A synthetic epigenetic silencing system for large-scale multi-gene expression regulation, a method for achieving reversible gene silencing, and its applications.

Abstract

This invention discloses a synthetic epigenetic silencing system for large-scale multi-gene expression regulation and its applications, belonging to the fields of synthetic biology and metabolic engineering. The invention first integrates a DNA target sequence into the chromosome of a eukaryotic host cell; then, it constructs and expresses fusion proteins of a writer, reader, and reading effector. The writer specifically binds to the target sequence and catalyzes the generation of H3K9me2 / 3 modifications; the reader recognizes existing modifications and autonomously diffuses along the chromosome via a positive feedback mechanism, forming a large-scale modification domain of 50–200 kb; the reading effector locates the modified region and inhibits gene transcription within the region; further, small molecules induce the reading effector to enter and exit the cell nucleus, achieving dynamic and reversible control of the system. This invention eliminates the need for individual gene gRNA design, significantly reducing genetic load, avoiding off-target accumulation risks, and achieving simultaneous transcriptional silencing of large chromosomal fragments of multiple genes, making it suitable for metabolic engineering scenarios such as segmented fermentation.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a synthetic epigenetic silencing system for large-scale multi-gene expression regulation and its applications. Background Technology

[0002] In the fields of metabolic engineering and synthetic biology, precisely regulating the expression levels of multiple genes within host cells is a core technical challenge for optimizing biosynthetic pathways and increasing product yield. Early gene expression regulation methods mainly relied on modifying single gene promoters or using inducible expression systems. These methods performed well in regulating single or a few genes, but struggled to achieve synergistic and programmed regulation of multiple genes. With the development of gene editing and transcriptional regulation technologies, researchers began to seek strategies capable of simultaneously regulating multiple target genes.

[0003] Currently, the technology most closely associated with multigene expression regulation is transcriptional regulation based on the CRISPR / dCas9 system. This technology uses Cas9 protein (dCas9) with inactivated nuclease activity. dCas9 loses its ability to cleave DNA but retains its ability to target specific DNA sequences under the guidance of guide RNA (gRNA). By fusing transcriptional repression domains (such as KRAB) or activation domains (such as VP64) to dCas9, these effectors can be localized to the promoter regions of target genes under the guidance of gRNA, thereby achieving the repression or activation of single-gene transcription. To achieve multigene regulation, existing technologies further employ a "multiple gRNA" strategy, simultaneously designing and expressing multiple gRNAs targeting different gene promoters. For example, in a study published in Cell (Vol. 160, Issues 1-2, pp. 339-350, 2015), Zalatan et al. used engineered multiple gRNA scaffolds to simultaneously recruit regulatory factors, achieving simultaneous activation or repression of three genes in yeast. The essence of this approach is to use multiple gRNA-dCas9 complexes in parallel to target and regulate multiple genomic sites.

[0004] However, the above-mentioned CRISPR / dCas9 multiple regulation scheme has the following technical drawbacks in practical applications: First, there are limitations in the scope and efficiency of regulation. This approach regulates multiple genes in a "point-to-point" manner, typically requiring the expression of at least N gRNAs to regulate N genes. As the number of target genes increases, the number of required gRNAs also increases, placing a huge transcriptional and translational burden on the host cell, consuming a large amount of cellular resources, often leading to plasmid instability and severely inhibited cell growth, making it difficult to achieve truly large-scale regulation of gene clusters spanning tens to hundreds of kilobase regions.

[0005] Second, there is a cumulative risk of off-target effects. Each gRNA has a certain off-target binding potential. When a large number of gRNAs are introduced simultaneously, their off-target effects will accumulate, leading to a significant increase in the risk of non-specific regulation across the entire genome, affecting the normal physiological state of cells, and reducing the specificity and reliability of regulation.

[0006] Third, it is difficult to achieve overall silencing at the chromosome region level. This technology lacks a mechanism to create an inhibitory chromatin environment in a specific chromosome region (such as a heterochromatin region) like natural epigenetic silencing, thereby permanently shutting down the transcription of all genes in that region. It cannot directly establish a diffuse and sustainable silencing state.

[0007] Fourth, the dynamic reversible regulatory capacity is insufficient. Although the CRISPR system can control the expression of dCas9 or gRNA through inducible promoters to achieve switching, its on / off dynamics are often affected by transcriptional and translational delays, making it difficult to achieve a rapid, uniform, and completely reversible response.

[0008] Therefore, a new technical solution needs to be developed that can achieve synchronous and holistic transcriptional regulation of multiple genes in a large chromosomal region without relying on a large number of parallel gRNAs, and has dynamic and reversible on / off control capabilities to meet the needs of precise temporal control in complex metabolic engineering. Summary of the Invention

[0009] In order to overcome the shortcomings of the prior art, the present invention aims to provide a synthetic epigenetic silencing system for large-scale multi-gene expression regulation and its application, which aims to solve the problems of high load, high off-target risk, lack of regional stable silencing and lag in dynamic regulatory response of the existing CRISPR multi-gene regulation system. To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a synthetic epigenetic regulatory system for large-scale multi-gene expression regulation, comprising: a DNA targeting sequence integrated into the host cell chromosome; a writer, which is a fusion protein of a DNA binding domain and an H3K9 methyltransferase catalytic domain; a reader, which is a fusion protein of an H3K9me recognition domain and an H3K9 methyltransferase catalytic domain; and a reading effector, which is a fusion protein of an H3K9me recognition domain and a transcriptional repressor.

[0010] The writer is specifically bound to the DNA target sequence and generates H3K9me2 / 3 modification via the H3K9 methyltransferase catalytic domain. The reader recognizes existing H3K9me2 / 3 modification via the H3K9me recognition domain and propagates the modification along the chromosome via the H3K9 methyltransferase catalytic domain, forming a large-scale H3K9me2 / 3 modification domain. The reading effector locates the H3K9me2 / 3 modification domain via the H3K9me recognition domain and inhibits the transcription of genes within the modification domain via a transcriptional repressor.

[0011] This system integrates the functional components of the above methods into a complete composition, which can be used as a tool system for multi-gene expression regulation.

[0012] Furthermore, the writer is integrated into the host cell chromosome, and the reader and effector exist in plasmid form. This combination ensures stable expression of the writer while providing flexibility for the expression regulation of the reader and effector.

[0013] Furthermore, the reading effector incorporates a small molecule-inducible nuclear localization domain. By incorporating this domain, the system gains reversible controllability, expanding its application scenarios.

[0014] Furthermore, the expression molar ratio of the writer, reader, and reading effector can typically be adjusted based on the promoter strength.

[0015] Furthermore, in a preferred embodiment of the present invention, the expression molar ratio of the writer, reader, and reading effector is 1:2 to 5:5 to 10. This expression ratio is optimized to ensure the effective establishment and propagation of the modification while avoiding overexpression that could burden the cells.

[0016] Furthermore, the writer, reader, and reader effector all contain a nuclear localization signal sequence; the writer incorporates a Flag tag, the reader incorporates an HA tag, and the reader effector incorporates a Myc tag.

[0017] Furthermore, the length of the large-scale H3K9me2 / 3 modification domain is 50-200 kb.

[0018] A second aspect of the present invention discloses a method for constructing a synthetic epigenetic silencing system in eukaryotic host cells to achieve large-scale multi-gene expression regulation, comprising the following steps: Integrate DNA targeting sequences onto the chromosomes of the host cell; A writer was constructed and expressed, which is a fusion protein of a DNA-binding domain and an H3K9 methyltransferase catalytic domain; A reader / writer was constructed and expressed, wherein the reader / writer is a fusion protein of the H3K9me recognition domain and the H3K9 methyltransferase catalytic domain; A reading effector was constructed and expressed, which is a fusion protein of the H3K9me recognition domain and a transcriptional repressor.

[0019] The writer specifically binds to the target DNA sequence through its DNA binding domain and catalyzes the generation of H3K9me2 / 3 modification at the site through its H3K9 methyltransferase catalytic domain, thereby establishing an epigenetic marker de novo at a specific genomic site. The reader recognizes existing H3K9me2 / 3 modification through its H3K9me recognition domain and propagates the modification along the chromosome through its H3K9 methyltransferase catalytic domain, forming a large-scale H3K9me2 / 3 modification domain, thus achieving the diffusion and localization of the modification. The reading effector locates the H3K9me2 / 3 modification domain through its H3K9me recognition domain and inhibits the transcription of genes within the modification domain through its transcriptional repressor, thus converting the epigenetic marker into actual gene silencing function.

[0020] Through the above technical solution, this invention artificially constructs an orthogonal, inducible epigenetic silencing system in *Saccharomyces cerevisiae*. This system mimics the heterochromatin formation and gene silencing mechanism based on histone H3K9me2 / 3 in eukaryotes, and through engineering modification, it is precisely controlled by a specific DNA sequence. Unlike existing technologies that target individual gene promoters, this invention establishes a repressive chromatin state in the target chromosomal region, thereby silencing all transcription units within that region at once, eliminating the need to design gRNAs for each gene and significantly reducing the genetic load.

[0021] Furthermore, the DNA targeting sequence is a TetO sequence, and the DNA binding domain is a TetR protein. The TetO-TetR system is a mature orthogonal DNA binding system that does not cross-react with endogenous regulatory systems in yeast, ensuring the specificity of the targeting.

[0022] Furthermore, the catalytic domain of the H3K9 methyltransferase is derived from human G9A methyltransferase, which can specifically catalyze the methylation of lysine at position 9 of histone H3. G9A mainly catalyzes H3K9me1 and H3K9me2 in vivo, but can further catalyze the production of H3K9me3 in vitro and in Saccharomyces cerevisiae (disclosed for the first time in this invention).

[0023] Furthermore, the H3K9me recognition domain is the chromatin-binding domain of the human HP1 family protein CBX1. It can specifically recognize and bind to H3K9me2 / 3 modifications, providing readers and effectors with the ability to locate the modified region.

[0024] Furthermore, the transcriptional repressor is a protein such as Sir2, Mig1, or Tup1 from Saccharomyces cerevisiae. Sir2 is an endogenous deacetylase in Saccharomyces cerevisiae that can alter chromatin conformation to achieve transcriptional repression; Mig1 and Tup1 are also effective transcriptional repressors in Saccharomyces cerevisiae, providing the system with a variety of optional silencing effectors.

[0025] Furthermore, the length of the aforementioned large-scale H3K9me2 / 3 modification domain ranges from 50 to 200 kilobases. This range can cover multiple genes or even entire biosynthetic pathways, achieving truly large-scale regional regulation, which is significantly superior to the point-to-point regulation methods of existing technologies.

[0026] Furthermore, the host cell is a diploid cell of *Saccharomyces cerevisiae*. Using diploid cells avoids the problem of cell death caused by silencing essential genes, and simultaneously allows for the construction of a synthetic epigenetic regulatory system on only one homologous chromosome, while the other chromosome retains a wild-type copy, ensuring normal cell growth.

[0027] A third aspect of the present invention also discloses a method for achieving reversible gene silencing, comprising the following steps: In host cells where a synthetic epigenetic silencing system has been constructed, a small molecule inducible nuclear localization domain is fused to the reading effector to form an inducible reading effector. When the host cells are cultured without an inducer, the small molecule inducible nuclear localization domain retains the inducible reading effector in the cytoplasm, and the gene silencing system is in a closed state. An inducer is added to the culture medium, and the inducer binds to the small molecule inducible nuclear localization domain, causing the inducible reading effector to be transported into the cell nucleus; after entering the nucleus, the inducible reading effector is localized to the H3K9me2 / 3 modification domain and initiates gene silencing in that region; Upon removal of the inducer, the inducible reading effector exits the nucleus or re-enters the cytoplasm, thus ending gene silencing.

[0028] By coupling a small molecule-induced nuclear localization system with an epigenetic silencing system, this invention achieves precise temporal and spatial control of the silencing system. The control point is placed directly at the crucial step of nuclear localization of effector proteins. By directly controlling the intracellular concentration of functional proteins through small molecules, a faster, more uniform, and completely reversible response can be achieved compared to inducible promoter-controlled expression.

[0029] Furthermore, the small molecule-induced nuclear localization domain is the estrogen-binding domain EBD. In the absence of a ligand, EBD binds to cytosolic chaperone proteins such as heat shock protein Hsp90, retaining the fusion protein in the cytoplasm; when β-estradiol binds to EBD, EBD is released from the cytosolic chaperone and exposes the nuclear localization signal, allowing the fusion protein to rapidly enter the nucleus.

[0030] Furthermore, the inducer is β-estradiol. β-estradiol is a specific ligand for EBD, exhibits low cytotoxicity to yeast cells, and is an ideal inducer for achieving reversible regulation.

[0031] Furthermore, the concentration of the inducer is 10 to 100 micromoles. This concentration range effectively induces the reading effector to enter the nucleus and initiate silencing without causing significant toxicity to the cell; it is an optimized process parameter.

[0032] Furthermore, the change in gene silencing state after the addition or removal of the inducer is completed within 6 to 72 hours. This response time is suitable for metabolic engineering applications requiring temporal control and offers a faster response compared to transcriptional-translational regulation.

[0033] A fourth aspect of the present invention also provides the application of the above-described method or system in metabolic engineering, including: Multiple genes targeting metabolic pathways are integrated into downstream chromosomal regions of the DNA targeting sequence; the synthetic epigenetic regulatory system is constructed. At different stages of the fermentation process, the nuclear localization of the reading effector is controlled by adding or removing an inducer, thereby controlling the silencing state of the metabolic pathway genes. By dynamically switching the expression of genes in the metabolic pathways, the temporal redirection of metabolic flux can be achieved, thereby increasing the yield of target products.

[0034] This application method provides a novel cluster control strategy for metabolic engineering, which can regulate multiple functionally related genes as a functional cluster in a unified manner. By switching the expression of metabolic pathway genes on and off at different fermentation stages, it can achieve precise guidance and optimization of metabolic flux.

[0035] Furthermore, the target metabolic pathway is the violacein biosynthesis pathway, including the vioA, vioB, vioC, vioD, and vioE genes. As a typical multi-gene biosynthesis pathway, the violacein biosynthesis pathway has a total length of approximately 12 kilobases across its five genes, making it suitable for verifying the wide-ranging multi-gene regulatory capabilities of the system of this invention.

[0036] Furthermore, these multiple genes are integrated downstream of the Bar1 gene on chromosome 9 of *Saccharomyces cerevisiae*. This location has been validated as suitable for constructing synthetic epigenetic regulatory systems capable of effectively establishing and disseminating H3K9me2 / 3 modifications.

[0037] Furthermore, without the addition of an inducer in the early stage of fermentation, metabolic pathway genes are expressed normally; while the addition of an inducer in the later stage of fermentation silences these genes. This segmented fermentation strategy allows for the accumulation of intermediate products or biomass through metabolic pathways in the early stage, and the redirection of metabolic flux to the synthesis of the target product by silencing the pathway in the later stage, thereby optimizing the yield.

[0038] Furthermore, the applications also include regulating competitive metabolic pathways, optimizing metabolic flux, or improving strain stability. The system of this invention is not limited to a single metabolic pathway and can be widely used in various metabolic engineering scenarios requiring multi-gene synergistic regulation.

[0039] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention enables regionalized regulation with a lighter burden and higher efficiency: Existing technologies require the expression of at least one gRNA for each target gene, and regulating multiple discrete genes increases the genetic and metabolic burden, with efficiency decreasing as the number of targets increases. In contrast, this invention establishes a continuous chromatin silencing domain, covering all genes within the region at once. Whether the region contains 5 or 50 genes, only one set of fixed epigenetic elements is needed to achieve overall silencing, reducing cellular burden and improving the feasibility and efficiency of large-scale regulation.

[0040] 2. Lower off-target risk and higher specificity: The CRISPR / dCas9 system relies on the sequence complementarity of gRNAs, and each gRNA has the potential for off-target effects, with risks compounding when multiple gRNAs are used. The targeting specificity of the system in this invention is determined by a single, precisely designed DNA landing site, and subsequent modification propagation and silencing effects are strictly limited to this chromosomal region. This locus-based regulatory approach reduces non-specific interference to other sites throughout the genome, resulting in higher overall specificity.

[0041] 3. More stable and heritable regulatory state: dCas9-mediated regulation requires sustained expression of effector proteins to maintain. The H3K9me2 / 3 modification established in this invention is a stable epigenetic marker. Once established, even without sustained expression of the reader / writer, it can be passed down during cell division through positive feedback from the reader, providing a more durable and stable silencing effect. This is particularly important for long-term fermentation processes or scenarios requiring a stable silencing state.

[0042] 4. Dynamic and Reversible "On / Off" Control: While CRISPR systems can also control dCas9 or gRNA expression via inducible promoters to achieve on / off switching, their kinetics are often affected by transcriptional and translational delays. This invention places the control point directly at the crucial step of nuclear localization of effector proteins, directly controlling the intranuclear concentration of functional proteins using the small molecule β-estradiol. This design enables a faster, more uniform, and completely reversible response, providing a superior tool for complex metabolic engineering applications requiring precise timing control.

[0043] 5. Providing a novel cluster control strategy for metabolic engineering: The greatest advantage of this invention lies in its ability to regulate multiple functionally related genes as a "functional cluster." For example, in synthetic biology, all enzyme genes involved in a competing pathway can be organized into the same region of the genome and shut down with a single switch, thereby efficiently redirecting metabolic flow. This regionalized cluster control strategy is difficult to achieve with existing point-to-point regulation techniques, opening up new pathways for the global optimization of metabolic networks. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the synthetic epigenetic silencing system of the present invention; Figure 2 A schematic diagram showing the integration of three different fluorescent reporter genes at different locations on chromosome 9; Figure 3 A ChIP-seq result showing the distribution of H3K9me2 modification on chromosome 9; Figure 4 The image shows the RT-qPCR results for detecting the expression levels of three fluorescent reporter genes. Figure 5 A schematic diagram showing the integration location of the gene for the violacein biosynthesis pathway on chromosome 9; Figure 6 Phenotypic diagram of a yeast strain containing the violacein synthesis pathway; Figure 7 Figure 1 shows the ChIP-qPCR results of H3K9me2 modification enrichment in the gene region of the violacein synthesis pathway. Figure 8 Figure 1 shows the RT-qPCR results of the expression levels of five genes in the violacein synthesis pathway. Figure 9 Phenotypic diagram of color changes in yeast strains before and after the addition and removal of β-estradiol. Detailed Implementation

[0045] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0046] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0047] It should be noted that the technical terms in the following embodiments are defined as follows: The DNA targeting sequence described in this invention refers to a specific DNA sequence that is artificially designed and integrated into the host cell chromosome for recruiting programmer proteins; The writer described in this invention is a protein formed by the fusion of a DNA-binding domain and an H3K9 methyltransferase catalytic domain, which is responsible for initiating the methylation modification of the 9th lysine residue of histone H3 at the target site.

[0048] The reader / writer described in this invention refers to a protein formed by the fusion of the H3K9me recognition domain and the H3K9 methyltransferase catalytic domain, which is responsible for recognizing existing H3K9 methylation modifications and propagating them along the chromosome.

[0049] The reading effector described in this invention refers to a protein formed by the fusion of the H3K9me recognition domain and a transcriptional repressor, which is responsible for converting epigenetic markers into gene silencing functions.

[0050] The H3K9me2 / 3 mentioned in this invention refers to the dimethylation or trimethylation modification of lysine at position 9 of histone H3, which is a repressive epigenetic marker.

[0051] The β-estradiol described in this invention is a small molecule compound that can bind to the estrogen-binding domain and induce protein nuclear translocation.

[0052] The technical solution of this invention specifically includes the following three core components, which together constitute a complete epigenetic switch for regulating gene expression: 1. De novo establish a large-scale, heritable H3K9me2 / 3 modification domain at a specific genomic locus.

[0053] This step aims to create a persistent epigenetic marker in the target chromosomal region using an orthogonal synthetic epigenetic system, which mainly consists of two modules: (1) The compiler module consists of the protein of the orthogonal system and the catalytic domain of histone H3K9 methyltransferase, which de novo establishes H3K9me2 / 3 modification at a specific genomic site.

[0054] (2) Reader module: In the brewer yeast with existing writer, the reader recognizes the de novo H3K9me2 / 3 modification and continues to catalyze the generation of new H3K9me2 / 3 modifications from both ends starting from the established site.

[0055] 2. Construct a reading effector targeting H3K9me2 / 3 modification to perform gene silencing function.

[0056] After establishing the H3K9me2 / 3 modification domain, it needs to be converted into actual gene transcription repression. This invention designs and expresses a reading effector, in which the reading protein recognizes and binds to the H3K9me2 / 3 modification, and then regulates gene expression in the modified region through effector proteins.

[0057] To avoid the problem of yeast growth restriction caused by the suppression of essential genes in haploid yeast, this invention uses diploid yeast and constructs a synthetic epigenetic regulatory system on only one homologous chromosome. Furthermore, to avoid the compensatory effect between the two copies of endogenous genes in diploid yeast, the exogenous genes better reflect the multi-gene regulatory effect of this invention.

[0058] 3. Achieve dynamic and reversible control of the system through a small molecule-induced nuclear localization system.

[0059] To achieve precise temporal and tertiary control of the aforementioned silencing system, this invention integrates a small molecule-induced nuclear input switch onto the reading effector. The estrogen-binding domain (EBD) is fused into the reading effector, forming an EBD-reading effector fusion protein. EBD binds to cytoplasmic chaperone proteins such as the heat shock protein Hsp90, retaining the EBD-reading effector in the cytoplasm and preventing it from entering the nucleus to contact chromatin; thus, the silencing system is in a "closed" state. When exogenous small molecule β-estradiol is added, β-estradiol binds to EBD, causing a conformational change that releases it from the cytoplasmic chaperone and exposes nuclear localization signals. This allows the EBD-reading effector protein to rapidly transport into the nucleus, where it localizes to the pre-established H3K9me2 / 3 modification via its reading protein domain and initiates gene silencing. Upon removal of β-estradiol, the EBD-reading effector protein is either re-exported or retained in the cytoplasm, thus releasing the silencing effect. Therefore, reversible switching control of the expression state of a wide range of gene clusters can be achieved by simply adding or removing β-estradiol.

[0060] The invention will be further described in detail below with reference to specific experimental examples and accompanying drawings: Example 1 like Figure 1 The diagram illustrates the synthetic epigenetic regulatory system of the present invention, including a target sequence, an orthogonal writer, a reader / writer, and a reading effector / estrogen-binding domain-reading effector. This embodiment provides a method for constructing a synthetic epigenetic silencing system in *Saccharomyces cerevisiae* to achieve large-scale multi-gene expression regulation. *Saccharomyces cerevisiae* is a single-celled eukaryotic microorganism with a well-defined genome structure and mature genetic manipulation capabilities, widely used in metabolic engineering and synthetic biology research. This embodiment uses *Saccharomyces cerevisiae* as the host cell, integrating artificial DNA target sequences at specific sites in its genome and constructing orthogonal epigenetic regulatory elements to achieve synchronous transcriptional repression of multiple genes across a large chromosomal region.

[0061] 1. Target sequences of integrated orthogonal systems The DNA targeting sequence 7xTetO and the downstream reporter gene ADE2 were integrated downstream of the Bar1 gene on chromosome 9 of *Saccharomyces cerevisiae*. The 7xTetO sequence is a DNA sequence consisting of seven TetO repeat units, approximately 140 base pairs in length, which can be specifically recognized and bound by the TetR protein. The ADE2 reporter gene is a key gene in the yeast adenine synthesis pathway, and its expression level can be phenotypically screened by colony color. The sequence is as follows:

[0062] The DNA fragment integration method is PEG / LiAc transformation, and the specific operation is as follows (the subsequent plasmid transformation method is the same): 1) The integrated fragment is approximately 3,000 base pairs in length and was synthesized by the company. An upstream primer, TetO-ADE2-F, and a downstream primer, TetO-ADE2-R, each with genomic homologous arms, were designed. The homologous arms are each 50 base pairs long and are perfectly complementary to the downstream region of the Bar1 gene on chromosome 9. Details are shown below: TetO-ADE2-F: 5'-gactgttaataataataagattgtcaagacaccccggtattactcgagccGTAAAACGACGGCCAGTGAATT-3' (SEQ ID NO: 2); TetO-ADE2-R: 5'-tctcataagttatgtcatcatcttaacaacgtatatgataatatattgatCATGATTACGCCAAGCTCGG-3' (SEQ ID NO: 3).

[0063] 2) After obtaining the linear DNA fragment by PCR, it was transformed into haploid Saccharomyces cerevisiae cells using the PEG / LiAc method. The specific transformation steps are as follows: 2.1) Inoculate the yeast strain into 3 mL of liquid culture medium and incubate overnight at 220 rpm and 30°C. The next day, inoculate the overnight cultured yeast into a fresh 3 mL culture medium with an initial concentration of OD (optical density at 600 nm) of 0.1. 2.2) Continue culturing for 4-6 hours until the OD is 0.6-0.8, centrifuge at 3000 rpm at room temperature for 5 minutes, collect the bacterial cells, and discard the supernatant; 2.3) Resuspend the bacterial cells in 1 mL ddH2O, transfer to a new 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 minute, and discard the supernatant; 2.4) Resuspend the bacterial cells in 1 mL of 0.1 M LioAc buffer, centrifuge at 3000 rpm for 1 minute, and discard the supernatant; 2.5) Resuspend the bacterial cells in 100 μL of 0.1 M LioAc buffer, and add 2-25 μL of transformation fragment to the resuspended bacterial solution (the volume should be based on the content of linear fragment or expression plasmid of 100-200 ng). 2.6) Add the transformation solution, mix well, and incubate at 30°C for 30 minutes. Each tube of transformation solution contains 50% PEG4000 (312 μL), 1M LioAc (41 μL), and 10 mg / mL ssDNA (25 μL). Mix thoroughly. The ssDNA needs to be heated at 100°C for 10 minutes and then placed on ice for 5 minutes before use. 2.7) Add 50 μL DMSO, mix by inversion, and heat shock the sample at 42°C for 15 minutes. Centrifuge at 3000 rpm for 2 minutes and discard the supernatant; resuspend in 1 mL of 5 mM CaCl2, centrifuge for 1 minute, and discard the supernatant. 2.8) Cultivation and Screening. Add 100 μL of 5 mM CaCl2 to resuspend the yeast culture. Spread the resuspended yeast culture onto an appropriate selective medium and incubate at 30°C for 2-3 days to screen out successfully transformed yeast strains.

[0064] A yeast strain that successfully integrated the gene was obtained through genotyping and screening. This strain carries a 7xTetO-ADE2 integration fragment approximately 500 base pairs downstream of the Bar1 gene on chromosome 9, providing a specific DNA landing site for the subsequent construction of an epigenetic regulatory system.

[0065] 2. Construct and express a writer to synthesize, de novo, position-specific histone H3K9me modification in Saccharomyces cerevisiae. The coding mechanism is composed of the catalytic domain of the orthogonal protein TetR and the human histone H3K9 methyltransferase G9A, integrated into the yeast genome. The TetR protein originates from the tetracycline resistance regulatory system of the E. coli Tn10 transposon, has a molecular weight of approximately 23 kilodaltons, and can specifically bind to the TetO sequence in a dimer form. G9A is a histone lysine methyltransferase belonging to the SET domain protein superfamily, primarily catalyzing the methylation of lysine at position 9 of histone H3. The specific steps are as follows: 1) Synthesize the optimized writer fragment, the sequence is as follows:

[0066] This embodiment uses the catalytic domain of the human G9A protein, located at the C-terminus. This domain contains the SET domain and its flanking Pre-SET and Post-SET domains, totaling approximately 350 amino acids in length, and possesses complete methyltransferase activity. A Flag tag is added to the N-terminus of the fusion protein to facilitate subsequent detection of protein expression and localization using Western blotting or immunofluorescence methods. The writer gene, approximately 1800 base pairs in length, was synthesized by the company.

[0067] 2) Design an upstream primer X3-WF and a downstream primer X3-WR with genomic homologous arms. The homologous arms have 50 base pairs of homologous sequences with the predetermined integration site on chromosome 10. Simultaneously, design a gRNA sequence X3-gRNA. This gRNA targets the genomic sequence near the integration site, guiding the Cas9 protein to generate a double-strand break at that site, promoting homologous recombination-mediated gene integration.

[0068] X3-WF: 5'-ataaggtttaaaggcactgaaacaataggcaagaagtaggcgagagccgacatacgagaTTTTCCACCGTACACACACG-3' (SEQ ID NO: 5); X3-WR: 5'-cgctcttgagctcgtccttttactagcatatcaatatccgtttcattgaaaagtggcctGCAAATTAAAGCCTTCGAGCG-3' (SEQ ID NO: 6); X3-gRNA: 5'-ctaatgtgtccgcgtttcta-3' (SEQ ID NO: 7).

[0069] The gRNA plasmid is the pML107 vector, which carries the Cas9 and Leu proteins.

[0070] 3) The linear DNA fragment of the writer was co-transformed with the pML107 plasmid carrying the Cas9 protein and the Leu selection marker into a yeast strain that had integrated the 7xTetO sequence. The transformation was performed using the PEG / LiAc method described above and plated on SC-Leu conditioned auxotrophic medium. 4) After successfully integrating the strain through genotyping and screening, single clones were picked and streaked onto YPD complete medium to allow the strain to grow under non-selective pressure, thereby promoting the loss of pML107 plasmid.

[0071] 5) Continue to select single clones and streak them simultaneously on YPD and SC-Leu media to screen for strains that can grow in YPD but not in SC-Leu, confirming that the plasmid has been lost. The finally obtained strains stably integrate the writer gene into their genome. The writer protein is expressed under the control of a constitutive promoter, specifically localizing to the 7xTetO sequence through its TetR domain, and catalyzing the production of H3K9me2 / 3 modification at this site through its G9A catalytic domain.

[0072] 3. Construct a reader / writer and establish a wide range of H3K9me2 / 3 modifiers. The reader / writer is formed by fusing the H3K9me recognition domain with the H3K9 methyltransferase catalytic domain. The specific steps are as follows: 1) The H3K9me recognition domain is selected from the chromatin-binding domain of human CBX1 protein. CBX1 is a member of the heterochromatin protein 1 family. Its N-terminal chromatin-binding domain contains about 60 amino acids and can specifically recognize and bind to H3K9me2 and H3K9me3 modifications.

[0073] The binding domain of the human CBX1 protein was selected as the reading protein for H3K9me. The reading protein and the G9A catalytic domain from the aforementioned writer were fused together and constructed into the pRS414 expression vector of *Saccharomyces cerevisiae* to create the reader / writer. Figure 1 The optimized sequence of this invention is as follows:

[0074] 2) The pRS414-reader plasmid was transformed into a yeast strain that had integrated the 7xTetO sequence and the writer using the PEG / LiAc method and then plated on SC-Trp conditionally deficient medium.

[0075] 3) The diffusion of H3K9me modification was subsequently detected by ChIP-qPCR or ChIP-seq technology.

[0076] 4. Construct and express reading effectors to establish H3K9me modification-dependent multigene silencing. 1) The reading effector is formed by fusing the H3K9me recognition domain with a transcriptional repressor. The H3K9me recognition domain is also selected from the chromatin-binding domain of the human CBX1 protein, and its sequence is the same as the recognition domain in the reader. The transcriptional repressor is the endogenous Sir2 protein from Saccharomyces cerevisiae. Sir2 is an NAD-dependent histone deacetylase belonging to the Sirtuin protein family, with a molecular weight of approximately 63 kDa. It can catalyze deacetylation reactions at multiple sites of histone H3 and H4, leading to chromatin densification and inhibiting transcription initiation and elongation. The CBX1 chromatin-binding domain and the Sir2 protein are connected by a flexible linker, and the linker sequence is also a Gly-Gly-Gly-Gly-Ser repeat sequence. A nuclear localization signal sequence is added to the N-terminus of the fusion protein, and a Myc tag is added to the C-terminus for detection. The total length of the reading effector gene is approximately 2400 base pairs, and it was synthesized by the company after codon optimization. The reading effector gene was cloned into the yeast expression vector pRS415, which carries the LEU2 selection marker and a 2-micron origin of replication. The reading effector was expressed under the control of a constitutive promoter. The pRS415-reading effector plasmid was transformed into different mating types of *Saccharomyces cerevisiae* haploid strains W303 using the PEG / LiAc method. The W303 strain was a standard laboratory strain with the genotype MATα and was plated on SC-Leu conditioned auxotrophic medium.

[0077] 2) The obtained W303 strain carries a reading effector expressed by a plasmid. The reading effector protein localizes to the H3K9me2 / 3 modified region through its CBX1 chromatin-binding domain and catalyzes the deacetylation of histones in this region through its Sir2 domain, thereby altering the chromatin structure and inhibiting the transcription of genes in this region.

[0078] 5. Construct a synthetic epigenetic regulatory system based on histone H3K9me2 / 3 modification 1) Pair the haploid yeast strain carrying both a writer and a reader with the W303 haploid strain carrying a reading effector. The specific procedure is as follows: 1.1) Pick appropriate amounts of cells from the two haploid strains and place them in 50 μL of sterile deionized water, then mix thoroughly by pipetting. 1.2) Drop the mixed bacterial solution onto the surface of YPD solid culture medium, let it air dry in a clean bench, and then place it in a 30°C incubator; 1.3) After culturing for 3 to 4 hours, take a small amount of cells and observe them under a microscope. When clover-shaped yeast cells appear, it indicates that mating has been successful. Clover-shaped cells are composed of diploid cells formed by the fusion of two haploid cells and daughter cells produced by the first budding. 1.4) Continue culturing for 2 to 3 hours, then streak onto SC-Leu-Trp dual-selection medium and incubate at 30 degrees Celsius for 2 to 3 days.

[0079] 2) The selected diploid yeast strains simultaneously carry a writer, a reader, and a reading effector, forming a complete synthetic epigenetic silencing system. In this system, the writer specifically locates to the 7xTetO sequence downstream of the Bar1 gene on chromosome 9 via the TetR domain, and catalyzes the methylation of H3K9 at this site via the G9A catalytic domain on the nucleosome, producing H3K9me2 and H3K9me3 modifications; 3) The reader recognizes the H3K9me2 / 3 modification generated by the writer through the CBX1 chromatin-binding domain, and catalyzes the generation of new H3K9me2 / 3 modifications on adjacent nucleosomes through its G9A catalytic domain. This positive feedback mechanism causes the modification to diffuse laterally along the chromosome, eventually forming a large-scale H3K9me2 / 3 modification domain. The reading effector locates the H3K9me2 / 3 modification domain through the CBX1 chromatin-binding domain, and catalyzes the deacetylation of histones in this region through the Sir2 domain, resulting in chromatin densification. RNA polymerase II cannot effectively bind to the promoter region, and transcription initiation is inhibited, thereby achieving synchronous transcriptional silencing of all genes within the modification domain.

[0080] Compare with Example 1 To verify that the gene silencing effect of the synthetic epigenetic silencing system indeed depends on the establishment of H3K9me2 / 3 modification, a control strain was constructed. The only difference between the control strain and the experimental strain in Example 1 was the inactivation of the G9A catalytic domain of the writer. Specifically, a site-directed mutagenesis was performed to mutate the key catalytic residue in the SET domain of the G9A catalytic domain from histidine to alanine. This mutation resulted in the complete loss of methyltransferase activity in G9A, but did not affect protein folding and stability. The inactivated writer could still locate to the 7xTetO sequence via the TetR domain, but could not catalyze the generation of H3K9me2 / 3 modification at that site. The construction process of the control strain was exactly the same as in Example 1, including integrating the 7xTetO sequence and inactivating the writer on the chromosome, transforming the pRS414 empty vector to replace the reader plasmid, and mating it with the W303 strain carrying the reading effector to generate a diploid. The control strain served as a negative control in the subsequent experiments of Example 2 and Example 4 to compare and verify the gene silencing effect of the experimental group.

[0081] Example 2 Based on the synthetic epigenetic silencing system constructed in Example 1, this embodiment integrates a remote fluorescent reporter gene verification system to verify the silencing ability of a wide range of multiple genes, and uses chromatin immunoprecipitation sequencing technology and real-time quantitative polymerase chain reaction to detect the distribution range of H3K9me2 modification and the expression level of reporter genes.

[0082] like Figure 2 As shown, on chromosome 9, which already has the 7xTetO sequence integrated, three different fluorescent reporter genes—Venus, mCherry, and BFP—were integrated at positions approximately 20 kilobase pairs upstream and 50 kilobase pairs upstream of the 7xTetO sequence, respectively. The expression of the three fluorescent reporter genes is controlled by constitutive promoters with similar promoter intensities. Using the same PEG / LiAc transformation method and Cas9-mediated genome editing technology as in Example 1, the three reporter genes were sequentially integrated into predetermined sites. The successfully integrated strain carries the 7xTetO sequence and the nearby Venus gene, the mCherry gene 20 kilobase pairs upstream, and the BFP gene 50 kilobase pairs upstream on chromosome 9. The writer and reader plasmids were integrated into this strain, and it was crossbred with the haploid strain carrying the reading effector constructed in Example 1 to obtain a diploid strain carrying all three fluorescent reporter genes and a complete epigenetic silencing system.

[0083] Diploid strains in the experimental group and the control group (Example 1) were cultured to the logarithmic growth phase, and bacterial cells were collected for chromatin immunoprecipitation sequencing. The specific procedures were as follows: Chromatin was fixed by cross-linking with 1% formaldehyde at room temperature for 20 minutes, and the cross-linking reaction was quenched by adding glycine to a final concentration of 125 mmol. Bacterial cells were collected and washed twice with cold phosphate buffer. The cells were resuspended in lysis buffer, and a protease inhibitor was added. Chromatin was fragmented into 200-500 base pairs using micrococcal nuclease MNase and an ultrasonic disruptor. The supernatant was collected by centrifugation, and anti-H3K9me2 antibody was added. The cells were incubated overnight at 4°C. Protein A / G magnetic beads were added and incubated at 4°C for 2 hours. The magnetic beads were washed sequentially with low-salt wash buffer, high-salt wash buffer, lithium chloride wash buffer, and TE buffer. The bound chromatin was eluted with elution buffer, and the cross-linking was reversed overnight at 65°C. Proteins were removed by proteinase K treatment, and DNA was purified by phenol-chloroform extraction. The purified DNA was used for high-throughput sequencing library construction and sequencing. After alignment and normalization, the enrichment fold of each genomic locus was calculated using the input DNA as a control.

[0084] The results are as follows Figure 3 As shown, chromatin immunoprecipitation sequencing results revealed that in the experimental group strains, a region of approximately 200 kilobase pairs in length, enriched by H3K9me2 modification, formed around the 7xTetO site on chromosome 9. This modification domain extended upstream and downstream of 7xTetO for approximately 100 kilobase pairs, with the enrichment fold remaining high within this region, averaging 8 to 10 times that of the input control. The three fluorescent reporter genes, Venus, mCherry, and BFP, were all located within this modification domain, with H3K9me2 modification enrichment folds in their promoter regions of approximately 10-fold, 6-fold, and 4-fold, respectively. The boundaries of the modification domain were relatively clear; within approximately 10 kilobase pairs outside the domain, the H3K9me2 modification level rapidly decreased to background levels. In the control group strains, due to the inactivation of the writer, the initial H3K9me2 modification could not be generated, and the H3K9me2 modification level across chromosome 9 remained at background levels, showing no significant difference from the input control. The results demonstrate that the synthetic epigenetic silencing system of the present invention can de novo establish H3K9me2 modification at specific chromosomal sites, and through the positive feedback mechanism of the reader, the modification spreads along the chromosome to form a large-scale modification domain of about 200 kilobase pairs. The length of this modification domain far exceeds the regulatory range of existing CRISPR / dCas9 technology, and can cover multiple genes or even complete biosynthetic pathways.

[0085] Cells from the experimental and control groups were collected, and total RNA was extracted. The mRNA expression levels of three fluorescent reporter genes were detected using real-time quantitative polymerase chain reaction (qPCR). The specific procedures were as follows: Total RNA was extracted using the phenol method, and DNase I treatment was used to remove genomic DNA contamination. One microgram of total RNA was used for reverse transcription to synthesize the first strand of cDNA. Using cDNA as a template, real-time quantitative polymerase chain reaction was performed using SYBR Green fluorescent dye and gene-specific primers. The reaction system was 20 microliters, and the cycling conditions were 95 degrees Celsius pre-denaturation for 3 minutes, followed by 95 degrees Celsius denaturation for 10 seconds and 60 degrees Celsius annealing extension for 30 seconds, for a total of 40 cycles.

[0086] The results are as follows Figure 4 As shown, the results of real-time quantitative polymerase chain reaction (qPCR) revealed that in the experimental group strains, the relative mRNA expression levels of the three fluorescent reporter genes Venus, mCherry, and BFP were reduced to approximately 30%, 40%, and 35% of those in the control group, respectively, meaning their expression levels were inhibited by approximately 70%, 60%, and 65%. Statistical analysis showed that the differences between the experimental and control groups were significant, with P values ​​less than 0.01. These results indicate that the synthetic epigenetic silencing system of this invention can effectively inhibit the transcription of genes within the H3K9me2 modification domain. Even the BFP gene, located 50 kilobase pairs away from the initial establishment site 7xTetO, showed significant inhibition of its expression. The mechanism of this inhibition is as follows: H3K9me2 modification, through the propagation of the reader, covers the promoter region of the reporter gene. After the reading effector locates the modified region, Sir2 catalyzes histone deacetylation, leading to chromatin densification. This prevents transcription factors and RNA polymerase II from effectively accessing the promoter, thus inhibiting transcription initiation. Notably, genes closer to 7xTetO exhibit higher levels of H3K9me2 modification and stronger expression repression, consistent with the mechanism of modification diffusion from the initial site. This result demonstrates that the present invention can achieve synchronous, holistic transcriptional repression of multiple genes across a large chromosomal region without requiring the design of individual gRNAs for each gene, significantly reducing genetic load and operational complexity.

[0087] Example 3 This embodiment, based on the synthetic epigenetic silencing system constructed in Example 1, establishes a small molecule-induced reversible gene silencing switch by fusing the estrogen-binding domain to the N-terminus of the reading effector, thereby achieving dynamic regulation of gene silencing state. This embodiment applies it to the regulation of the violacein biosynthesis pathway, validating the system's application value in metabolic engineering.

[0088] 1. Construction of a reversible gene silencing switch An estrogen-binding domain-reading effector fusion protein was constructed. The estrogen-binding domain was selected from the ligand-binding domain of the human estrogen receptor α, which is approximately 250 amino acids long and can specifically bind β-estradiol. In the ligand-free state, the estrogen-binding domain forms a complex with cytotoxic chaperone proteins such as heat shock protein Hsp90, retaining the fusion protein in the cytoplasm. When β-estradiol binds to the estrogen-binding domain, the domain undergoes a conformational change, dissociates from the cytotoxic chaperone protein, and exposes the nuclear localization signal sequence, enabling the fusion protein to be transported into the nucleus through the nuclear pore complex.

[0089] The estrogen-binding domain gene was cloned to the N-terminus of the reading effector gene, with the two linked by a short peptide linker. A nuclear localization signal sequence was added to the N-terminus of the fusion protein, while the Myc tag was retained at the C-terminus. The total length of this estrogen-binding domain-reading effector gene was approximately 3100 base pairs. It was synthesized after codon optimization and cloned into the pRS415 vector, replacing the reading effector plasmid in Example 1.

[0090] The pRS415-estrogen-binding domain-reading effector plasmid was transformed into the W303 haploid strain using the PEG / LiAc method, and strains carrying inducible reading effectors were screened. This strain was then crossbred with the haploid strain carrying a writer and a reader from Example 1 to obtain a diploid strain carrying a complete reversible silencing system.

[0091] 2. Integration of the violacein biosynthesis pathway and strain construction like Figure 5 As shown, the viobin biosynthesis pathway consists of five genes, vioA, vioB, vioC, vioD, and vioE, which encode tryptophan oxidase, viobin synthase, viobin hydroxylase, viobin reductase, and viobin transporter, respectively, catalyzing the complete biosynthetic pathway from tryptophan to viobin. The total length of the five genes is approximately 12 kilobase pairs.

[0092] An integrated fragment (approximately 15 kilobase pairs in total length) containing the aforementioned five genes, the Venus fluorescent reporter gene, and the TRP1 selection marker gene was synthesized. Primers with genomic homologous arms were designed, and the integrated fragment was inserted approximately 1 kilobase pair downstream of the 7xTetO sequence on chromosome 9 using the same PEG / LiAc transformation method and Cas9-mediated genome editing technology as in Example 1. The successfully integrated strain was able to grow on SC-Trp conditionally deficient medium, and Venus fluorescence signals could be observed under a microscope.

[0093] The strains that integrate the violacein synthesis pathway were backcrossed with the haploid strains carrying the writer and reader in Example 1 (to obtain diploid strains carrying the basic silencing system and the violacein pathway) and mated with the haploid strains carrying the inducible reading effector (to obtain diploid strains carrying the complete reversible silencing system and the violacein pathway) for subsequent verification of silencing effect and reversibility.

[0094] 3. Silencing effect, reversibility verification and mechanism analysis 1) Holistic Silence Verification of the Basic Silence System Diploid strains carrying the basal silencing system and the violacein pathway in the experimental group, as well as the diploid strain in the control group (Example 1), were cultured and subjected to liquid shake-flask culture and solid medium plate culture, respectively. Color changes in the strains were observed. Figure 6 The results showed that the colonies and bacterial solutions of the wild-type control group strains were both dark purple, indicating that the violacein synthesis pathway was normally expressed; the colonies of the experimental group strains were significantly lighter in color, turning light pink, and the bacterial solutions were almost colorless, indicating that the violacein synthesis pathway was significantly inhibited.

[0095] The enrichment of H3K9me2 modification in pathway gene regions was detected by chromatin immunoprecipitation-quantitative polymerase chain reaction (the experimental procedure was the same as in Example 2, with the ACT1 gene as a negative control, and specific primers designed for the 7xTetO site and the promoter regions of each vio gene). Figure 7 The results showed that the enrichment fold of H3K9me2 modification at the 7xTetO site in the experimental group was approximately 8.2 times that of the input control. The enrichment folds of the promoter regions of vioA, vioB, vioC, vioD, and vioE genes were approximately 1.5, 6.5, 3.0, 1.2, and 0.5 times, respectively, while the ACT1 gene site was at the background level. In the control group, no significant enrichment was found at any of the detected sites, indicating that H3K9me2 modification spread downstream from the 7xTetO site, covering the entire violacein synthesis pathway, and that the modification levels at different gene sites varied (related to gene location, chromatin structure, etc.).

[0096] The expression levels of pathway genes mRNA were detected using real-time quantitative polymerase chain reaction (the experimental procedure was the same as in Example 2, with ACT1 gene used as an internal control). Figure 8 The results showed that the relative mRNA expression levels of vioA, vioB, vioC, vioD, and vioE genes in the experimental group were reduced to about 5% of those in the control group, with an inhibition efficiency as high as 95%, and the statistical difference was extremely significant (P<0.001). This demonstrates that the synthetic epigenetic silencing system can simultaneously inhibit the transcription of all genes in the pathway, achieving overall silencing, and does not require the design of gRNA for each gene separately, greatly reducing the genetic load and operational complexity.

[0097] 2) Verification of dynamic control of reversible silent switch Add β-estradiol (β-est) inducer (stock solution prepared with dimethyl sulfoxide, concentration 100 mmol, stored at -20℃, added at a ratio of 1:2000 to a final concentration of 50 μmol) to the culture medium of a diploid strain carrying a complete reversible silencing system and the violacein pathway. After β-estradiol binds to the estrogen-binding domain of the fusion protein, it induces a conformational change and dissociation from the cytosolic chaperone protein, exposing the nuclear localization signal sequence, allowing the fusion protein to be transported into the nucleus. The fusion protein localizes to the H3K9me2 / 3 modification domain via the CBX1 chromatin-binding domain, and catalyzes histone deacetylation via the Sir2 domain, leading to chromatin compaction, repression of gene transcription, and activation of the silencing system. After 2-3 days of culture, the strain color lightens to near colorless, indicating that the violacein synthesis pathway has been silenced.

[0098] The β-estradiol-induced strains were transformed with an initial OD600 of 0.1 and cultured in fresh β-estradiol-free medium for 2-3 days. As the intracellular β-estradiol concentration decreased, the fusion protein dissociated from β-estradiol, rebinded to cytosol chaperone proteins, and remained in the cytoplasm. The concentration of nuclear reading effectors decreased, histone deacetylation weakened, chromatin relaxed, gene transcription resumed, silencing was lifted, and the strain color returned to deep purple. Figure 9 As shown, the wild-type control group strain remained deep purple throughout the experiment and was unaffected by β-estradiol; the experimental group strain remained deep purple without the inducer, became lighter after the inducer was added, and recovered after the inducer was removed. This directly demonstrates that the reversible silencing system can achieve dynamic control of gene silencing state, with a response time of 2-3 days (48-72 hours), meeting the needs of temporal regulation in metabolic engineering.

[0099] 4. Application Scenarios of Metabolic Engineering This system can be applied to segmented fermentation scenarios: β-estradiol is not added in the early stage of fermentation, allowing the violacetin synthesis pathway to be expressed normally, accumulating intermediate metabolites or maintaining normal cellular metabolism; β-estradiol is added in the later stage of fermentation to silence the violacetin synthesis pathway, redirecting metabolic flux to the target product synthesis pathway and improving the yield of the target product. This strategy is applicable to various metabolic engineering scenarios, such as regulating competitive metabolic pathways, optimizing metabolic flux allocation, and improving strain stability. Compared with the control group, strains using this strategy have significant advantages in target product yield, growth stability, and fermentation controllability, providing a powerful tool for complex metabolic engineering.

[0100] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A synthetic epigenetic silencing system for large-scale multi-gene expression regulation, characterized in that, include: DNA targeting sequences integrated into the host cell chromosome; The compiler is a fusion protein of the DNA-binding domain and the H3K9 methyltransferase catalytic domain; The reader / writer is a fusion protein of the H3K9me recognition domain and the H3K9 methyltransferase catalytic domain; The reading effector is a fusion protein of the H3K9me recognition domain and a transcriptional repressor. The writer is capable of specifically binding to the DNA target sequence and generating H3K9me2 / 3 modification through the H3K9 methyltransferase catalytic domain. The reader recognizes existing H3K9me2 / 3 modifications through the H3K9me recognition domain, and propagates the modification along the chromosome through the H3K9 methyltransferase catalytic domain, forming a large-scale H3K9me2 / 3 modification domain. The reading effector locates the H3K9me2 / 3 modification domain through the H3K9me recognition domain and inhibits the transcription of genes within the modification domain through a transcriptional repressor.

2. The synthetic epigenetic silencing system for large-scale multi-gene expression regulation according to claim 1, characterized in that, The writer is integrated into the host cell chromosome, and the writer and reading effector exist in the form of plasmids.

3. The synthetic epigenetic silencing system for large-scale multi-gene expression regulation according to claim 1, characterized in that, The writer, reader, and reader effector all contain a nuclear localization signal sequence.

4. The synthetic epigenetic silencing system for large-scale multi-gene expression regulation according to claim 1, characterized in that, The writer incorporates the Flag tag, the reader incorporates the HA tag, and the reader effector incorporates the Myc tag.

5. The synthetic epigenetic silencing system for large-scale multi-gene expression regulation according to claim 1, characterized in that, The length of the large-scale H3K9me2 / 3 modification domain is 50-200 kb.

6. A method for achieving reversible gene silencing, based on the synthetic epigenetic silencing system for large-scale multi-gene expression regulation as described in any one of claims 1-5, characterized in that, include: S1. In a host cell that has been constructed with the synthetic epigenetic silencing system for large-scale multi-gene expression regulation, a small molecule inducible nuclear localization domain is fused into the reading effector to form an inducible reading effector. S2. The host cells are cultured, and the small molecule-induced nuclear localization domain retains the inducible reading effector in the cytoplasm, and the gene silencing system is in a closed state. S3. Add an inducer to the culture medium. The inducer binds to the small molecule inducible nuclear localization domain, causing the inducible reading effector to be transported into the cell nucleus. After entering the nucleus, the inducible reading effector is localized to the H3K9me2 / 3 modification domain and initiates gene silencing in that region. S4. Remove the inducer, and the inducible reading effector exits the nucleus or re-enters the cytoplasm, thus silencing the gene.

7. The method for achieving reversible gene silencing according to claim 6, characterized in that, The small molecule-induced nuclear localization domain is the estrogen-binding domain EBD.

8. The method for achieving reversible gene silencing according to claim 6, characterized in that, The inducer is β-estradiol, and the concentration of the inducer is 10-100 μM.

9. The application of the synthetic epigenetic silencing system for large-scale multi-gene expression regulation as described in any one of claims 1-5 in metabolic engineering, characterized in that, include: By integrating multiple genes of the target metabolic pathway into the downstream chromosomal region of the DNA target sequence, a synthetic epigenetic regulatory system containing multiple genes of the target metabolic pathway is constructed. At different stages of the fermentation process, the nuclear localization of the reading effector is controlled by adding or removing the inducing agent, thereby controlling the silencing state of multiple genes in the target metabolic pathway; By dynamically switching the expression of target metabolic pathways, the temporal control of metabolic flux can be achieved.

10. The application according to claim 9, characterized in that, This system is applied to the metabolic engineering of brewer's yeast.

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