KR102310518B1 - Cytochrome P450 mutant protein and its applications - Google Patents

Abstract

The present invention provides a cytochrome P450 mutant protein and its applications. Specifically, the present invention can significantly improve PPD production and PPD/DM ratio after remodeling key sites in cytochrome P450 CYP716A47.

Description

Cytochrome P450 mutant protein and its applications

The present invention relates to the fields of biotechnology, plant biology, and natural product drugs, and more particularly, to a cytochrome P450 mutant protein and its applications.

, Shenyi Capsule)은 종양 환자의 기허 증상을 개선하고, 생물체 면역 기능을 향상시킬 수 있다. 진세노사이드 Rh2 단량체를 주요 성분으로 하는 진싱 캡슐(

, Jinxing capsule)은 보건 약품으로서 생물체 면역력을 향상시키고 질병 저항력을 강화시키기 위한 것이다.Ginsenoside is a major active substance in ginseng plants of the Araliaceae (eg, ginseng, ginseng, western ginseng, etc.), and in recent years, it is also Some ginsenosides were found. Currently, domestic and foreign scientists have isolated at least 100 kinds of ginsenosides from plants such as ginseng and stone, and the difference in the content of these saponins among ginseng is very large. Here, the triterpenoid saponin, which has some remarkable therapeutic effects, has a very low content in the total natural saponin (also called rare saponin), but the extraction cost is very high, so the price is also very high. Currently, various saponins have already been used in clinical practice, for example, Samil capsule (

, Shenyi Capsule) can improve the symptoms of qiheo in oncology patients and enhance the immune function of the organism. Jinxing capsules containing ginsenoside Rh2 monomer as the main ingredient

, Jinxing capsule) as a health medicine, is intended to enhance the organism's immunity and enhance disease resistance.

Since rare ginsenosides often have unique biological activity or more remarkable therapeutic effects, all of the existing rare ginsenosides are manufactured by chemical hydrolysis, enzymatic hydrolysis and microbial hydrolysis of a large amount of saponin extracted from ginseng or ginseng. do. Since wild ginseng resources have already been basically depleted, the current main source of total saponin resources of ginseng is by artificial cultivation of ginseng or ginseng, and the artificially grown growth cycle is long (usually 5 to 7 years or more are required) ), also subject to regional restrictions, and because a large amount of pesticides are often used due to pests and diseases, artificial cultivation of ginseng or ginseng is a serious problem of continuous cropping There is), so the production, quality and safety of ginsenosides all face difficulties. On the other hand, a single component of saponin is produced using the entire saponin of ginseng as a raw material, which is a target ginsenoside monomer (for example, protopanaxatriol saponin) in which a large amount of the saponin is converted into a target ginsenoside monomer. Because it cannot be used because it cannot be used, not only waste of resources, but also increase the cost of extraction and purification.

Advances in synthetic biology provide new opportunities for the heterogeneous synthesis of natural products of plant origin. Artemisinic acid or Dihydroartemisinin was fermented and synthesized as a low-cost monosaccharide through assembly and optimization by metabolic pathways with yeast as a chassis. produced mycinin, which means that synthetic biology has huge potential in the field of drug synthesis from natural products. Rare ginsenoside monomers are heterogeneously synthesized through synthetic biological methods using yeast chassis cells, raw materials are inexpensive monosaccharides, and the manufacturing process is a fermentation process with controllable safety, so that any external contamination (e.g., Agrochemicals used in artificial cultivation of raw materials) are prevented, so by producing rare ginsenoside monomers through synthetic biological technology, it is possible to not only have a cost advantage but also to secure the quality and safety of the finished product. By using synthetic biology technology to prepare various high-purity rare ginsenoside monomers in a satisfactory amount and use them for activity measurement and clinical trials, innovative drug R&D of rare ginsenosides was promoted.

In order to artificially synthesize ginsenosides with medicinal activity using a synthetic biological method, it is necessary to first analyze and reconstruct the synthetic metabolic pathway with protopanaxadiol (PPD). As ginsenosides belong to triterpenoid compounds, the MVA and MEP metabolic pathways in plants provide the co-precursors IPP and DMAPP of the terpenoid compounds to the triterpenoid compound precursors Squalene and 2,3-epoxysqualene. Since the catalytic activity of cytochrome P450 protein to catalyze dammarenediol to synthesize protopanaxadiol is very low, It is a rate-limiting step, which not only limited the improvement of PPD production, but also resulted in the accumulation of intermediate DM.

Therefore, in the present art, more research and modification of cytochrome P450 is carried out to obtain more efficient cytochrome P450 protein element, thereby promoting ginsenoside cell factory synthesis efficiency.

The present invention provides a cytochrome P450 mutant protein, and a very high PPD production and PPD/DM ratio can be generated using the mutant protein.

According to a first aspect of the present invention, there is provided a mutant protein of cytochrome P450, wherein the mutant protein is a non-natural protein, the mutant protein has a catalytic activity catalyzing the production of protopanaxadiol, the mutant protein corresponds to SEQ ID NO.: 1 of the wild-type cytochrome P450, site 91 proline (P), site 87 leucine (L), site 235 lysine (K), site 349 lysine (K), site 366 Deletion of site valine (V), site 231 asparagine (N), site 285 serine (S), site 113 glutamine (Q), site 18 leucine (L), and/or site 1 to 4 Mutations are made in one or a plurality of key amino acids associated with enzymatic catalytic activity selected from one, two, three or four amino acids of those listed.

In another preferred embodiment, the mutant protein is of SEQ ID NO.: 1, first site methionine (M), second site alanine (A), third site alanine (A), fourth site alanine (A) One or a plurality of amino acids selected from is deleted.

In another preferred embodiment, in the mutant protein, the first to fourth sites in SEQ ID NO.: 1 are deleted, and a mutation occurs in the 18th site leucine (L).

In another preferred embodiment, the mutant protein has the first to fourth sites in SEQ ID NO.: 1 deleted, and the 18th site leucine (L) is mutated to isoleucine (I).

In another preferred embodiment, the 87 site leucine (L) is mutated to isoleucine (I); and/or site 235 lysine (K) is mutated to arginine (R); and/or site 349 lysine (K) is mutated to arginine (R); and/or site 366 valine (V) is mutated to isoleucine (I); and/or site 231 asparagine (N) is mutated to tyrosine (Y); and/or site 285 serine (S) is mutated to cysteine (C); and/or site 91 proline (P) is mutated to histidine (H); and/or site 113 glutamine (Q) is mutated to arginine (R); and/or the 18th site leucine (L) is mutated to isoleucine (I).

In another preferred embodiment, the mutation is L87I; K235R; K349R and V366I; N231Y and S285C; P91H; Q113R.

In another preferred embodiment, the amino acid sequence of the cytochrome P450 mutant protein is as shown in SEQ ID NO.: 2-8.

In another preferred embodiment, the mutant protein comprises the mutant protein (eg, 87 sites, 235 sites, 349 sites, 366 sites, 231 sites, 285 sites, 91 sites, 113 sites, 18 sites, and/or 1 to The rest of the amino acid sequence except for the 4 site amino acids) is identical to or essentially identical to the sequence shown in SEQ ID NO.: 1.

In another preferred embodiment, the basically same thing differs by at most 50 (relatively preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5) amino acids, wherein the different These include substitutions, deletions or additions of amino acids, and the mutant protein still has catalytic activity to catalyze the production of protopanaxadiol.

In another preferred embodiment, the homology with the sequence shown in SEQ ID NO.: 1 is at least 80%, relatively preferably at least 85% or 90%, more preferably at least 95%, most preferably at least 98% and homology ≤ 485/486 or 99.79%.

In another preferred embodiment, the mutant protein of cytochrome P450 catalyzes the reaction of damarendiol (DM) to produce protopanaxadiol (PPD).

In another preferred embodiment, the mutant protein of cytochrome P450 catalyzes the hydroxylation of the C12 site of damarendiol (DM) to produce protopanaxadiol (PPD).

In another preferred embodiment, the mutant protein of cytochrome P450 catalyzes the following reaction.

In another preferred embodiment, the reaction has one or a plurality of characteristics selected from the following.

(i) The pH of the reaction system is 5.0 to 9.0, relatively preferably 7.0 to 8.0, and more preferably 7.4 to 7.5.

(ii) The reaction temperature is 20-40°C, relatively preferably 25-35°C, more preferably 26-33°C, and most preferably 30°C.

(iii) The reaction time is 0.5 h to 36 h, relatively preferably 2 h to 12 h, and more preferably 2 h to 3 h.

In another preferred embodiment, the catalytic activity of the mutant protein of cytochrome P450 to damarendiol (DM) to produce protopanaxadiol (PPD) is 125 of wild-type P450 (SEQ ID NO.: 1). ~250%.

In another preferred embodiment, the cytochrome P450 mutant protein has one or a plurality of characteristics selected from the following.

(a) compared to wild-type cytochrome P450 protein, the catalytically obtained protophanaxadiol production/damarendiol (PPD/DM) ratio ≥ 20%, relatively preferably 23 to 250%, more preferably 25 to 250% or 30-200%.

(b) Compared with the wild-type cytochrome P450 protein, the production (mg/L) of protopanaxadiol (PPD) obtained by catalysis is ≥ 300, relatively preferably 303 to 600, and more preferably 305 to 500.

According to a second aspect of the present invention, there is provided a polynucleotide encoding the mutant protein according to the first aspect of the present invention.

In another preferred embodiment, the polynucleotide comprises:

(a) a polynucleotide encoding a polypeptide as shown in any one of SEQ ID NO.: 2-8;

(b) a polynucleotide whose sequence is as shown in any one of SEQ ID NO.: 15-21;

(c) ≥95% (relatively preferably ≥98%) homology between the nucleotide sequence and the sequence shown in any one of SEQ ID NOs: 15-21, and SEQ ID NO.: 1 or SEQ ID NO.: 2- a polynucleotide encoding the polypeptide shown in any one of 8;

(d) a polynucleotide complementary to the polynucleotide according to any one of (a) to (c).

In another preferred embodiment, the polynucleotide has an auxiliary element selected from a signal peptide, a secretory peptide, a tag sequence (eg, 6His), or a combination thereof in the flank of the ORF of the cytochrome P450 mutant protein. more included.

In another preferred embodiment, the polynucleotide is selected from a DNA sequence, an RNA sequence, or a combination thereof.

According to a third aspect of the present invention, there is provided a carrier containing the polynucleotide according to the second aspect of the present invention.

In another preferred embodiment, the carrier comprises an expression carrier, a shuttle carrier (shuttle vector), an integration carrier.

According to a fourth aspect of the present invention, there is provided a host cell containing the carrier according to the third aspect of the present invention or in which the polynucleotide according to the second aspect of the present invention is integrated in its genome.

In another preferred embodiment, the host cell is a eukaryotic cell such as a yeast cell or a plant cell.

In another preferred embodiment, the host cell is a prokaryotic cell such as E. coli.

In another preferred embodiment, the host cell is a ginseng cell.

According to a fifth aspect of the present invention, there is provided a method for generating a cytochrome P450 mutant protein according to the first aspect of the present invention, wherein the method comprises the host cell according to the fourth aspect of the present invention under conditions suitable for expression. culturing to express a mutant protein of cytochrome P450; and isolating the mutant protein of the cytochrome P450.

According to a sixth aspect of the present invention, there is provided an enzyme preparation comprising the cytochrome P450 mutant protein according to the first aspect of the present invention.

In another preferred embodiment, the enzyme preparation includes an injection, and/or a freeze-dried preparation.

According to a seventh aspect of the present invention, the cytochrome P450 mutant protein according to the first aspect is contacted with a reaction substrate to perform a catalytic reaction to obtain the protopanaxadiol (i); and optionally, isolating and purifying the protopanaxadiol (ii).

In another preferred embodiment, the reaction substrate is damarenediol.

In another preferred embodiment, in step (i), the catalytic reaction time is 0.5 h to 36 h, relatively preferably 2 h to 12 h, and more preferably 2 h to 3 h.

In another preferred embodiment, in step (i), the catalytic reaction temperature is 20 to 40 °C, relatively preferably 25 to 35 °C, more preferably 26 to 33 °C, and most preferably 30 °C.

According to an eighth aspect of the present invention, there is provided the use of the mutant protein according to the first aspect of the present invention, wherein the mutant protein catalyzes damarendiol (DM) to produce protopanaxadiol (PPD). used or used to catalyze damarendiol (DM) to prepare catalyst formulations to produce protopanaxadiol (PPD).

According to a ninth aspect of the present invention, there is provided a use of the mutant protein according to the first aspect of the present invention or a host cell of the present invention, characterized in that for producing protopanaxadiol (PPD).

According to a tenth aspect of the present invention, there is provided a method for producing a transgenic plant, comprising regenerating the host cell according to the fourth aspect of the present invention into a plant, wherein the host cell is a plant cell. .

According to an eleventh aspect of the present invention, there is provided a mutant promoter sequence, wherein the promoter sequence has a transcriptional activity of at least 30% (eg, 40%) of the P450 protein compared to the wild-type P450 promoter (SEQ ID NO.: 9). ~100%) is improved.

In another preferred embodiment, the promoter sequence is the sequence shown in any one of SEQ ID NO.: 10-13.

In another preferred embodiment, the mutant promoter is promoter 1D1, which can significantly improve the expression level of P450 protein.

The mutant promoter is associated with a transcriptional strength wherein the 28th site nucleotide is T deleted, the 417 site nucleotide is G, the 445 site nucleotide is G, the 654 site nucleotide is A, and the 655th site nucleotide is A. and a key nucleotide, wherein the nucleotide position number is based on the sequence set forth in SEQ ID NO.: 9.

In another preferred embodiment, the promoter 1D1 of the mutant comprises a key nucleotide associated with transcriptional strength, wherein the 417 site nucleotide is G and the 445 site nucleotide is G, wherein the nucleotide position number is SEQ ID NO .: is based on the sequence shown in 9.

In another preferred embodiment, the remaining nucleotides except for nucleotides at site 417 and site 445 in the mutant promoter 1D1 are identical to or basically identical to the sequence shown in SEQ ID NO.: 9.

In another preferred embodiment, the mutant promoter 1D1 has the sequence shown in SEQ ID NO.: 10.

In another preferred embodiment, the promoter of the mutant is promoter 9C1-2, and the 28th site nucleotide comprises a key nucleotide associated with transcriptional strength in which T is deleted, wherein the nucleotide position number is SEQ ID NO.: 9 It is based on the sequence shown in

In another preferred embodiment, the remaining nucleotides except for the 28 site nucleotides in the mutant promoter 9C1-2 are identical to or basically identical to the sequence shown in SEQ ID NO.: 9.

In another preferred embodiment, the mutant promoter 9C1-2 has the sequence shown in SEQ ID NO.: 11.

In another preferred embodiment, the promoter of the mutant is promoter 11B5, and the 654 site nucleotide contains a key nucleotide related to transcriptional strength that is A, wherein the nucleotide position number is in the sequence shown in SEQ ID NO. it will be based

In another preferred embodiment, the remaining nucleotides except for the 654 site nucleotides in the mutant promoter 11B5 are identical to or basically identical to the sequence shown in SEQ ID NO.: 9.

In another preferred embodiment, the mutant promoter 11B5 has the sequence shown in SEQ ID NO.: 12.

In another preferred embodiment, the promoter of the mutant is promoter 15F1, and contains a key nucleotide related to transcriptional strength, where the 655th site nucleotide is A, wherein the nucleotide position number is in the sequence shown in SEQ ID NO. it will be based

In another preferred embodiment, the remaining nucleotides except for the 655 site nucleotides in the mutant promoter 15F1 are identical to or basically identical to the sequence shown in SEQ ID NO.: 9.

In another preferred embodiment, the mutant promoter 15F1 has the sequence shown in SEQ ID NO.: 13.

According to a twelfth aspect of the present invention, there is provided an expression cassette comprising the promoter of the present invention and a nucleotide sequence encoding the P450 mutant protein of the present invention linked to the promoter operability.

It should be understood that within the scope of the present invention, all of the above technical features of the present invention and each of the technical features specifically described below (for example, examples) may be mutually combined, whereby new or preferred technical features construct a solution. Due to the limitation of the side width, it will not be explained in detail here.

1 shows a schematic diagram of the construction of a recombinant budding yeast strain WP8 producing damarendol.
2 shows histograms of wild-type and 1G4, 2D8, 3B9, 8G7, 9C1, 16F8 and 24A10 mutant strains PPD/DM.
Figure 3 shows the HPLC detection degree of the recombinant budding yeast strain producing protopanaxadiol.
Figure 4 shows a schematic diagram of the production of a recombinant budding yeast strain producing protopanaxadiol.

After extensive in-depth studies, the present inventors unexpectedly selected key amino acid sites capable of remarkably improving the catalytic activity of cytochrome P450 mutant proteins by mass selection. According to the present invention, after remodeling the key site of cytochrome P450 CYP716A47, the PPD production and PPD/DM ratio can be significantly improved. In addition, the present inventors have found that the first to fourth site amino acids of wild-type cytochrome P450 CYP716A47 are deleted, and one key site (eg, the 18th site amino acid) is simultaneously mutated to significantly improve its catalytic activity. It can be improved, specifically, it was found that the ratio of PPD/DM can be improved up to 125.6%.

In addition, the present inventors further constructed a budding yeast chassis cell WP8 that synthesizes damarendiol (DM), and selected Stratagene's GeneMorph II Random Mutagenesis Kit random mutagenesis kit to prepare a mutant library of cytochrome P450 (CYP716A47). In addition, one CYP716A47 yeast mutant library was constructed by transforming the budding yeast chassis cell WP8 into a single copy insertion into the yeast genome and synthesizing protopanaxadiol (PPD). Based on this, the present invention was completed.

Terms

As used herein, the term “AxxB” denotes changing amino acid A at site xx to amino acid B, for example, “L87I” refers to mutation of amino acid L at site 87 to I, and , inferred in this way.

Mutant protein of the present invention and its encoding nucleic acid

As used herein, the terms “mutant protein”, “mutant protein of the present invention”, and “cytochrome P450 mutant protein of the present invention” are used interchangeably and refer to all non-naturally occurring cytochrome P450 mutant proteins, , wherein the mutant protein is a protein that has undergone artificial modification based on the protein shown in SEQ ID NO.: 1, wherein the mutant protein comprises a key amino acid related to enzyme catalytic activity, and at least one of the key amino acids is artificially modified will go through In addition, the mutant protein of the present invention has an enzymatic activity that catalyzes the C12 hydroxylation of damarendiol (DM) to form protopanaxadiol (PPD).

The term “key amino acid” is based on SEQ ID NO.: 1 and has at least 80% homology with SEQ ID NO.: 1, for example, 84%, 85%, 90%, 92%, 95 %, the corresponding site in 98% of the sequence is a specific amino acid described in the text, for example, the key amino acid based on the sequence shown in SEQ ID NO.: 1 is the 91st site proline (P); site 87 leucine (L); site 235 lysine (K); site 349 lysine (K); site 366 valine (V); site 231 asparagine (N); site 285 serine (S); site 113 glutamine (Q); site 18 leucine (L); and/or 1, 2, 3 or 4 amino acids in which the first to fourth sites are deleted, and a mutant protein obtained by mutating the key amino acid or the first to fourth site amino acids are deleted The resulting mutant protein has an enzymatic activity that catalyzes the C12 hydroxylation of damarendiol (DM) to form protopanaxadiol (PPD).

Preferably, in the present invention, the following mutations are performed for the key amino acids of the present invention.

site 87 leucine (L) is mutated to isoleucine (I); and/or site 235 lysine (K) is mutated to arginine (R); and/or site 349 lysine (K) is mutated to arginine (R); and/or site 366 valine (V) is mutated to isoleucine (I); and/or site 231 asparagine (N) is mutated to tyrosine (Y); and/or site 285 serine (S) is mutated to cysteine (C); and/or site 91 proline (P) is mutated to histidine (H); and/or site 113 glutamine (Q) is mutated to arginine (R); and/or the 18th site leucine (L) is mutated to isoleucine (I).

It should be understood that the amino acid number in the mutant protein of the present invention is formed based on SEQ ID NO.: 1, and the homology between any one specific mutant protein and the sequence shown in SEQ ID NO.: 1 is 80% or more , the amino acid number of the mutant protein may be misaligned with respect to the amino acid number of SEQ ID NO. Using conventional sequence comparison techniques of %), and a mutant protein having the same or similar catalytic activity to produce protopanaxadiol (PPD) should not fall within the scope of the mutant protein of the present invention.

Mutant proteins of the invention are synthetic or recombinant proteins, ie, either chemically synthesized products or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants). Depending on the host used in the recombinant production scheme, the mutant protein of the present invention may be glycosylated or aglycosylated. Mutant proteins of the invention may or may not further comprise an initial methionine residue.

The present invention further includes fragments, derivatives and analogs of the mutant protein. For example, as used herein, the terms “fragment”, “derivative” and “analog” refer to a protein that essentially retains the same biological function or activity as the mutant protein.

A fragment, derivative or analog of a mutant protein of the present invention may be a mutant protein substituted by (i) one or a plurality of conserved and non-conserved amino acid residues (preferably conserved amino acid residues), wherein the substituted amino acid residue is a genetic code may or may not be encoded by, or (ii) a mutant protein having a substitution radical at one or multiple amino acid residues, or (iii) a compound other than the mature mutant protein (e.g., a mutant It may be a mutant protein formed by fusion of a compound with an extended protein half-life, for example, polyethylene glycol, or (iv) a mutant protein (eg, leader) formed by fusion of an additional amino acid sequence to the mutant protein sequence sequence or secretory sequence, or a sequence for purifying the mutant protein, or a proteogen sequence, or a fusion protein formed with an antigenic IgG fragment). In accordance with the teachings herein, such fragments, derivatives and analogs are within the scope known to those skilled in the art. In the present invention, the conservatively substituted amino acid is preferably generated by amino acid substitution according to Table 1.

initial residue representative substitution preferred substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu

The active mutant protein of the present invention has an enzymatic activity that catalyzes the C12 hydroxylation of damarendiol (DM) to form protopanaxadiol (PPD).

Preferably, the mutant protein is as shown in SEQ ID NO.: 2-8. It should be understood that the mutant protein of the present invention usually has relatively high homology (identity) compared to the sequence shown in SEQ ID NO.: 2-8, preferably, the mutant protein and SEQ ID NO.: 2 The homology of the sequence indicated in -8 is at least 80%, relatively preferably at least 85% to 90%, more preferably at least 95%, most preferably at least 98%, most preferably > 485/486 (99.79%) )am.

In addition, the mutant protein of the present invention may be further modified. Modification (generally not changing the primary structure) forms include chemically derived forms of the mutant protein in vivo or ex vivo, such as acetylation or carboxylation. The modification further includes glycosylation, such as a mutant protein produced by undergoing glycosylation modifications in the synthesis and processing or further processing steps of the mutant protein. This modification can be accomplished by exposing the mutant protein to an enzyme that undergoes glycosylation (eg, mammalian glycosylase or diglycosylase). Modified forms further include sequences having phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). It further includes a mutant protein that has been modified to improve its resistance proteolytic hydrolysis performance or optimize its dissolution performance.

The term “polynucleotide encoding a mutant protein” may include a polynucleotide encoding a mutant protein of the present invention, and may also be a polynucleotide further comprising additional coding and/or non-coding sequences.

The present invention also relates to variants of said polynucleotides, which encode fragments, analogues and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. Such nucleotide variants include substitutional variants, deletional variants and insertional variants. As known in the art, an allelic variant is an alternate form of one polynucleotide, and may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded mutant protein.

The present invention also relates to polynucleotides that hybridize with said sequences and have at least 50%, relatively preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates to a polynucleotide capable of hybridizing with a polynucleotide according to the invention under particularly stringent (or stringent) conditions. In the present invention, "stringent conditions" refer to (1) hybridization and elution under relatively low ionic strength and relatively high temperature, such as 0.2 × SSC, 0.1 % SDS, 60°C, or (2) 50% (v/ v) indicates the addition of a denaturant during hybridization, such as formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc., or (3) the homology between the two sequences is at least 90% or more, more preferably 95% or more indicates that hybridization occurs only in

The mutant protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

The full-length polynucleotide sequence of the present invention can generally be obtained through PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers are designed according to the nucleotide-related sequences disclosed in the present invention, in particular according to an open reading frame sequence, and cDNA libraries purchased on the market or general methods known by those skilled in the art. The prepared cDNA library is used as a template and amplified to obtain a related sequence. If the sequence is relatively long, it is often necessary to perform PCR amplification twice or several times, and then combine the amplified fragments in the correct order each time.

Once the relevant sequences are obtained, large quantities of the relevant sequences can be obtained by recombinant methods. Usually, it is cloned into a carrier, transferred to a cell, and then isolated from a host cell after propagation by a general method to obtain a related sequence.

In addition, related sequences can be synthesized by artificial synthesis, and in particular, the fragment length is relatively short. In general, fragments with very long sequences can be obtained by first synthesizing a plurality of small fragments and then ligating them again.

Currently, a DNA sequence encoding a protein (or a fragment thereof, or a derivative thereof) of the present invention can be completely obtained through chemical synthesis. The DNA sequence of interest can then be incorporated into cells and various existing DNA molecules known in the art (or, for example, carriers). In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. In particular, when it is difficult to obtain full-length cDNA from the library, it may be preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and the primers used for PCR are appropriate according to the sequence information of the present invention disclosed herein. can be selected, and can be synthesized by a general method. DNA/RNA fragments can be isolated and purified through general methods such as gel electrophoresis.

wild-type cytochrome P450

As used herein, “wild-type cytochrome P450” refers to a naturally occurring, non-artificially modified cytochrome P450, whose nucleotides can be obtained through genetic engineering techniques such as genome sequencing, polymerase chain reaction (PCR), etc. and the amino acid sequence can be obtained by being derived from the nucleotide sequence. The amino acid sequence of the wild-type cytochrome P450 is as shown in SEQ ID NO.: 1.

The sequence information of the wild protein and the mutant protein of the present invention is as shown in Table 2 (see Examples).

expression carrier

The present invention also relates to a carrier comprising the polynucleotide of the present invention, and a host cell in which the sequence encoded by the carrier of the present invention or the mutant protein of the present invention is generated through genetic engineering, and the polypeptide according to the present invention through recombinant technology It is about how to create

The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins through general recombinant DNA techniques. In general, it has the following steps.

(1) Transformation or transduction into a suitable host cell using the polynucleotide (or variant) of the present invention encoding the mutant protein of the present invention, or a recombinant expression carrier containing the polynucleotide.

(2) Cultivate the host cells in an appropriate medium.

(3) Separation and purification of proteins from media or cells.

In the present invention, a polynucleotide sequence encoding a mutant protein may be inserted into a recombinant expression carrier. The term “recombinant expression carrier” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, reverse transcriptase virus or other carriers known in the art. Any plasmid and carrier may be used as long as they replicate and are stable in the host body. One important characteristic of an expression carrier generally includes an origin of replication, a promoter, a marker gene and translational control elements.

Methods known to those skilled in the art are used to construct the expression carrier of the coding DNA sequence comprising the mutant protein of the present invention and suitable transcriptional/translational control signals. Such methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, and the like. The DNA sequence is effectively linked onto an appropriate promoter in an expression carrier to direct mRNA synthesis. Representative examples of such promoters include E. coli lac or trp promoters; λ phage PL promoter; There are eukaryotic promoters, which include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of reverse transcriptase virus and some other known controllable genes in prokaryotic or eukaryotic cells or their viruses. It contains a promoter that expresses it. The expression carrier further comprises a ribosome binding site and a transcription terminator used for initiation of translation.

In addition, the expression carrier preferably selects transformed host cells such as dihydrogen folate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance used in E. coli. one or a plurality of selectable marker genes for providing a phenotypic trait for

A carrier comprising the appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to express the protein.

The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Representative examples include E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; There are fungal cells such as yeast and plant cells (eg, ginseng cells).

When the polynucleotides of the present invention are expressed in higher eukaryotes, insertion of an enhancer sequence in the carrier will amplify transcription. Amplifiers are cis-acting factors of DNA, usually about 10 to 300 base pairs, and act on promoters to amplify the transcription of genes. For example, it includes an SV40 amplifier of 100 to 270 base pairs on one side of the replication starting point, a polyoma amplifier and an adenovirus amplifier on one side of the replication starting point.

Selection of appropriate carriers, promoters, enhancers and host cells will be apparent to one of ordinary skill in the art.

Transformation of host cells by recombinant DNA can be carried out by conventional techniques known to those skilled in the art. When the host is a prokaryote such as E. coli, a soluble cell capable of absorbing DNA can be obtained after the exponential growth phase _{, treated with CaCl 2} method, and the steps used are as well known in the art. Another method is to use _{MgCl 2 .} Depending on the demand, transformation can also be carried out by electroporation. When the host is a eukaryote, a DNA transfection method, which is a general mechanical method such as calcium phosphate co-precipitation and microscopic injection, electroporation, and liposome packaging, may be selected.

The obtained transformant can be cultured by a conventional method, and the polypeptide encoded by the gene of the present invention is expressed. Depending on the host cell used, the medium used during culturing is selected from a variety of common media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the cells are re-cultured for some time by inducing the selected promoter by a suitable method (eg, temperature conversion or chemical induction).

The recombinant polypeptide in the method described above can be expressed intracellularly, in the cell membrane, or secreted extracellularly. Depending on the demand, its physical, chemical, and other properties can isolate and purify the recombinant protein by a variety of isolation methods. Such methods are known to those skilled in the art. Examples of such methods include general circular reproduction treatment, treatment with a protein precipitating agent (salting-out method), centrifugation, osmosis, excess treatment, excess centrifugation, molecular selection chromatography (gel filtration), adsorption chromatography, ion exchange chromatography chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

The main advantages of the present invention are as follows.

(i) After a large amount of selection and modification, the present invention first discovered the catalytic activity site of cytochrome P450 (CYP716A47) and the key site of its promoter. can be significantly improved, and the PPD production and PPD/DM ratio can be significantly improved.

(ii) In the present invention, for the first time, the first to fourth site amino acids of wild-type cytochrome P450 CYP716A47 are deleted, and at the same time, one of the key sites (eg, the 18th site amino acid) is mutated to significantly improve its catalytic activity. In particular, it was found that the ratio of PPD/DM can be improved up to 125.6%.

(iii) In the present invention, the catalytic activity of other sites of wild-type cytochrome P450 CYP716A47, for example, 235/349/366/231/285/91/113, etc. is mutated even if one or more amino acids are mutated, the catalytic activity is remarkable. It was found that, specifically, the ratio of PPD/DM can be improved by 26.1 to 80.2%.

The present invention will be described in more detail below in conjunction with specific examples. It should be understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Experimental methods that do not specify specific conditions in the examples below generally follow general conditions, for example, human and molecular clones of Sambrook et al. follow the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989). or follow the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.

Unless otherwise specified, all reagents and materials in the examples of the present invention are commercially available products.

Example 1. Construction of a recombinant budding yeast strain producing damarendol

In order to screen the library of cytochrome P450 CYP716A47, the present invention first constructs a budding yeast chassis cell capable of producing the protopanaxadiol precursor compound damarendiol.

Damarendiol can be synthesized by introducing the damarendiol synthase gene PgDDS into wild-type germination yeast, and using 2,3-epoxysqualene synthesized by the mevalonic acid pathway of germination yeast itself. For the artificially constructed damarendiol synthesis pathway, optimization including synthesis limiting-rate step optimization and precursor supply optimization was performed to obtain damarendiol high budding yeast strain WP8, and as shown in FIG. 1 , the budding yeast chassis used as cells.

Example 2. Acquired highly efficient cytochrome P450 mutant protein by forward evolution

(1) Using the cytochrome P450 gene sequence (optPPDS, SEQ ID NO.: 14, named as the amino acid sequence encoding SEQ ID NO.: 1) as a template, primer EP-1 (5''-ATGGCTGCGGCCATGGTCTTAT-3' '(SEQ ID NO.: 22)) and EP-2 (5''-GTTATGTGGATGCAGATGGATT-3'' (SEQ ID NO.: 23)) to perform error prone PCR. The error-inducing PCR is used by selecting a GeneMorph II Random Mutagenesis Kit random mutation kit from Stratagene. The PCR procedure was 95 °C for 2 min; 95 °C 10 s, 55 °C 15 s, 72 °C 2 min, total of 28 cycles; Decrease to 10 °C for 10 min at 72 °C, and the template used is 50 ng. The PCR product is obtained by recovering the cytochrome P450 mistake-induced PCR product after undergoing agarose gel electrophoresis.

(2) Using the budding yeast genome as a template, using the lithium acetate transformation method in the general method for molecular cloning, the mistake-induced PCR product in step (1) was introduced into the budding yeast chassis cells prepared in Example 1 and transformed A transformation product (ie a transformant) is obtained.

(3) The transformation product is uniformly coated on a YPD+200mg/L G418 antibiotic selection plate, and cultured by standing at 30°C for 2-3 days. All clones were picked with a toothpick and transferred to a 96-well plate, cultured with shaking at 30°C for 1 day, then transferred to a new 96-well plate and fermented for 4 days. The same volume of n-butanol solvent is added to the fermentation broth, extracted for 1 hour, and the upper organic phase is absorbed, and then HPLC is performed to detect the production and ratio of damarendol and protopanaxadiol of each transformant.

(4) Through selection of 24 pieces of 96-well plates, a total of 7 clones that improved the ratio of protophanaxadiol production/damarendiol (PPD/DM) by 20% or more were obtained, and the numbers were 1G4 and 2D8, respectively. , 3B9, 8G7, 9C1, 16F8 and 24A10. Using each cloned genome as a template, PCR was performed using primers EP-1 and EP-2 to obtain a cytochrome P450 fragment of each clone, and sequencing detection was performed to determine the nucleotide sequence and protein sequence of each mutant. get

Each wild-type and mutant protein sequence information and PPD/DM obtained above are shown in FIG. 2 and Table 2.

SEQ ID NO. note PPD/DM Enhancements optPPDS One wild type 0 1G4 2 L87I 53.2% 2D8 3 K235R 57.7% 3B9 4 K349R,V366I 80.2% 8G7 5 N231Y,S285C 26.1% 9C1 6 P91H 84.20% 16F8 7 Q113R 30.6% 24A10 8 Site 1 to Site 4 amino acid deletion, L18I 125.6%

(5) In the selection process for the cytochrome P450 mutant library, a series of mutant promoter sequences are obtained by further screening, and this mutated promoter sequence improves the expression level of cytochrome P450 to improve the efficiency and production of protopanaxadiol. It is advantageous. There are 4 mutant promoters in which the ratio of protopanaxadiol production/damarendiol (PPD/DM) is improved by 20% or more, and the numbers are 1D1, 9C1-2, 11B5 and 15F1, respectively. Using the genomes of clones 1D1, 9C1-2, 11B5 and 15F1, respectively, as a template, PCR is performed using primers to obtain promoter fragments of each clone, and sequencing is performed to obtain mutant promoter nucleotide sequences.

The obtained wild-type and mutant promoter nucleotide sequence information and PPD/DM are as shown in Table 3.

Different promoter+wild-type P450 SEQ ID NO. note PPD/DM Enhancements GAL1 9 wild-type promoter 0 1D1 10 Mutations in SEQ ID NO.: 9: C417G, A445G 44.1% 9C1-2 11 Mutations in SEQ ID NO.: 9: Site 28 T deletion 80.1% 11B5 12 Mutations in SEQ ID NO.: 9: G654A 62.2% 15F1 13 Mutations in SEQ ID NO.: 9: G655A 44.1%

According to the above results, this mutated promoter can further improve the expression level of the wild-type P450 protein, thereby improving the ratio of protopanaxadiol production/damarendiol. In addition, this mutated promoter can also be used in combination with the coding sequence of the P450 mutant protein of the present invention to further improve the ratio of protopanaxadiol production/damarendiol.

Example 3. High-efficiency heterologous synthesis of protopanaxadiol using the cytochrome P450 mutant protein

(1) Recombinant budding yeast strain WP8 using the budding yeast genome as a template and using the method of Example 1 to produce protopanaxadiol by transforming the budding yeast strain WP8 with SEQ ID NO.: 14 -get WT.

(2) Similarly, the mutant genes 1G4, 2D8, 3B9, 8G7, 9C1, 16F8 and 24A10, respectively, (the nucleotide sequence is SEQ ID NO.: 15-21, and the corresponding amino acid sequence is SEQ ID NO.: 2-8 ) as a template by replacing the wild-type optPPDS gene. Recombinant budding yeast strains WP8-1G4, WP8-2D8, WP8 to obtain each PCR fragment by performing the PCR, and transform into each budding yeast strain WP8 to produce protopanaxadiol containing each mutant protein -3B9, WP8-8G7, WP8-9C1, WP8-16F8 and WP8-24A10 are obtained.

(3) Place a solid medium, that is, 1 % Yeast Extract, 2 % Peptone, 2 % Dextrose (glucose), and 2 % agar powder.

Place the liquid medium, ie, 1 % Yeast Extract, 2 % Peptone, and 2 % Dextrose (glucose) (glucose).

Recombinant budding yeast strains WP8-1G4, WP8-2D8, WP8-3B9, WP8-8G7, WP8-9C1, WP8-16F8 and WP8-24A10 separated by lines from a solid medium plate were selected, each containing 5 mL of liquid medium. Incubate overnight (30 °C, 250 rpm, 16 h) in a test tube. The cells are collected by centrifugation and transferred to a 50 mL Erlenmeyer flask of 10 mL liquid medium, OD600 is adjusted to 0.05, and cultured with shaking at 30°C and 250 rpm for 4 days to obtain a fermentation product. This method sets up one parallel experiment for each recombinant yeast simultaneously.

Extraction and detection of damarendiol and protopanaxadiol: Absorb 100 μL fermentation broth from 10 mL fermentation broth, shake and pyrolyze yeast with Fastprep, add the same volume of normal butanol for extraction, and evaporate normal butanol under vacuum conditions. make it After dissolving in 100 μL methanol, the production of target products PPD and DM (Fig. 3, Fig. 4 and Table 4) is detected through HPLC.

Each of the obtained recombinant budding yeast strains protopanaxadiol and damarendiol production amounts are as shown in Table 4.

PPD (mg/L) DM (mg/L) PPD/DM WP8-WT 290 1058 27.42% WP8-1G4 312 715 43.62% WP8-2D8 316 699 45.23% WP8-3B9 330 635 51.95% WP8-8G7 335 925 36.22% WP8-9C1 435 848 51.29% WP8-16F8 386 1041 37.09% WP8-24A10 490 777 63.08%

As a result, compared to the wild-type cytochrome P450, the cytochrome P450 mutant protein of the present invention significantly increased the production of PPD (achievable at a maximum of 490 mg/L) and a ratio of PPD/DM (a maximum of 63%). can be significantly improved.

All publications mentioned herein are incorporated herein by reference as if each publication were independently incorporated by reference. In addition, after reading the above-described contents of the present invention, those skilled in the art can make various changes or modifications to the present invention, and such equivalent forms are also within the scope defined by the claims of the present invention. you will have to understand

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Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 2 <211> 486 <212> PRT <213> Panax ginseng <400> 2 Met Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Ile Leu Gly Glu Pro Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Gln Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 3 <211> 486 <212> PRT <213> Panax ginseng <400> 3 Met Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Gln Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Arg Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 4 <211> 486 <212> PRT <213> Panax ginseng <400> 4 Met Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Gln Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Arg Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Ile Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 5 <211> 486 <212> PRT <213> Panax ginseng <400> 5 Met Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Gln Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Tyr Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Cys Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 6 <211> 486 <212> PRT <213> Panax ginseng <400> 6 Ile Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Leu Leu Gly Glu His Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Gln Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 7 <211> 486 <212> PRT <213> Panax ginseng <400> 7 Met Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro 20 25 30 Gln Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly 35 40 45 Trp Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser 50 55 60 Gly Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro 65 70 75 80 Lys Val Phe Arg Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys 85 90 95 Gly Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val 100 105 110 Arg Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His 115 120 125 Gly Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met 130 135 140 Phe Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp 145 150 155 160 Arg Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln 165 170 175 Ile Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys 180 185 190 Arg Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly 195 200 205 Ser Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn 210 215 220 Ile Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu 225 230 235 240 Thr Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu 245 250 255 Glu Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu 260 265 270 Leu Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala 275 280 285 Ser His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly 290 295 300 Thr Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr 305 310 315 320 Asn Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro 325 330 335 Lys Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp 340 345 350 Asn Val Ala Gln Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr 355 360 365 Phe Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro 370 375 380 Lys Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro 385 390 395 400 Thr Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly 405 410 415 Asn Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg 420 425 430 Met Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met 435 440 445 His Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu 450 455 460 Lys Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile 465 470 475 480 His Leu His Pro His Asn 485 <210> 8 <211> 482 <212> PRT <213> Panax ginseng <400> 8 Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Pro Ile Leu Leu 1 5 10 15 Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro Gln Lys Glu Asn 20 25 30 Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly Trp Pro Leu Ile 35 40 45 Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser Gly Val Ser Glu 50 55 60 Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro Lys Val Phe Arg 65 70 75 80 Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys Gly Pro Glu Gly 85 90 95 Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val Gln Val Trp Phe 100 105 110 Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His Gly Glu Ser Asn 115 120 125 Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met Phe Leu Leu Lys 130 135 140 Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp Arg Val Met Lys 145 150 155 160 Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln Ile Asn Val His 165 170 175 Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys Arg Val Phe Met 180 185 190 Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly Ser Ser Ile Gln 195 200 205 Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn Ile Pro Gly Thr 210 215 220 Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu Thr Arg Glu Val 225 230 235 240 Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu Glu Asn Lys Gln 245 250 255 Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu Leu Thr Ala Asn 260 265 270 Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala Ser His Leu Ile 275 280 285 Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly Thr Ile Thr Phe 290 295 300 Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr Asn Gln Val Leu 305 310 315 320 Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro Lys Glu Leu Leu 325 330 335 Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp Asn Val Ala Gln 340 345 350 Glu Val Leu Arg Ile Ile Pro Gly Val Gly Thr Phe Arg Glu Ala 355 360 365 Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro Lys Gly Trp Lys 370 375 380 Met His Leu Ile Pro His Asp Thr His Lys Asn Pro Thr Tyr Phe Pro 385 390 395 400 Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn Gly Pro Ala 405 410 415 Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly 420 425 430 Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met His Asn Val Val 435 440 445 Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu Lys Ile Leu Thr 450 455 460 Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile His Leu His Pro 465 470 475 480 His Asn <210> 9 <211> 668 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 9 ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60 atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240 acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420 gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600 tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660 aaactata 668 <210> 10 <211> 668 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 10 ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60 atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240 acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatgaac 420 gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg 480 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600 tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660 aaactata 668 <210> 11 <211> 667 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 11 ttatattgaa ttttcaaaaa ttcttacttt tttttggatg gacgcaaaga agtttaataa 60 tcatattaca tggcaatacc accatataca tatccatatc taatcttact tatatgttgt 120 ggaaatgtaa agagccccat tatcttagcc taaaaaaacc ttctctttgg aactttcagt 180 aatacgctta actgctcatt gctatattga agtacggatt agaagccgcc gagcgggcga 240 cagccctccg acggaagact ctcctccgtg cgtcctggtc ttcaccggtc gcgttcctga 300 aacgcagatg tgcctcgcgc cgcactgctc cgaacaataa agattctaca atactagctt 360 ttatggttat gaagaggaaa aattggcagt aacctggccc cacaaacctt caaatcaacg 420 aatcaaatta acaaccatag gataataatg cgattagttt tttagcctta tttctggggt 480 aattaatcag cgaagcgatg atttttgatc tattaacaga tatataaatg caaaagctgc 540 ataaccactt taactaatac tttcaacatt ttcggtttgt attacttctt attcaaatgt 600 cataaaagta tcaacaaaaa attgttaata tacctctata ctttaacgtc aaggagaaaa 660 aactata 667 <210> 12 <211> 668 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 12 ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60 atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240 acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420 gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600 tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaagagaaa 660 aaactata 668 <210> 13 <211> 668 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 13 ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60 atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240 acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420 gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600 tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caagaagaaa 660 aaactata 668 <210> 14 <211> 1458 <212> DNA <213> Panax ginseng <400> 14 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 15 <211> 1458 <212> DNA <213> Panax ginseng <400> 15 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctat tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcataccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agacttgtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 16 <211> 1458 <212> DNA <213> Panax ginseng <400> 16 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaggacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 17 <211> 1458 <212> DNA <213> Panax ginseng <400> 17 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagcgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaggtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtatcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 18 <211> 1458 <212> DNA <213> Panax ginseng <400> 18 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg tacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aatgtgatat cgcatcccat ttgataggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcca agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 19 <211> 1458 <212> DNA <213> Panax ginseng <400> 19 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacctgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa catatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 20 <211> 1458 <212> DNA <213> Panax ginseng <400> 20 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc acttcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcgag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 21 <211> 1458 <212> DNA <213> Panax ginseng <400> 21 atggctgcgg ccatggtctt attcttttcc cttagtttat tgttgttgcc aattcttcta 60 ctctttgctt atttctcata cactaagaga atcccacaaa aagagaatga ttcaaaagct 120 cctttacctc caggccaaac aggttggcca ttgattggag agacactcaa ttacttgagt 180 tgtgtcaagt caggtgtttc agaaaacttc gtgaagtaca gaaaggaaaa gtactcccca 240 aaggttttta gaacatctct tttaggggaa cctatggcaa ttctttgcgg accagaaggt 300 aataagtttc tctactcaac tgagaaaaag ttggttcaag tttggtttcc atcttcagta 360 gaaaagatgt tcccacgtag ccatggtgag tcaaacgccg acaacttttc taaggttaga 420 ggtaagatga tgttcctact aaaagttgac gggatgaaaa agtatgttgg tctaatggat 480 agagtgatga aacagttctt ggaaacagat tggaacaagac agcaacaaat caatgttcat 540 aacactgtca aaaagtacac tgttactatg tcctgcagag tattcatgtc tatcgatgat 600 gaggaacaag tcacaagatt gggttcttct attcaaaaca tagaggctgg ccttttagca 660 gttccaatca acattcctgg aactgcaatg aacagagcca tcaagacagt taaactctta 720 actagagaag ttgaggcagt cattaagcag agaaaggttg acttattgga aaacaagcaa 780 gcctctcagc cacaggatct tttaagccac ctactattaa cagctaatca agatggtcaa 840 ttcttatcag aaagtgatat cgcatcccat ttgattggtt tgatgcaagg aggctacaca 900 actctaaatg gtacaattac cttcgttttg aattacttgg cagaattccc tgatgtttac 960 aaccaagtgt taaaagagca agtagaaata gccaactcta agcatccaaa ggaactgctt 1020 aactgggaag atttgagaaa aatgaagtac tcttggaatg tggcgcaaga ggtactgaga 1080 atcattccac ctggtgtcgg gacatttaga gaagctatta ccgatttcac ctacgctggt 1140 tatttgattc ctaaagggtg gaagatgcat ttgattccac acgacactca caaaaaccca 1200 acctacttcc cttctcctga gaagttcgac ccaacaagat tcgaaggaaa tggcccagca 1260 ccatacacat ttacaccatt tggcggcgga ccacgtatgt gtcctggtat cgaatacgct 1320 agactagtca ttttgatctt tatgcacaac gtggtaacaa acttccgttg ggaaaaactg 1380 atccctaatg aaaagatact gaccgatcca atacctagat tcgcacacgg tttaccaatc 1440 catctgcatc cacataac 1458 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 atggctgcgg ccatggtctt at 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gttatgtgga tgcagatgga tt 22

Claims (10)

In the mutant protein of cytochrome P450,
The mutant protein is a non-natural protein, and the mutant protein has a catalytic activity catalyzing the production of protopanaxadiol, wherein the mutant protein comprises at least one of a substitution mutation and a deletion mutant, an enzyme catalytic activity and It is characterized by the occurrence of mutations in the relevant key amino acids,
The substitution mutation is
mutation of proline (P) to histidine (H) at site 91, corresponding to SEQ ID NO.: 1 of wild-type cytochrome P450; site 87 mutation of leucine (L) to isoleucine (I); site 235 lysine (K) to arginine (R) mutation; site 349 lysine (K) to arginine (R) mutation; site 366 mutation of valine (V) to isoleucine (I); site 231 mutation of asparagine (N) to tyrosine (Y); mutation of site 285 serine (S) to cysteine (C); site 113 glutamine (Q) to arginine (R) mutation; and a mutation of the 18th site leucine (L) to isoleucine (I); at least one selected from the group consisting of,
The deletion mutation is
One of a first site methionine (M), a second site alanine (A), a third site alanine (A) and a fourth site alanine (A), corresponding to SEQ ID NO.: 1 of the wild-type cytochrome P450 to 4 amino acids are deleted,
SEQ ID NO.: represented by any one of 2 to 8
Mutant protein of cytochrome P450. In the polynucleotide,
A polynucleotide encoding the mutant protein according to claim 1 . In the vector,
A vector comprising the polynucleotide according to claim 2. In a host cell,
A host cell containing the vector according to claim 3. In the method for producing the mutant protein of cytochrome P450 according to claim 1,
culturing the host cell according to claim 4 under conditions suitable for expression to express the mutant protein of cytochrome P450; and
A method for producing a mutant protein of cytochrome P450, comprising the step of isolating the mutant protein of cytochrome P450. In the enzyme preparation,
An enzyme preparation comprising the cytochrome P450 mutant protein according to claim 1. In the method for preparing protopanaxadiol,
(i) obtaining the protopanaxadiol by contacting the cytochrome P450 mutant protein according to claim 1 with a reaction substrate to perform a catalytic reaction; and
A method for producing protopanaxadiol, comprising the step (ii) of isolating and purifying the protopanaxadiol. A composition for preparing a catalyst for producing protopanaxadiol (PPD) comprising the mutant protein according to claim 1,
The mutant protein is used to catalyze the hydroxylation of the C12 site of damarenediol (DM) to produce protopanaxadiol (PPD) or catalyze the hydroxylation of the C12 site of damarenediol (DM). A composition that is used to prepare a catalyst agent that functions to produce protopanaxadiol (PPD). A composition for producing protopanaxadiol (PPD) comprising the mutant protein according to claim 1 or the host cell according to claim 4. In the method for producing a transgenic plant,
A method for producing a transgenic plant, comprising regenerating the host cell according to claim 4 into a plant, wherein the host cell is a plant cell.

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