z +} 319.0827 at 4.45 minutes of retention time (tR) is shown in FIG. 4a(i). This was the same as the theoretical molecular weight (319.0818) of _{carboxylic acid atrochrysome carboxylic acid (molecular formula C 16} H ₁₄ O ₇ ), which is a precursor of emodin synthesis among the results of the polyketide synthetase reaction of FIG. 1 . Therefore, it can be seen that the substance produced by the enzymatic reaction is atrochrysome carboxylic acid. In addition, it was ^{confirmed that the theoretical mass value of the product reacted with the isotope-treated substrate ( 13} C ₃ -malonyl-CoA) and the mass value of the experimental result using the actual isotope exactly match (Fig. 4a(ii)) ).

In addition, ESI-MS spectrum analysis of the result of the enzymatic reaction to which 1 mM NADPH cofactor was added. As a result of the analysis, it showed a result value of ^{m/z +} 301.0715 at a retention time of 4.62 minutes other than atrochrysome carboxylic acid. This was the same as the theoretical molecular weight (301.0712) of _{endocrocin anthrone (molecular formula C 16} H ₁₂ O ₆ ), which is an emodin synthesis precursor, among the results of the polyketide synthetase reaction of FIG. 1 . Therefore, the substance produced by the enzymatic reaction is endocrocin anthrone (Fig. 4b(i)). In addition, it was ^{confirmed that the theoretical mass value of the product reacted with the isotope-treated substrate ( 13} C ₃ -malonyl-CoA) and the actual mass value of the experimental result using the isotope exactly match (Fig. 4b(ii)) ).

From the above results, it was confirmed that CHS-L (Sto07g228250) has polyketide synthetase activity and synthesizes atrochrysome carboxylic acid or endocrocin anthrone, which is an emodin precursor, by using it as a malonyl-CoA substrate.

In addition, emodin anthrone, endocrocin, emodin, which require additional reactions of decarboxylation and oxidation except for atrochrysome carboxylic acid and endocrocin anthrone metabolites; No products such as chrysophanol or islandicin were detected.

In conclusion, it was confirmed that the CHS-L (Sto07g228250) protein derived from Kaleidoscope was involved in the biosynthesis pathway of emodin, a type of anthraquinone, through the biosynthesis pathway of type III polyketide, atrochrysome carboxylic acid and endocrocin anthrone, which are precursors of emodin. .

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) Industry-University Cooperation Foundation Sunmoon University <120> Senna tora-derived gene having anthoraquinone biosynthesis function and use thereof <130> DP20200066 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> CHS-L gene <400> 1 atggagagtg ctgcagaaaa gaagggattc gccacagtgc ttgctattgg cactgcaaat 60 cccccaaaca tttatcttca atctgagttt cctgatttct tcttcagagt caccaatagt 120 gagcatcatg tccaactcaa gcagaagttc aagcgcatgt gtgacaactc caatatcagg 180 aagcgccatt ttcttgttga tgaagatatt cttaaagagc atccaaacat cagcacctat 240 ggagctcctt cgttggatac acgcagagaa atagtaacta agtacattcc taagcttggg 300 aaggaagcag ccttgaaatg tattaaagaa tggggccaac ctttatctaa gatcacacat 360 ctcatcttct gcacatcttc atgcatcaac agcattcctg gacctgattt ctaccttgcc 420 agagaaattg gcctcccagc cacttgtaac cgccttgtga tttatgatca tggttgtcac 480 gctggtggtt cagtcattcg tgtagccaag gctcttgctg agagcatccc tggctcacgt 540 gtgctcactg tgtgtgctga gaccatgctt acttccttcc aagccccaag tccgtcacat 600 atggacattg tggttgggca cgccttgttt ggagatggtg ctgctgccat gattgtcggt 660 acagatccta tccccaatgt tgaacgtcca ctgtttgagt ttgtgttggc tgcacaacag 720 acagtgggag gatctgagga ggcaattcat ggacatgcaa ctgaaagagg tcaaacttac 780 tttttaggaa aagagattcc aaatgttgtt gctggtaatg tgaagaagtg tatgcatgat 840 gcatttggga cgcttggtat gacaatcaat gaaggagagt ggaattcgtt gttctacgtg 900 gtgcatcctg gtgggaaggc agtactgaat gggatggaag aggtgcttga gttgaaggaa 960 gagaagcttg ctgcgagtag gactattttg agagagtacg gaaacatgtg gagtccatgt 1020 gtgttcttcg tgttggatga aatgagaaag aaatcttcca acgaagggaa gtccacaacc 1080 ggagaaggac atgactgggg tgctttgatg acctttggac caggtttgac ggttgaaaca 1140 attgttttgc attccatttc cctgaaggac tag 1173 <210> 2 <211> 390 <212> PRT <213> Artificial Sequence <220> <223> CHS-L protein <400> 2 Met Glu Ser Ala Ala Glu Lys Lys Gly Phe Ala Thr Val Leu Ala Ile 1 5 10 15 Gly Thr Ala Asn Pro Pro Asn Ile Tyr Leu Gln Ser Glu Phe Pro Asp 20 25 30 Phe Phe Phe Arg Val Thr Asn Ser Glu His His Val Gln Leu Lys Gln 35 40 45 Lys Phe Lys Arg Met Cys Asp Asn Ser Asn Ile Arg Lys Arg His Phe 50 55 60 Leu Val Asp Glu Asp Ile Leu Lys Glu His Pro Asn Ile Ser Thr Tyr 65 70 75 80 Gly Ala Pro Ser Leu Asp Thr Arg Arg Glu Ile Val Thr Lys Tyr Ile 85 90 95 Pro Lys Leu Gly Lys Glu Ala Ala Leu Lys Cys Ile Lys Glu Trp Gly 100 105 110 Gln Pro Leu Ser Lys Ile Thr His Leu Ile Phe Cys Thr Ser Ser Cys 115 120 125 Ile Asn Ser Ile Pro Gly Pro Asp Phe Tyr Leu Ala Arg Glu Ile Gly 130 135 140 Leu Pro Ala Thr Cys Asn Arg Leu Val Ile Tyr Asp His Gly Cys His 145 150 155 160 Ala Gly Gly Ser Val Ile Arg Val Ala Lys Ala Leu Ala Glu Ser Ile 165 170 175 Pro Gly Ser Arg Val Leu Thr Val Cys Ala Glu Thr Met Leu Thr Ser 180 185 190 Phe Gln Ala Pro Ser Pro Ser His Met Asp Ile Val Val Gly His Ala 195 200 205 Leu Phe Gly Asp Gly Ala Ala Ala Met Ile Val Gly Thr Asp Pro Ile 210 215 220 Pro Asn Val Glu Arg Pro Leu Phe Glu Phe Val Leu Ala Ala Gln Gln 225 230 235 240 Thr Val Gly Gly Ser Glu Glu Ala Ile His Gly His Ala Thr Glu Arg 245 250 255 Gly Gln Thr Tyr Phe Leu Gly Lys Glu Ile Pro Asn Val Val Ala Gly 260 265 270 Asn Val Lys Lys Cys Met His Asp Ala Phe Gly Thr Leu Gly Met Thr 275 280 285 Ile Asn Glu Gly Glu Trp Asn Ser Leu Phe Tyr Val Val His Pro Gly 290 295 300 Gly Lys Ala Val Leu Asn Gly Met Glu Glu Val Leu Glu Leu Lys Glu 305 310 315 320 Glu Lys Leu Ala Ala Ser Arg Thr Ile Leu Arg Glu Tyr Gly Asn Met 325 330 335 Trp Ser Pro Cys Val Phe Phe Val Leu Asp Glu Met Arg Lys Lys Ser 340 345 350 Ser Asn Glu Gly Lys Ser Thr Thr Gly Glu Gly His Asp Trp Gly Ala 355 360 365 Leu Met Thr Phe Gly Pro Gly Leu Thr Val Glu Thr Ile Val Leu His 370 375 380 Ser Ile Ser Leu Lys Asp 385 390 <210> 3 <211> 1170 <212> DNA <213> Artificial Sequence <220> <223> CHS gene <400> 3 atggtgaatg tggaagagat ccgtaaggca caacgagcgg aaggtgctgc tacagtaatg 60 gctatcggta cagcgacccc aacaaactgt atagaacaaa gtacctatcc agattactat 120 tttcgtatta caaacagtga gcacatgaca gagttgaaag agaaattcca gcgcatgtgt 180 gataagtcaa tgataaaaaa gagatatatg cacttgacag aggagatctt aaaggagaac 240 cctaatatgt gtgcttacat ggcaccttct attgatgcaa ggcaagatat agtggttttg 300 gaagtaccaa agcttggaaa agaggctgca acaaaggcca ttaaggaatg gggccaaccc 360 aaatccaaaa ttacgcactt gatcttttgc actacaagtg gtgtggacat gccaggagct 420 gactaccaac tcacaaagct cttaggtctt cgcccatctg tgaagcgata catgatgtac 480 caacaaggtt gctttgcagg cggtacggtg ctccgtttag ccaaagactt ggctgagaat 540 aacaaaggag cacgtgtact tgtggtttgt tctgagatta ctgcagttac attccgtggg 600 cctactgata cccatcttga cagccttgtg ggccaagcat tgtttggcga tggggcagct 660 gctgttattg ttggatctga cccaatccca caagttgaga agcctttatt tgaacttgta 720 tggacagcac aaactattct tcccgatagt gaaggggcta ttgatgggca tcttcgtgaa 780 gttgggctca cattccatct tctgaaagat gttcctgggc tcatctcaaa gaacattgag 840 aaagccttgg ttgaagcctt caaaccattg ggaatttctg attataactc aattttctgg 900 attgctcacc caggtgggcc tgcaattttg gaccaagttg aggccaaatt agagttgaag 960 cccgaaaaga tgcaggccac tagacacgtg cttagtgagt atggaaacat gtccagtgca 1020 tgcgtgttat tcattttgga tgaaatgagg agaaaatcag caaaagatgg acttggcaca 1080 acaggtgaag gacttgagtg gggtgttcta tttggatttg ggcctggcct tactgttgag 1140 accgttgtgc tccacagtat tgctatttaa 1170 <210> 4 <211> 389 <212> PRT <213> Artificial Sequence <220> <223> CHS protein <400> 4 Met Val Asn Val Glu Glu Ile Arg Lys Ala Gln Arg Ala Glu Gly Ala 1 5 10 15 Ala Thr Val Met Ala Ile Gly Thr Ala Thr Pro Thr Asn Cys Ile Glu 20 25 30 Gln Ser Thr Tyr Pro Asp Tyr Tyr Phe Arg Ile Thr Asn Ser Glu His 35 40 45 Met Thr Glu Leu Lys Glu Lys Phe Gln Arg Met Cys Asp Lys Ser Met 50 55 60 Ile Lys Lys Arg Tyr Met His Leu Thr Glu Glu Ile Leu Lys Glu Asn 65 70 75 80 Pro Asn Met Cys Ala Tyr Met Ala Pro Ser Ile Asp Ala Arg Gln Asp 85 90 95 Ile Val Val Leu Glu Val Pro Lys Leu Gly Lys Glu Ala Ala Thr Lys 100 105 110 Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His Leu Ile 115 120 125 Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp Tyr Gln Leu 130 135 140 Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Tyr Met Met Tyr 145 150 155 160 Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu Ala Lys Asp 165 170 175 Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val Cys Ser Glu 180 185 190 Ile Thr Ala Val Thr Phe Arg Gly Pro Thr Asp Thr His Leu Asp Ser 195 200 205 Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala Val Ile Val 210 215 220 Gly Ser Asp Pro Ile Pro Gln Val Glu Lys Pro Leu Phe Glu Leu Val 225 230 235 240 Trp Thr Ala Gln Thr Ile Leu Pro Asp Ser Glu Gly Ala Ile Asp Gly 245 250 255 His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys Asp Val Pro 260 265 270 Gly Leu Ile Ser Lys Asn Ile Glu Lys Ala Leu Val Glu Ala Phe Lys 275 280 285 Pro Leu Gly Ile Ser Asp Tyr Asn Ser Ile Phe Trp Ile Ala His Pro 290 295 300 Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Ala Lys Leu Glu Leu Lys 305 310 315 320 Pro Glu Lys Met Gln Ala Thr Arg His Val Leu Ser Glu Tyr Gly Asn 325 330 335 Met Ser Ser Ala Cys Val Leu Phe Ile Leu Asp Glu Met Arg Arg Lys 340 345 350 Ser Ala Lys Asp Gly Leu Gly Thr Thr Gly Glu Gly Leu Glu Trp Gly 355 360 365 Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Val Glu Thr Val Val Leu 370 375 380 His Ser Ile Ala Ile 385 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CHS-L forward primer <400> 5 aaggatccat ggagagtgct ggag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CHS-L reverse primer <400> 6 aactcgagct agtctctcag aggg 24 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CHS forward primer <400> 7 ggatccatgg tgaatgtgga agatc 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CHS reverse primer <400> 8 gaattcttag acagccacac tatggag 27

Claims (11)

A polyketide synthase (polyketide synthase) derived CHS-L protein represented by the amino acid sequence of SEQ ID NO: 2 having activity. The method of claim 1,
The polyketide synthase (polyketide synthase) is to synthesize the emodin precursor using a malonyl-CoA (malonyl-CoA) substrate, protein. 3. The method of claim 2,
The emodin precursor is atrochrysome carboxylic acid (atrochrysome carboxylic acid) or endocrosine anthrone (endocrocin anthrone) will, the protein. The CHS-L gene represented by SEQ ID NO: 1 encoding the CHS-L protein of claim 1. A recombinant vector comprising the CHS-L gene of claim 4. A transformant transformed with the recombinant vector of claim 5 . 7. The method of claim 6,
The transformant is an Escherichia coli BL21 (DE3) strain, the transformant. A primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6 capable of specifically amplifying the CHS-L gene represented by SEQ ID NO: 1. A method for producing an emodin precursor, comprising the step of reacting the protein of claim 1 with malonyl-CoA. 10. The method of claim 9,
Further comprising the step of adding a NADPH cofactor. 10. The method of claim 9,
The emodin precursor is atrochrysome carboxylic acid (atrochrysome carboxylic acid) or endocrosine anthrone (endocrocin anthrone) will, the production method.

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