CN106566815A

CN106566815A - Saccharomyces cerevisiae engineered strain used for producing glycyrrhetinic acid and precursor thereof and construction method of saccharomyces cerevisiae engineered strain

Info

Publication number: CN106566815A
Application number: CN201610965315.9A
Authority: CN
Inventors: 李春; 朱明�; 王彩霞; 孙文涛; 周安琪
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2017-04-19
Anticipated expiration: 2036-11-04
Also published as: CN106566815B

Abstract

The invention discloses a saccharomyces cerevisiae engineered strain used for producing glycyrrhetinic acid and a precursor of glycyrrhetinic acid, namely, 11-hydroxy-beta-amyrin, or 11-oxo-beta-amyrin, or 30-hydroxy-beta-amyrin, or 11,30-hydroxy-beta-amyrin, or 30-hydroxy-11-oxo-beta-amyrin, or 30-aldehydo-11-oxo-beta-amyrin and a construction method of the saccharomyces cerevisiae engineered strain. The construction method comprises the following steps: constructing a beta-amyrin synthase expression box, an oxidase expression box of the 11th site of carbon of beta-amyrin, an oxidase expression box of the 30th site of carbon of 11-oxo-beta-amyrin, and an oxidordeuctase expression box of cytochrome P450, and importing the expression boxes to a saccharomyces cerevisiae genome. According to the saccharomyces cerevisiae engineered strain and the construction method, the synthesis of glycyrrhetinic acid is coupled with the growth of the yeast, so that the yeast synthesis of glycyrrhetinic acid or the precursor of glycyrrhetinic acid is realized, for the method, the inducing of an effector is not needed, the process is simple, and the method can be used for fermentation production of glycyrrhetinic acid and the key precursor of glycyrrhetinic acid.

Description

It is a kind of to produce enoxolone or the saccharomyces cerevisiae engineered yeast and construction method of its precursor

Technical field

The present invention relates to a kind of structure of saccharomyces cerevisiae engineered yeast and fermenting and producing enoxolone or its precursor：11- hydroxyls 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 technology

Terpene substances are one kind of Secondary metabolites, with diversified biological activity and pharmacology value.Medicine Verified triterpenoid compound such as glycyrrhizic acid (Glycyrrhizin, GL) of science is in liver protection, anti-inflammatory, antitumor, antiviral Etc. aspect have very important effect, while also can be used as sweeting agent or food additive.Enoxolone (Glycyrrhetinic acid, GA) is the precursor substance of glycyrrhizic acid synthesis, and compared with glycyrrhizic acid, enoxolone lacks 2 molecules Glucal acidic group, so polarity is weaker, be easier to permeates cell membranes, and remain the physiologically active consistent with glycyrrhizic acid with Function, it is easier to play its biological activity and function in the cell.In liquorice root, the content of enoxolone is extremely low, mainly With the presence of glycyrrhizic acid form.There is the shortcomings of low cycle length, efficiency, high cost, big environmental disruption in plant extract.Enoxolone is answered Miscellaneous molecular structure, harsh synthesis condition are also difficult to its chemosynthesis.With the development of synthetic biology, using micro- The compound with great pharmaceutical value such as biosynthesiss enoxolone has become a kind of development trend.Microbe synthesis have week Phase is short, and yield is high, mild condition, the features such as process control, therefore can effectively alleviate the dependence to plant resourceses, so as to have Higher economic benefit and social value.

In saccharomyces cerevisiae engineered yeast, enoxolone or its precursor 11- hydroxy-betas-Amyrin, or 11- oxygen-β-perfume Resinol, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β-balsam Alcohol, or the synthesis of 30- aldehyde radical -11- oxygen-β-Amyrin is Jing mevalonate pathways synthesis zamene, then Jing 2,3- oxidation sharks Alkene monooxygenase, β-Amyrin synthase is catalyzed to form β-Amyrin.This research be on this basis, further excavate find β- Amyrin is common by two oxidase C YP88D6 and CYP72A154 (or CYP72A63) orders to the synthesis of enoxolone Catalysis is completed, and both enzymes is not found in microorganism, accordingly, it would be desirable to two enzymes in introduced plant Radix Glycyrrhizae are in Saccharomyces cerevisiae Inside expressed, so as to realize the biosynthesiss of enoxolone.

The content of the invention

It is an object of the invention to provide a kind of CYP450 lysyloxidase gene of 11, the carbon of oxidation of beta-Amyrin.

Second object of the present invention is to provide a kind of CYP450 lysyloxidase gene of 11, the carbon of oxidation of beta-Amyrin The protein of coding.

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

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

5th purpose of the present invention is to provide a kind of production enoxolone or its precursor 11- hydroxy-betas-Amyrin, Or 11- oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- Oxygen-β-Amyrin, or the saccharomycetic construction method of 30- aldehyde radical -11- oxygen-β-Amyrin.

6th purpose of the present invention is to provide a kind of using production enoxolone or its precursor 11- hydroxy-betas-XIANGSHU Lipidol, or 11- oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyls Base -11- oxygen-β-Amyrin, or the wine brewing ferment that the saccharomycetic construction method of 30- aldehyde radical -11- oxygen-β-Amyrin builds Female engineering bacteria.

Technical scheme is summarized as follows：

A kind of CYP450 lysyloxidase gene of 11, the carbon of oxidation of beta-Amyrin, it is SEQ ID No.5 in sequence table Described nucleotide sequence.

A kind of protein of the CYP450 lysyloxidase gene coding of 11, the carbon of oxidation of beta-Amyrin, it is sequence table Aminoacid sequence described in SEQ ID No.6.

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

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

One kind production enoxolone or its precursor 11- hydroxy-betas-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 radicals - The construction method of the saccharomyces cerevisiae engineered yeast of 11- oxygen-β-Amyrin, comprises the steps：

(1) structure of β-Amyrin synthase expression box：

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

(2) structure of CYP450 oxidase (I) expression cassette of 11, the carbon of oxidation of beta-Amyrin：

By Yeast promoter, the CYP450 oxidase (I) of 11, the carbon of oxidation of beta-Amyrin, yeast terminator application weight The method connection of folded extension PCR, obtains the oxidasic expression cassettes of CYP450 of 11, the carbon of oxidation of beta-Amyrin.

(3) structure of CYP450 oxidase (II) expression cassette of 30, the carbon of 11- oxygen-β-Amyrin is aoxidized：

By Yeast promoter, CYP450 oxidase (II), the yeast termination of 30, the carbon of oxidation 11- oxygen-β-Amyrin Son connects using the method for Overlap extension PCR, and the CYP450 for obtaining 30, the carbon of oxidation 11- oxygen-β-Amyrin is oxidasic Expression cassette.

(4) structure of cytochrome P450 reductase expression cassette

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

(5) structure of selection markers expression cassette

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

(6) by above-mentioned β-Amyrin synthase expression box, and/or the CYP450 oxidations of 11, the carbon of oxidation of beta-Amyrin Enzyme (I) expression cassette, and/or CYP450 oxidase (II) expression cassette of 30, the carbon of oxidation 11- oxygen-β-Amyrin, and/or carefully Born of the same parents' cytochrome p 450 oxidoreductase 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 sites, obtain produce enoxolone or Its precursor：11- hydroxy-betas-Amyrin, or 11- oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- Hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β-Amyrin, or the wine brewing ferment of 30- aldehyde radical -11- oxygen-β-Amyrin 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 HO sites, and/or YPRCtau3 sites, and/or URA3 sites, and/or TRP1 Site, and/or LEU2 sites, and/or HIS3 sites, and/or delta sites, and/or rDNA sites.

The nucleotide sequence Genbank registration sequences number of the β-Amyrin synthase are AB037203.

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

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

The cytochrome P450 reductase is the nucleotide sequence in sequence table described in SEQ ID No.10, or carefully Born of the same parents' cytochrome p 450 oxidoreductase LjCPR1, its nucleotide sequence Genbank registration sequences number are AB433810, and/or nucleoside Acid sequence Genbank registration sequences number for BT008426.1 cytochrome P450 reductase AtCPR1, and/or protein 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-betas-Amyrin, or 11- oxygen-β-Amyrin, Or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β-Amyrin, or 30- The saccharomyces cerevisiae engineered yeast that the saccharomycetic construction method of aldehyde radical -11- oxygen-β-Amyrin builds.

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

Description of the drawings

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

The matter of Fig. 2 saccharomyces cerevisiae engineered yeasts fermenting and producing 11- hydroxy-beta-Amyrin (11-hydroxy- β-amyrin) Spectrogram.

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

The mass spectrum of Fig. 4 saccharomyces cerevisiae engineered yeast fermenting and producing 11- oxygen-β-Amyrin (11-oxo- β-amyrin).

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

The matter of Fig. 6 saccharomyces cerevisiae engineered yeasts fermenting and producing 30- hydroxy-beta-Amyrin (30-hydroxy- β-amyrin) Spectrogram.

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

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

Fig. 9 saccharomyces cerevisiae engineered yeast fermenting and producing 30- hydroxyl -11- oxygen-β-Amyrin (30-hydroxy-11-oxo- β-amyrin) component analyses 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 spectrum.

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

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

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

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

Enoxolone synthesis path figure in Figure 15 saccharomyces cerevisiaes.

Specific embodiment

Below by synthesis enoxolone and its key precursor thing preferred embodiment and combine accompanying drawing and illustrate this 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, and not It is to limit the scope of the present 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 fall within protection scope of the present invention.For example, by institute in this experimental example The promoter or terminator for using replaces with other promoteres commonly used in the art or terminator, is the other technologies people of this area Member it will be appreciated that and realize.

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

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

The acquisition of A, β-Amyrin synthase gene

It is excellent by codon according to β-Amyrin synthase gene (Genbank registration sequences number are AB037203) sequence Change, the codon for making β-Amyrin synthase gene has yeast Preference, and the optimized gene sequence of generation is SEQ ID No.1。

The acquisition of B, the CYP450 lysyloxidase gene of 11, the carbon of oxidation of beta-Amyrin

According to CYP450 oxidase C YP88D6 genes (Genbank registration sequences number are AB433179) sequence, by password Son optimization, the codon for making CYP88D6 genes has yeast Preference, and the optimized gene sequence of generation is SEQ ID No.2.

According to CYP450 oxidase C YP88D6 genes (Genbank registration sequences number are AB433179) primers, SEQ ID No.3：5 '-ATGGAAGTACATTGGGTTTGC-3 ' and SEQ ID No.4：5’- CTAAGCACATGAGACCTTCATCACC-3 ', with Glycyrrhiza uralensis Fisch. cDNA as masterplate, using ExTaq enzymes performing PCR amplification is entered, and is obtained To 1482bp fragments, shown in SEQ ID No.5, to be cloned into pMD18-T carriers, sequencing is proved and the CYP88D6 phases reported Than the change that there occurs 23 bases, aminoacid there occurs 11 variations.The aminoacid sequence of fragment expression shown in SEQ ID No.5 Row are with shown in SEQ ID No.6.

The acquisition of C, the CYP450 lysyloxidase gene of 30, the carbon of oxidation 11- oxygen-β-Amyrin

According to CYP450 oxidase C YP72A154 genes (Genbank registration sequences number are AB558153) sequence, by close Numeral optimizes, and the codon for making CYP72A154 genes has yeast Preference, and the optimized gene sequence of generation is SEQ ID No.7。

According to CYP450 oxidase C YP72A63 genes (Genbank registration sequences number are AB558146) sequence, by close Numeral optimizes, and the codon for making CYP72A63 genes has yeast Preference, and the optimized gene sequence of generation is SEQ ID No.8。

The acquisition of D, cytochrome P450 reductase gene

Screened according to Radix Glycyrrhizae transcript profile data base, obtain cytochrome P450 reductase gene, design primer, SEQ ID No.9：5 '-ATGACTTCGAATTCCGATTTGG-3 ' and SEQ ID No.10：5’- TCACCAGACATCCCTGAGGTAACG-3 ', with Glycyrrhiza uralensis Fisch. cDNA as masterplate, using ExTaq enzymes performing PCR amplification is entered, and is obtained To 2076bp fragments, shown in SEQ ID No.11, to be cloned into pMD18-T carriers, sequencing determines its complete sequence.SEQ ID The aminoacid sequence of fragment expression shown in No.11 is with shown in SEQ ID No.12.

The acquisition of E, riddled basins

According to Geneticin (G418) expression cassette sequence in pRS42K carriers, primer SEQ ID No.13 are designed：5’- GACATGGAGGCCCAGAATACCC-3 ' and SEQ ID No.14：5 '-TCGACACTGGATGGCGGCGT-3 ', with pRS42K matter Grain is masterplate, and using ExTaq enzymes performing PCR amplification is entered, and 1394bp fragments is obtained, shown in SEQ ID No.15, to be cloned into PMD18-T carriers, sequencing proves that it is not undergone mutation.

The acquisition of F, genome homology arm

According to the left homology arm HOL in HO sites in saccharomyces cerevisiae genome (SEQ ID No.16) and right homology arm sequence HOR (SEQ ID No.17), designs primer SEQ ID No.18：5 '-AATTATCCTGGGCACGAGTGAAACAAAGC-3 ' and SEQ ID No.19：5 '-ACGCCATTTTAAGTCCAAAGGCACAATTTTAC-3 ' (as shown in SEQ ID No.53), HO is left for amplification Homology arm HOL；Primer SEQ ID No.20：5 '-CTGGGGGAACAACTTCACAG-3 ' and SEQ ID No.21：5’- ACTGTAAGATTCCGCCACATTTTATAC-3 ' expands right homology arm HOR, with INVSc1 genomes as masterplate, using ExTaq Enzyme enters performing PCR amplification, is cloned into pMD18-T carriers, and sequencing proves that it is not undergone mutation.

Experimental example 2：The structure of expression cassette

The structure of A, β-Amyrin synthase expression box

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

The structure of B, the CYP450 Oxidase Expression boxes of 30, the carbon of oxidation 11- oxygen-β-Amyrin

By yeast terminator T_CYC1, Yeast promoter P_GPD, CYP450 lysyloxidase gene CYP72A154 or CYP72A63, ferment Female terminator T_ADH1Connected with this using the method for Overlap extension PCR, obtain aoxidizing 30, the carbon of 11- oxygen-β-Amyrin CYP450 oxidase T_CYC1-P_GPD–CYP72A154-T_ADH1Or T_CYC1-P_GPD-CYP72A63-T_ADH1

The structure of C, the CYP450 Oxidase Expression boxes of 11, the carbon of oxidation of beta-Amyrin

By yeast terminator T_ADH1, Yeast promoter P_ENO2, oxidation of beta-Amyrin 11, carbon CYP450 oxidase bases Because of UNI47 or CYP450 lysyloxidase gene CYP88D6, yeast terminator T_TYS1Connected with this using the method for Overlap extension PCR, Obtain β-Amyrin synthase expression box T_ADH1-P_ENO2-UNI47-T_TYS1Or T_ADH1-P_ENO2-CYP88D6-T_TYS1

The structure of D, cytochrome P450 reductase expression cassette

By yeast terminator T_TYS1, Yeast promoter P_PGK1, cytochrome P450 reductase gene C PR, yeast termination Sub- T_PGK1Connected with this using the method for Overlap extension PCR, obtain cytochrome P450 reductase expression cassette T_TYS1-P_PGK1- CPR-T_PGK1

The structure of E, selection markers expression cassette

By yeast terminator T_PGK1, Geneticin expression cassette gene P_TEF1-kanMX-T_TEF1, right homology arm HOR application overlaps 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 production enoxolone

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

Using the homologous recombination function of yeast itself, the above-mentioned expression cassette for building all is proceeded to into saccharomyces cerevisiae jointly The HO sites of INVSc1 genomes.Screened using the YPD solid plates of addition Geneticin (G418) after conversion, obtained Transformant is transferred in the YPD fluid mediums of addition Geneticin (G418) carries out culture 1-2 days, extracts genome, PCR mirror Determine positive colony, plate streaking or glycerol stock are preserved.

Exist on the basis of MVA approach using yeast itself, be exogenously introduced the synzyme of enoxolone, acquisition possesses production 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 thing

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

Fermenting experiment：In 100ml YPD culture medium being inoculated into according to 1% inoculum concentration (absorbance about 0.1), 30 DEG C, 200rpm shaken cultivation 120 hours, per 24 hours, sampling once, determined containing for enoxolone and its key precursor thing in cell Amount, as a result shows, ferment final Determination of glycyrrhetic acid of saccharomyces cerevisiae engineered yeast is 1.515mg/L, its precursor (11- hydroxy-betas- Amyrin content is for 23mg/L, 30- hydroxy-beta-Amyrin content for 6.9mg/L, 11- oxygen-β-Amyrin content It is 40mg/ that 25mg/L, 11,30- hydroxy-beta-Amyrin content is 15mg/L, 30- hydroxyl -11- oxygen-β-Amyrin content 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, precipitation is resuspended with 1.5ml saturated sodium-chlorides, and pearl is broken broken 20 minutes, isopyknic ethyl acetate extraction, rotary evaporation in being transferred to gas phase bottle, added 500 μ l N, O- to powder Double (trimethyl silicon substrate) three fluoro acetamides and 500 μ l pyridines, 80 DEG C are reacted 30 minutes, are cooled to room temperature.Using Shimadzu GC2010QP gas chromatograph-mass spectrometer is analyzed to extract.Heating schedule：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, the μ l of sample introduction 1, chromatographic mass spectrometry interface temperature is 290 ℃.As a result show, total ion chromatogram combination defines the characteristic peak of enoxolone with mass spectrum, illustrates saccharomyces cerevisiae engineered yeast Enoxolone and its precursor can successfully be synthesized：11- hydroxy-betas-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 radicals - 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 in GAL7p, and/or GAL1p, and/or GAL10p replacement experimental examples 2, or GPD, or ENO2p, or PGK1p, 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 TPI1t replaces the PGK1t in experimental example 2, or ADH1t, or CYC1t, or TYS1t, it is possible to obtain corresponding expression cassette.

Using 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 strains in experimental example 3.

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

SEQUENCE LISTING

<110>Beijing Institute of Technology

<120>It is a kind of to produce enoxolone or the saccharomyces cerevisiae engineered yeast and construction method of its precursor

<130> 1

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 2298

<212> DNA

<213>Artificial sequence

<400> 1

atgtggagat tgaagatcgc tgaaggtggt aaggacccat acatctactc tactaacaac 60

ttcgttggta gacaaacttg ggaatacgac ccagacggtg gtactccaga agaaagagct 120

caagttgacg ctgctagatt gcacttctac aacaacagat tccaagttaa gccatgtggt 180

gacttgttgt ggagattcca aatcttgaga gaaaacaact tcaagcaaac tatcgcttct 240

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

aactacatct gtatgagaat cttgggtgaa ggtccagacg gtggtcaaga caacgcttgt 600

gctagagcta gaaagtggat ccacgaccac ggtggtgtta ctcacatccc atcttggggt 660

aagacttggt tgtctatctt gggtgttttc gactggtgtg gttctaaccc aatgccacca 720

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

gaaacttcta gatacatcac tatcggttgt gttgaaaagg ttttgtgtat gttggcttgt 1140

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

tggttcgctt tgggtggttt ggctgctgct ggtaagactt tcgctaactg tgctgctatc 1920

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 CYP450 oxidase, its aminoacid sequence is shown in SEQ ID No.6.

2. the oxidasic genetic fragments of CYP450 described in claim 1 are encoded.

3. genetic fragment according to claim 2, its nucleotide sequence is as shown in SEQ ID No.5.

4. a kind of cytochrome P450 reductase, its aminoacid sequence is shown in SEQ ID No.12.

5. the genetic fragment of the cytochrome P450 reductase described in claim 4 is encoded.

6. genetic fragment according to claim 5, its nucleotide sequence is as shown in SEQ ID No.11.

7. it is a kind of to produce enoxolone or its precursor：11- hydroxy-betas-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 radicals - The construction method of the saccharomyces cerevisiae engineered yeast of 11- oxygen-β-Amyrin, is characterized in that comprising the steps：

(1) structure of β-Amyrin synthase expression box：

Left side homology arm, Yeast promoter, β-Amyrin synthase, the method for yeast terminator application Overlap extension PCR are connected Connect, obtain the expression cassette of β-Amyrin synthase；

By Yeast promoter, the CYP450 oxidase of 11, the carbon of oxidation of beta-Amyrin, yeast terminator application overlap extension The method connection of PCR, obtains the oxidasic expression cassettes of CYP450 of 11, the carbon of oxidation of beta-Amyrin.

By Yeast promoter, CYP450 oxidase, the yeast terminator application weight of 30, the carbon of oxidation 11- oxygen-β-Amyrin The method connection of folded extension PCR, obtains the oxidasic expression cassettes of CYP450 of 30, the carbon of oxidation 11- oxygen-β-Amyrin.

(4) structure of cytochrome P450 reductase expression cassette：

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

(5) by above-mentioned β-Amyrin synthase expression box, and/or the CYP450 oxidase of 11, the carbon of oxidation of beta-Amyrin (I) expression cassette, and/or CYP450 oxidase (II) expression cassette of 30, the carbon of oxidation 11- oxygen-β-Amyrin, and/or cell Cytochrome p 450 oxidoreductase 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 sites, obtain produce enoxolone or Its precursor：11- hydroxy-betas-Amyrin (11-hydroxy- β-amyrin), or 11- oxygen-β-Amyrin (11-oxo- β- Amyrin), or 30- hydroxy-betas-Amyrin (30-hydroxy- β-amyrin), or 11,30- hydroxy-beta-Amyrin (11, 30-hydroxy- β-amyrin), or 30- hydroxyl -11- oxygen-β-Amyrin (30-hydroxy-11-oxo- β-amyrin), Or the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin (Glycyrrhetaldehyde).

8. one kind according to claim 7 produces enoxolone or its precursor：11- hydroxy-betas-Amyrin or 11- Oxygen-β-Amyrin or 30- hydroxy-betas-Amyrin or 11,30- hydroxy-betas-Amyrin or 30- hydroxyl -11- oxygen-β-perfume The construction method of the saccharomyces cerevisiae engineered yeast of resinol or 30- aldehyde radical -11- oxygen-β-Amyrin, is characterized in that the yeast is opened Mover 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.

9. one kind according to claim 7 produces enoxolone or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the construction method of the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin, is characterized in that the ferment Female 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.

10. one kind according to claim 7 produces enoxolone or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the construction method of the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin, is characterized in that the base Because group integration site is HO sites, and/or YPRCtau3 sites, and/or URA3 sites, and/or TRP1 sites, and/or LEU2 Site, and/or HIS3 sites, and/or delta sites, and/or rDNA sites.

A kind of 11. production enoxolone according to claim 7 or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the construction method of the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin, it is characterized in that the β - The nucleotide sequence Genbank registration sequences number of Amyrin synthase are AB037203.

A kind of 12. production enoxolone according to claim 7 or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the construction method of the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin, is characterized in that the oxygen The CYP450 oxidase for changing 11, the carbon of β-Amyrin is the CYP450 oxidase described in right 1, and/or described in right 2 or 3 Genetic fragment coding oxidation of beta-Amyrin 11, carbon CYP450 oxidase, and/or nucleotide sequence Genbank note The CYP450 oxidase C YP88D6 of volume Serial No. AB433179.

A kind of 13. production enoxolone according to claim 7 or its precursor：30- hydroxy-betas-Amyrin, or 11, 30- hydroxy-betas-Amyrin, or 30- hydroxyl -11- oxygen-β-Amyrin, or the wine of 30- aldehyde radical -11- oxygen-β-Amyrin The construction method of brewer yeast engineering bacteria, is characterized in that the CYP450 oxidase of 30, the carbon of the oxidation 11- oxygen-β-Amyrin For CYP450 oxidase C YP72A154, its nucleotide sequence Genbank registration sequences number are AB558153, and/or nucleotides sequence Row Genbank registration sequences number are the CYP450 oxidase C YP72A63 of AB558146.

A kind of 14. production enoxolone according to claim 7 or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the construction method of the saccharomyces cerevisiae engineered yeast of 30- aldehyde radical -11- oxygen-β-Amyrin, is characterized in that described thin Born of the same parents' cytochrome p 450 oxidoreductase is the cytochrome P450 reductase described in right 4, or the gene described in right 5 or 6 The cytochrome P450 reductase of fragment coding, and/or nucleotide sequence Genbank registration sequences number are for AB433810's Cytochrome P450 reductase LjCPR1, and/or nucleotide sequence Genbank registration sequences number are the thin of BT008426.1 Born of the same parents' cytochrome p 450 oxidoreductase AtCPR1, and/or protein sequence Genbank registration sequences number are for NP_001328167.1's Cytochrome P450 reductase AtCPR2.

A kind of production enoxolone of one of 15. claim 7-14 or its precursor：11- hydroxy-betas-Amyrin, or 11- Oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyl -11- oxygen-β - Amyrin, or the saccharomyces cerevisiae engineering that the construction method of the saccharomyces cerevisiae of 30- aldehyde radical -11- oxygen-β-Amyrin builds Bacterium.

Saccharomyces cerevisiae engineered yeast described in 16. claim 15 is in production enoxolone or its precursor：11- hydroxy-betas-XIANGSHU Lipidol, or 11- oxygen-β-Amyrin, or 30- hydroxy-betas-Amyrin, or 11,30- hydroxy-beta-Amyrin, or 30- hydroxyls Application in base -11- oxygen-β-Amyrin, or 30- aldehyde radical -11- oxygen-β-Amyrin.