Saccharomyces cerevisiae pyruvic acid response biosensor as well as construction method and application thereof
By constructing a pyruvate response biosensor in Saccharomyces cerevisiae, the problem of difficulty in regulating Saccharomyces cerevisiae is solved, dynamic regulation of the Saccharomyces cerevisiae metabolic network is achieved, and the metabolic efficiency of Saccharomyces cerevisiae is improved.
Patent Information
- Application Number
- CN202510661302.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art, the pyruvate response gene circuit of Saccharomyces cerevisiae in eukaryotic organisms is insufficiently developed, which leads to difficulty in metabolic regulation of Saccharomyces cerevisiae cells in synthetic biology, and there is difficulty in application of prokaryotic regulatory elements in eukaryotic cells.
Saccharomyces pyruvate-responsive biosensor was constructed. By fusing nuclear localization signal peptides at the N-terminus of the PdhR gene, the transcription factor PdhR and xylose transcription inhibitor XylR were transported to the cell nucleus, and glucose was used as an indirect activator to design pyruvate-activated, inhibited and bifunctional gene circuits to achieve regulation of Saccharomyces metabolic network.
The dynamic regulation of the metabolic network of Saccharomyces cerevisiae has been achieved, the ability to regulate metabolic flux is enhanced, the application reference for prokaryotic regulatory elements in eukaryotic cells is provided, and the metabolic efficiency of Saccharomyces cerevisiae has been improved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a pyruvic acid responsive biosensor for saccharomyces cerevisiae, a construction method and an application thereof. Background Art
[0002] To achieve efficient biosynthesis, metabolic engineering allows cells to be reprogrammed and metabolic fluxes to be directed towards the biosynthesis of target chemicals. However, the synthesis efficiency of most microbial cell factories is still low, and a major limiting factor is the trade-off between product synthesis flux and cell growth flux. Simple static modification methods, such as knocking out competing pathways and overexpressing rate-limiting enzymes, may lead to disorders in the intracellular metabolic system, thereby reducing the synthesis efficiency of the target product. These bottlenecks hinder the efficient production of natural products by microorganisms. Mimicking natural regulatory systems, gene circuits have been designed to sense signal molecules or metabolite concentrations to control the expression of key genes in the metabolic network, thereby autonomously controlling and balancing cellular metabolic fluxes, giving microbial cell factories the ability to self-learn and make decisions.
[0003] Pyruvate is a key central metabolite connecting the glycolysis pathway and the tricarboxylic acid cycle. It provides a carbon skeleton for product synthesis and, through its entry into the tricarboxylic acid cycle, provides energy and reducing power for cell growth. It is a key node in the central carbon metabolism network. The synthesis of its related products competes with cell growth, allowing it to be designed to fine-tune the synthesis of various central metabolic products. With the advancement of synthetic biology, several pyruvate-responsive gene circuits have been designed and developed based on the pyruvate-responsive transcription factor PdhR. In 2019, Zhang Dawei et al. from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, published a patent for the construction of a pyruvate gene circuit in Escherichia coli based on PdhR and the promoter Pap, which was used for high-throughput screening of high-pyruvate-producing strains. In 2020, Liu Long et al. from Jiangnan University published a patent for the construction of a pyruvate gene circuit in Bacillus subtilis based on PdhR and the promoter P43, which was used to control downstream gene expression. Although pyruvate-responsive gene circuits show great potential for regulating carbon flux, currently, there is a lack of developed and exploited chassis cells.
[0004] Saccharomyces cerevisiae is a model eukaryotic microorganism and an important industrial microorganism and synthetic biology chassis. It is widely used in the synthesis of a variety of high-value chemicals. At present, the research on the gene circuits of the eukaryotic model organism Saccharomyces cerevisiae is still lagging behind. Gene circuits that respond to substances such as malonyl CoA and xylose have been developed in Saccharomyces cerevisiae, and progress has been made in metabolic flux regulation and high-throughput screening. However, response switches related to the control of central metabolism such as pyruvate have not been widely developed in eukaryotic cell chassis. At present, pyruvate-responsive gene circuits have only been developed and applied in Bacillus subtilis and Escherichia coli. Given its role as a node in the central carbon metabolism network, the development of a responsive switch for the key metabolite pyruvate in the model organism Saccharomyces cerevisiae has important reference and application value.
[0005] The gene circuits that have been developed are generally concentrated in prokaryotic hosts, and the development and application of regulatory elements in eukaryotes is still lacking. This is because the genetic regulatory elements of eukaryotes are often larger and more complex than those of prokaryotes, making their rational design and evolution more difficult. The existing strategy is to introduce prokaryotic regulatory elements into eukaryotic hosts, but due to the strict compartmentalization and complex regulation of eukaryotic systems, it is still difficult to use prokaryotic regulatory elements to construct functional genetic circuits in eukaryotic hosts. For example, gene transcription in eukaryotic systems involves the coordinated action of multiple transcription factors, and related proteins need to be recruited into the cell nucleus; its signal transduction requires multiple phosphorylases and dephosphorylases as bridges; the transmembrane transport of small molecules in eukaryotic systems is more complex, etc. At present, there are no universal design principles to guide the construction of gene circuits using prokaryotic elements in eukaryotic cells. Summary of the Invention
[0006] The first object of the present invention is to provide a Saccharomyces cerevisiae pyruvate-responsive biosensor.
[0007] To achieve the above object, the present invention adopts the following technical solutions: A pyruvate-responsive biosensor of Saccharomyces cerevisiae, comprising a pyruvate-activated gene circuit, a pyruvate-repressed gene circuit, or a bifunctional gene circuit; The pyruvate-activated gene circuit comprises a transcription factor PdhR, a target gene to be activated, a PdhR expression promoter, and a pyruvate-activated promoter, all constructed on the same vector; The pyruvate-repressible gene circuit comprises a transcription factor PdhR, a target gene to be repressed, a PdhR expression promoter, a pyruvate-activated promoter, a xylose transcription repressor XylR, and a pyruvate-repressible promoter, all constructed on the same vector; The bifunctional gene circuit comprises a target gene to be activated, a target gene to be repressed, a transcription factor PdhR, a PdhR expression promoter, a pyruvate-activated promoter, a xylose transcription repressor XylR, and a pyruvate-repressible promoter, all constructed on the same vector; The pyruvate-activated promoter is a constitutive promoter with a PdhR binding sequence PdhO inserted into it; the ketoacid-repressible promoter is a constitutive promoter with a XylR binding sequence xylO inserted into it; and the N-termini of the PdhR and XylR are fused with nuclear localization signal peptides, respectively.
[0008] Unlike prokaryotes, eukaryotic cells have a strictly compartmentalized structure. Transcription factors must be transported into the nucleus to recognize and bind to modified promoters, thereby exerting their regulatory effects. This invention, specifically targeting the Saccharomyces cerevisiae system, links a nuclear localization signal peptide to the N-terminus of the PdhR gene, thereby fully transporting PdhR into the nucleus.
[0009] In some embodiments of the present invention, the nucleotide sequence of the nuclear localization signal peptide is shown as SEQ ID NO.1.
[0010] In some embodiments of the present invention, the C-terminal fusion protein degradation tags of the transcription factor PdhR and the xylose transcription repressor XylR avoid the continuous expression and accumulation of the transcription factors and enhance the dynamic regulation ability.
[0011] Preferably, the nucleotide sequence of the protein degradation tag fused to the C-terminus of the transcription factor PdhR is shown as SEQ ID NO.2, and the nucleotide sequence of the protein degradation tag fused to the C-terminus of the xylose transcription repressor XylR is shown as SEQ ID NO.10.
[0012] In some embodiments of the present invention, the constitutive promoter inserted into the pyruvate-activated gene circuit is P TDH3 The constitutive promoter inserted into the xylO in the pyruvate repressible gene circuit is P TEF1 .
[0013] Preferably, the PdhO is inserted into P TDH3 -150 site, -128 site or -5 site of the promoter; The XylO is inserted into the P TEF1 The -105 site of the promoter.
[0014] In some embodiments of the present invention, the PdhR expression promoter is selected from P with weak promoter strength. PRM promoter; The expression promoter of the xylose transcription repressor XylR is a pyruvate-activated promoter.
[0015] A second object of the present invention is to provide a method for constructing the above-mentioned pyruvate-responsive biosensor, comprising: Connecting the pyruvate-activated promoter to the target gene to be activated and introducing the promoter into a vector to construct an expression vector for the target gene to be activated; Connecting the PdhR expression promoter to the PdhR gene with a nuclear localization signal peptide fused to its N-terminus to construct a PdhR expression cassette, and constructing the PdhR expression cassette into the expression vector of the target gene to be activated to obtain a recombinant expression vector, i.e., a pyruvate-responsive sensor containing a pyruvate-activated gene circuit; or; Connecting the pyruvate-repressible promoter to the target gene to be repressed and introducing the promoter into a vector to construct an expression vector for the target gene to be repressed; Connecting a pyruvate-activated promoter to the XylR gene with a nuclear localization signal peptide fused to its N-terminus to construct an expression cassette of XylR, and constructing the XylR expression cassette into an expression vector of the target gene to be inhibited to obtain a first recombinant expression vector; constructing the PdhR expression cassette into the first recombinant expression vector to obtain a second recombinant expression vector, namely a pyruvate-responsive sensor containing a pyruvate-inhibitory gene circuit; or; The pyruvate-activated promoter is connected to the target gene to be activated to construct an expression cassette of the target gene to be activated, and the expression cassette of the target gene to be activated is constructed into the second recombinant expression vector to obtain a third recombinant expression vector, i.e., a pyruvate-responsive sensor containing a dual-function gene circuit.
[0016] The third object of the present invention is to provide the application of the above-mentioned pyruvate-responsive biosensor in the metabolic regulation of Saccharomyces cerevisiae.
[0017] In some embodiments of the present invention, the pyruvate-responsive sensor uses glucose as an activator to regulate the metabolic network of Saccharomyces cerevisiae.
[0018] Unlike the simple transmembrane transport system of bacteria, the transmembrane transport system of Saccharomyces cerevisiae is more complex, resulting in the inability of adding extracellular pyruvate to significantly increase pyruvate levels in the nucleus. The present invention uses glucose as an indirect activator, which can effectively alter intracellular pyruvate levels and facilitate the regulation of metabolic networks.
[0019] This invention constructs a pyruvate-responsive biosensor for eukaryotic cells (Saccharomyces cerevisiae) by designing the PdhR structure (fusion of a nuclear localization signal peptide at the N-terminus of the PdhR gene), selecting an activator, and optimizing PdhR expression levels. The technical solution of this invention has the following advantages: (1) The pyruvate-responsive gene circuit constructed in the present invention uses the eukaryotic model organism Saccharomyces cerevisiae as the host. In view of the compartmentalized structure of Saccharomyces cerevisiae and the difficulty of pyruvate uptake, the PdhR design and indirect activator selection are improved, providing a reference for the construction of gene circuits based on prokaryotic regulatory elements in eukaryotic cells.
[0020] (2) This invention constructs an orthogonalized bifunctional pyruvate gene circuit, which facilitates the comprehensive regulation of complex metabolic networks. Given that pyruvate is a node substance in central carbon metabolism, this gene circuit can be used to dynamically regulate carbon metabolic flux in yeast cell factories. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 These are fluorescence microscopy images of Saccharomyces cerevisiae expressing GFP, PdhR-GFP, and NLS-PdhR-GFP.
[0022] Figure 2 This is a schematic diagram of the design and construction of a pyruvate-activated gene circuit.
[0023] Figure 3 Figure 2 is a performance test diagram of the pyruvate-activated circuit at different pdhO insertion sites.
[0024] Figure 4 This is a performance test diagram of the pyruvate-activated gene circuit at different PdhR expression levels.
[0025] Figure 5 This is a schematic diagram of the design and construction of a pyruvate-repressible gene circuit.
[0026] Figure 6 This is a performance test diagram of the pyruvate-repressible gene circuit.
[0027] Figure 7 This is a schematic diagram of the design and construction of a bifunctional pyruvate gene circuit.
[0028] Figure 8 This is a test diagram of the orthogonality of the bifunctional pyruvate gene circuit in yeast cells. DETAILED DESCRIPTION
[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, which may help to better understand the present invention. The contents described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.
[0030] The Saccharomyces cerevisiae BY4741 (starting strain) used in the examples is a commercial strain and can be purchased from commercial channels.
[0031] Unless otherwise specified, the experimental materials, reagents, etc. used in the examples of the present invention can be obtained from commercial channels.
[0032] The experimental methods in the following examples are conventional methods unless otherwise specified.
[0033] Fluorescence detection methods involved: The strain to be tested was inoculated into a SC-Ura tube containing 1 mM xylose and cultured overnight at 30°C. The bacterial solution was washed twice with sterile water and concentrated to obtain the initial OD value. 600 0.5 was transferred to inorganic salt medium, followed by the addition of 10 g / L glucose. After shaking culture at 30°C for 8 h, the cells were detected on a fluorescence microplate reader. Detection parameters included green fluorescence intensity of GFP (excitation: 488 nm; emission: 528 nm), red fluorescence intensity of mCherry (excitation: 550 nm; emission: 620 nm), and cell density (600 nm).
[0034] Example 1: Functional verification of nuclear localization signal peptide Amplification of the constitutively strong promoter P by existing methods TDH3 , green fluorescent protein gene GFP, terminator T CYC1 and transcription factor PdhR (nucleotide sequence shown in SEQ ID NO.3). The GFP expression cassette P was obtained by assembling multiple fragments through fusion PCR overlap extension. TDH3 -gfp-T CYC1 , the expression cassette P of the transcription factor pdhR connected to the reporter gene GFP TDH3 -pdhR-GFP-T CYC1 and the expression cassette P of the transcription factor pdhR connected to the reporter gene GFP and fused to the nuclear localization signal peptide NLS (nucleotide sequence as shown in SEQ ID NO.1) at the N-terminus. TDH3 -NLS-pdhR-gfp-T CYC1 The three expression cassettes were constructed into the centromere expression vector pRS416 to obtain recombinant plasmids pRS416-GFP, pRS416-PdhR-GFP and pRS416-NPdhR-GFP, which were respectively transformed into Saccharomyces cerevisiae BY4741 for the localization verification of PdhR and nuclear localization signal peptide.
[0035] The results are as follows Figure 1 As shown, the unmodified fluorescent protein GFP is uniformly dispersed in the cytoplasm after expression. The PdhR-GFP from E. coli is partially localized to the nucleus, while a portion remains free in the cytoplasm. After modification with the nuclear localization signal peptide, the fluorescence of the NLS-PdhR-GFP is concentrated and single, indicating that PdhR is completely and strictly transported to the nucleus.
[0036] Example 2: Construction of a pyruvate-activated gene circuit The PdhR binding sequence (pdhO) was inserted into the constitutive promoter P TDH3 The -150, -128 and -5 sites of the pyruvate-activated promoter P TDH3-1 、P TDH3-2 and P TDH3-3 , pdhO, promoter P TDH3-1 、P TDH3-2 、P TDH3-3 The nucleotide sequences are shown in SEQ ID NO.5-8 respectively.
[0037] The expression cassettes P were constructed by overlapping extension PCR and connecting the reporter gene GFP. TDH3-1 -gfp-T CYC1 、P TDH3-2 -gfp-T CYC1 and P TDH3-3 -gfp-T CYC1 The expression cassettes were constructed into the vector pRS416 to obtain recombinant plasmids pRS416-GFP1, pRS416-GFP2 and pRS416-GFP3, which were respectively transformed into Saccharomyces cerevisiae BY4741. The constitutive promoter P was amplified using the genome of Saccharomyces cerevisiae BY4741 as a template. TEF1 The transcription factor pdhR N-terminal was fused with the nuclear localization tag NLS, and the C-terminal was fused with the protein degradation tag PEST to obtain NLS-pdhR-PEST. The nucleotide sequence of PEST is shown in SEQ ID NO.2. The expression cassette P was obtained by overlapping extension PCR. TEF1 -NLS-pdhR-PEST-T ADH1 The above expression cassettes were constructed into recombinant plasmids pRS416-GFP1, pRS416-GFP2 and pRS416-GFP3 with different pdhO insertion sites, respectively, to obtain recombinant plasmids pRS416-TPdhR-GFP1, pRS416-TPdhR-GFP2 and pRS416-TPdhR-GFP3, which were respectively transformed into Saccharomyces cerevisiae BY4741 to construct the following Figure 2 The pyruvate-activated gene circuit shown in Figure 1 is a pyruvate-activated gene circuit. The transcription factor PdhR can specifically recognize pdhO and bind to the modified pyruvate-activated promoter, inhibiting the transcription of the downstream reporter gene gfp. When pyruvate is present, the transcription factor PdhR preferentially binds to pyruvate and detaches from the modified promoter, allowing the downstream gene to resume normal transcriptional expression. Figure 3 As shown, PdhR can bind to the promoter P TDH3-2 On the other hand, PdhR inhibits the fluorescence expression. pdhO The inhibition folds of the insertion sites were 5.7, 22.0, and 8.5, respectively. Site No. 2 (-128) was selected for subsequent investigation and evaluation.
[0038] Amplify the weak promoter P by existing methods PRM and P AGA , expression cassettes with different promoter strengths were obtained by overlapping extension PCR. PRM / P AGA -NLS-pdhR-PEST-T ADH1 The expression cassettes with different promoter strengths were constructed into the recombinant plasmid pRS416-GFP2 to obtain the recombinant plasmid pRS416-PPdhR-GFP2 (pdhR promoter is P PRM , nucleotide sequence as shown in SEQ ID NO.4) and pRS416-APdhR-GFP2 (pdhR promoter is P AGA ), were transformed into Saccharomyces cerevisiae BY4741. Figure 4 As shown, further reducing the expression level of the transcription factor PdhR, under weak PdhR expression, the pyruvate-activated gene circuit was turned on, with dynamic multiples of 3.7 times and 3.4 times respectively.
[0039] Example 3: Construction of a pyruvate-repressible gene circuit The XylR binding sequence (xylO) was inserted into the constitutive promoter P TEF1 -105 site of the synthesized pyruvate-repressible promoter P TEF1-1 , xylO and promoter P TEF1-1 The nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12 respectively. The expression cassette of the reporter gene mCherry was obtained by overlapping extension PCR. TEF1-1 -mcherry-T CYC1 The expression cassette was constructed into the vector pRS416 to obtain the recombinant plasmid pRS416-mCherry1.
[0040] The nuclear localization tag NLS was fused to the N-terminus of the xylose transcription factor XylR, and the protein degradation tag CLN2 was fused to the C-terminus to synthesize the codon-optimized xylose transcription factor NLS-xylR-CLN2 containing the nuclear localization signal peptide and the degradation tag. The nucleotide sequences of XylR, NLS and CLN2 are shown in SEQ ID NO.9, SEQ ID NO.1 and SEQ ID NO.10 respectively. The expression cassette of XylR was obtained by overlap extension PCR. TDH3-2 -NLS-xylR-CLN2-T CYC1 The expression cassette was constructed into the recombinant plasmid pRS416-mCherry1 to obtain the recombinant plasmid pRS416-XylR-mCherry1. PRM-NLS-pdhR-PEST-T ADH1 , constructed into the vector pRS416-XylR-mCherry1, and obtained the pyruvate-repressed recombinant plasmid pRS416-PdhR-XylR-mCherry1, which was transformed into Saccharomyces cerevisiae BY4741 to obtain the following Figure 5 The pyruvate-repressible gene circuit shown in Figure 1 activates the expression of XylR, which further inhibits the promoter P. TEF1-1 The inhibition factor of the pyruvate repressible gene circuit was tested using different concentrations of glucose. The results are as follows: Figure 6 As shown, under the condition of glucose concentration of 10 g / L, the inhibition factor of the pyruvate-repressible gene circuit was 3.1 times.
[0041] Example 4: Construction of a bifunctional pyruvate-responsive gene circuit Amplified expression cassette P TDH3-2 -gfp-T CYC1 The expression cassette was constructed into the recombinant plasmid pRS416-PdhR-XylR-mCherry1 to obtain the recombinant plasmid pRS416-PdhR-XylR-mCherry1-GFP2, which was then transformed into Saccharomyces cerevisiae BY4741. Figure 7 As shown in Figure 2, a Saccharomyces cerevisiae cell containing both a pyruvate-activated gene circuit and a pyruvate-repressed gene circuit was obtained. Using glucose as an activator, the performance of the bifunctional pyruvate-responsive gene circuit was tested using different concentrations of glucose. The results are shown in Figure 2. Figure 8 As shown, glucose activated GFP expression while simultaneously repressing mCherry expression. Under 10 g / L glucose conditions, the activation factor was 3.6-fold and the repression factor was 3.0-fold, similar to the performance of the two gene circuits tested separately. Therefore, the constructed pyruvate-activated and repressive gene circuits have good orthogonality and can be used simultaneously to regulate the metabolic network of Saccharomyces cerevisiae.
Claims
1. A pyruvate-responsive biosensor for Saccharomyces cerevisiae, characterized in that: The biosensor comprises a pyruvate-activated gene circuit, a pyruvate-repressed gene circuit, or a bifunctional gene circuit; The pyruvate-activated gene circuit comprises a transcription factor PdhR, a target gene to be activated, a PdhR expression promoter, and a pyruvate-activated promoter, all constructed on the same vector; The pyruvate-repressible gene circuit comprises a transcription factor PdhR, a target gene to be repressed, a PdhR expression promoter, a pyruvate-activated promoter, a xylose transcription repressor XylR, and a pyruvate-repressible promoter, all constructed on the same vector; The bifunctional gene circuit comprises a target gene to be activated, a target gene to be repressed, a transcription factor PdhR, a PdhR expression promoter, a pyruvate-activated promoter, a xylose transcription repressor XylR, and a pyruvate-repressible promoter, all constructed on the same vector; The pyruvate-activated promoter is a constitutive promoter inserted with the PdhR binding sequence pdhO; the ketoacid-repressible promoter is a constitutive promoter inserted with the XylR binding sequence xylO; and the N-termini of the PdhR and XylR are fused with nuclear localization signal peptides respectively.
2. The pyruvate-responsive biosensor according to claim 1, wherein The nucleotide sequence of the nuclear localization signal peptide is shown in SEQ ID NO.
1.
3. The pyruvate-responsive biosensor according to claim 1, wherein The C-terminal fusion protein degradation tags of the transcription factor PdhR and the xylose transcription repressor XylR.
4. The pyruvate-responsive biosensor according to claim 3, wherein The nucleotide sequence of the protein degradation tag fused to the C-terminus of the transcription factor PdhR is shown in SEQ ID NO.2, and the nucleotide sequence of the protein degradation tag fused to the C-terminus of the xylose transcription repressor XylR is shown in SEQ ID NO.
10.
5. The pyruvate-responsive yeast of claim 1, wherein The constitutive promoter of pdhO inserted into the pyruvate-activated gene circuit is P TDH3 The constitutive promoter inserted into the xylO in the pyruvate repressible gene circuit is P TEF1 .
6. The pyruvate-responsive yeast of claim 5, wherein The PdhO inserts into P TDH3 -150 site, -128 site or -5 site of the promoter; The XylO is inserted into the P TEF1 The -105 site of the promoter.
7. The pyruvate-responsive yeast of claim 1, wherein The PdhR expression promoter is selected from P PRM promoter; The expression promoter of the xylose transcription repressor XylR is a pyruvate-activated promoter.
8. The method for constructing a pyruvate-responsive biosensor according to any one of claims 1 to 7, characterized in that: include: Connecting the pyruvate-activated promoter to the target gene to be activated and introducing the promoter into a vector to construct an expression vector for the target gene to be activated; Connecting the PdhR expression promoter to the PdhR gene with a nuclear localization signal peptide fused to its N-terminus to construct a PdhR expression cassette, and constructing the PdhR expression cassette into the expression vector of the target gene to be activated to obtain a recombinant expression vector, i.e., a pyruvate-responsive sensor containing a pyruvate-activated gene circuit; or; Connecting the pyruvate-repressible promoter to the target gene to be repressed and introducing the promoter into a vector to construct an expression vector for the target gene to be repressed; Connecting a pyruvate-activated promoter to the XylR gene with a nuclear localization signal peptide fused to its N-terminus to construct an expression cassette of XylR, and constructing the XylR expression cassette into an expression vector of the target gene to be inhibited to obtain a first recombinant expression vector; constructing the PdhR expression cassette into the first recombinant expression vector to obtain a second recombinant expression vector, namely a pyruvate-responsive sensor containing a pyruvate-inhibitory gene circuit; or; The pyruvate-activated promoter is connected to the target gene to be activated to construct an expression cassette of the target gene to be activated, and the expression cassette of the target gene to be activated is constructed into the second recombinant expression vector to obtain a third recombinant expression vector, i.e., a pyruvate-responsive sensor containing a dual-function gene circuit.
9. Use of the pyruvate-responsive biosensor according to any one of claims 1 to 7 in regulating the metabolism of Saccharomyces cerevisiae.
10. The use according to claim 9, characterized in that The pyruvate-responsive sensor uses glucose as an activator to regulate the metabolic network of Saccharomyces cerevisiae.