CN106566815B

CN106566815B - A kind of saccharomyces cerevisiae engineered yeast and construction method producing enoxolone or its precursor

Info

Publication number: CN106566815B
Application number: CN201610965315.9A
Authority: CN
Inventors: 李春; 朱明�; 王彩霞; 孙文涛; 周安琪
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2019-11-08
Anticipated expiration: 2036-11-04
Also published as: CN106566815A

Abstract

The present invention discloses a kind of production enoxolone or its precursor: 11- hydroxy-beta-amyrin, or 11- oxygen-β-amyrin, or 30- hydroxy-beta-amyrin, or 11, 30- hydroxy-beta-amyrin, or 30- hydroxyl -11- oxygen-β-amyrin, or 30- aldehyde radical -11- oxygen-β-amyrin saccharomyces cerevisiae engineered yeast and its construction method, its production stage is to construct β-amyrin synthase expression box respectively, the Oxidase Expression box of 11, β-amyrin carbon, the Oxidase Expression box that 11- oxygen-β-amyrin is 30, carbon, cytochrome P450 reductase expression cassette, and it imports in saccharomyces cerevisiae genome jointly.The present invention couples the synthesis of enoxolone with the growth of yeast, realizes enoxolone or the yeast synthesis of its precursor, this method do not need the induction of effector agent, simple process, can be used for fermenting and producing enoxolone and its key precursor object.

Description

A kind of saccharomyces cerevisiae engineered yeast and construction method producing enoxolone or its precursor

Technical field

The present invention relates to a kind of building of saccharomyces cerevisiae engineered yeast and fermenting and producing enoxolones or its precursor: 11- hydroxyl Base-β-amyrin (11-hydroxy- β-amyrin) or 11- oxygen-β-amyrin (11-oxo- β-amyrin) or 30- Hydroxy-beta-amyrin (30-hydroxy- β-amyrin) or 11,30- hydroxy-beta-amyrin (11,30-hydroxy- β- ) or 30- hydroxyl -11- oxygen-β-amyrin (30-hydroxy-11-oxo- β-amyrin) or 30- aldehyde radical -11- amyrin The method of oxygen-β-amyrin (Glycyrrhetaldehyde), belongs to bioengineering field.

Background technique

Terpene substances are one kind of Secondary metabolites, have diversified bioactivity and pharmacology value.Medicine Verified triterpene compound such as glycyrrhizic acid (Glycyrrhizin, GL) of science is in liver protection, anti-inflammatory, antitumor, antiviral Etc. have very important effect, while also can be used as sweetener or food additives.Enoxolone (Glycyrrhetinic acid, GA) is the precursor substance of glycyrrhizic acid synthesis, and compared with glycyrrhizic acid, enoxolone lacks 2 molecules Glucuronic acid base be easier to penetrating cell film so polarity is weaker, and remain with the consistent physiological activity of glycyrrhizic acid with Function, it is easier to play its bioactivity and function in the cell.In liquorice root, the content of enoxolone is extremely low, mainly Exist in the form of glycyrrhizic acid.Plant extract there are period length, low efficiency, it is at high cost, environmental disruption is big the disadvantages of.Enoxolone is multiple Miscellaneous molecular structure, harsh synthesis condition also make its chemical synthesis be difficult to carry out.With the development of synthetic biology, utilization is micro- There is biosynthesis enoxolone etc. the compound of great pharmaceutical value to have become a kind of development trend.Microbe synthesis has week The features such as phase is short, and yield is high, mild condition, process control, therefore the dependence to plant resources can be effectively relieved, to have Higher economic benefit and social value.

In saccharomyces cerevisiae engineered yeast, enoxolone or its precursor 11- hydroxy-beta-amyrin or 11- oxygen-β-perfume Resinol or 30- hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl -11- oxygen-β-botany bar gum Alcohol or the synthesis of 30- aldehyde radical -11- oxygen-β-amyrin are that squalene is synthesized through mevalonate pathway, then aoxidize shark through 2,3- Alkene monooxygenase, β-amyrin synthase are catalyzed to form β-amyrin.This research is on this basis, further to excavate discovery β- The synthesis of amyrin to enoxolone is common by two oxidase C YP88D6 and CYP72A154 (or CYP72A63) sequences Catalysis is completed, and does not find both enzymes in microorganism, therefore, it is necessary to two enzymes in introduced plant Radix Glycyrrhizae in Saccharomyces cerevisiae It is inside expressed, to realize the biosynthesis of enoxolone.

Summary of the invention

The object of the present invention is to provide a kind of 11, carbon CYP450 oxidase genes of oxidation of beta-amyrin.

A second object of the present invention is to provide a kind of 11, carbon CYP450 oxidase genes of oxidation of beta-amyrin The protein of coding.

Third object of the present invention is to provide a kind of cytochrome P450 reductase genes.

Fourth object of the present invention is to provide a kind of protein of cytochrome P450 reductase gene coding.

Fifth object of the present invention is to provide a kind of production enoxolone or its precursor 11- hydroxy-beta-amyrin, Or 11- oxygen-β-amyrin or 30- hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl -11- The construction method of oxygen-β-amyrin or 30- aldehyde radical -11- oxygen-β-amyrin saccharomycete.

It is set sixth object of the present invention is to provide a kind of using production enoxolone or its precursor 11- hydroxy-beta-perfume Lipidol or 11- oxygen-β-amyrin or 30- hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl The wine brewing ferment of the construction method of base -11- oxygen-β-amyrin or 30- aldehyde radical -11- oxygen-β-amyrin saccharomycete building Female engineering bacteria.

Technical solution of the present invention is summarized as follows:

A kind of 11, carbon CYP450 oxidase genes of oxidation of beta-amyrin, it is SEQ ID No.5 in sequence table The nucleotide sequence.

A kind of protein of 11, the carbon CYP450 oxidase genes coding of oxidation of beta-amyrin, it is sequence table Amino acid sequence described in SEQ ID No.6.

A kind of cytochrome P450 reductase gene, it is nucleotides sequence described in SEQ ID No.11 in sequence table Column.

A kind of protein of cytochrome P450 reductase gene coding, it is described in sequence table SEQ ID No.12 Amino acid sequence.

A kind of production enoxolone or its precursor 11- hydroxy-beta-amyrin or 11- oxygen-β-amyrin or 30- Hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl -11- oxygen-β-amyrin or 30- aldehyde radical - The construction method of 11- oxygen-β-amyrin saccharomyces cerevisiae engineered yeast, includes the following steps:

(1) β-amyrin synthase expression box building:

By the method for left homology arm, Yeast promoter, β-amyrin synthase, yeast terminator application Overlap extension PCR Connection, obtains β-amyrin synthase expression cassette；

(2) building of 11, oxidation of beta-amyrin carbon CYP450 oxidizing ferment (I) expression cassette:

By Yeast promoter, the CYP450 oxidizing ferment (I) of 11, oxidation of beta-amyrin carbon, yeast terminator application weight The method connection of folded extension PCR, obtains the expression cassette of the CYP450 oxidizing ferment of 11, oxidation of beta-amyrin carbon.

(3) building of 11- oxygen-β-amyrin 30, carbon CYP450 oxidizing ferment (II) expression cassette is aoxidized:

By Yeast promoter, the CYP450 oxidizing ferment (II) of oxidation 11- oxygen-β-amyrin 30, carbon, yeast termination Son is connected using the method for Overlap extension PCR, obtains the CYP450 oxidizing ferment of oxidation 11- oxygen-β-amyrin 30, carbon Expression cassette.

(4) building of cytochrome P450 reductase expression cassette

The method of Yeast promoter, cytochrome P450 reductase, yeast terminator application Overlap extension PCR is connected It connects, obtains the expression cassette of cytochrome P450 reductase.

(5) building of selection markers expression cassette

By yeast terminator T_PGK1, Geneticin expression cassette gene P_TEF1-kanMX-T_TEF1, right homology arm HOR application overlapping The method of extension PCR is connected with this, obtains selection markers expression cassette.

(6) CYP450 of above-mentioned β-amyrin synthase expression box and/or 11, oxidation of beta-amyrin carbon is aoxidized Enzyme (I) expression cassette, and/or CYP450 oxidizing ferment (II) expression cassette of oxidation 11- oxygen-β-amyrin 30, carbon, and/or it is thin Born of the same parents' cytochrome p 450 oxidoreducing enzyme expression cassette is inserted into saccharomyces cerevisiae INVSc1 or W303a or W303 ɑ or BJ2168, or In BY4742 or BY4700 or CEN.PK2-1C or CEN.PK2-1D genomic integration site, obtain production enoxolone or Its precursor: 11- hydroxy-beta-amyrin or 11- oxygen-β-amyrin or 30- hydroxy-beta-amyrin or 11,30- Hydroxy-beta-amyrin or 30- hydroxyl -11- oxygen-β-amyrin or 30- aldehyde radical -11- oxygen-β-amyrin wine brewing ferment Female engineering bacteria.

The Yeast promoter is GPDp and/or PGK1p and/or TDH1p and/or TEF1p and/or ADH1p, and/ Or PGI1p and/or TDH2p and/or HXT2p and/or HXT7p and/or TEF2p and/or PYK1p and/or ENO2p, and/ Or PDC1p and/or FBA1p and/or GPM1p and/or TPI1p and/or GAL7p and/or GAL1p and/or GAL10p.

The yeast terminator is TDH3t and/or PGK1t and/or TDH1t and/or TEF1t and/or ADH1t, and/ Or PGI1t and/or TDH2t and/or HXT2t and/or HXT7t and/or TEF2t and/or PYK1t and/or ENO2t, and/ Or PDC1t and/or FBA1t and/or GPM1 and/or CYC1t and/or TPI1t.

The genomic integration site is the site HO and/or the site YPRCtau3 and/or the site URA3 and/or TRP1 Site and/or the site LEU2 and/or the site HIS3 and/or the site delta and/or the site rDNA.

The β-amyrin synthase nucleotide sequence Genbank registration sequence number is AB037203.

CYP450 oxidizing ferment (I) gene of 11, carbon of the oxidation of beta-amyrin is SEQ ID No.16 in sequence table The nucleotide sequence or CYP450 oxidase C YP88D6, nucleotide sequence Genbank registration sequence number are AB433179。

CYP450 oxidizing ferment (II) gene of oxidation 11- oxygen-β-amyrin 30, carbon is CYP450 oxidizing ferment CYP72A154, nucleotide sequence Genbank registration sequence number are AB558153 and/or CYP450 oxidase C YP72A63, Its nucleotide sequence Genbank registration sequence number is AB558146.

The cytochrome P450 reductase is nucleotide sequence described in SEQ ID No.10 in sequence table, or thin Born of the same parents cytochrome p 450 oxidoreducing enzyme LjCPR1, nucleotide sequence Genbank registration sequence number are AB433810 and/or nucleosides Acid sequence Genbank registration sequence number is the cytochrome P450 reductase AtCPR1 and/or protein of BT008426.1 Sequence Genbank registration sequence number is the cytochrome P450 reductase AtCPR2 of NP_001328167.1.

A kind of above-mentioned production enoxolone or its precursor: 11- hydroxy-beta-amyrin or 11- oxygen-β-amyrin, Or 30- hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl -11- oxygen-β-amyrin or 30- The saccharomyces cerevisiae engineered yeast of the construction method building of aldehyde radical -11- oxygen-β-amyrin saccharomycete.

The present invention is exactly the Radix Glycyrrhizae using metabolic engineering and synthetic biology technology introduced plant source in saccharomyces cerevisiae body The route of synthesis of hypo acid realizes the Microbe synthesis of enoxolone and its key precursor object.Entire production process is convenient, fast It is prompt, pollution-free, there is boundless application prospect.The saccharomyces cerevisiae of production enoxolone and its key precursor object of the invention Engineering bacteria provides a kind of practicable method for the heterologous production of enoxolone and its key precursor object.

Detailed description of the invention

Fig. 1 saccharomyces cerevisiae engineered yeast fermenting and producing 11- hydroxy-beta-amyrin (11-hydroxy- β-amyrin) group Analysis total ion chromatogram.

Fig. 2 saccharomyces cerevisiae engineered yeast fermenting and producing 11- hydroxy-beta-amyrin (11-hydroxy- β-amyrin) matter Spectrogram.

The component analysis of Fig. 3 saccharomyces cerevisiae engineered yeast fermenting and producing 11- oxygen-β-amyrin (11-oxo- β-amyrin) Total ion chromatogram.

Fig. 4 saccharomyces cerevisiae engineered yeast fermenting and producing 11- oxygen-β-amyrin (11-oxo- β-amyrin) mass spectrogram.

Fig. 5 saccharomyces cerevisiae engineered yeast fermenting and producing 30- hydroxy-beta-amyrin (30-hydroxy- β-amyrin) group Analysis total ion chromatogram.

Fig. 6 saccharomyces cerevisiae engineered yeast fermenting and producing 30- hydroxy-beta-amyrin (30-hydroxy- β-amyrin) matter Spectrogram.

Fig. 7 saccharomyces cerevisiae engineered yeast fermenting and producing 11,30- hydroxy-beta-amyrin (11,30-hydroxy- β- Amyrin component analysis total ion chromatogram).

Fig. 8 saccharomyces cerevisiae engineered yeast fermenting and producing 11,30- hydroxy-beta-amyrin (11,30-hydroxy- β- Amyrin mass spectrogram).

Fig. 9 saccharomyces cerevisiae engineered yeast fermenting and producing 30- hydroxyl -11- oxygen-β-amyrin (30-hydroxy-11-oxo- β-amyrin) component analysis total ion chromatogram.

Figure 10 saccharomyces cerevisiae engineered yeast fermenting and producing 30- hydroxyl -11- oxygen-β-amyrin (30-hydroxy-11-oxo- β-amyrin) mass spectrogram.

Figure 11 saccharomyces cerevisiae engineered yeast fermenting and producing 30- aldehyde radical -11- oxygen-β-amyrin (Glycyrrhetaldehyde) component analysis total ion chromatogram.

Figure 12 saccharomyces cerevisiae engineered yeast fermenting and producing 30- aldehyde radical -11- oxygen-β-amyrin (Glycyrrhetaldehyde) mass spectrogram.

The component analysis total ion chromatogram of Figure 13 saccharomyces cerevisiae engineered yeast fermenting and producing enoxolone.

The mass spectrogram of Figure 14 saccharomyces cerevisiae engineered yeast fermenting and producing enoxolone.

Enoxolone synthesis path figure in Figure 15 saccharomyces cerevisiae.

Specific embodiment

Below by the preferred embodiment of synthesis enoxolone and its key precursor object and this hair is illustrated in conjunction with attached drawing Bright various aspects and feature.It should be appreciated by those skilled in the art the purpose of, specific experiment example simply to illustrate that, without It is to limit the scope of the invention.Under conditions of without departing substantially from Claims scope, those skilled in the art can be for this The various aspects of invention are modified, but these modifications also belong to protection scope of the present invention.For example, by institute in this experimental example The promoter or terminator used replaces with other promoters or terminator commonly used in the art, is the other technologies people of this field Member is it will be appreciated that with realization.

In addition, it should be noted that unless otherwise indicated, a variety of materials and reagent employed in example are equal in detail below For material commonly used in the art and reagent, can be obtained by normal commercial sources；Used method is art technology Conventional method well known to personnel.

Experimental example 1: the acquisition of related gene in enoxolone route of synthesis

A, β-amyrin synthase gene acquisition

It is excellent by codon according to β-amyrin synthase gene (Genbank registration sequence number is AB037203) sequence Change, make β-amyrin synthase gene codon that there is yeast Preference, the optimization gene sequence of generation is SEQ ID No.1。

B, the acquisition of 11, oxidation of beta-amyrin carbon CYP450 oxidase genes

According to CYP450 oxidase C YP88D6 gene (Genbank registration sequence number is AB433179) sequence, pass through password Son optimization, makes the codon of CYP88D6 gene have yeast Preference, and the optimization gene sequence of generation is SEQ ID No.2.

According to CYP450 oxidase C YP88D6 gene (Genbank registration sequence number be AB433179) primers, SEQ ID No.3:5 '-ATGGAAGTACATTGGGTTTGC-3 ' and SEQ ID No.4:5 '- CTAAGCACATGAGACCTTCATCACC-3 ' carries out PCR amplification using ExTaq enzyme, obtains using Glycyrrhiza Uralensis cDNA as template To 1482bp segment, shown in SEQ ID No.5, to be cloned into pMD18-T carrier, sequencing is proved and reported CYP88D6 phase Than the variation that 23 bases have occurred, 11 variations are had occurred in amino acid.The amino acid sequence of fragment expression shown in SEQ ID No.5 Column are shown in SEQ ID No.6.

C, the acquisition of 11- oxygen-β-amyrin 30, carbon CYP450 oxidase genes is aoxidized

According to CYP450 oxidase C YP72A154 gene (Genbank registration sequence number is AB558153) sequence, by close Numeral optimization, makes the codon of CYP72A154 gene have yeast Preference, and the optimization gene sequence of generation is SEQ ID No.7。

According to CYP450 oxidase C YP72A63 gene (Genbank registration sequence number is AB558146) sequence, by close Numeral optimization, makes the codon of CYP72A63 gene have yeast Preference, and the optimization gene sequence of generation is SEQ ID No.8。

D, the acquisition of cytochrome P450 reductase gene

It is screened according to Radix Glycyrrhizae transcript profile database, obtains cytochrome P450 reductase gene, design primer, SEQ ID No.9:5 '-ATGACTTCGAATTCCGATTTGG-3 ' and SEQ ID No.10:5 '- TCACCAGACATCCCTGAGGTAACG-3 ' carries out PCR amplification using ExTaq enzyme, obtains using Glycyrrhiza Uralensis cDNA as template It is sequenced shown in SEQ ID No.11, to be cloned into pMD18-T carrier to 2076bp segment and determines its complete sequence.SEQ ID The amino acid sequence of fragment expression shown in No.11 is shown in SEQ ID No.12.

E, the acquisition of riddled basins

According to Geneticin (G418) expression cassette sequence in pRS42K carrier, design primer SEQ ID No.13:5 '- GACATGGAGGCCCAGAATACCC-3 ' and SEQ ID No.14:5 '-TCGACACTGGATGGCGGCGT-3 ', with pRS42K matter Grain is template, carries out PCR amplification using ExTaq enzyme, 1394bp segment is obtained, to be cloned into shown in SEQ ID No.15 PMD18-T carrier, sequencing prove that it does not mutate.

F, the acquisition of genome homology arm

According to the left homology arm HOL in the site HO in saccharomyces cerevisiae genome (SEQ ID No.16) and right homology arm sequence HOR (SEQ ID No.17), design primer SEQ ID No.18:5 '-AATTATCCTGGGCACGAGTGAAACAAAGC-3 ' and SEQ ID No.19:5 '-ACGCCATTTTAAGTCCAAAGGCACAATTTTAC-3 ' (as shown in SEQ ID No.53), amplification HO are left Homology arm HOL；Primer SEQ ID No.20:5 '-CTGGGGGAACAACTTCACAG-3 ' and SEQ ID No.21:5 '- ACTGTAAGATTCCGCCACATTTTATAC-3 ' expands right homology arm HOR and uses ExTaq using INVSc1 genome as template Enzyme carries out PCR amplification, is cloned into pMD18-T carrier, and sequencing proves that it does not mutate.

Experimental example 2: the building of expression cassette

A, β-amyrin synthase expression box building

By left homology arm HOL, Yeast promoter P_FBA1, β-amyrin synthase gene, yeast terminator T_CYC1Using overlapping The method of extension PCR is connected with this, obtains β-amyrin synthase expression box P_FBA1-bAS-T_CYC1

B, the building of 11- oxygen-β-amyrin 30, carbon CYP450 Oxidase Expression boxes is aoxidized

By yeast terminator T_CYC1, Yeast promoter P_GPD, CYP450 oxidase gene CYP72A154 or CYP72A63, ferment Female terminator T_ADH1It is connected using the method for Overlap extension PCR with this, obtains oxidation 11- oxygen-β-amyrin 30, carbon CYP450 oxidizing ferment T_CYC1-P_GPD–CYP72A154-T_ADH1Or T_CYC1-P_GPD-CYP72A63-T_ADH1

C, the building of 11, oxidation of beta-amyrin carbon CYP450 Oxidase Expression boxes

By yeast terminator T_ADH1, Yeast promoter P_ENO2, 11, oxidation of beta-amyrin carbon CYP450 oxidizing ferment base Because of UNI47 or CYP450 oxidase gene CYP88D6, yeast terminator T_TYS1It is connected using the method for Overlap extension PCR with this, Obtain β-amyrin synthase expression box T_ADH1-P_ENO2-UNI47-T_TYS1Or T_ADH1-P_ENO2-CYP88D6-T_TYS1

D, the building of cytochrome P450 reductase expression cassette

By yeast terminator T_TYS1, Yeast promoter P_PGK1, cytochrome P450 reductase gene C PR, yeast termination Sub- T_PGK1It is connected using the method for Overlap extension PCR with this, obtains cytochrome P450 reductase expression cassette T_TYS1-P_PGK1- CPR-T_PGK1

E, the building of selection markers expression cassette

By yeast terminator T_PGK1, Geneticin expression cassette gene P_TEF1-kanMX-T_TEF1, right homology arm HOR application overlapping The method of extension PCR is connected with this, obtains selection markers expression cassette T_PGK1–P_TEF1-kanMX-T_TEF1-HOR

Experimental example 3: the acquisition of the saccharomyces cerevisiae engineered yeast of enoxolone is produced

The yeast conversion of segment is carried out using Li-acetate method

Using the homologous recombination function of yeast itself, the above-mentioned expression cassette built is all transferred to saccharomyces cerevisiae jointly The site HO of INVSc1 genome.It is screened, is obtained using the YPD solid plate of addition Geneticin (G418) after conversion Transformant is transferred in the YPD fluid nutrient medium of addition Geneticin (G418) and carries out culture 1-2 days, extracts genome, PCR mirror Determine positive colony, plate streaking or glycerol stock save.

Has production there are the synzyme of enoxolone, acquisition on the basis of MVA approach, is exogenously introduced using yeast itself The saccharomyces cerevisiae engineered yeast strain of enoxolone function.

Experimental example 4: the acquisition of the saccharomyces cerevisiae engineered yeast of production enoxolone and its key precursor object

The culture of seed liquor: being inoculated into 2ml YPD culture medium, 30 DEG C from picking positive colony in screening flat board, 200rpm shaken cultivation 18 hours.

Fermenting experiment: being inoculated into 100ml YPD culture medium, 30 DEG C according to 1% inoculum concentration (absorbance about 0.1), 200rpm shaken cultivation 120 hours, sampling in every 24 hours was primary, measured containing for enoxolone in cell and its key precursor object Amount, the results show that saccharomyces cerevisiae engineered yeast ferments, final glycyrrhetic acid content is 1.515mg/L, precursor (11- hydroxy-beta- Amyrin content is that 6.9mg/L, 11- oxygen-β-amyrin content are that 23mg/L, 30- hydroxy-beta-amyrin content are 25mg/L, 11,30- hydroxy-beta-amyrin content are that 15mg/L, 30- hydroxyl -11- oxygen-β-amyrin content are 40mg/ L, 30- aldehyde radical -11- oxygen-β-amyrin content is 2.8mg/L) there is different yield.

Experimental example 5: the identification of saccharomyces cerevisiae engineered yeast tunning

Tunning 8000rpm is centrifuged 5 minutes, supernatant is removed, precipitating is resuspended with 1.5ml saturated sodium-chloride, and pearl is broken broken 20 minutes, isometric ethyl acetate extraction, rotary evaporation was transferred in gas phase bottle, adds 500 μ l N, O- to powdered Bis- (trimethyl silicon substrate) three fluoro acetamides and 500 μ l pyridines, 80 DEG C are reacted 30 minutes, are cooled to room temperature.Utilize Shimadzu GC2010QP gas chromatography-mass spectrometry analyzes extract.Temperature program: 80 DEG C are kept for 1 minute, with 20 DEG C/min 290 DEG C are risen to, is kept for 38 minutes；250 DEG C of injector temperature, split ratio 5:1,1 μ l of sample introduction, chromatographic mass spectrometry interface temperature is 290 ℃.The results show that total ion chromatogram and mass spectrogram combination define the characteristic peak of enoxolone, illustrate saccharomyces cerevisiae engineered yeast Enoxolone and its precursor: 11- hydroxy-beta-amyrin or 11- oxygen-β-amyrin or 30- can successfully be synthesized Hydroxy-beta-amyrin or 11,30- hydroxy-beta-amyrin or 30- hydroxyl -11- oxygen-β-amyrin or 30- aldehyde radical - 11- oxygen-β-amyrin.

Experiment shows using TDH1p and/or TEF1p and/or ADH1p and/or PGI1p and/or TDH2p, and/or HXT2p and/or HXT7p and/or TEF2p and/or PYK1p and/or PDC1p and/or GPM1p and/or TPI1p, and/or FBA1p or GPD or ENO2p or PGK1p in GAL7p and/or GAL1p and/or GAL10p replacement experimental example 2, can obtain Obtain corresponding expression cassette.

Using TDH3t and/or TDH1t and/or TEF1t and/or PGI1t and/or TDH2t and/or HXT2t, and/ Or HXT7t and/or TEF2t and/or PYK1t and/or ENO2t and/or PDC1t and/or FBA1t and/or GPM1, and/ Or PGK1t or ADH1t or CYC1t or TYS1t in TPI1t replacement experimental example 2, corresponding expression cassette can be obtained.

Utilize Wine brewing yeast strain W303a or W303 ɑ or BJ2168 or BY4742 or BY4700 or CEN.PK2- 1C or CEN.PK2-1D can substitute the INVSc1 bacterial strain in experimental example 3.

With the following site YPRCtau3 and/or URA3 and/or TRP1 and/or LEU2 and/or HIS3 of genome, And/or the site delta and/or rDNA can substitute the site HO in experimental example 3.

SEQUENCE LISTING

<110>Beijing Institute of Technology

<120>a kind of saccharomyces cerevisiae engineered yeast and construction method for producing enoxolone or its precursor

<130> 1

<160> 21

<170> PatentIn version 3.5

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atgtggagat tgaagatcgc tgaaggtggt aaggacccat acatctactc tactaacaac 60

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caagttgacg ctgctagatt gcacttctac aacaacagat tccaagttaa gccatgtggt 180

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gttaagatcg gtgacggtga agaaatcact tacgaaaagg ctactactgc tgttagaaga 300

gctgctcacc acttgtctgc tttgcaaact tctgacggtc actggccagc tcaaatcgct 360

ggtccattgt tcttcttgcc accattggtt ttctgtatgt acatcactgg tcacttggac 420

tctgttttcc cagaagaata cagaaaggaa atcttgagat acatctacta ccaccaaaac 480

gaagacggtg gttggggttt gcacatcgaa ggtcactcta ctatgttctg tactgctttg 540

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gaattctgga tcttgccatc tttcttgcca atgcacccag ctaagatgtg gtgttactgt 780

agattggttt acatgccaat gtcttacttg tacggtaaga gattcgttgg tccaatcact 840

ccattgatct tgcaattgag agaagaattg ttcactgaac catacgaaaa ggttaactgg 900

aagaaggcta gacaccaatg tgctaaggaa gacttgtact acccacaccc attgttgcaa 960

gacttgatct gggactcttt gtacttgttc actgaaccat tgttgactag atggccattc 1020

aacaagttgg ttagagaaaa ggctttgcaa gttactatga agcacatcca ctacgaagac 1080

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tgggttgaag acccaaacgg tgacgctttc aagaagcact tggctagagt tccagactac 1200

ttgtgggttt ctgaagacgg tatgactatg caatctttcg gttctcaaga atgggacgct 1260

ggtttcgctg ttcaagcttt gttggctact aacttggttg aagaaatcgc tccaactttg 1320

gctaagggtc acgacttcat caagaagtct caagttagag acaacccatc tggtgacttc 1380

aagtctatgt acagacacat ctctaagggt tcttggactt tctctgacca agaccacggt 1440

tggcaagttt ctgactgtac tgctgaaggt ttgaagtgtt gtttgttgtt gtctatgttg 1500

ccaccagaaa tcgttggtga aaagatggaa ccagaaagat tgtacgactc tgttaacgtt 1560

ttgttgtctt tgcaatctaa gaagggtggt ttgtctgctt gggaaccagc tggtgctcaa 1620

gaatggttgg aattgttgaa cccaactgaa ttcttcgctg acatcgttgt tgaacacgaa 1680

tacgttgaat gtactggttc tgctatccaa gctttggttt tgttcaagaa gttgtaccca 1740

ggtcacagaa agaaggaaat cgaaaacttc atcgctaacg ctgttagatt cttggaagac 1800

actcaaactg ctgacggttc ttggtacggt aactggggtg tttgtttcac ttacggttct 1860

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agaaaggctg ttaagttctt gttgactact caaagagaag acggtggttg gggtgaatct 1980

tacttgtctt ctccaaagaa gatctacgtt ccattggaag gttctagatc taacgttgtt 2040

cacactgctt gggctttgat gggtttgatc cacgctggtc aagctgaaag agacccagct 2100

ccattgcaca gagctgctaa gttgatcatc aactctcaat tggaagaagg tgactggcca 2160

caacaagaaa tcactggtgt tttcatgaag aactgtatgt tgcactaccc aatgtacaga 2220

gacatctacc caatgtgggc tttggctgaa tacagaagaa gagttccatt gccatctact 2280

ccagtttgtt tgacttaa 2298

<210> 2

<211> 1482

<212> DNA

<213>artificial sequence

<400> 2

atggaagttc actgggtttg tatgtctgct gctactttgt tggtttgtta catcttcggt 60

tctaagttcg ttagaaactt gaacggttgg tactacgacg ttaagttgag aagaaaggaa 120

cacccattgc caccaggtga catgggttgg ccattgatcg gtgacttgtt gtctttcatc 180

aaggacttct cttctggtca cccagactct ttcatcaaca acttggtttt gaagtacggt 240

agatctggta tctacaagac tcacttgttc ggtaacccat ctatcatcgt ttgtgaacca 300

caaatgtgta gaagagtttt gactgacgac gttaacttca agttgggtta cccaaagtct 360

atcaaggaat tggctagatg tagaccaatg atcgacgttt ctaacgctga acacagattg 420

ttcagaagat tgatcacttc tccaatcgtt ggtcacaagg ctttggctat gtacttggaa 480

agattggaag aaatcgttat caactctttg gaagaattgt cttctatgaa gcacccagtt 540

gaattgttga aggaaatgaa gaaggtttct ttcaaggcta tcgttcacgt tttcatgggt 600

tcttctaacc aagacatcat caagaagatc ggttcttctt tcactgactt gtacaacggt 660

atgttctcta tcccaatcaa cgttccaggt ttcactttcc acaaggcttt ggaagctaga 720

aagaagttgg ctaagatcgt tcaaccagtt gttgacgaaa gaagattgat gatcgaaaac 780

ggtccacaag aaggttctca aagaaaggac ttgatcgaca tcttgttgga agttaaggac 840

gaaaacggta gaaagttgga agacgaagac atctctgact tgttgatcgg tttgttgttc 900

gctggtcacg aatctactgc tacttctttg atgtggtcta tcacttactt gactcaacac 960

ccacacatct tgaagaaggc taaggaagaa caagaagaaa tcactagaac tagattctct 1020

tctcaaaagc aattgtcttt gaaggaaatc aagcaaatgg tttacttgtc tcaagttatc 1080

gacgaaactt tgagatgtgc taacatcgct ttcgctactt tcagagaagc tactgctgac 1140

gttaacatca acggttacat catcccaaag ggttggagag ttttgatctg ggctagagct 1200

atccacatgg actctgaata ctacccaaac ccagaagaat tcaacccatc tagatgggac 1260

gactacaacg ctaaggctgg tactttcttg ccattcggtg ctggttctag attgtgtcca 1320

ggtgctgact tggctaagtt ggaaatctct atcttcttgc actacttctt gagaaactac 1380

agattggaaa gaatcaaccc agaatgtcac gttacttctt tgccagtttc taagccaact 1440

gacaactgtt tggctaaggt tatcaaggtt tcttgtgctt aa 1482

<210> 3

<211> 21

<212> DNA

<213>artificial sequence

<400> 3

atggaagtac attgggtttg c 21

<210> 4

<211> 25

<212> DNA

<213>artificial sequence

<400> 4

ctaagcacat gagaccttca tcacc 25

<210> 5

<211> 1482

<212> DNA

<213>Radix Glycyrrhizae (Glycyrrhiza uralensis)

<400> 5

atggaagtac attgggtttg catgtgcgct gccactttgt tggtatgcta catttttgga 60

agcaagtttg tgaggaattt gaatgggtgg tattatgatg taaaactaag aaggaaagaa 120

cacccactac ccccaggtga catgggatgg cctcttatgg gcaatctatt gtccttcatc 180

aaagatttct catcgggtca ccctgattca ttcatcaaca accttgttct caaatatgga 240

cgaagtggta tctacaagac tcacttgttt gggaatccaa gcatcattgt ttgcgagcct 300

cagatgtgta ggcgagttct cactgatgat gtgaacttta agcttggtta tccaaaatct 360

atcaaagagt tggcacgatg tagacccatg attgatgtct ctaatgcgga acataggctt 420

tttcgacgcc tcattacttc cccaatcgtg ggtcacaagg cgctagcaat gtacctagaa 480

cgtcttgagg aaattgtgat caattcgttg gaagaattgt ccagcatgaa gcaccccgtt 540

gagctcttga aagagatgaa gaaggtttcc tttaaagcca ttgtccacgt tttcatgggc 600

tcttccaatc aggacatcat taaaaaaatt ggaagttcgt ttactgattt gtacaatggc 660

atgttctcta tccccattaa cgtacctggt tttacattcc acaaagcact cgaggcacgt 720

aagaagctag ccaaaatagt tcaacccgtt gtggatgaaa ggcggttgat gatagaaaat 780

ggtcaacaag aaggggacca aagaaaagat cttattgata ttcttttgga agtcaaagat 840

gagaatggac gaaaattgga ggacgaggat attagcgatt tattaatagg gcttttgttt 900

gctggccatg aaagtacagc aaccagttta atgtggtcaa ttacatatct tacacagcat 960

ccccatatct tgaaaaaggc taaggaagag caggaagaaa taatgaggac aagattgtcc 1020

tcgcagaaac aattaagttt taaggaaatt aaacaaatgg tttatctttc tcaggtaatt 1080

gatgaaactt tacgatgtgc caatattgcc tttgcaactt ttcgagaggc aactgctgat 1140

gtgaacatca atggttatat cataccaaag ggatggagag tgctaatttg ggcaagagcc 1200

attcatatgg attctgaata ttacccaaat ccagaagaat ttaatccatc gagatgggat 1260

gattacaatg ccaaagcagg aaccttcctt ccttttggag caggaagtag actttgtcct 1320

ggagccgact tggcgaaact tgaaatttcc atatttcttc attatttcct ccttaattac 1380

aggttggagc gagtaaatcc agaatgtcat gttaccagct taccagtatc taagcccaca 1440

gacaattgcc tcgctaaggt gatgaaggtc tcatgtgctt ag 1482

<210> 6

<211> 493

<212> PRT

<213>artificial sequence

<400> 6

Met Glu Val His Trp Val Cys Met Cys Ala Ala Thr Leu Leu Val Cys

1 5 10 15

Tyr Ile Phe Gly Ser Lys Phe Val Arg Asn Leu Asn Gly Trp Tyr Tyr

20 25 30

Asp Val Lys Leu Arg Arg Lys Glu His Pro Leu Pro Pro Gly Asp Met

35 40 45

Gly Trp Pro Leu Met Gly Asn Leu Leu Ser Phe Ile Lys Asp Phe Ser

50 55 60

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

65 70 75 80

Arg Ser Gly Ile Tyr Lys Thr His Leu Phe Gly Asn Pro Ser Ile Ile

85 90 95

Val Cys Glu Pro Gln Met Cys Arg Arg Val Leu Thr Asp Asp Val Asn

100 105 110

Phe Lys Leu Gly Tyr Pro Lys Ser Ile Lys Glu Leu Ala Arg Cys Arg

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Lys His Pro Val Glu Leu Leu Lys Glu Met Lys Lys Val Ser Phe Lys

180 185 190

Ala Ile Val His Val Phe Met Gly Ser Ser Asn Gln Asp Ile Ile Lys

195 200 205

Lys Ile Gly Ser Ser Phe Thr Asp Leu Tyr Asn Gly Met Phe Ser Ile

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Asp Ile Leu Leu Glu Val Lys Asp Glu Asn Gly Arg Lys Leu Glu Asp

275 280 285

Glu Asp Ile Ser Asp Leu Leu Ile Gly Leu Leu Phe Ala Gly His Glu

290 295 300

Ser Thr Ala Thr Ser Leu Met Trp Ser Ile Thr Tyr Leu Thr Gln His

305 310 315 320

Pro His Ile Leu Lys Lys Ala Lys Glu Glu Gln Glu Glu Ile Met Arg

325 330 335

Thr Arg Leu Ser Ser Gln Lys Gln Leu Ser Phe Lys Glu Ile Lys Gln

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ile His Met Asp Ser Glu Tyr Tyr Pro Asn Pro Glu Glu Phe Asn Pro

405 410 415

Ser Arg Trp Asp Asp Tyr Asn Ala Lys Ala Gly Thr Phe Leu Pro Phe

420 425 430

Gly Ala Gly Ser Arg Leu Cys Pro Gly Ala Asp Leu Ala Lys Leu Glu

435 440 445

Ile Ser Ile Phe Leu His Tyr Phe Leu Leu Asn Tyr Arg Leu Glu Arg

450 455 460

Val Asn Pro Glu Cys His Val Thr Ser Leu Pro Val Ser Lys Pro Thr

465 470 475 480

Asp Asn Cys Leu Ala Lys Val Met Lys Val Ser Cys Ala

485 490

<210> 7

<211> 1572

<212> DNA

<213>artificial sequence

<400> 7

atggacgctt cttctactcc aggtgctatc tgggttgttt tgactgttat cttggctgct 60

atcccaatct gggtttgtca catggttaac actttgtggt tgagaccaaa gagattggaa 120

agacacttga gagctcaagg tttgcacggt gacccataca agttgtcttt ggacaactct 180

aagcaaactt acatgttgaa gttgcaacaa gaagctcaat ctaagtctat cggtttgtct 240

aaggacgacg ctgctccaag aatcttctct ttggctcacc aaactgttca caagtacggt 300

aagaactctt tcgcttggga aggtactgct ccaaaggtta tcatcactga cccagaacaa 360

atcaaggaag ttttcaacaa gatccaagac ttcccaaagc caaagttgaa cccaatcgct 420

aagtacatct ctatcggttt ggttcaatac gaaggtgaca agtgggctaa gcacagaaag 480

atcatcaacc cagctttcca cttggaaaag ttgaagggta tgttgccagc tttctctcac 540

tcttgtcacg aaatgatctc taagtggaag ggtttgttgt cttctgacgg tacttgtgaa 600

gttgacgttt ggccattctt gcaaaacttg acttgtgacg ttatctctag aactgctttc 660

ggttcttctt acgctgaagg tgctaagatc ttcgaattgt tgaagagaca aggttacgct 720

ttgatgactg ctagatacgc tagaatccca ttgtggtggt tgttgccatc tactactaag 780

agaagaatga aggaaatcga aagaggtatc agagactctt tggaaggtat catcagaaag 840

agagaaaagg ctttgaagtc tggtaagtct actgacgacg acttgttggg tatcttgttg 900

caatctaacc acatcgaaaa caagggtgac gaaaactcta agtctgctgg tatgactact 960

caagaagtta tggaagaatg taagttgttc tacttggctg gtcaagaaac tactgctgct 1020

ttgttggctt ggactatggt tttgttgggt aagcacccag aatggcaagc tagagctaga 1080

caagaagttt tgcaagtttt cggtaaccaa aacccaaact tcgaaggttt gggtagattg 1140

aagatcgtta ctatgatctt gtacgaagtt ttgagattgt acccaccagg tatctacttg 1200

actagagctt tgagaaagga cttgaagttg ggtaacttgt tgttgccagc tggtgttcaa 1260

gtttctgttc caatcttgtt gatccaccac gacgaaggta tctggggtaa cgacgctaag 1320

gaattcaacc cagaaagatt cgctgaaggt atcgctaagg ctactaaggg tcaagtttgt 1380

tacttcccat tcggttgggg tccaagaatc tgtgttggtc aaaacttcgc tttgttggaa 1440

gctaagatcg ttttgtcttt gttgttgcaa aacttctctt tcgaattgtc tccaacttac 1500

gctcacgttc caactactgt tttgactttg caaccaaagc acggtgctcc aatcatcttg 1560

cacaagttgt aa 1572

<210> 8

<211> 1575

<212> DNA

<213>artificial sequence

<400> 8

atggaagttt tcatgttccc aactggtact actgttatca tctctgtttt gtctgttttg 60

ttggctgtta tcccatggta cttgttgaac aagttgtggt tgaagccaaa gagattcgaa 120

aagttgttga aggctcaagg tttccaaggt gaaccataca acttgtctgt tttgaaggac 180

aagtctaagc aaaactacat gttgaagttg caacaagaag acaagtctaa gtctatcggt 240

ttgtctaagg aagctgctcc atctatcttc actccagttc accaaactgt tagaaagtac 300

ggtaacaact ctttcttgtg ggaaggtact actccaagag ttatcatcac tgacccagac 360

caaatcaagg acgttttcaa caagatcgac gacttcccaa agccaaagtt gagatctatc 420

gctaagtact tgtctgttgg tatcttggac cacgaaggta agaagtgggc taagcacaga 480

aagatcgcta acccagcttt ccacttggaa aagttgaagg ttatgttgcc agctttctct 540

cactcttgta acgaaatgat ctctaagtgg aaggaattgt tgtcttctga cggtacttgt 600

gaaatcgacg tttggccatc tttgcaaaac ttcacttgtg acgttatctc tagaactgct 660

ttcggttctt cttacgctga aggtactaag ttgttccaat tgttgaagaa gcaaggtttc 720

ttgttgatga ctggtagaca cactaacaac ccattgtggg gtttgttggc tactactact 780

aagactaaga tgaaggaaat cgacagagaa atccacgact ctttggaagg tatcatcgaa 840

aagagagaaa aggctttgaa gaacggtgaa actactaacg acgacttgtt gggtatcttg 900

ttgcaatcta accacgctga aaagcaaggt caaggtaact ctaagaacat cggtatgact 960

actcaagacg ttatcgacga atgtaagttg ttctacttgg ctggtcaaga aactacttct 1020

tctttgttgg tttggactat ggttttgttg ggtagatacc cagaatggca agctagagct 1080

agagaagaag ttttgcaagt tttcggtaac caaaacccaa acaacgaagg tttgtctcaa 1140

ttgaagatcg ttactatgat cttgtacgaa gttttgagat tgttcccacc attgatctac 1200

ttcaacagag ctttgagaaa ggacttgaag ttgggtaact tgttgttgcc agaaggtact 1260

caaatctctt tgccaatctt gttgatccac caagaccacg acttgtgggg tgacgacgct 1320

aaggaattca agccagaaag attcgctgaa ggtatcgcta aggctactaa gggtcaagtt 1380

tcttacttcc cattcggttg gggtccaaga atctgtttgg gtcaaaactt cgctttgttg 1440

gaagctaaga tcgctgtttc tttgttgttg caaaacttct ctttcgaatt gtctccaaac 1500

tacgttcacg ttccaactac tgttttgact ttgcaaccaa agaacggtgc ttctatcatc 1560

ttgcacaagt tgtaa 1575

<210> 9

<211> 22

<212> DNA

<213>artificial sequence

<400> 9

atgacttcga attccgattt gg 22

<210> 10

<211> 24

<212> DNA

<213>artificial sequence

<400> 10

tcaccagaca tccctgaggt aacg 24

<210> 11

<211> 2076

<212> DNA

<213>Radix Glycyrrhizae (Glycyrrhiza uralensis)

<400> 11

atgacttcga attccgattt ggttcgcacc attgagtcgg tgctgggcgt ttcactcggc 60

gattcggtct ccgattcgct cgttctgata gcgactacct ccgtcgccgt cataattggg 120

ctccttgtgt tcctgtggaa gaaatcttcg gatcggagca gggaggtgag gccggtgatt 180

gtgccgaagt cgttggtgaa ggatgaagac gatgatgtcg acgttgcctc cgggaagact 240

aaggttactg ttttcttcgg tactcagact ggtactgctg agggcttcgc taaggcattg 300

gcggacgaga tcaaggcaag atatgaaaaa gcatatgtca aagttgttga tttggatgac 360

tatgcaatgg atgatgatca atatgaggag aagctgaaga aagaaactct tgcatttttc 420

atgctggcaa cttatggaga tggagagcca actgacaatg ctgcaagatt ctacaaatgg 480

tttactgagg gtaaagagga gaggggcacc tggcttcaac agctcacaca tggagttttt 540

ggcctaggta acaagcaata tgaacatttt aataagatag gtaaagttgt tgatgaagac 600

cttagtgaac aaggtgcaaa gcgtcttgtt ccacttggac taggtgatga tgatcaatcc 660

attgaggatg atttttctgc ctggaaagaa tctctgtggc ctgagttgga tcagttgctc 720

cgagatgagg atgatgtgaa tactgtttct actccctata ctgctgctat tcctgaatat 780

cgagtagtga ttcatgactc cactgtcaca ccatcctatg ataatcaatt cagcgcagca 840

aatgggggtg ctgtatttga tattcatcat ccttgcaggg taaatgttgc tgttaaaagg 900

gagcttcaca aacctcagtc tgaccgttcc tgcatacatt tggagtttga tatatcgggg 960

actggcataa catatgaaac tggagaccat gtgggtgttt atgctgagaa ctgtgatgaa 1020

actgttgaag aagctgggaa gttgttgggt caaaatttag atttgctgtt ttctcttcac 1080

actgataacg aggatggcac ttcccttgga ggctctctgc tacctccttt ccctggtcct 1140

tgcacactgc gtacggcgtt agcacgttat gcagatctct tgaaccctcc acgaaaggct 1200

gctttagttg tattagctgc tcatgcttct gaacctagtg aggcagaaag attgaagttc 1260

ctctcctctc ctcaggggaa ggatgagtac tccaaatggg tggttggaag ccagagaagt 1320

ctccttgagg tgatggctga gtttccatca gcaaaacctc cacttggtgt gtttttcgct 1380

gccatagccc ctcgtttaca gcctcgttat tattctattt catcctctcc taggtttgcc 1440

tcacaaaggg tacatgtaac ttgtgccctg gtgtatggtc caactcccac tggcagaatt 1500

cacaaaggag tatgctcaac ctggatgaag aatgctattc ccttagagga aagccgtgac 1560

tgtggctggg ctcccatttt tatcaggcca tcaaatttca agctaccagc cgatcattcg 1620

attcctatta ttatggttgg acctggtact ggtttggcac cttttagggg atttttacag 1680

gaaagatttg ccctcaaaga ggatggtgtt caacttggtc cttcattact cttctttgga 1740

tgcaggaacc gtcaaatgga ttttatttat gaggatgagc taaagaattt tgtggaacaa 1800

ggttctctgt cagagttgat agttgcattc tctagagagg ggcctgaaaa ggagtatgtt 1860

caacacaaga tgatggataa agcagcatac ctgtggagtc tgatttctca gggaggttat 1920

ctttatgtct gtggtgatgc caagggtatg gccagagatg ttcatcgaat tcttcatacc 1980

attgtccagc agcaggaaaa tgtggagtcg tcaaaggcgg aggctatagt gaaaaaactc 2040

cagatggatg gacgttacct cagggatgtc tggtga 2076

<210> 12

<211> 691

<212> PRT

<213>artificial sequence

<400> 12

Met Thr Ser Asn Ser Asp Leu Val Arg Thr Ile Glu Ser Val Leu Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Asp Arg Ser Arg Glu Val Arg Pro Val Ile Val Pro Lys Ser

50 55 60

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

65 70 75 80

Lys Val Thr Val Phe Phe Gly Thr Gln Thr Gly Thr Ala Glu Gly Phe

85 90 95

Ala Lys Ala Leu Ala Asp Glu Ile Lys Ala Arg Tyr Glu Lys Ala Tyr

100 105 110

Val Lys Val Val Asp Leu Asp Asp Tyr Ala Met Asp Asp Asp Gln Tyr

115 120 125

Glu Glu Lys Leu Lys Lys Glu Thr Leu Ala Phe Phe Met Leu Ala Thr

130 135 140

Tyr Gly Asp Gly Glu Pro Thr Asp Asn Ala Ala Arg Phe Tyr Lys Trp

145 150 155 160

Phe Thr Glu Gly Lys Glu Glu Arg Gly Thr Trp Leu Gln Gln Leu Thr

165 170 175

His Gly Val Phe Gly Leu Gly Asn Lys Gln Tyr Glu His Phe Asn Lys

180 185 190

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

195 200 205

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

210 215 220

Phe Ser Ala Trp Lys Glu Ser Leu Trp Pro Glu Leu Asp Gln Leu Leu

225 230 235 240

Arg Asp Glu Asp Asp Val Asn Thr Val Ser Thr Pro Tyr Thr Ala Ala

245 250 255

Ile Pro Glu Tyr Arg Val Val Ile His Asp Ser Thr Val Thr Pro Ser

260 265 270

Tyr Asp Asn Gln Phe Ser Ala Ala Asn Gly Gly Ala Val Phe Asp Ile

275 280 285

His His Pro Cys Arg Val Asn Val Ala Val Lys Arg Glu Leu His Lys

290 295 300

Pro Gln Ser Asp Arg Ser Cys Ile His Leu Glu Phe Asp Ile Ser Gly

305 310 315 320

Thr Gly Ile Thr Tyr Glu Thr Gly Asp His Val Gly Val Tyr Ala Glu

325 330 335

Asn Cys Asp Glu Thr Val Glu Glu Ala Gly Lys Leu Leu Gly Gln Asn

340 345 350

Leu Asp Leu Leu Phe Ser Leu His Thr Asp Asn Glu Asp Gly Thr Ser

355 360 365

Leu Gly Gly Ser Leu Leu Pro Pro Phe Pro Gly Pro Cys Thr Leu Arg

370 375 380

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

385 390 395 400

Ala Leu Val Val Leu Ala Ala His Ala Ser Glu Pro Ser Glu Ala Glu

405 410 415

Arg Leu Lys Phe Leu Ser Ser Pro Gln Gly Lys Asp Glu Tyr Ser Lys

420 425 430

Trp Val Val Gly Ser Gln Arg Ser Leu Leu Glu Val Met Ala Glu Phe

435 440 445

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

450 455 460

Arg Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Phe Ala

465 470 475 480

Ser Gln Arg Val His Val Thr Cys Ala Leu Val Tyr Gly Pro Thr Pro

485 490 495

Thr Gly Arg Ile His Lys Gly Val Cys Ser Thr Trp Met Lys Asn Ala

500 505 510

Ile Pro Leu Glu Glu Ser Arg Asp Cys Gly Trp Ala Pro Ile Phe Ile

515 520 525

Arg Pro Ser Asn Phe Lys Leu Pro Ala Asp His Ser Ile Pro Ile Ile

530 535 540

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

545 550 555 560

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

565 570 575

Leu Phe Phe Gly Cys Arg Asn Arg Gln Met Asp Phe Ile Tyr Glu Asp

580 585 590

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

595 600 605

Ala Phe Ser Arg Glu Gly Pro Glu Lys Glu Tyr Val Gln His Lys Met

610 615 620

Met Asp Lys Ala Ala Tyr Leu Trp Ser Leu Ile Ser Gln Gly Gly Tyr

625 630 635 640

Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Arg Asp Val His Arg

645 650 655

Ile Leu His Thr Ile Val Gln Gln Gln Glu Asn Val Glu Ser Ser Lys

660 665 670

Ala Glu Ala Ile Val Lys Lys Leu Gln Met Asp Gly Arg Tyr Leu Arg

675 680 685

Asp Val Trp

690

<210> 13

<211> 22

<212> DNA

<213>artificial sequence

<400> 13

gacatggagg cccagaatac cc 22

<210> 14

<211> 20

<212> DNA

<213>artificial sequence

<400> 14

tcgacactgg atggcggcgt 20

<210> 15

<211> 1394

<212> DNA

<213>artificial sequence

<400> 15

gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 60

tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 120

cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag 180

cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 240

aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 300

ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaggaaaaga 360

ctcacgtttc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat 420

gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg 480

atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 540

agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta 600

tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggcaaa acagcattcc 660

aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc 720

tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc 780

gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg 840

acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataag cttttgccat 900

tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg 960

aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg 1020

atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt 1080

ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg 1140

atgagttttt ctaatcagta ctgacaataa aaagattctt gttttcaaga acttgtcatt 1200

tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg tgatttatat 1260

tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc 1320

atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat actaacgccg 1380

ccatccagtg tcga 1394

<210> 16

<211> 906

<212> DNA

<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 16

aattatcctg ggcacgagtg aaacaaagct aaaaccttta tttagcatgg ccattgaatg 60

taacaattat atatatcgca agcacaaaaa atcaaggaga gagaactacc actttgttca 120

tgtgtacaat gttcattatc tccataagca aaaaaaaaaa atagaaaaca tatgctataa 180

ggttgatatt ctcacgagta agcggcactt gctacttatt gacattgcag atttttggct 240

acagaaatag tatattagag attataattg ctaatcaaat caaaatataa aattagtaaa 300

ccaaaccatt tatacccttc cttagtagtt atggattgtt ttttaatgat atttctgcaa 360

accaaagaaa gattgttatc cagatagaat ttagttttga tattcatttt tttgttgaag 420

attgaacgcc atatctgggc ctcataattc aaaagacggt gccattatcg gtagcgtttc 480

gcattgtact ggatttcaga aatttcacag ttgatgaatc gaaaagaatg gtctcattgc 540

aacacgtaag gttaagatgt ccctttttac cattataggc aataaatgaa tcataaaacg 600

accgtatact ggtgaaatag tagggagaac gagtacctgt agtaaaaagt ataaatcata 660

gttaatcggg caatgtccct cgatcaagga gtattgtgtc atgttcgaga caaacgccaa 720

catttttgtt tcttttggac aaatgttgtt tgcatttatg atccgttata ttttgatcta 780

atgtagagtt gcacgtagtt cttactggca aagaaatcga tgcataccaa aaaagaataa 840

aggtgatatt tgatctttac cgtttagttc caacgtaaaa ttgtgccttt ggacttaaaa 900

tggcgt 906

<210> 17

<211> 501

<212> DNA

<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 17

ctgggggaac aacttcacag aatgttttgt catattgtcg aagtggtcac aaaacaagag 60

aagttccgcc aattataaaa agggaacccg tatatttcag cttcacggat gatttccagg 120

gtgagagtac tgtatatggg cttacgatag aaggccataa aaatttcttg cttggcaaca 180

aaatagaagt gaaatcatgt cgaggctgct gtgtgggaga acagcttaaa atatcacaaa 240

aaaagaatct aaaacactgt gttgcttgtc ccagaaaggg aatcaagtat ttttataaag 300

attggagtgg taaaaatcga gtatgtgcta gatgctatgg aagatacaaa ttcagcggtc 360

atcactgtat aaattgcaag tatgtaccag aagcacgtga agtgaaaaag gcaaaagaca 420

aaggcgaaaa attgggcatt acgcccgaag gtttgccagt taaaggacca gagtgtataa 480

aatgtggcgg aatcttacag t 501

<210> 18

<211> 29

<212> DNA

<213>artificial sequence

<400> 18

aattatcctg ggcacgagtg aaacaaagc 29

<210> 19

<211> 32

<212> DNA

<213>artificial sequence

<400> 19

acgccatttt aagtccaaag gcacaatttt ac 32

<210> 20

<211> 20

<212> DNA

<213>artificial sequence

<400> 20

ctgggggaac aacttcacag 20

<210> 21

<211> 27

<212> DNA

<213>artificial sequence

<400> 21

actgtaagat tccgccacat tttatac 27

Claims

1. a kind of construction method for the saccharomyces cerevisiae engineered yeast for producing enoxolone or its precursor substance, which is characterized in that utilize The β cloned from glycyrrhiza glabra-amyrin synthase (bAS) gene, the β-obtained from Glycyrrhiza Uralensis by clone Its gene order of 11, amyrin carbon oxidizing ferment UNI47 gene is as shown in SEQ IDNo.5, CYP450 oxidoreducing enzyme CPR base Because its gene order is as shown in SEQ ID No.11,11- oxygen-β -30, amyrin carbon oxidizing ferment for being cloned from clover Its gene order of CYP72A63 gene carries out codon optimization and chemical synthesis to it, then utilizes as shown in SEQ ID No.8 Saccharomyces cerevisiae constitutive promoter and terminator construct respectively, P_FBA1-bAS-T_CYC1,T_ADH1-P_ENO2-UNI47-T_TYS1,T_CYC1- P_GPD-CYP72A63-T_ADH1,T_TYS1-P_PGK1-CPR-T_PGK1Expression cassette, in a manner of genome conformity in saccharomyces cerevisiae body Enoxolone and its precursor route of synthesis are reconstructed, realizes saccharomyces cerevisiae synthesis enoxolone or its precursor.

2. application of the saccharomyces cerevisiae as described in claim 1 in synthesis enoxolone or its precursor, precursor includes 11- Hydroxy-beta-amyrin or 11- oxygen-β-amyrin or 30- hydroxy-beta-amyrin or 11,30- hydroxy-beta-botany bar gum Alcohol or 30- hydroxyl 11- oxygen-β-amyrin or 30- aldehyde radical -11- oxygen-β-amyrin.