A yeast quorum sensing dynamic regulation system and method and application thereof

Abstract

This invention belongs to the fields of genetic engineering and microbial technology, specifically relating to a yeast quorum sensing dynamic regulation system, method, and its applications. This invention successfully constructs a functional, bacterial-derived RpaI / RpaR quorum sensing system in yeast for the first time. The constructed yeast quorum sensing dynamic regulation system possesses advantages such as low background leakage, high sensitivity, and wide dynamic range. By flexibly coupling the core system with a signal amplification module (Gal4) and a signal conversion module (CRISPRi), a bifunctional cascade platform integrating activation and inhibition is constructed, achieving multidimensional and precise dynamic regulation of metabolic pathways. Furthermore, the system has been applied to the biosynthesis of several important compounds, including cordycepin, geraniol, and 3-hydroxypropionic acid, significantly increasing yields and effectively balancing the conflict between cell growth and product synthesis, demonstrating its great potential as a universal dynamic regulation tool.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial technology, specifically relating to a yeast quorum sensing dynamic regulation system, method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] brewing yeast ( Saccharomyces cerevisiae As an important microbial cell factory, cell growth is widely used in the synthesis of various high-value compounds. However, in metabolic engineering, there is often a conflict between cell growth and product synthesis. Overexpression of heterologous pathways can lead to metabolic burden, resulting in impaired cell growth and thus limiting efficient product production. Therefore, achieving dynamic allocation of metabolic flux is crucial for optimizing product synthesis.

[0004] Quorum sensing (QS)-based dynamic regulation strategies utilize cell-generated signaling molecules to sense population density and autonomously regulate gene expression. These strategies offer advantages such as independence from specific metabolites, high autonomy, and broad applicability, and have been widely applied in bacterial metabolic engineering. However, in *Saccharomyces cerevisiae*, existing quorum sensing systems are mostly based on α-pheromones or cytokinins, which involve complex signal transduction pathways and have limited dynamic ranges. Introducing a simple and high-performance bacterial quorum sensing system into yeast is an ideal dynamic regulation strategy, but so far there have been no successful examples. Studies have shown that eukaryotic cells cannot synthesize typical bacterial AHL-type signaling molecules, which is a significant reason for the failure of previous attempts. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a yeast quorum sensing dynamic regulation system, method, and its applications. Specifically, the present invention establishes a programmable population density-dependent regulation platform in yeast by engineering an unconventional bacterial quorum sensing system: the RpaI / RpaR system. This system uses p-hydroxycoumaryl-HSL (… p -coumaroyl-HSL, pC-HSL, as a signaling molecule, overcomes the fundamental metabolic barrier preventing eukaryotes from achieving functional bacterial quorum sensing. This engineered circuit features low leakage, high sensitivity, and a wide dynamic range. By coupling the QS with a signal amplification and CRISPRi module, a bifunctional cascade system with both autonomous transcriptional activation and inhibition functions is constructed. This QS platform achieves decoupling of growth and product, significantly increasing the yields of cordycepin, geraniol, and 3-hydroxypropionic acid. Based on the above research results, this invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows: A first aspect of the present invention provides a yeast quorum sensing dynamic regulation system, the yeast quorum sensing dynamic regulation system comprising at least an RpaI / RpaR quorum sensing system; the RpaI / RpaR quorum sensing system comprising a signal molecule synthesis module and a signal response module; wherein the signal molecule synthesis module is capable of synthesizing and secreting signal molecules. p C-HSL; the signal response module includes at least one fusion protein RpaR-AD (RpaR fused with the Gal4 transcription activation domain) and its recognized specific DNA binding sequence RpaO.

[0007] In a second aspect, the present invention provides a recombinant cell comprising at least the yeast quorum sensing dynamic regulation system described above.

[0008] A third aspect of the present invention provides a method for dynamic regulation of yeast quorum sensing, the method comprising at least introducing the above-mentioned yeast quorum sensing dynamic regulation system into cells.

[0009] A fourth aspect of the present invention provides the use of the above-described yeast quorum sensing dynamic regulation system, recombinant cells, or methods in any one or more of the following: (a) Balancing cell growth and metabolite synthesis; (b) Increase the production of metabolites.

[0010] The beneficial technical effects of one or more of the above technical solutions are as follows: The aforementioned technical solution successfully constructed a functional, bacterial-derived RpaI / RpaR quorum sensing system in yeast for the first time, overcoming the long-standing technical obstacle of eukaryotes' inability to synthesize AHL signaling molecules. Simultaneously, the constructed yeast quorum sensing dynamic regulation system possesses advantages such as low background leakage, high sensitivity, and wide dynamic range. By flexibly coupling the core system with the signal amplification module (Gal4) and the signal conversion module (CRISPRi), a bifunctional cascade platform integrating activation and inhibition was constructed, achieving multidimensional and precise dynamic regulation of metabolic pathways. Furthermore, the system was applied to the biosynthesis of several important compounds, including cordycepin, geraniol, and 3-hydroxypropionic acid, significantly increasing yields and effectively balancing the conflict between cell growth and product synthesis, demonstrating its enormous potential as a universal dynamic regulation tool. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1 In brewer's yeast p Design and optimization schematic diagram of C-HSL QS.

[0013] (A) Schematic diagram of the pC circuit. This quorum sensing (QS) system comprises two modules: a signal molecule synthesis module (containing...) p C-HSL synthesis pathway) and response module (including sensing) p (The response circuit of C-HSL). As cell density increases... p The concentration of C-HSL gradually increased. p C-HSL interacts with RpaR-AD, subsequently binding to RpaO and activating transcription. (B) Regulating RpaI expression using different promoters affects the synthetic characteristics of pC-HSL in yeast and (C) cell density (OD). 60 0) Characterization. (D) Characterization diagram of response module. (E) Regulating RpaR-AD expression using different promoters to reduce missed expression and enhance the dynamic range of the circuit. (F) Performance characterization of circuits with different RpaO copy numbers in the promoter. (G) Dose response curves of pC loop genetic circuits with different RpaO copy numbers.

[0014] Figure 2 Characterization of cell density-dependent pC loop genetic circuitry.

[0015] (A) Schematic diagram of sender-receiver cell communication. Sender cells containing the synthesis module are cultured in liquid culture medium, and cell-free supernatant is collected. To evaluate cell-cell communication, the collected fermentation broth is mixed with an equal volume of fresh culture medium, and cells containing... p The receiving cell of the C-HSL response module. (B) The sending cell generates... p Correlation between C-HSL concentration and fluorescence intensity in receiving cells. (C) In intercellular communication, the sending cell... p (D) Relationship between C-HSL concentration, cell density, and fluorescence intensity of receiving cells.

[0016] Figure 3 Construction and application of cascaded amplification dynamic control system.

[0017] (A) Characterization of the signal amplification circuit. Signal amplification circuit: pC loop control. GAL4 The expression of Gal4 further regulates GAL Promoter. (B) Schematic diagram of dynamic regulation of geraniol biosynthesis pathway based on signal amplification circuit. The gene encoding the mevalonate pathway enzyme is... GAL1 / 10 Promoter regulation. tCrGES–ERG20 ww by GAL 1. Controlled by promoter, pC loop, or signal amplification loop. (C) Geraniol content in different strains. (D) Biomass accumulation (OD). 600 All data are the averages of three repeated experiments, and the error bars represent the standard deviation of each measurement.

[0018] Figure 4 Multidimensional dynamic regulation of metabolic pathways (A) Characterization of the signal conversion circuit. This circuit is constructed by coupling a signal amplification circuit with the CRISPRi system. (B) Schematic diagram of the bifunctional dynamic regulation of the 3-hydroxyphenylpropionic acid biosynthetic pathway. MCR expression is controlled by a QS-based signal amplification system. FAS1 The expression of MCRN was inhibited by a QS-based inhibition system. (C) 3-HP yield of different MCR-expressing strains. Signal amplifying circuit: A QS-based signal amplification system was used, with MCRN and MCRC dynamically controlled by GAL1 and GAL10; Gal1p-MCRN-Gal10p-MCRC: without p Strains with the C-HSL biosynthetic pathway, MCRN and MCRC controlled by the GAL1 and GAL10 promoters; Gal1p-MCRN-Gal10p-MCRC plus 0.02mM pC-HSL: Strains expressing MCRN and MCRC under the control of the Gal1 and Gal10 promoters, respectively, were treated with 0.02 mM... p C-HSL titration. (D) Different strategies for suppression. FAS1 Signal-transition circuit: In strains containing a QS signal amplification system, crFAS1 by GAL1p Control. Constitutive expression of CRISPRi: In strains containing a QS signal amplification system, FAS1 It is continuously suppressed by SN52p-crRNA.

[0019] Figure 5 Dynamic regulation of the 3-HP biosynthetic pathway in high-yielding strains.

[0020] (A) Schematic diagram of genetic modification in the high-yielding 3-HP strain. Overexpression of malonyl-CoA was used to increase the supply of malonyl-CoA and intracellular bicarbonate levels. xPK , PTA , ACC1S659AS1157A , STB5 , YHM2 , CIT1 and SUL 1. MCR- C N941VK1107WS1115R and YdfG Controlled by a signal amplification circuit; FAS1 Genes are dynamically suppressed through signal conversion circuits. (B) 3-HP yield in different strains.

[0021] Figure 6 Optimization of the pC loop.

[0022] (A) Comparison of different promoter strengths. Background leakage was reduced by altering RpaR-AD transcript levels. (B) Dose-response curves of pC circuit sensing different RpaO copies.

[0023] Figure 7 Characterization of intercellular communication.

[0024] (A) Schematic design of communication between the sending and receiving cells. The sending cell contains signaling molecules. p The C-HSL synthesis pathway. Receptor cells contain sensory receptors. p C-HSL response module. (B) Methods for simultaneously adding sending and receiving cells in a co-culture system using different initial ratio strategies. Total cell content represents the total amount of both types of cells in the culture medium.

[0025] Figure 8 Dynamically regulate the cordycepin biosynthesis pathway.

[0026] (A) Schematic diagram of the dynamic regulation of the cordycepin biosynthesis pathway based on QS. The CNS1 and CNS2 genes are regulated by the pC circuit. 3'-AMP: adenosine-3'-monophosphate; 2'-C-3'-dA: 2'-carbonyl-3'-deoxyadenosine. (B) Cordycepin production. (C) Biomass accumulation (OD). 600 (D) p C-HSL concentration. (E) Dynamic regulation of the cordycepin biosynthesis pathway by regulating Gal4 expression. Cordycepin production (F) and OD were correlated when CNS1 / 2 expression was dynamically induced in yeast strains via the GAL1 promoter, pC circuit, or signal amplification circuit. 600 Quantitative analysis of GAL1p-CNS1-CNS2: without GAL1p-CNS1-CNS2. p In strains with the C-HSL synthesis pathway, CNS1 / 2 is regulated by GAL1p; pC circuit-CNS1-CNS2: contains p The C-HSL synthesis pathway, CNS1 / 2 is regulated by strains with a pC loop; Signal-amplification circuit-CNS1-CNS2: containing p The C-HSL synthesis pathway, CNS1-CNS2, is regulated by a signal amplification loop in the strains. All data are the average of three replicates, and error bars represent the standard deviation of each measurement.

[0027] Figure 9 Comparison of the 6×RpaO QS circuit with the TEF1 promoter strength.

[0028] Figure 10 Characterization of signal suppression circuit based on pC loop.

[0029] (A) Schematic diagram of the signal inhibition circuit. Under high cell density conditions, RpaR-AD and... p C-HSL interaction activates CRISPRi expression controlled by the 6×RpaO promoter, thereby downregulating GFP expression. (B) Optical density value (OD) 600 ) and quantitative analysis of fluorescence values. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available.

[0033] In a typical embodiment of the present invention, a yeast quorum sensing dynamic regulation system is provided, the yeast quorum sensing dynamic regulation system comprising at least an RpaI / RpaR quorum sensing system; the RpaI / RpaR quorum sensing system includes a signal molecule synthesis module and a signal response module; wherein, the signal molecule synthesis module is capable of synthesizing and secreting signal molecules. p C-HSL; the signal response module comprises at least one fusion protein RpaR-AD (RpaR fused to the Gal4 transcriptional activation domain) and its recognized specific DNA-binding sequence RpaO. The copy number of RpaO can be one or more, further ranging from 1 to 6. When the copy number of RpaO is 6, this high-performance genetic circuit exhibits the highest sensitivity and the largest dynamic response range.

[0034] High concentrations of transcriptional regulatory proteins can uncontrolledly activate transcription, resulting in the expression of downstream genes being initiated without binding to signaling molecules. Therefore, by altering... RpaR-AD This reduces transcriptional levels, lowers background leakage, and improves the dynamic response range of genetic circuits. In one specific embodiment of the invention, promoters of different strengths are used to control... RpaR-AD Transcription, the promoter including but not limited to TEF1p, LEU1p, CYC1p and REV1p Preferred CYC1p .

[0035] Specifically, the signal molecule synthesis module includes at least genes encoding tyrosine ammonia-lyase (TAL), coumaroyl-CoA ligase (4CL), and RpaI synthase (RpaI), thereby enabling the synthesis of signal molecules. p C-HSL.

[0036] Furthermore, the yeast quorum sensing dynamic regulation system also includes Gal4 or other transcription factors, thereby coupling with the aforementioned RpaI / RpaR quorum sensing system to amplify transcriptional activation signals.

[0037] Furthermore, the yeast quorum sensing dynamic regulation system also includes CRISPRi, which, by regulating the expression of CRISPRi components (such as crRNA), couples with the aforementioned RpaI / RpaR quorum sensing system, converting transcriptional activation signals into transcriptional repression signals to achieve population density-dependent repression of the target gene. In this invention, regulating the expression of CRISPRi components (such as crRNA) can be achieved through promoters (such as 6×RpaO or Gal1).

[0038] In another specific embodiment of the present invention, a recombinant cell is provided, wherein the recombinant cell at least includes the above-mentioned yeast quorum sensing dynamic regulation system; The recombinant cells may be eukaryotic cells, and more particularly, fungal cells, wherein the fungi may be molds and yeasts; wherein the molds include, but are not limited to, Aspergillus flavus (…). Aspergillus flavus Aspergillus niger ( ) Aspergillus niger ) and Trichoderma reesei ( Trichoderma reesei The yeast mentioned includes, but is not limited to, brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Max Kluyveromycin ( Kluyveromyces marxianus Pichia pastoris () Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ) and Debali yeast ( Debaryomyces hansenii ).

[0039] In this invention, the recombinant cell can be a single recombinant cell, that is, the same recombinant cell simultaneously contains the above-mentioned signal molecule synthesis module and signal response module; or the recombinant cell can be composed of a transmitting cell and a receiving cell, wherein the transmitting cell contains only the signal molecule synthesis module and the receiving cell contains only the signal response module. The sending and receiving cells can be cultured independently or co-cultured to achieve quorum sensing communication between cells.

[0040] In another specific embodiment of the present invention, the signal molecule synthesis module is capable of synthesizing and secreting the signal molecule pC-HSL; the signal response module includes at least a fusion protein RpaR-AD and its recognized specific DNA binding sequence RpaO.

[0041] In another specific embodiment of the present invention, a method for dynamic regulation of yeast quorum sensing is provided, the method comprising at least introducing the above-mentioned yeast quorum sensing dynamic regulation system into cells.

[0042] In one specific embodiment of the invention, autonomous regulation dependent on population density or the addition of external signaling molecules is employed. p The C-HSL method increases the production of metabolites.

[0043] The cells may be eukaryotic cells, and more particularly, fungal cells, wherein the fungi may be molds and yeasts; wherein the molds include, but are not limited to, Aspergillus flavus (…). Aspergillus flavus Aspergillus niger ( ) Aspergillus niger ) and Trichoderma reesei ( Trichoderma reesei The yeast mentioned includes, but is not limited to, brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Max Kluyveromycin ( Kluyveromyces marxianus Pichia pastoris () Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ) and Debali yeast ( Debaryomyces hansenii ).

[0044] In another specific embodiment of the present invention, the application of the above-described yeast quorum sensing dynamic regulation system, recombinant cells, or method in any one or more of the following: (a) Balancing cell growth and metabolite synthesis; (b) Increase the production of metabolites.

[0045] The metabolites include, but are not limited to, nucleoside antibiotics, terpenoids, and bio-based platform compounds; further, the nucleoside antibiotic is cordycepin; the terpenoid is geraniol; and the bio-based platform compound is 3-hydroxypropionic acid.

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0047] Example I. Experimental Methods 1. Microbial strains and culture conditions Plasmids were constructed and amplified using *Escherichia coli* Trans5α strain (Beijing Transgenic Biotechnology Co., Ltd.). Recombinant *E. coli* was cultured in Luria-Bertani medium (10 g / L NaCl, 10 g / L tryptophan, 5 g / L yeast extract) supplemented with 100 μg / mL ampicillin at 37°C and 220 rpm. *Saccharomyces cerevisiae* CEN.PK102-5B was used as the parent strain and cultured in YPD medium containing 1% yeast extract, 2% peptone, and 2% glucose at 30°C and 200 rpm. Transformed strains were cultured on basal (SC) medium. The SC medium contained 0.5% ammonium sulfate, 0.17% yeast nitrogen base (BBI Life Sciences, China), a complete supplement mixture (excluding uracil, histidine, or leucine) (Sunrise Science Products, USA), and 2% glucose.

[0048] 2. Plasmid and strain construction All plasmid structures are listed in Table 1. High-fidelity DNA amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co. Ltd.). Yeast transformation was performed using the LiAc method. Engineered strains are listed in Table 2. All genomic modifications were performed using the CRISPR / Cas9 system. All plasmids were constructed using the Gibson assembly method. All exogenous genes were synthesized with codon optimization by Genscript (Nanjing).

[0049] Table 1. Plasmids involved in this study

[0050] Table 2. Strains involved in this study

[0051] Gal: Recombinant strain with a cascade amplification system; Geraniol: Recombinant strain for geraniol production; Cordycepin: Recombinant strain for cordycepin production; 3-HP: Recombinant strain for 3-HP production.

[0052] In order to produce p C-HSL, we optimized the codons of Flavobacterium junceum for tyrosine aminolysin (C-HSL). TAL Arabidopsis thaliana 4-coumarate coenzyme a ligase ( 4CL ) and signal molecule synthase ( RPAI This was integrated into the CEN.PK102-5B genome. (This was done to construct the response...) pThe C-HSL promoter inserts seven combinatorial variants (n = 0-6 RapOs) upstream of the Gal1p core region of the 2μ plasmid pJFE3. The reporter gene GFP is present in the above... p Under the control of C-HSL-sensing promoters, the Gal4 transcriptional activation domain (AD) and RpaR are fused in the 2μ plasmid pIYC04, and expression is controlled by promoters of varying strengths.

[0053] In terms of signal amplification, bidirectional... GAL1 / 10 Promoters control gene expression.

[0054] To facilitate signal transduction, we constructed crRNAs driven by either the 6×RpaO or Gal1 promoter.

[0055] To synthesize cordycepin, the protein-coding gene of the Cordyceps militaris oxidoreductase domain was encoded using the GGGGS linker. CNS1 (Genbank accession number: CCM_04436) and the SAICAR synthase encoding gene ( CNS2 The plasmids pJFE3-TEF1p-Cns1-Cns2, pJFE3-6×RpaO-Cns1-Cns2, and pJFE3-6×RpaO-Gal4-Galp-Cns1-Cns2 were constructed by fusing with Genbank accession number CCM_04437.

[0056] In order to synthesize geraniol, Catharanthus roseus The truncated geraniol synthase (tGES) and the double mutant ERG20 from this source The N-terminal fusion of (F96W–N127W) was used to construct pJFE3-Gal1p-tCrGES-ERG20. -PGK1t vector.

[0057] For the biosynthesis of 3-HP, by GAL1 The promoter is controlled by Chrysophagus chinensis (Golden Green Grass) Chloroflexus aurantiacus The N-terminal region of ) (MCRN: amino acids 1 ~ 549) and GAL10 The promoter-controlled C-terminal region (MCRC: amino acids 550-1219) was fused together and integrated into the HO site of CEN.PK102-5B. To obtain high-yield 3-HP, the recombinant plasmid pNLW11-Ga1 was transformed into the recombinant strain QLW19. This recombinant plasmid carries the... GAL1 Starter-controlled MCRC N941VK1107WS1115R and E. coli Ydf G gene.

[0058] 3. Measurement of fluorescence intensity Real-time fluorescence monitoring was performed using a multi-detector microplate reader (Synergy HT, Biotek, USA). GFP excitation wavelength was 485 nm, and emission wavelength was 528 nm. Fluorescence intensity (au) was normalized to OD. 600 In the send-receive experiment, the sending cell (sending cell: producing...) will be used. p C-HSL cells were cultured to different cell densities, and then the fermentation broth was collected. Receiving cells (receiving cells: for...) p C-HSL-responsive strains were cultured in fresh medium, and an equal volume of fermentation broth collected from the sending cells was added. Fluorescence intensity was then measured. In co-culture experiments, senders and receivers with different inoculation ratios were co-cultured in 48-well plates, and fluorescence was detected every 1 hour.

[0059] 4. Cultivation conditions In all biosynthesis processes, transformants were inoculated in 5 ml of medium for approximately 2 days, followed by inoculation in 40 ml of medium (initial OD). 600 = 0.2), and cultured at 30°C and 200 rpm for 96 h. Samples were taken at different time points, centrifuged at 10,625×g for 5 min, and analyzed by high performance liquid chromatography (HPLC).

[0060] 5. Quantitative analysis using high performance liquid chromatography (HPLC) The determination was performed using high performance liquid chromatography (HPLC, Shimadzu Corporation, Japan). p Concentrations of C-HSL, cordycepin, and 3-HP. p C-HSL and cordycepin were detected using a Hypersil C18 column (4.6 × 250 mm, 5 μm; Elite Analytical Instruments) at 30°C. 1 mL of fermentation broth was centrifuged at 12,000 rpm for 5 min. The supernatant was then filtered through a 0.22 μm filter membrane with 500 μl methanol. The mobile phase was 35% acetonitrile aqueous solution. p C-HSL) or 20% methanol aqueous solution (cordycepin); flow rate: 1 mL / min; detection wavelengths: 300 nm and 260 nm, respectively.

[0061] 3-HP was detected using an Aminex HPX-87H column (Bio-Rad, Hercules, USA). The column oven temperature was 50℃, the mobile phase was 5 mm H2SO4, the flow rate was 0.5 mL / min, and the PDA detector wavelength was 210 nm.

[0062] 6. GC Quantification Geraniol content was determined by high-performance gas chromatography (HPLC). The fermentation sample was centrifuged at 12,000 rpm for 5 min to promote phase separation. The resulting organic phase was collected, diluted 1:10 (v / v) with n-dodecane, and then transferred to a glass GC vial for analysis. Geraniol concentration was determined using a Shimadzu GC-FID system equipped with an SH-Wax capillary column (30 m × 0.25 mm, 0.2 μm). Nitrogen was used as the carrier gas. The GC column temperature program was set as follows: initial isotherm at 60°C for 2 min, followed by ramping to 150°C at a rate of 10°C / min (isotherm at 10 min), and finally ramping to 230°C at a rate of 20°C / min (isotherm at 5 min). The injector and flame ionization detector (FID) temperatures were maintained at 260°C and 280°C, respectively.

[0063] II. Experimental Results 1. Design and optimization of the pC loop genetic circuit in Saccharomyces cerevisiae To introduce a quorum sensing system from bacteria with a simple regulatory pathway into Saccharomyces cerevisiae, we selected the unconventional Rhodopseudomonas palustris (…). Rhodopseudomonas palustris The RpaI / RpaR swarm sensing system (from which the RpaI / RpaR source is located) Figure 1 A). RpaI synthesizes signal molecules using aromatic amino acids as substrates. p C-HSL, thus circumventing the problem that eukaryotes cannot synthesize AHL signaling molecules. Introducing C-HSL into yeast... Flavobacterium johnsoniae of Tyrosine ammonia lyase (TAL) , Arabidopsis thaliana of p-coumaroyl-CoA ligase (4CL) as well as R. palustris of LuxI-type synthase (RpaI) Genes were used to construct a synthetic module for use in [the following contexts]. p Synthesis of C-HSL. For example... Figure 1 As shown in BC, yeast cells with a synthesis module can synthesize signal molecules, and p The concentration of C-HSL increased with prolonged fermentation time in the sending cells. By regulating RpaI expression with promoters of different strengths, corresponding gradients could be obtained. p The C-HSL yield demonstrates that the output signal is tunable. A key characteristic of quorum sensing is the extracellular accumulation of signaling molecules.

[0064] RpaR is a type of... p Transcriptional activators that interact with C-HSL. p Following C-HSL interaction, RpaR further binds to RpaO, activating transcription. In the response module, two copies of the RpaO sequence are inserted... GAL1Upstream of the core promoter, green fluorescent protein is expressed. To achieve transcriptional activation in yeast, [the following is mentioned:] TEF1 Driven by the promoter, the active domain (AD) of Gal4 is fused to the C-terminus of RpaR (RpaR-AD). Then, different concentrations of [unspecified ingredient] are added. p C-HSL, a module characterizing the response. We found that RpaR-AD can be used to characterize the response. p C-HSL activates GFP expression when p At a C-HSL concentration of 1 mM, GFP expression increased by 1.74-fold. These results indicate that the response module is capable of sensing... p C-HSL regulates gene expression ( Figure 1 D).

[0065] High concentrations of transcriptional regulatory proteins can uncontrolledly activate transcription, resulting in the expression of downstream genes without binding to signaling molecules. Therefore, we attempted to modify... RpaR-AD This reduces transcriptional levels, lowers background leakage, and improves the dynamic response range of genetic circuits. Promoters of varying strengths are used to control transcriptional levels. RpaR-AD Transcription (promoter strength: TEF1p >LEU1p>CYC1p>REV1p () Figure 6 ).like Figure 1 As shown in D, except REV1p Furthermore, all promoters exhibited significant activation effects. Specifically, at 0.1 mM p Under C-HSL conditions, the promoters respectively activate yeGFP 1.7 times (TEF1p), 2.5 times (LEU1p), and 38.1 times (CYC1p). When RpaR-AD is powered by a weak promoter (such as... CYC1p When driven by a CYC1 promoter, the circuit exhibits low leakage and achieves a high dynamic response range. Therefore, we use a CYC1 promoter to control the expression of the RpaR-AD. Furthermore, we cascaded RpaOs with different copy numbers. As the RpaO copy number increases, the circuit's sensitivity and dynamic output range also increase. Figure 1 EG and Figure 6 B). Specifically, the dynamic output ranges from 1×RpaO to 6×RpaO were 18.5, 38.1, 42.4, 41.8, 42.0, and 48.3 times, respectively. Among these, 6×RpaO exhibited a lower response threshold, with an initial response concentration of 0.004 mM, an order of magnitude lower than 1×RpaO. Furthermore, 6×RpaO demonstrated the highest sensitivity and the largest dynamic response range, at 48.3 times. Based on these results, we successfully constructed... p C-HSL-mediated high-performance genetic circuits.

[0066] 2. QS-based yeast cell communication and autonomous induction Furthermore, we verified whether the above genetic circuit is cell density dependent. First, we characterized the intercellular communication of the 6×RpaO system ( Figure 2 A). We constructed sending cells containing a signal molecule synthesis module and receiving cells containing a response module. The sending cells were fermented, the yield of signal molecules was measured, and the fermentation broth was collected. The fermentation broth was mixed with an equal volume of fresh culture medium, and the receiving cells were cultured. Fluorescence intensity was measured, and the correlation between signal molecule content and fluorescence intensity was analyzed. Figure 2 As shown in B, strains with different promoters produce p The concentration of C-HSL differs. When the receiving cells are cultured together with the sending cell culture, the fluorescence intensity differs from that produced by the sending cells. p C-HSL concentration showed a strong positive correlation. Furthermore, fermentation supernatants from sending cells cultured for different times were collected and incubated with an equal volume of fresh culture medium to assess cell-cell communication. We observed... p The concentration of C-HSL increases with increasing cell density. Correspondingly, the fluorescence intensity of the receiver cells also gradually increases, indicating that the receiver cells can effectively sense changes in the density of the sending cells and accordingly regulate gene expression. Figure 2 C).

[0067] To further characterize the population density dependence of the pC circuit, we will p The C-HSL biosynthesis module and response module were introduced into the same yeast strain. Both the 2×RpaO and 6×RpaO systems exhibited cell density dependence, and different response modules showed different initial response densities and dynamic response ranges. The initial response density of the 2×RpaO system was OD0. 600= The initial response density of the 6×RpaO system was 0.90, while the initial response density was 0.57, and the dynamic range was 21.9 times wider. This dynamic range is significantly higher than that of other reported yeast QS systems.

[0068] To demonstrate the function of QS-based dynamic regulation, we applied this circuit to control the biosynthesis of cordycepin, a toxic ATP analog that interferes with mRNA synthesis, thereby preventing the formation of 3',5'-phosphodiester bonds, leading to cell damage and inhibiting cell growth. Regulation of the biosynthetic pathway via the 6×RpaO circuit increased cordycepin production by 1.7-fold and reduced the growth inhibition caused by cordycepin production. Figure 8 These results demonstrate that this QS-based circuit can autonomously balance cell growth and product production.

[0069] 3. Construction of a cascaded signal amplification system for dynamic activation Although the pC circuit developed here exhibits a wide dynamic range, its maximum output under full induction remains relatively modest. Figure 9 This limits its application in pathways requiring high expression levels. In electronic systems, signal amplifiers are typically used to improve the signal-to-noise ratio. To simulate this principle and further increase the dynamic output range of the QS system, we attempted to couple the pC loop with the GAL system, designing a cell density-responsive GAL regulation loop to regulate Gal4 expression in Gal80-deficient strains. Figure 3 A). For example Figure 3 As shown in Figure A, the cascade system significantly improved the activation efficiency of GFP, increasing it by 3.86 times compared to the original pC circuit, with a dynamic range reaching 35.5 times. These results indicate that adding Gal4 as an intermediate activator can effectively amplify the transcriptional output of the QS system.

[0070] We then applied the signal amplification circuit to the multigene regulation of terpene biosynthesis pathways and compared the system with the commonly used GAL induction system. Geraniol is a monoterpene with wide applications in the fragrance and pharmaceutical industries and possesses antibacterial and antifungal properties. The truncated periwinkle geraniol synthase (tCrGES) and geraniol pyrophosphate (GPP) synthase ERG20ww (ERG20) were also discussed. F96W-N127W ) respectively in GAL1 Fusion expression under the control of promoter or pC loop ( Figure 3 B). mvaE, mvaS, IDI1, ERG19 / 8 / 12 Key genes in the mevalonate pathway are GAL1 or GAL10 Promoter control activates these genes through cascaded amplifier circuits to enhance precursor supply. For example... Figure 3 As shown in C, compared to using alone GAL Compared to promoter-induced induction, the introduction of the pC circuit increased the geraniol titer by 2.1 times (to 173 mg / L), while the cascaded amplifier circuit further increased the geraniol titer by 3.0 times, to 242 mg / L. Figure 3 (C), and its impact on growth is relatively small (Figure 3D). The results indicate that the signal amplification circuit provides an effective strategy for the regulation of multi-gene expression.

[0071] 4. Multidimensional dynamic regulation of metabolic pathways Besides gene activation, QS-dependent dynamic inhibition is also crucial for mitigating the competition between cell growth and product formation. To achieve this regulatory modality, we further designed a signal conversion system that can convert activation signals into inhibition signals. We combined the pC loop, or the aforementioned cascaded amplifier, with our previously constructed protein scaffold-mediated CRISPR interference (CRISPRi) system. GAL1 The signal-transition circuit (Figure 4A) or the 6×RpaO promoter (pC-CRISPRi-Circuit) is used. Figure 10 A ribozyme-mediated crRNA expression cassette was constructed under the control of [the specific technology / mechanism]. For example... Figure 4 A and Figure 10 As shown in Figure B, both QS-based CRISPRi designs effectively suppressed GFP expression, reducing fluorescence intensity by 72% and 64.6%, respectively, compared to the control group. Unlike constitutive expression, the QS-based regulatory system achieved tunable, population-density-dependent suppression. In summary, we established a QS-based... p A bifunctional regulatory system for C-HSL signaling, supporting both transcriptional activation and repression. This platform provides a means to fine-tune metabolic pathways in response to population density, paving the way for more complex dynamic regulatory strategies in yeast.

[0072] To verify p To demonstrate the versatility and application potential of the C-HSL cascade system, we employed signal amplification and signal conversion circuits to dynamically regulate the biosynthesis of 3-hydroxypropionic acid (3-HP) as a proof-of-concept. Figure 4 B). 3-HP uses malonyl-CoA as a precursor, derived from... Chloroflexus aurantiacus The bifunctional enzyme MCR catalyzes synthesis. We compared the synthesis when... MCR-N and MCR-C respectively by GAL The production capacity of the strain under promoter-driven or signal amplification circuit control ( Figure 4 B). Our results show that, compared to [the previous method], GAL1 / 10 Regarding the promoter control, the signal amplification circuit increased the yield of 3-HP by 3.1 times (Figure 4C). It is noteworthy that the signal amplification system gradually accumulates after 24 hours. p C-HSL triggers pathway activation, thereby autonomously delaying the start of production and effectively separating cell growth from product synthesis (Figure 4C-D).

[0073] To further increase 3-HP yield, we addressed the trade-off between precursor allocation and biomass synthesis. Malonyl-CoA is a direct precursor of 3-HP and is essential for fatty acid biosynthesis and cell membrane formation. While inhibiting fatty acid biosynthesis can increase 3-HP yield, compositional inhibition negatively impacts cell viability. We hypothesized that a population density-dependent inhibition strategy could mitigate this issue. To verify this, we utilized signal transduction circuitry to control targeting. FAS1Transcription of the Hammerhead ribozyme-crFAS1-HDV ribozyme. In utilizing... SN52 promoter constitutive expression crFAS1 Continuous inhibition FAS1 In this case, cell growth and 3-HP production were reduced. In contrast, the signal conversion circuit... FAS1 Population density-dependent suppression increased 3-HP titers by 34.1% ( Figure 4 E). Importantly, we observed that strains containing a population density-dependent inhibition system maintained a growth rate comparable to strains containing only signal amplification circuits. Figure 4 F). By simultaneously activating 3-HP biosynthesis and inhibiting the competing pathway, the total yield of 3-HP increased by 4.1 times.

[0074] To evaluate the robustness and versatility of the system, we further applied this bifunctional cascade system to previously reported high-yield 3-HP strains ( Figure 5 Against this backdrop, the MCR variant MCR-C, regulated by a QS-driven signal amplification loop, is... N941VK1107WS1115R The expression of this enzyme increased the yield of 3-HP by 25.2%, reaching 3.98 g / L. Figure 5 B), while the yield was only 1.57 g / L when using only the GAL1 promoter for regulation. After implementing additional dynamic repression of the FAS1 gene through the signal transduction circuit, the 3-HP yield was further increased by 30.2%, reaching 4.32 g / L (B). Figure 5 B). These results demonstrate that the bifunctional cascaded QS loop has broad applicability to producing strains and provides a robust population response framework for the multidimensional dynamic regulation of yeast metabolic pathways.

[0075] Table 3. Structure and sequence of Gal-cRNA

[0076] Table 4. DNA Sequence

[0077] Note: RpaO sequences are shown in blue, Gal1 cores are shown in red, and GFP sequences are shown in green.

[0078]

[0079] Note: RPAR sequences are shown in blue, the activation domain (AD) of Gal4 is marked in red, and the linkage sequence is shown in black.

[0080] Table 5. Protein Sequences

[0081] References: 1. Chen, Y. et al. (2013) Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng 15, 48-54. 10.1016 / j.ymben.2012.11.002 2. Zhang, GC et al. (2014) Construction of a quadruple auxotrophicmutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. Appl Environ Microbiol 80, 7694-7701. 10.1128 / aem.02310-14 3. Zhai, H. et al. (2022) CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in Saccharomyces cerevisiae. Nucleic Acids Res 50, 5988-6000. 10.1093 / nar / gkac463 4. Qin, N. et al. (2024) Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast. Nature Communications 15.10.1038 / s41467-024-45557-9 The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A yeast quorum sensing dynamic control system, characterized in that, The yeast quorum sensing dynamic regulation system includes at least an RpaI / RpaR quorum sensing system; the RpaI / RpaR quorum sensing system includes a signal molecule synthesis module and a signal response module; wherein, the signal molecule synthesis module is capable of synthesizing and secreting signal molecules. p C-HSL; the signal response module includes at least one fusion protein RpaR-AD and its recognized specific DNA-binding sequence RpaO.

2. The yeast quorum sensing dynamic control system as described in claim 1, characterized in that, The number of copies of RpaO is one or more, and more specifically 1 to 6; Use promoters of different strengths for control RpaR-AD Transcription, the promoter including but not limited to TEF1p, LEU1p, CYC1p and REV1p Preferred CYC1p .

3. The yeast quorum sensing dynamic control system as described in claim 1, characterized in that, The signal molecule synthesis module contains at least the genes encoding tyrosine ammonia-lyase (TAL), coumaroyl-CoA ligase (4CL), and RpaI synthase (RpaI).

4. The yeast quorum sensing dynamic control system as described in claim 1, characterized in that, The yeast quorum sensing dynamic regulation system also includes Gal4 or other transcription factors, thereby amplifying transcriptional activation signals; Furthermore, the yeast quorum sensing dynamic regulation system also includes CRISPRi, which converts transcriptional activation signals into transcriptional repression signals.

5. A recombinant cell, characterized in that, The recombinant cells comprise at least the yeast quorum sensing dynamic regulation system as described in any one of claims 1-4; The recombinant cells are eukaryotic cells, further comprising fungal cells, wherein the fungi are molds and yeasts; wherein the molds include Aspergillus flavus (…). Aspergillus flavus Aspergillus niger ( ) Aspergillus niger ) and Trichoderma reesei ( Trichoderma reesei The yeast includes Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Max Kluyveromycin ( Kluyveromyces marxianus Pichia pastoris () Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ) and Debali yeast ( Debaryomyces hansenii ).

6. A method for dynamic regulation of yeast quorum sensing, characterized in that, The method includes at least introducing the yeast quorum sensing dynamic regulation system according to any one of claims 1-4 into the cell.

7. The method as described in claim 6, characterized in that, The cells are eukaryotic cells, and more specifically, fungal cells, wherein the fungi are molds and yeasts; wherein the molds include Aspergillus flavus (…). Aspergillus flavus Aspergillus niger ( ) Aspergillus niger ) and Trichoderma reesei ( Trichoderma reesei The yeast includes Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Max Kluyveromycin ( Kluyveromyces marxianus Pichia pastoris () Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ) and Debali yeast ( Debaryomyces hansenii ).

8. The use of the yeast quorum sensing dynamic regulation system according to any one of claims 1-4, the recombinant cell according to claim 5, or the method according to any one of claims 6-7 in any one or more of the following: (a) Balancing cell growth and metabolite synthesis; (b) Increase the production of metabolites.

9. The application as described in claim 8, characterized in that, The metabolites include nucleoside antibiotics, terpenoids, and bio-based platform compounds.

10. The application as described in claim 9, characterized in that, The nucleoside antibiotic is cordycepin; the terpene compound is geraniol; and the bio-based platform compound is 3-hydroxypropionic acid.

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