Glycyrrhetinic acid biosensor and application thereof

Abstract

The invention relates to the field of microbial engineering, in particular to a glycyrrhetinic acid biosensor and application thereof. The glycyrrhetinic acid biosensor comprises a PDR5 promoter. The glycyrrhetinic acid biosensor has high specific response to glycyrrhetinic acid, and the specific response can be adjusted by inserting a transcription factor binding sequence into a PDR5 promoter. The biosensor can be applied to high-throughput detection and screening of glycyrrhetinic acid high-yield strains and stable inheritance and adaptive evolution of the high-yield strains. The glycyrrhetinic acid biosensor and the application of the glycyrrhetinic acid biosensor provide an effective method for rapid breeding of saccharomyces cerevisiae strains with high yield of glycyrrhetinic acid.

Description

Technical Field

[0001] The invention relates to the field of microbial engineering, and in particular to a glycyrrhetinic acid biosensor and application thereof. Background Art

[0002] Glycyrrhizic acid is the main active ingredient of the traditional Chinese medicine licorice. It is a typical pentacyclic triterpene compound and is widely used in medicine, food, cosmetics and health products. At present, the main method for obtaining glycyrrhizic acid is to extract glycyrrhizic acid from the root of licorice, and then obtain glycyrrhizic acid through acid hydrolysis or enzyme hydrolysis. The process includes drying, grinding, crushing, hydrolysis and other steps, and the production process is complicated. In addition, licorice has a well-developed root system, which can prevent wind and sand, and has important ecological application value. The acquisition mode of "digging roots to extract acid" seriously damages the vegetation distribution of licorice and the local ecological environment, which is not conducive to environmental protection.

[0003] In recent years, the development of synthetic biology and metabolic engineering has endowed microorganisms with the ability to synthesize heterologous terpene compounds. At the same time, the advantages of using microorganisms to synthesize natural products have become increasingly obvious, becoming a potential green and environmentally friendly alternative method. Researchers have constructed a Saccharomyces cerevisiae cell factory capable of synthesizing glycyrrhetinic acid, with the authorization number: ZL201610965315.9. At the same time, the development of iterative omics technology, gene editing technology and in vitro assembly technology has promoted the construction and optimization of cell factories, providing favorable conditions for further improving the production efficiency of terpene compounds. However, the current detection method of glycyrrhetinic acid mainly relies on gas chromatography, which requires cell samples to be crushed, extracted, derivatized, chromatographically detected, etc. The steps are cumbersome, the sample processing efficiency is low, and the repeatability is poor. The detection process has become the biggest bottleneck hindering the high-throughput construction and optimization of the cell factory for synthesizing glycyrrhetinic acid.

[0004] Therefore, a rapid and high-throughput method for detecting glycyrrhizic acid is urgently needed in the relevant technology. Summary of the invention

[0005] The present invention solves one of the problems of the related art from at least the following aspects.

[0006] The first aspect of the present application provides a glycyrrhizic acid biosensor, comprising: a PDR5 promoter.

[0007] In some embodiments, the glycyrrhetinic acid biosensor further comprises a reporter gene.

[0008] In some embodiments, the reporter gene is a gene encoding a fluorescent protein or a gene encoding histidine synthetase.

[0009] In some embodiments, the glycyrrhizic acid biosensor further comprises: a transcription factor binding sequence inserted into one or more sites in the PDR5 promoter, wherein the transcription factor binding sequence is selected from the group consisting of: a first motif (TCCGCGGA), a second motif (TTACTAA) and a third motif (CGGAACGA).

[0010] In some embodiments, the one or more sites include: a first site, a second site, and a third site, wherein the first site is located at any one of the 501st to 525th nucleotides from the 3' end of the PDR5 promoter, the second site is located at any one of the 374th to 419th nucleotides from the 3' end of the PDR5 promoter, and the third site is located at any one of the 92nd to 239th nucleotides from the 3' end of the PDR5 promoter.

[0011] In some embodiments, the first site is located at the 513th nucleotide from the 3' end of the PDR5 promoter, the second site is located at the 407th nucleotide from the 3' end of the PDR5 promoter, and the third site is located at the 220th nucleotide from the 3' end of the PDR5 promoter.

[0012] In some embodiments, there is no inserted sequence at the first site, or the first sequence inserted at the first site is: the second motif, the third motif, or a combination of the second motif and the third motif.

[0013] In some embodiments, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif, the second motif, the third motif, a combination of the first motif and the third motif, a combination of the second motif and the third motif, a combination of the first motif and the second motif, and a combination of the first to third motifs.

[0014] In some embodiments, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the second motif, the third motif, or a combination of the second motif and the third motif.

[0015] In some embodiments, at least two of the one or more sites are inserted with the transcription factor binding sequence.

[0016] In some embodiments, there is no inserted sequence at the first site, or the first sequence inserted at the first site is a first motif or a second motif.

[0017] In some embodiments, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif.

[0018] In some embodiments, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the first motif.

[0019] In some embodiments, at least one of the one or more sites is inserted with the transcription factor binding sequence.

[0020] The second aspect of the present application provides a Saccharomyces cerevisiae comprising the glycyrrhetinic acid biosensor described in any one of the embodiments of the first aspect.

[0021] In some embodiments, the starting strain of Saccharomyces cerevisiae is selected from the group consisting of: S288C, SynV, GA183, GA184.

[0022] In some embodiments, the Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or molecular chaperone protein (SSZ1), wherein the nucleotide sequence of the gene encoding RDR1 is shown in SEQ ID NO:8, and the nucleotide sequence of the gene encoding SSZ1 is shown in SEQ ID NO:9.

[0023] The third aspect of the present application provides the use of the glycyrrhetinic acid biosensor described in any embodiment of the first aspect or the brewer's yeast described in any embodiment of the second aspect in obtaining brewer's yeast with high glycyrrhetinic acid production.

[0024] Compared with the related art, the embodiments of the present invention achieve at least the following beneficial effects:

[0025] The glycyrrhetinic acid biosensor of the embodiment of the present invention has a high specific response to glycyrrhetinic acid and the specific response can be adjusted by inserting a transcription factor binding sequence into the PDR5 promoter. The biosensor can be applied to high-throughput detection and screening of glycyrrhetinic acid high-producing strains, as well as the stable inheritance and adaptive evolution of high-producing strains.

[0026] The glycyrrhetinic acid biosensor and its application in the embodiment of the present invention provide a simple and effective method for the rapid breeding of brewer's yeast strains with high glycyrrhetinic acid production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The composition and working principle of the glycyrrhetinic acid biosensor in the embodiment of the present invention;

[0029] Figure 2 Schematic diagram of the PDR5 promoter and the mutation site in the embodiment of the present invention, wherein the -800 end is the 5' end, the -1 end is the 3' end, and the numerical values ​​are the same as the nucleotide values;

[0030] Figure 3 The fermentation test results of the strains selected in Example 5 of the present invention are as follows;

[0031] Figure 4 This is a schematic diagram of the application of the glycyrrhizic acid biosensor in the rapid evolution of strains in Example 6 of the present invention;

[0032] Figure 5 The glycyrrhizic acid biosensor in Example 6 of the present invention is applied to the rapid evolution of strains, wherein Figure 5 a is the yield of fermentation product of yeast strain GA184 without glycyrrhizic acid biosensor after passage in YPD medium, Figure 5 b is the yield of the fermentation product of the strain (E0) after subculturing in SD-HIS medium using the glycyrrhizic acid biosensor;

[0033] Figure 6 The change of fluorescence intensity of reporter gene expression in the strain after adding different concentrations of glycyrrhetinic acid in the characterization test of the glycyrrhetinic acid biosensor of the present invention;

[0034] Figure 7 The strain SynV (SynV-P that does not produce glycyrrhetinic acid) containing the gene circuit of the PDR5 promoter-mCherry reporter gene in the characterization test of the glycyrrhetinic acid biosensor of the present invention is PDR5 ) and GA183 (GA183-P producing glycyrrhetinic acid PDR5 ) Fluorescence intensity detection results during the fermentation process;

[0035] Figure 8 The results of adjusting the response intensity of the glycyrrhetinic acid biosensor by modifying the glycyrrhetinic acid response sequence in Example 3 of the present invention;

[0036] Fig. 9 The results of the modification and optimization experiment of the glycyrrhetinic acid biosensor of the present invention by knocking out the endogenous gene of Saccharomyces cerevisiae to adjust the response intensity of the glycyrrhetinic acid biosensor;

[0037] Fig.10 To explore P in Example 3 of the present invention PDR5 - Experimental results of PE3 response characteristics.

[0038] In order to facilitate the understanding of the present invention, the implementation process of the present invention is further described below in conjunction with specific implementation examples. These descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0040] In the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0041] The present invention is made based on the following knowledge of the inventors:

[0042] With the rapid development of synthetic biology in recent years, genetically encoded biosensors have gradually developed into convenient tools for qualitative and quantitative analysis and detection of compounds. The high-throughput screening method constructed based on this can quickly and accurately screen high-yield strains from large mutant libraries.

[0043] The first aspect of the present invention provides a glycyrrhetinic acid biosensor, comprising: a PDR5 promoter.

[0044] As an example, the nucleotide sequence of the PDR5 promoter is shown in SEQ ID NO:1.

[0045] An embodiment of the present invention provides a genetically encoded glycyrrhetinic acid biosensor. By performing transcriptome analysis on brewer's yeast that synthesizes glycyrrhetinic acid, it is discovered that the expression level of gene PDR5 is significantly increased in brewer's yeast that synthesizes glycyrrhetinic acid. Therefore, the PDR5 promoter and reporter gene are assembled into a glycyrrhetinic acid biosensor to indicate the synthesis level of glycyrrhetinic acid.

[0046] In some embodiments, the glycyrrhetinic acid biosensor further comprises a reporter gene.

[0047] In some embodiments, the reporter gene is a gene encoding a fluorescent protein or a gene encoding histidine synthetase.

[0048] As an example, the fluorescent protein gene includes, but is not limited to: mCherry, mOrange, YFP, EGFP, ECFP or EBFP, and the amino acid sequences of the fluorescent proteins are shown in SEQ ID NOs: 2-7, respectively.

[0049] As an example, the nucleotide sequence of the gene encoding histidine synthetase (HIS3) is shown in SEQ ID NO:10.

[0050] In some embodiments, the glycyrrhetinic acid biosensor further comprises: a transcription factor binding sequence inserted into one or more sites of the PDR5 promoter, wherein the transcription factor binding sequence is selected from the group consisting of: a first motif (TCCGCGGA), a second motif (TTACTAA), and a third motif (CGGAACGA).

[0051] In some embodiments, the one or more sites include: a first site, a second site, and a third site, wherein the first site is located at any one of the 501st to 525th nucleotides from the 3' end of the PDR5 promoter, the second site is located at any one of the 374th to 419th nucleotides from the 3' end of the PDR5 promoter, and the third site is located at any one of the 92nd to 239th nucleotides from the 3' end of the PDR5 promoter.

[0052] In some embodiments, the first site is located at the 513th nucleotide from the 3' end of the PDR5 promoter, the second site is located at the 407th nucleotide from the 3' end of the PDR5 promoter, and the third site is located at the 220th nucleotide from the 3' end of the PDR5 promoter.

[0053] It is understandable that the above-mentioned sites and insertion sequences can be combined.

[0054] It can be understood that the above sites are counted according to the nucleotide positions of the original PDR5 promoter (ie, SEQ ID NO: 1).

[0055] In some embodiments, there is no inserted sequence at the first site, or the first sequence inserted at the first site is: the second motif, the third motif, or a combination of the second motif and the third motif.

[0056] In some embodiments, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif, the second motif, the third motif, a combination of the first motif and the third motif, a combination of the second motif and the third motif, a combination of the first motif and the second motif, and a combination of the first to third motifs.

[0057] In some embodiments, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the second motif, the third motif, or a combination of the second motif and the third motif.

[0058] In some embodiments, at least two of the one or more sites are inserted with the transcription factor binding sequence.

[0059] In some embodiments, there is no insertion sequence at the first site, or the first sequence inserted at the first site is: the second motif, the third motif, or a combination of the second and third motifs; there is no insertion sequence at the second site, or the second sequence inserted at the second site is: the first motif, the second motif, the third motif, a combination of the first and third motifs, a combination of the second and third motifs, a combination of the first and second motifs, or a combination of the first to third motifs; there is no insertion sequence at the third site, or the third sequence inserted at the third site is: the second motif, the third motif, or a combination of the second and third motifs; and the transcription factor binding sequence is inserted into at least two of the one or more sites.

[0060] In some embodiments, there is no inserted sequence at the first site, or the first sequence inserted at the first site is a first motif or a second motif.

[0061] In some embodiments, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif.

[0062] In some embodiments, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the first motif.

[0063] In some embodiments, at least one of the one or more sites is inserted with the transcription factor binding sequence.

[0064] In some embodiments, there is no insertion sequence at the first site, or the first sequence inserted at the first site is a first motif and a second motif; there is no insertion sequence at the second site, or the second sequence inserted at the second site is: the first motif; there is no insertion sequence at the third site, or the third sequence inserted at the third site is: the first motif; and at least one of the one or more sites has the transcription factor binding sequence inserted therein.

[0065] A second aspect of the present invention provides a Saccharomyces cerevisiae comprising the glycyrrhetinic acid biosensor according to any one of the first aspects.

[0066] In some embodiments, the starting strain of Saccharomyces cerevisiae is selected from the group consisting of: S288C, SynV, GA183, GA184.

[0067] In some embodiments, the Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or molecular chaperone protein (SSZ1), wherein the nucleotide sequence of the gene encoding RDR1 is shown in SEQ ID NO:8, and the nucleotide sequence of the gene encoding SSZ1 is shown in SEQ ID NO:9.

[0068] It is understandable that "the Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or molecular chaperone protein (SSZ1)" means: in the Saccharomyces cerevisiae, RDR1 and / or SSZ1 are fully or partially mutated (mutations include: insertion, deletion or substitution), that is, in the Saccharomyces cerevisiae, RDR1 and / or SSZ1 (or their variants) are not contained, or the RDR1 and / or SSZ1 (or their variants) contained therein cannot perform the functions of RDR1 and / or SSZ1.

[0069] A third aspect of the present invention provides a use of the glycyrrhetinic acid biosensor of any embodiment of the first aspect or the brewer's yeast of any embodiment of the second aspect in obtaining brewer's yeast with high glycyrrhetinic acid production.

[0070] It is understandable that the glycyrrhetinic acid biosensor of any embodiment of the first aspect or the Saccharomyces cerevisiae of any embodiment of the second aspect can be used to obtain Saccharomyces cerevisiae with high glycyrrhetinic acid production through high-throughput screening; or Saccharomyces cerevisiae with high glycyrrhetinic acid production can be obtained through rapid evolution.

[0071] As an example, one application method is strain high-throughput screening: the fluorescence intensity of the brewer's yeast containing the glycyrrhetinic acid biosensor is detected, and the fluorescence detection method can be an ELISA method or flow cytometry, and the cells with high fluorescence intensity are selected as strains with high glycyrrhetinic acid production; another application method is strain rapid evolution: the glycyrrhetinic acid response sequence and the HIS3 gene are assembled into a gene circuit, and the synthetic glycyrrhetinic acid brewer's yeast strain containing the gene circuit is cultured or subcultured in a nutrient-deficient medium lacking histidine, thereby obtaining an engineered bacterium with increased glycyrrhetinic acid production ( Figure 3 ), the nucleic acid sequence of HIS3 is shown in SEQ ID NO:10.

[0072] The present invention constructs a glycyrrhetinic acid biosensor through synthetic biology to achieve the screening or evolution of high-yield glycyrrhetinic acid strains. The method is simple and rapid, and is an effective method for rapid breeding of high-yield glycyrrhetinic acid strains.

[0073] the term

[0074] As used herein, the "engineered bacteria" refers to a fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0075] As used herein, the "starting strain" refers to the original strain used for transformation.

[0076] As used herein, the "gene circuit" refers to a gene expression system that contains all the necessary elements required to express a target polypeptide, and generally includes the following elements: a promoter, an encoded reporter gene, and a terminator.

[0077] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0078] The term “include” or “comprising” described in the present invention is an open description, which contains the specified components or steps described and other specified components or steps that will not be substantially affected.

[0079] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0080] Strain source

[0081] SynV: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, synthesized chromosome V; (Literature source: Xie ZX, Li BZ, Mitchell LA, et al. "Perfect" designer chromosome V and behavior of a ring derivative. Science. 2017, 355(6329): eaaf4704. doi: 10.1126 / science.aaf4704.) The public can obtain the bacterium from the applicant and can only be used to repeat the experiments of the present invention.

[0082] GA183: SynV, ΔHO::FBA1p-GgbAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-Gal7p-Uni25647(F222Y)-TYS1t-PGK1p-GuCPR1-PGK1t-HSP12p-ERG1-PYK1t-G418, ΔGAL80::URA3; (Literature source: Sun W, Wan S, Liu C, et al. Establishing cell suitability for high-level production of licorice triterpenoids in yeast. Acta Pharm Sin B. 2024, 14(9): 4134-4148. doi: 10.1016 / j.apsb.2024.04.032.) The public can obtain the bacteria from the applicant and can only be used to repeat the experiments of the present invention.

[0083] GA184: GA183, Δleu2::Gal2p-NHMGR-Leu2; (Literature source: Sun W, Wan S, Liu C, et al. Establishing cell suitability for high-level production of licoricetriterpenoids in yeast. Acta Pharm Sin B. 2024, 14(9): 4134-4148. doi: 10.1016 / j.apsb.2024.04.032.) The public can obtain the bacteria from the applicant and can only be used to repeat the experiments of the present invention.

[0084] Saccharomyces cerevisiae S288C is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.22135. The deposit date is April 6, 2021, and the deposit location is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0085] Construction of glycyrrhetinic acid biosensor

[0086] The PDR5 promoter derived from Saccharomyces cerevisiae and a reporter gene are assembled into a gene circuit and expressed in Saccharomyces cerevisiae that synthesizes glycyrrhetinic acid, so that the level of glycyrrhetinic acid synthesized by Saccharomyces cerevisiae can be detected. Specifically, the nucleotide sequence of the PDR5 promoter is shown in SEQ ID NO: 1. In addition, the embodiment of the present invention exemplarily provides a reporter gene with an amino acid sequence as shown in SEQ ID NO: 2-7.

[0087] According to the nucleotide sequence encoding the corresponding enzyme and its source provided by the present invention, the corresponding target gene is obtained by PCR amplification, recombination or artificial synthesis. In order to stably express the target gene in yeast, a gene circuit including the above target gene is constructed, such as Figure 1 As shown. The gene circuit is constructed in the order of PDR5 promoter, reporter gene, and terminator. The selection of terminator and the connection method of gene can be carried out according to conventional technical means in the art. For example, the PDR5 promoter, mCherry reporter gene, and CYC1 terminator are connected by overlapping extension PCR to obtain the gene circuit of glycyrrhetinic acid biosensor; the PDR5 promoter, EGFP reporter gene, and ADH1 terminator are connected by Gibson assembly to obtain the gene circuit of glycyrrhetinic acid biosensor.

[0088] The terminator used in the present invention is not required, and the sequences are all known sequences that can be retrieved from the database, which is Saccharomyces Genome Database|SGD (yeastgenome.org).

[0089] The above gene circuit can be expressed by plasmid or integrated into the genome for expression. When expressing the genome, it is necessary to construct homology arms upstream and downstream of the gene circuit, insert the target gene into the yeast genome through the homologous recombination ability of the yeast itself, and stably express the target gene in the yeast. The specific operation can be carried out according to conventional technical means in the art.

[0090] The above gene circuit is transformed into a starting strain of Saccharomyces cerevisiae to obtain an engineered bacterium containing a glycyrrhetinic acid biosensor. The yeast transformation operation can be performed according to conventional techniques in the art.

[0091] Characterization of glycyrrhetinic acid biosensor

[0092] In order to confirm the ability of the PDR5 promoter to respond to the product glycyrrhetinic acid in the starting strain SynV of Saccharomyces cerevisiae that synthesizes glycyrrhetinic acid, a gene circuit with the PDR5 promoter, mCherry reporter gene, and terminator CYC1 was constructed and integrated into the Saccharomyces cerevisiae strain to obtain the strain SynV-P PDR5The strain was fermented, and different concentrations of glycyrrhetinic acid (0, 10, 25, 50 mg / L) were added after 6 hours of fermentation. Samples were taken after another 6 hours of culture, and the fluorescence intensity of the cells was measured by flow cytometry. Figure 6 As shown, with the increase of glycyrrhetinic acid concentration, the fluorescence intensity of the cells showed an upward trend, which was manifested as a rightward shift in the histogram of fluorescence intensity-cell number, indicating that the PDR5 promoter can respond to changes in the concentration of glycyrrhetinic acid.

[0093] To further confirm whether there is a correlation between the response intensity of the PDR5 promoter and the production of endogenously synthesized glycyrrhetinic acid in the strain, the SynV strain containing the gene circuit of the PDR5 promoter-mCherry reporter gene (SynV-P that does not produce glycyrrhetinic acid) was tested. PDR5 ) and GA183 strain (GA183-P producing glycyrrhetinic acid PDR5 ), respectively, fermentation experiments were carried out, samples were taken at 24h intervals, and the fluorescence intensity of the cells was measured. The results are shown in Figure 7 As the fermentation time increased, the control group strain SynV-P PDR5 The fluorescence intensity of the experimental group strain GA183-P PDR5 The fluorescence intensity of strain GA183-P PDR5 There was a good linear correlation between the fluorescence intensity and the intracellular glycyrrhetinic acid production (R 2 =0.9958).

[0094] Modification and Optimization of Glycyrrhetinic Acid Biosensor

[0095] In order to adjust the response intensity of the glycyrrhetinic acid biosensor, the number of transcription activator DNA binding motifs was increased in the PDR5 promoter to modify it. Three positions were selected in the sequence of the PDR5 promoter to insert different transcription factor binding motifs, the insertion positions were -220bp, -407bp, and -513bp, and the transcription factor binding motifs were Motif1: Pdr1 / Pdr3 (also expressed as Pdr1 / 3) (TCCGCGGA), Motif2: Yap1 (TTACTAA), and Motif3: Stb5 (CGGAACGA). 31 modified promoters (PE1 to PE31) were designed, and the response intensity of the glycyrrhetinic acid biosensor was characterized by the above-mentioned "Characterization of Glycyrrhetinic Acid Biosensor" method, and a biosensor with enhanced response intensity was obtained. The results are shown in Figure 8 (This is the test result of sampling at 120 hours of fermentation), where P PDR5 In the group: Gray bars contain P PDR5 -mCherry (without insertion motif) fluorescence values ​​of SynV strains, pink bars are P PDR5- Fluorescence value of GA183 strain with mCherry (without insertion motif), blue dots are the ratio of pink bar graph to gray bar graph, which is the dynamic range; P PDR5 -PEXX group: The gray bars are P PDR5 -mCherry fluorescence values ​​of SynV strains, and the pink bar graphs are P PDR5 -The fluorescence value of the GA183 strain expressing mCherry. The blue dots are the ratio of the pink bar graph to the gray bar graph, which is the response range (Dynamic range).

[0096] In addition, knockout of some endogenous genes in Saccharomyces cerevisiae can also regulate the response intensity of glycyrrhetinic acid biosensor, including RDR1, SSZ1 ( Fig. 9 ), among which, in the Control group: the gray bar graph contains P PDR5 -mCherry (without insertion motif) fluorescence values ​​of SynV strains, pink bars are P PDR5 -mCherry (without insertion motif) fluorescence value of GA183 strain; In the △Rdr1 group: the gray bar graph is the fluorescence value of the strain containing P PDR5 -mCherry (without insertion motif) and the fluorescence values ​​of the SynV strain with Rdr1 knocked out. The pink bar graph is the fluorescence value of the strain containing P PDR5 -mCherry (without insertion motif) and the fluorescence value of GA183 strain with Ssz1 knocked out; In the △Ssz group: the gray bar graph contains P PDR5 -mCherry (without insertion motif) and the fluorescence values ​​of the SynV strain with Ssz1 knocked out; the pink bar graph is the fluorescence value of the strain containing P PDR5 -mCherry (without insertion motif), and the fluorescence values ​​of the GA183 strain in which Ssz1 was knocked out.

[0097] High-throughput screening of glycyrrhetinic acid-producing strains using glycyrrhetinic acid biosensor

[0098] The glycyrrhetinic acid biosensor was used to detect the level of glycyrrhetinic acid synthesized by Saccharomyces cerevisiae. There were two detection methods: (1) the strain was spread on a solid culture medium, a single colony was picked and inoculated into a 96-well plate containing a liquid culture medium, and the fluorescence intensity of the strain was detected using an ELISA reader; (2) the strain was detected and sorted using a flow cytometer, and the cells with high fluorescence were sorted out, spread on a solid culture medium, and a single colony was picked.

[0099] Glycyrrhetinic acid biosensor for adaptive evolution of glycyrrhetinic acid-sensitive strains

[0100] In order to improve the ability of engineered bacteria to synthesize glycyrrhetinic acid, the expression of HIS3 gene was regulated by glycyrrhetinic acid biosensor, and glycyrrhetinic acid production was coupled with bacterial growth to screen high-yield bacteria, such as Figure 4 As shown. With the increase of intracellular glycyrrhetinic acid synthesis accumulation, the expression intensity of PDR5 promoter is enhanced, and more His3 protein is expressed to catalyze the production of histidine from imidazole-glycerol-3-phosphate, so that the cells can resume growth in the nutrient-deficient medium SD-His (glucose 20g / L, amino acid-free yeast nitrogen source containing ammonium sulfate 6.7g / L, and amino acid mixture without histidine 1.3g / L). The glycyrrhetinic acid synthesis ability is related to cell growth, so that the accumulation of glycyrrhetinic acid is converted into an easily detectable growth parameter, and glycyrrhetinic acid high-producing bacteria are separated according to cell growth conditions.

[0101] The present invention is further explained below by specific examples. It should be understood that these examples are merely used to describe and help understand the present invention in an exemplary manner, and are not intended to limit the present invention to these examples.

[0102] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0103] Example 1 Construction of Glycyrrhetinic Acid Biosensor

[0104] To construct HOL-P PDR5 -mcherry-T CYC1 -KanMX-HOR gene circuit (HOL and HOR are homology arms integrated into the genome, P PDR5 is the promoter, mcherry is the red fluorescent protein gene, T CYC1 The target gene fragments were amplified by PCR, HOL, P PDR、 T CYC1 , HOR were amplified by PCR using the genome of Saccharomyces cerevisiae S288C as a template, and mcherry and KanMX were amplified by PCR using plasmids pmCherry-C1 and pFA6-kanMX4 as templates, respectively. Then, the fragments were connected by Gibson assembly, the connected gene circuits were amplified by PCR, and the gene circuits were transformed into Saccharomyces cerevisiae using the lithium acetate transformation method, cultured in solid YPD medium containing 300 μg / mL G418, and positive clones were screened.

[0105] Example 2 Characterization of Glycyrrhetinic Acid Biosensor

[0106] The constructed glycyrrhetinic acid biosensor was transformed into Saccharomyces cerevisiae GA183 (experimental group) that synthesizes glycyrrhetinic acid and Saccharomyces cerevisiae SynV (control group) that does not synthesize glycyrrhetinic acid. The strain was inoculated into 20 mL YPD liquid culture medium and cultured in a shaker at 200 rpm and 30°C for 72 hours.

[0107] Take 1 mL of the fermented sample, centrifuge the fermented sample to be tested at 5000 rpm for 2 minutes, and discard the supernatant. Resuspend the bacteria in 0.01 M PBS buffer (pH = 7.4), centrifuge at 5000 rpm for 2 minutes, and discard the supernatant. Add an appropriate volume of 0.01 M PBS buffer to the centrifuge tube to resuspend the bacteria so that the OD600 of the sample is between 0.2-0.8. Take 100 μL of the sample and add it to the 96-well ELISA plate in turn. Set the detection parameters of the ELISA instrument: 1) absorbance at 600 nm; 2) excitation wavelength 580 nm, emission wavelength 610 nm. When analyzing the test results, compare the fluorescence intensity of the unit cell (fluorescence intensity / OD600).

[0108] The test results showed that the [fluorescence intensity / OD600] of Saccharomyces cerevisiae that does not synthesize glycyrrhetinic acid was 178 (see Figure 7 SynV-P PDR5 72 hours data), the [fluorescence intensity / OD600] of Saccharomyces cerevisiae synthesizing glycyrrhetinic acid = 336 (see Figure 7 Medium GA183-P PDR5 72 hours data). This indicates that the glycyrrhetinic acid biosensor can respond to glycyrrhetinic acid.

[0109] Example 3 Modification of Glycyrrhetinic Acid Biosensor

[0110] With the help of the online tool YEASTRACT+ (http: / / www.yeastract.com / index.php), the “Find TF Binding Site(s)” under the “Pattern Matching” module was used to predict the DNA binding motifs of the transcription activators Pdr1, Pdr3, Yap1, Stb5 and the transcription repressor Rdr1 contained in the promoter PDR5.

[0111] The above DNA binding motifs were inserted into the PDR5 promoter using the point mutation method. The insertion positions were -220bp, -407bp, and -513bp. The specific transcription factor binding sequences inserted were: TCCGCGGA (Pdr1 / 3), TTACTAA (Yap1), and CGGAACGA (Stb5). Since the insertion positions of the transcription factor DNA binding motifs were far apart from each other, only one DNA fragment could be inserted in each round of experiments. If different DNA binding motifs were inserted at multiple sites, iterative construction was required. The primers used are shown in Table 1.

[0112] Table 1 Pair of promoters PDR5 Primers used for transformation

[0113]

[0114] The above primers were used to construct 31 promoter mutants from PE1 to PE31. The above promoter mutants were characterized by the method of Example 2. The mutant sequences and detection results are shown in Tables 2 and Figure 8 The results showed that the mutants had lower or higher response ranges (defined as: fluorescence intensity of Saccharomyces cerevisiae that synthesizes glycyrrhetinic acid / fluorescence intensity of Saccharomyces cerevisiae that does not synthesize glycyrrhetinic acid). For example, the response range of the original promoter PDR5 was 2.22, and after modification, P PDR5 -PE20 response range was increased to 2.96; the response range of the starting promoter PDR5 was 2.22. PDR5 -The response range of PE3 decreased to 0.94.

[0115] Furthermore, the response capacity of P PDR5 -PE3 was used to explore its response characteristics. After the strain was cultured for 6 hours, different concentrations of glycyrrhetinic acid (0, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500 mg / L) were added, and samples were taken after another 6 hours of culture. The fluorescence intensity of the cells was measured using an ELISA instrument. The results are as follows: Fig.10 When the concentration of glycyrrhetinic acid was between 10-50 mg / L, the fluorescence intensity of the cells was positively correlated with the amount of glycyrrhetinic acid added; when the concentration of glycyrrhetinic acid was higher than 50 mg / L, the response intensity of the element tended to saturation. The results showed that P PDR5 -PE3 still has the ability to respond to glycyrrhetinic acid, and the response concentration is within 50 mg / L.

[0116] Table 2

[0117]

[0118]

[0119] It should be understood that the motif combination shown in Table 2 above is a nucleotide sequence in which the motifs are sequentially connected in order, for example, Yap1+Stb5, which represents TTACTAACGGAACGA (SEQ ID NO: 34).

[0120] It should be understood that the insertion sites in Table 2 above are based on the original P PDR5 The number of nucleotides (i.e., P without any motif inserted) PDR5 (SEQ ID NO: 1)). As an example, P PDR5 -PE23 (EQ ID NO: 35) was constructed by PDR5 (SEQ ID NO: 1) Stb5 (CGGAACGA), Pdr1 / 3 (TCCGCGGA) and Stb5 (CGGAACGA) were inserted before the 220th, 407th and 513th nucleotides (see the underlined nucleotides in SEQ ID NO: 1 below) from the 3' end (ie, in the 5' direction).

[0121] >SEQ ID NO:1(5'-3')

[0122] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGT TCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCAACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGA A ACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGGTGTGGGCTCACCCGCGGGTCGT GATCACGATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGAT G TGCATAACCTTATGGCTGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0123] >SEQ ID NO:35(5’-3’)

[0124] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGTTCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCAACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGACGGAACGA A ACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGTGTGGGCTCACCCGCGGGTCGTTCCGCGGA GATCACGATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGATCGGAACGA G TGCATAACCTTATGGCTGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0125] Example 4 High-throughput screening of glycyrrhizic acid-producing strains using an ELISA instrument

[0126] Using room temperature plasma (ARTP) to characterize the glycyrrhetinic acid biosensor P PDR5 -PE21 was used to mutate the brewer's yeast GA183 that synthesizes glycyrrhetinic acid. The ARTP treatment time was 1 minute. After mutagenesis, the bacterial solution was spread on YPD solid culture medium and cultured at 30°C for 3 days. Single colonies on the plate were picked up and placed in a 96-well plate containing YPD culture medium and cultured at 30°C for 3 days. The characterization method of the glycyrrhetinic acid biosensor in Example 2 was used to perform fluorescence detection on the cultured strains, and the [fluorescence intensity / OD600] value was calculated. The strains with high [fluorescence intensity / OD600] values ​​were selected, and the glycyrrhetinic acid production was checked by liquid chromatography. The results showed that the [fluorescence intensity / OD600] value had a significant corresponding relationship with the glycyrrhetinic acid production, and the selected high-yield strain had a 23.6% increase in glycyrrhetinic acid production compared to the control strain GA183.

[0127] Example 5 High-throughput screening of glycyrrhetinic acid-producing strains using flow cytometry

[0128] Using room temperature plasma (ARTP) to characterize the glycyrrhetinic acid biosensor P PDR5 Synthesis of glycyrrhetinic acid by Saccharomyces cerevisiae GA183 (GA183-P PDR5-PE28) was induced by ARTP, the treatment time was 1 minute, and the bacterial solution was spread on YPD solid culture medium after mutagenesis and cultured at 30°C for 3 days.

[0129] The above bacterial solution was tested and sorted by flow cytometry: centrifuge the test bacterial solution at 5000rpm for 2min and discard the supernatant. Resuspend the bacteria with 0.01M PBS buffer (pH=7.4), centrifuge at 5000rpm for 2min and discard the supernatant. Add an appropriate volume of 0.01M PBS buffer to the centrifuge tube to resuspend the bacteria. Use a 70μm cell sieve to filter the sample into a flow tube for testing. The steps for using a flow cytometer are as follows:

[0130] (1) Before starting the instrument, check whether the amount of waste liquid in the waste liquid bucket and the amount of liquid in the 8× sheath liquid bucket and distilled water bucket are sufficient. (2) After opening the software, click "start-up" and the instrument will enter the startup program and automatically calibrate. After the startup is completed, add 6 drops of calibration microspheres to the flow tube and run the "Run QC" program. (3) After the Quality Control passes, place the filtered sample on the sample stage and push it to the running position. (4) Open the detection method, click "Start" to start loading, and save the analyzed data file. (5) Channel parameters: FSC 205, SSC 282, FL3 850; number of cells detected: 50,000.

[0131] The results showed that the relative fluorescence intensity of the control strain GA183 was 2.03; the relative fluorescence intensity of the experimental group was 85.52.

[0132] Use the "Gate" function to circle the cells with high fluorescence and sort them into new test tubes. Spread them on YPD solid culture medium and culture at 30°C for 3 days. Pick up single colonies on the plate and transfer them to a 96-well plate containing YPD culture medium and culture at 30°C for 3 days. Use the characterization method of the glycyrrhetinic acid biosensor in Example 2 to perform fluorescence detection on the cultured strains, calculate the [fluorescence intensity / OD600] value, select the strains with high [fluorescence intensity / OD600] values, and check the glycyrrhetinic acid production by liquid chromatography. The results showed that the selected high-yield strain had a 40.1% increase in glycyrrhetinic acid production compared to the control strain GA183 (for specific information on the production of each product of this strain, see Figure 3 ).

[0133] Figure 3 The results of fermentation detection of screened strains after applying glycyrrhetinic acid biosensor to high-throughput screening. Highly fluorescent cells were sorted by flow cytometry and then plated. 50 single colonies were selected on the plate and inoculated into YPD medium for fermentation for 5 days to detect glycyrrhetinic acid production. The strain with the highest glycyrrhetinic acid production increased by 40.1% compared with the control.

[0134] The detection method of glycyrrhetinic acid is:

[0135] (1) After fermentation, take 5 mL of fermentation broth into a 10 mL centrifuge tube and centrifuge at 5000 rpm for 10 min to separate the bacterial precipitate and the fermentation broth supernatant.

[0136] (2) The bacterial pellet was resuspended in 400 μL of 6 M HCl solution and transferred to a centrifuge tube for bead beating. An appropriate amount of 0.5 mm glass beads was added and the cells were broken using a homogenizer (75 Hz, 60 s ON / 15 s PAUSE, 5 cycles).

[0137] (3) The disrupted cell solution was transferred to a 10 mL centrifuge tube, 6 mL of ethyl acetate was added, and the mixture was vortexed and fully extracted. The mixture was centrifuged at 5000 rpm for 10 min, and the upper ethyl acetate layer was transferred to a centrifuge tube and evaporated to dryness using a vacuum concentrator.

[0138] (4) Add 6 mL of ethyl acetate to the supernatant of the fermentation broth, vortex and oscillate, centrifuge at 5000 rpm for 10 min after sufficient extraction, and draw the upper ethyl acetate layer into a centrifuge tube and evaporate to dryness.

[0139] (5) Add 300 μL of chromatography-grade methanol to reconstitute the precipitate and supernatant extracted and evaporated samples.

[0140] (6) The reconstituted sample was filtered through a 0.22 μm organic phase filter membrane into a liquid phase vial and detected using an ultra-high performance liquid chromatography (UHPLC).

[0141] UPLC detection method of fermentation products:

[0142] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent technologies 1260). A Poroshell 120EC-C18 column with a particle size of 2.7 μm and 3.0 × 100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination.

[0143] Example 6 Adaptive evolution of glycyrrhizic acid strains using glycyrrhizic acid biosensor

[0144] Building HOL-P PDR5 -HIS3-T CYC1 -HOR gene circuit, and transformed it into the Saccharomyces cerevisiae strain GA184 that synthesizes glycyrrhetinic acid, which is named E0. The method is the same as in Example 1.

[0145] The strain E0 was subjected to adaptive laboratory evolution. The operation process was as follows: inoculate it into the nutrient-deficient medium SD-His, with an initial inoculation volume of OD600 = 0.2, and culture it in a test tube for 2 days, with the shaking conditions of 200rpm and 30℃. After that, transfer it every 3 days and take it out on the 18th day. Save the bacteria in the evolution process in a glycerol tube. Inoculate the evolved strain into YPD medium, ferment it for 5 days, and detect the production of glycyrrhetinic acid. The results are as follows Figure 5 As shown in b, after 6 days of evolution, the glycyrrhizic acid production reached the maximum, which was 17.5% higher than that of the starting strain, reaching 174.2 mg / L.

[0146] >SEQ ID NO: 1

[0147] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGTTCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCA ACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGAAACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGTGTGGGCTCACCCGCGGGTCGTGATCACG ATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGATGTGCATAACCTTATGGCGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0148] >SEQ ID NO:2

[0149] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0150] >SEQ ID NO:3

[0151] MSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0152] >SEQ ID NO:4

[0153] MSYSKHGIVQEMKTKYHMEGSVNGHEFTIEGVGTGYPYEGKQMSELVIIKPAGKPLPFSFDILSSVFQYGNRCFTKYPADMPDYFKQAFPDGMSYERSFLFEDGAVATASWNIRLEGNCFIHKSIFHGVNFPADGPVMKKKTIDWDKSFEKMTVSKEVLRGDVTMFLMLEGGGSHRCQFHSTYKTEKPVTLPPNHVVEHQIVRTDLGQSAKGFTVKLEAHAAAHVNPLKVK

[0154] >SEQ ID NO:5

[0155] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0156] >SEQ ID NO: 6

[0157] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0158] >SEQ ID NO:7MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLIKEANNETYVEQHEVAVARYCDLPSKLGHKLN

[0159] >SEQ ID NO: 8

[0160]

[0161] >SEQ ID NO:9

[0162]

[0163] >SEQ ID NO: 10

[0164] ATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAGTGATTAACGTC CACACAGGTATAGGGTTTCTGGACCATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTTTAAAGAGGCCCTACTGGCG CGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGC TTTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAG

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0166] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0167] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A glycyrrhetinic acid biosensor, characterized in that: Contains: PDR5 promoter.

2. The glycyrrhizic acid biosensor according to claim 1, characterized in that: Also contains a reporter gene.

3. The glycyrrhizic acid biosensor according to claim 2, characterized in that: The reporter gene is a gene encoding a fluorescent protein or a gene encoding histidine synthetase.

4. The glycyrrhizic acid biosensor according to claim 1, characterized in that: It also comprises: a transcription factor binding sequence inserted into one or more sites in the PDR5 promoter, wherein the transcription factor binding sequence is selected from the group consisting of: a first motif (TCCGCGGA), a second motif (TTACTAA) and a third motif (CGGAACGA).

5. The glycyrrhetinic acid biosensor according to claim 4, characterized in that: The one or more sites include: a first site, a second site and a third site, wherein the first site is located at any one of the 501st to 525th nucleotides from the 3' end of the PDR5 promoter, the second site is located at any one of the 374th to 419th nucleotides from the 3' end of the PDR5 promoter, and the third site is located at any one of the 92nd to 239th nucleotides from the 3' end of the PDR5 promoter; Preferably, the first site is located at the 513th nucleotide from the 3' end of the PDR5 promoter, the second site is located at the 407th nucleotide from the 3' end of the PDR5 promoter, and the third site is located at the 220th nucleotide from the 3' end of the PDR5 promoter.

6. The glycyrrhizic acid biosensor according to claim 5, characterized in that: There is no inserted sequence at the first site, or the first sequence inserted at the first site is: the second motif, the third motif, or a combination of the second motif and the third motif; Optionally, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif, the second motif, the third motif, a combination of the first motif and the third motif, a combination of the second motif and the third motif, a combination of the first motif and the second motif, a combination of the first to third motifs; Optionally, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the second motif, the third motif, or a combination of the second motif and the third motif; Optionally, at least two of the one or more sites are inserted with the transcription factor binding sequence.

7. The glycyrrhizic acid biosensor according to claim 5, characterized in that: There is no inserted sequence at the first site, or the first sequence inserted at the first site is a first motif or a second motif; Optionally, there is no inserted sequence at the second site, or the second sequence inserted at the second site is: the first motif; Optionally, there is no inserted sequence at the third site, or the third sequence inserted at the third site is: the first motif; Optionally, at least one of the one or more sites is inserted with the transcription factor binding sequence.

8. Saccharomyces cerevisiae comprising the glycyrrhetinic acid biosensor according to any one of claims 1 to 7.

9. The brewer's yeast according to claim 8, characterized in that The starting strain of Saccharomyces cerevisiae is selected from the group consisting of: S288C, SynV, GA183, GA184; Optionally, the Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or molecular chaperone protein (SSZ1), wherein the nucleotide sequence of the gene encoding RDR1 is shown in SEQ ID NO:8, and the nucleotide sequence of the gene encoding SSZ1 is shown in SEQ ID NO:

9.

10. Use of the glycyrrhetinic acid biosensor according to any one of claims 1 to 7 or the Saccharomyces cerevisiae according to claim 8 or 9 in obtaining Saccharomyces cerevisiae with high glycyrrhetinic acid production.

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