Yeast cell model responding to umami signal as well as preparation method and application thereof

Abstract

The invention belongs to the field of microbial engineering, and particularly relates to a yeast cell model responding to umami signals and a preparation method and application thereof. The construction method comprises the following specific construction steps: firstly, constructing a basic model strain mffi-y1 by knocking out 12 genes related to an endogenous GPCR (Glucagon Polymerase Chain Reaction) pathway of saccharomyces cerevisiae BY4741; a functional expression system of two subunits of umami receptors T1R1 and T1R3 is constructed on the basis of an mffi-y1 strain; and then, the GPA1-gust5 chimera module, the transcription factor STE12 module and the signal output module are sequentially integrated into the genome of the mffi-y1 strain. The invention discloses a flavor signal mode strain based on yeast cells for the first time, and the flavor signal mode strain will become a powerful platform of a complementary mammalian cell model.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering, and in particular relates to a yeast cell model responding to umami signals and a preparation method and application thereof. Background Art

[0002] Umami often represents the intake of protein or L-amino acids. Humans mainly recognize potential umami components in food through the umami receptor heterodimer T1R1 / T1R3 (class c G protein-coupled receptor, GPCR). Rapid and accurate acquisition of ligand-mediated umami signals is of great significance to human health and physiology. Mammalian cells have long been a common model for analyzing T1R1 / T1R3 signal activation, but there are also some challenges in using this model. Mammalian cells express dozens of endogenous GPCRs. For example, the widely used HEK 293 cell model has 75 different endogenous GPCRs, and the intracellular signaling pathways are complex. Therefore, when determining the phenotype of a specific GPCR activated by a certain ligand, the same Gα transduction pattern may exist, resulting in unclear signal output and other problems; in addition, mammalian cells have long passage and growth cycles, harsh culture environments, and relatively expensive analysis costs. The above limiting factors severely restrict large-scale, multi-scenario umami signal analysis.

[0003] In contrast, haploid Saccharomyces cerevisiae has only two native GPCRs: STE2 or STE3 respond to pheromone-mediated yeast mating behavior, while GPR1 senses glucose, which provides a simple and predictable system for studying GPCR signaling. The pheromone response pathway of Saccharomyces cerevisiae is one of the most studied examples of eukaryotic GPCR signaling. Similar to mammalian cells, Saccharomyces cerevisiae also has similar G protein heterotrimers, in which the Gα protein is GPA1 and the Gβγ is STE4 / STE18. Activation of STE2 or STE3 receptors by α-factor pheromones leads to the dissociation of STE4 / STE18 from GPA1. The STE4 / STE18 dimer binds to the downstream target STE20, prompting the latter to activate the MAPK signaling cascade (STE5: STE11-STE7-Fus3); after signal activation, phosphorylated Fus3 mediates the downstream transcription factor STE12, inducing the expression of mating-specific genes.

[0004] By expressing exogenous GPCRs on the surface of Saccharomyces cerevisiae, using the yeast pheromone GPCR signaling pathway, and expressing reporter genes (such as auxotrophic, green fluorescent protein, luciferase, etc.) under the control of a mating pathway-responsive promoter, ligand-mediated target GPCR signal analysis can be achieved. However, a yeast-based cell model has not yet been developed to characterize umami signals to make up for the limitations of mammalian cell models. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for preparing a yeast cell model that responds to umami taste signals, comprising the following steps:

[0006] (1) Knockout genes related to non-essential natural regulation and feedback regulation in the endogenous GPCR pathway of Saccharomyces cerevisiae BY4741 to construct the basic model strain mffi-y1;

[0007] (2) Based on the mffi-y1 strain in step (1), functional expression of T1R1 / T1R3 in a yeast system was performed;

[0008] (3) Based on the strain in step (2), the genome was reconstructed into a GPA1-gust5 chimera module, a transcription factor STE12 module and a signal output module.

[0009] (4) Response analysis and fluorescence signal processing of the umami yeast cell model: After genome reconstruction in steps (1) to (3), an umami yeast cell model is obtained; after activation and culture of the cell model, OD 600 0.5 was inoculated into fresh SD medium containing ligand (96-deep well plate), and the fluorescence value of each well was collected by fluorescence imaging plate reader or flow cytometer after culturing at 30℃ for 8h. The excitation wavelength and emission wavelength of fluorescence collection were set to 485 and 535nm, respectively. The selected stimuli included the medium stimulation group without ligand, the non-umami control group and the umami stimulation group. The cell fluorescence values ​​at different concentrations of each ligand were used to fit the dose-effect curve to solve the EC50.

[0010] Furthermore, the genes related to non-essential natural regulation and feedback regulation in the endogenous GPCR pathway in step (1) include STE2, STE3, GPA1, GPR1, GPA2, SST2, FAR1, MF(ALPHA)1, MF(ALPHA)2, MFA1, MFA2 and STE12.

[0011] Furthermore, the specific method for functional expression of T1R1 / T1R3 in the yeast system in step (2) is a plasmid vector method, and the plasmid is selected as a high-copy free plasmid pESC-Ura3-pGal 1,10 and low-copy centromeric plasmid pRS316-4M5.3R-pGal 1,10 , using the galactose bidirectional promoter pGal 1,10 Also mediates the expression of T1R1 / T1R3.

[0012] Furthermore, the specific method for functional expression of T1R1 / T1R3 in the yeast system in step (2) is a genome integration method, wherein the genome integration uses the CRISPR / Cas9 tool to integrate the p-SS-kozak-T1R1 / T1R3-t element into sites such as X2, X3, XI-2, XII-5, URA3, LEU2, STE2 and STE3; wherein pt is a promoter-terminator sequence, and three pairs of promoter-terminator elements, pTDH3-tTDH1, pTEF1-tCYC1 and pCCW12-tSSA1, are used to mediate the expression of T1R1 and T1R3; SS is a signal peptide sequence, and the mating factor α secretion signal is used to assist the expression and membrane localization of T1R1 / T1R3; a kozak sequence is added before the start codons of T1R1 and T1R3; and the codon preference of T1R1 and T1R3 expressed in Saccharomyces cerevisiae is optimized.

[0013] Furthermore, the nucleotide sequence of the kozak sequence is AAAAAAATGTCT.

[0014] Furthermore, the genomic reconstruction of the GPA1-gust5 chimera module in step (3) includes the following steps: replacing the five sequences corresponding to the C-terminus of the yeast endogenous Gα protein with the five amino acid residues at the C-terminus of the Gα protein coupled to T1R1 / T1R3, thereby constructing a GPA1-gust5 chimera that couples the active T1R1 / T1R3 signal to the yeast mating pathway.

[0015] Furthermore, the genome reconstruction of the transcription factor STE12 module in step (3) includes the following steps: integrating a natural transcription factor or a synthetic transcription factor-synthetic promoter module into the HO site.

[0016] Furthermore, the signal output module in step (3) is composed of a promoter-reporter gene mediated by STE12, the former is selected as pFUS1 or pFIG1, and the latter is selected as the fluorescent protein GFP / ZsGreen.

[0017] The present invention also provides a yeast cell model responding to umami taste signals, which is prepared by the above preparation method.

[0018] The present invention also provides application of the yeast cell model responding to umami taste signals in analyzing umami taste signals.

[0019] The present invention has the following beneficial effects:

[0020] The present invention constructs a yeast cell model that specifically responds to umami signals for the first time. All signal elements of the model cells are integrated into the genome, and the heterogeneity between cells is low; because the endogenous yeast GPCR and the non-essential natural regulation and feedback regulation elements involved are knocked out, and the SFT-synthetic promoter module is used, the umami signal output is single and highly specific; at the same time, the cell model has simple cultivation requirements, short growth and passage cycles, low cultivation and analysis costs, and has the potential to be widely promoted and applied to actual production; the cell model can be applied to high-throughput umami detection, potential umami component screening and matching molecular simulation flux T1R1 / T1R3-ligand recognition interaction research and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is a schematic diagram of the Saccharomyces cerevisiae pheromone pathway and the engineered umami signaling pathway;

[0023] Figure 2 This is the electrophoresis diagram of PCR verification of 12 gene knockout colonies in Saccharomyces cerevisiae BY4741. Several single colonies were picked for each strain, and the control / c was BY4741 genome PCR;

[0024] Figure 3 This is a diagram of T1R1-GFP / T1R3-mCherry (pESC-Ura3-pGal1,10) expression analysis. Figure 3 A- Figure 3 B) Confocal laser scanning microscopy was used to characterize the expression of T1R1-GFP and T1R3-mCherry; ( Figure 3 C) Quantitative characterization of receptor expression by microplate reader;

[0025] Figure 4 is qPCR analysis of the mRNA levels of T1R1 / T1R3 mediated by different transcription elements;

[0026] Figure 5 This is the expression of T1R1-GFP / T1R3-mCherry integrated into the genome. Figure 5 A- Figure 5 G) Laser confocal microscopy characterization results, ( Figure 5 A) pCCW12-MfαSS-GF P-tSSA1 (STE2 site); ( Figure 5B) pCCW12-T1R1-GFP-tSSA1 (X2 site); Figure 5 C) pCCW12-MfαSS-T1R1-ZsGreen-tSSA1(STE2 site); Figure 5 D) mCherry (STE3 site); Figure 5 E) T1R3-mCherry (STE3 site); Figure 5 F) pCCW12-MfαSS-T1R3-mCherry-tSSA1(URA3 site); Figure 5 G) pCCW12-MfαSS-T1R3-mCherry-tSSA1(STE3 site); Figure 5 H- Figure 5 1) Quantitative characterization of fluorescence intensity by microplate reader;

[0027] Figure 6 is the immunoblot analysis of T1R1 / T1R3, HEK 293(T1R1 / T1R3) refers to the expression of T1R1 / T1R3 in HEK 293 cells (positive control);

[0028] Figure 7 For the GPA1-gust5 chimera, genomic reconstruction analysis of the GFP signaling output module;

[0029] Figure 8 Schematic diagram of the GPA1-gust5-STE12-GFP series module knocked into the HO site;

[0030] Fig. 9 Schematic diagram of STE12 composition, GFP expression and STF mediation;

[0031] Fig.10 To analyze the growth and optimal incubation conditions of the umami yeast cell model. Fig.10 A) Optimal incubation temperature; Fig.10 B) Optimal incubation time; Fig.10 C) Growth rate analysis;

[0032] Fig.11 This is the dose-response analysis of the mffi-y4 strain under stimulation of different ligands. Fig.11 A)MSG; Fig.11 B) WSA; Fig.11 C)IMP; Fig.11 D) sucrose; Fig.11 E) BMP; Fig.11 F)MSG+IMP; Fig.11 G) NaCl; Fig.11 H) Quinine sulfate; Fig.111) Citric acid. DETAILED DESCRIPTION

[0033] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The purpose of the present invention is to provide a yeast cell model that responds to umami signals, which is implemented according to the following technical scheme. The specific steps are as follows:

[0039] (1) Construction of the basic model strain mffi-y1: Saccharomyces cerevisiae BY4741 was selected as the initial strain, and the CRISPR / Cas9 gene editing tool was used to sequentially knock out 12 genes related to non-essential natural regulation and feedback regulation in the endogenous GPCR pathway of BY4741 to form the basic model strain mffi-y1; mffi-y1 only retained the MAPK cascade signal processing module. The specific genes and functional characteristics knocked out included: 1) removal of the negative feedback regulator SST2 in the signal pathway that regulates the sensitivity of pheromone receptors or exogenous GPCR response signals; 2) removal of the yeast cell division cycle arrest factor FAR1; 3) deletion of the yeast endogenous pheromone receptors STE2 and STE3;

[0040] 4) Deletion of pheromone communication mechanisms MF(ALPHA)1, MF(ALPHA)2, MFA1, and MFA2;

[0041] 5) Knockout of the yeast glucose sensing receptor GPR1 and the coupled Gα protein signal GPA2;

[0042] (2) Functional expression of T1R1 / T1R3: Based on the mffi-y1 strain in step (1), functional expression of T1R1 / T1R3 was carried out. The expression of T1R1 / T1R3 was carried out in two forms: plasmid vector and genome integration. The plasmid selected was a high-copy free plasmid (pESC-Ura3-pGal 1,10 ), using the galactose bidirectional promoter pGal 1,10 Simultaneously mediate the expression of T1R1 / T1R3; for genomic integration, the CRISPR / Cas9 tool was used to integrate the p-SS-kozak-T1R1 / T1R3-t element into the X2, X3, XI-2, XII-5, URA3, LEU2, STE2 and STE3 sites; pt is the promoter-terminator sequence, and three pairs of promoter-terminator elements, pTDH3-tTDH1, pTEF1-tCYC1 and pCCW12-tSSA1, were used to mediate the expression of T1R1 and T1R3; SS is the signal peptide sequence, and MfαSS was used to assist the expression and membrane localization of T1R1 / T1R3; the kozak sequence (AAAAAAATGTCT) was added before the start codons of T1R1 and T1R3; the codon preference of T1R1 and T1R3 in Saccharomyces cerevisiae was optimized;

[0043] (3) Genome reconstruction of the GPA1-gust5 chimera module, transcription factor STE12 module, and signal output module: Based on the mffi-y1 strain in step (1), three rounds of systematic genome reconstruction were performed to complete the construction of the GPA1-gust5 chimera module, transcription factor STE12 module, and signal output module at different levels. In the first round, a minimal reconstruction operation was implemented. First, the GPA1-gust5 chimera was knocked into the GPA1 site to replace GPA1. The expression of the GPA1-gust5 chimera was regulated by the original transcription element of GPA1 (pGPA1-tGPA1). STE12 was not changed. Finally, GFP was knocked into the FUS1 site to replace the FUS1 gene. The pFUS1 gene was used to regulate the expression of the GPA1-gust5 chimera. In the second round of genome reconstruction, the transcription elements of GPA1-gust5 and STE12 were optimized, and the three modules of GPA1-gust5-STE12-GFP were placed adjacent to each other. pPGK1-GPA1-gust5-tENO2, pRAD27-STE12-tENO1 and pFUS1-ZsGreen-tTDH1 were knocked into the HO site in sequence, and the three modules were connected by spacer sequences. In the third round of gene reconstruction, the STE12-pFUS1 module was replaced by the STF-synthetic promoter (LexA-PRD-LexO(6x)-pLEU2m) module, and the others were consistent with the second round.

[0044] (4) Response analysis of umami yeast cell model: The order of steps (2) and (3) was adjusted according to the needs. Based on the basic model strain mffi-y1, modular and customized genome reconstruction of steps (2) and (3) was performed to construct umami signal yeast cell models mffi-y3 and mffi-y4. The former used the second round of gene reconstruction operation of step (3), and the latter used the third round of gene reconstruction operation of step (3). The other modules were the same. The umami yeast cell model was activated and cultured in SD medium overnight, and the OD 600 0.5 was inoculated into SD medium containing ligand (96-deep well plate), and the fluorescence value (FI) and OD of each well were collected by fluorescence imaging plate reader after culturing at 30°C for 8 h. 620 Value, fluorescence data using OD 620 Value calibration (FI / OD 620), the excitation wavelength and emission wavelength of fluorescence collection were set to 485 and 535 nm respectively; the selected stimuli included a culture medium stimulation group without ligand, a non-umami control group (NaCl, sucrose, citric acid and quinine sulfate) and an umami stimulation group (monosodium glutamate MSG, sodium inosinate IMP, disodium succinate WSA, beef cinnamon peptide BMP and MSG+IMP binary mixture), ensuring that the highest concentration of the ligand did not affect the normal growth of the engineered strain. Each group of experiments was designed with an empty control strain mffi-y2 (a strain based on mffi-y1 but only step (3) was implemented) without T1R1 / T1R3 to ensure the reliability of the response results; because the OD value after strain culture exceeded the linear range of the instrument's photoelectric detector, it was calibrated by the formula:

[0045]

[0046] Among them, OD meas OD is the measured optical density value. sat is the saturation value of the instrument (photoelectric detector), and k is the actual optical density value when the detector reaches half-saturation of the measured optical density.

[0047] The relationship between the calibrated fluorescence value and the logarithmic concentration of the ligand was fitted using a four-parameter logarithmic curve to calculate the EC50. The formula is as follows:

[0048]

[0049] Where Y is the fluorescence value after calibration, Bottom is the fluorescence value of the bottom platform of the fitting curve, Top is the fluorescence value of the top platform of the fitting curve, X is the logarithmic concentration of the ligand (log mM), and Hillslope is the Hill coefficient.

[0050] Example

[0051] The present invention is further described below with specific embodiments to illustrate the implementation process of the invention.

[0052] In this example, Saccharomyces cerevisiae BY4741 was selected as a model strain, and 12 genes including STE2, STE3, GPA1, GPR1, GPA2, SST2, FAR1, MF (ALPHA) 1, MF (ALPHA) 2, MFA1, MFA2 and STE12 were knocked out in sequence using CRISPR / Cas9 tools ( Figure 2 ), the basic model strain mffi-y1 was constructed. The gRNA sequences of the experimental design are shown in Table 1.

[0053] Table 1 gRNA sequence information involved in knocking out 12 genes in the BY4741 genome

[0054]

[0055]

[0056] In this example, two forms of expression of T1R1 / T1R3 were attempted: 1) plasmid vector and 2) genome integration. Plasmid selection: high-copy free plasmid (pESC-Ura3-pGal 1,10 ), using the galactose bidirectional promoter pGal 1,10 At the same time, it mediates the expression of T1R1 / T1R3. The results showed that most of T1R1 and T1R3 were expressed in the cytoplasm, some may be accumulated in the endoplasmic reticulum, and only a small amount was located on the cell membrane. The fluorescence intensity was analyzed by an ELISA instrument and it was found that the expression levels of the two receptor proteins were extremely low ( Figure 3 ), indicating that plasmid vectors cannot effectively assist the functional expression of T1R1 / T1R3. The genome integration mode was then explored. Four sites, X2, X3, XI-2, and XII-5, were selected for the first round of genome integration, and three pairs of promoter-terminator elements were used to regulate the expression of umami taste receptors. qPCR analysis found that the mRNA level of T1R1 / T1R3 mediated by pCCW12-tSSA1 was higher than that of pTDH3-tTDH1 and pTEF1-tCYC1( Figure 4 ); therefore, the pCCW12-tSSA1 element was selected for subsequent T1R1 / T1R3 expression analysis. Unfortunately, after knocking in pCCW12-T1R1-GFP-tSSA1 and pCCW12-T1R3-mCherry-tSSA1 into the X2 and XII-5 or STE2 and STE3 sites, no expression of fluorescent protein-labeled umami taste receptors was observed. The above results indicate that the functional expression of T1R1 / T1R3 in yeast is not as easy as imagined, and a simple genome integration (including regulatory elements) strategy is not feasible.

[0057] In this example, in order to promote high expression and effective plasma membrane localization of T1R1 / T1R3, an additional signal peptide or leader sequence was added to the N-terminus of the umami taste receptor. In this example, the MfαSS sequence was selected. MfαSS-GFP, MfαSS-mCherry, MfαSS-T1R1-GFP and MfαSS-T1R3-mCherry sequences were constructed, and the three sites STE2, STE3 and URA3 were selected for knock-in. The expression of the protein is shown in Figure 2. Figure 5 As shown. After the MfαSS-T1R1-ZsGreen fragment was knocked into the STE2 site, the fusion protein was not only expressed in the cytoplasm, but also partially fluorescent on the cell membrane ( Figure 5 C); at the same time, the fluorescence intensity of MfαSS-T1R1-ZsGreen was close to that of MfαSS-GFP, and was significantly different from that of the basic strain mffi-y1 (p<0.001, Figure 5H). The expression of T1R3 is similar to that of T1R1. MfαSS-T1R3-mCherry was knocked into URA3 ( Figure 5 F) or STE3 locus ( Figure 5 G), the accumulation of T1R3-mCherry in the cytoplasm decreased, especially at the STE3 site, and MfαSS-T1R3-mCherry was partially localized to the cell membrane ( Figure 5 G); at the same time, the fluorescence intensity of MfαSS-T1R3-mCherry (STE3 site) and MfαSS-T1R3-mCherry (URA3 site) was significantly higher than that of the basic model strain mffi-y1 (p<0.05), especially the former (p<0.01, Figure 5 I), the fluorescence intensity at this time is 13-15 times that of the plasmid system ( Figure 3 F, Figure 5 H / I). The above results indicate that MfαSS combined with the strong promoter pCCW12 and STE2 / STE3 integration sites contribute to the functional expression and partial membrane localization of T1R1 / T1R3. This is the first time that T1R1 / T1R3 has been functionally expressed in yeast. After the MfαSS-T1R1 and MfαSS-T1R3 fragments were knocked into the STE2 and STE3 sites, respectively, protein immunoblot analysis showed that the expression level of T1R1 / T1R3 in yeast was higher than that in the HEK 293 cell system ( Figure 6 ).

[0058] In this example, the optimal GPA1-gust5 chimeric protein, transcription factor STE12 and signal output module were determined through three rounds of genome reconstruction. In the first round, a minimal reconstruction operation was performed. First, the constructed GPA1-gust5 chimera was knocked into the GPA1 site to replace GPA1 ( Figure 7 A), the expression of the GPA1-gust5 chimera was regulated by the original transcription element of GPA1 (pGPA1-tGPA1); STE12 was not modified; finally, GFP was knocked into the FUS1 locus to replace the FUS1 gene ( Figure 7 B), GFP expression was regulated by pFUS1 (pFUS1-GFP-tFUS1). The second round of genome reconstruction optimized the transcriptional elements of GPA1-gust5 and STE12 ( Figure 8 ). The three modules of "Gα-STE12-GFP" were placed next to each other ( Figure 8), pPGK1-GPA1-gust5-tENO2, pRAD27-STE12-tENO1 and pFUS1-ZsGreen-tTDH1 were knocked into the HO site in sequence, and the three modules were connected by spacer sequences. In the third round of gene reconstruction, the STE12-pFUS1-ZsGreen module was replaced with the STF (pRAD27-LexA-PRD-tENO1)-synthetic promoter-ZsGreen (LexO (6x)-pTEF2m-ZsGreen-tTDH1) module ( Fig. 9 ). After the above modular and customized genome reconstruction, the umami signal yeast cell models mffi-y3 and mffi-y4 were constructed. The former used the natural transcription factor STE12, and the latter used the STF-synthetic promoter module, and the other modules were consistent.

[0059] In this example, the optimal incubation time and temperature for the umami yeast cell model to respond to ligand stimulation were optimized. The results showed that the incubation temperature of 20°C was too low, the strain grew slowly, and the fluorescence after 24 hours of induction with MSG was weaker than that at 30°C (p<0.05); on the contrary, the temperature of 37°C was too high, the strain grew too fast, and the density was too high, resulting in an average fluorescence value weaker than that at 30°C (p<0.05); the fluorescence values ​​at 25°C and 30°C were close, and the incubation time of 30°C was selected after comprehensive consideration ( Fig.10 A). In terms of incubation time, after adding MSG, the fluorescence value increased significantly from 0 to 8 h (p<0.05), remained basically constant from 8 to 14 h, and gradually decreased after 14 h ( Fig.10 B). After comprehensive consideration, 8 h was selected as the optimal incubation time. This condition not only achieved high fluorescence values, but also shortened the ligand-mediated umami signal response time.

[0060] In this example, the effect of multiple rounds of gene editing on strain growth was analyzed by growth rate parameters ( Fig.10 C), the results showed that compared with the original strain BY4741, the absolute values ​​of the growth rates of mffi-y3 and mffi-y4 showed a decreasing trend. Except for the mffi-y4 strain, the growth rates of mffi-y3 and mffi-y2 (without integration of T1R1 / T1R3 genes, and the other reconstruction processes were the same as mffi-y3) were not significantly different from BY4174. In general, gene reconstruction did not cause abnormal growth of the strain.

[0061] In this example, the umami stimulation dose-effect analysis of the engineered strain was carried out under the above optimal conditions ( Fig.11As expected, the response value of the mffi-y2 strain was basically at the baseline level under the stimulation of different concentrations of MSG; mffi-y5 is a derivative of mffi-y2 (pCCW12-T1R1 / T1R3-tSSA1 sequences were introduced into the X3 and X1-2 sites, respectively), and no dose-response curve was observed after MSG stimulation ( Fig.11 A), which corresponds to the previous result ( Figure 4 ), under this condition, the expression level of T1R1 / T1R3 was low and not effectively localized to the cell membrane. Typical dose-response curves were observed for mffi-y3 and mffi-y4 after MSG stimulation; in particular, mffi-y4 had a higher maximum response value than mffi-y3 due to the use of the STF-synthetic promoter module, and the EC50 value was also reduced from 17.88 to 16.33mM ( Fig.11 A). The umami response performance of mffi-y4 was better than that of mffi-y3, and the former was selected for dose-response analysis of various umami and non-umami components ( Fig.11 When non-umami ingredients such as sucrose ( Fig.11 D), NaCl( Fig.11 G), Quinine sulfate ( Fig.11 H) and citric acid ( Fig.11 I) When stimulating mffi-y4, the change of fluorescence value is basically the same as that of mffi-y2, and the fluorescence value does not increase with the increase of stimulus concentration. Fig.11 B), IMP( Fig.11 C), BMP( Fig.11 E) and MSG+IMP( Fig.11 F) stimulation, a typical dose-response "S" curve was observed. The above results show that the umami yeast cell model has good specificity. In particular, the EC50 of MSG alone is 16.35mM, and when IMP is added, the EC50 of MSG decreases to 10.77mM ( Fig.11 F), indicating that the umami yeast cell model constructed in this study can be used for the analysis of umami synergistic effects.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a yeast cell model that responds to umami signals, characterized in that: The following steps are involved: (1) Knockout genes related to non-essential natural regulation and feedback regulation in the endogenous GPCR pathway of Saccharomyces cerevisiae BY4741 to construct the basic model strain mffi-y1; (2) Based on the mffi-y1 strain in step (1), functional expression of T1R1 / T1R3 in a yeast system was performed; (3) Based on the strain in step (2), the genome was reconstructed into a GPA1-gust5 chimera module, a transcription factor STE12 module and a signal output module.

2. The preparation method according to claim 1, characterized in that: The genes related to non-essential natural regulation and feedback regulation in the endogenous GPCR pathway in step (1) include STE2, STE3, GPA1, GPR1, GPA2, SST2, FAR1, MF(ALPHA)1, MF(ALPHA)2, MFA1, MFA2 and STE12.

3. The preparation method according to claim 1, characterized in that: The specific method for functional expression of T1R1 / T1R3 in the yeast system in step (2) is a plasmid vector method, and the plasmid is selected as a high-copy free plasmid pESC-Ura3-pGal 1,10 and low-copy centromeric plasmid pRS316-4M5.3R-pGal 1,10 , using the galactose bidirectional promoter pGal 1,10 Also mediates the expression of T1R1 / T1R3.

4. The preparation method according to claim 1, characterized in that: The specific method for functional expression of T1R1 / T1R3 in the yeast system in step (2) is a genome integration method, wherein the p-SS-kozak-T1R1 / T1R3-t element is integrated into sites such as X2, X3, XI-2, XII-5, URA3, LEU2, STE2 and STE3 using the CRISPR / Cas9 tool for genome integration; wherein pt is a promoter-terminator sequence, and three pairs of promoter-terminator elements, pTDH3-tTDH1, pTEF1-tCYC1 and pCCW12-tSSA1, are used to mediate the expression of T1R1 and T1R3; SS is a signal peptide sequence, and a mating factor α secretion signal is used to assist the expression and membrane localization of T1R1 / T1R3; a kozak sequence is added before the start codons of T1R1 and T1R3; and the codon preference of T1R1 and T1R3 expressed in Saccharomyces cerevisiae is optimized.

5. The preparation method according to claim 4, characterized in that: The nucleotide sequence of the kozak sequence is AAAAAAATGTCT.

6. The preparation method according to claim 1, characterized in that: The genomic reconstruction of the GPA1-gust5 chimera module in step (3) includes the following steps: using the C-terminal 5 amino acid residues of the Gα protein coupled to T1R1 / T1R3 to replace the 5 sequences corresponding to the C-terminus of the yeast endogenous Gα protein, to construct a GPA1-gust5 chimera that couples the active T1R1 / T1R3 signal to the yeast mating pathway.

7. The preparation method according to claim 1, characterized in that: The genomic reconstruction of the transcription factor STE12 module in step (3) comprises the following steps: integrating a natural transcription factor or a synthetic transcription factor-synthetic promoter module into the HO site.

8. The preparation method according to claim 1, characterized in that: The signal output module in step (3) is composed of a promoter-reporter gene mediated by STE12, the former is selected as pFUS1 or pFIG1, and the latter is selected as the fluorescent protein GFP / ZsGreen.

9. A yeast cell model that responds to umami taste signals, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. Use of the yeast cell model that responds to umami taste signals as claimed in claim 9 in analyzing umami taste signals.

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