A glycyrrhetinic acid biosensor and its application

Abstract

This invention relates to the field of microbial engineering, specifically to a glycyrrhetinic acid biosensor and its applications. The glycyrrhetinic acid biosensor comprises a PDR5 promoter. This biosensor exhibits a high specificity response to glycyrrhetinic acid, and this specificity can be modulated by inserting a transcription factor binding sequence into the PDR5 promoter. This biosensor can be applied to high-throughput detection and screening of high-yield glycyrrhetinic acid strains, as well as the stable inheritance and adaptive evolution of high-yield strains. The glycyrrhetinic acid biosensor and its applications according to this invention provide an effective method for the rapid selection of high-yield glycyrrhetinic acid-producing Saccharomyces cerevisiae strains.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering, specifically to a glycyrrhetinic acid biosensor and its applications. Background Technology

[0002] Glycyrrhetinic acid is the main active ingredient of licorice, a traditional Chinese medicine. It is a typical pentacyclic triterpenoid compound widely used in medicine, food, cosmetics, and health products. Currently, the main method for obtaining glycyrrhetinic acid is to extract glycyrrhizic acid from the roots of licorice, followed by acid or enzymatic hydrolysis. This process involves multiple steps, including drying, grinding, pulverizing, and hydrolysis, making it complex. Furthermore, licorice has a well-developed root system that helps prevent wind erosion and sandstorms. In my country, it is mainly distributed in western desert and semi-desert areas such as Gansu, Xinjiang, and Inner Mongolia, possessing significant ecological value. The "root extraction" method severely damages the vegetation distribution of licorice and the local ecological environment, which is detrimental to environmental protection.

[0003] In recent years, advancements in synthetic biology and metabolic engineering have endowed microorganisms with the ability to synthesize heterologous terpenes. Simultaneously, the advantages of utilizing microorganisms to synthesize natural products are becoming increasingly apparent, making it a potential green and environmentally friendly alternative. Researchers have already constructed a Saccharomyces cerevisiae cell factory capable of synthesizing glycyrrhetinic acid (license number: ZL201610965315.9). Furthermore, the iterative development of omics technologies, gene editing technologies, and in vitro assembly technologies has promoted the construction and optimization of cell factories, providing favorable conditions for further improving the production efficiency of terpenes. However, current methods for detecting glycyrrhetinic acid mainly rely on gas chromatography, requiring processes such as cell sample disruption, extraction, derivatization, and chromatographic detection. These steps are cumbersome, have low sample processing efficiency, and poor reproducibility. The detection process has become the biggest bottleneck hindering the high-throughput construction and optimization of glycyrrhetinic acid synthesis cell factories.

[0004] Therefore, there is an urgent need for a rapid and high-throughput method for the detection of glycyrrhetinic acid in related technologies. Summary of the Invention

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

[0006] The first aspect of this application provides a glycyrrhetinic 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 synthase.

[0009] In some embodiments, the glycyrrhetinic acid biosensor further comprises: a transcription factor binding sequence inserted at 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 of the 501st to 525th nucleotides from the 3' end of the PDR5 promoter, the second site is located at any of the 374th to 419th nucleotides from the 3' end of the PDR5 promoter, and the third site is located at any 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: a second motif, a 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: a first motif, a second motif, a 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, or a combination of the first to the 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: a second motif, a third motif, or a combination of a second motif and a third motif.

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

[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 a 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 a 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 this application provides a brewer's yeast comprising the glycyrrhetinic acid biosensor described in any of the embodiments of the first aspect above.

[0021] In some embodiments, the starting strain of the brewing yeast is selected from the group consisting of: S288C, SynV, GA183, and 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 this application provides the application of the glycyrrhetinic acid biosensor described in any of the embodiments of the first aspect or the brewing yeast described in any of the embodiments of the second aspect in brewing yeast that produces high levels of glycyrrhetinic acid.

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

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

[0026] The glycyrrhetinic acid biosensor and its application in this invention provide a simple and effective method for the rapid selection of high-yielding Saccharomyces cerevisiae strains. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This describes the composition and working principle of the glycyrrhetinic acid biosensor in this embodiment of the invention.

[0029] Figure 2 This is a schematic diagram of the PDR5 promoter and mutation site in an 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 are for the strains screened in Example 5 of this invention;

[0031] Figure 4 This is a schematic diagram illustrating the application of the glycyrrhetinic acid biosensor to the rapid evolution of bacterial strains in Example 6 of the present invention.

[0032] Figure 5 This is the result of applying the glycyrrhetinic acid biosensor to the rapid evolution of bacterial strains in Example 6 of the present invention, wherein... Figure 5 a represents the yield of fermentation products from yeast strain GA184, which does not contain a glycyrrhetinic acid biosensor, after passage in YPD medium. Figure 5 b represents the yield of the fermentation product of strain (E0) after passage in SD-HIS medium using a glycyrrhetinic acid biosensor.

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

[0034] Figure 7 The glycyrrhetinic acid biosensor of this invention was characterized using strain SynV (SynV-P, which does not produce glycyrrhetinic acid), which contains the gene circuit of the PDR5 promoter-mCherry reporter gene. PDR5 ) and GA183 (GA183-P, which produces glycyrrhetinic acid) PDR5 Fluorescence intensity detection results during fermentation;

[0035] Figure 8 This is the result of modifying the glycyrrhetinic acid response sequence to adjust the response intensity of the glycyrrhetinic acid biosensor in Example 3 of the present invention;

[0036] Figure 9 This is the result of the modification and optimization experiment of the glycyrrhetinic acid biosensor of the present invention, which modifies the response intensity of the glycyrrhetinic acid biosensor by knocking out the endogenous gene of Saccharomyces cerevisiae.

[0037] Figure 10 To investigate P in Embodiment 3 of the present invention PDR5 Experimental results of the PE3 response characteristics.

[0038] To facilitate understanding of the present invention, the implementation process of the present invention will be further described below with reference to specific embodiments. These descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] In this invention, the term "comprising" is an open-ended expression, meaning it includes the content specified in this invention, but does not exclude other aspects.

[0041] This invention is based on the inventor's following understanding:

[0042] In recent years, with the rapid development of synthetic biology, gene-encoded biosensors have gradually become convenient tools for qualitative and quantitative analysis and detection of compounds. Based on this, high-throughput screening methods can quickly and accurately screen high-yielding strains from large mutant libraries.

[0043] A 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] This invention provides a gene-encoded glycyrrhetinic acid biosensor. By performing transcriptomic analysis on Saccharomyces cerevisiae, which synthesizes glycyrrhetinic acid, it was found that the expression level of the gene PDR5 was significantly increased in Saccharomyces cerevisiae that synthesizes glycyrrhetinic acid. Therefore, by assembling the PDR5 promoter and reporter genome into a glycyrrhetinic acid biosensor, the synthesis level of glycyrrhetinic acid can be indicated.

[0046] In some embodiments, the glycyrrhetinic acid biosensor also includes a reporter gene.

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

[0048] As an example, fluorescent protein genes include, but are not limited to: mCherry, mOrange, YFP, EGFP, ECFP, or EBFP, the amino acid sequences of which are shown in SEQ ID NO: 2-7, respectively.

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

[0050] In some embodiments, the glycyrrhetinic acid biosensor further comprises: a transcription factor binding sequence inserted at one or more sites on 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 of the 501st to 525th nucleotides from the 3' end of the PDR5 promoter, the second site is located at any of the 374th to 419th nucleotides from the 3' end of the PDR5 promoter, and the third site is located at any 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] Understandably, the above-mentioned sites can be combined with the inserted sequence.

[0054] Understandably, the above sites are counted according to the number of nucleotides in the original PDR5 promoter (i.e., 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: a second motif, a 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: a first motif, a second motif, a 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, or a combination of the first to the 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: a second motif, a third motif, or a combination of a second motif and a third motif.

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

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

[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 a 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 a 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 inserted 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 inserted sequence at the second site, or the second sequence inserted at the second site is a first motif; there is no inserted sequence at the third site, or the third sequence inserted at the third site is a first motif; and at least one of the one or more sites has the transcription factor binding sequence inserted.

[0065] A second aspect of the present invention provides a brewer's yeast comprising a glycyrrhetinic acid biosensor according to any of the embodiments of the first aspect described above.

[0066] In some embodiments, the starting strain of the brewing yeast is selected from the group consisting of: S288C, SynV, GA183, and 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] Understandably, the statement "the Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or molecular chaperone protein (SSZ1)" means that in the Saccharomyces cerevisiae, RDR1 and / or SSZ1 are all or partially mutated (mutations include insertion, deletion or substitution), that is, the Saccharomyces cerevisiae does not contain RDR1 and / or SSZ1 (or its variants), or the RDR1 and / or SSZ1 (or its variants) contained therein cannot perform the functions of RDR1 and / or SSZ1.

[0069] The third aspect of the present invention provides the application of the glycyrrhetinic acid biosensor of any of the first aspects or the brewing yeast of any of the second aspects in obtaining high-yield glycyrrhetinic acid brewing yeast.

[0070] It is understood that high-glycyrrhetinic acid-producing Saccharomyces cerevisiae can be obtained by using the glycyrrhetinic acid biosensor of any embodiment of the first aspect above or the Saccharomyces cerevisiae of any embodiment of the second aspect above through high-throughput screening; or by obtaining high-glycyrrhetinic acid-producing Saccharomyces cerevisiae through rapid evolution.

[0071] As an example, one application method is high-throughput screening of strains: the fluorescence intensity of *Saccharomyces cerevisiae* containing a glycyrrhetinic acid biosensor is detected using methods such as ELISA or flow cytometry; cells with high fluorescence intensity are selected as strains producing high levels of glycyrrhetinic acid. Another application method is rapid strain evolution: a gene circuit is constructed using the glycyrrhetinic acid response sequence and the HIS3 genome; strains of *Saccharomyces cerevisiae* containing this gene circuit that synthesize glycyrrhetinic acid are cultured or subcultured in a histidine-deficient auxotrophic medium to obtain engineered strains with increased glycyrrhetinic acid production. Figure 3 The nucleic acid sequence of HIS3 is shown in SEQ ID NO: 10.

[0072] This invention utilizes synthetic biology to construct a glycyrrhetinic acid biosensor, enabling the screening or evolution of high-yielding glycyrrhetinic acid strains. The method is simple and rapid, making it an effective approach for the rapid breeding of high-yielding glycyrrhetinic acid strains.

[0073] the term

[0074] As used in this article, "engineered bacteria" refers to bacterial cell lines that have been genetically engineered to express exogenous genes efficiently.

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

[0076] As used herein, a “genetic circuit” refers to a gene expression system containing all the necessary elements required to express a target polypeptide, typically including the following elements: a promoter, a coding reporter gene, and a terminator.

[0077] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, 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 use of "comprising" or "including" in this invention is an open-ended description, containing the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0079] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As 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; (Source: Xie ZX, Li BZ, Mitchell LA, et al. "Perfect" designer chromosome Vand behavior of a ring derivative. Science. 2017, 355(6329):eaaf4704. doi:10.1126 / science.aaf4704.) This bacterium is available to the public from the applicant and may only be used for repeating experiments of this 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; (Source: Sun W, Wan S, Liu C, et al. Establishing cellsuitability 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.) This bacterium can be obtained from the applicant and is only for repeating experiments of this invention.

[0083] GA184:GA183,Δleu2::Gal2p-NHMGR-Leu2; (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.) This bacterium can be obtained from the applicant and is only for repeating experiments of this invention.

[0084] brewing yeast ( Saccharomyces cerevisiae S288C is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22135, on April 6, 2021, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0085] Construction of glycyrrhetinic acid biosensor

[0086] From brewer's yeast Saccharomyces cerevisiaeThe PDR5 promoter and reporter genome are assembled into a gene circuit and expressed in Saccharomyces cerevisiae, which synthesizes glycyrrhetinic acid, enabling the detection of glycyrrhetinic acid synthesis levels in Saccharomyces cerevisiae. Specifically, the nucleotide sequence of the PDR5 promoter is shown in SEQ ID NO: 1. In addition, the present invention provides, exemplarily, reporter genes with amino acid sequences shown in SEQ ID NO: 2-7.

[0087] Based on the nucleotide sequence encoding the corresponding enzyme provided by this invention and its source, the corresponding target gene is obtained by PCR amplification, recombination, or artificial synthesis. To ensure stable expression of the target gene in yeast, a gene circuit including the aforementioned target gene is constructed, such as... Figure 1 As shown. Gene circuits are constructed in the order of PDR5 promoter, reporter gene, and terminator. The selection of the terminator and the gene ligation method can be performed using conventional techniques in this field. For example, the PDR5 promoter, mCherry reporter gene, and CYC1 terminator can be ligated using overlap extension PCR to obtain the gene circuit for the glycyrrhetinic acid biosensor; the PDR5 promoter, EGFP reporter gene, and ADH1 terminator can be ligated using Gibson assembly to obtain the gene circuit for the glycyrrhetinic acid biosensor.

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

[0089] The aforementioned gene pathways can be expressed using plasmids or integrated into the genome. For genome expression, homologous arms need to be constructed upstream and downstream of the gene pathway. Through yeast's own homologous recombination ability, the target gene is inserted into the yeast genome, enabling stable expression within the yeast. Specific procedures can be performed using conventional techniques in this field.

[0090] The aforementioned gene circuitry was transformed into a starting strain of *Saccharomyces cerevisiae* to obtain an engineered strain containing a glycyrrhetinic acid biosensor. The yeast transformation process can be performed using conventional techniques in this field.

[0091] Characterization of glycyrrhetinic acid biosensor

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

[0093] To further confirm whether there is a correlation between the response intensity of the PDR5 promoter and the production of glycyrrhetinic acid endogenously synthesized by the strain, the SynV strain (SynV-P, which does not produce glycyrrhetinic acid) containing the PDR5 promoter-mCherry reporter gene pathway was investigated. PDR5 ) and GA183 strain (GA183-P, which produces glycyrrhetinic acid) PDR5 Fermentation experiments were conducted separately, with samples taken at 24-hour intervals to measure cell fluorescence intensity. The results are as follows: Figure 7 As shown. With prolonged fermentation time, the control group strain SynV-P PDR5 The fluorescence intensity did not change significantly, while the experimental group strain GA183-P PDR5 The fluorescence intensity showed a significant increasing trend in strain GA183-P. PDR5 In the study, fluorescence intensity showed a good linear correlation with intracellular glycyrrhetinic acid production (R0). 2 =0.9958).

[0094] Modification and optimization of glycyrrhetinic acid biosensor

[0095] To modulate the response intensity of the glycyrrhetinic acid (GLA) biosensor, the number of transcription factor DNA-binding motifs was increased in the PDR5 promoter, thus modifying it. Three different transcription factor binding motifs were inserted into the PDR5 promoter sequence at positions of -220bp, -407bp, and -513bp, respectively. These motifs were: Motif1: Pdr1 / Pdr3 (also represented as Pdr1 / 3) (TCCGCGGA), Motif2: Yap1 (TTACTAA), and Motif3: Stb5 (CGGAACGA). Thirty-one modified promoters (PE1 to PE31) were designed, and the response intensity of the GLA biosensor was characterized using the methods described above for "characterization of the GLA biosensor," resulting in a biosensor with enhanced response intensity. Results are shown below. Figure 8 (The results were obtained from samples taken after 120 hours of fermentation), where P PDR5 Within the group: gray bars represent bars containing P. PDR5 The fluorescence values ​​of the -mCherry (without inserted motif) SynV strain are shown in the pink bar graph, which contains P.PDR5 The fluorescence values ​​of the GA183 strain with -mCherry (without inserted motif) are shown. The blue dots represent the ratio of the pink bars to the gray bars, which represents the dynamic range. PDR5 In the PEXX group: the gray bars represent P bars containing the inserted corresponding motif. PDR5 The fluorescence values ​​of the SynV strain of -mCherry are shown in the pink bar graph, which represents P strains containing the inserted motif. PDR5 The fluorescence values ​​of the GA183 strain of -mCherry are shown. The blue dots represent the ratio of the pink bars to the gray bars, which is the dynamic range.

[0096] In addition, knockout of some endogenous genes in Saccharomyces cerevisiae can also modulate the response intensity of glycyrrhetinic acid biosensors, including RDR1 and SSZ1. Figure 9 In the Control group, the gray bars represent bars containing P. PDR5 The fluorescence values ​​of the -mCherry (without inserted motif) SynV strain are shown in the pink bar graph, which contains P. PDR5 Fluorescence values ​​of GA183 strain with -mCherry (without inserted motif); In group △Rdr1: the gray bars represent those containing P PDR5 -mCherry (without inserted motif) and the fluorescence value of SynV strains with Rdr1 knocked out; the pink bars represent strains containing P. PDR5 -mCherry (without inserted motif), and the fluorescence value of GA183 strain with Ssz1 knocked out; △In the Ssz group: the gray bars are those containing P PDR5 -mCherry (without inserted motif), and the fluorescence value of the SynV strain with Ssz1 knocked out; the pink bar graph represents the presence of P PDR5 -mCherry (without inserted motif), and the fluorescence value of GA183 strain with Ssz1 knocked out.

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

[0098] The glycyrrhetinic acid biosensor was used to detect the level of glycyrrhetinic acid synthesis in Saccharomyces cerevisiae. There are 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 liquid culture medium, and the fluorescence intensity of the strain was detected by an enzyme-linked immunosorbent assay (ELISA) reader; (2) the strain was detected and sorted by flow cytometer, the cells with high fluorescence were sorted out, spread on a solid culture medium, and a single colony was picked.

[0099] Glycyrrhetinic acid biosensors for the adaptive evolution of glycyrrhetinic acid strains

[0100] To enhance the ability of engineered bacteria to synthesize glycyrrhetinic acid, a glycyrrhetinic acid biosensor was used to regulate the expression of the HIS3 gene, thereby coupling glycyrrhetinic acid production with bacterial growth to screen for high-yielding strains. Figure 4 As shown, with the increase in intracellular glycyrrhetinic acid synthesis and accumulation, the expression intensity of the PDR5 promoter is enhanced, leading to the expression of more His3 protein catalyzing the production of histidine from imidazole-glycerol-3-phosphate, thus enabling cells to recover growth in the auxotrophic medium SD-His (20 g / L glucose, 6.7 g / L ammonium sulfate as the amino acid-free yeast nitrogen source, and 1.3 g / L of a histidine-free amino acid mixture). By correlating glycyrrhetinic acid synthesis capacity with cell growth, the accumulation of glycyrrhetinic acid can be converted into an easily detectable growth parameter, and high-yielding glycyrrhetinic acid-producing bacteria can be isolated based on cell growth status.

[0101] The present invention will be further explained below through specific embodiments. It should be understood that these embodiments are merely illustrative and helpful in understanding the present invention, and are not intended to limit the present invention to these embodiments.

[0102] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0103] Example 1: Construction of a glycyrrhetinic acid biosensor

[0104] In order to build HOL-P PDR5 -mcherry-T CYC1 -KanMX-HOR gene pathway (HOL and HOR are homologous arms integrated into the genome, P PDR5 The promoter is T, mcherry is the red fluorescent protein gene, and T is the promoter. CYC1 (KanMX is the antibiotic resistance selection marker gene), and the target gene fragment was amplified by PCR. HOL and P were used as the terminator. PDR、 T CYC1 HOR uses brewer's yeast Saccharomyces cerevisiae Using the S288C genome as a template, PCR amplification was performed. mcherry and KanMX were amplified using plasmids pmCherry-C1 and pFA6-kanMX4, respectively. Fragment ligation was then performed via Gibson assembly. The ligated gene circuits were amplified by PCR and transformed into *Saccharomyces cerevisiae* using lithium acetate conversion. The transformed cells were cultured in solid YPD medium containing 300 μg / mL G418 and positive clones were selected.

[0105] Example 2 Characterization of glycyrrhetinic acid biosensor

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

[0107] Take 1 mL of the fermented sample and centrifuge at 5000 rpm for 2 min, discarding the supernatant. Resuspend the cells in 0.01 M PBS buffer (pH=7.4), centrifuge at 5000 rpm for 2 min, and discard the supernatant. Add an appropriate volume of 0.01 M PBS buffer to the centrifuge tube to resuspend the cells, ensuring the OD600 of the sample is between 0.2 and 0.8. Add 100 μL of sample sequentially to a 96-well microplate. Set the microplate reader's detection parameters: 1) absorbance at 600 nm; 2) excitation wavelength 580 nm, emission wavelength 610 nm. When analyzing the results, compare the fluorescence intensity per unit cell (fluorescence intensity / OD600).

[0108] The test results showed that the fluorescence intensity / OD600 of the *Saccharomyces cerevisiae* that does not synthesize glycyrrhetinic acid was 178 (see [link to relevant documentation]). Figure 7 SynV-P PDR5 (72-hour data), the fluorescence intensity / OD600 of the Saccharomyces cerevisiae that synthesizes glycyrrhetinic acid is 336 (see...). Figure 7 GA183-P PDR5 (Data from 72 hours). This indicates that the glycyrrhetinic acid biosensor is responsive to glycyrrhetinic acid.

[0109] Example 3 Modification of glycyrrhetinic acid biosensor

[0110] Using the online tool YEASTRACT+ (http: / / www.yeastract.com / index.php), the DNA binding motifs of transcriptional activators Pdr1, Pdr3, Yap1, Stb5 and transcriptional repressor Rdr1 contained in promoter PDR5 were predicted using the "Find TF Binding Site(s)" function under the "Pattern Matching" module.

[0111] The aforementioned DNA-binding motifs were inserted into the PDR5 promoter using point mutation. The insertion sites were -220 bp, -407 bp, and -513 bp. The specific transcription factor binding sequences inserted were: TCCGCGGA (Pdr1 / 3), TTACTAA (Yap1), and CGGAACGA (Stb5), respectively. Because the insertion sites of the transcription factor DNA-binding motifs were spaced far apart, only one DNA fragment could be inserted at each site 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. For promoter P PDR5 Primers used for modification

[0113]

[0114] Thirty-one promoter mutants, PE1 to PE31, were constructed using the primers described above. These mutants were characterized using the method described in Example 2. The mutation sequences and detection results are shown in Table 2 below. Figure 8 The results showed that the mutants had either a lower or higher response range (defined as the fluorescence intensity of *Saccharomyces cerevisiae* that synthesizes glycyrrhetinic acid / the fluorescence intensity of *Saccharomyces cerevisiae* that does not synthesize glycyrrhetinic acid). For example, the response range of the initiator promoter PDR5 was 2.22, and after modification, P... PDR5 The response range of PE20 has been improved to 2.96; the response range of the starter PDR5 is 2.22, and after modification, P... PDR5 - The response range of PE3 decreased to 0.94.

[0115] Furthermore, for P with reduced response capability PDR5 -PE3 was used to investigate its response characteristics. After culturing the strain for 6 h, different concentrations of glycyrrhetinic acid (0, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500 mg / L) were added, and the cells were cultured for another 6 h. Samples were then taken, and the fluorescence intensity of the cells was measured using a microplate reader. The results are as follows: Figure 10 As shown, 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 saturate. The results indicate that P... PDR5 -PE3 still has the ability to respond to glycyrrhetinic acid, with a response concentration of up to 50 mg / L.

[0116] Table 2

[0117]

[0118] It should be understood that the motif combinations shown in Table 2 above are nucleotide sequences in which the motifs are linked together in sequence, for example, Yap1+Stb5, which represents TTACTAACGGAACGA (SEQ ID NO: 34).

[0119] It should be understood that the insertion sites in Table 2 above are based on the original P PDR5 Nucleotide number (i.e., P without any inserted motif) PDR5 (SEQ ID NO: 1) is counted. As an example, P PDR5 The construction scheme for -PE23 (EQ ID NO: 35) is as follows: [The following text appears to be incomplete and requires further context: "in the original P..."] PDR5 (SEQ ID NO: 1) Stb5(CGGAACGA), Pdr1 / 3(TCCGCGGA) and Stb5(CGGAACGA) are inserted before the nucleotides at positions 220, 407 and 513 (see the underlined nucleotides in SEQ ID NO: 1 below) from the 3' end (i.e., in the 5' direction).

[0120] >SEQ ID NO: 1 (5'-3')

[0121] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGT TCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCAACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGA A ACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGGTGTGGGCTCACCCGCGGGTCGT GATCACGATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGAT G TGCATAACCTTATGGCTGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0122] >SEQ ID NO: 35 (5’-3’)

[0123] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGTTCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCAACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGACGGAACGA A ACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGTGTGGGCTCACCCGCGGGTCGTTCCGCGGA GATCACGATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGATCGGAACGA G TGCATAACCTTATGGCTGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0124] Example 4: High-throughput screening of high-yield glycyrrhetinic acid strains using an enzyme-linked immunosorbent assay (ELISA) reader.

[0125] Using ambient temperature plasma (ARTP) to study the P biosensor containing glycyrrhetinic acid PDR5 -PE21 was used to mutagenesis of Saccharomyces cerevisiae GA183, which synthesizes glycyrrhetinic acid. The ARTP treatment time was 1 minute. After mutagenesis, the bacterial culture was plated on YPD solid medium and incubated at 30°C for 3 days. Single colonies from the plates were transferred to 96-well plates containing YPD medium and incubated at 30°C for 3 days. The cultured strains were characterized using the glycyrrhetinic acid biosensor characterization method described in Example 2, and the fluorescence intensity / OD600 value was calculated. Strains with high fluorescence intensity / OD600 values ​​were selected, and their glycyrrhetinic acid yield was examined by liquid chromatography. The results showed a significant correlation between fluorescence intensity / OD600 value and glycyrrhetinic acid yield. The selected high-yielding strains showed a 23.6% increase in glycyrrhetinic acid yield compared to the control strain GA183.

[0126] Example 5: High-throughput screening of high-yield glycyrrhetinic acid-producing strains using flow cytometry

[0127] Using ambient temperature plasma (ARTP) to study the P biosensor containing glycyrrhetinic acid PDR5 -PE28 synthesizes glycyrrhetinic acid in Saccharomyces cerevisiae GA183 (GA183-P PDR5Mutagenesis was performed using PE28, with ARTP treatment time of 1 minute. After mutagenesis, the bacterial culture was spread on YPD solid medium and cultured at 30°C for 3 days.

[0128] The above bacterial suspensions were analyzed and sorted using a flow cytometer: The bacterial suspension was centrifuged at 5000 rpm for 2 min, and the supernatant was discarded. The bacterial cells were resuspended in 0.01 M PBS buffer (pH=7.4), centrifuged at 5000 rpm for 2 min, and the supernatant was discarded. An appropriate volume of 0.01 M PBS buffer was added to the centrifuge tube to resuspend the bacterial cells. The sample was filtered into a flow cytometer tube using a 70 μm cell sieve for analysis. The steps for using a flow cytometer are as follows:

[0129] (1) Before starting the instrument, check the amount of waste liquid in the waste liquid tank and whether the liquid volume in the 8× sheath liquid tank and distilled water tank is sufficient. (2) After opening the software, click “start-up”, the instrument will enter the startup program and automatically calibrate. After startup, add 6 drops of calibration microspheres to the flow cytometer 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 the sample, and save the data file after analysis. (5) Channel parameters: FSC 205, SSC 282, FL3 850; Detection cell count: 50000.

[0130] The results showed that the relative fluorescence intensity of the control strain GA183 was 2.03, while the relative fluorescence intensity of the experimental group was 85.52.

[0131] Highly fluorescent cells were selected using the "Gate" function and sorted into new test tubes. These tubes were then plated on YPD solid medium and incubated at 30°C for 3 days. Single colonies from the plates were transferred to 96-well plates containing YPD medium and incubated at 30°C for 3 days. The cultured strains were characterized using the glycyrrhetinic acid biosensor characterization method described in Example 2, and the fluorescence intensity / OD600 value was calculated. Strains with high fluorescence intensity / OD600 values ​​were selected, and their glycyrrhetinic acid yield was examined by liquid chromatography. The results showed that the selected high-yielding strains increased glycyrrhetinic acid production by 40.1% compared to the control strain GA183 (see [link to relevant documentation] for specific yields of each product of this strain). Figure 3 ).

[0132] Figure 3 This study presents the fermentation detection results of screened strains after applying a glycyrrhetinic acid biosensor to high-throughput screening. High-fluorescence cells were sorted by flow cytometry and plated. Fifty single colonies were selected from each plate and inoculated into YPD medium for fermentation for 5 days. Glycyrrhetinic acid production was then measured. The strain with the highest glycyrrhetinic acid production increased by 40.1% compared to the control.

[0133] The detection method for glycyrrhetinic acid is as follows:

[0134] (1) After fermentation, take 5 mL of fermentation broth into a 10 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, and separate the cell precipitate and fermentation broth supernatant.

[0135] (2) The bacterial pellet was resuspended in 400 μL of 6 M HCl solution and transferred to a bead-disrupting centrifuge tube. An appropriate amount of 0.5 mm glass beads were added, and the cells were disrupted using a homogenizer (75 Hz, 60 s ON / 15 s PAUSE, 5 cycles).

[0136] (3) Transfer the lysed cell solution to a 10 mL centrifuge tube, add 6 mL of ethyl acetate, vortex and extract thoroughly, centrifuge at 5000 rpm for 10 min, and transfer the upper ethyl acetate layer to a centrifuge tube for evaporation using a vacuum concentrator.

[0137] (4) Add 6 mL of ethyl acetate to the supernatant of the fermentation broth, vortex and extract thoroughly, centrifuge at 5000 rpm for 10 min, and transfer the upper ethyl acetate layer to a centrifuge tube to evaporate to dryness.

[0138] (5) Add 300 μL of chromatographic grade methanol to reconstitute the precipitate and the supernatant after extraction and evaporation.

[0139] (6) The reconstituted sample was filtered through a 0.22 μm organic phase filter membrane into a liquid phase vial and detected by ultra-high performance liquid chromatography (UHPLC).

[0140] UPLC detection method for fermentation products:

[0141] The fermentation products of the engineered strain were quantitatively determined using an UPLC instrument (Agilent Technologies 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and a diameter of 3.0 × 100 mm was used. The detection wavelength was 254 nm, the column temperature was 35 °C, and the mobile phases were: A: 0.1% formic acid aqueous solution and B: acetonitrile. The gradient elution process was as follows: 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. Qualitative and quantitative determinations were performed using the external standard method.

[0142] Example 6: Adaptive evolution of glycyrrhetinic acid strains using a glycyrrhetinic acid biosensor

[0143] Building HOL-P PDR5 -HIS3-T CYC1 The -HOR gene circuit was extracted and transformed into the Saccharomyces cerevisiae strain GA184, which synthesizes glycyrrhetinic acid, and named E0. The method was the same as in Example 1.

[0144] The strain E0 underwent adaptive laboratory evolution as follows: It was inoculated into auxotrophic medium SD-His with an initial inoculum size of OD600 = 0.2 and cultured in test tubes for 2 days at 200 rpm and 30°C. Subcultures were performed every 3 days thereafter, and the culture was removed on day 18. The evolved strain was preserved in glycerol tubes. The evolved strain was then inoculated into YPD medium and fermented for 5 days. The yield of glycyrrhetinic acid was then measured. The results are as follows: Figure 5 As shown in b, after 6 days of evolution, the glycyrrhetinic acid production reached its maximum, increasing by 17.5% compared to the starting strain, reaching 174.2 mg / L.

[0145] >SEQ ID NO: 1

[0146] AAGCAGCACCTCGTTGGCGCAGTCCCTTACATAGTACACAACATTTATCACTTCACACAATCAGGAGTGGAACTCAATGGAAAACAACACCACACGTATGATCTTACTAATAAAAGAACATGAACGTTCCTCAGCGCGAACGTTCGCATTCTGCGCCTTCGAGCACAGGATAAGTTGCAGGAAGCCATCACATCTATGCA ACGATTATCACGACACAACCTTGCCGCCGAGAAAACGTCCGTGGAGAACCATTCGGTCGATTGCTTCCCACGGAACGAGTGGACTGAAACTTAAGACTGCCCCTCTCTTTCCGCGGAATCGCTCATGCCGCGGTGCCACAACATTTTCAGATTTACTAAGACTCCGGTGAGTGTGGGCTCACCCGCGGGTCGTGATCACG ATTCAGCACCCTTTGGACTCGTGATTCCGTGGAAAGGTCAGATCTGTATTCCTACTTATGGTAATGTGCTAAAAAAAGAGAAATGTCTCCGCGGAACTCTTCTACGCCGTGGTACGATATCTGTTGAACGTAATCTGAGCAATACAAACAAGGCCTCTCCTATACATATATAATTGTGATGTGCATAACCTTATGGCTGTTCGCTTTTATTATCATACCTTAGAATGAAATCCAAAAGAAAAAAGTCACGCAAAGTTGCAAACATATAACAACTGTGTTAGTTATCACTCGACTTTGTTATTCTAATTATAAATAAATTGGCAACTAGGAACTTTCGAAAAAGAAATTAAAGACCCTTTTAAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACAAAA

[0147] SEQ ID NO:2

[0148] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

[0149] >SEQ ID NO:3

[0150] MSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0151] >SEQ ID NO:4

[0152] MSYSKHGIVQEMKTKYHMEGSVNGHEFTIEGVGTGYPYEGKQMSELVIIKPAGKPLPFSFDILSSVFQYGNRCFTKYPADMPDYFKQAFPDGMSYERSFLFEDGAVATASWNIRLEGNCFIHKSIFHGVNFPADGPVMKKKTIDWDKSFEKMTVSKEVLRGDVTMFLMLEGGGSHRCQFHSTYKTEKPVTLPPNHVVEHQIVRTDLGQSAKGFTVKLEAHAAAHVNPLKVK

[0153] >SEQ ID NO:5

[0154] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0155] >SEQ ID NO: 6

[0156] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0157] >SEQ ID NO: 7

[0158] MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILASFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN

[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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0167] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A glycyrrhetinic acid biosensor, characterized by, Comprises: a PDR5 promoter, 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, wherein the one or more sites comprise: 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.

2. The glycyrrhetinic acid biosensor according to claim 1, wherein Further comprises a reporter gene.

3. The glycyrrhetinic acid biosensor according to claim 2, wherein The reporter gene is a gene encoding a fluorescent protein or a gene encoding a histidine synthase.

4. The glycyrrhetinic acid biosensor according to claim 1, characterized by, 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.

5. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the first site, or a first sequence inserted at the first site is: the second motif, the third motif, a combination of the second motif and the third motif.

6. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the second site, or a 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.

7. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the third site, or a third sequence inserted at the third site is: the second motif, the third motif, a combination of the second motif and the third motif.

8. The glycyrrhetinic acid biosensor according to claim 1, wherein At least two of the one or more sites are inserted with the transcription factor binding sequence.

9. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the first site, or a first sequence inserted at the first site is the first motif, the second motif.

10. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the second site, or a second sequence inserted at the second site is: the first motif.

11. The glycyrrhetinic acid biosensor according to claim 1, wherein No insertion sequence is inserted at the third site, or a third sequence inserted at the third site is: the first motif.

12. The glycyrrhetinic acid biosensor of claim 1, wherein, At least one of the one or more sites is inserted with the transcription factor binding sequence.

13. Saccharomyces cerevisiae comprising the glycyrrhetinic acid biosensor of any one of claims 1 to 12.

14. The Saccharomyces cerevisiae of claim 13, wherein, The Saccharomyces cerevisiae is selected from the group consisting of: S288C, SynV, GA183, and GA184.

15. The Saccharomyces cerevisiae of claim 13, wherein, The Saccharomyces cerevisiae does not express RNA-dependent RNA polymerase (RDR1) and / or chaperone protein (SSZ1), wherein the nucleotide sequence of the gene encoding RDR1 is shown as SEQ ID NO: 8, and the nucleotide sequence of the gene encoding SSZ1 is shown as SEQ ID NO:

9.

16. Use of a glycyrrhetinic acid biosensor according to any one of claims 1 to 12 or of a Saccharomyces cerevisiae according to claim 13 or 14 for obtaining a Saccharomyces cerevisiae producing high amounts of glycyrrhetinic acid.

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