CN112852763B - Application of Pn3-32-i5 protein and coding gene thereof in production of notoginsenoside R1 - Google Patents

Abstract

The invention discloses a Pn3-32-i5 protein and application of a coding gene thereof in production of notoginsenoside R1. The amino acid sequence of the Pn3-32-i5 protein is shown as SEQ ID NO: 5, respectively. The recombinant yeast Rg1-XM + Pn3-32-i5 is obtained BY introducing the coding gene of phosphoglucomutase 1, the coding gene of alpha-phosphoglucomutase, the coding gene of uridine diphosphate glucose pyrophosphorylase, the coding gene of Arabidopsis UDP-glucose dehydrogenase 1, the coding gene of Arabidopsis UDP-glucuronic acid decarboxylase 3, the coding gene of ginseng UDP-glycosyltransferase 101 and the Pn3-32-i5 gene into BY-PPD-PPT. The recombinant yeast Rg1-XM + Pn3-3-i5 can simultaneously produce notoginsenoside R1 and ginsenoside Rg1, and has good industrial application prospect. The invention has important application value.

Description

Application of Pn3-32-i5 protein and coding gene thereof in production of notoginsenoside R1

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Pn3-32-i5 protein and application of a coding gene thereof in production of notoginsenoside R1.

Background

The notoginsenoside R1 (chemical formula shown in formula (I)) is a dammarane type triterpenoid saponin compound, is mainly distributed in plants of Araliaceae such as Ginseng radix, Notoginseng radix, and radix Panacis Quinquefolii, has neuroprotective, antiinflammatory, anti-apoptosis, blood circulation promoting, and blood stasis removing effects, and is a chemical substance with high medicinal value. Notoginsenoside R1 is mainly separated and extracted from notoginseng, but the separation and extraction method has many defects, such as low content, large difference, difficult product purification, long plant growth period, serious damage to biological resources, especially wild resources, and the like.

At present, the design and engineering of microbial strains to produce natural products using the principles of synthetic biology has been internationally recognized as one of the most promising approaches, such as producing the precursor taxadiene of paclitaxel in E.coli to 1000mg/L (Parayil Kumaran Ajikumar et al, 2010, Science, 330: 70-74); l-pinosylidene (Levopimaradiene) which is a precursor of Ginkgolides (Ginkgolides) achieves the yield of 700mg/L in the transformed engineering bacteria of escherichia coli (Effenti Leonard et al, 2010, PNAS, 107(31): 13654-13659); the precursor arteannuic acid (Artemisinic acid) for producing Artemisinin (Artemisinin) in the yeast engineering bacteria reaches up to 25g/L (Paddon CJ et al, 2013, Nature, 2013, 496: 528-. At present, the biosynthesis of artemisinin, paclitaxel, ginsenoside, tanshinone and other drug molecules is researched in China.

The clear biosynthesis process of natural drugs is a prerequisite for the creation of artificial cell factories for fermentative production of target compounds using synthetic biology techniques. Currently, there have been related advances in the study of ginsenoside biosynthetic pathways, including some key enzymes in the ginsenoside synthetic pathway, such as Squalene Synthase (SS) catalyzing the isoprenoid pathway towards sterol and triterpene saponins, Squalene Epoxidase (SE) catalyzing the production of 2, 3-oxidosqualene, Dammarenediol Synthase (DS) catalyzing the production of dammarenediol, and cytochrome P450 enzymes responsible for hydroxylation, but the synthetic pathway of notoginsenoside R1 has not been fully explored.

Disclosure of Invention

The invention aims to produce notoginsenoside R1.

The invention provides a protein which is derived from pseudo-ginseng and named as Pn3-32-i5 protein, and can be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is SEQ ID NO: 5;

a2) in SEQ ID NO: 5, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) the protein with the activity of notoginsenoside R1 synthetase is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a 2);

a4) protein with 80% or more identity with protein shown in a1) or a2), derived from Panax notoginseng, and having notoginsenoside R1 synthetase activity.

Wherein, SEQ ID NO: 5 consists of 446 amino acid residues.

To facilitate purification and detection of the protein, the protein may be identified in the sequence set forth by SEQ ID NO: 5, the amino terminal or the carboxyl terminal of the protein Pn3-32-i5 consisting of the amino acid sequence shown in the table 5 is connected with the label shown in the table 5.

TABLE 5 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by converting the amino acid sequence of SEQ ID NO: 1, and/or is missense mutated by one or more base pairs, and/or is obtained by linking the coding sequence of the tag shown in table 5 above at its 5 'end and/or 3' end.

Nucleic acid molecules encoding the Pn3-32-i5 protein are also within the scope of the invention.

The nucleic acid molecule encoding the Pn3-32-i5 protein can be a DNA molecule shown in the following b1) or b2) or b3) or b 4):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from pseudo-ginseng and encodes the Pn3-32-i5 protein;

b4) DNA molecules which are derived from pseudo-ginseng and code for the Pn3-32-i5 protein, which hybridize under stringent conditions with the nucleotide sequences defined under b1) or b 2).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 1341 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence shown in SEQ ID NO: 5.

The nucleotide sequence of the Pn3-32-i5 protein of the invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified and which have 80% or more identity to the nucleotide sequence of the Pn3-32-i5 protein isolated according to the invention, as long as they encode the Pn3-32-i5 protein, are derived from and identical to the nucleotide sequence according to the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 5, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the Pn3-32-i5 protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The nucleic acid molecule for coding the Pn3-32-i5 protein can be specifically a gene for coding the Pn3-32-i5 protein and is named as Pn3-32-i5 gene.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines comprising any of the above-described nucleic acid molecules are also within the scope of the present invention.

The recombinant vector containing any one of the nucleic acid molecules can be obtained by inserting the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

The vector may be a plasmid vector, cosmid vector, phage vector or viral vector.

The vector may be an expression vector or a cloning vector.

The expression vector may be plasmid pRS425-LEU2-PTEF1-STpGMAS-TCYC 1.

The recombinant vector containing any one of the above nucleic acid molecules can be specifically a recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC 1. The recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC1 can replace a small DNA fragment between restriction enzymes SexAI and AscI of the plasmid pRS425-LEU2-PTEF1-STpGMAS-TCYC1 with a DNA fragment shown in SEQ ID NO: 1.

The recombinant microorganism containing any of the above-described nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-described nucleic acid molecules into a starting microorganism.

The starting microorganism can be yeast, bacteria, algae or fungi. The yeast may be Saccharomyces cerevisiae (Saccharomyces cerevisiae), Yarrowia lipolytica (Yarrowia lipolytica), Pichia pastoris (Pichia pastoris), Phaffia rhodozyma (Phaffia rhodozyma), or Cryptococcus aereus.

The recombinant microorganism containing any one of the nucleic acid molecules can be specifically recombinant yeast Rg1-XM + Pn3-32-i 5. The recombinant yeast Rg1-XM + Pn3-32-i5 can be a recombinant bacterium obtained by introducing a recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC1 into recombinant yeast Rg 1-XM.

The transgenic cell line can be a transgenic plant cell line or a transgenic animal cell line.

The transgenic cell line does not include propagation material.

The invention also protects the application of any one of the Pn3-32-i5 proteins, which can be c1), c2), c3), c4), c5) or c 6):

c1) producing notoginsenoside R1;

c2) preparing a product for producing notoginsenoside R1;

c3) producing ginsenoside Rg 1;

c4) preparing a product for producing the ginsenoside Rg 1;

c5) as notoginsenoside R1 synthase;

c6) preparing the product with the function of the notoginsenoside R1 synthetase.

The invention also protects the application of any one of the nucleic acid molecules, which can be c1), c2), c3), c4), c5) or c6) as follows:

c1) producing notoginsenoside R1;

c2) preparing a product for producing notoginsenoside R1;

c3) producing ginsenoside Rg 1;

c4) preparing a product for producing the ginsenoside Rg 1;

c5) as notoginsenoside R1 synthase;

c6) preparing the product with the function of the notoginsenoside R1 synthetase.

The invention also discloses a method for preparing the engineering bacteria (hereinafter referred to as engineering bacteria I) for producing the notoginsenoside R1 and/or the ginsenoside Rg1, which comprises the following steps: improving the expression and/or activity of phosphoglucomutase 1(GeneID 853732), alpha-phosphoglucomutase (GeneID 855131), uridine diphosphate glucose pyrophosphorylase (GeneID 853830), Arabidopsis UDP-glucose dehydrogenase 1, Arabidopsis UDP-glucuronic acid decarboxylase 3, ginseng UDP-glycosyltransferase 101 and any one of the Pn3-32-i5 proteins in a recipient yeast, thereby obtaining the engineering bacteria for producing notoginsenoside R1 and/or ginsenoside Rg 1;

the receptor yeast is obtained by improving the expression quantity and/or activity of hydroxyglutaryl-CoA reductase 1 (obtained by removing amino acid residues from positions 2 to 528 of the N terminal of a target protein; the GeneID of the target protein is 854900), farnesyl pyrophosphate synthase (GeneID is 853272), squalene synthase (GeneID is 856597), squalene epoxidase (GeneID is 853086), dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and cytochrome P450 reductase (GeneID is 826869) in saccharomyces cerevisiae.

In the above method, the step of "increasing the expression level and/or activity of phosphoglucomutase 1, alpha-phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, Arabidopsis thaliana UDP-glucose dehydrogenase 1, Arabidopsis thaliana UDP-glucuronic acid decarboxylase 3, Ginseng UDP-glycosyltransferase 101 and any one of the Pn3-32-i5 proteins" in the recipient yeast is carried out by introducing a gene encoding phosphoglucomutase 1 (i.e., PGM1 gene, Genbank No. Z28127.1), a gene encoding alpha-phosphoglucomutase (i.e., PGM2 gene, Genbank No. AY723853.1), a gene encoding uridine diphosphate glucose pyrophosphorylase (i.e., UGP1 gene, Genbank No. NM-001179601.3), a gene encoding Arabidopsis thaliana UDP-glucose dehydrogenase 1 (i.e., Arabidopsis thaliana SynUGD 1 gene), a gene encoding UDP-glucuronic acid decarboxylase 3 (i.e., SynAtUXS3 gene), a gene, A coding gene of the ginseng UDP-glycosyltransferase 101 (namely UGTPg101 gene) and a coding gene of any one of the Pn3-32-i5 proteins (namely Pn3-32-i5 gene).

The invention also discloses a method for preparing the engineering bacteria (hereinafter referred to as engineering bacteria II) for producing the ginsenoside Rg1, which comprises the following steps: improving the expression and/or activity of phosphoglucomutase 1, alpha-phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, Arabidopsis UDP-glucose dehydrogenase 1, Arabidopsis UDP-glucuronic acid decarboxylase 3 and ginseng UDP-glycosyltransferase 101 in the recipient yeast, thereby obtaining the engineering bacteria for producing the ginsenoside Rg 1;

the receptor yeast is obtained by improving the expression level and/or activity of hydroxymethylglutaryl coenzyme A reductase 1, farnesyl pyrophosphate synthase, squalene epoxidase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and cytochrome P450 reductase in saccharomyces cerevisiae.

In the above method, the "improving the expression level and/or activity of phosphoglucomutase 1, alpha-phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, Arabidopsis UDP-glucose dehydrogenase 1, Arabidopsis UDP-glucuronic acid decarboxylase 3 and ginseng UDP-glycosyltransferase 101 in the recipient yeast" is carried out by introducing a gene encoding phosphoglucomutase 1 into the recipient yeast, alpha-phosphoglucomutase coding gene, uridine diphosphate glucose pyrophosphorylase coding gene, Arabidopsis UDP-glucose dehydrogenase 1 coding gene (namely SynAtUGD1 gene), Arabidopsis UDP-glucuronic acid decarboxylase 3 coding gene (namely SynAtUXS3 gene) and ginseng UDP-glycosyltransferase 101 coding gene (namely UGTPg101 gene).

Any of the above-mentioned recipient yeasts obtained by increasing the expression level and/or activity of hydroxymethylglutaryl-CoA reductase 1, farnesyl pyrophosphate synthase, squalene epoxidase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase, cytochrome P450 reductase in Saccharomyces cerevisiae "is obtained by introducing a gene encoding hydroxymethylglutaryl-CoA reductase 1 (obtained by removing the 4 th to 1584 th positions from the 5' end of the target gene; Genbank No. NM-001182434.1 for the target gene), a gene encoding farnesyl pyrophosphate synthase (Genbank No. NM-001181600.1), a gene encoding squalene synthase (Genbank No. NM-001179321.1), a gene encoding squalene synthase (Genbank No. EPM-001181304.1), a gene encoding damenediol-II synthase (i.e., SynPgDDS gene), a gene encoding protopanaxadiol synthase (i.e., SynPgS gene), and the like into Saccharomyces cerevisiae, A coding gene of protopanaxatriol synthase (namely SynPgPPTS gene) and a coding gene of cytochrome P450 reductase (Genebank number NM-001084908.2).

The nucleotide sequence of any one of the SynAtUXS3 genes can be shown as SEQ ID NO: 2, respectively.

The nucleotide sequence of any one of the SynAtUGD1 genes can be shown as SEQ ID NO: 3, respectively.

The nucleotide sequence of any UGTPg101 gene can be shown as SEQ ID NO: 4, respectively.

The nucleotide sequence of any one of the SynPgDDS genes can be shown as SEQ ID NO: and 6.

The nucleotide sequence of any one of the SynPgPPDS genes can be as shown in SEQ ID NO: shown at 7.

The nucleotide sequence of any of the SynPgPPTS genes can be as shown in SEQ ID NO: shown in fig. 8.

The amino acid sequence of any one of the above arabidopsis UDP-glucuronic acid decarboxylase 3 can be as set forth in SEQ ID NO: shown at 9.

The amino acid sequence of any one of the above-mentioned Arabidopsis UDP-glucose dehydrogenases 1 can be represented by SEQ ID NO: shown at 10.

The amino acid sequence of any one of the above-mentioned ginseng UDP-glycosyltransferases 101 may be as set forth in SEQ ID NO: shown at 11.

The amino acid sequence of any of the dammarenediol-II synthases described above may be as set forth in SEQ ID NO: shown at 12.

The amino acid sequence of any of the protopanoxadiol synthases described above may be as set forth in SEQ ID NO: shown at 13.

The amino acid sequence of any of the protopanaxatriol synthases can be as set forth in SEQ ID NO: as shown at 14.

Any one of the above saccharomyces cerevisiae may be saccharomyces cerevisiae BY 4742.

In any of the above methods, the recipient yeast may be yeast BY-PPD-PPT.

The engineering bacteria can be specifically recombinant yeast Rg1-XM + Pn3-32-i5 mentioned in the embodiment of the invention.

The second engineering bacterium can be specifically the recombinant yeast Rg1-XM mentioned in the embodiment of the invention.

The invention also protects the engineering bacterium I which is prepared by any one of the methods and is used for producing the notoginsenoside R1 and/or the ginsenoside Rg 1.

The invention also protects the second engineering bacterium which is prepared by any one of the methods and is used for producing the ginsenoside Rg 1.

The invention also protects the application of any one of the engineering bacteria I in producing notoginsenoside R1 and/or ginsenoside Rg 1.

The invention also protects the application of any one of the engineering bacteria II in the production of ginsenoside Rg 1.

The invention also provides a method for producing the notoginsenoside R1 and/or the ginsenoside Rg1, which comprises the following steps: fermenting and culturing any one of the engineering bacteria I, collecting fermentation products, and obtaining notoginsenoside R1 and/or ginsenoside Rg1 from the fermentation products.

In the above process, the medium of the fermentation culture may be an aqueous solution containing 0.6-1.0% (e.g., 0.6-0.8%, 0.8-1.0%, 0.6%, 0.8%, or 1.0%) of the yeast selection medium SD-Trp-Leu-His-Ura, 1-3% (e.g., 1-2%, 2-3%, 1%, 2%, or 3%) of glucose, and 0.005-0.015% (e.g., 0.005-0.010%, 0.010-0.015%, 0.005%, 0.010%, or 0.015%) of Leu.

In the above method, the fermentation culture conditions may be 28-32 deg.C (such as 28-30 deg.C, 30-32 deg.C, 28 deg.C, 30 deg.C or 32 deg.C), 200-300rpm (such as 200-250rpm, 250-300rpm, 200rpm, 250rpm or 300rpm) shaking culture.

In the above method, the step of obtaining notoginsenoside R1 and/or ginsenoside Rg1 from the fermentation product may be:

(1) collecting the precipitate in the fermentation product;

(2) crushing and collecting supernatant.

The invention also provides a method for producing the ginsenoside Rg1, which comprises the following steps: fermenting and culturing any one of the engineering bacteria II, collecting a fermentation product, and obtaining the ginsenoside Rg1 from the fermentation product.

In the above method, the medium for the fermentation culture may be an aqueous solution containing 0.6-1.0% (e.g., 0.6-0.8%, 0.8-1.0%, 0.6%, 0.8%, or 1.0%) of the yeast selection medium SD-Trp-Leu-His-Ura and 1-3% (e.g., 1-2%, 2-3%, 1%, 2%, or 3%) of glucose.

In the above method, the fermentation culture conditions may be 28-32 deg.C (such as 28-30 deg.C, 30-32 deg.C, 28 deg.C, 30 deg.C or 32 deg.C), 200-300rpm (such as 200-250rpm, 250-300rpm, 200rpm, 250rpm or 300rpm) shaking culture.

In the above method, the step of obtaining ginsenoside Rg1 from the fermentation product may be:

(1) collecting the precipitate in the fermentation product;

(2) crushing and collecting supernatant.

In the above, after the step (1) is completed and before the step (2) is performed, the method further comprises the step (a): and (5) cleaning the precipitate. The cleaning was with ddH 2O.

In the above step (2), the crushing method may be adding an extraction liquid, shaking for crushing, and then ultrasonic crushing. Glass beads may also be added at the same time as the extraction solution. The extract can be prepared by mixing 1 volume part of methanol and 1 volume part of acetone. The time for shaking and crushing can be 8-12min (such as 8-10min, 10-12min, 8min, 10min or 12 min). The ultrasonication time may be 25-35min (such as 25-30min, 30-35min, 25min, 30min or 35 min).

In the above, after the step (2) is completed, the method further comprises the step (B): and filtering and sterilizing the supernatant.

The recombinant yeast Rg1-XM is obtained BY introducing a coding gene of phosphoglucomutase 1, a coding gene of alpha-phosphoglucomutase, a coding gene of uridine diphosphate glucose pyrophosphorylase, a coding gene of Arabidopsis UDP-glucose dehydrogenase 1, a coding gene of Arabidopsis UDP-glucuronic acid decarboxylase 3 and a coding gene of ginseng UDP-glycosyltransferase 101 into a strain BY-PPD-PPT for producing PPT; the recombinant yeast Rg1-XM can produce ginsenoside Rg 1. The coding gene (namely Pn3-32-i5 gene) of notoginsenoside R1 synthetase is introduced into recombinant yeast Rg1-XM to obtain recombinant yeast Rg1-XM + Pn3-32-i 5. The recombinant yeast Rg1-XM + Pn3-3-i5 can simultaneously produce notoginsenoside R1 and ginsenoside Rg1, and has good industrial application prospect. The invention provides a method for producing notoginsenoside, which has important application value.

Drawings

FIG. 1 shows the results of LC-MS analyses of the standards, Rg1-XM solutions, and Rg1-XM + Pn3-32-i5 solutions.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Saccharomyces cerevisiae BY4742(Saccharomyces cerevisiae BY4742) is described in the following references: carrie baker brachhmann et al, 1998, YEAST, 14: 115-. Hereinafter, Saccharomyces cerevisiae BY4742 is referred to as Saccharomyces cerevisiae for short.

The yeast selection medium SD-Trp-Leu-His-Ura is a product of Beijing Pankeno science and technology Co. The pUC57 vector is a product of Kinry Biotechnology, Inc. The pEASY-Blunt Simple plasmid is a product of Beijing all-purpose gold biotechnology, Inc.

PCR SuperMix (+ dye) is a product of Beijing Quanjin Biotechnology, Inc. The PCR product purification kit is a product of Shanghai biological engineering Co.

HS DNA polymerase is a product of TAKARA. 5 times PS Buffer is

A component in HS DNA polymerase.

The reverse transcription kit is a product of Thermo company. Oligo (dT)18 primer, 5 × RT Buffer and Maxima H Minus Enzyme Mix were all components of the reverse transcription kit.

The plasmid pM3-ERG9 is disclosed in the Chinese patent publication CN 103484389B.

The plasmids pM9-Pn1-31, pM16-Pn3-31, pM2-PGM1, pM8-PGM2, pM11-UGP1 and TRP1-PGK are all disclosed in CN 110438099A.

The plasmid pRS425-LEU2-PTEF1-STpGMAS-TCYC1 is disclosed in the Chinese patent publication CN 108060092A.

The plasmid information referred to in the following examples is shown in Table 1.

TABLE 1 plasmid information

Information on the strains referred to in the following examples is shown in Table 2.

TABLE 2 Strain information

The GeneID of phosphoglucomutase 1 was 853732. The Genebank number of the gene encoding phosphoglucomutase 1 (i.e., the PGM1 gene) is Z28127.1.

The GeneID of alpha-phosphoglucomutase was 855131. The Genebank number of the gene encoding alpha-phosphoglucomutase (i.e., the PGM2 gene) is AY 723853.1.

The GeneID of uridine diphosphate-glucose pyrophosphorylase was 853830. The Genebank number of the gene encoding uridine diphosphate glucose pyrophosphorylase (i.e., UGP1 gene) is NM-001179601.3.

The nucleotide sequence of the encoding gene (namely SynAtUXS3 gene) of the UDP-glucuronic acid decarboxylase 3 of Arabidopsis is shown as SEQ ID NO: 2, respectively.

The nucleotide sequence of the coding gene (namely SynAtUGD1 gene) of the UDP-glucose dehydrogenase 1 of arabidopsis thaliana is shown as SEQ ID NO: 3, respectively.

The nucleotide sequence of the coding gene (namely UGTPg101 gene) of the ginseng UDP-glycosyltransferase 101 is shown as SEQ ID NO: 4, respectively.

Example 1 cloning of the Pn3-32-i5 Gene

1. And extracting the total RNA of the panax notoginseng leaves by using a QIAGEN plant RNA extraction kit.

2. And (3) after the step 1 is finished, carrying out reverse transcription on the total RNA of the pseudo-ginseng leaves by adopting a reverse transcription kit to obtain the cDNA of the pseudo-ginseng.

The reaction system was 15. mu.L, and included 3. mu.L of Panax notoginseng cDNA, 1. mu.L of Oligo (dT)18 primer (concentration 10. mu.M), 1. mu.L of dNTP Mix (concentrations of dATP, dTTP, dGTP and dCTP were all 10mM), 4. mu.L of 5 XT Buffer, 1. mu.L of Maxima H Minus Enzyme Mix, and 10. mu.L of RNase-free water.

Reaction procedure: 50min at 50 ℃, 5min at 85 ℃ and keeping the temperature at 4 ℃.

3. Using cDNA of pseudo-ginseng as a template, and adopting a primer SexA1-Pn3-32-i 5: 5' -GCGACCTGGTATGGATAACCAAGAAGCTAGAATCAG-3' (recognition sites for the restriction enzyme SexAI are underlined) and primer Pn3-32-i5-Asc 1: 5' -GCGGCGCGCCCTATTGTGCATCTTTCTTCTTCTTAC-3' (recognition sites for the restriction enzyme AscI are underlined) was subjected to PCR amplification, and a PCR amplification product of about 1360bp was recovered and then purified using a PCR product purification kit.

The reaction system is 50 μ L, including 10 μ L of 5 XPS Buffer, 4 μ L of dNTP mix, 1 μ L of each primer, 0.5 μ L of cDNA of Panax notoginseng, and 0.5 μ L of cDNA

HS polymerase (2.5U/. mu.L concentration) and distilled water.

Reaction procedure: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 8 min.

4. And (3) connecting the PCR amplification product purified in the step (3) with pEASY-Blunt Simple plasmid to obtain the recombinant plasmid p-Pn3-32-i 5.

The recombinant plasmid p-Pn3-32-i5 was sequenced. Sequencing results show that the recombinant plasmid p-Pn3-32-i5 contains nucleotide sequences shown as SEQ ID NO: 1, Pn3-32-i5 gene.

The Pn3-32-i5 gene codes Pn3-32-i5 protein, and the amino acid sequence of Pn3-32-i5 protein is shown as SEQ ID NO: 5, respectively.

Example 2 application of protein Pn3-32-i5 in preparation of notoginsenoside R1

Construction of recombinant plasmid pM3-SynAtUGD1, recombinant plasmid pM9-SynAtUXS3, recombinant plasmid pM16-UGTPg101 and recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC1

1. Construction of recombinant plasmid p-SynAtUXS3, recombinant plasmid p-SynAtUGD1 and recombinant plasmid p-UGTPg101

SEQ ID NOs: 2, and the sequence shown in SEQ ID NO: 3 and the SynAtUGD1 gene shown in SEQ ID NO: 4, UGTPg101 gene.

The SynAtUXS3 gene was inserted into the multiple cloning site of pUC57 vector to obtain recombinant plasmid p-SynAtUXS 3.

The SynAtUGD1 gene was inserted into the multiple cloning site of pUC57 vector to obtain recombinant plasmid p-SynAtUGD 1.

The UGTPg101 gene is inserted into the multiple cloning site of the pUC57 vector to obtain the recombinant plasmid p-UGTPg 101.

2. Construction of recombinant plasmid pM3-SynAtUGD1

(1) The recombinant plasmid p-SynAtUGD1 was digested with restriction enzymes SexA I and Asc I, and the digested product of about 1460bp was recovered.

(2) The plasmid pM3-ERG9 was digested with the restriction enzymes SexA I and Asc I, and the vector backbone of about 4598bp was recovered.

(3) And (3) connecting the enzyme digestion product recovered in the step (1) with the vector skeleton recovered in the step (2) to obtain the recombinant plasmid pM3-SynAtUGD 1.

The recombinant plasmid pM3-SynAtUGD1 was sequenced. Sequencing results show that the recombinant plasmid pM3-SynAtUGD1 is a recombinant plasmid obtained by replacing a small DNA fragment between SexA I and Asc I of restriction enzymes of the plasmid pM3-ERG9 with a DNA molecule A; the DNA molecule A contains SEQ ID NO: 3, and the SynAtUGD1 gene. The recombinant plasmid pM3-SynAtUGD1 expresses Arabidopsis UDP-glucose dehydrogenase 1.

3. Construction of recombinant plasmid pM9-SynAtUXS3

(1) The recombinant plasmid p-SynAtUXS3 was digested with restriction enzymes SexA I and Asc I, and the digested product of about 1043bp was recovered.

(2) The plasmid pM9-Pn1-31 was digested with restriction enzymes SexA I and Asc I, and a vector backbone of about 5136bp was recovered.

(3) And (3) connecting the enzyme digestion product recovered in the step (1) with the vector skeleton recovered in the step (2) to obtain a recombinant plasmid pM9-SynAtUXS 3.

The recombinant plasmid pM9-SynAtUXS3 was sequenced. Sequencing results show that the recombinant plasmid pM9-SynAtUXS3 is a recombinant plasmid obtained by replacing a small DNA fragment between restriction enzymes SexA I and Asc I of the plasmid pM9-Pn1-31 with a DNA molecule B; the DNA molecule B contains SEQ ID NO: 2, and the SynAtUXS3 gene. The recombinant plasmid pM9-SynAtUXS3 expresses Arabidopsis UDP-glucuronic acid decarboxylase 3.

4. Construction of recombinant plasmid pM16-UGTPg101

(1) The recombinant plasmid p-UGTPg101 is cut by restriction enzymes SexA I and Asc I, and a cut product with the length of 1442bp is recovered.

(2) The plasmid pM16-Pn3-31 was digested with the restriction enzymes SexA I and Asc I, and a vector backbone of about 5136bp was recovered.

(3) And (3) connecting the enzyme digestion product recovered in the step (1) with the vector skeleton recovered in the step (2) to obtain the recombinant plasmid pM16-UGTPg 101.

The recombinant plasmid pM16-UGTPg101 was sequenced. Sequencing results show that the recombinant plasmid pM16-UGTPg101 is a recombinant plasmid obtained by replacing a small DNA fragment between the restriction enzymes SexA I and Asc I of the plasmid pM16-Pn3-31 with a DNA molecule C; the DNA molecule C contains NO: 4, UGTPg101 gene. The recombinant plasmid pM16-UGTPg101 expresses ginseng UDP-glycosyltransferase 101.

5. Recombinant plasmid pRS425-LEU2-P_TEF1-Pn3-32-i5-T_CYC1

(1) The recombinant plasmid p-Pn3-32-I5 was digested with restriction enzymes SexA I and Asc I, and a digested product of about 1355bp was recovered.

(2) The plasmid pRS425-LEU2-PTEF1-STpGMAS-TCYC1 was digested with restriction enzymes SexA I and Asc I, and a vector backbone of about 7602bp was recovered.

(3) And (3) connecting the enzyme digestion product recovered in the step (1) with the vector skeleton recovered in the step (2) to obtain a recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC 1.

The recombinant plasmid pRS425-LEU2-P_TEF1-Pn3-32-i5-T_CYC1And (4) sequencing. The sequencing result shows that the recombinant plasmid pRS425-LEU2-P_TEF1-Pn3-32-i5-T_CYC1To plasmid pRS425-LEU2-P_TEF1-STpGMAS-T_CYC1The small DNA fragment between the restriction enzymes SexA I and Asc I of (1) is replaced with SEQ ID NO: 1. Recombinant plasmid pRS425-LEU2-P_TEF1-Pn3-32-i5-T_CYC1Expresses Pn3-32-i5 protein.

Second, construction of recombinant yeast Rg1-XM and recombinant yeast Rg1-XM + Pn3-32-i5

1. Construction of recombinant yeast Rg1-XM

(1) The plasmids or yeast genomic DNAs shown in column 2 of Table 3 were used as templates, respectively, and primers shown in columns 3 and 4 of Table 3 were used to perform PCR amplification, and the corresponding PCR amplification products were recovered, thereby obtaining modules M1 ', M2 ', M3 ', M4 ', M5 ', M6 ', M7 ', M8 ' and M9 '.

The reaction system is 50 μ L, including 5 XPSBuffer 10 μ L, dNTP mix 4 μ L, primers each 1 μ L, template 0.5 μ L,

HS polymerase (2.5U/. mu.L) 0.5. mu.L and distilled water.

The reaction conditions are as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.

TABLE 3 primer sequences

(2) BY using the method for preparing competent cells in example 2 of chinese patent publication CN 102925376B, BY-PPD-PPT (disclosed in chinese patent publication CN 110438099 a) competent cells were prepared.

(3) After 0.1. mu.g of module M1 ', 0.1. mu.g of module M2 ', 0.1. mu.g of module M3 ', 0.1. mu.g of module M4 ', 0.1. mu.g of module M5 ', 0.1. mu.g of module M6 ', 0.1. mu.g of module M7 ', 0.1. mu.g of module M8 ' and 0.1. mu.g of module M9 ' were added to BY-PPT competent cells, 2.7kV electric shock was applied, 1mL of 1Mol/L sorbitol aqueous solution was added, and the mixture was thawed at 30 ℃ for 1h and spread on screening medium 1 to obtain several single clones.

Screening medium 1: solid medium containing 0.8% (m/m) yeast selection medium SD-Trp-Leu-His-Ura, 2% (m/m) glucose, 0.01% (m/m) Leu and 2% (m/m) agar powder.

The screening culture conditions are as follows: culturing at 30 deg.C for over 36 hr.

(4) PCR identification

The monoclonal obtained in each step (3) was identified as follows:

(1-1) extracting the monoclonal genome DNA, taking the monoclonal genome DNA as a template, and respectively carrying out PCR amplification by using a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6, a primer pair 7, a primer pair 8, a primer pair 9, a primer pair 10, a primer pair 11, a primer pair 12 and a primer pair 13 to obtain corresponding PCR amplification products.

The reaction system is 10. mu.L, and the reaction system is composed of 5. mu.L

PCR Supermix (+ dye), 0.5. mu.L each of primers, 1. mu.L of template, and distilled water.

The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.

The names of the primers and the nucleotide sequences of the primers constituting each primer pair are shown in Table 4.

TABLE 4

(1-2) subjecting the PCR amplification products obtained in the step (1-1) to 1% (m/m) agarose gel electrophoresis, and then judging as follows: if a certain single clone contains a DNA fragment with the size of 1098bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 1, a DNA fragment with the size of 2492bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 2, a DNA fragment with the size of 2870bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 3, a DNA fragment with the size of 1860bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 4, a DNA fragment with the size of 2459bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 5, a DNA fragment with the size of 2459bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 6, a DNA fragment with the size of 2678bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 7, a DNA fragment with the size of 2561bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 8, a DNA fragment with the size of 2940bp in a PCR amplification product obtained by amplifying the single clone by using a primer pair 9, and a DNA fragment with the single clone by amplifying the single clone by using a primer pair 9, The PCR amplification product obtained by amplification of the primer pair 10 contains a DNA fragment with the size of 2332bp, the PCR amplification product obtained by amplification of the primer pair 11 contains a DNA fragment with the size of 2343bp, the PCR amplification product obtained by amplification of the primer pair 12 contains a DNA fragment with the size of 1887bp, and the PCR amplification product obtained by amplification of the primer pair 13 contains a DNA fragment with the size of 2298bp, so that the monoclonal antibody is a positive clone.

One positive clone is named as recombinant yeast Rg 1-XM.

The recombinant yeast Rg1-XM is a recombinant bacterium obtained BY introducing a PGM1 gene, a PGM2 gene, a UGP1 gene, a SynAtUGD1 gene, a SynAtUXS3 gene and a UGTPg101 gene into BY-PPD-PPT.

2. Construction of recombinant yeast Rg1-XM + Pn3-32-i5

(1) Rg1-XM competent cells were prepared by the method for preparing competent cells in example 2 of CN 102925376B.

(2) Adding 1 μ g of recombinant plasmid pRS425-LEU2-P into Rg1-XM competent cells_TEF1-Pn3-32-i5-T_CYC1After 2.7kV electric shock, 1mL of 1Mol/L sorbitol aqueous solution is added, the mixture is revived at 30 ℃ for 1h, and the mixture is coated on a screening culture medium 2 to obtain a plurality of monoclonals.

Screening medium 2: solid medium containing 0.8% (m/m) yeast selection medium SD-Trp-Leu-His-Ura, 2% (m/m) glucose and 2% (m/m) agar powder.

The screening culture conditions are as follows: culturing at 30 deg.C for over 36 hr.

(3) PCR identification

The monoclonal obtained in each step (2) was identified as follows:

(1-1) extracting a monoclonal genomic DNA and taking the genomic DNA as a template, and carrying out PCR by using a primer SacII-pTEF 1: 5'-GCGCCGCGGAGTGATCCCCCACACACCATAGCTT-3' and primer Pn3-32-i 5-Asc: 5'-GCGGCGCGCCCTATTGTGCATCTTTCTTCTTCTTAC-3' to obtain PCR amplification product.

The reaction system is 10. mu.L, and the reaction system is composed of 5. mu.L

PCR Supermix (+ dye), 0.5. mu.L each of primers, 1. mu.L of template, and distilled water.

The reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 10 min.

(1-2) subjecting the PCR amplification product obtained in the step (1-1) to 1% (m/m) agarose gel electrophoresis, and then judging as follows: if the PCR amplification product of a certain monoclonal contains a DNA fragment with the size of 1782bp, the monoclonal is a positive clone.

One positive clone is named as recombinant yeast Rg1-XM + Pn3-32-i 5.

The recombinant yeast Rg1-XM + Pn3-32-i5 is a recombinant bacterium obtained by introducing Pn3-32-i5 gene into recombinant yeast Rg 1-XM.

Third, shake flask fermentation and LC-MS detection

1. Shake flask fermentation

The solute of the solid selective medium 1 and the mass percent concentration thereof are as follows: 0.8% yeast selection medium SD-Trp-Leu-His-Ura, 2% glucose, 0.01% Trp, 0.01% Leu and 2% agar powder; the solvent is water.

The liquid selective medium 1 comprises the following solutes in percentage by mass: 0.8% yeast selection medium SD-Trp-Leu-His-Ura, 2% glucose, 0.01% Trp and 0.01% Leu; the solvent is water.

The solute of the solid selective medium 2 and the mass percentage concentration thereof are as follows: 0.8% of yeast selection medium SD-Trp-Leu-His-Ura, 2% of glucose, 0.01% of Leu and 2% of agar powder; the solvent is water.

The solute of the liquid selective medium 2 and the mass percentage concentration thereof are as follows: 0.8% yeast selection medium SD-Trp-Leu-His-Ura, 2% glucose and 0.01% Leu; the solvent is water.

The solute of the solid selective medium 3 and the mass percent concentration thereof are as follows: 0.8% of yeast selection medium SD-Trp-Leu-His-Ura, 2% of glucose and 2% of agar powder; the solvent is water.

The solute of the liquid selective culture medium 3 and the mass percentage concentration thereof are as follows: 0.8% yeast selection medium SD-Trp-Leu-His-Ura and 2% glucose; the solvent is water.

(1) Activating BY-PPD-PPT on a solid selective culture medium 1, then inoculating the monoclonal on 4mL of liquid selective culture medium 1, and culturing at 30 ℃ and 250rpm for 16h to obtain a seed solution; 150 mu L of the seed solution is inoculated into a triangular flask (the specification is 100mL) filled with 15mL of liquid selection medium 1, and the mixture is subjected to shaking culture at 250rpm and 30 ℃ for 6 days to obtain BY-PPD-PPT fermentation liquor.

(2) Activating recombinant yeast Rg1-XM on a solid selective culture medium 2, then inoculating the single clone into 4mL of liquid selective culture medium 2, and culturing at 30 ℃ and 250rpm for 16h to obtain a seed solution; inoculating 150 μ L of the seed solution into a triangular flask (specification of 100mL) containing 15mL of liquid selection medium 2, and performing shaking culture at 30 deg.C and 250rpm for 6 days to obtain Rg1-XM fermentation liquid.

(3) Activating recombinant yeast Rg1-XM + Pn3-32-i5 on a solid selective culture medium 3, then inoculating the monoclonal into 4mL of liquid selective culture medium 3, and culturing at 30 ℃ and 250rpm for 16h to obtain a seed solution; inoculating 150 μ L of the seed solution into a triangular flask (specification of 100mL) containing 15mL of liquid selection medium 3, and performing shaking culture at 30 deg.C and 250rpm for 6 days to obtain Rg1-XM + Pn3-32-i5 fermentation liquid.

2. Extraction of compounds from fermentation broths

(1) Taking BY-PPD-PPT fermentation liquor, centrifuging at 5000rpm for 5min, and collecting precipitate;

(2) after completion of step (1), the precipitate was taken and treated with ddH₂Cleaning O, transferring to a crushing tube, centrifuging at 12000rpm for 2min, and removing supernatant;

(3) after the step (2) is finished, adding glass beads (the diameter is 0.5mm) and 1mL of extraction liquid (formed by mixing 1 volume part of methanol and 1 volume part of acetone) into the crushing tube, vibrating and crushing for 10min, and ultrasonically crushing for 30 min;

(4) after completion of step (3), the mixture was centrifuged at 13000rpm for 2min, and the supernatant was collected.

(5) After the step (4) is finished, filtering the supernatant by using an organic filter membrane (the aperture is 0.22 mu m), and collecting filtrate; the filtrate is BY-PPD-PPT solution.

According to the method, the BY-PPD-PPT fermentation broth is replaced BY the Rg1-XM fermentation broth, and other steps are not changed, so that the Rg1-XM solution is obtained.

According to the method, the BY-PPD-PPT fermentation broth is replaced BY Rg1-XM + Pn3-32-i5 fermentation broth, and the Rg1-XM + Pn3-32-i5 solution is obtained without changing other steps.

3. LC-MS qualitative analysis

LC-MS respectively detects a standard substance, a BY-PPD-PPT solution, an Rg1-XM solution and an Rg1-XM + Pn3-32-i5 solution. The standard product is prepared by mixing notoginsenoside R1 and ginsenoside Rg1 in mass. Notoginsenoside R1 and ginsenoside Rg1 are both products of Shanghai-sourced leaf Biotech limited.

The instrument comprises the following steps: the liquid chromatogram-tandem mass spectrum (LC-MS) instrument consists of an Agilent 1200 high performance liquid chromatograph and a Bruker-microOTOF-II mass spectrometer; MicroOTOF control Version 3.0/Data analysis Version 4.0 Data acquisition and processing system.

Mass spectrum conditions: electrospray ionization source positive ion mode (ESI)⁺) Spray voltage (4.5kV), atomization gas flow (6L/h), atomizer temperature (180 ℃), collision gas is nitrogen, pressure is 1.0Bar, data acquisition frequency is 1.0 HZ: the collision energy was 8.0 eV.

LC conditions: a DAD detector, detecting a wavelength of 203nm,

PG-C18 column (250mm × 4.6mm, 5 μm), mobile phase a (formic acid: water ═ 1: 999); mobile phase B (formic acid: acetonitrile 1: 999); gradient elution with a flow rate of 1 mL/min; the column temperature is 25 ℃; sample volume (20. mu.L). The elution mode was as follows: the volume percent concentration of the mobile phase A is kept at 81% and the volume percent concentration of the mobile phase B is kept at 19% in 0-12min (including 12 min); the volume percentage concentration of the mobile phase A is 81-74% and the volume percentage concentration of the mobile phase B is 19-26% in 12-32 min (including 32 min); the volume percentage concentration of the mobile phase A is 74-10% and the volume percentage concentration of the mobile phase B is 26-90% within 32-34 min (including 34 min); 34-44 min (including 44min), keeping the volume percent concentration of the mobile phase A at 10%, and keeping the volume percent concentration of the mobile phase B at 90%; the volume percentage concentration of the mobile phase A is 10-81% in 44-46 min (including 46min), and the volume percentage concentration of the mobile phase B is 90-19%; the volume percentage concentration of the mobile phase A is kept at 81% and the volume percentage concentration of the mobile phase B is kept at 19% in 46-50 min (including 50 min).

Part of the detection results are shown in figure 1(A is LC-MS ion diagram of the standard, Rg1-XM solution and Rg1-XM + Pn3-32-i5 solution, B is high resolution mass spectrum molecular weight of corresponding peak, C is pseudo-ginseng source Pn3-32-i5 protein participating in synthesis of pseudo-ginseng saponin R1). The result shows that the Rg1-XM solution contains the ion peak diagram of the high-resolution ginsenoside Rg1 mass spectrum which is the same as that of the standard product. The Rg1-XM + Pn3-32-i5 solution contains the ion peak pattern of the mass spectrum of the notoginsenoside R1 and the ginsenoside Rg1 which have the same high resolution as the standard substance.

The above results show that BY-PPD-PPT is introduced into a gene encoding phosphoglucomutase 1 (i.e., PGM1 gene), a gene encoding alpha-phosphoglucomutase (i.e., PGM2 gene), a gene encoding uridine diphosphate glucose pyrophosphorylase (i.e., UGP1 gene), a gene encoding Arabidopsis UDP-glucose dehydrogenase 1 (i.e., SynAtUGD1 gene), a gene encoding Arabidopsis UDP-glucuronic acid decarboxylase 3 (i.e., SynAtUXS3 gene), and a gene encoding ginseng UDP-glycosyltransferase 101 (i.e., UGTPg101 gene), thereby obtaining recombinant yeast Rg 1-XM. The recombinant yeast Rg1-XM can produce ginsenoside Rg 1. The gene (namely Pn3-32-i5 gene) for coding the Pn3-32-i5 protein is introduced into the recombinant yeast Rg1-XM to obtain the recombinant yeast Rg1-XM + Pn3-32-i 5. The recombinant yeast Rg1-XM + Pn3-32-i5 can simultaneously produce notoginsenoside R1 and ginsenoside Rg 1.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> Pn3-32-i5 protein and application of coding gene thereof in production of notoginsenoside R1

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 1341

<212> DNA

<213> Artificial sequence

<400> 1

atggataacc aagaagctag aatcagtata gttatgctgc catttttagc ccatggccac 60

atttctccat tctttgagct agccaagcat ctctcaaaaa gaaattgcaa tatattcctc 120

tgttctaccc caatcaatct tagctccatc aagaacagag tatctgataa ggattcctct 180

gcttctataa aactagtaga gcttcatctt ccatcttccc ctgatcttcc tcctcactac 240

cacaccacaa atggcctccc ttcccatctc atggtcccac tcagaaacgc ctttgaaaca 300

gcaggcccca ccttctctga aatccttaaa accttaaacc ccgatttgct tatttatgat 360

ttcaatccct catgggcacc ggagatcgct tcgtctcaca atattccggc agtttgtttc 420

ataattgggg gagcagcctc ctcttccatg agcctacata gtttcaaaaa cccaggtgaa 480

aaatacccat ttctagattt taatgagaac agtaatatta cccctgaacc accttcagca 540

gataacatga agctatttct tgattttatg acttgtttcg aacgatcttg cgacattatt 600

ttgattaaga gttttagaga actagaaggg aaatattttg attttttttc cactttatct 660

gataaaactg tggttcctgt tggtccactc gttcaagatc ctatgggcca taatgaagat 720

ccaaaaacag agcagtttat aaactggctt gacaaaaggg ctgaatctac agtggtgttt 780

gtctgctttg gaagtgagta ttttctctcc aatgaggaat tggaagaagt agcaattggg 840

ctagagatta gcatggttaa tttcatatgg gctgtgagat taattgaagg agagaaaaaa 900

ggggttttac cagaggggtt tgttcaaagg gtaggagaca gaggattggt tgtggagggg 960

tgggctccac aggcaagaat tttaggacat tcaagcaccg gtgggtttgt gagccattgt 1020

gggtggagtt ctattgcgga gagtatgaag tttggggttc cagtaattgc catggctagg 1080

catcttgatc agcctttgaa tgctaagctg gcggcggagg ttggtgtggg catggaggtt 1140

gtgagagatg ataatgggaa atataagagg gaagggattg cagaggtaat aagaaaagtc 1200

gttgtggaga aaagtgggga ggttatcagg aggaaagcaa gggagttgag tgagaaaatg 1260

aaagagaaag gagagcaaga gattgatagg gcagtggagg agctagtaca aatttgtaag 1320

aagaagaaag atgcacaata g 1341

<210> 2

<211> 1029

<212> DNA

<213> Artificial sequence

<400> 2

atggctgcaa cttctgaaaa gcaaaacact acaaaaccac caccatctcc atcaccattg 60

agaaactcaa agttctgtca accaaacatg agaattttaa tttctggtgg tgctggtttt 120

attggttcac atttggttga taaattgatg gaaaacgaaa agaatgaagt tgttgttgca 180

gataactact tcactggttc taaggaaaat ttgaagaaat ggatcggtca tccaagattc 240

gaattgatca gacatgatgt tacagaacca ttgttgatcg aagttgatag aatctatcat 300

ttggcttgtc cagcatcacc aattttctat aagtacaacc cagttaagac tattaaaaca 360

aatgttattg gtacattgaa catgttgggt ttggctaaga gagttggtgc aagaattttg 420

ttgacttcta catcagaagt ttatggtgac ccattgattc atccacaacc agaatcttac 480

tggggtaatg ttaatccaat tggtgttaga tcatgttatg atgaaggtaa aagagttgct 540

gaaactttga tgttcgatta ccatagacaa catggtatcg aaatcagaat cgcaagaatt 600

ttcaatacat acggtccaag aatgaacatc gatgatggta gagttgtttc taacttcatc 660

gctcaagcat tgagaggtga agcattgact gttcaaaagc caggtactca aacaagatct 720

ttttgttacg tttcagatat ggttgatggt ttgatcagat tgatggaggg taacgataca 780

ggtccaatta atatcggtaa tcctggtgaa ttcactatgg ttgaattggc tgaaacagtt 840

aaggaattga ttaatccatc tatcgaaatt aaaatggttg aaaatactcc agatgatcca 900

agacaaagaa agccagatat ctcaaaggca aaggaagttt tgggttggga accaaaagtt 960

aaattgagag aaggtttgcc attgatggaa gaagatttca gattgagatt aaatgttcca 1020

agaaattaa 1029

<210> 3

<211> 1446

<212> DNA

<213> Artificial sequence

<400> 3

atggttaaaa tttgttgtat tggtgctggt tatgttggtg gtccaactat ggctgttatg 60

gcattgaaat gtccagaaat cgaagttgtt gttgttgata tctctgaacc aagaattaat 120

gcttggaact cagatagatt gccaatctat gaaccaggat tagaagatgt tgttaagcaa 180

tgtagaggta aaaatttgtt tttctctact gatgttgaaa agcatgtttt cgaatctgat 240

atcgtttttg tttcagttaa tactccaaca aaaactcaag gtttgggtgc tggtaaagct 300

gcagatttga catattggga atctgctgca agaatgattg cagatgtttc aaagtcttca 360

aagatcgttg ttgaaaaatc aactgttcca gttagaacag ctgaagcaat tgaaaagatt 420

ttgactcata actctaaggg tatcgaattc caaatcttgt caaatccaga atttttagct 480

gaaggtactg caattaaaga tttgtacaac ccagatagag ttttaattgg tggtagagat 540

acagctgcag gtcaaaaggc tattaaagca ttgagagatg tttacgctca ttgggttcca 600

gttgaacaaa tcatctgtac aaatttgtgg tctgcagaat tgtcaaagtt ggctgcaaac 660

gcatttttgg cacaaagaat ctcttcagtt aatgctatgt ctgcattatg tgaagctact 720

ggtgcagatg ttacacaagt tgctcatgca gttggtacag atactagaat cggtccaaag 780

ttcttgaatg cttctgttgg tttcggtggt tcatgtttcc aaaaggatat cttgaatttg 840

atctatatct gtgaatgtaa cggtttgcca gaagctgcaa actactggaa gcaagttgtt 900

aaggttaacg attaccaaaa gattagattc gctaacagag ttgtttcttc aatgttcaac 960

acagtttctg gtaaaaagat tgctatcttg ggtttcgctt ttaagaaaga tacaggtgac 1020

actagagaaa caccagctat tgatgtttgt aacagattgg ttgctgataa ggcaaagttg 1080

tctatctatg atccacaagt tttggaagaa caaatcagaa gagatttgtc aatggctaga 1140

tttgattggg atcatccagt tccattgcaa caaattaaag cagaaggtat ctctgaacaa 1200

gttaacgttg tttcagatgc ttacgaagca actaaagatg ctcatggttt gtgtgttttg 1260

acagaatggg atgaattcaa atctttggat ttcaagaaaa ttttcgataa catgcaaaaa 1320

ccagctttcg ttttcgatgg tagaaacgtt gttgatgctg ttaagttgag agaaatcggt 1380

tttattgttt actctatcgg taaaccattg gattcatggt tgaaggatat gccagctgtt 1440

gcataa 1446

<210> 4

<211> 1428

<212> DNA

<213> Artificial sequence

<400> 4

atgaagtccg aattgatttt tttgccagct ccagctattg gtcatttggt tggtatggtt 60

gaaatggcca agttgttcat ttctaggcac gaaaacttgt ccgttaccgt tttgattgct 120

aagttctaca tggataccgg tgttgacaat tacaacaagt ccttgttgac taacccaact 180

ccaagattga ctatcgttaa cttgccagaa actgacccac aaaactatat gttgaaacct 240

agacatgcca tctttccatc cgttattgaa actcaaaaga cccacgttag agacatcatt 300

tctggtatga ctcaatccga atctaccaga gttgttggtt tgttggctga tttgttgttc 360

atcaacatta tggatatcgc caacgaattc aacgttccaa cttatgttta ttctccagct 420

ggtgctggtc acttgggttt agcttttcac ttgcaaactc tgaacgataa gaagcaagac 480

gttaccgaat tcagaaactc tgataccgaa ttattggttc cctcatttgc taatccagtt 540

ccagctgaag ttttgccatc tatgtatgtt gacaaagaag gtggttacga ctacctgttt 600

tctttgttca gaagatgcag agaatccaag gccattatta tcaacacctt cgaagaattg 660

gaaccctacg ctattaactc cttgagaatg gattctatga tcccaccaat ctatccagtt 720

ggtccaattt tgaatttgaa cggtgatggt caaaactccg atgaagctgc tgttatttta 780

ggttggttgg atgatcaacc accatcctct gttgtttttt tgtgttttgg ttcctacggc 840

accttccaag aaaatcaagt aaaagaaatc gccatgggtc tagaaagatc tggtcataga 900

tttttgtggt ctttgaggcc atctattcca aagggtgaaa ctaagttgca gttgaagtac 960

tctaacctgg aagaaatttt gccagttggt ttcttggata gaacctcttg tgttggtaaa 1020

gttattggtt gggctccaca agttgctgtt ttgggtcatg aagctgttgg tggttttttg 1080

tctcattgtg gttggaactc taccttggaa tctgtttggt gtggtgttcc agttgctact 1140

tggccaatgt atggtgaaca gcaattgaat gctttcgaaa tggtcaaaga attgggtatc 1200

gccgttgaaa tcgaagttga ttacaagaac gaatacttca acatgaacaa cgacttcatc 1260

gttagagccg aagaaatcga aacgaagatc aaaaagttga tgatggacga gaagaactcc 1320

gagattcgta agaaagtcaa agagatgaag gaaaagtcca gattggctat gtctgaaaac 1380

ggttcttctt acaactcttt ggccaagctg tttgaagaga tcatgtga 1428

<210> 5

<211> 446

<212> PRT

<213> Artificial sequence

<400> 5

Met Asp Asn Gln Glu Ala Arg Ile Ser Ile Val Met Leu Pro Phe Leu

1 5 10 15

Ala His Gly His Ile Ser Pro Phe Phe Glu Leu Ala Lys His Leu Ser

20 25 30

Lys Arg Asn Cys Asn Ile Phe Leu Cys Ser Thr Pro Ile Asn Leu Ser

35 40 45

Ser Ile Lys Asn Arg Val Ser Asp Lys Asp Ser Ser Ala Ser Ile Lys

50 55 60

Leu Val Glu Leu His Leu Pro Ser Ser Pro Asp Leu Pro Pro His Tyr

65 70 75 80

His Thr Thr Asn Gly Leu Pro Ser His Leu Met Val Pro Leu Arg Asn

85 90 95

Ala Phe Glu Thr Ala Gly Pro Thr Phe Ser Glu Ile Leu Lys Thr Leu

100 105 110

Asn Pro Asp Leu Leu Ile Tyr Asp Phe Asn Pro Ser Trp Ala Pro Glu

115 120 125

Ile Ala Ser Ser His Asn Ile Pro Ala Val Cys Phe Ile Ile Gly Gly

130 135 140

Ala Ala Ser Ser Ser Met Ser Leu His Ser Phe Lys Asn Pro Gly Glu

145 150 155 160

Lys Tyr Pro Phe Leu Asp Phe Asn Glu Asn Ser Asn Ile Thr Pro Glu

165 170 175

Pro Pro Ser Ala Asp Asn Met Lys Leu Phe Leu Asp Phe Met Thr Cys

180 185 190

Phe Glu Arg Ser Cys Asp Ile Ile Leu Ile Lys Ser Phe Arg Glu Leu

195 200 205

Glu Gly Lys Tyr Phe Asp Phe Phe Ser Thr Leu Ser Asp Lys Thr Val

210 215 220

Val Pro Val Gly Pro Leu Val Gln Asp Pro Met Gly His Asn Glu Asp

225 230 235 240

Pro Lys Thr Glu Gln Phe Ile Asn Trp Leu Asp Lys Arg Ala Glu Ser

245 250 255

Thr Val Val Phe Val Cys Phe Gly Ser Glu Tyr Phe Leu Ser Asn Glu

260 265 270

Glu Leu Glu Glu Val Ala Ile Gly Leu Glu Ile Ser Met Val Asn Phe

275 280 285

Ile Trp Ala Val Arg Leu Ile Glu Gly Glu Lys Lys Gly Val Leu Pro

290 295 300

Glu Gly Phe Val Gln Arg Val Gly Asp Arg Gly Leu Val Val Glu Gly

305 310 315 320

Trp Ala Pro Gln Ala Arg Ile Leu Gly His Ser Ser Thr Gly Gly Phe

325 330 335

Val Ser His Cys Gly Trp Ser Ser Ile Ala Glu Ser Met Lys Phe Gly

340 345 350

Val Pro Val Ile Ala Met Ala Arg His Leu Asp Gln Pro Leu Asn Ala

355 360 365

Lys Leu Ala Ala Glu Val Gly Val Gly Met Glu Val Val Arg Asp Asp

370 375 380

Asn Gly Lys Tyr Lys Arg Glu Gly Ile Ala Glu Val Ile Arg Lys Val

385 390 395 400

Val Val Glu Lys Ser Gly Glu Val Ile Arg Arg Lys Ala Arg Glu Leu

405 410 415

Ser Glu Lys Met Lys Glu Lys Gly Glu Gln Glu Ile Asp Arg Ala Val

420 425 430

Glu Glu Leu Val Gln Ile Cys Lys Lys Lys Lys Asp Ala Gln

435 440 445

<210> 6

<211> 2310

<212> DNA

<213> Artificial sequence

<400> 6

atgtggaagt taaaggtagc tcaaggtaat gacccttact tatactcaac caacaatttc 60

gtcggtagac aatactggga atttcaacca gatgctggta cacctgaaga aagagaagaa 120

gtcgaaaagg caagaaagga ctacgtaaac aacaaaaagt tacatggtat tcacccatgt 180

tcagatatgt tgatgagaag acaattgata aaagaatcag gtatcgactt gttatccatt 240

ccacctttga gattggatga aaacgaacaa gttaactacg acgccgtcac tacagctgtt 300

aaaaaggctt tgagattaaa tagagcaatt caagcccatg atggtcactg gccagctgaa 360

aacgcaggta gtttgttgta caccccacct ttgataatag ctttgtacat ctctggtact 420

atagatacaa tcttaaccaa gcaacataaa aaggaattga tcagattcgt ctacaaccac 480

caaaacgaag atggtggttg gggtagttac atcgaaggtc attctactat gattggttcc 540

gttttgagtt acgtcatgtt gagattgttg ggtgaaggtt tagccgaatc agatgacggt 600

aatggtgctg ttgaaagagg tagaaaatgg atcttggatc atggtggtgc tgcaggtatt 660

ccatcttggg gtaaaacata tttggctgta ttgggtgttt acgaatggga aggttgtaat 720

ccattaccac ctgaattttg gttgttccct tcttcatttc cattccatcc tgcaaaaatg 780

tggatctatt gtagatgcac ctacatgcca atgtcatatt tgtacggtaa aagataccac 840

ggtcctataa ctgatttggt tttatccttg agacaagaaa tctataacat cccatacgaa 900

caaattaaat ggaaccaaca aagacacaac tgttgcaagg aagatttgta ttaccctcac 960

actttagtac aagatttggt ttgggacggt ttgcattact tctctgaacc attcttgaag 1020

agatggcctt ttaataagtt gagaaagaga ggtttgaaga gagttgtcga attaatgaga 1080

tacggtgcta cagaaactag attcattacc actggtaatg gtgaaaaagc attgcaaatc 1140

atgtcatggt gggccgaaga tccaaacggt gacgaattca agcatcactt agccagaatt 1200

cctgatttct tgtggatagc tgaagacggt atgacagttc aatcttttgg ttcacaattg 1260

tgggattgta tattggccac tcaagctatc attgcaacaa atatggtcga agaatatggt 1320

gacagtttga agaaagctca tttctttatc aaggaatctc aaatcaagga aaacccacgt 1380

ggtgactttt tgaaaatgtg tagacaattc accaagggtg catggacttt ttcagatcaa 1440

gaccacggtt gtgtagtttc cgattgcacc gcagaagcct tgaagtgctt gttgttgttg 1500

tctcaaatgc cacaagacat tgtaggtgaa aagcctgaag ttgaaagatt gtacgaagcc 1560

gttaacgtct tgttgtactt gcaatccaga gttagtggtg gtttcgctgt ttgggaacca 1620

cctgtcccaa aaccttattt ggaaatgttg aacccatcag aaatctttgc tgatatagtc 1680

gtagaaagag aacatatcga atgtacagct tccgtaatca aaggtttgat ggcttttaaa 1740

tgcttgcatc caggtcacag acaaaaggaa atagaagata gtgttgctaa ggcaatcaga 1800

tatttggaaa gaaaccaaat gcctgacggt tcttggtatg gtttttgggg tatatgtttc 1860

ttatacggta ctttctttac attgagtggt tttgcctctg ctggtagaac atacgataat 1920

tcagaagcag tcagaaaagg tgtaaagttt ttcttatcca cccaaaacga agaaggtggt 1980

tggggtgaat ctttggaatc atgcccatcc gaaaaattca ctcctttgaa gggtaacaga 2040

acaaacttgg ttcaaacctc ttgggcaatg ttaggtttga tgtttggtgg tcaagccgaa 2100

agagatccaa ctcctttgca tagagccgct aaattgttga ttaatgcaca aatggataac 2160

ggtgacttcc cacaacaaga aatcacaggt gtttactgta agaactctat gttgcactac 2220

gccgaataca gaaacatttt tcctttgtgg gccttgggtg aatacagaaa aagagtttgg 2280

ttacctaagc atcaacaatt aaagatatga 2310

<210> 7

<211> 1461

<212> DNA

<213> Artificial sequence

<400> 7

atggcagccg ctatggtttt gttcttttca ttgtccttat tgttgttacc tttgttattg 60

ttgtttgctt atttctctta cactaaaaga ataccacaaa aagaaaatga ttccaaggct 120

cctttacctc caggtcaaac cggttggcca ttgatcggtg aaactttgaa ctatttgtca 180

tgtgttaagt ccggtgtcag tgaaaacttc gtaaagtaca gaaaggaaaa gtactctcca 240

aaggttttca gaacttcatt gttaggtgaa ccaatggcca ttttatgcgg tcctgaaggt 300

aataagttct tgtactctac agaaaagaaa ttggtacaag tttggtttcc atcttcagtt 360

gaaaagatgt tccctagatc tcatggtgaa tcaaacgcag ataacttctc taaagttaga 420

ggtaaaatga tgttcttgtt aaaggtcgat ggtatgaaaa agtatgtagg tttgatggac 480

agagttatga agcaattctt ggaaacagat tggaacagac aacaacaaat taatgtacac 540

aacaccgtta aaaagtacac cgtcactatg tcctgtagag tattcatgag tatagatgac 600

gaagaacaag ttaccagatt gggttccagt attcaaaaca tagaagctgg tttgttagca 660

gtcccaatca atattcctgg tacagccatg aacagagcta tcaaaacagt aaagttgtta 720

accagagaag tcgaagccgt aattaaacaa agaaaggttg acttgttgga aaataagcaa 780

gcatctcaac cacaagattt gttgagtcat ttgttgttga ctgctaacca agatggtcaa 840

tttttatctg aatcagacat cgcatcacac ttaattggtt tgatgcaagg tggttacact 900

acattgaacg gtacaatcac cttcgtcttg aactatttgg cagaattccc tgacgtctac 960

aatcaagtat tgaaggaaca agttgaaatc gccaactcta agcatccaaa ggaattgttg 1020

aactgggaag atttgagaaa gatgaagtac tcatggaacg ttgctcaaga agtcttgaga 1080

attatacctc caggtgttgg tacttttaga gaagcaatta ccgatttcac ttatgccggt 1140

tacttaattc ctaaaggttg gaagatgcac ttgataccac atgacactca caagaatcct 1200

acatacttcc catctcctga aaagttcgat cctactagat tcgagggtaa cggtccagct 1260

ccttatactt ttacaccatt cggtggtggt ccaagaatgt gccctggtat cgaatacgca 1320

agattagtta tattgatctt tatgcataat gttgtcacaa acttcagatg ggaaaaattg 1380

atcccaaacg aaaagatctt gactgaccct atcccaagat tcgcccacgg tttacctatc 1440

cacttacacc cacacaactg a 1461

<210> 8

<211> 1410

<212> DNA

<213> Artificial sequence

<400> 8

atggacttat tcatctcatc tcaattgttg ttattattag tattctgttt attcttattc 60

tggaacttca agccttcttc tcaaaataag ttgcctccag gtaaaaccgg ttggccaata 120

atcggtgaaa ctttggaatt catatcatgt ggtcaaaagg gtaacccaga aaagttcgtc 180

actcaaagaa tgaataagta ctctcctgat gtattcacta catcattggc tggtgaaaag 240

atggttgtct tttgcggtgc atccggtaat aagtttattt tctctaacga aaacaagttg 300

gtagtttctt ggtggccacc tgctatatca aagatcttga ctgcaacaat cccatctgtt 360

gaaaaatcaa aggccttgag atctttaatc gtcgaattct tgaagcctga agcattgcat 420

aagtttattt cagttatgga tagaaccact agacaacact tcgaagacaa atggaacggt 480

tctacagaag ttaaggcctt tgctatgtcc gaaagtttga ccttcgaatt ggcttgttgg 540

ttgttatttt ctattaatga tccagttcaa gtccaaaaat tgtcacattt gttcgaaaag 600

gttaaggcag gtttgttatc tttgccatta aattttcctg gtaccgcctt caacagaggt 660

atcaaggctg caaatttgat cagaaaggaa ttgtccgtcg taattaagca aagaagaagt 720

gataagttgc aaactagaaa ggacttgttg tcccatgtta tgttaagtaa cggtgaaggt 780

gaaaagtttt tctctgaaat ggatatagca gacgttgtct tgaatttgtt aatcgcatcc 840

cacgatacaa cctcttcagc catgggtagt gtagtttact ttttggccga ccatccacac 900

atctacgcta aagttttgac agaacaaatg gaaatagcaa agtctaaggg tgccgaagaa 960

ttgttgtcat gggaagatat caagagaatg aagtactcca gaaacgttat taatgaagct 1020

atgagattgg ttccacctag tcaaggtggt tttaaggtcg taacttctaa attttcatac 1080

gcaaacttca tcattccaaa aggttggaag attttctggt ccgtttatag tacacataaa 1140

gatcctaagt acttcaagaa cccagaagaa ttcgatcctt ccagattcga aggtgacggt 1200

ccaatgcctt ttacattcat accatttggt ggtggtccaa gaatgtgccc tggttcagaa 1260

ttcgctagat tggaagtctt gatctttatg catcacttag taaccaactt taagtgggaa 1320

aaagttttcc ctaatgaaaa gattatctac acaccattcc cattcccaga aaacggttta 1380

cctatcagat tatcaccttg taccttatga 1410

<210> 9

<211> 342

<212> PRT

<213> Artificial sequence

<400> 9

Met Ala Ala Thr Ser Glu Lys Gln Asn Thr Thr Lys Pro Pro Pro Ser

1 5 10 15

Pro Ser Pro Leu Arg Asn Ser Lys Phe Cys Gln Pro Asn Met Arg Ile

20 25 30

Leu Ile Ser Gly Gly Ala Gly Phe Ile Gly Ser His Leu Val Asp Lys

35 40 45

Leu Met Glu Asn Glu Lys Asn Glu Val Val Val Ala Asp Asn Tyr Phe

50 55 60

Thr Gly Ser Lys Glu Asn Leu Lys Lys Trp Ile Gly His Pro Arg Phe

65 70 75 80

Glu Leu Ile Arg His Asp Val Thr Glu Pro Leu Leu Ile Glu Val Asp

85 90 95

Arg Ile Tyr His Leu Ala Cys Pro Ala Ser Pro Ile Phe Tyr Lys Tyr

100 105 110

Asn Pro Val Lys Thr Ile Lys Thr Asn Val Ile Gly Thr Leu Asn Met

115 120 125

Leu Gly Leu Ala Lys Arg Val Gly Ala Arg Ile Leu Leu Thr Ser Thr

130 135 140

Ser Glu Val Tyr Gly Asp Pro Leu Ile His Pro Gln Pro Glu Ser Tyr

145 150 155 160

Trp Gly Asn Val Asn Pro Ile Gly Val Arg Ser Cys Tyr Asp Glu Gly

165 170 175

Lys Arg Val Ala Glu Thr Leu Met Phe Asp Tyr His Arg Gln His Gly

180 185 190

Ile Glu Ile Arg Ile Ala Arg Ile Phe Asn Thr Tyr Gly Pro Arg Met

195 200 205

Asn Ile Asp Asp Gly Arg Val Val Ser Asn Phe Ile Ala Gln Ala Leu

210 215 220

Arg Gly Glu Ala Leu Thr Val Gln Lys Pro Gly Thr Gln Thr Arg Ser

225 230 235 240

Phe Cys Tyr Val Ser Asp Met Val Asp Gly Leu Ile Arg Leu Met Glu

245 250 255

Gly Asn Asp Thr Gly Pro Ile Asn Ile Gly Asn Pro Gly Glu Phe Thr

260 265 270

Met Val Glu Leu Ala Glu Thr Val Lys Glu Leu Ile Asn Pro Ser Ile

275 280 285

Glu Ile Lys Met Val Glu Asn Thr Pro Asp Asp Pro Arg Gln Arg Lys

290 295 300

Pro Asp Ile Ser Lys Ala Lys Glu Val Leu Gly Trp Glu Pro Lys Val

305 310 315 320

Lys Leu Arg Glu Gly Leu Pro Leu Met Glu Glu Asp Phe Arg Leu Arg

325 330 335

Leu Asn Val Pro Arg Asn

340

<210> 10

<211> 481

<212> PRT

<213> Artificial sequence

<400> 10

Met Val Lys Ile Cys Cys Ile Gly Ala Gly Tyr Val Gly Gly Pro Thr

1 5 10 15

Met Ala Val Met Ala Leu Lys Cys Pro Glu Ile Glu Val Val Val Val

20 25 30

Asp Ile Ser Glu Pro Arg Ile Asn Ala Trp Asn Ser Asp Arg Leu Pro

35 40 45

Ile Tyr Glu Pro Gly Leu Glu Asp Val Val Lys Gln Cys Arg Gly Lys

50 55 60

Asn Leu Phe Phe Ser Thr Asp Val Glu Lys His Val Phe Glu Ser Asp

65 70 75 80

Ile Val Phe Val Ser Val Asn Thr Pro Thr Lys Thr Gln Gly Leu Gly

85 90 95

Ala Gly Lys Ala Ala Asp Leu Thr Tyr Trp Glu Ser Ala Ala Arg Met

100 105 110

Ile Ala Asp Val Ser Lys Ser Ser Lys Ile Val Val Glu Lys Ser Thr

115 120 125

Val Pro Val Arg Thr Ala Glu Ala Ile Glu Lys Ile Leu Thr His Asn

130 135 140

Ser Lys Gly Ile Glu Phe Gln Ile Leu Ser Asn Pro Glu Phe Leu Ala

145 150 155 160

Glu Gly Thr Ala Ile Lys Asp Leu Tyr Asn Pro Asp Arg Val Leu Ile

165 170 175

Gly Gly Arg Asp Thr Ala Ala Gly Gln Lys Ala Ile Lys Ala Leu Arg

180 185 190

Asp Val Tyr Ala His Trp Val Pro Val Glu Gln Ile Ile Cys Thr Asn

195 200 205

Leu Trp Ser Ala Glu Leu Ser Lys Leu Ala Ala Asn Ala Phe Leu Ala

210 215 220

Gln Arg Ile Ser Ser Val Asn Ala Met Ser Ala Leu Cys Glu Ala Thr

225 230 235 240

Gly Ala Asp Val Thr Gln Val Ala His Ala Val Gly Thr Asp Thr Arg

245 250 255

Ile Gly Pro Lys Phe Leu Asn Ala Ser Val Gly Phe Gly Gly Ser Cys

260 265 270

Phe Gln Lys Asp Ile Leu Asn Leu Ile Tyr Ile Cys Glu Cys Asn Gly

275 280 285

Leu Pro Glu Ala Ala Asn Tyr Trp Lys Gln Val Val Lys Val Asn Asp

290 295 300

Tyr Gln Lys Ile Arg Phe Ala Asn Arg Val Val Ser Ser Met Phe Asn

305 310 315 320

Thr Val Ser Gly Lys Lys Ile Ala Ile Leu Gly Phe Ala Phe Lys Lys

325 330 335

Asp Thr Gly Asp Thr Arg Glu Thr Pro Ala Ile Asp Val Cys Asn Arg

340 345 350

Leu Val Ala Asp Lys Ala Lys Leu Ser Ile Tyr Asp Pro Gln Val Leu

355 360 365

Glu Glu Gln Ile Arg Arg Asp Leu Ser Met Ala Arg Phe Asp Trp Asp

370 375 380

His Pro Val Pro Leu Gln Gln Ile Lys Ala Glu Gly Ile Ser Glu Gln

385 390 395 400

Val Asn Val Val Ser Asp Ala Tyr Glu Ala Thr Lys Asp Ala His Gly

405 410 415

Leu Cys Val Leu Thr Glu Trp Asp Glu Phe Lys Ser Leu Asp Phe Lys

420 425 430

Lys Ile Phe Asp Asn Met Gln Lys Pro Ala Phe Val Phe Asp Gly Arg

435 440 445

Asn Val Val Asp Ala Val Lys Leu Arg Glu Ile Gly Phe Ile Val Tyr

450 455 460

Ser Ile Gly Lys Pro Leu Asp Ser Trp Leu Lys Asp Met Pro Ala Val

465 470 475 480

Ala

<210> 11

<211> 475

<212> PRT

<213> Artificial sequence

<400> 11

Met Lys Ser Glu Leu Ile Phe Leu Pro Ala Pro Ala Ile Gly His Leu

1 5 10 15

Val Gly Met Val Glu Met Ala Lys Leu Phe Ile Ser Arg His Glu Asn

20 25 30

Leu Ser Val Thr Val Leu Ile Ala Lys Phe Tyr Met Asp Thr Gly Val

35 40 45

Asp Asn Tyr Asn Lys Ser Leu Leu Thr Asn Pro Thr Pro Arg Leu Thr

50 55 60

Ile Val Asn Leu Pro Glu Thr Asp Pro Gln Asn Tyr Met Leu Lys Pro

65 70 75 80

Arg His Ala Ile Phe Pro Ser Val Ile Glu Thr Gln Lys Thr His Val

85 90 95

Arg Asp Ile Ile Ser Gly Met Thr Gln Ser Glu Ser Thr Arg Val Val

100 105 110

Gly Leu Leu Ala Asp Leu Leu Phe Ile Asn Ile Met Asp Ile Ala Asn

115 120 125

Glu Phe Asn Val Pro Thr Tyr Val Tyr Ser Pro Ala Gly Ala Gly His

130 135 140

Leu Gly Leu Ala Phe His Leu Gln Thr Leu Asn Asp Lys Lys Gln Asp

145 150 155 160

Val Thr Glu Phe Arg Asn Ser Asp Thr Glu Leu Leu Val Pro Ser Phe

165 170 175

Ala Asn Pro Val Pro Ala Glu Val Leu Pro Ser Met Tyr Val Asp Lys

180 185 190

Glu Gly Gly Tyr Asp Tyr Leu Phe Ser Leu Phe Arg Arg Cys Arg Glu

195 200 205

Ser Lys Ala Ile Ile Ile Asn Thr Phe Glu Glu Leu Glu Pro Tyr Ala

210 215 220

Ile Asn Ser Leu Arg Met Asp Ser Met Ile Pro Pro Ile Tyr Pro Val

225 230 235 240

Gly Pro Ile Leu Asn Leu Asn Gly Asp Gly Gln Asn Ser Asp Glu Ala

245 250 255

Ala Val Ile Leu Gly Trp Leu Asp Asp Gln Pro Pro Ser Ser Val Val

260 265 270

Phe Leu Cys Phe Gly Ser Tyr Gly Thr Phe Gln Glu Asn Gln Val Lys

275 280 285

Glu Ile Ala Met Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Ser

290 295 300

Leu Arg Pro Ser Ile Pro Lys Gly Glu Thr Lys Leu Gln Leu Lys Tyr

305 310 315 320

Ser Asn Leu Glu Glu Ile Leu Pro Val Gly Phe Leu Asp Arg Thr Ser

325 330 335

Cys Val Gly Lys Val Ile Gly Trp Ala Pro Gln Val Ala Val Leu Gly

340 345 350

His Glu Ala Val Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ser Val Trp Cys Gly Val Pro Val Ala Thr Trp Pro Met Tyr

370 375 380

Gly Glu Gln Gln Leu Asn Ala Phe Glu Met Val Lys Glu Leu Gly Ile

385 390 395 400

Ala Val Glu Ile Glu Val Asp Tyr Lys Asn Glu Tyr Phe Asn Met Asn

405 410 415

Asn Asp Phe Ile Val Arg Ala Glu Glu Ile Glu Thr Lys Ile Lys Lys

420 425 430

Leu Met Met Asp Glu Lys Asn Ser Glu Ile Arg Lys Lys Val Lys Glu

435 440 445

Met Lys Glu Lys Ser Arg Leu Ala Met Ser Glu Asn Gly Ser Ser Tyr

450 455 460

Asn Ser Leu Ala Lys Leu Phe Glu Glu Ile Met

465 470 475

<210> 12

<211> 769

<212> PRT

<213> Artificial sequence

<400> 12

Met Trp Lys Leu Lys Val Ala Gln Gly Asn Asp Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Tyr Trp Glu Phe Gln Pro Asp Ala

20 25 30

Gly Thr Pro Glu Glu Arg Glu Glu Val Glu Lys Ala Arg Lys Asp Tyr

35 40 45

Val Asn Asn Lys Lys Leu His Gly Ile His Pro Cys Ser Asp Met Leu

50 55 60

Met Arg Arg Gln Leu Ile Lys Glu Ser Gly Ile Asp Leu Leu Ser Ile

65 70 75 80

Pro Pro Leu Arg Leu Asp Glu Asn Glu Gln Val Asn Tyr Asp Ala Val

85 90 95

Thr Thr Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

His Asp Gly His Trp Pro Ala Glu Asn Ala Gly Ser Leu Leu Tyr Thr

115 120 125

Pro Pro Leu Ile Ile Ala Leu Tyr Ile Ser Gly Thr Ile Asp Thr Ile

130 135 140

Leu Thr Lys Gln His Lys Lys Glu Leu Ile Arg Phe Val Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Ser Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Val Leu Ser Tyr Val Met Leu Arg Leu Leu Gly Glu

180 185 190

Gly Leu Ala Glu Ser Asp Asp Gly Asn Gly Ala Val Glu Arg Gly Arg

195 200 205

Lys Trp Ile Leu Asp His Gly Gly Ala Ala Gly Ile Pro Ser Trp Gly

210 215 220

Lys Thr Tyr Leu Ala Val Leu Gly Val Tyr Glu Trp Glu Gly Cys Asn

225 230 235 240

Pro Leu Pro Pro Glu Phe Trp Leu Phe Pro Ser Ser Phe Pro Phe His

245 250 255

Pro Ala Lys Met Trp Ile Tyr Cys Arg Cys Thr Tyr Met Pro Met Ser

260 265 270

Tyr Leu Tyr Gly Lys Arg Tyr His Gly Pro Ile Thr Asp Leu Val Leu

275 280 285

Ser Leu Arg Gln Glu Ile Tyr Asn Ile Pro Tyr Glu Gln Ile Lys Trp

290 295 300

Asn Gln Gln Arg His Asn Cys Cys Lys Glu Asp Leu Tyr Tyr Pro His

305 310 315 320

Thr Leu Val Gln Asp Leu Val Trp Asp Gly Leu His Tyr Phe Ser Glu

325 330 335

Pro Phe Leu Lys Arg Trp Pro Phe Asn Lys Leu Arg Lys Arg Gly Leu

340 345 350

Lys Arg Val Val Glu Leu Met Arg Tyr Gly Ala Thr Glu Thr Arg Phe

355 360 365

Ile Thr Thr Gly Asn Gly Glu Lys Ala Leu Gln Ile Met Ser Trp Trp

370 375 380

Ala Glu Asp Pro Asn Gly Asp Glu Phe Lys His His Leu Ala Arg Ile

385 390 395 400

Pro Asp Phe Leu Trp Ile Ala Glu Asp Gly Met Thr Val Gln Ser Phe

405 410 415

Gly Ser Gln Leu Trp Asp Cys Ile Leu Ala Thr Gln Ala Ile Ile Ala

420 425 430

Thr Asn Met Val Glu Glu Tyr Gly Asp Ser Leu Lys Lys Ala His Phe

435 440 445

Phe Ile Lys Glu Ser Gln Ile Lys Glu Asn Pro Arg Gly Asp Phe Leu

450 455 460

Lys Met Cys Arg Gln Phe Thr Lys Gly Ala Trp Thr Phe Ser Asp Gln

465 470 475 480

Asp His Gly Cys Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys

485 490 495

Leu Leu Leu Leu Ser Gln Met Pro Gln Asp Ile Val Gly Glu Lys Pro

500 505 510

Glu Val Glu Arg Leu Tyr Glu Ala Val Asn Val Leu Leu Tyr Leu Gln

515 520 525

Ser Arg Val Ser Gly Gly Phe Ala Val Trp Glu Pro Pro Val Pro Lys

530 535 540

Pro Tyr Leu Glu Met Leu Asn Pro Ser Glu Ile Phe Ala Asp Ile Val

545 550 555 560

Val Glu Arg Glu His Ile Glu Cys Thr Ala Ser Val Ile Lys Gly Leu

565 570 575

Met Ala Phe Lys Cys Leu His Pro Gly His Arg Gln Lys Glu Ile Glu

580 585 590

Asp Ser Val Ala Lys Ala Ile Arg Tyr Leu Glu Arg Asn Gln Met Pro

595 600 605

Asp Gly Ser Trp Tyr Gly Phe Trp Gly Ile Cys Phe Leu Tyr Gly Thr

610 615 620

Phe Phe Thr Leu Ser Gly Phe Ala Ser Ala Gly Arg Thr Tyr Asp Asn

625 630 635 640

Ser Glu Ala Val Arg Lys Gly Val Lys Phe Phe Leu Ser Thr Gln Asn

645 650 655

Glu Glu Gly Gly Trp Gly Glu Ser Leu Glu Ser Cys Pro Ser Glu Lys

660 665 670

Phe Thr Pro Leu Lys Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp

675 680 685

Ala Met Leu Gly Leu Met Phe Gly Gly Gln Ala Glu Arg Asp Pro Thr

690 695 700

Pro Leu His Arg Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asn

705 710 715 720

Gly Asp Phe Pro Gln Gln Glu Ile Thr Gly Val Tyr Cys Lys Asn Ser

725 730 735

Met Leu His Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu

740 745 750

Gly Glu Tyr Arg Lys Arg Val Trp Leu Pro Lys His Gln Gln Leu Lys

755 760 765

Ile

<210> 13

<211> 486

<212> PRT

<213> Artificial sequence

<400> 13

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> 14

<211> 469

<212> PRT

<213> Artificial sequence

<400> 14

Met Asp Leu Phe Ile Ser Ser Gln Leu Leu Leu Leu Leu Val Phe Cys

1 5 10 15

Leu Phe Leu Phe Trp Asn Phe Lys Pro Ser Ser Gln Asn Lys Leu Pro

20 25 30

Pro Gly Lys Thr Gly Trp Pro Ile Ile Gly Glu Thr Leu Glu Phe Ile

35 40 45

Ser Cys Gly Gln Lys Gly Asn Pro Glu Lys Phe Val Thr Gln Arg Met

50 55 60

Asn Lys Tyr Ser Pro Asp Val Phe Thr Thr Ser Leu Ala Gly Glu Lys

65 70 75 80

Met Val Val Phe Cys Gly Ala Ser Gly Asn Lys Phe Ile Phe Ser Asn

85 90 95

Glu Asn Lys Leu Val Val Ser Trp Trp Pro Pro Ala Ile Ser Lys Ile

100 105 110

Leu Thr Ala Thr Ile Pro Ser Val Glu Lys Ser Lys Ala Leu Arg Ser

115 120 125

Leu Ile Val Glu Phe Leu Lys Pro Glu Ala Leu His Lys Phe Ile Ser

130 135 140

Val Met Asp Arg Thr Thr Arg Gln His Phe Glu Asp Lys Trp Asn Gly

145 150 155 160

Ser Thr Glu Val Lys Ala Phe Ala Met Ser Glu Ser Leu Thr Phe Glu

165 170 175

Leu Ala Cys Trp Leu Leu Phe Ser Ile Asn Asp Pro Val Gln Val Gln

180 185 190

Lys Leu Ser His Leu Phe Glu Lys Val Lys Ala Gly Leu Leu Ser Leu

195 200 205

Pro Leu Asn Phe Pro Gly Thr Ala Phe Asn Arg Gly Ile Lys Ala Ala

210 215 220

Asn Leu Ile Arg Lys Glu Leu Ser Val Val Ile Lys Gln Arg Arg Ser

225 230 235 240

Asp Lys Leu Gln Thr Arg Lys Asp Leu Leu Ser His Val Met Leu Ser

245 250 255

Asn Gly Glu Gly Glu Lys Phe Phe Ser Glu Met Asp Ile Ala Asp Val

260 265 270

Val Leu Asn Leu Leu Ile Ala Ser His Asp Thr Thr Ser Ser Ala Met

275 280 285

Gly Ser Val Val Tyr Phe Leu Ala Asp His Pro His Ile Tyr Ala Lys

290 295 300

Val Leu Thr Glu Gln Met Glu Ile Ala Lys Ser Lys Gly Ala Glu Glu

305 310 315 320

Leu Leu Ser Trp Glu Asp Ile Lys Arg Met Lys Tyr Ser Arg Asn Val

325 330 335

Ile Asn Glu Ala Met Arg Leu Val Pro Pro Ser Gln Gly Gly Phe Lys

340 345 350

Val Val Thr Ser Lys Phe Ser Tyr Ala Asn Phe Ile Ile Pro Lys Gly

355 360 365

Trp Lys Ile Phe Trp Ser Val Tyr Ser Thr His Lys Asp Pro Lys Tyr

370 375 380

Phe Lys Asn Pro Glu Glu Phe Asp Pro Ser Arg Phe Glu Gly Asp Gly

385 390 395 400

Pro Met Pro Phe Thr Phe Ile Pro Phe Gly Gly Gly Pro Arg Met Cys

405 410 415

Pro Gly Ser Glu Phe Ala Arg Leu Glu Val Leu Ile Phe Met His His

420 425 430

Leu Val Thr Asn Phe Lys Trp Glu Lys Val Phe Pro Asn Glu Lys Ile

435 440 445

Ile Tyr Thr Pro Phe Pro Phe Pro Glu Asn Gly Leu Pro Ile Arg Leu

450 455 460

Ser Pro Cys Thr Leu

465

Claims (12)

1. The Pn3-32-i5 protein is a1) or a2) as follows:

   a1) the amino acid sequence is SEQ ID NO: 5;

   a2) in SEQ ID NO: 5, and/or the N-terminal and/or the C-terminal of the protein shown in the figure are connected with a label to obtain the fusion protein.
2. 2. A nucleic acid molecule encoding the Pn3-32-i5 protein of claim 1.
3. 3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as b1) or b 2):

   b1) the coding region is SEQ ID NO: 1;

   b2) the nucleotide sequence is SEQ ID NO: 1.
4. 4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.
5. 5. The use of the Pn3-32-i5 protein of claim 1 as c1) or c2) or c3) or c 4):

   c1) producing notoginsenoside R1;

   c2) preparing a product for producing notoginsenoside R1;

   c3) as notoginsenoside R1 synthase;

   c4) preparing the product with the function of the notoginsenoside R1 synthetase.
6. 6. The use of the nucleic acid molecule of claim 2 or 3 as c1) or c2) or c 3):

   c1) producing notoginsenoside R1;

   c2) preparing a product for producing notoginsenoside R1;

   c3) preparing the product with the function of the notoginsenoside R1 synthetase.
7. 7. The method for preparing the engineering bacteria for producing the notoginsenoside R1 comprises the following steps: improving the expression and/or activity of phosphoglucomutase 1, alpha-phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, Arabidopsis UDP-glucose dehydrogenase 1, Arabidopsis UDP-glucuronic acid decarboxylase 3, ginseng UDP-glycosyltransferase 101 and the Pn3-32-i5 protein in claim 1 in a recipient yeast, thereby obtaining the engineering bacteria for producing notoginsenoside R1;

   the receptor yeast is obtained by improving the expression level and/or activity of hydroxymethylglutaryl coenzyme A reductase 1, farnesyl pyrophosphate synthase, squalene epoxidase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and cytochrome P450 reductase in saccharomyces cerevisiae.
8. 8. The method of claim 7, wherein: the method for improving the expression level and/or activity of phosphoglucomutase 1, alpha-phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, Arabidopsis thaliana UDP-glucose dehydrogenase 1, Arabidopsis thaliana UDP-glucuronic acid decarboxylase 3, ginseng UDP-glycosyltransferase 101 and the Pn3-32-i5 protein in the acceptor yeast is characterized in that the coding gene of phosphoglucomutase 1 is introduced into the acceptor yeast, alpha-phosphoglucomutase coding gene, uridine diphosphate glucose pyrophosphorylase coding gene, Arabidopsis UDP-glucose dehydrogenase 1 coding gene, Arabidopsis UDP-glucuronic acid decarboxylase 3 coding gene, ginseng UDP-glycosyltransferase 101 coding gene, and Pn3-32-i5 protein coding gene as defined in claim 1.
9. 9. The method of claim 7, wherein: the recipient yeast is yeast BY-PPD-PPT.
10. 10. An engineering bacterium prepared by the method of any one of claims 7 to 9 and used for producing notoginsenoside R1.
11. 11. The use of the engineered bacterium of claim 10 in the production of notoginsenoside R1.
12. 12. A method for producing notoginsenoside R1 comprises the following steps: fermenting and culturing the engineering bacteria of claim 10, collecting the fermentation product, and obtaining the notoginsenoside R1 from the fermentation product.

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