A combined optimized optogenetic system with near-infrared light response

Abstract

The application discloses a mutant of a photosensitive protein BphP1. The application also provides a kind of optogenetic system, which comprises the fusion protein of the BphP1 mutant and the transcription activation domain (AD) of Gal4, the fusion protein of the photosensitive protein PpsR2 and the DNA binding domain (DBD) of Gal4, and the expression vector for expressing target genes through Gal1 promoter, wherein the PpsR2 can be wild type truncated variant Q-PpsR2. The induction intensity of the optimized optogenetic system of the application is 2.99 times that of the original light induction system.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and genetic engineering, and specifically relates to a near-infrared light-responsive combinatorial optimized optogenetic system. Background Technology

[0002] Saccharomyces cerevisiae is an excellent model microbial strain in the field of synthetic biology, possessing significant advantages such as a clear genetic background, short growth cycle, low culture cost, and the ability to modify proteins after secretion. It is currently widely used as a cell factory for the production of high-value products such as ethanol, flavonoids, lipids, and terpenes.

[0003] However, when heterologous genes are highly expressed in Saccharomyces cerevisiae, two major problems arise: firstly, it significantly increases the metabolic burden on the cells; secondly, the resulting product may be biotoxic, thus inhibiting the growth of Saccharomyces cerevisiae. In existing technologies, researchers typically use chemical inducers or temperature induction to regulate the induction process of Saccharomyces cerevisiae in an attempt to circumvent these problems. However, both methods have significant limitations: chemical inducer induction suffers from strong dose dependence, insufficient spatiotemporal resolution, and difficulty in removing the inducer, making precise and residue-free regulation impossible; while temperature induction solves the problems of difficult inducer removal and dose dependence, it still severely interferes with cell growth and is costly, making it unsuitable for large-scale applications.

[0004] Optogenetics is a novel biotechnology that integrates optics and genetics. Its core principle is to utilize natural photoreceptors and couple them to transcription factors through gene editing, thereby achieving precise regulation of target gene expression using light of specific wavelengths. Based on this principle, photoinducible systems with high spatiotemporal resolution, minimal cell damage, rapid response, and low cost can be constructed, providing a new direction for solving the challenges of induction regulation in Saccharomyces cerevisiae.

[0005] Currently, the reported photoinduction systems in Saccharomyces cerevisiae are mainly derived from natural photoreceptors such as cryptochromes or LOV domains, and both of these systems are photoinduction systems that respond to blue light. However, blue light has the characteristics of short wavelength and high photon energy density, which makes Saccharomyces cerevisiae prone to photo-oxidative damage during blue light irradiation, thus affecting the normal growth of the cells and failing to meet the requirements for efficient and low-damage induction regulation. Summary of the Invention

[0006] In view of the aforementioned deficiencies in existing Saccharomyces cerevisiae induction and regulation technologies, this application proposes to address these issues through the following technical approach: First, based on its strong penetrability and low photo-oxidative damage, Bphp1-PpsR2 is selected and identified as the natural photoreceptor for constructing a photoinduction system; second, using fission-type transcription factors, a fusion protein is constructed by combining the aforementioned natural photosensitive protein pair with the fragments of the fused transcription factor, thereby constructing a near-infrared light-mediated photoinduction system to achieve precise regulation of target gene expression in response to near-infrared light; simultaneously, by mutating key sites of the photosensitive protein chaperone and reconstructing the photosensitive protein domain, the interaction between related proteins is enhanced, further optimizing the regulatory efficiency of the photoinduction system.

[0007] Therefore, according to one aspect of the present invention, a mutant of the photosensitive protein BphP1 is provided, which differs from wild-type BphP1 in that it contains the mutant N499K or N499A, preferably N499K.

[0008] In a preferred embodiment, the wild-type BphP1 is derived from Rhodopseudomonas palustris (… Rhodopseudomonas palustris Its amino acid sequence is shown in SEQ ID No. 1, and its gene coding sequence is shown in SEQ ID No. 70.

[0009] Therefore, in a particularly preferred embodiment, the amino acid sequence of the mutant of the photosensitive protein BphP1 is shown in SEQ ID No. 2, and its gene coding sequence is shown in SEQ ID No. 71.

[0010] According to another aspect of the present invention, an optogenetic system is provided, comprising:

[0011] 1) A fusion protein A of the mutant according to the invention and the transcriptional activation domain (AD) of yeast cell transcription factor Gal4, or a vector expressing said fusion protein A, wherein a linker is optionally included between the mutant and AD; and

[0012] 2) A fusion protein B of the light-sensitive protein PpsR2 and the DNA-binding domain (DBD) of the yeast cell transcription factor Gal4, or a vector expressing said fusion protein B, wherein a linker is optionally included between said PpsR2 and DBD.

[0013] In a preferred embodiment, the gene sequence of the transcriptional activation domain (AD) of Gal4 is shown in SEQ ID No. 6.

[0014] In a preferred embodiment, the gene sequence of the DNA binding domain (DBD) of Gal4 is shown in SEQ ID No. 7.

[0015] In a preferred embodiment, the sequence of the connector is: ggggggggggggtca (SEQ ID No. 5).

[0016] In a preferred embodiment, the optogenetic system further comprises an expression vector for expressing a target gene, the expression vector containing a yeast cell Gal1 promoter operatively linked to the target gene, the Gal1 promoter including an upstream activation sequence (UAS) capable of binding to the yeast cell transcription factor Gal4.

[0017] In a preferred embodiment, the sequence of the Gal1 promoter is shown in SEQ ID No. 8.

[0018] In another preferred embodiment, the yeast cell Gal1 promoter is also operatively linked to a gene encoding a tag protein selected from mCherry, TrxA, MBP, DsRed.T3, or GFP, wherein TrxA represents thioredoxin A; MBP represents maltose-binding protein; and DsRed.T3 is a red fluorescent protein derived from... Discosoma sp. GFP stands for Green Fluorescent Protein; mCherry is a red fluorescent protein derived from coral.

[0019] In a preferred embodiment, the gene sequence of the mCherry is shown in SEQ ID No. 9.

[0020] In a preferred embodiment, the expression vector further comprises a GAL1 terminator, preferably having the sequence shown in SEQ ID No. 10.

[0021] In another preferred embodiment, the photosensitive protein PpsR2 is derived from wild-type PpsR2 or a truncated form of Rhodopseudomonas palustris, the amino acid sequence of which is shown in SEQ ID No. 3, its gene coding sequence is shown in SEQ ID No. 72, and the amino acid sequence of which is shown in SEQ ID No. 4.

[0022] In another preferred embodiment, the optogenetic system is responsive to 740 nm light induction.

[0023] According to another aspect of the present invention, the present invention provides a method for regulating gene expression in host cells, the method comprising:

[0024] 1) Provide host cells;

[0025] 2) Introducing the optogenetic system according to the present invention into the host cell; and

[0026] 3) The optogenetic system is induced with 740 nm light to regulate gene expression. In a preferred embodiment, the gene is the target gene on the expression vector.

[0027] In a preferred embodiment, the method is a non-disease treatment and diagnostic method.

[0028] In a preferred embodiment, the host cell is *Saccharomyces cerevisiae*.

[0029] The technical effects achieved by this invention are as follows:

[0030] By using the mutant Bphp1 of the photosensitive protein BphP1 of the present invention alone N499K It can increase the induction intensity of the photoinduction system to 1.41 times that of the wild-type strain (see...). Figure 4 However, by using the wild-type truncated variant Q-PpsR2 alone, the induction intensity of the photoinduction system was increased to 1.81 times that of the wild-type strain (see [link]). Figure 7 When Bphp1 N499K When used in combination with Q-PpsR2, the two have a synergistic effect, increasing the induction intensity of the photoinduction system to 2.99 times that of the wild-type strain (see [link]). Figure 8 ). Attached Figure Description

[0031] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0032] Figure 1 This is a schematic diagram of the construction of the expression box in the light-induced system;

[0033] Figure 2 This is a schematic diagram of the Bphp1-PpsR2 docking structure model;

[0034] Figure 3 This is the result of red fluorescence intensity in the Bphp1 alanine-scanning mutant strain;

[0035] Figure 4 These are the results of red fluorescence intensity from random mutant strains of Bphp1;

[0036] Figure 5 These are the domain prediction results from the InterPro and SMART databases;

[0037] Figure 6 The results show the red fluorescence intensity of the mutant strain with the PpsR2 domain removed.

[0038] Figure 7The results show the red fluorescence intensity of the mutant strain with the PpsR2 combinatorial domain.

[0039] Figure 8 The results show the red fluorescence intensity of the mutant strain modified by combining Bphp1 and PpsR2.

[0040] Figure 9 It is the optimized Bphp1 N499K Plasmid map of a new optogenetic system constructed by combining mutants with Q-PpsR2 reconstructors. Detailed Implementation

[0041] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0042] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0043] In this invention, the terms "comprising" or "including" are open-ended expressions, referring to a specific component or step described, without excluding other components or steps that do not substantially affect the meaning. When describing protein or nucleotide sequences, the sequences may constitute the target molecule on their own, or may have additional amino acids or nucleotides added to one or both ends, or may be engineered to resemble proteins, while retaining the functional activity described in this invention.

[0044] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0045] This invention aims to provide a near-infrared light-regulated optogenetic system. By modifying the Gal1 promoter through a transcription factor splitting strategy and combining protein mutation and domain reconstruction optimization, the system's induction intensity and regulatory range are significantly improved. This system can precisely regulate downstream gene expression through infrared light, solving the problems of low induction efficiency and narrow regulatory range in existing optogenetic systems.

[0046] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0047] Materials and methods

[0048] 1. PCR preparation of the target fragment

[0049] Primers were designed based on the gene sequence of the target fragment and synthesized by Suzhou Genewiz Co., Ltd. Using the corresponding plasmid or genome as a template, the reaction system was prepared according to the instructions of the polymerase used, and the PCR temperature program was set according to the instructions. PCR products were detected by agarose gel electrophoresis, and the correctly sized product bands were excised and recovered.

[0050] 2. Gibson Assembly

[0051] Add the backbone with homologous ends and the purified PCR product to one tube of Master Mix. Immediately after addition, gently rinse 5-10 times with a pipette, then place the reaction solution in a 50°C metal bath for 15-60 minutes. After the time is up, immediately remove the reaction solution and place it on ice for use in the transformation of competent cells; alternatively, it can be temporarily stored in a -20°C freezer. See Table 1 for details of the Gibson assembly system.

[0052]

[0053] 3. Chemical transformation of Escherichia coli

[0054] (1) Thaw competent cells stored at -80℃ on ice.

[0055] (2) When the competent cells are thawed to 2 / 3 thawed, add Gibson assembly product or plasmid (100-300 ng), mix gently, and then place in an ice bath for 30 minutes, avoiding shaking.

[0056] (3) After heating the reaction mixture in a 42°C water bath for 60-90 seconds, immediately transfer it to ice for a 2-minute ice bath. Do not shake during the process.

[0057] (4) Add 1 mL of antibiotic-free LB medium and incubate at 37°C and 200 rpm for 1 hour.

[0058] (5) Centrifuge at 4000 rpm for 2 minutes, remove 1 mL of supernatant and resuspend, and take an appropriate amount to spread on LB screening plates containing the corresponding resistance.

[0059] (6) Place the plate in a 37°C incubator overnight for incubation.

[0060] 4. Electroporation of Saccharomyces cerevisiae

[0061] (1) Streak the brewing yeast on YPD plates (or SD nutrient-deficient plates) and incubate overnight at 30°C.

[0062] (2) On the second day, select single colonies from the plate and inoculate them into 4 mL of YPD medium (or SD nutrient-deficient medium) and incubate overnight at 30°C and 200 rpm.

[0063] (3) Based on the OD of seed liquid 600 Value, based on initial OD 600 To obtain 0.1, the seed culture was transferred to 4 mL of YPD (or SD nutrient-deficient medium) liquid medium and cultured at 30°C and 200 rpm in a shaker until OD reached 0.1. 600 It is 0.6-0.8.

[0064] (4) Transfer the bacterial solution to a 1.5 mL sterile centrifuge tube in a clean bench, centrifuge at 5000 rpm and 4℃ for 5 min, and discard the supernatant.

[0065] (5) Transfer the bacterial culture to a 1.5 mL sterile centrifuge tube, centrifuge at 5000 rpm and 4℃ for 5 min, and discard the supernatant.

[0066] (6) Add 250 μL of cooled 1 M sorbitol, centrifuge at 5000 rpm and 4℃ for 5 minutes, and discard the supernatant. Then wash again with 1 M sorbitol.

[0067] (7) Add 100 μL of cooled 1 M sorbitol and dispense 50 μL into each of pre-cooled sterile 1.5 mL centrifuge tubes to obtain the prepared competent cells of Saccharomyces cerevisiae.

[0068] (8) Add less than 5 μL of DNA fragments or plasmids to 50 μL of competent cells of Saccharomyces cerevisiae, and transfer them together to an electric transfer cup (0.2 cm) using a pipette, taking care to avoid the generation of air bubbles.

[0069] (9) Set the electro-excitation instrument program to: 1.5 kV, 200 W, 25 μF (5 ms), and click the electro-excitation button to complete the electro-excitation process.

[0070] (10) Immediately after electroporation, inject 1 mL of YPD medium into the electroporation cup, and use a pipette to transfer the electroporated competent cells to a 1.5 mL centrifuge tube. Recover the cells at 30°C and 200 rpm for 1 hour.

[0071] (11) After revival, the cells were centrifuged at 5000 rpm for 2 minutes to collect the cells. The supernatant was discarded until 40 μL remained. The cells were then evenly spread on SD solid plates containing the corresponding defect type. The plates were inverted and incubated in a 30°C incubator for 2-3 days until a single colony was formed.

[0072] 5. Single-point mutation steps

[0073] (1) Primer design

[0074] The primer consists of two parts: a 5' overlap region and a 3' extension region. Each primer is approximately 25 to 30 nucleotides long, with the 5' overlap region consisting of about 15 to 20 bases and the 3' extension region containing at least ten bases. The two mutation sites are located on the two primers, one downstream of the overlap region of the forward mutation primer, adjacent to the overlap region, and the other at the 5' end of the reverse mutation primer.

[0075] (2) PCR system

[0076]

[0077] (3) Single-point mutation RCR program

[0078]

[0079] (4) Electrophoresis detection: Take 10 μL of PCR product and detect it by 1% agarose gel electrophoresis.

[0080] (5) PCR product digestion: Add 1 μL of FastDigest DpnI enzyme to the PCR product, mix well, and incubate at 37°C for 1 h.

[0081] (6) E. coli transformation, bacterial P verification and sequencing.

[0082] 6. Near-infrared induction

[0083] (1) Equipment deployment

[0084] Connect the positive and negative terminals of a 2 m long 24 W / h near-infrared LED strip to the relay, then connect the relay to a 220V-12V transformer. Connect the two contacts of the transformer to the power supply line to complete the assembly of the near-infrared generator. Securely fix the infrared LED strip to the edge of the spring on the shaker stand using metal paperclips; at this point, the illumination distance is approximately 27 mm.

[0085] (2) Induction conditions

[0086] Turn on the near-infrared generator in a 30℃, 200 rpm shaker, keep the light strip coiled and fixed on the shaker support at equal intervals, and perform light induction for 15 minutes as needed.

[0087] 7. Gene Synthesis

[0088] Gene synthesis and vector construction: The Bphp1-PpsR2 gene is derived from Rhodopseudomonas palustris ( Rhodopseudomonas palustris Wild-type Bphp1 gene (SEQ ID No. 70) and wild-type PpsR2 gene (SEQ ID No. 72) were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. The synthesized gene fragments were cloned into the universal vector pRS413 plasmid (Jiutian Gene Technology (Tianjin) Co., Ltd.) to construct recombinant vectors pRS413-Bphp1 and pRS413-PpsR2. Using these recombinant vectors as templates, sufficient optimized Bphp1 and PpsR2 gene fragments were obtained by PCR amplification. The polymerase used for PCR was Phanta DNA Polymerase (a commercially available high-fidelity polymerase).

[0089] 8. Homology modeling and molecular docking of Bphp1 and PpsR2 proteins

[0090] 8.1 Homologous Modeling

[0091] 8.1.1 Homology Modeling of Bphp1: Using the PDB structure of the bacterial plant pigment photostimulated protomer protein (PDB ID: 4gw9.1.A) as a reference template, the three-dimensional structure of the Bphp1 protein was constructed using the homology modeling method. The model quality was evaluated by Seq Identity (amino acid sequence similarity) and GMQE (global quality assessment). The results showed that the Seq Identity of the model was 91.68% and the GMQE was 0.84.

[0092] 8.1.2 PpsR2 Homology Modeling: Since the Seq Identity of other candidate templates is less than 30% (not meeting the reliable modeling threshold), the protein structure corresponding to the UniProtKB accession number A0A495V416.1 predicted by AlphaFold 2 (labeled as A0A495V416.1.A) was selected as the reference template; the evaluation after modeling showed that the Seq Identity of the model was 90.41% and the GMQE was 0.80.

[0093] 8.1.3 Model reliability standard: It is generally accepted in the field that when the Seq Identity is greater than 30%, the homology modeling structure has high reliability; the GMQE score ranges from 0 to 1, and the higher the score, the higher the consistency between the model and the real structure. In this embodiment, the GMQE of both protein models is greater than 0.8, which meets the requirements of subsequent analysis.

[0094] 8.2 Molecular docking and results analysis

[0095] 8.2.1 Docking tools and parameters: GRAMM Docking Web Server (https: / / gramm.compbio.ku.edu / ) and Autodock Vina 1.1.2 software were used for Bphp1-PpsR2 protein-protein molecular docking, and the docking parameters were set to default.

[0096] 8.2.2 Screening of docking results: The conformation with the highest confidence level was selected from the docking results and its structure was compared and verified in the PDB protein docking result database. Finally, the conformation with the lowest binding energy after docking was selected as the core research object.

[0097] 8.2.3 Visualization and Interaction Analysis: Pymol software was used to perform visualization analysis on the screened docking conformations.

[0098] 9. Construction and Validation of Bphp1 Mutant Plasmid

[0099] 9.1 Construction of Bphp1 single-point mutant plasmid

[0100] 9.1.1 Primer design: Based on the Bphp1 gene sequence, single-point mutation primers targeting the target amino acid sites were designed.

[0101] 9.1.2 Circular PCR Amplification: Using the recombinant vector pRS413-Bphp1 as a template, circular PCR (Loop-PCR) was performed using the above-mentioned mutant primers to introduce the target amino acid mutation; the PCR reaction system was 50 μL, containing 100 ng of template plasmid, 0.5 μmol / L each of forward and reverse primers, 200 μmol / L of dNTP mixture, and 1 U of Phanta DNA Polymerase.

[0102] 9.1.3 Plasmid Digestion and Transformation: Circular PCR products were digested with DpnI restriction endonuclease and incubated at 37°C for 1 h to remove the template plasmid; the digested product was then transformed into plasmid using chemical transformation. E. coli In DH5α competent cells (Beijing Qingke Biotechnology Co., Ltd.), the cells were incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, incubated on ice for 2 min, and then 800 μL of LB liquid culture medium was added. The cells were then thawed at 37℃ and 200 rpm for 1 h.

[0103] 9.1.4 Screening and identification of positive clones: The revived bacterial culture was plated on LB-Amp solid medium containing 1‰ (w / v) ampicillin and incubated upside down at 37℃ for 18-24 h; single colonies were picked and streaked onto fresh LB-Amp solid medium and incubated at 37℃ for 12 h. Positive clones were verified by colony PCR (using universal primers for the vector); 1-2 positive clones were selected and inoculated into LB-Amp liquid medium containing 1‰ ampicillin and incubated at 37℃ and 200 rpm for at least 8 h. Plasmids were extracted using a plasmid extraction kit and sequenced for verification by Suzhou Genewiz Biotechnology Co., Ltd. The plasmids of each Bphp1 mutant with the correct sequence were screened and named pRS413-Bphp1*.

[0104] 9.2 Transformation and Functional Detection of Mutant Plasmids in Saccharomyces cerevisiae

[0105] 9.2.1 Yeast transformation: The pRS413-Bphp1* plasmid, which was verified by sequencing, was transformed into Saccharomyces cerevisiae BY4741 competent cells by electroporation (Brachmann CB, Davies A, Cost GJ, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications[J]. Yeast, 1998, 14(2): 115-132.).

[0106] 9.2.2 Screening of positive yeast: The revived bacterial culture was spread on histidine-deficient SD-ΔHIS solid medium and cultured at 30℃ for 2-3 days; single colonies were picked and streaked onto new SD-ΔHIS solid medium and cultured for 12 h. Positive clones were verified by colony PCR, and 21 Saccharomyces cerevisiae strains containing the Bphp1 mutant were finally obtained.

[0107] 9.2.3 Functional assay: Positive yeast strains were inoculated into 4 mL of SD-ΔHIS liquid medium and cultured at 30℃ and 200 rpm for 24 h; 1 mL of seed culture was transferred to a 100 mL Erlenmeyer flask containing 20 mL of SD-ΔHIS liquid medium and cultured at 30℃ and 200 rpm for 48 h; after sampling, the samples were induced with 740 nm wavelength light for 15 min, and the fluorescence intensity was measured using a microplate reader to analyze the photoresponse activity of the mutants.

[0108] 9.3 Construction and Screening of Bphp1 Random Saturation Mutants

[0109] 9.3.1 Design of saturation mutation primers: Single-point mutation primers containing NNN degenerate codons (NNN covers all 20 amino acids) were designed to construct a library of random saturation mutants of Bphp1.

[0110] 9.3.2 Plasmid construction and transformation: Using pRS413-Bphp1 as a template, random mutations were introduced by circular PCR. After DpnI digestion for 1 h, the product was transformed into Saccharomyces cerevisiae BY4741 competent cells by electroporation. After recovery, the cells were plated on SD-ΔHIS solid medium and cultured at 30℃ for 2-3 days.

[0111] 9.3.3 Screening and activity detection of positive clones: Single colonies were streaked and preserved, and positive clones were verified by colony PCR; the positive strains were cultured under the culture conditions of "9.2.3" and induced by 740nm light, and the fluorescence intensity was measured; 5 mutant strains with significantly increased fluorescence intensity compared with wild type (positive effect) were screened, and Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to sequence them to determine the specific mutation sites.

[0112] 10. PpsR2 Domain Prediction and Truncation Verification

[0113] 10.1 Domain Prediction

[0114] The domains and functional annotations of PpsR2 were predicted using the InterPro database (http: / / www.ebi.ac.uk / interpro / ) and the SMART database (https: / / smart.embl.de / ). The results showed that both databases predicted that PpsR2 contains two PAS domains (PAS domains are universal signal sensing modules that can respond to oxygen tension, redox potential, or light intensity and mediate protein-protein interactions), but there were differences in the prediction results for the CC domain (coil-coil domain) and the HTH domain (helix-turn-helix domain).

[0115] 10.2 Construction of PpsR2 truncated somatic plasmids

[0116] 10.2.1 Amplification of truncated fragments: Based on the domain prediction results, truncated primers targeting different domains were designed. Using pRS413-PpsR2 as a template, the truncated PpsR2 gene fragment was amplified with Phanta DNA Polymerase, while the pRS413 plasmid backbone was amplified by circular PCR.

[0117] 10.2.2 Vector Assembly and Transformation: The truncated gene fragment was ligated to the pRS413 plasmid backbone using the Gibson assembly method to construct a series of truncated somatic plasmids (named pRS413-ΔPpsR2); the assembly product was then transformed into vectors using chemical transformation. E. coli DH5α competent cells were used to screen for positive clones and extract plasmids. Sequencing was then used to verify the correctness of fragment insertion.

[0118] 10.2.3 Transformation and Functional Validation of Truncated Yeast

[0119] The correctly sequenced pRS413-ΔPpsR2 plasmid was transformed into Saccharomyces cerevisiae BY4741 to obtain a yeast strain containing a truncated PpsR2. The strain was cultured and treated with 740 nm light induction for 15 min and natural light exposure for 15 min, respectively. The fluorescence intensity was measured to verify the effect of different domains on PpsR2 function and to determine the core functional domain.

[0120] 10.3 PpsR2 Refactoring and Functional Verification

[0121] 10.3.1 Design and amplification of reconstructed fragments: Based on the truncation validation results, two functional PAS domains and one HTH domain were selected for combination. Specific primers were designed to amplify each domain fragment and perform overlap extension PCR (OE-PCR) ligation to obtain the reconstructed PpsR2 gene (RePpsR2); at the same time, the pRS413 plasmid backbone was amplified by circular PCR.

[0122] 10.3.2 Construction of recombinant plasmid: The RePpsR2 gene was ligated to the pRS413 plasmid backbone using the Gibson assembly method to construct the recombinant plasmid pRS413-RePpsR2; it was transformed into E. coli DH5α and positive clones were screened and sequenced for verification.

[0123] 10.3.3 Yeast Transformation and Functional Detection: The pRS413-RePpsR2 plasmid was transformed into Saccharomyces cerevisiae BY4741 to obtain a yeast strain containing reconstructed PpsR2; light-induced treatment and fluorescence intensity measurement were performed to verify the light response function of reconstructed PpsR2 and screen out the reconstructed strain with the best function.

[0124] 11. Assembly and functional verification of the light-induced system

[0125] 11.1 System Component Expansion

[0126] Amplification of Q-PpsR2 gene and Bphp1 N499K Gene fragments.

[0127] 11.2 Construction of plasmids for the photoinduced system

[0128] The pRS413 plasmid backbone was amplified by circular PCR, and all the above elements (Bphp1) were assembled using the Gibson assembly method. N499K The recombinant plasmid was constructed by linking Q-PpsR2, promoter, terminator, Gal4 AD / DBD, and mCherry with the pRS413 backbone and transforming it. E. coli DH5α was used to screen for positive clones, and sequencing was performed to verify the correctness of element assembly. See also Figure 9 .

[0129] 11.3 Systemic yeast transformation and functional validation

[0130] The recombinant plasmid was transformed into Saccharomyces cerevisiae BY4741 to obtain a yeast strain containing a complete photoinduction system. The strain was cultured and induced by 740 nm light for 15 min. The mCherry fluorescence intensity was measured to prove that the photoinduction system constructed in this invention can respond efficiently to 740 nm light signals and initiate reporter gene expression.

[0131] Example 1: System Strength Optimization Based on Bphp1 Mutation

[0132] This invention constructs a light-induced gene expression system and a method for detecting its expression level using a transcription factor splitting method. The specific steps are as follows:

[0133] Fusion protein construction: The transcriptional activation domain (AD, encoding gene as shown in SEQ ID No. 6) of the transcription factor Gal4 of the Gal1 gene was fused with the light-sensitive protein Bphp1 (SEQ ID No. 1) via a linker to obtain the Bphp1-AD fusion protein; the DNA binding domain (DBD, encoding gene as shown in SEQ ID No. 7) of Gal4 was fused with the light-sensitive protein PpsR2 (SEQ ID No. 3) via a linker to obtain the PpsR2-DBD fusion protein. The linker sequences were all (5'→3'): ggggggggggggtca (SEQ ID No. 5). The expression plasmid for the fusion protein was pRS413 (Jiutian Gene Technology (Tianjin) Co., Ltd.), and the host was Saccharomyces cerevisiae BY4741 (Brachmann CB, Davies A, Cost GJ, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: auseful set of strains and plasmids for PCR-mediated gene disruption and other applications[J]. Yeast, 1998, 14(2): 115-132.).

[0134] Construction of the expression vector for the target gene: The Gal1 promoter (SEQ ID No. 8) was operatively linked with the tag protein mCherry (encoding gene SEQ ID No. 9) and the GAL1 terminator (SEQ ID No. 10) and integrated into the expression plasmid.

[0135] The above expression plasmid was introduced into Saccharomyces cerevisiae BY4741 to obtain the original NLS system strain.

[0136] Regulation under non-light conditions: Under non-light conditions, the PpsR2-DBD fusion protein is active only in the cell nucleus; at this time, the Gal4 transcription factor can bind to the UAS (upstream activation sequence) of the Gal1 promoter, but cannot initiate transcription, and the downstream gene is in a silent state because the AD and DBD are separated.

[0137] Regulation under illumination: When the system receives 740 nm infrared light, Bphp1-AD undergoes a conformational change mediated by the chromophore, exposing the nuclear localization sequence (NLS). This sequence guides Bphp1-AD into the nucleus, where it binds to the PpsR2-DBD fusion protein. AD, linked by a flexible peptide, synergistically performs the full function of the Gal4 transcription factor, specifically binding to the UAS region of the Gal1 promoter and activating downstream gene expression (see [link to relevant documentation]). Figure 1 The expression of downstream genes can be determined by measuring the fluorescence intensity of tag proteins such as mCherry, as described above.

[0138] To enhance the binding affinity between Bphp1 and PpsR2 (both are chaperone proteins, and their interaction is crucial for system activation), this invention optimizes Bphp1 through amino acid mutation screening. The specific scheme is as follows:

[0139] Mutation site screening: Homology modeling was performed on Bphp1 and PpsR2 to analyze their protein-protein interaction patterns. Based on the interaction forces and the amino acid distribution within 4 Å after docking, 17 candidate amino acid sites were screened (SER47, VAL48, GLY49, SER52, ASP59, ASP62, SER63, PHE68, GLN70, GLN110, THR111, GLN253, ARG326, ARG366, LEU415, ​​ARG417, ASN499) (Reference Figure 2 ).

[0140] Alanine scanning mutation verification: Alanine scanning mutations were performed on the above 21 sites. The primers used for mutation are shown in SEQ ID Nos. 11-44. Fluorescence intensity was detected using the original photoinduction system (denoted as the NLS system) as a control: the fluorescence intensity of mutants THR111 (T111), ASP62 (D62), ASN499 (N499), and ASP253 (D253) was higher than that of the control, among which Bphp1 N499AThe mutant strain exhibited the best photoinduction intensity, 1.34 times that of the NLS system strains. The fluorescence intensity of mutants ARG366 (R366) and ARG417 (R417) was lower than that of the control; the fluorescence intensity of the remaining mutants showed no significant difference from the control (reference). Figure 3 ).

[0141] Saturation mutation screening for optimal sites: Saturation mutations were performed on the above 6 differentially expressed sites, and the results showed that Bphp1 N499K The mutant (i.e., ASN499 mutated to LYS, sequence shown in SEQ ID No. 2) exhibited the best photoinduction intensity, with an induction intensity 1.41 times that of the NLS system strain (reference). Figure 4 ).

[0142] Example 2: System Strength Optimization Based on PpsR2 Domain Reconstruction

[0143] To reduce spatial steric hindrance and further improve system strength, this invention reconstructs the minimum functional domain of PpsR2 through structural domain prediction and truncation verification. The specific scheme is as follows:

[0144] PpsR2 domain prediction: The domains and functions of PpsR2 were predicted using the UniProt, SMART, and InterPro databases. Results showed that the core functional region contains two PAS domains (widely found in biological proteins, serving as sensing modules for oxygen tension, redox potential, or light intensity, and mediating protein-protein interactions upon stimulation); the predicted results for the CC and HTH domains differed and require verification through truncation experiments (see reference). Figure 5 ).

[0145] PpsR2 domain truncation verification: The predicted domains (including CC domain (108-123), PAS domain (5-103, 257-366), PAS_2 domain (131-202, 257-324), PAS_Like domain (132-201, 254-326), PAS_8 domain (133-166, 260-307), HTH domain (407-424, 425-444), HTH_8 domain (403-441), and HTH_fi8 domain (403-444)) were truncated. The primers used for truncation are shown in SEQ ID Nos. 45-68.

[0146] The performance of the detection system is as follows:

[0147] Deletion of the CC domain: Significantly increased expression leakage in the strain, indicating that the CC domain is responsible for oligomerization in the inactive state and is an essential domain; Removal of the PAS domain: Decreased induction intensity but still retains regulatory function. Combined with the PAS domain's predicted location upstream of the non-overlapping domain, this confirms that PpsR2 contains more than two PAS-type domains; Removal of the PAS_2, PAS_Like, and PAS_8 domains: The degree of decrease in induction intensity is consistent with the regulatory range, proving that the removal of these three domains disrupts the same PAS-type domain; Removal of HTH-type domains: Slight decrease in induction intensity; among them, the regulatory range is smallest when the HTH_fi8 domain is removed, suggesting a more accurate estimation of its length and location (see [link]). Figure 6 ).

[0148] PpsR2 domain reconstruction and screening: Based on the truncation validation results, PpsR2 was reconstructed using a combination of two PAS-like domains and one HTH domain: the "PAS-like domain-X-HTH_fi8" combination showed the highest fluorescence intensity, reaching 1.81 times that of the NLS system strains (reference). Figure 7 The PAS_8 domain in this combination has the shortest length within the second PAS domain sequence interval. Therefore, the combination of "PAS domain-PAS_8 domain-HTH_fi8 domain" is selected as the reconstructed PpsR2 and named Q-PpsR2 (the sequence is shown in SEQ ID No. 4).

[0149] Example 3: System performance after combined optimization

[0150] The optimized Bphp1 obtained above N499K The mutant was combined with the Q-PpsR2 reconstructor to construct a new optogenetic system, and the plasmid map is shown below. Figure 9 As shown (sequence as shown in SEQ ID No. 69). A light-induced gene expression system was constructed according to the method of Example 1, and its expression level was detected.

[0151] Analysis revealed that by using the mutant Bphp1 of the photosensitive protein BphP1 of this invention alone... N499K It can increase the induction intensity of the photoinduction system to 1.41 times that of the wild-type strain (see...). Figure 4 Furthermore, by using the truncated variant Q-PpsR2 alone, the induction intensity of the photoinduction system was increased to 1.81 times that of the wild-type strain (see [link to original text]). Figure 7 When Bphp1 N499K When used in combination with Q-PpsR2, the two have a synergistic effect, increasing the induction intensity of the photoinduction system to 2.99 times that of the wild-type strain (see [link]). Figure 8 ).

[0152] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A mutant of the photosensitive protein BphP1, the amino acid sequence of which is shown in SEQ ID No.

2.

2. An optogenetic system comprising: 1) A fusion protein A of the mutant of claim 1 and the transcriptional activation domain of yeast transcription factor Gal4, or a vector expressing said fusion protein A, wherein the mutant and said transcriptional activation domain optionally include a linker; and 2) A fusion protein B of the photosensitive protein PpsR2 and the DNA-binding domain of the yeast transcription factor Gal4, or a vector expressing the fusion protein B, wherein the photosensitive protein PpsR2 and the DNA-binding domain optionally include a linker, wherein the photosensitive protein PpsR2 is wild-type PpsR2 of Rhodopseudomonas palustris or a truncated form thereof, the amino acid sequence of the wild-type PpsR2 is shown in SEQ ID No. 3, and the amino acid sequence of the truncated form is shown in SEQ ID No.

4.

3. The optogenetic system of claim 2, further comprising an expression vector for expressing a target gene, the expression vector comprising a yeast cell Gal1 promoter operatively linked to the target gene.

4. The optogenetic system of claim 3, wherein the yeast cell Gal1 promoter is further operatively linked to a gene encoding a tag protein selected from mCherry, TrxA, MBP, DsRed.T3, or GFP.

5. The optogenetic system according to claim 2, wherein the amino acid sequence of the photosensitive protein PpsR2 is shown in SEQ ID No.

4.

6. The optogenetic system according to any one of claims 2 to 5, which is responsive to light induction at 740 nm.

7. A method for regulating gene expression in host cells, the method comprising: 1) Provide host cells; 2) Introducing the optogenetic system according to any one of claims 2 to 6 into the host cell; and 3) The optogenetic system was induced by 740 nm light to regulate gene expression.

8. The method according to claim 7, wherein the host cell is Saccharomyces cerevisiae.

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