CN106635853B - Recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, and construction method and application thereof - Google Patents

Abstract

The invention relates to recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, a construction method and application thereof. Specifically, the invention relates to a recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid with the preservation number of CGMCC13126, a construction method thereof and application thereof in producing the glycyrrhetinic acid.

Description

Recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, and construction method and application thereof

Technical Field

The invention relates to the technical field of glycyrrhetinic acid production. Specifically, the invention relates to recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, a construction method and application thereof.

Background

The liquorice (Glycyrrhiza uralensis Fisch) is a perennial herb of liquorice of leguminous, takes roots and stems as medicines, has the effects of tonifying spleen and qi, clearing heat and detoxicating, eliminating phlegm and stopping cough, relieving spasm and pain, harmonizing the medicines and the like, and is the most widely applied traditional Chinese medicine in China. At present, more than 200 compounds are separated from liquorice and mainly divided into triterpenes, flavonoids, polysaccharides and the like. Wherein glycyrrhizic acid (also called glycyrrhizin) is an oleanane type pentacyclic triterpene saponin, which is the main active component of licorice and accounts for 2-8% of dry weight of licorice. Modern pharmacological research proves that glycyrrhizic acid has various pharmaceutical activities, and has the effects of resisting bacteria and inflammation, regulating the immunity of organisms, resisting ulcer and the like. Glycyrrhizic acid has obvious inhibiting effect on various DNA and RNA viruses, such as immunodeficiency virus (HIV), acute respiratory coronavirus (SARS), hepatitis virus, etc., wherein the inhibiting rate on HIV is as high as 90%. Glycyrrhizic acid also has effects of reducing blood lipid, resisting atherosclerosis, and preventing and resisting cancer. In addition to the above effects, glycyrrhizic acid has a sweetness about 150 times that of sucrose, and thus is widely used as a natural sweetener in the fields of food, tobacco, and the like.

The glycyrrhetinic acid is aglycon of glycyrrhizic acid, and pharmacological experiments show that the glycyrrhizic acid cannot be directly absorbed by gastrointestinal tracts after entering a human body, but is firstly decomposed into the glycyrrhetinic acid by intestinal flora in the human body and then enters blood to play a role after being absorbed by intestinal mucosa. Therefore, glycyrrhizic acid actually plays its pharmacodynamic action in the form of its aglycone, i.e. glycyrrhetinic acid. The research of glycyrrhetinic acid proves that the glycyrrhetinic acid has better pharmacological activity than glycyrrhizic acid, for example, the in vitro experiment effect of the glycyrrhetinic acid is better than that of the glycyrrhizic acid in the aspect of resisting platelet aggregation. In addition, glycyrrhetinic acid has good cytotoxicity, so that the glycyrrhetinic acid has a certain inhibition effect on tumor cells, viruses and the like. modifying-COOH and-OH functional groups in the structure of glycyrrhetinic acid to obtain various derivatives with activity. Up to now, more than 400 derivatives are obtained by modification with glycyrrhetinic acid as a skeleton, and 128 of the derivatives with cytotoxicity with IC50<30 μ M are obtained. In addition to being used as a medicine, glycyrrhetinic acid is widely used in the field of cosmetics for its anti-inflammatory and whitening effects. The content of glycyrrhetinic acid in licorice is far lower than that of licorice, which accounts for about 0.6-1.6% of the dry weight of licorice, and the difference between different populations in different regions is large, for example, the content of glycyrrhetinic acid in licorice in Asia region is generally not more than 0.7%. Therefore, glycyrrhetinic acid is generally obtained by hydrolysis of glycyrrhizic acid to remove two glucuronic acids, and at present, a chemical acidolysis method is mainly adopted, and an enzymatic hydrolysis method is also adopted, but the enzymatic hydrolysis method is still in a laboratory exploration stage. Because of its low content in licorice and the glycyrrhizic acid used as raw material is further hydrolyzed, the price of glycyrrhetinic acid is 2-3 times of that of glycyrrhizic acid, about $ 500-.

Due to the wide application of glycyrrhizic acid and glycyrrhetinic acid, the liquorice is in great demand at home and abroad. The annual demand of China is about 6 to 7 ten thousand tons in China in recent two years, and the export is also continuously increased. The global licorice market trade amount in 2007 was about $42.1 million. Compared with the huge demand of liquorice, the liquorice resource is seriously insufficient. Due to the disordered mining and digging under huge demand, the wild liquorice resource is seriously damaged and endangered to be extinct, and the domestic wild liquorice reserve is less than 20 ten thousand tons at present. The wild glycyrrhiza community has extremely strong wind prevention and sand fixation functions. Excessive mining causes wild liquorice, and vegetation damage caused by excessive mining aggravates sand storm disasters. In 2000, the government of China issued the prohibited digging order of wild liquorice and managed the export of liquorice. The current liquorice resource mainly comprises planting liquorice, but the planting of the liquorice has the main problem that the content of glycyrrhizic acid serving as an effective component is lower than 2 percent and cannot meet the requirements of pharmacopoeia.

Glycyrrhetinic acid has a complex structure, and cannot be chemically synthesized at present. The extraction and separation of liquorice are limited by limited liquorice resources and low content of liquorice, and large-scale liquorice excavation can cause ecological environment damage and desertification. Synthetic biology is based on engineering theories to design and synthesize new biological elements or to design and modify existing biological systems. Therefore, based on the synthetic biology thought, the method realizes the heterologous synthesis of the medicinal effective components in the mode strains such as escherichia coli or yeast and the like by assembling the synthetic route of the medicinal plant effective components, and provides a new development opportunity for realizing the sustainable utilization and development of the traditional Chinese medicine resources. The constructed yeast strain producing artemisinic acid is fermented to produce 25g/L of artemisinic acid by a Keasling research team (Paddon C J, Westfall P J, Pitera D J, et al, high-level semi-synthetic production of the potential and timing industrial artemisinine [ J ]. Nature,2013,496(7446):528.) of the university of California through transferring an artemisinic acid synthesis path in a yeast cell and regulating and controlling the metabolism, and then the artemisinin is successfully synthesized through 4 steps of chemical catalysis, so that the production of the artemisinic acid is greatly promoted. At present, the achievement authorizes international pharmaceutical companies to carry out industrial production. In addition, the synthesis of paclitaxel, tanshinone, ginsenoside, morphine, vinblastine or their precursors has advanced to some extent. Stephanopoulos (Ajikumar P K, Xiao W H, Tyo K E J, et al, isopreoid pathway engineering for Taxol precursor over production in Escherichia coli [ J ] Science,2010,330(6000):70.) through a modular pathway engineering strategy, the synthesis pathway of taxane (taxadiene) is divided into two modules for regulation, one is an MEP pathway upstream of Escherichia coli to increase carbon flow into terpenoid precursor IPP, the other is integration of heterologous taxane synthesis pathway into Escherichia coli, and the final yield of taxane precursor component of paclitaxel reaches 1g/L by regulating the metabolic flow of the two modules. The synthesis of Strobilantinin, an important precursor of catharanthine in microorganisms, was achieved by a series of gene rearrangements by the Sarah E.O' Connor team (Brown S.Clastre M.Cordavault V.et al.2015.Denovo production of the plant-derived aldaloid strictinine in yeast. Proc Natl Acad Sci U S.A.112 (11): 3205. 3210.) promoter was optimized in addition to some essential gene-introducing strains, to add 5 additional copies of genes tHMW, MAF1, IDI GR 1, SAM2, ZWF1, deletion of ERG20, ATF1, OYE2, yield of isochrroside of 0.5 mg/L. Dai et al (Dai Z, Yi L, Zhang X, et al. Metabolic engineering of Saccharomyces cerevisiae, for production of ginsenosides) introduce a ginseng-derived dammaradiene synthase gene (PgDDS), a protopanaxadiol synthase gene (CYP716A47) and an Arabidopsis thaliana (A. thaliana) -derived AtCPR1 gene into yeast strains, systematically regulate and control related genes tHMG1, ERG20, ERG9, ERG1 and the like, optimize related codons to construct an engineered strain producing protopanaxadiol, wherein the yield of the protopanaxadiol is 1.2g/L, which is 262 times higher than that of an original strain. This is the first time that protopanaxadiol, an important precursor of ginsenosides, is synthesized from glucose in yeast.

The excavation and identification of functional genes are the front problems of realizing the synthetic biology of functional components of natural products. Some progress has been made at home and abroad in the research on the glycyrrhizic acid biosynthesis pathway. The synthesis route is basically clear, and the glycyrrhizic acid is synthesized by cyclization, hydroxylation and glycosylation of 2, 3-oxidation-squalene which is a common precursor of triterpenoids, as a typical oleanane-type triterpenoid. Specifically, 2, 3-oxidosqualene generates beta-balsamic alcohol under the catalysis of beta-balsamic alcohol synthase (beta-AS), and a beta-AS gene is an important branch point for catalyzing the generation of glycyrrhizic acid and plays an important role in forming a pentacyclic triterpene mother nucleus skeleton of glycyrrhizic acid. Subsequently, the formed mother nucleus is subjected to hydroxylation modification by a series of CYP450 genes. An article published by Toshiya Muranaka team of Japan in 2008 on PNAS (Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, SudoH, Akashi T, Aoki T, Saito K, Muranaka T. Licorice beta-amyrin 11-oxidase, acytochrome P450with a key roll in the biosynthesis of the tripereneseneter glycidyl. Proc Natl Acad Sci U S.2008, 105:14204 and 14209.) successfully identified CYP88D6 using the licorice transcriptome information and successfully expressed in the yeast system, which hydroxylated and further carboxylated C-11 of beta-resinol. An article by The team in 2011 was published on The Plant Cell (Seki H, Sawai S, Ohyama K, Mizutani M, Ohnishi T, Sudo H, Fukushima EO, Akashi T, Aoki T, Saito K, Muranaka T.Triterpen functional genes in availability for identification of CYP72A154involved in The biochemical of Glycyrrhiza cell.2011,23:4112-4123.) and The genes CYP72A154 and CYP72A63, which catalyze The formation of carboxylic acids by C-30 of beta-resinol to glycyrrhetinic acid, were also identified by The Glycyrrhiza transcriptome. So far, all catalytic genes of the glycyrrhetinic acid synthesis pathway are identified. These work lay the foundation for the synthesis of glycyrrhetinic acid.

The method has the advantages of short growth period, low cost of fermentation raw materials, high utilization rate, high efficiency and single synthesis of fermentation products, few byproducts, mild conditions, little environmental pollution and convenient separation.

Disclosure of Invention

Based on the application market and huge demand of glycyrrhetinic acid and the limitation of limited liquorice resource at present, the invention provides a glycyrrhetinic acid producing yeast strain and a construction method thereof, and the glycyrrhetinic acid can be produced by yeast fermentation finally through technologies such as over-expression of 10 genes of yeast, deletion of exogenous genes in a synthetic way of 3 glycyrrhetinic acids, deletion of the genes of the yeast and the like.

In one aspect, the invention provides a recombinant yeast strain that produces glycyrrhetinic acid, which overexpresses the yeast genes acetoacetyl-CoA thiolase gene (ERG10), mevalonate kinase gene (ERG12), HMG-CoA synthase gene (ERG13), mevalonate diphosphate decarboxylase gene (ERG19), farnesyl pyrophosphate synthase (ERG20), squalene synthase (ERG9), squalene epoxidase (ERG1), mevalonate phosphate kinase gene (ERG8), isopentenyl diphosphate isomerase gene (IDI1), and truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tmgh); and the recombinant yeast strain expresses amyrin synthase (beta-AS), CYP88D6 and CYP72A154 genes in a glycyrrhetinic acid synthesis pathway and cytochrome P450 reductase (CPR1) gene of arabidopsis thaliana; simultaneously the recombinant yeast strain deleted the GGPP synthase gene (BTS 1).

The recombinant yeast strain is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 21 days in 2016, and has the following addresses: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; and E, postcode: 100101; the preservation number is CGMCC 13126; it is classified and named as Saccharomyces cerevisiae.

In another aspect, the present invention provides a method for preparing a recombinant yeast strain that produces glycyrrhetinic acid, the method comprising the steps of:

a) obtaining beta-AS, CYP88D6 and CYP72A154 genes participating in a glycyrrhetinic acid synthesis pathway from a glycyrrhiza plant, and obtaining a CPR1 gene fragment from Arabidopsis thaliana;

b) constructing Ppgk 1-beta-AS-Tadh 1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 gene expression clusters from the beta-AS, CYP88D6, CYP72A154 and CPR1 gene fragments obtained in the step a), and integrating the four gene expression clusters into the rDNA locus of yeast chromosome Cen.pk2-1D;

c) carrying out codon optimization on the CYP88D6 and CYP72A154 genes in the step b) to obtain OPCYP88D6 and OPCYP72A154 genes, constructing a Ptdh3-OPCYP88D6-Tcyc1 and a Padh1-OPCYP72A154-adh1 gene cluster, and integrating the gene clusters together with the Ppgk 1-beta-AS-Tadh 1 and the Ptdh3-CPR1-Tcyc1 gene cluster into an rDNA locus of the saccharomyces cerevisiae Cen.pk2-1D;

d) carrying out PCR amplification on ERG9, ERG20, ERG1 and tHMG (recombinant human growth hormone) fragments of a yeast strain obtained from yeast, fusing ERG20 and ERG9 genes to obtain a fused fragment ERG20+9 or ERG9+20, constructing corresponding gene expression clusters Ptdh3-E20+9-Tcyc1, Ptdh3-E9+20-Tcyc1, Ptef1-ERG1-Tpgk1 and Ppgk1-tHMG-Tadh1 together with ERG1 and tHMG, and integrating the gene clusters into the chromosomal site of the strain constructed in the step c);

e) carrying out PCR amplification on the ERG10, ERG8 and ERG13 of the yeast strain from the yeast, constructing corresponding gene clusters of the gene clusters of Padh1-E10-Tadh1, Ptdh3-ERG8-Ttdh3 and Padh1-E13-Tadh1, and integrating the three gene expression clusters to the position of +314bp of the trp site on the yeast chromosome in the step d) to ensure that the genes of ERG10, ERG8 and ERG13 are overexpressed; and

f) obtaining ERG12, ERG19 and IDI1 genes of yeast strains by PCR amplification from yeast, obtaining a coding gene (CYB5) of cytochrome b5 of liquorice by amplification from the liquorice, constructing corresponding gene expression clusters of Padh1-ERG12-Tadh1, Ptef2-ERG19-cyc1, Ppgk1-IDI-Tpgk1 and Ptdh3-CYB5-Ttdh3, and integrating the four gene expression clusters to the BTS1 gene locus of the yeast chromosome in the step e) to obtain the final recombinant saccharomyces cerevisiae GA-5.

According to the method for preparing the recombinant yeast strain for producing the glycyrrhetinic acid, the sequences of the codon-optimized CYP88D6 and CYP72A154 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2 and the encoding gene of CYB5 is shown as SEQ ID NO: 3, respectively.

In another aspect, the invention provides the use of a recombinant yeast strain according to the invention for the production of glycyrrhetinic acid.

In another aspect, the present invention relates to a method for producing glycyrrhetinic acid, the method comprising the step of fermenting a recombinant yeast strain producing glycyrrhetinic acid according to the present invention under suitable fermentation conditions.

The method for producing glycyrrhetinic acid according to the present invention comprises a step of fermenting a recombinant yeast strain producing glycyrrhetinic acid according to the present invention in a medium consisting of 50g/L glucose, 10g/L tryptone, 20g/L yeast extract, 0.2g/L uracil, 0.04mol/L methyl- β -cyclodextrin at a culture temperature of 30 ℃ at a pH of 5; after the glucose is exhausted, ethanol is fed and fermented, and the ethanol is fed every 12 hours to ensure that the concentration of the ethanol is 7 g/L.

Drawings

FIGS. 1A to 1F show the integration sequences of the gene expression clusters constructed in examples 3 to 7, respectively. Wherein the abbreviation E10 represents gene ERG10, E8 represents gene ERG8, E19 represents gene ERG19, E13 represents gene ERG13, and the abbreviation E12 represents gene ERG 12; 72A represents the gene CYP72A154, 88D6 represents the gene CYP88D6, OP88D6 represents the codon-optimized CYP88D6 gene, and OP72A represents the codon-optimized CYP72A154 gene.

FIG. 2A-FIG. 2C are schematic diagrams of a plasmid construction method in the embodiment of the present invention.

FIG. 2A, construction of cloning vectors with promoter-restriction site-terminator by seamless ligation scheme puc19L-Ptef1-Tpgk1, puc19L-Ptdh3-Tcyc1, puc19L-Ppgk1-Tadh1, puc19L-Ppgk1-Tpgk1, puc19L-Ptef2-Tcyc1 and puc19L-Ptdh3-Ttdh 3;

FIG. 2B shows that the construction of gene expression clusters is performed by means of enzyme digestion and ligation, and the ERG8, ERG12, IDI, ERG10, CYP72A154, OPCYP72A154 and Cyb5 genes in the patent are all expression clusters constructed by the means;

FIG. 2C shows the construction of gene expression clusters by seamless connection, in this patent Ptef2-ERG19-cyc, Ptdh3-CYP88D6-Tcyc1, Ptdh3-OPCYP88D6-Tcy, Ppgk 1-beta-AS-Tadh 1, Ppgk1-tHMG-Tadh1, Ptdh3-ERG20+9-Tcyc1, Ptdh3-ERG9+20-Tcyc1, Ptef1-ERG1-Tpgk1, Ptdh3-CPR1-Tcyc 1.

FIG. 3 shows LC-MS chromatograms. The upper diagram: LC-MS chromatogram of glycyrrhetinic acid standard product, wherein the abscissa is retention time, the ordinate is abundance, and the peak-appearance time is 6.72 min; the following figures: LC-MS chromatogram of engineering strain GA-5 fermentation sample with peak emergence time of 6.76 min.

FIG. 4 shows the LC-MS mass spectrum with M/Z on the abscissa and abundance on the ordinate. The upper diagram: LC-MS mass spectrum of 6.72min peak of glycyrrhetinic acid standard; the following figures: LC-MS mass spectrogram of the peak of the engineering strain GA-5 fermentation sample at 6.76 min.

FIG. 5 shows a GCMS detection map of beta-amyrin produced by fermentation of the engineered strain GA-5. The upper diagram: GCMS detects a beta-amyrin chromatogram of a fermentation sample, the beta-amyrin chromatogram is at the peak-off time of 16.44min, the horizontal coordinate is retention time, and the vertical coordinate is abundance; the following figures: GCMS detects the beta-amyrin essence spectrogram of a fermentation sample, wherein the abscissa is M/Z, and the ordinate is abundance.

FIG. 6 shows a GCMS detection map of glycyrrhetinic acid produced by fermentation of the engineered strain GA-5. The upper diagram: GCMS detects a glycyrrhetinic acid chromatogram of a fermentation sample, wherein the glycyrrhetinic acid chromatogram is at the peak-out time of 22.5min, the abscissa is retention time, and the ordinate is abundance; the following figures: GCMS detects the glycyrrhetinic acid mass spectrogram of a fermentation sample, wherein the abscissa is M/Z, and the ordinate is abundance.

FIG. 7 shows a schematic diagram of the chromosomal integration pattern of yeast.

Detailed Description

The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way. Those skilled in the art will appreciate that variations and other uses are included within the spirit and scope of the invention as defined by the scope of the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and the genes mentioned in the examples are all the entire coding frame regions of the genes unless otherwise specified. All are available from NCBI, and the promoter and terminator sequences mentioned are also available from NCBI download, and the specific sequence start positions can be known from primers in the primer tables.

Example 1 acquisition of genes required for Glycyrrhetinic acid Synthesis and Gene elements of Yeast itself in Modular control

Fresh Glycyrrhiza uralensis is used as a material, and RNA is extracted and reverse transcription is carried out by adopting a method known in the field to obtain cDNA. Designing primers of beta-AS, CYP88D6, CYP72A154 and Cyb5 genes according to a conventional method in the field, and amplifying the fragments; using genome of Saccharomyces cerevisiae CEN. PK2-1D as template, designing primers of ERG10, ERG12, ERG13, ERG19, ERG20, ERG9, ERG1, ERG8, IDI1 and tHMG gene segments, and amplifying the above-mentioned gene segments in yeast.

Example 2 acquisition of expression clusters consisting of genes required for Glycyrrhetinic acid Synthesis and Gene elements of Yeast itself in Modular control

Specifically, the expression cluster refers to a promoter + a gene fragment sequence + a terminator. The construction of the further expression cluster is divided into two steps: the constitutive promoters and terminator sequences commonly used in yeast were first amplified, where the amplified promoters include P-TEF1, P-TEF2, P-TDH3, P-ADH1 and P-PGK1 and the terminator sequences include: T-PGK1, T-CYC1, T-TDH3 and T-ADH1, and the primers used for amplifying the promoter and terminator sequences are shown in Table 1. The amplified promoter sequence and terminator sequence are connected to a cloning vector puc19L in the sequence of promoter-restriction enzyme site-terminator to construct puc19L-Ptef1-Tpgk1, puc19L-Ptdh3-Tcyc1, puc19L-Ppgk1-Tadh1, puc19L-Ppgk1-Tpgk1, puc19L-Ptef2-Tcyc1 and puc19L-Ptdh3-Ttdh3, and the connection of the promoter sequence and the terminator sequence is carried out in a seamless connection mode.

The genes amplified in example 1 were linked to the middle of the promoter and terminator by seamless linkage (AS shown in FIG. 2C) to construct expression clusters of the corresponding genes, specifically, Ptef2-ERG19-Tcyc1, Ptdh3-ERG9+20-Tcyc1, Ptdh 1-ERG1 +9-Tcyc1, Ptef1-ERG1-Tpgk1, Ppgk1-tHMG-Tadh1, Ppgk 1-beta-AS-Tadh 1, Ptdh 1-CYP 88D 1-Tcyc1, Ptdh 1-CPR 1-Tcyc1, ERG1, and IDI1, which were linked by enzyme digestion (AS shown in FIG. 2B) to the promoters of the Ptdh 1-Ppgh 1, the genes of the PtdTpdg 1-Ppgh 1, the genes of the Ptdh 1-Ppgh 1, the promoters of the genes of the TPdTpdh 1-1, the genes of the promoters of the genes of the TPdTpdh 1-1, the genes of, Ptdh3-Cyb5-Ttdh 3.

TABLE 1

Example 3 creation of a synthetic pathway for Glycyrrhetinic acid in Yeast

Ppgk 1-beta-AS-Tadh 1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 are integrated into the rDNA locus of a yeast chromosome in a homologous recombination mode (shown in figure 7), positive clones are detected by PCR, the capacity of the positive clone strains for producing glycyrrhetinic acid is detected, and the yeast strain GA named-1 with the highest glycyrrhetinic acid production is obtained. Wherein, the sequence of integration of the exogenous gene expression cluster fragments is shown in FIG. 1A.

Example 4 obtaining Glycyrrhetinic acid-producing Yeast Strain GA-2

CYP88D6, CYP72a154 were codon optimized, and the sequences of the codon optimized CYP88D6 and CYP72a15 are shown in table 1. Connecting a promoter and a terminator to form expression clusters Ptdh3-OPCYP88D6-Tcyc1 and Padh1-OPCYP72A154-adh1, integrating gene expression clusters Ppgk 1-beta-AS-Tadh 1, Ptdh3-OPCYP88D6-Tcyc1, Padh1-OPCYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 into rDNA sites of yeast Cen.pk2-1D bodies in a homologous recombination mode (shown in figure 7), detecting positive clones by PCR (polymerase chain reaction), detecting the glycyrrhetinic acid production capability of the positive clone strains, obtaining yeast strains with the highest glycyrrhetinic acid production named AS GA-1, and obtaining yeast strains with the highest glycyrrhetinic acid production named AS GA-2. The sequence in which the gene expression cluster fragments are integrated is shown in FIG. 1B.

TABLE 2

Example 5 obtaining Glycyrrhetinic acid-producing Yeast Strain GA-3

The yeast strain GA-2 is used as an initial strain, gene expression clusters of Ptdh3-E9+20-Tcyc1(Ptdh3-E20+9-Tcyc1), Ptef1-ERG1-Tpgk1 and Ppgk1-tHMG-Tadh1 are amplified, the fragments are integrated at delta sites of Saccharomyces cerevisiae GA-2) in a homologous recombination mode, positive clones are selected, the content of glycyrrhetinic acid is detected, and the engineering strain GA-3 with the highest glycyrrhetinic acid production is obtained. Wherein the sequence of integration of the gene expression cluster fragments is shown in FIG. 1C or FIG. 1D.

Example 6 obtaining Glycyrrhetinic acid-producing Yeast Strain GA-4

The method comprises the steps of using saccharomyces cerevisiae GA-3 as an initial strain, amplifying gene expression clusters of Ptdh3-ERG8-Ttdh3, Padh1-E13-Tadh1 and Padh1-E10-Tadh1, using his3 as a screening marker, integrating the gene expression clusters into Trp1 locus of saccharomyces cerevisiae GA-3 chromosome, selecting positive clone, detecting content of glycyrrhetinic acid, and using the strain with highest content of glycyrrhetinic acid as saccharomyces cerevisiae GA-4. The sequence in which the gene expression cluster fragments are integrated is shown in FIG. 1E.

Example 7 obtaining Glycyrrhetinic acid-producing Yeast Strain GA-5

By taking saccharomyces cerevisiae GA-4 as an initial strain, amplifying gene expression clusters of Padh1-ERG12-Tadh1, Ptdh3-Cyb5-Ttdh3, Ptef2-ERG19-cyc1 and Ppgk1-IDI-Tpgk1, respectively taking the sequences of-210-502 and 530-1008 of BTS1 as homologous sequences, taking trp1 as a screening marker, jointly integrating the homologous sequences into a chromosome of saccharomyces cerevisiae GA-4, and overexpressing the genes of ERG12, Cyb5 and ERG19 on the basis of breaking a coding frame of BTS1 gene to enhance the metabolic flow of a strain MVA pathway. Selecting positive clones, detecting the content of glycyrrhetinic acid, and taking the strain with the highest content of glycyrrhetinic acid as saccharomyces cerevisiae GA-5. The sequence in which the gene expression cluster fragments are integrated is shown in FIG. 1F. The GA-5 is preserved in the China general microbiological culture Collection center on 2016, 10 months and 21 days, and the preservation number is CGMCC 13126.

Example 8 Glycyrrhetinic acid production by fermentation of Yeast Strain GA-5

Through the metabolic engineering optimization, a high-yield glycyrrhetinic acid yeast strain GA-5 is obtained, and the fermentation conditions are further optimized, wherein the specific fermentation conditions are pH 5, the culture medium comprises 50g/L of glucose, 10g/L of tryptone, 20g/L of yeast extract, and uracil: 0.2g/L, methyl- β -cyclodextrin: 0.04 mol/L; after the glucose is exhausted, adopting an ethanol feeding fermentation strategy, and feeding ethanol every 12 hours to ensure that the concentration of the ethanol is 7 g/L; the culture temperature is 30 ℃, and finally the concentration of the glycyrrhetinic acid produced by shake flask fermentation reaches 445mg/L through GCMS detection.

Example 9 Glycyrrhetinic acid and intermediate metabolite production by Yeast cells samples and detection treatment methods

Sample treatment: taking 4ml of saccharomyces cerevisiae engineering strain, centrifuging for 10min at 12000rmp, removing supernatant, adding sterile water for cleaning for 3 times, centrifuging for 10min at 12000rmp, removing supernatant, adding 1ml of methanol acetone extracting agent (methanol: acetone (v/v) ═ 1:1), ultrasonically crushing for 10min, centrifuging for 10min at 12000rmp, collecting supernatant, taking 100ul, adding a lining tube, performing nitrogen blowing and drying, adding N-methyl-N-trimethylsilyltrifluoroacetamide, and performing derivatization treatment at 80 ℃ for 30 min.

Sample detection: the sample is qualitatively and quantitatively determined by Agilent 7890GC-7000MS/MS gas chromatography-triple tandem quadrupole mass spectrometry, and the injection port temperature is 300 ℃; the chromatographic column is as follows: 2 HP-5ms UI 15m × 0.25mm × 0.25 μm, connected in the middle by a Purge Ultimate Union (PUU); adopting a standard mode of 3.0mL/min to perform spacer purging; flow rate of the column: constant current, column 1: 1.1 mL/min; column 2: 1.3 mL/min; column oven temperature program: keeping at 80 deg.C for 1min, and heating to 310 deg.C at 20 deg.C/min for 17.5 min; the temperature of the transmission line is 300 ℃; back flushing setting: carrying out back flushing on the column incubator at 310 ℃ for 7min, wherein the auxiliary gas pressure is 50psi, and the sample inlet pressure is 2 psi; the ion source temperature is 280 ℃; the four-stage rods Q1 and Q2 are both 150 ℃; collision cell gas flow rate: helium flow 2.25mL/min, nitrogen flow 1.5 mL/min; the sample injection volume is 2ul, the data acquisition mode is adopted, the squalene, ergosterol, lanosterol and beta-balsamic alcohol adopt a full scan mode, the glycyrrhetinic acid is in an MRM mode, and specific qualitative ions are shown in the following table:

TABLE 3

A method for qualitatively detecting glycyrrhetinic acid in a sample by LCMS (liquid Crystal display System) comprises the following steps: an apparatus Agilent LCMS 6420, wherein a chromatographic column is an Agilent C18 chromatographic column, the column temperature is 25 ℃, the flow rate is 0.2ml/min, the mobile phase is methanol and water, and the gradient elution condition is 0-0.5 min and 1% of methanol; 0.5-1.0 min, 1-80% methanol; 1.0-2.0 min, 80-90% methanol; 2.0-3.5 min, 90-1% methanol, the sample injection volume is 2ul, and the collection m/z of the glycyrrhetinic acid qualitative mass spectrum is set as 469.44-355.38.

Sequence listing

<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine

<120> recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, and construction method and application thereof

<130> king Caesalpinia japonica

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 1482

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized CYP88D6

<400> 1

atggaagttc attgggtttg tatgtcagct gcaactttgt tggtttgtta catcttcggt 60

tctaagttcg ttagaaattt gaacggttgg tactacgatg ttaagttgag aagaaaggaa 120

catccattac caccaggtga catgggttgg ccattaattg gtgacttgtt gtcttttatt 180

aaggatttct cttcaggtca tccagattct tttattaaca atttggtttt gaagtacggt 240

agatcaggta tctataagac tcatttgttc ggtaacccat ctatcatcgt ttgtgaacca 300

caaatgtgta gaagagtttt gacagatgat gttaacttca agttgggtta cccaaagtct 360

attaaagaat tggctagatg tagaccaatg attgatgttt caaacgcaga acatagattg 420

tttagaagat taatcacatc tccaatcgtt ggtcataaag ctttagcaat gtacttggaa 480

agattggaag aaatcgttat taattcattg gaagaattgt cttcaatgaa gcatccagtt 540

gaattgttga aggaaatgaa gaaagtttct tttaaagcta tcgttcatgt ttttatgggt 600

tcttcaaacc aagatattat taagaaaatt ggttcttctt ttactgattt gtacaacggc 660

atgttctcta tcccaattaa tgttccaggt tttacattcc ataaagcttt ggaagcaaga 720

aagaaattgg ctaagatcgt tcaaccagtt gttgatgaaa gaagattaat gatcgaaaat 780

ggtccacaag aaggttcaca aagaaaggat ttgatcgata tcttgttaga agttaaagat 840

gaaaatggta gaaaattaga agatgaagat atttctgatt tgttaattgg tttgttattt 900

gctggtcatg aatctactgc aacatcattg atgtggtcta tcacttactt aacacaacat 960

ccacatatct tgaagaaagc taaggaagaa caagaagaaa tcactagaac aagattttct 1020

tcacaaaaac aattgtcatt gaaggaaatt aaacaaatgg tttatttgtc tcaagttatt 1080

gatgaaactt tgagatgtgc aaacattgct tttgcaactt ttagagaagc tacagcagat 1140

gttaacatca acggttacat catcccaaaa ggttggagag ttttgatttg ggctagagca 1200

atccatatgg attcagaata ctacccaaat ccagaagagt ttaatccatc tagatgggat 1260

gattacaatg ctaaagcagg tacattttta ccatttggtg ctggttcaag attgtgtcca 1320

ggtgctgatt tggcaaagtt ggaaatctct attttcttgc attacttttt gttaaattac 1380

agattggaaa gaattaatcc agaatgtcat gttacttcat taccagtttc taaaccaaca 1440

gataactgtt tggctaaagt tattaaagtt tcttgtgcat aa 1482

<210> 2

<211> 1572

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized CYP72A154 sequence

<400> 2

atggatgctt cttcaactcc aggtgcaatt tgggttgttt tgacagttat tttagctgca 60

attccaattt gggcttgtca tatggttaac actttgtggt tgagaccaaa gagattggaa 120

agacatttga gagcacaagg tttgcatggt gacccataca agttgtcttt ggataactca 180

aagcaaacat acatgttgaa gttgcaacaa gaagctcaat ctaagtcaat cggtttgtct 240

aaggatgatg ctgcaccaag aattttctct ttggcacatc aaactgttca taagtacggt 300

aaaaattctt ttgcttggga aggtactgca ccaaaagtta ttattacaga tccagaacaa 360

attaaagaag tttttaataa gattcaagat ttcccaaagc caaagttgaa cccaattgct 420

aagtacatct caatcggttt gatccaatac gaaggtgaca agtgggctaa gcatagaaag 480

attattaacc cagcattcca tttggaaaag ttgaagggca tgttgccagc tttttctcat 540

tcatgtcatg aaatgatctc taagtggaag ggtttgttgt cttcagatgg tacatgtgaa 600

gttgatgttt ggccattttt gcaaaatttg acttgtgatg ttatctcaag aacagctttt 660

ggttcttcat acgctgaagg tgcaaagatc ttcgaattgt tgaagagaca aggttacgca 720

ttgatgactg ctagatacgc aagaattcca ttatggtggt tgttgccatc tactacaaag 780

agaagaatga aggaaatcga tagaggtatc agagattcat tggaaggtat catcagaaag 840

agagaaaagg ctttgaagtc tggtaaatca acagatgatg atttgttagg tatcttgttg 900

caatctaacc atatcgaaaa taagggtgac gaaaactcta aatcagctgg tatgactaca 960

caagaagtta tggaagagtg taagttgttt tatttggctg gtcaagaaac tacagctgca 1020

ttgttagcat ggactatggt tttgttaggt aaacatccag aatggcaagc tagagcaaag 1080

caagaagttt tgcaaatctt cggtaaccaa aacccaaact tcgaaggttt gggtagattg 1140

aagatcgtta ctatgatctt gtacgaagtt ttgagattgt acccaccagg tatatatttg 1200

acaagagctt tgagaaagga tttgaagttg ggtaatttgt tattgccagc aggtgttcaa 1260

ttttctgttc caatcttgtt gatccatcat gatgaaggta tttggggtaa tgatgctaaa 1320

gaattcaatc cagaaagatt tgcagatggt attgctaagg caacaaaggg tcaagtttgt 1380

tacttcccat ttggttgggg tccaagaatc tgtgttggtc aaaacttcgc tttgttggaa 1440

gcaaagattg ttttgtcttt gttgttgcaa aacttctctt tcgaattatc accaacttac 1500

gctcatgttc caactacagt tttgacatta caaccaaaac atggtgcacc aattattttg 1560

cataaattat aa 1572

<210> 3

<211> 405

<212> DNA

<213> Glycyrrhiza uralensis

<400> 3

atggcttcag atccaaagct tcacactttc gatgaggtgg caaagcacaa ccagaccaaa 60

gattgctggc ttatcatttc tgggaaggtg tatgatgtca ccccttttat ggaggatcat 120

cccggaggtg atgaggtttt gttatctgca acagggaaag atgcaaccaa tgattttgaa 180

gatgtggggc acagtgattc tgctagagaa atgatggaca agtactatat tggtgagatt 240

gattcctcaa ctgtcccact aaagcgtacc tacgttccac ctcagcaaac ccagtacaac 300

cctgacaaga cctcggaatt cgtgatcaag atcttgcagt tcctggtccc tctcctcata 360

ttgggcttag cctttgttgt ccgacactac accaagaatg agtag 405

<210> 4

<211> 1579

<212> DNA

<213> Glycyrrhiza uralensis

<400> 4

tgcagaccaa ttggtgaaaa ctgaagtcac caagaagtct tttactgctc ctgtacaaaa 60

ggcttctaca ccagttttaa ccaataaaac agtcatttct ggatcgaaag tcaaaagttt 120

atcatctgcg caatcgagct catcaggacc ttcatcatct agtgaggaag atgattcccg 180

cgatattgaa agcttggata agaaaatacg tcctttagaa gaattagaag cattattaag 240

tagtggaaat acaaaacaat tgaagaacaa agaggtcgct gccttggtta ttcacggtaa 300

gttacctttg tacgctttgg agaaaaaatt aggtgatact acgagagcgg ttgcggtacg 360

taggaaggct ctttcaattt tggcagaagc tcctgtatta gcatctgatc gtttaccata 420

taaaaattat gactacgacc gcgtatttgg cgcttgttgt gaaaatgtta taggttacat 480

gcctttgccc gttggtgtta taggcccctt ggttatcgat ggtacatctt atcatatacc 540

aatggcaact acagagggtt gtttggtagc ttctgccatg cgtggctgta aggcaatcaa 600

tgctggcggt ggtgcaacaa ctgttttaac taaggatggt atgacaagag gcccagtagt 660

ccgtttccca actttgaaaa gatctggtgc ctgtaagata tggttagact cagaagaggg 720

acaaaacgca attaaaaaag cttttaactc tacatcaaga tttgcacgtc tgcaacatat 780

tcaaacttgt ctagcaggag atttactctt catgagattt agaacaacta ctggtgacgc 840

aatgggtatg aatatgattt ctaaaggtgt cgaatactca ttaaagcaaa tggtagaaga 900

gtatggctgg gaagatatgg aggttgtctc cgtttctggt aactactgta ccgacaaaaa 960

accagctgcc atcaactgga tcgaaggtcg tggtaagagt gtcgtcgcag aagctactat 1020

tcctggtgat gttgtcagaa aagtgttaaa aagtgatgtt tccgcattgg ttgagttgaa 1080

cattgctaag aatttggttg gatctgcaat ggctgggtct gttggtggat ttaacgcaca 1140

tgcagctaat ttagtgacag ctgttttctt ggcattagga caagatcctg cacaaaatgt 1200

tgaaagttcc aactgtataa cattgatgaa agaagtggac ggtgatttga gaatttccgt 1260

atccatgcca tccatcgaag taggtaccat cggtggtggt actgttctag aaccacaagg 1320

tgccatgttg gacttattag gtgtaagagg cccgcatgct accgctcctg gtaccaacgc 1380

acgtcaatta gcaagaatag ttgcctgtgc cgtcttggca ggtgaattat ccttatgtgc 1440

tgccctagca gccggccatt tggttcaaag tcatatgacc cacaacagga aacctgctga 1500

accaacaaaa cctaacaatt tggacgccac tgatataaat cgtttgaaag atgggtccgt 1560

cacctgcatt aaatcctaa 1579

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> P-PGK1-F primer

<400> 5

aaagatgccg atttgggc 18

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> P-PGK1-R primer

<400> 6

gttttatatt tgttgtaaaa agtagataat 30

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> P-TDH3-F primer

<400> 7

gacacaaggc aattgaccca c 21

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> P-TDH3-R primer

<400> 8

tttgtttgtt tatgtgtgtt tattc 25

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> P-TEF2-F primer

<400> 9

gataggtcaa gatcaatgta aacaattac 29

<210> 10

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> P-TEF2-R primer

<400> 10

aaacgtttag ttaattatag ttcgttga 28

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> T-CYC1-F primer

<400> 11

gtttaaacac aggccccttt tc 22

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> T-CYC1-R primer

<400> 12

aaaatatgca catgaggcga a 21

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> T-PGK1-F primer

<400> 13

gaaataaatt gaattgaatt gaaatc 26

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> T-PGK1-R primer

<400> 14

agctttaacg aacgcagaat tttc 24

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> T-ADH1F primer

<400> 15

gcgaatttct tatgatttat g 21

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> T-ADH1R primer

<400> 16

gaatgacgat gaagatagag c 21

<210> 17

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> P-TEF1-F primer

<400> 17

tcaatagtca tacaacagaa agc 23

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> P-TEF1-R primer

<400> 18

tttgtaatta aaacttagat tagat 25

<210> 19

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> P-ADH1-F primer

<400> 19

aaggaaaaaa gcggccgcgt tgtcctctga ggacataaaa taca 44

<210> 20

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> P-ADH1-R primer

<400> 20

ccggaattcc ccatacatcg ggattcctat aata 34

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> T-TDH3-F primer

<400> 21

gtgaatttac tttaaatctt gca 23

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> T-TDH3-R primer

<400> 22

tcctggcgga aaaaattcat t 21

Claims (9)

1. A recombinant yeast strain that produces glycyrrhetinic acid that overexpresses the yeast genes ERG10, ERG12, ERG13, ERG19, ERG20, ERG9, ERG1, ERG8, IDI1, and tmgh; the recombinant yeast strain expresses beta-AS, CYP88D6 and CYP72A154 genes in a glycyrrhetinic acid synthesis pathway and a cytochrome P450 reductase gene of arabidopsis; while the recombinant yeast strain deleted BTS 1.

2. The recombinant yeast strain producing glycyrrhetinic acid according to claim 1, wherein the CYP88D6 and CYP72a154 genes are codon optimized, and the codon optimized CYP88D6 and CYP72a154 sequences are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

3. A recombinant yeast strain for producing glycyrrhetinic acid is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 21 days in 2016, the preservation number is CGMCC13126, and the recombinant yeast strain is classified and named as Saccharomyces cerevisiae (Saccharomyces cerevisiae).

4. A method of making the recombinant yeast strain producing glycyrrhetinic acid of claim 1 or 2, the method comprising the steps of:

a) obtaining beta-AS, CYP88D6 and CYP72A154 genes participating in a glycyrrhetinic acid synthesis pathway from a glycyrrhiza plant, and obtaining a CPR1 gene fragment from Arabidopsis thaliana;

b) constructing Ppgk 1-beta-AS-Tadh 1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 gene expression clusters from the beta-AS, CYP88D6, CYP72A154 and CPR1 gene fragments obtained in the step a), and integrating the four gene expression clusters into the rDNA locus of yeast chromosome Cen.pk2-1D;

c) carrying out codon optimization on the CYP88D6 and CYP72A154 genes in the step b) to obtain OPCYP88D6 and OPCYP72A154 genes, constructing a Ptdh3-OPCYP88D6-Tcyc1 and a Padh1-OPCYP72A154-adh1 gene cluster, and integrating the gene clusters together with the Ppgk 1-beta-AS-Tadh 1 and the Ptdh3-CPR1-Tcyc1 gene cluster into an rDNA locus of the saccharomyces cerevisiae Cen.pk2-1D;

d) carrying out PCR amplification on ERG9, ERG20, ERG1 and tHMG (recombinant human growth hormone) fragments of a yeast strain obtained from yeast, fusing ERG20 and ERG9 genes to obtain a fused fragment ERG20+9 or ERG9+20, constructing corresponding gene expression clusters Ptdh3-E20+9-Tcyc1, Ptdh3-E9+20-Tcyc1, Ptef1-ERG1-Tpgk1 and Ppgk1-tHMG-Tadh1 together with ERG1 and tHMG, and integrating the gene clusters into the chromosomal site of the strain constructed in the step c);

e) carrying out PCR amplification on the ERG10, ERG8 and ERG13 of the yeast strain from the yeast, constructing corresponding gene clusters of the gene clusters of Padh1-E10-Tadh1, Ptdh3-ERG8-Ttdh3 and Padh1-E13-Tadh1, and integrating the three gene expression clusters to the position of +314bp of the trp site on the yeast chromosome in the step d) to ensure that the genes of ERG10, ERG8 and ERG13 are overexpressed; and

f) obtaining ERG12, ERG19 and IDI1 genes of yeast strains by PCR amplification from yeast, obtaining a CYB5 encoding gene of liquorice by amplification from the liquorice, constructing corresponding gene expression clusters of Padh1-ERG12-Tadh1, Ptef2-ERG19-cyc1, Ppgk1-IDI-Tpgk1 and Ptdh3-Cyb5-Ttdh3, and integrating the four gene expression clusters to the BTS1 gene locus of the yeast chromosome in the step e) to obtain the final recombinant saccharomyces cerevisiae.

5. The method of claim 4, wherein the sequences of the codon-optimized CYP88D6 and CYP72A154 are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, the encoding gene of CYB5 is shown as SEQ ID NO: 3, respectively.

6. The method according to claim 4 or 5, wherein the recombinant yeast strain for producing glycyrrhetinic acid obtained by the method is deposited at the China general microbiological culture Collection center (CGMCC) at 21/10/2016, with the collection number of CGMCC13126, and is classified and named as Saccharomyces cerevisiae (Saccharomyces cerevisiae).

7. Use of a recombinant yeast strain producing glycyrrhetinic acid according to any one of claims 1-3 in the production of glycyrrhetinic acid.

8. A method of producing glycyrrhetinic acid comprising the step of fermenting a recombinant yeast strain producing glycyrrhetinic acid according to any one of claims 1-3 under suitable fermentation conditions.

9. The method for producing glycyrrhetinic acid according to claim 8, comprising a step of fermenting a recombinant yeast strain producing glycyrrhetinic acid according to any one of claims 1-3 in a medium consisting of 50g/L glucose, 10g/L tryptone, 20g/L yeast extract, 0.2g/L uracil, 0.04mol/L methyl- β -cyclodextrin at a pH of 5 at a culture temperature of 30 ℃; after the glucose is exhausted, ethanol is fed and fermented, and the ethanol is fed every 12 hours to ensure that the concentration of the ethanol is 7 g/L.