CN114774503B - Squalene epoxidase and coding gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly discloses squalene epoxidase as well as a coding gene and application thereof. The invention discloses application of squalene epoxidase or a coding gene thereof, or a biological material containing the coding gene thereof in biological fermentation synthesis of 11-oxo-beta-resinol or a triterpene compound; the amino acid sequence of the squalene epoxidase is shown in SEQ ID No:9 or SEQ ID No: shown at 10. The invention discovers that the gene of squalene epoxidase is over-expressed in a fermentation strain, can improve the yield of 11-oxo-beta-resinol or triterpene compound synthesized by the fermentation strain, and further provides the application of the squalene epoxidase or the coding gene thereof, or the biological material containing the coding gene thereof in the biological fermentation synthesis of 11-oxo-beta-resinol or triterpene compound. Provides a new way for efficiently synthesizing the triterpene compound.

Description

Squalene epoxidase and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to squalene epoxidase and an encoding gene and application thereof.

Background

The triterpenoid is a kind of compound with 6 isoprene as structural unit (C) ₅ H ₈ ) The natural product containing 30 carbon atoms widely exists in nature, is an important secondary metabolite in plants, and has the characteristics ofVarious medicinal activities, such as glycyrrhizic acid, glycyrrhetinic acid, 11-Oxo-beta-resinol (11-Oxo-beta-amyrin) in licorice, oleanolic acid and ursolic acid in medicago truncatula, ginsenoside in ginseng, etc., have pharmaceutical activities of resisting inflammation, resisting tumor, resisting virus, protecting liver, etc. Some triterpene compounds are also important insect-resistant agents, natural sweeteners and emulsifiers, and also have very important application and research values, such as azadirachtin, which has good insecticidal effect under the concentration of 1-10 ppm and has obvious repellent and insecticidal effects on more than 200 pests; monoglucuronic acid glycyrrhetinic acid and mogroside are also good sweeteners, and the sweetness is 941 times and 300 times that of sucrose respectively. Due to the complex chemical structure of the triterpenoid, the low metabolic yield in the source plant, the existence of various structural analogs and other factors, the triterpenoid is difficult to obtain by chemical synthesis and extraction of the source plant. The development of synthetic biology provides a new idea for acquiring a large amount of high-value triterpenoid components. The saccharomyces cerevisiae has the advantages of clear genetic background, short growth cycle, good safety, easy operation and the like, and contains all genes required by synthesizing triterpenoids, so the saccharomyces cerevisiae can be used as a good host for terpenoid cell factories.

The plant-derived metabolic pathway of triterpene compounds is basically well-understood, and the biosynthesis of triterpene compounds can be generally divided into three parts: namely, active forms of isopentenyl pyrophosphate (IPP) and dimethyl propenyl Diphosphate (DMAPP) from a general C5 composition module isoprene of a mevalonate pathway (MVA) and a 2-methylerythritol-4-phosphate (MEP) pathway are condensed by farnesene pyrophosphate synthase (FPPS) to generate farnesene pyrophosphate (FPP), two molecules of FPP are catalyzed by squalene synthase (FPPS) to synthesize squalene, and the squalene is further catalyzed by squalene epoxidase (SQE) to generate common precursor 2, 3-oxidosqualene of triterpenoids; followed by cyclization at oxidosqualene cyclase (OSCs) to form a polycyclic triterpene skeleton. Then the skeleton compound is further modified under the catalytic action of corresponding enzymes such as CYP450s and the like to finally form the corresponding triterpene compound.

In the above process, SQE plays an important role in the triterpene compound biosynthesis pathway, and is a key branch point of triterpene compound biosynthesis. SQE is a non-cytochrome P450 enzyme system epoxidised olefin enzyme, under the combined action of molecular oxygen and cofactors FAD, NAD (P) H, 1 oxygen atom is inserted between squalene C-C to change into singlet squalene, and the intermediate can generate 2, 3-oxidized squalene under the action of strong light and ultraviolet; 2, 3-oxidosqualene is a common substrate for the synthesis of triterpenoid production, and sufficient substrate supply is a key factor for high yield of its final product. SQE has been identified in several species, such as luo han guo, candida albicans, gynostemma pentaphylla, arabidopsis thaliana, oriental alisma orientale, panax notoginseng, panax japonicus, tobacco, ginseng, etc. Because the specific activity of SQE in the basal disc cells of yeast and the like is very low, and the catalytic product 2, 3-oxidosqualene has multiple branch metabolism in the total metabolic flux of microorganisms, SQE is still considered to be one of the key rate-limiting enzymes in the biosynthetic pathway of plant triterpenoids, and further research on SQE is necessary.

Disclosure of Invention

The invention aims to provide a SEQ and a gene with high catalytic activity to promote more squalene to flow to triterpene metabolic flow (including but not limited to glycyrrhetinic acid, glycyrrhizic acid, ginsenoside, mogroside and the like).

The invention obtains squalene epoxidase genes with high catalytic activity from different plant sources through research, and particularly, the squalene epoxidase genes (and the squalene epoxidase genes) are over-expressed in Chassis cells of high-yield squalene through chromosome integrationUni25647The gene,GuCPR1Genes andβ-Asgene coexpression), the determination of the yield of 11-oxo-beta-resinol in the fermentation product shows that the squalene epoxidase gene has higher catalytic activity and lays a foundation for the subsequent construction of glycyrrhetinic acid strains. Meanwhile, the microbial inoculum obtained by the invention can be used as a platform strain for producing 11-oxo-beta-resinol, and the 11-oxo-beta-resinol also has various effects.

The present invention overexpresses squalene epoxidase gene (and) in the underpan cells (Y81) producing 11-oxo-beta-resinol by chromosomal integrationUni25647Gene, gene,GuCPR1Gene, gene,Mut72A63Andβ-Asgene co-expression) and determining the yield of the glycyrrhetinic acid in the fermentation product, the squalene epoxidase gene can be used for improving the yield of the glycyrrhetinic acid produced by fermentation, and a glycyrrhetinic acid high-yield strain can be obtained according to the method.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides the use of a squalene epoxidase or a gene encoding it, or a biological material comprising a gene encoding it, in the biofermentation synthesis of 11-oxo- β -resinol or a triterpene compound; the amino acid sequence of the squalene epoxidase is shown in SEQ ID No:9 or SEQ ID No: 10. as shown.

The nucleic acid sequence of the squalene epoxidase coding gene is shown as SEQ ID No:1 or SEQ ID No:7 is shown in the specification;

and/or, the biological material is an expression cassette, a vector, or a host cell.

Preferably, the triterpene compound is glycyrrhetinic acid, glycyrrhizic acid, ginsenoside or mogroside.

In the present invention, the strain used in the biological fermentation can produce squalene.

The strain of the invention can produce squalene in metabolism, and is further used for realizing the synthesis of triterpene compounds.

The strain used in the biological fermentation is saccharomycetes, and the saccharomycetes also co-express the squalene epoxidase coding gene on the basis of overexpressionUni25647Gene, gene,GuCPR1Genes andβ-Asa gene.

When the glycyrrhetinic acid is produced by biological fermentation, the microzyme further co-expresses the encoding gene of the squalene epoxidaseUni25647Gene、GuCPR1Gene、Mut72A63Genes andβ-Asa gene.

Uni25647Genes can be found, for example, in NCBI: the method has the advantages of providing a new method in KY499143.1,GuCPR1genes can be found, for example, in NCBI: KY798117;β-Asgenes can be found, for example, in NCBI: the material is AB037203 in the specification,Mut72A63the gene consists of CYPC72A63 (NCBI: AB 5581)46 Modified, see in particular the literature: control Chemo-and Regiospectivity of a Plant P450 in Yeast Cell tissue Biosynthesis Wentao Sun, haijie Xue, hu Liu, bo Lv, yang Yu, ying Wang, meilan Huang, and Chun Li, ACS Catalysis 2020 (7), 4253-4260, DOI: 10.1021/acscatal.0c00128.Mut72A63The amino acid sequence of (1) is shown as SEQ ID No: shown at 11.

In a second aspect, the invention provides a squalene epoxidase, the amino acid sequence of which is as shown in SEQ ID No: shown at 9.

In a third aspect, the invention provides a gene encoding squalene epoxidase, which has a nucleic acid sequence as shown in SEQ ID No:1 or SEQ ID No: shown at 7.

In a fourth aspect, the invention provides a method for increasing the yield of glycyrrhetinic acid synthesized by biological fermentation, which comprises the steps of over-expressing a gene of squalene epoxidase in a fermentation strain, and increasing the yield of glycyrrhetinic acid synthesized by the fermentation strain; the gene of the squalene epoxidase is shown as SEQ ID No:1 or SEQ ID No: shown at 7.

In the method, the fermentation strain is yeast capable of producing squalene, and the yeast overexpresses a gene shown as SEQ ID No:1, and co-expressingUni25647Gene, gene,GuCPR1Genes andβ-Asa gene.

The fermentation strain further overexpresses a polypeptide as shown in SEQ ID No:1 or SEQ ID No:7, and co-expressingUni25647Gene, gene,GuCPR1The gene,Mut72A63Andβ-Asa gene.

The invention has the beneficial effects that:

the SQE discovered by the invention has high catalytic activity, can promote squalene to flow to triterpene metabolic flow (including but not limited to glycyrrhetinic acid, glycyrrhizic acid, ginsenoside, mogroside and the like), improves the biosynthesis yield of triterpene compounds, and provides new help and thinking for the modification of other triterpene substances.

Drawings

FIG. 1 is a schematic diagram of the metabolic pathway for producing terpenoids using squalene in yeast as a substrate according to the present invention;

FIG. 2 is a schematic diagram showing the metabolic pathway for producing glycyrrhetinic acid using 2, 3-oxidosqualene as a substrate in the yeast of the present invention;

FIG. 3 shows squalene epoxidase genes from different sources according to the present inventionUni25647The gene,GuCPR1Genes andβ-Asa chromosomal integration scheme for a gene;

FIG. 4 is a schematic diagram showing the integration of YPRC sites upon construction of a glycyrrhetinic acid-producing strain of the present invention;

FIG. 5 shows the results of 11-oxo- β -resinol fermentation by a strain transformed with squalene epoxidase gene from different sources according to the present invention;

FIG. 6 shows the results of squalene production by fermentation of a strain transformed with squalene epoxidase gene from a different source according to the present invention;

FIG. 7 shows the OD of the strains transformed with squalene epoxidase genes from different sources according to the present invention ₆₀₀ A value;

FIG. 8 shows the results of fermentation of the strain transformed with squalene epoxidase gene from different sources to produce glycyrrhetinic acid according to the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. In the present invention, unless otherwise specified, the OD values are all the results of detection at a wavelength of 600 nm.

Example 1 obtaining of Squalene epoxidase genes from different sources

In this example, arabidopsis thaliana, glycyrrhiza glabra, glycyrrhiza uralensis, tripterygium wilfordii, ginseng, neem and saccharomyces cerevisiae 7 source species were selected as experimental samples for subsequent SQE gene research, and in this example, the source of plant material was selectedIn the plant institute of Beijing, china academy of sciences. Cleaning the plants with flowing water, removing water on the surface with absorbent paper, cutting off the used parts with scissors, wrapping with tin foil paper, marking, treating with liquid nitrogen, and storing at-80 deg.C. Grinding a target sample by liquid nitrogen, extracting RNA, taking the extraction kit as a plant RNA rapid extraction kit (Tiangen Biochemical technology Co., ltd., product number: DP 432), carrying out reverse transcription by taking the RNA as a template according to a reverse transcription kit specification (the kit is from Beijing Pan Kagaku Kogyo, catalog number: AT 311-02), and synthesizing cDNA of each tissue for later use; designing primers according to the primer design principle and carrying out corresponding SQE gene amplification (the amplification primers are shown in Table 1), wherein the amplification sequences are shown as SEQ ID NO 1-8, in the SEQ ID NO 1-8, the SEQ ID NO 1 is derived from Neem (Chinaberry fruit)AiSQE) The sequence of the encoded protein is shown as SEQ ID NO. 9, and SEQ ID NO. 2 is derived from arabidopsis thaliana (A) and (B)AtSQE) SEQ ID NO 3 from Saccharomyces cerevisiae (SEQ ID NO)ERG1），SEQ ID NO:4（GuSQE1) Derived from Glycyrrhiza uralensis Fisch, SEQ ID NO. 5 derived from Glycyrrhiza glabra (L.) Merr. (GgSQE) SEQ ID NO 6 is derived from ginseng (ginseng)PgSQE) SEQ ID NO:7 is derived from Tripterygium wilfordii hook (F)TwSQE) The sequence of the encoded protein is shown as SEQ ID NO. 10, and SEQ ID NO. 8: (GuSQE2) Is derived from Glycyrrhiza uralensis Fisch. And respectively connecting the two ends of the gene with a promoter and a terminator to form a gene expression cluster. The gene expression cluster of each gene isPtef1-TwSQE-Tpgk1，Ptef1-AtSQE-Tpgk1，Ptef1-ERG1-Tpgk1，Ptef1-PgSQE-Tpgk1，Ptef1-GgSQE-Tpgk1，Ptef1- AiSQE-Tpgk1，Ptef1-GuSQE1-Tpgk1，Ptef1-GuSQE2-Tpgk1。

To construct NeemPtef1-AiSQE-Tpgk1For example, the specific construction method is as follows:

1. amplifying a target gene AiSQE by using primer AiSQE- (PTEF 1) -F and AiSQE- (TPGK 1) -R with gene homology arms by taking neem cDNA obtained by reverse transcription in the step as a template; the vector plasmid of Ptef1-Tpgk1-PUC19L preserved in a laboratory is taken as a template, and the vector is reversely amplified by primers TPGK1- (AiSQE) -F and PTEF1- (AiSQE) -R with homologous arms.

Ptef1-Tpgk1-PUC19L is described in: grand mengchu, chaulingyu, su xin 22575, cinnabar, suoyong, qian guang, old vaseline, wang Cabernet, xue Jianping, cell construction and high density fermentation of Saccharomyces cerevisiae producing beta-amyrin alcohol [ J ]. Chinese J.Medicine J.2019, 44 (07): 1341-1349. DOI. In particular, the plasmid pUC19L-PTEF1-ERG1-TPGK1 shown in Table 1 of the document.

2. And respectively obtaining a Ptef1-Tpgk1-PUC19L vector PCR product and a AiSQE gene PCR product, and respectively placing the PCR products on ice for later use after gel electrophoresis, gel cutting recovery and purification.

3. The AiSQE fragment and the Ptef1-Tpgk1-PUC19L carrier gel recovered product are seamlessly connected by using seamless ligase (seamless connection is specifically carried out by carrying out seamless connection through a homologous arm between a linear carrier and the fragment, the method comprises the steps of uniformly mixing 2 mu L of target gene, 0.5 mu L of fragment and 2.5 mu L of seamless ligase, and then carrying out 50 ℃ for 30min, and then adding 50 mu L of Escherichia coli competence into 5 mu L of the connected product to carry out large intestine transformation operation) to construct the plasmid Ptef1-AiSQE-Tpgk1-PUC19L.

4. And selecting the recombinant escherichia coli single colony for colony PCR, and screening positive colonies.

5. Successfully constructed escherichia coli Ptef1-AiSQE-Tpgk1-pUC19L is extracted into a plasmid for later use.

6. The plasmid was used as a template, and integrated primers S (PGK 1P) TEF1P-R, S (ADH 1P) -PGK1T-F (see Table 3) with homology arms were used to amplify to obtain PCR products (target gene cluster with promoter and terminator) of the desired fragmentPtef1-AiSQE-Tpgk1) The glue is recovered and then placed at-20 ℃ for standby.

Other SQE gene expression clusters were prepared as described above, and the primers used are shown in Table 1.

Ptef1-ERG1-Tpgk1See: construction and high-density fermentation of Saccharomyces cerevisiae cells producing beta-resinol [ J ] by Simmonchu, chaudoukun, suxin 22575]The Chinese traditional medicine journal, 2019,44 (07) 1341-1349. DOI.

TABLE 1 primers used for construction of plasmids (SEQ ID NO: 12-55)

EXAMPLE 2 construction of 11-oxo-beta-amyrin yeast strains

In this example, a high-yield squalene yeast strain Y41 (which is the yeast strain Z-CB-9-5 in example 2 of the Chinese patent CN 114107332A) is used as an underpinning strain, and the specific information of the strain is shown in Table 2 (see the integrated fragment of Y41 in Table 2 also in the Chinese patent CN 114107332A). The 8 strips obtained in example 1 were combinedSQEGene expression cluster (Ptef1-AiSQE-Tpgk1，Ptef1-AtSQE-Tpgk1，Ptef1-ERG1-Tpgk1，Ptef1-GuSQE1-Tpgk1，Ptef1-GgSQE-Tpgk1，Ptef1-PgSQE-Tpgk1，Ptef1-TwSQE-Tpgk1， Ptef1-GuSQE2-Tpgk1) Respectively associated with gene expression clustersPadh1-Uni25647-Tadh1, Pthd3-GuCPR1-Ttdh3, Ppgk1-β-As-Tadh1(those skilled in the art can routinely construct according to the general knowledge in the art) a GAL80 site (squalene epoxidase gene and/or gene) which is integrated into the chromosome of the Chassis strain Y41 by homologous recombinationUni25647The gene,GuCPR1Genes andβ-Asthe chromosomal integration of the genes is schematically shown in FIG. 3), positive clones are detected by PCR, and the resulting yeast strains are named separatelyY81、Y82、Y83、Y84、Y85、Y86、Y87、Y88. The information on the construction of the integration strain is shown in Table 2, and the integration primers are shown in Table 3.

TABLE 2 construction of the strains

TABLE 3 primers used for integration (SEQ ID NO: 56-71)

In the table, P _tef1 Different SQE-T _pgk1 Reference is made to the 8 strips obtained in example 1SQEA gene expression cluster. Taxonomy ID of the yeast genome 559292.

The specific operations for constructing the strain are as follows:

(1) Strain activation: streaking a chassis strain Y41 stored at minus 80 ℃ in a four-deficient culture medium SD-Leu-Trp-His-Ura, standing in a 30 ℃ constant temperature incubator for culture, selecting a single colony, placing in a 50 ml centrifuge tube filled with 5ml of deficient culture medium, placing at 30 ℃, culturing at 220 rpm until OD reaches 0.8-1.2, and taking the single colony as a seed solution for subsequent activation; inoculating the seed solution in the tube into a 100ml triangular flask filled with 20 ml of defective culture medium, wherein the inoculation amount is 2-5%, and the seed solution is cultured at 30 ℃ and 220 rpm until the OD reaches 0.8-1.2; the strain was activated 3 times.

(2) And (3) competent preparation: inoculating 200 μ L of the activated yeast liquid into 100mL triangular flask containing 20 mL of defective culture medium, and culturing in a shaker at 30 deg.C for 200 r/min until OD600 nm value reaches 0.8-1.2; transferring the bacterial liquid into a sterile 50 mL centrifuge tube, centrifuging for 5min at the speed of 3000r/min, and removing the supernatant; adding a precooled 10 mL sterile water gun head, blowing, beating and mixing uniformly, centrifuging for 5min at the speed of 3000r/min, and discarding the supernatant (the step is to clean the residual culture medium in the thalli, and repeating for 2-3 times); adding 1mL of precooled 100 mmol/L LiAc into the thalli, blowing and resuspending a gun head, centrifuging at 13000r/min for 15s, and discarding the supernatant; and adding the precooled LiAc with the concentration of 100 mmol/L to 400 mu L again, and subpackaging the mixture into precooled 2 ml centrifuge tubes with the concentration of 50 ul per tube.

(3) Yeast transformation: to the prepared competence were added 240. Mu.L of 50% PEG3350, 36. Mu.L of 1mol/L LiAc and 5. Mu.L of salmon sperm (ssDNA, denatured by heating in a 100 ℃ metal bath for 10min, immediately after denaturation on ice for use). Make it possible toObtaining gene segments required for integration by PCR using the integration amplification primers in Table 3 and the templates corresponding to the primers in Table 3GAL80-up、KANMX、ADH1 _P -Uni25647-ADH1t、TDH3p-GuCPR1-TDH3t、ADH1 _P -Uni25647- ADH1t、TEF1p-SQE-PGK1t、PGK1 _P -β-AS-ADH1t、GAL80-down(the dosage of each fragment is 400 ng, and the negative control is corresponding water volume), blowing and beating the gun head and mixing uniformly; ice-cooling for 30min, heat-shocking for 30min at 42 deg.C, centrifuging for 2 min at 3000r/min, discarding supernatant, adding 800 μ L YPD liquid culture medium into thallus, and shake-culturing for 2 hr at 30 deg.C with shaking table 200 r/min; the transformed strain was spread evenly on a plate of SD-Leu-Trp-His-Ura + G418 (G418 concentration in the plate was 200. Mu.g/mL, G418 was an antibiotic, which served as a screening), and the plate was inverted in an incubator at 30 ℃ for 4 to 6 days.

(4) And (3) positive detection: selecting the monoclonal, extracting the genome according to a Tiangen yeast genome extraction kit, and carrying out PCR detection by using the genome as a target template and primers in the table 3.

Example 3 construction of Glycyrrhetinic acid-producing Yeast strains

Using yeast strain Y81 of example 2 as a base strain, 8 of the strains obtained in example 1 were culturedSQEGene expression cluster (Ptef1-AiSQE-Tpgk1，Ptef1-AtSQE-Tpgk1，Ptef1-ERG1-Tpgk1，Ptef1-GuSQE1-Tpgk1，Ptef1-GgSQE-Tpgk1，Ptef1-PgSQE-Tpgk1，Ptef1-TwSQE-Tpgk1， Ptef1-GuSQE2-Tpgk1) Respectively associated with gene expression clustersPadh1-Uni25647-Tadh1, Pthd3-GuCPR1-Ttdh3, Padh1-Mut72A63-Tadh1，Ppgk1-β-As-Tadh1(those skilled in the art can routinely construct according to the general knowledge in the art) each gene was integrated into the YPRC site of the chromosome of the chassis strain by the yeast integration method and homologous recombination in example 2 (see FIG. 4 for schematic integration diagram), positive clones were detected by PCR, and the resulting yeast strains were named individuallyY91、Y92、Y93、Y94、Y95、Y96、Y97、Y98. The information on the construction of the integration strain is shown in Table 4, and the integration primers are shown in Table 5. Wherein Hyg represents hygromycin B and plays a role in screening.

TABLE 4 Glycyrrhetinic acid-producing strains

TABLE 5 Glycyrrhetinic acid-producing Strain primers (SEQ ID NO: 72-79)

Wherein, with P _adh1 -Mut72A63-T _adh1 When the primer is used as a template, the upstream primer and the downstream primer are used together with P _adh1 -Uni25647-T _adh1 The same applies to the template.

Gene expression clusterTEF1p-SQE-PGK1t、Pthd3-GuCPR1-Ttdh3、Ppgk1-β-As-Tadh1For primers and templates used for integration see table 3.

Taxonomy ID of the yeast genome 559292.pEA6-HphMX6 was purchased from AddGene, addgene: pFA6a-A1-FLAG: hphMX6.

Example 4 fermentation and product testing

The SQE catalytic step in the yeast cell is a key branch point for synthesizing a triterpene compound, and the introduction of the SQE gene with high catalytic activity greatly promotes the metabolic flux of squalene to glycyrrhetinic acid. A schematic diagram of the metabolic pathway for producing terpenoids using squalene in yeast as a substrate is shown in FIG. 1. A schematic diagram of the metabolic pathway for the production of glycyrrhetinic acid in yeast using 2, 3-oxidosqualene as a substrate is shown in FIG. 2.

In this example, SQE gene with high catalytic activity was discovered by shake flask fermentation and product detection.

The specific fermentation conditions were as follows:

(1) Strain activation: marking the target strain constructed in the embodiment on a corresponding screening marker flat plate, standing in a constant temperature incubator at 30 ℃ for culturing for 4 to 6 days, selecting a single colony, putting the single colony in a 50 ml centrifuge tube filled with 5ml of defective culture medium, culturing at 30 ℃ and 220 rpm until OD reaches 0.8-1.2, and taking the single colony as seed liquid for subsequent activation; inoculating the seed solution in the tube into a 100ml triangular flask filled with 20 ml of defective culture medium, wherein the inoculation amount is 2-5%, and the seed solution is cultured at 30 ℃ and 220 rpm until the OD reaches 0.8-1.2; the strain was activated 3 times.

(2) Fermenting the strain: the activated strain was inoculated at 5% inoculum size into a 100mL flask containing 40mL of a culture medium (YPD medium: 20 g/L peptone, 10 g/L yeast powder, 20 g/L glucose), 2% glucose content, 3 per strain in parallel, fermented at 30 ℃ and 220 rpm for 7 days, treated with a bacterial solution, and tested on a computer.

Example 5 treatment and detection methods for recombinant Saccharomyces cerevisiae fermentation product

(1) Sample treatment: 1mL of recombinant saccharomyces cerevisiae engineering strain (bacterial liquid obtained after fermentation in example 4) is taken, centrifuged at 12000rmp for 10min and the supernatant is discarded, sterilized water is added for cleaning for 3 times, centrifuged at 12000rmp for 10min and the supernatant is discarded, 0.5g of glass beads and 1mL of ethyl acetate are added, shaking is carried out for 15min, ultrasonic treatment is carried out for 30min, centrifuged at 12000rmp for 10min, the supernatant is collected, and 100 mu L of recombinant saccharomyces cerevisiae engineering strain is taken after a filter membrane is added into a lining tube.

(2) Sample detection: and analyzing and identifying the ethyl acetate extraction product of the saccharomyces cerevisiae by using a gas chromatography-mass spectrometer (GC-MS). The chromatograph is Agilent gas chromatography-mass spectrometer GCMS-7000, the chromatographic column is SE-30 (0.25 μm × 0.25m × 30 m), and the flow rate of carrier gas helium is 1.5 mL/min ^-1 (ii) a The temperature of a sample inlet is 300 ℃, split-flow sample injection is not carried out, and the sample injection amount is 1 mu L; the column box was programmed to a starting temperature of 80 ℃ for 1min and then at 20 ℃ min ^-1 Heating to 280 deg.C, maintaining for 15min, and cooling at 20 deg.C/min ^-1 Heating to 300 deg.C, and maintaining for 5min; the mass spectrum scanning range m/z is 40-550, and the injection volume is 2 mu L. And (3) data acquisition mode: TIC mode. The metabolite ion pairs are shown in table 6.

TABLE 6 GCMS detection conditions for the substances

The results of the fermentation production of 11-oxo- β -resinol by the strains transformed with squalene epoxidase genes from different sources are shown in FIG. 5; the results of squalene production by fermentation of strains transformed with squalene epoxidase genes from different sources are shown in FIG. 6.

OD of each recombinant yeast solution obtained 7 days after fermentation in example 4 was measured ₆₀₀ The results are shown in FIG. 7.

The results of the fermentation of 11-oxo- β -amyrin producing yeast are shown in fig. 5 to 7, from which it is known that: after 4 SQE gene expression clusters AiSQE (SEQ ID NO: 1), guSQE1 (SEQ ID NO: 4), pgSQE (SEQ ID NO: 6) and TwSQE (SEQ ID NO: 7) are integrated with chromosomes of other 4 gene clusters (Uni 25647, guCPR1, beta-As), squalene can be better promoted to flow to 11-oxo-beta-balsamic alcohol metabolic flux; wherein the 11-oxo-beta-balsamic alcohol yield in the yeast strain Y81 of the gene cluster AiSQE (SEQ ID NO: 1) is the highest and is improved by 5.8 times compared with the blank (Y41), the intermediate metabolite squalene is reduced by 5.7 times, and the strain is selected as the underplate cell of the subsequent glycyrrhetinic acid-producing Saccharomyces cerevisiae.

The results of fermentation production of glycyrrhetinic acid by strains transformed with squalene epoxidase genes from different sources are shown in FIG. 8, from which it can be seen that: the 4 SQE gene expression clusters AiSQE (SEQ ID NO: 1), guSQE1 (SEQ ID NO: 4), pgSQE (SEQ ID NO: 6), twSQE (SEQ ID NO: 7) of the present invention were combined with the other 4 gene clusters (Uni 25647, guCPR1,Mut72A63beta-As) can better promote squalene to flow to glycyrrhetinic acid metabolic flux after chromosome integration; wherein the glycyrrhetinic acid yield of the yeast strain Y91 of the gene cluster AiSQE (SEQ ID NO: 1) is the highest and is 5.8 times higher than that of the blank yeast strain Y81.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

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<120> squalene epoxidase and coding gene and application thereof

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ggagattcgt cgcagagcaa gagtgcgtgc gtgaagactg ccttcgatgc tgttaatgga 180

gaatgcagat ccggtggttt cgccgatgct gacgtcatcg tcgtcggagc tggcgtcgct 240

ggtgccgctc tcgctcacac tctcggcaag gatggacgtc gagtgtgcat gattgaaaga 300

gatttgtcag agcctgaccg aattgttggt gaattgctgc aaccaggagg ctacctcaaa 360

ttgattgagc taggacttga agattgtgtg gagaaaattg atgcacaacg ggttttcggt 420

tatgcacttt tcatgaatgg aagaaatacc cgactatcat atccattgga gaagtttcac 480

tcagatgttg ctgggaggag ctttcataat ggacgtttca tacagaggct gcgggagaaa 540

gctgcttccc ttccaaatgt acgattggag caaggaacag taacttccct gattgaagaa 600

aaagggactg ttaaaggtgt gcaatacaag actaaagctg gcgaagaact gactgcatat 660

gctcctttga caattgtgtg tgatggctgt ttttcaaact tgcgtcgctc actttgcaac 720

cctaaggtag aggtgccctc ctgttttgtt ggtctggtcc tagaaaattg caatcttcca 780

tttgcaaatc atgggcatgt tatactagca gacccttccc ccattttgtg ttatcccatc 840

agtagcaatg aggttcggtg tctggttgat gtacctggtc aaaaggttcc ttccatttcc 900

aacggtgaaa tggcaaacta tttgaagact gtcgtggctc ctcagattcc acccgaaatc 960

tatgattcct ttgtagctgc agttgacaaa ggaaatatta gaacaatgcc aaatagaagc 1020

atgccagctt ctccttatcc cactcctgga gctcttttga tgggggatgc gttcaacatg 1080

cgccatccat taactggagg aggaatgact gttgcactgt ctgatattgt tattctgcgc 1140

aatcttctta ggcctttgcg taacctgaat aatgcaccag ctctctgcaa ataccttgaa 1200

tccttttata ccttgcgtaa gcctgtgtca tcgactatca atacattggc tggtgccttg 1260

taccaggtgt tttctgcttc ccctgatgaa gcaaggaagg aaatgcgtga ggcttgcttt 1320

gactatctaa gtcttggagg tatttgctcg tcaggaccag tctctctgct ctcgggtttg 1380

aatcctcgcc cattaagctt ggttcttcat ttctttgctg tcgcagtata tggtgttggc 1440

cgattagtac tgccatatcc ttcaccttat cgcatctgga ttggagctag aataatttca 1500

agtgcatcag gaatcatctt ccccatcatg aacgcggaag ggttaaggca aatgttcttc 1560

cctgcaactg ttcctgctta ttacagagct cctcctgtta aatctagttg a 1611

<210> 2

<211> 2324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaaaccat tcgtaatcag gaaccttcca agatttcaat caactcttag atcttcgctt 60

ctctacacaa atcatcgccc ctcttctcga ttctctctct ctactcgtcg attcaccacc 120

ggagccacct acattcgccg atggaaagca acggcggcgc agacactcaa actctccgcc 180

gtgaactcca cggtgatgat gaaaccggcg aagattgcgt tggatcaatt tatagcttct 240

ctatttacgt ttctgcttct ctacattttg cgtcggagta gtaataagaa taagaagaat 300

cgtggactcg tcgtttccca aaacgatacc gtatccaaaa atcttgaaac ggaggttgat 360

tctggcactg atgtgatcat cgtcggagct ggtgtcgccg gttccgctct tgctcatact 420

ctcggcaagg caagtctata atctcaattt ttgatttttg catttctcca aatttattca 480

tgaaaattta tttttctaaa ttcatttcaa taaattttaa catttatttt gtaatataat 540

ttgtttttga ttatattttt tgtaataaga tcacaaaaat gatgtgatat tagttcccgg 600

aacattggag atatatactt gtctgatttt tgtgtgttgg agacgtaagt tcttgtctga 660

aattttggcc tttgtgtggt tattgaagga aggaagaaga gtgcacgtta tagaaagaga 720

tttttctgag caagacagaa tcgttggtga attgcttcaa cctggtggtt atttgaagtt 780

aatagaactt ggacttgaag gtagagacta actaatgagc ttaaactgtt atatatatat 840

atacttggaa tcgatttttg ttctatagtt gcgttttgtg attgtagatt gtgtgaagaa 900

gattgatgct caacgagttc ttggttatgt tctctttaaa gatgggaagc atactaaact 960

tgcttacccc ttggaaacgt ttgattcgga tgtagccggg agaagtttcc ataatgggag 1020

atttgtacag agaatgcgag aaaaagccct tactctttca aagtaatctt tatattgatt 1080

gtttctttaa ttgaatccag cttgactcta acggtttgtt tgtgaatcag tgtacgattg 1140

gaacaaggaa cggttacgtc gttgcttgaa gaacacggga caattaaagg ggttcgatac 1200

agaacaaaag agggcaatga gtttagatca tttgctcctc tcacaattgt atgtgatggt 1260

tgtttctcca acttgcgtcg ctctctttgc aaacctaagg tggatgtgcc atctactttt 1320

gtgggtcttg tcttggagaa ctgtgaactt ccatttgcaa atcacgggca cgttgttctc 1380

ggtgacccat cacccatctt aatgtatccc atcagcagtt ctgaagtccg ttgcttagta 1440

gatgtaccgg gtcaaaaact tcctcccatt gcaaatggtg aaatggcaaa gtatctgaaa 1500

acacgggttg cgcctcaagt accaaccaag gtccgtgaag cattcatcac cgctgttgag 1560

aaaggtaata tcagaaccat gccaaaccga agcatgccag ctgatccgat tcctactcct 1620

ggagctcttc ttcttggtga tgcattcaac atgagacatc ctttaaccgg tggtgggatg 1680

accgttgcat tggcggatat agttgtactc cgtgatcttc taaggccaat tcgcaacctt 1740

aatgacaaag aagctttgtc taagtatatt gaatcctttt acacactacg aaaagtaagc 1800

atctctttag ttaaaagtaa acgcttcttt atgttttgat tagtggactt atgtgtcgcc 1860

ttgcaacagc ctgtagcttc caccattaat acattggcgg atgcgttgta taaggtcttt 1920

ttagcatctt cagatgaagc aagaacggaa atgcgtgaag cttgcttcga ctatcttagc 1980

cttggaggtg ttttctcatc tggtccagtt gcattgctct ctggtttaaa ccctcgtcct 2040

ctgagtttag ttctccactt ctttgctgtg gcgatctacg ctgtttgtcg tttaatgcta 2100

ccatttcctt cgattgagag cttttggctt ggagctagga taatctcggt atgtggctct 2160

tcccattgtg gtatatacac aacatcgcaa agaagtttga tctttgatat ttttcttttt 2220

cacagagtgc ttcaagcatc atctttccaa taattaaagc agagggagtt agacaaatgt 2280

tcttccctcg tacaatccct gccatatacc gtgctcctcc ttaa 2324

<210> 3

<211> 1491

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat tacctacgat 60

gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct agcaagaaag 120

ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat tgttggtgaa 180

ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca atctatcaac 240

aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga acaagttgat 300

attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga cttggtcaaa 360

gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta cgaagatgat 420

gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt gagaaacatt 480

actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat attgaaggat 540

gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa ggtggaattc 600

aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa ggaattgcac 660

ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt caatgctaag 720

aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc aatcttggtt 780

taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc aaaggtccca 840

gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa gagtctacgt 900

ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa ctcctacttg 960

ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct aaatatgaga 1020

catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt gttgattaag 1080

aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt actagactac 1140

catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc tttgtattct 1200

ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt caaatatttc 1260

caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt cttgccaaag 1320

cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta cttgaacatg 1380

gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat gattttgatc 1440

acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta a 1491

<210> 4

<211> 1575

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggtggatc cgtycgttct cggttggatt ctaagctccg ttctgagcct ctttgctctg 60

tacagtttgg ttttcgccgg aaagggaagt cgtgcttcac cggagaaggc gactcagttc 120

gaggatagcg taactaccac cgctggagaa tgcagatcgg aaaaacttaa cggcgacgct 180

gacatcatca ttgtcggagc cggtgttgcc ggctccgctc tggctcacac tctcggcaag 240

gagggacgtc gagtgcttgt catcgaaaga gatctgagtg agccagaccg aattgttgga 300

gagctgctac aacctggggg ctatctcaaa ttaattgaac tgggacttga agattgtgtg 360

gagaatattg atgctcagca agtgtttggt tatgctcttt tcaaggatgg gaaacatact 420

cgtctctctt atcccttgga gaagtttcac tcagatgtct ctggcagaag ctttcacaat 480

gggcgtttta ttcagaggat gcgggagaaa gctgcctccc ttcccaatgt gcgactggag 540

caagggacag taacttccct acttgaagag aagggaacaa tcaaaggtgt gcaatacaag 600

accaaagatg gtcaggaatc gacatcatat gctcctctta ccattgtttg tgatggctgt 660

ttctcaaact tacggcgttc tctttgtaat cctaaggtag atattccctc ttgttttgtt 720

ggcttagttt tagagaactg tgaacttcct tgtgccaatc atggccacgt catactcgga 780

gatccttcac cagttctgtt ctatcctata agtagcacag aggttcgttg tctggtcgat 840

gtacctggtc agaaggttcc ttctatttca aatggtgata tggcaaagta tttgaagaac 900

acggtggctc cgcaggtacc ccctgagctt tatgatgcct tcatagccgc agtggacaag 960

ggcaacataa ggacaatgcc aaacagaagc atgccagcag atcctcatcc tacgcctgga 1020

gcccttctga tgggagatgc attcaacatg cggcatccac taacaggggg tggaatgact 1080

gtagcattgt ctgatattgt ggtgctgagg aatcttctca ggcctttgcg tgacctgaac 1140

gatgcaccca cactttgcaa ataccttgaa tccttttata ccttgcgtaa gcctgtggca 1200

gccactataa atacgttggc aggagccctt tacagggttt tttgtgcytc ccctgatcaa 1260

gctaggaagg aaatgcggca agcttgcttc gattatctga gccttggagg cctattctcg 1320

gaaggaccag tctctttact rtcaggatta aaccctcgcc ccttgagctt ggttctccat 1380

ttctttgctg ttgcagtatt tggtgttggc cgtttactat taccatttcc ttcacctaag 1440

cggatatgga tgggagcccg attactctct ggtgcatctg caatcatctt gcctatagtt 1500

aaggctgaag gaatccgcca gatgtttttc cctgccactg ttccagctta ttacagagcc 1560

cccccggctt cataa 1575

<210> 5

<211> 1101

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagagaga aggcttcctc ccttcccaat gtacgattgg agcaaggaac agtgacttcc 60

ctacttgaag agaaggggac aattaaaggt gtgcagtaca aaacgaaaga tgctcaagaa 120

ttatcagcat atggtcctct taccattgtt tgtgatggct gtttctcaaa cttgcgccgt 180

tctctttgta atcctaaggt agatattccc tcttgttttg ttggcttagt tttggaaaat 240

tgtgaacttc catgtgcaaa tcatggccac gtcatactgg gagatccttc gccagttctg 300

ttctatccca taagtagcac agaaattcgc tgtctggtgg atgttcctgg tcagaaagtt 360

ccatctattt ccaatggtga aatggcgaag tatttgaaaa cagtggtagc tccacaggtt 420

ccccctgagc tttatgatgc tttcatagct gcagtggaca aaggcaacat aaggacaatg 480

ccaaacagaa gcatgccagc agctccttat cccactcccg gagcccttct gatgggagat 540

gcattcaaca tgcgccatcc cctaactggg ggtggaatga ctgtggcatt atctgatata 600

gtagtgttgc gaaatcttct caggcctttg cgtgacctga atgatgcacc cagcctttgc 660

aaataccttg aatcctttta taccttgcgt aagcccgtgg catccaccat aaacactttg 720

gcaggagcac tttacaaggt tttttgtgca tcacctgatc ctgcgagaaa ggaaatgcgt 780

caagcttgct tcgattatct gagtcttgga ggtcaattct cggaagggcc agtctctttg 840

ctttcaggat taaaccctcg gcccctgagc ttggttcttc atttctttgc cgttgcaata 900

tatggcgttg gccgcttact gctaccattt ccttccccta aacgcttatg gattggagtc 960

cgattaatca ctgtaagttg gaatgttttt gccttcattg ttatttttgt cggaataaat 1020

tgccgggaaa cccattttaa ttgcaaaaac caaactcata ttttgacaga gtgcatccgg 1080

aatcatcttg cccataatta a 1101

<210> 6

<211> 1620

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaattcat cttcttctac tactactact gatacgttgc attcttttat ggaagctagc 60

gctctgctca ttgatcaata ttttcttgga tggatctttg ctttcctttt tgggttcctg 120

ctcttgctta atttcaaaag gaagagagag aaaaataatt ccacggagtt tggaacagat 180

gatagcaacg gttactacac accggaaaat attgccggaa gtacggacgt catcatcgtc 240

ggcgccgggg ttgctggctc tgctcttgct tatacgcttg ccaacgatgg ccggagagtt 300

catgtaattg agagggactt aactgagcaa gacagaattg taggtgaact tctacaacca 360

ggaggctact tgaaattgat tgaattaggg ctagaggatt gtgtgaatga aatcgatgct 420

caacgagttt ttggatatgc cctttacatg gatggtaaaa acaccaggct ttcttacccc 480

ttggagaaat ttcattcgga tgtagctgga agaagctttc ataacgggcg ttttgttcaa 540

cgaatgaggg agaaagctgc atcacttcca aatgtaagaa tggaacaggg gacagttaca 600

tctctggttg agaaaaaggg aagtgtaaaa ggggtgcaat acaaaaccaa ggatggccaa 660

gaattgtctg catttgctcc acttacaatt gtttgtgatg gttgtttttc gaatctccgt 720

cgctccctct gcaatcccaa ggtggaggtg ccttcgtgtt ttgttggttt gattttggaa 780

aatattgatc ttccacatat aaaccatggc catgtcattc tagcagatcc ttctccaatc 840

ttgttttata aaattagtag taccgagatt cgctgtttgg ttgatgtgcc tggacaaaag 900

gtgccttgta tttctaatgg ggaattggct aattatctca agacagtagt agctcctcag 960

gttccaaaac agctatataa ctctttcata gcagcagttg acaaaggaaa cattagaacc 1020

atgccaaaca gaagcatgcc agccgatcct catccaactc cgggtgcact tttattaggg 1080

gatgctttca atatgcgcca tcctttaacc ggcgggggaa tgacagttgc tctgtccgat 1140

attgtcttga tccgggatct tcttagacct ttacgcgatc tccatgactc gtcaaccctc 1200

tgcaaatatc tcgaatcctt ttacaccctt cgtaagcccg tggcgtctac tataaataca 1260

ttggcaggtg ccctttataa agttttttgt gcatcacctg ataaagcaag gcaagaaatg 1320

cgcaatgctt gttttgatta tctgagcctc ggaggaattt gttctcaagg gccaattgct 1380

ttactttctg gcctaaaccc gcgtccaatt agcctatttc tccacttctt tgccgtggct 1440

atctatggcg ttggccgctt gttgattcct tttccttcac caaaacgaat gtggctcggc 1500

gctagattga ttttgggtgc atctggaatt attttcccta ttataaagtc agaaggactt 1560

cgacaaatgt tcttccctgc aattgttcct gcttactaca gagctccccc cattcactaa 1620

<210> 7

<211> 1583

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggagatgg aatatcagca cattttggaa ggaattctag cttttgtatt ggggtttgtt 60

ctgttttcca ttttcagagc aaagaagagg accaagattg atgttcatcg caagaaaact 120

tccatcaata acacccgtga aaatggcatg tggaggccgg aggtcggagg gacaaccacc 180

gatgtaatcg tcgttggtgc cggcgtcgct ggttctgctc ttgcttacac tcttgccaag 240

gaaggtcgca gagttcatgt gattgagagg gacttaaacg aacctgatcg tattgtcggt 300

gagctgctac aaccaggcgg atatttgaag ttaatcgaat tgggtcttga agattgtgtg 360

aatgaaatcg atgctcaacg cgtgtcggga tacgctctct acaaggatgg gaagagtact 420

aaactgccat atcccctgca aagtttcagt tctgatgtgg ctggaagaag ctttcacaac 480

ggtcgattca tccagagaat gagagaaaag gctgcatctt tgtccaatgt gagactagag 540

caaggcacag taacatctct gattgaagag agagggatta ttaaaggagt tgtatacaaa 600

tccaagactg gccaggagct gacagtatcc gctcccctta cgatagtctg cgatgggtgc 660

ttctcgaatt tacgacgatc tctctgccat cccaaggttg atgtcccttc ttgctttgtt 720

ggtttgattt tggagaactg tgaaatccca tttgcgaatc acggacatgt agttctggcc 780

gatccttctc ccatcttgtt ttaccagata agtagtactg agattcgttg tttggtcgac 840

atacctggcc agaaagtacc ttcagtttct aatggcgaaa tggctcatta tctgaaaact 900

atggtcgctc cccaggttcc gcgagaactt tacgatgcat ttttacgcgc aattgagaaa 960

ggaaacataa gaacaatgcc taacagaagc atgcctgcct caccctattc aactccaggt 1020

gcacttctga taggagacgc gttcaacatg cgccatcctc taacgggagg aggtatgacg 1080

gtggcactct ccgatatcgt tgtactgaga gaccttctaa gacctctaga tgatttaaac 1140

gattcagctt cactttgcaa gtatcttgag tccttctaca cattgcgcaa gcccgtggcg 1200

tcgacaataa acacacttgc aggtgctttg tacaaagtct tcagtgcaca cctgatccgg 1260

caagaaaaga aatgcgacaa gcctgtttcg actacttgag cctaggaggt gttttcacca 1320

acggaccaat atctctcctt tctggcctaa acccgcgtcc attaatcttg gtgctacatt 1380

tctttgcggt ggcaatctat ggcgttggac gcttatttct tccattccca tcgccatatc 1440

gagtgtggat cggtgccaga ctgatctcgg gtgcatcagg gatcattttt ccgataataa 1500

aagccgaagg agttagacaa atgttcttcc ctgcaacagt tccagcatat tacagagccc 1560

cacctgttca taaacaactc tga 1583

<210> 8

<211> 1575

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgatggacc cctacgcgct tggatgcatc atatgctccg tgctgagcct cgttgcgctc 60

tacaatctgg ttttcgtacg gaagaaatat cccgcttctt ccgctgccgc gactccgagc 120

acggagaaca tcacaaccgc cgccggagaa tgcagatcct tcaacsgcca cggcgacgtt 180

gacatcatca ttgttggagc aggtgtcgct ggctctgctc tcgctcacac tctcggcaag 240

gatggacgtc gagtacttgt cattgaaaga gatctgaatg aaccagaccg aattgttgga 300

gagttgctac aacctggagg ctatctcaag ttaattgagc tgggacttga agattgtgtc 360

gagaaaattg atgctcagca agtggttggt tatgctcttt tcaaggatgg gaaacacaca 420

cgactctctt atcccttgga aaagtttcac tcagatattg ccggcagaag ctttcacaat 480

gggcgtttca ttcagaggat gagagagaag gcttcctccc ttcccaatgt acgattggag 540

caaggaacag tgacttccct acttgaagag aaggggacaa ttaaaggtgt gcagtacaaa 600

acgaaagatg ctcaagaatt atcagcatat ggtcctctta ccatwgtttg tgatggctgt 660

ttctcaaact tgcgccgttc tctttgtaat cctaaggtag atgttccctc ttgttttgtt 720

ggcttagttt tggaaaattg tgaacttcca tgtgcaaatc atggccacgt catactggga 780

gatccttcmc cagttctgtt ctatcccata agtagcacag aaattcgctg tctggtggat 840

gttcctggtc agaaagttcc atctatttcc aatggtgaaa tggcgaagta tttgaaaaca 900

gtggtagctc cacaggttcc ccctgagctt tatgatgctt tcatagctgc agtggacaaa 960

ggcaacataa ggacaatgcc aaacagaagc atgccagcag ctccttatcc cactcccgga 1020

gcccttctga tgggagatgc attcaacatg cgccatcccc taactggggg tggaatgact 1080

gtggcattat ctgatatagt agtgttgcga aatcttctca ggcctttgcg tgacctgaat 1140

gatgcaccca gcctttgcaa ataccttgaa tccttttata ccttgcgtaa gcccgtggca 1200

tccaccataa acactttggc aggagcactt tacaaggttt tttgcgcatc acctgatcct 1260

gcgagaaagg aaatgcgtca agcttgcttc gattatctga gtcttggagg tcaattctcg 1320

gaagggccag tctctttgct ttcaggatta aaccctcggc ccctgagctt ggttcttcat 1380

ttctttgccg ttgcaatata tggcgttggc cgcttactgc taccatttcc ttcccctaaa 1440

cgcttgtgga ttggagtccg attaatcact agtgcatccg gaatcatctt gcccataatt 1500

aaggcagaag gagtccgtca gatgttcttc cctgcaacag ttccagccta ttacagaact 1560

cccccggctg catag 1575

<210> 9

<211> 536

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Asp Gln Tyr Thr Trp Gly Leu Ile Leu Gly Ala Val Leu Gly

1 5 10 15

Leu Val Ala Phe Tyr Asn Leu Gly Leu Leu Ala Val Tyr Asn Phe Val

20 25 30

Met Lys Asn Lys Gly Asp Ala Asp Gly Asp Ser Ser Gln Ser Lys Ser

35 40 45

Ala Cys Val Lys Thr Ala Phe Asp Ala Val Asn Gly Glu Cys Arg Ser

50 55 60

Gly Gly Phe Ala Asp Ala Asp Val Ile Val Val Gly Ala Gly Val Ala

65 70 75 80

Gly Ala Ala Leu Ala His Thr Leu Gly Lys Asp Gly Arg Arg Val Cys

85 90 95

Met Ile Glu Arg Asp Leu Ser Glu Pro Asp Arg Ile Val Gly Glu Leu

100 105 110

Leu Gln Pro Gly Gly Tyr Leu Lys Leu Ile Glu Leu Gly Leu Glu Asp

115 120 125

Cys Val Glu Lys Ile Asp Ala Gln Arg Val Phe Gly Tyr Ala Leu Phe

130 135 140

Met Asn Gly Arg Asn Thr Arg Leu Ser Tyr Pro Leu Glu Lys Phe His

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Tyr Lys Thr Lys Ala Gly Glu Glu Leu Thr Ala Tyr Ala Pro Leu Thr

210 215 220

Ile Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ser Leu Cys Asn

225 230 235 240

Pro Lys Val Glu Val Pro Ser Cys Phe Val Gly Leu Val Leu Glu Asn

245 250 255

Cys Asn Leu Pro Phe Ala Asn His Gly His Val Ile Leu Ala Asp Pro

260 265 270

Ser Pro Ile Leu Cys Tyr Pro Ile Ser Ser Asn Glu Val Arg Cys Leu

275 280 285

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

290 295 300

Ala Asn Tyr Leu Lys Thr Val Val Ala Pro Gln Ile Pro Pro Glu Ile

305 310 315 320

Tyr Asp Ser Phe Val Ala Ala Val Asp Lys Gly Asn Ile Arg Thr Met

325 330 335

Pro Asn Arg Ser Met Pro Ala Ser Pro Tyr Pro Thr Pro Gly Ala Leu

340 345 350

Leu Met Gly Asp Ala Phe Asn Met Arg His Pro Leu Thr Gly Gly Gly

355 360 365

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

370 375 380

Pro Leu Arg Asn Leu Asn Asn Ala Pro Ala Leu Cys Lys Tyr Leu Glu

385 390 395 400

Ser Phe Tyr Thr Leu Arg Lys Pro Val Ser Ser Thr Ile Asn Thr Leu

405 410 415

Ala Gly Ala Leu Tyr Gln Val Phe Ser Ala Ser Pro Asp Glu Ala Arg

420 425 430

Lys Glu Met Arg Glu Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly Ile

435 440 445

Cys Ser Ser Gly Pro Val Ser Leu Leu Ser Gly Leu Asn Pro Arg Pro

450 455 460

Leu Ser Leu Val Leu His Phe Phe Ala Val Ala Val Tyr Gly Val Gly

465 470 475 480

Arg Leu Val Leu Pro Tyr Pro Ser Pro Tyr Arg Ile Trp Ile Gly Ala

485 490 495

Arg Ile Ile Ser Ser Ala Ser Gly Ile Ile Phe Pro Ile Met Asn Ala

500 505 510

Glu Gly Leu Arg Gln Met Phe Phe Pro Ala Thr Val Pro Ala Tyr Tyr

515 520 525

Arg Ala Pro Pro Val Lys Ser Ser

530 535

<210> 10

<211> 527

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Glu Met Glu Tyr Gln His Ile Leu Glu Gly Ile Leu Ala Phe Val

1 5 10 15

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

20 25 30

Ile Asp Val His Arg Lys Lys Thr Ser Ile Asn Asn Thr Arg Glu Asn

35 40 45

Gly Met Trp Arg Pro Glu Val Gly Gly Thr Thr Thr Asp Val Ile Val

50 55 60

Val Gly Ala Gly Val Ala Gly Ser Ala Leu Ala Tyr Thr Leu Ala Lys

65 70 75 80

Glu Gly Arg Arg Val His Val Ile Glu Arg Asp Leu Asn Glu Pro Asp

85 90 95

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

100 105 110

Glu Leu Gly Leu Glu Asp Cys Val Asn Glu Ile Asp Ala Gln Arg Val

115 120 125

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

130 135 140

Pro Leu Gln Ser Phe Ser Ser Asp Val Ala Gly Arg Ser Phe His Asn

145 150 155 160

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

165 170 175

Val Arg Leu Glu Gln Gly Thr Val Thr Ser Leu Ile Glu Glu Arg Gly

180 185 190

Ile Ile Lys Gly Val Val Tyr Lys Ser Lys Thr Gly Gln Glu Leu Thr

195 200 205

Val Ser Ala Pro Leu Thr Ile Val Cys Asp Gly Cys Phe Ser Asn Leu

210 215 220

Arg Arg Ser Leu Cys His Pro Lys Val Asp Val Pro Ser Cys Phe Val

225 230 235 240

Gly Leu Ile Leu Glu Asn Cys Glu Ile Pro Phe Ala Asn His Gly His

245 250 255

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

260 265 270

Thr Glu Ile Arg Cys Leu Val Asp Ile Pro Gly Gln Lys Val Pro Ser

275 280 285

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

290 295 300

Gln Val Pro Arg Glu Leu Tyr Asp Ala Phe Leu Arg Ala Ile Glu Lys

305 310 315 320

Gly Asn Ile Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ser Pro Tyr

325 330 335

Ser Thr Pro Gly Ala Leu Leu Ile Gly Asp Ala Phe Asn Met Arg His

340 345 350

Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp Ile Val Val

355 360 365

Leu Arg Asp Leu Leu Arg Pro Leu Asp Asp Leu Asn Asp Ser Ala Ser

370 375 380

Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala

385 390 395 400

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

405 410 415

Ser Pro Asp Pro Ala Arg Lys Glu Met Arg Gln Ala Cys Phe Asp Tyr

420 425 430

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

435 440 445

Gly Leu Asn Pro Arg Pro Leu Ile Leu Val Leu His Phe Phe Ala Val

450 455 460

Ala Ile Tyr Gly Val Gly Arg Leu Phe Leu Pro Phe Pro Ser Pro Tyr

465 470 475 480

Arg Val Trp Ile Gly Ala Arg Leu Ile Ser Gly Ala Ser Gly Ile Ile

485 490 495

Phe Pro Ile Ile Lys Ala Glu Gly Val Arg Gln Met Phe Phe Pro Ala

500 505 510

Thr Val Pro Ala Tyr Tyr Arg Ala Pro Pro Val His Lys Gln Leu

515 520 525

<210> 11

<211> 764

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Glu Val Phe Met Phe Pro Thr Gly Thr Thr Val Ile Ile Ser Val

1 5 10 15

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

20 25 30

Trp Leu Lys Pro Lys Arg Phe Glu Lys Leu Leu Lys Ala Gln Gly Phe

35 40 45

Gln Gly Glu Pro Tyr Asn Leu Ser Val Leu Lys Asp Lys Ser Lys Gln

50 55 60

Asn Tyr Met Leu Lys Leu Gln Gln Glu Asp Lys Ser Lys Ser Ile Gly

65 70 75 80

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

85 90 95

Val Arg Lys Tyr Gly Asn Asn Ser Phe Leu Trp Glu Gly Thr Thr Pro

100 105 110

Arg Val Ile Ile Thr Asp Pro Asp Gln Ile Lys Asp Val Phe Asn Lys

115 120 125

Ile Asp Asp Phe Pro Lys Pro Lys Leu Arg Ser Ile Ala Lys Tyr Leu

130 135 140

Ser Val Gly Ile Leu Asp His Glu Gly Lys Lys Trp Ala Lys His Arg

145 150 155 160

Lys Ile Ala Asn Pro Ala Phe His Leu Glu Lys Leu Lys Val Met Leu

165 170 175

Pro Ala Phe Ser His Ser Cys Asn Glu Met Ile Ser Lys Trp Lys Glu

180 185 190

Leu Leu Ser Ser Asp Gly Thr Cys Glu Ile Asp Val Trp Pro Ser Leu

195 200 205

Gln Asn Phe Thr Cys Asp Val Ile Ser Arg Thr Ala Phe Gly Ser Ser

210 215 220

Tyr Ala Glu Gly Thr Lys Leu Phe Gln Leu Leu Lys Lys Gln Gly Phe

225 230 235 240

Leu Leu Met Thr Gly Arg His Thr Asn Asn Pro Leu Trp Gly Leu Leu

245 250 255

Ala Thr Thr Thr Lys Thr Lys Met Lys Glu Ile Asp Arg Glu Ile His

260 265 270

Asp Ser Leu Glu Gly Ile Ile Glu Lys Arg Glu Lys Ala Leu Lys Asn

275 280 285

Gly Glu Thr Thr Asn Asp Asp Leu Leu Gly Ile Leu Leu Leu Met Thr

290 295 300

Gly Arg His Thr Asn Asn Pro Leu Trp Gly Leu Leu Ala Thr Thr Thr

305 310 315 320

Lys Thr Lys Met Lys Glu Ile Asp Arg Glu Ile His Asp Ser Leu Glu

325 330 335

Gly Ile Ile Glu Lys Arg Glu Lys Ala Leu Lys Asn Gly Glu Thr Thr

340 345 350

Asn Asp Asp Leu Leu Gly Ile Leu Leu Gln Ser Asn His Ala Glu Lys

355 360 365

Gln Gly Gln Gly Asn Ser Lys Asn Ile Gly Met Thr Thr Gln Asp Val

370 375 380

Ile Asp Glu Cys Lys Leu Phe Tyr Leu Ala Gly Gln Glu Thr Thr Ser

385 390 395 400

Ser Leu Leu Val Trp Thr Met Val Leu Leu Gly Arg Tyr Pro Glu Trp

405 410 415

Gln Ala Arg Ala Leu Gln Ser Asn His Ala Glu Lys Gln Gly Gln Gly

420 425 430

Asn Ser Lys Asn Ile Gly Met Thr Thr Gln Asp Val Ile Asp Glu Cys

435 440 445

Lys Leu Phe Tyr Leu Ala Gly Gln Glu Thr Thr Ser Ser Leu Leu Val

450 455 460

Trp Thr Met Val Leu Leu Gly Arg Tyr Pro Glu Trp Gln Ala Arg Ala

465 470 475 480

Arg Glu Glu Val Leu Gln Val Phe Gly Asn Gln Asn Pro Asn Asn Glu

485 490 495

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

500 505 510

Arg Leu Phe Pro Pro Leu Ile Tyr Phe Asn Arg Ala Leu Arg Lys Asp

515 520 525

Leu Lys Leu Gly Asn Leu Leu Leu Pro Glu Gly Thr Arg Glu Glu Val

530 535 540

Leu Gln Val Phe Gly Asn Gln Asn Pro Asn Asn Glu Gly Leu Ser Gln

545 550 555 560

Leu Lys Ile Val Thr Met Ile Leu Tyr Glu Val Leu Arg Leu Phe Pro

565 570 575

Pro Leu Ile Tyr Phe Asn Arg Ala Leu Arg Lys Asp Leu Lys Leu Gly

580 585 590

Asn Leu Leu Leu Pro Glu Gly Thr Gln Ile Ser Leu Pro Ile Leu Leu

595 600 605

Ile His Gln Asp His Asp Leu Trp Gly Asp Asp Ala Lys Glu Phe Lys

610 615 620

Pro Glu Arg Phe Ala Glu Gly Ile Ala Lys Ala Thr Lys Gly Gln Val

625 630 635 640

Ser Tyr Phe Pro Phe Gly Trp Gly Pro Arg Ile Cys Leu Gly Gln Asn

645 650 655

Phe Ala Leu Leu Gln Ile Ser Leu Pro Ile Leu Leu Ile His Gln Asp

660 665 670

His Asp Leu Trp Gly Asp Asp Ala Lys Glu Phe Lys Pro Glu Arg Phe

675 680 685

Ala Glu Gly Ile Ala Lys Ala Thr Lys Gly Gln Val Ser Tyr Phe Pro

690 695 700

Phe Gly Trp Gly Pro Arg Ile Cys Leu Gly Gln Asn Phe Ala Leu Leu

705 710 715 720

Glu Ala Lys Ile Ala Val Ser Leu Leu Leu Gln Asn Phe Ser Phe Glu

725 730 735

Leu Ser Pro Asn Tyr Val His Val Pro Thr Thr Val Leu Thr Leu Gln

740 745 750

Pro Lys Asn Gly Ala Ser Ile Ile Leu His Lys Leu

755 760

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggagatgg aatatcagca c 21

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcagagttgt ttatgaacag gt 22

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgaaaccat tcgtaatcag g 21

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ttaaggagga gcacggtata tg 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgtctgctg ttaacgttgc ac 22

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttaaccaatc aactcaccaa ac 22

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgaattcat cttcttctac t 21

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ttagtgaatg gggggagctc tgtag 25

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atggcggatc agtacacgtg gg 22

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcaactagat ttaacaggag gag 23

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggtggatc cgtycgttct c 21

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ttatgaagcc gggggggctc tg 22

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atgatggacc cctacgcgct tg 22

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctatgcagcc gggggagttc tg 22

<210> 26

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

atgagagaga aggcttcctc c 21

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ttaattatgg gcaagatgat tc 22

<210> 28

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gttttaatta caaaatggag atggaatatc ag 32

<210> 29

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

caattcaatt caattcagag ttgtttatga acag 34

<210> 30

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cataaacaac tctgaattga attgaattga aatcga 36

<210> 31

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

cataaacaac tctgaattga attgaattga aatcga 36

<210> 32

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gttttaatta caaaatgaat tcatcttctt ctacta 36

<210> 33

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

caattcaatt caatttagtg aatgggggga gctctgtag 39

<210> 34

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

tccccccatt cactaaattg aattgaattg aaatcga 37

<210> 35

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gaagaagatg aattcatttt gtaattaaaa cttagattag 40

<210> 36

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gttttaatta caaaatgaga gagaaggctt cctcc 35

<210> 37

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

caattcaatt caattcaatt aattatgggc aagatgattc 40

<210> 38

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

catcttgccc ataattaaat tgaattgaat tgaaatcga 39

<210> 39

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ggaagccttc tctctcattt tgtaattaaa acttagatta g 41

<210> 40

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gttttaatta caaaatggcg gatcagtaca cgtg 34

<210> 41

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

caattcaatt caattcaact agatttaaca ggaggag 37

<210> 42

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ctgttaaatc tagttgaatt gaattgaatt gaaatcga 38

<210> 43

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gtactgatcc gccattttgt aattaaaact tagattag 38

<210> 44

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

gttttaatta caaaatggtg gatccgtycg ttctc 35

<210> 45

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

caattcaatt caatttatga agccgggggg gctct 35

<210> 46

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

cccggcttca taaattgaat tgaattgaaa tcga 34

<210> 47

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

acggatccac cattttgtaa ttaaaactta gattag 36

<210> 48

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gttttaatta caaaatgatg gacccctacg cgc 33

<210> 49

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

caattcaatt caatctatgc agccggggga gttc 34

<210> 50

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

cccccggctg catagattga attgaattga aatcga 36

<210> 51

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

taggggtcca tcattttgta attaaaactt agattag 37

<210> 52

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

gttttaatta caaaatggat gaaaccattc gtaatcagga 40

<210> 53

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

caattcaatt caatttatgt ataccgtgct cctccttaa 39

<210> 54

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

tataccgtgc tcctccttaa attgaattga attgaaatcg a 41

<210> 55

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

ctgattacga atggtttcat attttgtaat taaaacttag attag 45

<210> 56

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ttgggtgcct ctatgatggg tat 23

<210> 57

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

aatcgtatgt gaatgctggt cgctatactg ggaaagaacg ggaaaccaac tatcgaga 58

<210> 58

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

caaggagggt attctgggcc tccatgtcag cacaaacccc atacatcggg attcctat 58

<210> 59

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

aatctcgata gttggtttcc cgttctttcc cagtatagcg accagcattc acatacga 58

<210> 60

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

gtcaaggagg gtattctggg cctccatgtc agcacaaacc ccatacatcg ggattcct 58

<210> 61

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

ttaaagttta caaatgaatt ttttccgcca ggagttgtcc tctgaggaca taaaata 57

<210> 62

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

ctcggtgtgt attttatgtc ctcagaggac aacaagaata cgtaaataat taatagtag 59

<210> 63

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

ctcggtgtgt attttatgtc ctcagaggac aactcctggc ggaaaaaatt catttgta 58

<210> 64

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

cagtatagtg tattcttcct gctccaagct agcacaaacc ccatacatcg ggattcct 58

<210> 65

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

gaaaatcact actattaatt atttacgtat tcttgttgtc ctctgaggac ataaaatac 59

<210> 66

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

ctgtgcgtct tgagttgaag tcaggaatct tcaatagtca tacaacagaa agcgacca 58

<210> 67

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

ataggaatcc cgatgtatgg ggtttgtgct agcttggagc aggaagaata cactatac 58

<210> 68

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

ggtggtcgct ttctgttgta tgactattga agattcctga cttcaactca agacgcac 58

<210> 69

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

gctgcactgg gggccaagca cagggcaaga gttgtcctct gaggacataa aataca 56

<210> 70

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

ggtgtgtatt ttatgtcctc agaggacaac tcttgccctg tgcttggccc ccagtgca 58

<210> 71

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

ccattcatcg tgttgttttg gcct 24

<210> 72

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

ccaggcgcct ttatatcata taatc 25

<210> 73

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

tgcgtcaatc gtatgtgaat gctggtcgct atactgtttg cgaaacccta tgctctgtt 59

<210> 74

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

ttcaagtccc aacaacagag catagggttt cgcaaacagt atagcgacca gcattcac 58

<210> 75

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

ggtattatag gaatcccgat gtatggggtt tgtgctgaca tggaggccca gaataccc 58

<210> 76

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

aagactgtca aggagggtat tctgggcctc catgtcagca caaaccccat acatcggga 59

<210> 77

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

cttagtgctt gtatatgctc atcccgacct tccattgttg tcctctgagg acataaaat 59

<210> 78

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

aatctcggtg tgtattttat gtcctcagag gacaacaatg gaaggtcggg atgagcat 58

<210> 79

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

ataaagcagc cgctaccaaa cag 23

Claims (9)

1. The application of squalene epoxidase or its coding gene, or biological material containing its coding gene in biological fermentation synthesis of 11-oxo-beta-resinol or triterpene compound; the triterpene compound is glycyrrhetinic acid, glycyrrhizic acid, ginsenoside or mogroside;

the amino acid sequence of the squalene epoxidase is shown in SEQ ID No:9 or SEQ ID No: shown at 10.

2. The use of claim 1, wherein the nucleic acid sequence of the gene encoding squalene epoxidase is as set forth in SEQ ID No:1 or SEQ ID No:7 is shown in the specification;

and/or, the biological material is an expression cassette, a vector, or a host cell.

3. Use according to any of claims 1-2, wherein the strain used in the biological fermentation is capable of producing squalene.

4. The use according to claim 3, wherein the strain used in the biological fermentation is a yeast which, on the basis of overexpression of the gene coding for squalene epoxidase, also co-expressesUni25647Gene, gene,GuCPR1Genes andβ-Asa gene.

5. The use according to claim 4, wherein the yeast further co-expresses the gene encoding squalene epoxidase when glycyrrhetinic acid is produced by biofermentationUni25647Gene、 GuCPR1Gene、Mut72A63Genes andβ-Asa gene.

6. A squalene epoxidase, the amino acid sequence of which is shown in SEQ ID No: shown at 9.

7. A gene for coding squalene epoxidase, wherein the nucleic acid sequence is shown in SEQ ID No:1 is shown.

8. A method for improving the yield of glycyrrhetinic acid synthesized by biological fermentation is characterized in that the yield of glycyrrhetinic acid synthesized by a fermentation strain is improved by over-expressing a gene of squalene epoxidase in the fermentation strain; the gene of the squalene epoxidase is shown as SEQ ID No:1 or SEQ ID No: shown at 7.

9. The method of claim 8, wherein the fermentation strain is a squalene-producing yeast, and the GAL80 site of the yeast chromosome is overexpressed as shown in SEQ ID No:1, and co-expressingUni25647Gene, gene,GuCPR1Genes andβ-Asa gene; and further overexpresses the YPRC locus of the yeast chromosome, such as the YPRC locus shown in SEQ ID No:1 or SEQ ID No:7, and co-expressingUni25647The gene,GuCPR1The gene,Mut72A63Andβ-Asa gene.

Images