WO2018120983A1

WO2018120983A1 - Glycyrrhetinic acid-producing recombinant saccharomyces cerevisiae, construction method for same, and uses thereof

Info

Publication number: WO2018120983A1
Application number: PCT/CN2017/105678
Authority: WO
Inventors: 陈士林; 王彩霞; 孙伟; 苏新尧; 张梦婷; 孙梦楚
Original assignee: 中国中医科学院中药研究所
Priority date: 2016-12-28
Filing date: 2017-10-11
Publication date: 2018-07-05
Also published as: CN106635853A; CN106635853B

Abstract

Provided are a Glycyrrhetinic acid-producing recombinant yeast strain, specifically the strain of deposit number CGMCC 13126, a preparation method for the strain, and uses of the strain in producing Glycyrrhetinic acid. The strain overexpresses yeast genes ERG10, ERG12, ERG13, ERG19, ERG20, ERG9, ERG1, ERG8, IDI1, and tHMG, expresses β-AS, CYP88D6, and CYP72A154 genes in a Glycyrrhetinic acid synthesis pathway and a cytochrome P450 reductase gene of Arabidopsis thaliana, and deletes BTS1 at the same time.

Description

Recombinant Saccharomyces cerevisiae producing glycyrrhetinic acid, its construction method and use thereof

Technical field

The present invention relates to the technical field of glycyrrhetinic acid production. In particular, the present invention relates to a recombinant Saccharomyces cerevisiae producing glycyrrhetinic acid, a method of constructing the same, and use thereof.

Background technique

Glycyrrhiza uralensis Fisch is a perennial herb of the genus Glycyrrhiza. It is rooted in stems and stems. It has the functions of replenishing spleen and replenishing qi, clearing away heat and detoxifying, relieving cough, relieving pain, reconciling and resolving various drugs. It is the most widely used tradition in China. Chinese herbal medicine. At present, about 200 kinds of compounds have been isolated from licorice, and are mainly classified into triterpenoids, flavonoids and polysaccharides. Among them, glycyrrhizic acid, also known as glycyrrhizin, is an oleanane-type pentacyclic triterpenoid saponin, which is the most active active ingredient of licorice, accounting for 2-8% of the dry weight of licorice. Modern pharmacological studies have confirmed that glycyrrhizic acid has a variety of pharmaceutically active activities, antibacterial and anti-inflammatory, regulating body immunity, and resisting ulcers. Glycyrrhizin has significant inhibitory effects on a variety of DNA and RNA viruses such as immunodeficiency virus (HIV), acute respiratory coronavirus (SARS), and hepatitis virus, with HIV inhibition rates as high as 90%. Glycyrrhizin also has antihyperlipidemic and anti-atherosclerotic effects, as well as anti-cancer and anti-cancer effects. In addition to having the above-mentioned effects, glycyrrhizic acid has a sweetness of about 150 times that of sucrose, and thus is widely used as a natural sweetener in the fields of food, tobacco, and the like.

Glycyrrhetinic acid is aglycone of glycyrrhizic acid. Pharmacological experiments show that glycyrrhizic acid can not be directly absorbed by the gastrointestinal tract after entering the human body. Instead, it is first decomposed into glycyrrhetinic acid by the intestinal flora in the human body, and is absorbed by the intestinal mucosa. The blood works. Therefore, glycyrrhizic acid actually exerts its pharmacological action in the form of its aglycone, namely glycyrrhetinic acid. The study of glycyrrhetinic acid confirmed that glycyrrhetinic acid has better pharmacological activity than glycyrrhizic acid. For example, in the anti-platelet aggregation, the in vitro test effect of glycyrrhetinic acid is better than that of glycyrrhizic acid. In addition, glycyrrhetinic acid also has good cytotoxicity, so it has a certain inhibitory effect on tumor cells and viruses. The -COOH and -OH functional groups in the structure of glycyrrhetinic acid are modified to obtain a variety of active derivatives. Up to now, more than 400 kinds of derivatives have been modified by using glycyrrhetinic acid as a skeleton, and 128 kinds of cytotoxic derivatives having an IC50 of <30 μM have been obtained. Except In addition to drugs, glycyrrhetinic acid is widely used in the field of cosmetics due to its anti-inflammatory and whitening effects. The content of glycyrrhetinic acid in licorice is much lower than that of licorice, accounting for 0.6%-1.6% of the dry weight of licorice. The difference between different populations in different regions is relatively large. For example, the content of glycyrrhetinic acid in licorice in Asia is generally less than 0.7%. . Therefore, glycyrrhetinic acid is generally obtained by hydrolysis of glycyrrhizic acid to remove two glucuronic acids. At present, the main method is to use a chemical acid hydrolysis method, and there is also a method of enzymatic hydrolysis, but the method of enzymatic hydrolysis is still at present. Laboratory exploration phase. Due to its low content in licorice and further hydrolysis with glycyrrhizic acid as raw material, the price of glycyrrhetinic acid is 2-3 times that of glycyrrhizic acid, which is about $500-700/kg.

Due to the wide range of uses of glycyrrhizic acid and glycyrrhetinic acid, the demand for licorice at home and abroad is very large. In the past two years, China's domestic demand has been about 60,000 to 70,000 tons per year, and exports have continued to grow. In 2007, the global licorice market transaction volume was approximately $42.1million. Compared with the huge demand of licorice, licorice resources are facing serious shortcomings. Due to the disorderly mining under huge demand, the wild licorice resources have been severely damaged and are on the verge of extinction. At present, the domestic wild licorice reserves are less than 200,000 tons. The wild licorice community has a strong wind and sand fixation function. Excessive mining caused wild licorice, and the vegetation damage caused by sandstorms increased. In 2000, the Chinese government issued a ban on excavation of wild licorice, while controlling the export of licorice. The current licorice resources are mainly planted with licorice, but the most important problem in the cultivation of licorice is that the glycyrrhizic acid content of its functional ingredient is less than 2%, which does not meet the requirements of the pharmacopoeia.

The structure of glycyrrhetinic acid is complex and it is not currently possible to carry out chemical synthesis. Extracting and separating from licorice is limited by limited licorice resources and its low content limit. At the same time, large-scale licorice excavation will bring about damage to the ecological environment and cause desertification. Synthetic biology is based on engineering theory, designing and synthesizing new biological components, or designing and transforming existing biological systems. Therefore, based on the idea of synthetic biology, by synthesizing the synthetic pathways of medicinal plant efficacy components, heterologous synthesis of medicinal efficacy components in Escherichia coli or yeast model strains provides new development for the sustainable, utilization and development of traditional Chinese medicine resources. opportunity. University of California, Keasling Research Group (Paddon C J, Westfall P J, Pitera D J, et al. High-level semi-synthetic production of the potent antimalarial artemisinin [J]. Nature, 2013, 496 (7446): 528.) By transferring the artemisinic acid synthesis pathway into yeast cells and regulating the metabolism, the constructed artemisinic acid-producing yeast strain ferments 25 g/L artemisinic acid, and then successfully synthesizes artemisinin after 4 steps of chemical catalysis. Greatly promoted the production of artemisinic acid. At present, this achievement has authorized international pharmaceutical companies to industrialize. produce. In addition, the synthesis of paclitaxel, tanshinone, ginsenoside, morphine, vinblastine or its precursor components has made some progress. Stephanopoulos (Ajikumar P K, Xiao W H, Tyo K E J, et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli [J]. Science, 2010, 330 (6000): 70.) Engineering through module access ( Modular pathway engineering), the taxadiene synthesis pathway is divided into two modules for regulation, one is the MEP pathway upstream of E. coli, which increases the IPP carbon flux into the steroidal precursor, and the other is heterogeneous The integration of the taxane synthesis pathway into E. coli, by regulating the metabolic flux of the two modules, ultimately yields the paclitaxel precursor component of paclitaxel to 1 g/L. Sarah E. O'Connor team (Brown S. Clastre M. Courtavault V. et al. 2015. De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci U S A.112(11):3205- 3210..) Through the series of gene rearrangements, the synthesis of isohumuline, an important precursor of vinblastine in microorganisms, was achieved. In addition to some necessary gene-introduced strains, the promoter was optimized and increased by 5 Additional copies of the genes tHMGR, MAF1, IDI1, SAM2, ZWF1, deletion of ERG20, ATF1, OYE2, iso-humulin production 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) ginseng-derived damadadiene synthase gene (PgDDS), proto-ginsengdiol synthase gene (CYP716A47) And the At CPR1 gene derived from Arabidopsis thaliana (A. thaliana) was introduced into the yeast strain, and the related genes tHMG1, ERG20, ERG9, ERG1, etc. were systematically regulated, and the relevant codons were optimized to construct the progeny. The engineering strain of panaxadiol, the yield of protopanaxadiol was 1.2g/L, which was 262 times higher than that of the original starting strain. This is the first time that ginseng saponin, an important precursor substance, has been synthesized from glucose in yeast.

The excavation and identification of functional genes is a premise for the realization of the synthetic biology of the functional components of natural products. For the research on glycyrrhizic acid biosynthesis pathway, some progress has been made at home and abroad. The synthesis route is basically clear. As a typical oleanane triterpenoid compound, the synthesis of glycyrrhizic acid is formed by cyclization, hydroxylation and glycosylation of the

common precursor

2,3 squalene of triterpenoids. Glycyrrhizinate. Specifically, 2,3 oxidized squalene is catalyzed by β-aromatic alcohol synthase (β-AS) to form β-aromatic alcohol, and β-AS gene is an important branch point for catalyzing the formation of glycyrrhizic acid. It has an important role in the core of the triterpenoid core. Next, the mother nucleus formed by a series of CYP450 genes is hydroxylated. Japan, 2008 Team Toshiya Muranaka at PNAS (Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K, Muranaka T. Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role In the biosynthesis of the triterpene sweetener glycyrrhizin. Proc Natl Acad Sci U S A. 2008, 105:14204-14209.) published an article using the licorice transcriptome information to successfully identify the CYP88D6 gene and achieve successful expression in the yeast system, This gene hydroxylates and further carboxylates C-11 of the β-aromatic alcohol. In 2011 the team was at 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. Triterpene functional genomics in licorice for identification of CYP72A154involved in the biosynthesis of glycyrrhizin. Plant Cell. 2011, 23:4112-4123.) published an article, also using the licorice transcriptome to further identify the CYP72A154 and CYP72A63 gene, the gene for the β-aromatic alcohol C-30 Catalytic formation of a carboxylic acid to form glycyrrhetinic acid. So far, all the catalytic genes of the glycyrrhetinic acid synthesis pathway have been identified. These work lay the foundation for the synthesis of glycyrrhetinic acid.

The use of microorganisms for the biosynthesis of natural products has many advantages, frees the dependence on raw materials, reduces land use, has a short growth cycle, low fermentation raw materials and high utilization rate, high efficiency of synthesis of fermentation products, few by-products, and conditions. It is mild, less polluting to the environment, and easy to separate.

Summary of the invention

Based on the application market of glycyrrhetinic acid and huge demand and limited limitation of current licorice resources, the present invention provides a strain of glycyrrhetinic acid producing yeast and a method for constructing the same, by overexpressing yeast 10 genes and 3 glycyrrhetinic acid synthesis The technology of exogenous genes and the deletion of yeast's own genes in the pathway can finally achieve the production of glycyrrhetinic acid by yeast fermentation.

In one aspect, the present invention provides a recombinant yeast strain producing glycyrrhetinic acid, the recombinant yeast strain overexpressing the yeast gene acetoacetyl-CoA thiolase gene (ERG10), mevalonate kinase gene (ERG12), HMG -CoA synthase gene (ERG13), mevalonate decarboxylase gene (ERG19), farnesyl pyrophosphate synthase (ERG20), squalene synthase (ERG9), squalene epoxidase (ERG1) a phosphomevalonate kinase gene (ERG8), an isopentenyl diphosphate isomerase gene (IDI1), and a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase (tHMG); Recombinant yeast strain expresses fragrant tree essence in the synthesis pathway of glycyrrhetinic acid The synthase (β-AS), CYP88D6 and CYP72A154 genes and the cytochrome P450 reductase (CPR1) gene of Arabidopsis thaliana; and the recombinant yeast strain deleted the GGPP synthase gene (BTS1).

The recombinant yeast strain according to the present invention is deposited at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on October 21, 2016, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101; the accession number is CGMCC13126; its classification is Saccharomyces cerevisiae.

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

a) obtaining the β-AS, CYP88D6 and CYP72A154 genes involved in the glycyrrhetinic acid synthesis pathway from licorice plants, and obtaining the CPR1 gene fragment from Arabidopsis thaliana;

b) constructing Ppgk1-β-AS-Tadh1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1, Ptdh3-CPR1-Tcyc1 gene expression clusters by using the β-AS, CYP88D6, CYP72A154 and CPR1 gene fragments obtained in step a) Integrating the above four gene expression clusters into the rDNA site of the yeast chromosome Cen.pk2-1D;

c) Codon-optimized the CYP88D6 and CYP72A154 genes in step b) to obtain the OPCYP88D6 and OPCYP72A154 genes, and construct the Ptdh3-OPCYP88D6-Tcyc1, Padh1-OPCYP72A154-adh1 gene cluster, together with Ppgk1-β-AS-Tadh1, Ptdh3-CPR1 -Tcyc1 gene cluster, integrated into the rDNA site of S. cerevisiae Cen.pk2-1D;

d) ERG9, ERG20, ERG1 and tHMG fragments of yeast strain were obtained by PCR amplification from yeast, and ERG20 and ERG9 genes were fused to obtain fusion fragment ERG20+9 or ERG9+20, and together with ERG1 and tHMG, the corresponding genes were constructed. The expression clusters Ptdh3-E20+9-Tcyc1, Ptdh3-E9+20-Tcyc1, Ptef1-ERG1-Tpgk1, Ppgk1-tHMG-Tadh1 integrate the above gene cluster into the chromosomal delta site of the strain constructed in step c);

e) ERG10, ERG8 and ERG13 of yeast strains were obtained by PCR amplification from yeast, and their corresponding gene clusters Padh1-E10-Tadh1, Ptdh3-ERG8-Ttdh3, Padh1-E13-Tadh1 were constructed, and these three gene expression clusters were integrated. To the +314 bp position of the trp site on the yeast chromosome to step d), the ERG10, ERG8 and ERG13 genes are over-subscribed Up; and

f) The ERG12, ERG19 and IDI1 genes of the yeast strain were obtained by PCR amplification from yeast, and the cytochrome b5 coding gene (CYB5) of licorice was obtained from the amplification of licorice, and the corresponding gene expression cluster Padh1-ERG12-Tadh1 was constructed. Ptef2-ERG19-cyc1, Ppgk1-IDI-Tpgk1, Ptdh3-Cyb5-Ttdh3, these four gene expression clusters were integrated into the BTS1 gene locus of the yeast chromosome in step e) to obtain the final recombinant Saccharomyces cerevisiae GA-5.

A method for producing a glycyrrhetic acid-producing recombinant yeast strain according to the present invention, wherein the sequences of the codon-optimized CYP88D6 and CYP72A154 are as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, of said CYB5 The coding gene is shown in SEQ ID NO: 3.

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 invention relates to a method of producing glycyrrhetinic acid, the method comprising the step of fermenting a glycyrrhetic acid-producing recombinant yeast strain according to the invention under suitable fermentation conditions.

A method for producing glycyrrhetinic acid according to the present invention, which comprises, at a pH of 5, at a culture temperature of 30 ° C, at 50 g/L glucose, 10 g/L tryptone, 20 g/L yeast extract, 0.2 a step of fermenting a glycyrrhetinic acid-producing recombinant yeast strain according to the present invention in a medium consisting of g/L uracil and 0.04 mol/L methyl-β-cyclodextrin; The fermentation was carried out, and ethanol was added every 12 hours so that the concentration of ethanol was 7 g/L.

DRAWINGS

1A to 1F show the integration order of the gene expression clusters constructed in Examples 3-7, respectively. The E10 abbreviation represents the gene ERG10, E8 represents the gene ERG8, E19 represents the gene ERG19, E13 represents the gene ERG13, E12 represents the gene ERG12; 72A represents the gene CYP72A154, 88D6 represents the gene CYP88D6, OP88D6 represents the codon-optimized CYP88D6 gene, OP72A represents the password Suboptimized CYP72A154 gene.

2A-2C are schematic views showing a method of constructing a plasmid in an embodiment of the present invention.

Figure 2A shows the construction of a cloning vector with a promoter-digestion site-terminator by seamless ligation, puc19L-Ptef1-Tpgk1, puc19L-Ptdh3-Tcyc1, puc19L-Ppgk1-Tadh1, puc19L-Ppgk1-Tpgk1 , puc19L-Ptef2-Tcyc1 and puc19L-Ptdh3-Ttdh3 are constructed by this method;

2B, the gene expression cluster is constructed by restriction enzyme ligation, and the ERG8, ERG12, IDI, ERG10, CYP72A154, OPCYP72A154 and Cyb5 genes in this patent are expression clusters constructed by this method;

Figure 2C, the construction of gene expression clusters by seamless ligation, in this patent Ptef2-ERG19-cyc, Ptdh3-CYP88D6-Tcyc1, Ptdh3-OPCYP88D6-Tcy, Ppgk1-β-AS-Tadh1, Ppgk1-tHMG-Tadh1, Ptdh3- ERG20+9-Tcyc1, Ptdh3-ERG9+20-Tcyc1, Ptef1-ERG1-Tpgk1, Ptdh3-CPR1-Tcyc1 are constructed by gene clustering in this manner.

Figure 3 shows the LC-MS chromatogram. Above: LC-MS chromatogram of glycyrrhetinic acid standard, the abscissa is retention time, the ordinate is abundance, the peak time is 6.72min; the following figure: LC-MS chromatogram of engineering strain GA-5 fermentation sample, peak Time 6.76min.

Figure 4 shows the LC-MS mass spectrum with the abscissa in M/Z and the ordinate as abundance. Upper panel: LC-MS mass spectrum of the peak of glycyrrhetinic acid standard at 6.72 min; lower panel: LC-MS mass spectrum of the peak of the engineered strain GA-5 fermentation sample at 6.76 min.

Figure 5 shows the GCMS detection pattern of β-香树精 produced by the engineering strain GA-5. Above: GCMS detects the β-fragrant extract chromatogram of the fermentation sample. The peak time is 16.44min, which is the chromatogram of β-香树精, the abscissa is the retention time, the ordinate is the abundance; the following figure: GCMS detects the fermentation sample β - Fragrant tree mass spectrum, the abscissa is M/Z, and the ordinate is abundance.

Figure 6 shows the GCMS detection of the production of glycyrrhetinic acid by the engineering strain GA-5. Above: GCMS detected the chromatogram of the glycyrrhetinic acid of the fermentation sample. The chromatogram of glycyrrhetinic acid was observed at 22.5 min. The abscissa was the retention time and the ordinate was the abundance. The following figure: GCMS detected the fermentation sample glycyrrhetinic acid mass spectrum The abscissa is M/Z and the ordinate is abundance.

Figure 7 shows a schematic diagram of yeast chromosome integration.

detailed description

The following examples are given for the purpose of illustrating the various embodiments of the 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 within the spirit of the invention as defined by the scope of the claims. The materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and the genes mentioned in the examples are, unless otherwise specified, the complete coding region of the gene. Both can be obtained from NCBI, and the mentioned promoter and terminator sequences are also available for download from the NCBI, and the specific sequence start position can be obtained from the primers in the primer table.

Example 1. Genes required for glycyrrhetinic acid synthesis and acquisition of yeast self-gene components in modular regulation

Using fresh ural licorice as a material, RNA is extracted and reverse transcribed to obtain cDNA by a method known in the art. According to the conventional methods in the art, the primers of β-AS, CYP88D6, CYP72A154 and Cyb5 genes were designed to amplify the above fragments; the genomes of Saccharomyces cerevisiae CEN.PK2-1D were used as templates to design ERG10, ERG12, ERG13, ERG19, ERG20, Primers of ERG9, ERG1, ERG8, IDI1, and tHMG gene fragments amplify the above gene fragments in yeast.

Example 2. Genes required for glycyrrhetinic acid synthesis and acquisition of expression clusters of yeast self-gene elements in modular regulation

Specifically, the expression cluster refers to a promoter + gene fragment sequence + terminator. The construction of further expression clusters is divided into two steps: first, the constitutive promoter and terminator sequences commonly used in yeast are amplified, and the promoters amplified here include P-TEF1, P-TEF2, P-TDH3, and P-ADH1. And P-PGK1, the terminator sequence includes: T-PGK1, T-CYC1, T-TDH3 and T-ADH1, and the primers used to amplify the above promoter and terminator sequences are shown in Table 1. The amplified promoter sequence and terminator sequence were ligated into the cloning vector puc19L in the order of the promoter-restriction site-terminator to construct puc19L-Ptef1-Tpgk1, puc19L-Ptdh3-Tcyc1, puc19L-Ppgk1-Tadh1 The puc19L-Ppgk1-Tpgk1, puc19L-Ptef2-Tcyc1, puc19L-Ptdh3-Ttdh3 vector, the promoter and terminator sequences were ligated in a seamless manner.

The gene amplified in Example 1 was ligated into the middle of the promoter and the terminator by seamless ligation (as shown in Fig. 2C), and the expression cluster of the corresponding gene was constructed, specifically, Ptef2-ERG19-Tcyc1, Ptdh3 -ERG9+20-Tcyc1, Ptdh3-ERG20+9-Tcyc1, Ptef1-ERG1-Tpgk1, Ppgk1-tHMG-Tadh1, Ppgk1-β-AS-Tadh1, Ptdh3-CYP88D6-Tcyc1, Ptdh3-CPR1-Tcyc1, ERG8, ERG10 The genes ERG12, ERG13, and IDI1 are ligated into the promoter and the terminator by restriction enzyme ligation (Fig. 2B) to form Ptdh3-ERG8-Ttdh3, Padh1-ERG10-Tadh1, Ptdh3-ERG12-Ttdh3, Padh1-ERG13-Tadh1, Padh1-CYP72A154-adh1, Padh1-Cyb5-adh1, Ppgk1-IDI1-Tpgk1, Ptdh3-Cyb5-Ttdh3.

Table 1

Example 3. Synthesis of glycyrrhetinic acid in yeast

The expression clusters of Ppgk1-β-AS-Tadh1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 were integrated into the rDNA locus of the yeast chromosome in the form of homologous recombination (shown in Figure 7). Positive clones were detected by PCR, and the ability of the positive clones to produce glycyrrhetinic acid was detected, and the yeast strain with the highest glycyrrhetinic acid was named GA-1. The sequence of integration of the exogenous gene expression cluster fragments is shown in FIG. 1A.

Example 4. Obtaining glycyrrhetinic acid yeast strain GA-2

The CYP88D6, CYP72A154 codon-optimized, codon-optimized sequences of CYP88D6 and CYP72A15 are shown in Table 1. The promoter and terminator are ligated to form expression clusters Ptdh3-OPCYP88D6-Tcyc1 and Padh1-OPCYP72A154-adh1, and Ppgk1-β-AS-Tadh1, Ptdh3-OPCYP88D6-Tcyc1 The expression clusters of Padh1-OPCYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 were integrated into the rDNA site of yeast-stained Cen.pk2-1D in a homologous recombination format (shown in Figure 7). Positive clones were detected by PCR and positive clones were detected. The ability of the strain to produce glycyrrhetinic acid, the yeast strain with the highest glycyrrhetinic acid was named GA-1, and the yeast strain with the highest glycyrrhetinic acid was named 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 yeast strain GA-3

The yeast gene strain GA-2 was used as the starting strain to amplify the gene expression clusters of Ptdh3-E9+20-Tcyc1 (Ptdh3-E20+9-Tcyc1), Ptef1-ERG1-Tpgk1, Ppgk1-tHMG-Tadh1, and these fragments were identical. The recombinant form was integrated into the delta site of Saccharomyces cerevisiae GA-2), and positive clones were selected to detect the content of glycyrrhetinic acid to obtain the yeast strain GA-3, which has the highest production of glycyrrhetinic acid. The order in which the gene expression cluster fragments are integrated is shown in Fig. 1C or Fig. 1D.

Example 6. Obtaining glycyrrhetinic acid yeast strain GA-4

Using Saccharomyces cerevisiae GA-3 as the starting strain, the gene expression clusters of Ptdh3-ERG8-Ttdh3, Padh1-E13-Tadh1, Padh1-E10-Tadh1 were amplified, and these gene expression clusters were integrated into Saccharomyces cerevisiae GA using his3 as a selection marker. The Trp1 site of the -3 chromosome was selected, and positive clones were selected to detect the content of glycyrrhetinic acid. The strain with the highest content of glycyrrhetinic acid was named 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 yeast strain GA-5

The S. cerevisiae GA-4 was used as the starting strain to amplify the gene expression clusters of Padh1-ERG12-Tadh1, Ptdh3-Cyb5-Ttdh3, Ptef2-ERG19-cyc1, Ppgk1-IDI-Tpgk1, and the sequence of BTS1-210-502 and The sequence of 530-1008 is a homologous sequence, and trp1 is used as a selection marker to integrate into the chromosome of Saccharomyces cerevisiae GA-4. The ERG12, Cyb5 and ERG19 genes are overexpressed based on the coding framework of the BTS1 gene, and the strain MVA is enhanced. The metabolic flux of the pathway. Positive clones were selected to detect the content of glycyrrhetinic acid, and the strain with the highest content of glycyrrhetinic acid was named Saccharomyces cerevisiae GA-5. The sequence in which the gene expression cluster fragments are integrated is shown in Fig. 1F. The GA-5 was deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 21, 2016, and the deposit number is CGMCC13126.

Example 8. Fermentation of Glycyrrhetinic Acid by Yeast Strains GA-5

Through the above metabolic engineering optimization, the high-yield glycyrrhetinic acid yeast strain GA-5 was obtained, and the fermentation conditions were further optimized. The specific fermentation conditions were pH=5, the composition of the medium was glucose: 50 g/L, and tryptone: 10 g/ L, yeast extract: 20g / L, uracil: 0.2g / L, methyl - β-cyclodextrin: 0.04mol / L; after glucose depletion, using ethanol feed fermentation strategy, every 12h Ethanol made the concentration of ethanol 7g / L; the culture temperature was 30 ° C, and finally passed the GCMS test, the concentration of glycyrrhetinic acid produced by shake flask fermentation reached 445 mg / L.

Example 9. Samples and detection sites for glycyrrhetinic acid and intermediate metabolites produced by yeast cells Method

Sample treatment: Take 4ml of S. cerevisiae engineering strain, centrifuge for 10min at 12000rmp, discard the supernatant, add 3 times with sterile water, centrifuge for 10min at 12000rmp, discard the supernatant, and add 1ml of methanol acetone extractant (methanol: acetone (v/v)= 1:1), sonication for 10min, centrifugation at 12000rmp for 10min, the supernatant was collected, 100ul was added to the inner liner for nitrogen blowing, and N-methyl-N-trimethylsilyltrifluoroacetamide was added to derivatize at 80 °C. Processed for 30 min.

Sample testing: The sample is qualitative and quantitative using the instrument model Agilent 7890GC-7000MS/MS gas chromatography-triple quadrupole mass spectrometer, the inlet temperature is 300 ° C; the column is: 2 HP-5ms UI 15m × 0.25mm × 0.25 μm, connected by Purged Ultimate Union (PUU) in the middle; septum purge using a standard mode of 3.0 mL/min; column flow rate: constant flow, column 1: 1.1 mL/min; column 2: 1.3 mL/min; Column oven heating program: 80 ° C for 1 min, at 20 ° C / min to 310 ° C for 17.5 min; transmission line temperature is 300 ° C; backflush setting: column oven at 310 ° C backflush for 7 min, auxiliary gas pressure 50 psi The inlet pressure is 2 psi; the ion source temperature is 280 ° C; the four-stage rods Q1 and Q2 are both 150 ° C; the collision cell gas flow rate: helium gas flow 2.25 mL/min, nitrogen flow rate 1.5 mL/min; injection volume 2 ul , data acquisition mode, squalene, ergosterol, lanosterol and β-aromatic alcohol use full scan scan mode, glycyrrhetinic acid is MRM mode, the specific qualitative ions are shown in the following table:

table 3

Qualitative determination of glycyrrhetinic acid LCMS in the sample: Agilent LCMS 6420, Agilent C18 column, column temperature 25 ° C, flow rate 0.2 ml / min, mobile phase methanol and water, gradient elution conditions, 0–0.5 min, 1% 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, injection volume 2 ul, licorice The secondary acid qualitative mass spectrometry acquisition m/z was set to 469.44-355.38.

Claims

A recombinant yeast strain producing glycyrrhetinic acid, the recombinant yeast strain overexpressing the yeast genes ERG10, ERG12, ERG13, ERG19, ERG20, ERG9, ERG1, ERG8, IDI1 and tHMG; and the recombinant yeast strain expressing glycyrrhetinic acid The β-AS, CYP88D6 and CYP72A154 genes in the synthetic pathway and the cytochrome P450 reductase gene of Arabidopsis thaliana; and the recombinant yeast strain deleted BTS1.
The glycyrrhetinic acid-producing recombinant yeast strain according to claim 1, wherein the CYP88D6 and CYP72A154 genes are codon-optimized, and the codon-optimized CYP88D6 and CYP72A154 sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Shown.
A recombinant yeast strain producing glycyrrhetinic acid, which was deposited on October 21, 2016 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, with the accession number CGMCC13126.
A method of preparing a glycyrrhetinic acid-producing recombinant yeast strain according to claim 1 or 2, the method comprising the steps of:

a) obtaining the β-AS, CYP88D6 and CYP72A154 genes involved in the glycyrrhetinic acid synthesis pathway from licorice plants, and obtaining the CPR1 gene fragment from Arabidopsis thaliana;

b) constructing Ppgk1-β-AS-Tadh1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1, Ptdh3-CPR1-Tcyc1 gene expression clusters by using the β-AS, CYP88D6, CYP72A154 and CPR1 gene fragments obtained in step a) Integrating the above four gene expression clusters into the rDNA site of the yeast chromosome Cen.pk2-1D;

c) Codon-optimized the CYP88D6 and CYP72A154 genes in step b) to obtain the OPCYP88D6 and OPCYP72A154 genes, and construct the Ptdh3-OPCYP88D6-Tcyc1, Padh1-OPCYP72A154-adh1 gene cluster, together with Ppgk1-β-AS-Tadh1, Ptdh3-CPR1 -Tcyc1 gene cluster, integrated into the rDNA site of S. cerevisiae Cen.pk2-1D;

d) PCR amplification of yeast to obtain ERG9, ERG20, ERG1 and yeast strains The tHMG fragment was fused with the ERG20 and ERG9 genes to obtain the fusion fragment ERG20+9 or ERG9+20. Together with ERG1 and tHMG, the corresponding gene expression clusters Ptdh3-E20+9-Tcyc1, Ptdh3-E9+20-Tcyc1 were constructed. Ptef1-ERG1-Tpgk1, Ppgk1-tHMG-Tadh1 integrate the above gene cluster into the chromosomal delta site of the strain constructed in step c);

e) ERG10, ERG8 and ERG13 of yeast strains were obtained by PCR amplification from yeast, and their corresponding gene clusters Padh1-E10-Tadh1, Ptdh3-ERG8-Ttdh3, Padh1-E13-Tadh1 were constructed, and these three gene expression clusters were integrated. The +314 bp position of the trp site on the yeast chromosome to step d) overexpresses the ERG10, ERG8 and ERG13 genes;

f) The ERG12, ERG19 and IDI1 genes of yeast strain were obtained by PCR amplification from yeast, and the coding gene of CYB5 of licorice was obtained from the amplification of licorice, and the corresponding gene expression clusters Padh1-ERG12-Tadh1, Ptef2-ERG19-cyc1 were constructed. , Ppgk1-IDI-Tpgk1, Ptdh3-Cyb5-Ttdh3, integrate these four gene expression clusters into the BTS1 gene locus of the yeast chromosome in step e) to obtain the final recombinant Saccharomyces cerevisiae.
The method according to claim 4, wherein the sequences of the codon-optimized CYP88D6 and CYP72A154 are as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the coding gene of CYB5 is as set forth in SEQ ID NO: Show.
The method according to claim 4 or 5, wherein the glycyrrhetinic acid-producing recombinant yeast strain obtained by the method is deposited on October 21, 2016 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, and the preservation number is For CGMCC13126.
Use of a glycyrrhetinic acid-producing recombinant yeast strain according to any one of claims 1 to 3 for producing glycyrrhetinic acid.
A method of producing glycyrrhetinic acid, the method comprising the step of fermenting a glycyrrhetic acid-producing recombinant yeast strain according to any one of claims 1-3 under suitable fermentation conditions.
The method for producing glycyrrhetinic acid according to claim 8, which comprises, at pH = 5, at a culture temperature of 30 ° C, at 50 g / L glucose, 10 g / L tryptone, 20 g / L yeast extract, Step of fermenting a glycyrrhetic acid-producing recombinant yeast strain according to any one of claims 1 to 3 in a medium consisting of 0.2 g/L uracil and 0.04 mol/L methyl-β-cyclodextrin; to be glucose After depletion, the ethanol was fed and fermented, and ethanol was added every 12 hours to make the concentration of ethanol 7 g/L.