KR20190129840A - Cytochrome P450 Mutant Proteins and Their Applications - Google Patents

Abstract

The present invention provides cytochrome P450 mutant proteins and their applications, and specifically, the present invention can significantly improve PPD production and PPD / DM ratio after modification of key sites in cytochrome P450 CYP716A47.

Description

Cytochrome P450 Mutant Proteins and Their Applications

FIELD OF THE INVENTION The present invention relates to the fields of biotechnology, plant biology, and natural product drugs, and in particular, to cytochrome P450 mutant proteins and their 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 the main active substance in Araliaceae ginseng plants (e.g. ginseng, samchil, western ginseng, etc.), and in recent years has also been found in the Cucurbitaceae plant Gynostemma pentaphyllum. Some ginsenosides have been found. At present, domestic and foreign scientists have separated at least 100 kinds of ginsenosides from plants such as ginseng, dol, etc., the difference in the saponin content of ginseng is very large. Triterpenoid saponins, which have some remarkable therapeutic effects here, are extremely low in natural total saponins (also called rare saponins), but the extraction cost is very high, and the price is very expensive. Currently, various saponins have already been used in the clinic, for example, a three-day capsule, which is a drug mainly composed of ginsenoside Rg3 monomers (

Shenyi Capsule can improve the symptoms of tumor patients and improve immune function. Ginsenoside capsules containing ginsenoside Rh2 monomer as a main component

Jinxing capsule is a health agent for improving immunity and disease resistance.

Since rare ginsenosides often have unique bioactivity or more pronounced therapeutic effects, all existing rare ginsenosides are prepared by chemical hydrolysis, enzymatic hydrolysis and microbial hydrolysis of large amounts of saponins extracted from ginseng or ginseng. do. Since wild ginseng resources are already depleted by default, the current main source of ginseng's total saponin resources is by artificial cultivation of ginseng or samchil, and the growing cycle of artificial cultivation is long (generally 5 to 7 years or more required) Also, due to local restrictions, and often pesticides, large amounts of pesticides are used, artificial cultivation of ginseng or samchil is a severe hereditary disorder (Ginseng or samchil plantation must be absent for more than 5 to 15 years before it can be overcome. Ginsenoside production, quality and safety all face difficulties. On the other hand, the whole saponin of ginseng is used as a raw material to produce a single component saponin, which is converted to a target ginsenoside monomer (for example, protopanaxatriol saponin) in a large amount of the total saponin. This is because not only can not be used and can not be used, but also wastes resources, and can increase the cost of extract purification.

The development of synthetic biology offers new opportunities for the heterogeneous synthesis of natural products of plant sources. Using yeast as a chassis and assembling and optimizing by metabolic pathways, Artemisinic acid or Dihydroartemisinin was fermented and synthesized with low-cost monosaccharides, and then Arte through a one-step chemical conversion method. The production of microcinin means that synthetic biology has huge potential in the field of drug synthesis of natural products. Heterogeneous rare ginsenoside monomers are synthesized by synthetic biological methods using yeast chassis cells, raw materials are inexpensive monosaccharides, and the manufacturing process is a fermentation process in which safety is controlled and controlled. Since pesticides used in artificial planting of raw materials are prevented, the rare ginsenoside monomer can be manufactured through synthetic biological techniques to ensure cost advantages and to ensure the quality and safety of the finished product. A variety of high purity rare ginsenoside monomers have been prepared using synthetic biology techniques and used in activity measurements and clinical trials to facilitate innovative drug research and development of rare ginsenosides.

In order to artificially synthesize ginsenosides with medicinal activity using synthetic biological methods, the synthetic metabolic pathways with protoparanaxadiol (PPD) must first be interpreted and reconstituted. Since 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 give the triterpenoid compound precursors Squalene and 2,3-epoxysqualene The catalytic activity of synthesizing protoparnaxadiol by the cytochrome P450 protein catalysis against dammarenediol is very low. Therefore, the process of producing PPD using microbial cell plant It is one rate limiting step, which not only limited the improvement of PPD production but also resulted in the accumulation of intermediate DM.

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

The present invention provides cytochrome P450 mutant proteins, which can be used to produce very high PPD yields and PPD / DM ratios.

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

In another preferred embodiment, the mutant protein is SEQ ID NO .: 1, site 1 methionine (M), site 2 alanine (A), site 3 alanine (A), site 4 alanine (A) One or a plurality of amino acids selected from are deleted.

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

In another preferred embodiment, the mutant protein is deleted from the first to fourth regions of SEQ ID NO .: 1, and the eighteenth site leucine (L) is mutated to isoleucine (I).

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

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

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

In another preferred embodiment, the mutant protein is the mutation (eg, 87 sites, 235 sites, 349 sites, 366 sites, 231 sites, 285 sites, 91 sites, 113 sites, 18 sites, and / or 1 ~ The remainder of the amino acid sequence, except for the four-site amino acid), is identical or basically identical to the sequence shown in SEQ ID NO .: 1.

In another preferred embodiment, the basically same thing is at most 50 (comparably preferably 1-20, more preferably 1-10, even more preferably 1-5) amino acids different, wherein the different It includes substitutions, deletions or additions of amino acids, the mutant proteins still having catalytic activity catalyzing the production of protopanaxadiol.

In another preferred embodiment, homology with the sequence set forth 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 damarenediol (DM) to produce protoparanaxadiol (PPD).

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

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

In another preferred embodiment, the reaction has one or more features selected from the following.

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

(ii) The reaction temperature is 20 to 40 ° C, relatively preferably 25 to 35 ° C, more preferably 26 to 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 producing protopanaxadiol (PPD) by catalyzing the dammardiol (DM) of the mutant protein of cytochrome P450 is 125 of wild type P450 (SEQ ID NO .: 1). To 250%.

In another preferred embodiment, the mutant protein of cytochrome P450 has one or more features selected from the following.

(a) The ratio of protoparanaxadiol production / damageddiol (PPD / DM) obtained by catalysis compared to wild-type cytochrome P450 protein is ≧ 20%, relatively preferably 23-250%, and more preferably 25- 250% or 30-200%.

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

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

In another preferred embodiment, the polynucleotide is,

(a) a polynucleotide encoding a polypeptide as shown in any one of SEQ ID NOs: 2-8;

(b) a polynucleotide having the sequence as set forth in any one of SEQ ID NOs: 15-21;

(c) the homology ≧ 95% (comparatively preferably ≧ 98%) of the nucleotide sequence and the sequence shown in any one of SEQ ID NO .: 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 is a flanking of the ORF of the mutant protein of cytochrome P450 is an auxiliary element selected from a signal peptide, secreted peptide, tag sequence (for example 6His), or a combination thereof It is included more.

In another preferred embodiment, the polynucleotide is selected from DNA sequences, RNA sequences, 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 includes an expression carrier, a shuttle vector, an integrated carrier.

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

In another preferred embodiment, said 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 producing a mutant protein of cytochrome P450 according to the first aspect of the present invention, which method is suitable for expression in a host cell according to the fourth aspect of the present invention. Incubating to express the mutated protein of cytochrome P450; And separating the mutant protein of the cytochrome P450.

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

In another preferred embodiment, the enzyme formulation comprises an injection and / or lyophilized formulation.

According to a seventh aspect of the present invention, the method comprises the steps of: (i) obtaining the protopanaxadiol by performing a catalytic reaction by contacting a mutant protein of cytochrome P450 according to the first aspect with a reaction substrate; And optionally, (ii) isolating and purifying the protopanaxadiol.

In another preferred embodiment, the reaction substrate is damarediol.

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

In another preferred embodiment, in step (i), the catalytic reaction temperature is 20-40 ° C, relatively preferably 25-35 ° C, more preferably 26-33 ° C, 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 the dama diol diol (DM) to produce protoparanaxadiol (PPD). It is used to prepare a catalyst formulation which is used or catalyzes for dimarylenediol (DM) to produce protoparanaxadiol (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 the host cell of the present invention, which comprises preparing protopanaxadiol (PPD).

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

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

In another preferred embodiment, the sequence of the promoter is the sequence set forth in any one of SEQ ID NOs .: 10-13.

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

The mutant promoter is associated with a transcriptional intensity of which the T 28th site nucleotide is deleted, the 417th site nucleotide is G, the 445th site nucleotide is G, the 654th site nucleotide is A, and the 655th site nucleotide is A 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 mutation comprises a key nucleotide associated with a transcription intensity of which the 417th site nucleotide is G and the 445th site nucleotide is G, wherein the nucleotide position number is SEQ ID NO .: Based on the sequence shown in FIG.

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

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

In another preferred embodiment, the promoter for the mutation is promoter 9C1-2, wherein the 28th site nucleotide comprises a key nucleotide associated with the transcription intensity at 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 of the mutant promoter 9C1-2 are identical or basically identical to the sequence shown in SEQ ID NO.:9.

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

In another preferred embodiment, the promoter for the mutation is promoter 11B5 and comprises a core nucleotide associated with a transcription intensity of which the 654 site nucleotide is A, wherein the nucleotide position number is in the sequence shown in SEQ ID NO .: 9 It is based.

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

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

In another preferred embodiment, the promoter of the mutation comprises a key nucleotide associated with a transcription intensity of promoter 15F1 and wherein the 655th site nucleotide is A, wherein the nucleotide position number is in the sequence shown in SEQ ID NO .: 9 It is based.

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

In another preferred embodiment, said 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 nucleotide sequence encoding the P450 mutant protein of the present invention comprising said promoter of the present invention and linked to said promoter functionality.

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, embodiments) may be combined with each other, thereby providing a new or preferred technology. Configure the solution. Due to the limitation of the width, no further explanation is given here.

Figure 1 shows the schematic diagram of the construction of the recombinant sprouted yeast strain WP8 to produce damarenediol.
2 shows wild type and 1G4, 2D8, 3B9, 8G7, 9C1, 16F8 and 24A10 mutant strain PPD / DM histograms.
Figure 3 shows the HPLC detection diagram of recombinant sprouted yeast strains producing Protopanaxanadiol.
Figure 4 shows a schematic of the yield of recombinant sprouted yeast strain producing Protopananacidol.

After extensive in-depth studies, the inventors unexpectedly selected key amino acid sites that could significantly improve cytochrome P450 mutant protein catalytic activity by large selection. According to the present invention, after modifying the core site of the cytochrome P450 CYP716A47, it is possible to significantly improve the PPD production and the PPD / DM ratio. In addition, the inventors have found that the first to fourth site amino acids of the wild-type cytochrome P450 CYP716A47 are deleted and one of the key sites (for example, the eighteenth site amino acid) is mutated at the same time, thereby significantly improving its catalytic activity. It was found that the ratio of PPD / DM can be improved up to 125.6%.

In addition, the present inventors further constructed a sprouted yeast chassis cell WP8 for synthesizing damarenediol (DM), and selected the MutMorph II Random Mutagenesis Kit random mutation kit from Stratagene to construct a mutant library of cytochrome P450 (CYP716A47). Germinated yeast chassis cell WP8 was also transformed to construct a single CYP716A47 yeast mutant library that was single copyed into the yeast genome and synthesized protoparanaxadiol (PPD). Based on this, the present invention has been completed.

Terms

As used herein, the term “AxxB” refers to changing amino acid A at site xx to amino acid B, for example “L87I” indicates that amino acid L at site 87 is mutated to I and Inferred in this way.

Mutant Proteins of the Present Invention and Coding Nucleic Acids thereof

As used herein, the terms “mutant protein”, “mutant protein of the invention”, “cytochrome P450 mutant protein of the invention” can be used interchangeably and all refer to cytochrome P450 mutant proteins that are non-naturally present. 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 associated with enzyme catalytic activity and at least one of the key amino acids is artificially modified Will be rough. In addition, the mutant protein of the present invention has an enzymatic activity that catalyzes C12 hydroxylation of damarenediol (DM) to form protoparanaxadiol (PPD).

The term “core amino acid” is based on SEQ ID NO .: 1 and also has at least 80% homology with SEQ ID NO .: 1, for example 84%, 85%, 90%, 92%, 95 Corresponding sites of the%, 98% sequence are the specific amino acids set forth in the text, for example, the key amino acids based on the sequences set forth in SEQ ID NO .: 1 are 91-position proline (P); The 87th site leucine (L); 235 site lysine (K); 349 site lysine (K); 366 site valine (V); 231 site asparagine (N); 28585 site serine (S); 113th site glutamine (Q); Eighteenth site leucine (L); And / or one, two, three, or four amino acids in which the first to fourth regions are deleted, and a mutant protein obtained by mutating the core amino acid or the first to fourth region amino acids is deleted. The resulting mutant protein has an enzymatic activity that catalyzes C12 hydroxylation of damarenediol (DM) to form protoparanaxadiol (PPD).

Preferably, in the present invention, the following mutations to the key amino acids of the present invention.

The 87th site leucine (L) is mutated to isoleucine (I); And / or 235 site lysine (K) is mutated to arginine (R); And / or the 349 site lysine (K) is mutated to arginine (R); And / or the 366 site valine (V) is mutated to isoleucine (I); And / or 231 site asparagine (N) is mutated to tyrosine (Y); And / or 285 site serine (S) is mutated to cysteine (C); And / or the 91st site proline (P) is mutated to histidine (H); And / or 113 th site glutamine (Q) is mutated to arginine (R); And / or the eighteenth 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 was formed based on SEQ ID NO .: 1, and the homology of any one specific mutant protein with the sequence shown in SEQ ID NO .: 1 is 80% or more. When is reached, the amino acid number of the mutant protein may be misaligned with respect to the amino acid number of SEQ ID NO .: 1, for example, if the N or C terminus of the amino acid is misaligned with 1-5 sites, Using conventional sequence comparison techniques, one of ordinary skill in the art would understand that such misalignment is within a reasonable range, and due to misalignment of amino acid numbers, the homology is 80% (eg, 90%, 95%, 98). Mutant proteins with the same or similar catalytic activity that do not reach%) and which produce Protoparanaxadiol (PPD) are within the range of mutant proteins of the present invention. It should not belong.

Mutant proteins of the invention are synthetic or recombinant proteins, ie chemically synthesized products or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeasts, plants) using recombinant techniques. Depending on the host used in the recombinant production approach, the mutant proteins of the invention may be glycosylated or unglycosylated. The mutant protein 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 proteins. For example, as used herein, the terms “fragment”, “derivative” and “analogue” refer to proteins that basically retain the same biological function or activity as the mutant protein.

Fragments, derivatives or analogs of the mutant proteins of the invention may be (i) mutant proteins substituted by one or a plurality of conservative and non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may be genetic code May or may not be encoded by (ii) a mutant protein having a substitution radical at one or a plurality of amino acid residues, or (iii) a compound other than a mature mutant protein (eg, a mutation A mutant protein formed by fusion of a compound having an extended protein half-life, such as polyethylene glycol, or (iv) a mutant protein formed by fusion of an additional amino acid sequence to that mutant protein sequence (eg, a leader To purify the sequence or secreted sequence or the corresponding mutant protein It may be a column or peuroteohjen sequence, or the antigen fusion protein is formed and IgG fragment). In accordance with the implications of the text, such fragments, derivatives and analogs fall within the range known to those skilled in the art. In the present invention, conservatively substituted amino acids are preferably produced 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 proteins of the present invention have enzymatic activity that catalyzes C12 hydroxylation of damarenediol (DM) to form protoparanaxadiol (PPD).

Preferably, said mutant protein is as shown in SEQ ID NO .: 2-8. It should be understood that the mutant proteins of the present invention typically have relatively high homology (identity) compared to the sequences shown in SEQ ID NO .: 2-8, and preferably, the mutant protein and SEQ ID NO .: 2 The homology of the sequences 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% )to be.

In addition, the mutant proteins of the present invention can be further modified. Modified (generally unchanging primary structures) forms include chemically derived forms of mutant proteins in or outside the body, such as acetylation or carboxylation. Modifications further include glycosylation, such as mutant proteins produced by undergoing glycosylation modification in the synthesis and processing of the mutant protein or further processing. Such modifications can be completed by exposing the mutant protein to enzymes that undergo glycosylation (eg, mammalian glycosylase or diglycosylase). The modified form further includes sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). It further comprises a mutant protein that has been modified to improve its resistance protein hydrolysis performance or optimize its dissolution performance.

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

The invention also relates to variants of said polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the invention. Such nucleotide variants include substitutional variants, deletion variants and insertional variants. As is known in the art, allelic variants are replacement forms of one polynucleotide and may be substitutions, deletions or insertions of one or a plurality of nucleotides, but cannot substantially alter the function of the encoded mutant protein.

The present invention also relates to a polynucleotide that is hybridized with said sequence and has at least 50%, relatively preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides capable of hybridizing with the polynucleotides according to the invention under stringent conditions (or dense conditions). In the present invention, “strict conditions” refer to (1) hybridization and elution under relatively high ionic strength and relatively high temperature, such as 0.2 × SSC, 0.1% SDS, 60 ° C., or (2) 50% (v / v) indicates denaturation at the time of hybridization, such as formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C., or (3) homology between the two sequences is at least 90% and more preferably at least 95% Only indicates that hybridization occurs.

The mutant proteins and polynucleotides of the present invention are preferably provided in an isolated form, more preferably homogeneously purified.

The polynucleotide full-length sequence of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers are designed according to the nucleotide related sequences disclosed herein, in particular according to an open reading frame sequence, and are available in the cDNA library or commercially known methods known in the art. The prepared cDNA library is templated and amplified to obtain related sequences. If the sequence is relatively long, it is often necessary to perform two or several PCR amplifications and then bind the amplified fragments again in the correct order.

Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. Typically, it is cloned into a carrier and transferred back to the cell, and then separated from the host cell after propagation through a general method to obtain the relevant sequence.

In addition, related sequences can be synthesized by artificial synthesis, in particular, the fragment length is relatively short. In general, a plurality of small fragments may be first synthesized and then relinked to obtain fragments with very long sequences.

Currently, chemical synthesis can fully obtain DNA sequences encoding proteins (or fragments thereof, or derivatives thereof) of the invention. The DNA sequence can then be introduced into a variety of existing DNA molecules (or carriers, for example) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

Methods of amplifying DNA / RNA using PCR techniques are preferably used to obtain polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from the library, it may be desirable to use the RACE method (RACE-cDNA terminal rapid amplification method), the primer used for PCR is appropriate according to the sequence information of the present invention disclosed in the text Can be selected and synthesized in a general manner. DNA / RNA fragments can be isolated and purified through common methods such as gel electrophoresis.

Wild type cytochrome P450

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

Sequence information of the wild protein, the mutant protein of the present invention is as shown in Table 2 (see Examples).

Expression carrier

The invention also provides a carrier comprising a polynucleotide of the invention, and a host cell in which a sequence encoded by the carrier of the invention or a mutant protein of the invention is produced through genetic engineering, and a polypeptide according to the invention via recombinant technology. It is about how to generate.

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

(1) A polynucleotide (or variant) of the present invention encoding a mutant protein of the present invention or a recombinant expression carrier containing the polynucleotide is transformed or transduced into a suitable host cell.

(2) Incubate the host cell in a suitable medium.

(3) Isolate and purify proteins from media or cells.

In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted in a recombinant expression carrier. The term “recombinant expression carrier” refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, reverse transcriptase viruses or other carriers as are known in the art. Any plasmid and carrier can be used, as long as they are replication and stable in the host body. One important feature of an expression carrier generally includes a replication starting point, a promoter, a marker gene and a translational control element.

Methods known to those of skill in the art are used to construct expression carriers of coding DNA sequences and suitable transcriptional / translational control signals comprising the mutant proteins of the invention. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence is effectively linked on the appropriate promoter in the expression carrier to direct mRNA synthesis. Representative examples of such promoters include Escherichia coli lac or trp promoters; lambda phage PL promoter; There are eukaryotic promoters, eukaryotic promoters are 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. Expressing promoters. The expression carrier further comprises a ribosome binding site and a transcription terminator used at the beginning of translation.

In addition, the expression carrier preferably selects transformed host cells such as dihydrofolate 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 phenotypic traits.

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

The host cell can 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 Escherichia coli, Streptomyces; Bacterial cells of Salmonella typhimurium; Fungal cells such as yeast, plant cells (eg, ginseng cells).

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be amplified when an amplifier sequence is inserted into the carrier. Amplifiers are cis agonists of DNA and typically have approximately 10 to 300 base pairs and act on a promoter to amplify the transcription of a gene. For example, the SV40 amplifier of 100 to 270 base pairs at one end of the replication start point, the polyoma amplifier and adenovirus amplifier at one end of the replication start point, and the like.

Choosing suitable carriers, promoters, amplifiers and host cells will be apparent to those skilled in the art.

Host cell transformation with recombinant DNA can proceed with conventional techniques known to those skilled in the art. If the host is a prokaryote, such as E. coli, water soluble cells capable of absorbing DNA can be obtained after the exponential growth phase, treated with CaCl ₂ method, and the steps used are well known in the art. Another method is to use MgCl ₂ . Depending on demand, transformation can also proceed with electroporation. If the host is a eukaryote, one can choose the DNA transfection method, which is a general mechanical method such as calcium phosphate coprecipitation, microscopy, electroporation, liposome packaging, and the like.

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

Recombinant polypeptides in the aforementioned methods can be expressed intracellularly, or in cell membranes, or secreted extracellularly. By demand, its physical, chemical, and other properties can separate and purify the recombinant protein by various separation methods. Such methods are known to those skilled in the art. Examples of such methods include general prototyping, treatment with protein precipitants (salting method), centrifugation, osmotic bacteria, excess treatment, excess centrifugation, molecular screening 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 screening and modification, the present invention first discovered the catalytic activity site of cytochrome P450 (CYP716A47) and the core site of its promoter, and after modifying the relevant site, the cytochrome P450 catalyst activity and expression level were remarkable. Can be improved and the PPD production and PPD / DM ratio can be significantly improved.

(ii) The present invention is the first time that 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 (e.g., the eighteenth site amino acid) is mutated to remarkably improve its catalytic activity. In particular, it was found that the ratio of PPD / DM can be improved to 125.6%.

(iii) The present invention also shows significant catalytic activity even when one or more amino acids in the wild-type cytochrome P450 CYP716A47 are mutated at one or more amino acids, such as at 235/349/366/231/285/91/113. It was found that it can be improved, and specifically, the ratio of PPD / DM can be improved by 26.1 to 80.2%.

The present invention will be described in more detail with reference to specific examples below. It is to 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 note specific conditions in the examples below generally follow general conditions, and for example, Sambrook et al., Human, molecular clones may be prepared using the conditions described in the Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989). Follow the manufacturer's recommendations. Unless stated otherwise, percentages and parts are by weight percentage and parts by weight.

Unless specifically stated, all reagents and materials in the examples of the present invention are products purchased on the market.

Example 1 Construction of Recombinant Sprouted Yeast Strains Producing Damarenediol

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

The damarenediol synthase gene PgDDS can be introduced into wild-type sprouted yeast, and 2,3-epoxysqualene synthesized by the mevalonic acid pathway of the sprouted yeast itself can be used to synthesize damarenediol. Optimization of the artificially constructed damalendiol synthesis pathway, including optimization of the speed limit step and optimization of precursor supply, is carried out to obtain a high-quality sprouted yeast strain WP8, which is shown in FIG. 1. Use as a cell.

Example 2. Clove evolution to obtain highly efficient cytochrome P450 mutant protein

(1) A primer EP-1 (5 ''-ATGGCTGCGGCCATGGTCTTAT-3 ') using a cytochrome P450 gene sequence (optPPDS, SEQ ID NO .: 14, designated as an amino acid sequence encoding SEQ ID NO .: 1) as a template Error prone PCR is performed using '(SEQ ID NO .: 22)) and EP-2 (5' '-GTTATGTGGATGCAGATGGATT-3' '(SEQ ID NO .: 23)). The real-induced PCR is used by selecting a GeneMorph II Random Mutagenesis Kit random mutation kit from Stratagene. PCR procedure was performed at 95 ° C. 2 min; 95 ° C. 10 s, 55 ° C. 15 s, 72 ° C. 2 min, total 28 cycles; Reduce to 10 ° C. for 10 min at 72 ° C. and use template 50 ng. PCR products are obtained by agarose gel electrophoresis, followed by recovery of cytochrome P450 real induced PCR products.

(2) Using the germ-type yeast genome as a template, the real-induced PCR product of step (1) was introduced into the germinated yeast chassis cells prepared in Example 1 using the lithium acetate transformation method in the molecular clone general method. Obtain the conversion product (ie transformant).

(3) The transformed product is uniformly coated on YPD + 200 mg / L G418 antibiotic selection plate, and incubated at 30 ° C. for 2-3 days. All clones are picked with toothpicks and transferred into 96 well plates, shaken for 1 day at 30 ° C., then transferred into one new 96 well plate and fermented for 4 days. The same volume of normal butanol solvent was added to the fermentation broth, extracted for 1 hour, the upper organic phase was absorbed, and then HPLC was performed to detect the amount and proportion of each transformant, dimarenediol and protopanaxadiol.

(4) Selection of 24 pieces of 96 well plates yielded a total of seven clones that improved the ratio of protoparnaxadiol production / damarenediol (PPD / DM) by at least 20%, numbered 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 cytochrome P450 fragments 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. Remarks PPD / DM upgrade 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 1st to 4th site amino acid deletion, L18I 125.6%

(5) One more step in the screening process for the cytochrome P450 mutant library to obtain a series of mutant promoter sequences, which enhance the expression level of cytochrome P450 to improve the efficiency and yield of protopanaxodiol synthesis. It is advantageous. There are four mutant promoters with a 20% or more improvement in the ratio of ProtoPanaxadiol Production / DamaryleneDiol (PPD / DM), numbered 1D1, 9C1-2, 11B5 and 15F1, respectively. The genomes of the 1D1, 9C1-2, 11B5 and 15F1 clones were used as templates, PCR was carried out using primers to obtain promoter fragments of each clone, and sequencing detection was performed to obtain mutant promoter nucleotide sequences.

Each wild type and mutant promoter nucleotide sequence information and PPD / DM obtained above are shown in Table 3.

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

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

Example 3. High-efficiency Heterogeneous Synthesis of Protoparnaxadiol Using the Cytochrome P450 Mutant Protein

(1) Recombinant sprouted yeast strain WP8, which uses the germinated yeast genome as a template and transforms the sprouted yeast strain WP8 with SEQ ID NO .: 14 to produce protopanaxadiol. Get WT

(2) Similarly, mutant genes 1G4, 2D8, 3B9, 8G7, 9C1, 16F8 and 24A10, respectively (nucleotide sequence is SEQ ID NO .: 15-21, and corresponding amino acid sequence is SEQ ID NO .: 2-8 To replace the wild-type optPPDS gene. The PCR was carried out to obtain respective PCR fragments, which were transformed with the germinated yeast strain WP8, respectively, to produce a protopanaxadiol containing each mutant protein, which was a recombinant germinated yeast strain WP8-1G4, WP8-2D8, and WP8. -3B9, WP8-8G7, WP8-9C1, WP8-16F8 and WP8-24A10 are obtained.

(3) A solid medium is placed, i.e. a medium which is 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose), 2% agapowder.

The liquid medium is placed, i.e. 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose).

Recombinant sprouted yeasts WP8-1G4, WP8-2D8, WP8-3B9, WP8-8G7, WP8-9C1, WP8-16F8 and WP8-24A10, divided into lines in solid medium plates, each containing 5 mL liquid medium Incubate overnight in a test tube (30 ° C., 250 rpm, 16 h). The cells were collected by centrifugation and transferred to a 50 mL Erlenmeyer flask of 10 mL liquid medium, the OD600 was adjusted to 0.05, and shaken for 4 days at 30 ° C. and 250 rpm to obtain a fermentation product. The method establishes one parallel experiment simultaneously for each recombinant yeast.

Extraction and Detection of Damarendiol and Protoparnaxadiol: Absorb 100 μL fermentation from 10 mL fermentation, shake pyrolyze yeast with Fastprep, extract with equal volume of normalbutanol, and evaporate normalbutanol under vacuum Let it be. After dissolving with 100 μL methanol the amount of production of the target product PPD and DM (Fig. 3, Fig. 4 and Table 4) is detected via HPLC.

Each of the obtained recombinant sprouted yeast strains, protoparnaxadiol and damarenediol production, is 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 wild-type cytochrome P450, the cytochrome P450 mutant protein of the present invention remarkably produced PPD production (up to 490 mg / L) and PPD / DM ratio (up to 63%). Can be improved.

All documents mentioned in the present invention are incorporated herein by reference in their entirety, such as the publications in the media are independently incorporated by reference. In addition, after reading the above-described contents of the present invention, those skilled in the art may make various modifications or modifications to the present invention, and such equivalent forms are within the scope defined by the claims of the present invention. You will have to understand.

<110> SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES          HONGGUAN BIO-PHARMA CO., LTD. <120> CYTOCHROME P450 MUTANT PROTEIN AND APPLICATIONS THEREOF <130> P2018-0080 <150> CN 201710057245.1 <151> 2017-01-20 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 486 <212> PRT <213> Panax ginseng <400> 1 Met 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 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 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   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 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   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 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   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 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   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 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 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   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 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 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 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 tggaacagac 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 tggaacagac 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 tggaacagac 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 tggaacagac 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 tggaacagac 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 tggaacagac 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 tggaacagac 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 tggaacagac 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)

For mutant proteins of cytochrome P450,
The mutant protein is an unnatural protein, the mutant protein has catalytic activity catalyzing the production of protopanaxadiol, and the mutant protein corresponds to SEQ ID NO .: 1 of wild-type cytochrome P450. , Site 91 Proline (P), Site 87 Leucine (L), Site 235 Lysine (K), Site 349 Lysine (K), Site 366 Valine (V), Site 231 Asparagine (N), 1, 2, 3 or 4 amino acids of 285 site serine (S), 113 site glutamine (Q), 18th site leucine (L), and / or the first to fourth sites deleted A mutant protein of cytochrome P450, characterized in that the mutation occurs in one or a plurality of key amino acids associated with the enzyme catalytic activity selected from. For polynucleotides,
A polynucleotide encoding a mutant protein according to claim 1. In the carrier,
A carrier comprising the polynucleotide according to claim 2. In host cells,
A host cell comprising the carrier according to claim 3 or the polynucleotide according to claim 2 integrated in the genome. In the method for producing a mutant protein of cytochrome P450 according to claim 1,
In conditions suitable for expression, culturing the host cell according to claim 4 to express the mutant protein of cytochrome P450; And
Method for producing a mutant protein of cytochrome P450 characterized in that it comprises the step of separating the mutant protein of cytochrome P450. In enzyme preparation,
An enzyme preparation comprising a mutant protein of cytochrome P450 according to claim 1. In the method for producing a protopananax diol,
(I) obtaining the protoparanaxadiol by performing a catalytic reaction by contacting the mutant protein of cytochrome P450 according to claim 1 with a reaction substrate; And
Optionally, (ii) separating and purifying the protopanaxadiol. In the use of the mutant protein according to claim 1,
The mutant protein is used to catalyze damarenediol (DM) to produce protoparanaxadiol (PPD) or to catalyze damarenediol (DM) to produce protoparanaxadiol (PPD). Use, characterized in that used to prepare. In the use of the mutant protein according to claim 1 or the host cell according to claim 4,
Use characterized by the preparation of Protopananax Diol (PPD). In the method of producing a transformed plant,
Regenerating the host cell according to claim 4, wherein the host cell is a plant cell production method characterized in that the plant cell.

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