Novel light-controlled repressor protein OptoLacI and its usage

Abstract

This method addresses the aforementioned problems in existing chemical induction methods for E. coli, such as cytotoxicity, high cost, and irreversible regulation, as well as the complexity of regulatory components, operational complexity, and incompleteness of bidirectional regulatory tools in photoregulated E. coli gene expression systems. [Solution] A novel light-regulated repressor protein OptoLacI and a light-regulated gene expression system constructed based on OptoLacI.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of a Chinese patent application with application number CN 202310707956.4 (filed on June 14, 2023), and the entire content of the said patent application is incorporated herein by reference. The present invention relates to the fields of protein engineering or synthetic biology in biotechnology, specifically to gene expression regulation, more specifically to a novel light - controlled repressor protein OptoLacI, a light - controlled gene expression system constructed by OptoLacI, and the use of the light - controlled expression system in protein production and metabolic engineering.

Background Art

[0002] In the practice of metabolic engineering, the introduction of foreign pathways causes problems such as non-uniform distribution of material and energy flows and imbalance of endogenous metabolic resources, resulting in growth impairment of the production strain and limitation of product yield. Therefore, in the practice of metabolic engineering, it is a common strategy to improve metabolic imbalance by regulating metabolic pathways, including enhancing precursor supply and substrate or product transport by gene overexpression, knocking out competing pathways, and overexpressing genes related to the target pathway. Escherichia coli is a preferred host for the production of many compounds and is widely applied in chemistry, food, pharmaceuticals, agriculture, and energy. In 1961, French scientists F. Jacob and J. Monod established the model of the lactose operon in Escherichia coli. The lactose operon induction system based on this model is the most commonly used induction system in Escherichia coli. When the lactose analog isopropyl-β-d-1-thiogalactoside (IPTG) is absent, the repressor protein LacI binds to the lac operon (LacO) upstream of the target gene, inhibiting the transcription of the target gene. When IPTG binds to the repressor protein LacI, the LacI repressor protein dissociates from the lacO site, thereby initiating the transcription of the target gene. In the practice of metabolic engineering, the addition of IPTG is the main method for regulating metabolic pathways. However, in metabolic engineering and industrial protein production, the chemical derivative IPTG has drawbacks such as cytotoxicity, high cost, and irreversible induction, and cannot achieve precise regulation in terms of time and space. Therefore, researchers have been continuously exploring the discovery of alternative means. With the continuous development of optogenetic tools, light has become an ideal means for inducing gene expression because of its low toxicity, easy availability, reversible regulation, and low cost. However, existing light-controlled gene expression systems have problems such as complexity of control components, complexity of operation, and incompleteness of bidirectional regulation tools.

Summary of the Invention

[0003] To address the aforementioned problems of existing chemical induction methods for E. coli, such as cytotoxicity, high cost, and irreversible regulation, as well as the complexity of regulatory components, operational complexity, and incompleteness of bidirectional regulatory tools in light-controlled E. coli gene expression systems, this invention primarily provides a novel light-controlled repressor protein, OptoLacI, and develops two light-controlled gene expression systems based on OptoLacI, thereby achieving bidirectional induction under blue light and in the dark. These systems possess high spatiotemporal resolution and exhibit excellent properties in protein expression and metabolic engineering.

[0004] In one embodiment, the present invention provides a light-controlled repressor protein comprising a LacI protein and an LOV domain inserted between adjacent amino acids in the loop 1, loop 2, or loop 3 region of the LacI protein.

[0005] In some embodiments, the LOV domain is the LOV2 domain; preferably, the LOV domain is selected from the LOV2 domain of Avena sativa phototropin 1 (AsLOV2), the photosensitive protein EL222 from Erythrobacter litoralis, the photosensitive domain LOV2 from Arabidopsis thaliana, or a variant thereof; preferably, the variant is a circular permutation variant or a variant with altered response rate; preferably, the LOV domain is AsLOV2 and includes, for example, the sequence described in SEQ ID NO: 1; preferably, the LOV domain is cpLOV27 and includes, for example, the sequence described in SEQ ID NO: 2.

[0006] In some embodiments, the insertion site is selected from the following amino acid positions of the LacI protein: between positions 335 and 336, between positions 314 and 315, between positions 315 and 316, between positions 316 and 317, between positions 334 and 335, between positions 336 and 337, between positions 337 and 338, between positions 338 and 339, or between positions 152 and 153; preferably, the insertion site is between positions 335 and 336 of the LacI protein. In some embodiments, the insertion site is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, or between positions 153 and 154; preferably, the insertion site is between positions 311 and 312 of the LacI protein. In some embodiments, the LacI protein is wild-type; preferably, the wild-type LacI protein contains the sequence described in SEQ ID NO: 26. In some embodiments, the LacI protein is a modified LacI protein that, compared to the wild-type LacI protein, comprises an amino acid substitution selected from (i) a substitution of the amino acid at position 220 with F, and / or (ii) a substitution of the amino acid at position 84 with E, C, S, T, or I; preferably, the wild-type LacI protein comprises the sequence described in SEQ ID NO: 26; preferably, the modified LacI protein comprises the sequence described in SEQ ID NO: 27 or 28, or, compared to SEQ ID NO: 27 or 28, comprises a sequence in which the amino acid at position 84 is substituted with C, S, T, or I.

[0007] In some embodiments, the N-terminus and / or C-terminus of the LOV domain are optionally linked to the LacI protein via a peptide linker. In some embodiments, the light-regulated repressor protein comprises the sequence described in any one of SEQ ID NOs: 11, 29, 6-8, 10, 12-14, 16, or the sequence described in SEQ ID NO: 3 or 17; preferably, the light-regulated repressor protein comprises the sequence described in SEQ ID NO: 11 or 29; preferably, the light-regulated repressor protein comprises the sequence described in SEQ ID NO: 3. In one embodiment, the present invention provides a nucleic acid construct comprising a nucleotide sequence encoding a light-controlled repressor protein as described above. In one embodiment, the present invention provides a vector comprising the nucleic acid construct described above. In some embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding a photoregulated repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a target gene or a cloning site for incorporating the target gene; preferably, the first and second nucleic acid constructs are located in different expression cassettes.

[0008] In some embodiments, the LacO operator gene included in the second nucleic acid construct is selected from LacO1, LacOid, or a combination thereof. In some embodiments, the LacO operator gene included in the second nucleic acid construct comprises at least one copy (e.g., 1 to 8 copies, such as 3 to 5 copies, such as 1, 2, 3, 4, 5, 6, 7, 8 copies) of the LacO1 operator sequence; preferably, the LacO1 operator sequence comprises the sequence described in SEQ ID NO: 24.

[0009] In some embodiments, the LacO operator gene included in the second nucleic acid construct further comprises LacOid; preferably, LacOid is located downstream of LacO1; and preferably, the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction. In some embodiments, the promoter included in the second nucleic acid construct is selected from the group consisting of the T7 promoter of T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter. In some embodiments, the first nucleic acid construct further comprises a promoter operably ligated to a nucleotide sequence encoding a photoregulated repressor protein; preferably, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In one embodiment, the present invention provides a host cell comprising the nucleic acid construct or vector described above; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli.

[0010] In some embodiments, the host cell is Escherichia coli, which has an exogenous nucleotide sequence encoding the above-described light-regulated repressor protein incorporated into its genome; preferably, the endogenous LacI gene of Escherichia coli is disrupted; preferably, both copies of the endogenous LacI gene of Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of Escherichia coli is replaced with the exogenous nucleotide sequence and the other copy of endogenous LacI gene 1 is knocked out. In some embodiments, the E. coli further comprises the above vector, which includes a first nucleic acid construct and a second nucleic acid construct. In one embodiment, the present invention provides a system for regulating the expression of a target gene, (1) A first nucleic acid construct comprising a nucleotide sequence encoding the above-described photoregulated repressor protein; (2) A second nucleic acid construct comprising a promoter, a LacO operator gene, and a target gene. The system includes providing a system.

[0011] In some embodiments, the second nucleic acid construct is defined in any embodiment of any of the above-described aspects. In some embodiments, the second nucleic acid construct comprises a plurality of target genes arranged in tandem; preferably, additional promoters are optionally inserted between the plurality of target genes. In some embodiments, the first nucleic acid construct further comprises a promoter operably ligated to a nucleotide sequence encoding a photoregulated repressor protein; preferably, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter.

[0012] In some embodiments, the system comprises at least two first nucleic acid constructs, one of which is integrated into the genome of a host cell, and the other, together with a second nucleic acid construct, on a vector (e.g., an expression vector); where the at least two first nucleic acid constructs are either identical or different from each other; preferably, the vector is the vector comprising the first and second nucleic acid constructs. In some embodiments, the first nucleic acid construct and the second nucleic acid construct reside on a vector (e.g., an expression vector); preferably, the vector is the vector comprising the first nucleic acid construct and the second nucleic acid construct. In some embodiments, the first nucleic acid construct is integrated into the genome of a host cell, and the second nucleic acid construct resides on a vector (e.g., an expression vector). In some embodiments, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli.

[0013] In one embodiment, the present invention provides a kit comprising host cells and an expression vector, wherein: (1) The host cell has an exogenous nucleotide sequence encoding the light-regulated repressor protein described above incorporated into its genome; the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises an exogenous nucleotide sequence encoding the light-regulated repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site for incorporating the target gene; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli; Or, (2) The host cell has an exogenous nucleotide sequence that encodes the above-mentioned light-regulated repressor protein, which is incorporated into its genome; the expression vector comprises a second nucleic acid construct; preferably the host cell is a prokaryotic cell; preferably the host cell is Escherichia coli; Or, (3) The host cell does not contain exogenous nucleotide sequences encoding light-regulated repressor proteins incorporated into its genome; the expression vector comprises a first nucleic acid construct and a second nucleic acid construct; preferably, the host cell is a prokaryotic cell such as Escherichia coli. In some embodiments, the second nucleic acid construct is defined in any embodiment of any of the aforementioned aspects.

[0014] In some embodiments, the first nucleic acid construct further comprises a promoter operably linked to a nucleotide sequence encoding a photoregulated repressor protein; preferably, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In some embodiments, the host cell described in (1) or (2) is Escherichia coli, which has an exogenous nucleotide sequence encoding the above-mentioned light-regulated repressor protein incorporated into its genome; preferably, the endogenous LacI gene of Escherichia coli is disrupted; preferably, both copies of the endogenous LacI gene of Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of Escherichia coli is replaced with the exogenous nucleotide sequence and the other copy of the endogenous LacI gene is knocked out.

[0015] In one embodiment, the present invention is a method for regulating the expression of a target gene, (1) To provide the above-mentioned system to host cells; (2) Inducing the expression of the target gene by culturing host cells under conditions that enable the expression of the target gene. This provides a method that includes [something].

[0016] In some embodiments, step (1) is: (i) To provide an expression vector comprising a first nucleic acid construct and a second nucleic acid construct of the system; comprising introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the above-mentioned photoregulatory repressor protein, which is incorporated into its genome; preferably, the expression vector is a vector comprising the first nucleic acid construct and the second nucleic acid construct, and preferably the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli; Or, (ii) To provide an expression vector comprising a second nucleic acid construct of the system; comprising introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding a photoregulatory repressor protein incorporated into its genome; preferably the host cell is a prokaryotic cell; preferably the host cell is Escherichia coli; Or, (iii) Provide an expression vector comprising a first nucleic acid construct and a second nucleic acid construct of the system; comprising introducing the expression vector into a host cell, wherein the host cell does not contain exogenous nucleotide sequences encoding a photoregulatory repressor protein incorporated into its genome; preferably, the expression vector is a vector comprising the first and second nucleic acid constructs described above; preferably, the host cell is a prokaryotic cell such as Escherichia coli.

[0017] In some embodiments, the host cell described in (i) or (ii) is Escherichia coli, which has an exogenous nucleotide sequence encoding the above-described light-regulated repressor protein incorporated into its genome; preferably, the endogenous LacI gene of Escherichia coli is disrupted; preferably, both copies of the endogenous LacI gene of Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of Escherichia coli is replaced with the exogenous nucleotide sequence and the other copy of the endogenous LacI gene is knocked out.

[0018] In some embodiments, the insertion site in the light-controlled repressor protein is selected from the following amino acid positions of the LacI protein: between positions 335 and 336, between positions 314 and 315, between positions 315 and 316, between positions 316 and 317, between positions 334 and 335, between positions 336 and 337, between positions 337 and 338, between positions 338 and 339, or between positions 152 and 153; preferably, the insertion site is between positions 335 and 336 of the LacI protein; the induction conditions in step (2) include culturing host cells in the dark to induce the expression of the target gene; preferably, the method further includes culturing recombinant host cells under blue light to inhibit the expression of the target gene. In some embodiments, the insertion site in the light-controlled repressor protein is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, or between positions 153 and 154; preferably, the insertion site is between positions 311 and 312 of the LacI protein; the induction conditions in step (2) include culturing host cells under blue light to induce the expression of the target gene; preferably, the method further includes culturing host cells under dark conditions to suppress the expression of the target gene.

[0019] In one embodiment, the present invention provides the use of the above-described photoregulated repressor proteins, nucleic acid constructs, vectors, host cells, systems, or kits for regulating the expression of a target gene; preferably, the use includes regulating protein expression; preferably, the use includes regulating metabolic pathways and / or biosynthesis. The present invention will be further described in conjunction with the following non-limiting drawings: [Brief explanation of the drawing]

[0020] [Figure 1] The results of the screening of light-controlled LacI mutants are shown. [Figure 2] A schematic diagram of the genome structure of light-controlled E. coli is shown. [Figure 3] This study shows the effect of the number of tandem LacO1 units on the expression of dark-induced genes in the strain. [Figure 4] This shows the effect of the number of tandem LacO1 cells on blue light-induced gene expression in the strain. [Figure 5] The results of optimizing the induction intensity of the dark-induced gene expression system are shown. [Figure 6] This paper demonstrates the effects of different promoters on dark-induced gene expression systems. [Figure 7] The results of optimizing the induction multiplier for the dark-induced gene expression system are shown. [Figure 8] This demonstrates the application of a dark-induced gene expression system to protein expression. (A) Expression results for alkalin protease (36.8 kD); (B) Expression results for PETase (29.0 kD); (C) Expression results for GDH (29.4 kD). M represents the protein marker (in kD units), and the numbers 0, 2, 4, 6, 9, and 12 indicate the sample numbers of cell lysates collected at different dark induction times (0 hours, 2 hours, 4 hours, 6 hours, 9 hours, 12 hours). [Figure 9] This demonstrates the application of a blue light-induced gene expression system to protein expression. (A) Expression results for FadA (41.6 KD); (B) Expression results for MdhII (35.9 KD). M represents the protein marker (in KD units), L represents the cell lysate sample collected 9 hours after blue light induction, and L represents the cell lysate sample collected 9 hours after dark culture. [Figure 10] A schematic diagram of the expression cassette of major genes in 1,3-propanediol biosynthesis is shown. [Figure 11]This demonstrates the application of a dark-induced system in metabolic pathway flux control. S1-IPTG: BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (no IPTG added during fermentation); S1-IPTG: BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (IPTG added at OD600=0.1~0.6); S2 LL: BL21_Dark_Dv1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (continuous blue light throughout the entire fermentation period); S2 LD: BL21_Dark_Dv1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (switched to dark conditions at OD600=0.1~0.6). [Figure 12] This paper compares the GFP expression effects of a light-controlled gene expression system and an IPTG-induced system. [Modes for carrying out the invention]

[0021] definition Unless otherwise specified, scientific and technical terms used herein have the meanings that are ordinarily understood by those skilled in the art. For a better understanding of the present invention, the definitions and explanations of the relevant terms are given below. As used herein, the terms “comprising” and its variations are used synonymously with “including” and its variations, and these are open, non-restrictive terms. The terms “comprising” and “including” are used herein to describe various embodiments, but the terms “basically consisting of” and “being composed of” may be used instead of “comprising” and “including” to describe more specific embodiments, and these are also disclosed.

[0022] As used herein, the articles "a," "an," and "the" mean "at least one" unless the context of their use clearly indicates otherwise. As used herein, the terms “Lac repressor protein (lactose operon repressor)” and “LacI protein” have the same meaning and are used interchangeably. The sequence of the LacI protein is well known to those skilled in the art and can be found in various public databases such as GenBank:CAF2498598.1. When referring herein to the amino acid position of the LacI protein, the sequence described in SEQ ID NO: 26 is referred to. For example, the statement “amino acid residue at position 335 of the LacI protein” refers to the 335th amino acid residue and its corresponding position in the sequence described in SEQ ID NO: 26. The corresponding position refers to the position in the sequence being compared that corresponds to a specific amino acid position in SEQ ID NO: 26 after optimal alignment with SEQ ID NO: 26 (i.e., to obtain the highest degree of agreement).

[0023] As used herein, the term “LOV domain” generally refers to a photooxygen voltage (LOV) domain derived from a photoreceptor protein of a plant, algae, or bacterium. Preferably, the LOV domain is an LOV2 domain, and preferably derived from Avena sativa (oat), Erythrobacter litoralis, or Arabidopsis thaliana. In some exemplary embodiments, the LOV domain has an amino acid sequence described in SEQ ID NOs. 1, 30, or 31, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity with those sequences.

[0024] As used herein, the term "circular permutation" refers to the introduction of a new terminology by linking the original N-terminus and C-terminus of a protein via a linker, thereby cleaving the original peptide bond of the protein. The resulting new protein is called a circular permutation mutant. As used herein, the term “gene” is used broadly to refer to DNA nucleic acids associated with biological function. As used herein, “target gene” refers to any nucleic acid sequence of interest and / or potentially meaningful in controlling its transcription level. As used herein, the term "transcription" refers to the synthesis of RNA from a DNA template; the term "translation" refers to the synthesis of polypeptides from an mRNA template. Transcription and translation together are referred to as "expression." As used herein, the term “operable linkage” refers to a functional connection between elements, where the elements are in a relationship that allows them to be manipulated in an intended manner. For example, in the case of a promoter, the promoter is functionally linked to a target gene, so that the promoter sequence can initiate transcription of the target gene.

[0025] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. If a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells by transformation, transduction, or transfection, making it possible for the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phage particles; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as λ phages or M13 phages; and animal viruses. One type of vector is the episomal vector, which is a nucleic acid capable of extrachromosomal replication. Another type of vector is the integrated vector, designed to recombine with the genetic material of a host cell. Vectors typically contain one or more restriction endonuclease recognition sites and / or sites for site-directed recombination, which allow exogenous DNA fragments to be cleaved and ligated to these sites within the vector. A vector may contain various elements that regulate gene expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may also contain an origin of replication.

[0026] As used herein, the term “expression cassette” refers to a combination of regulatory elements and genes to which they are operably ligated for expression. Regulatory elements include, for example, promoter sequences, start codons, stop codons, and terminators. An expression cassette may also include additional regulatory sequences such as enhancers, signal sequences, introns, IRES sequences, and other sequences.

[0027] As used herein, the term “promoter” refers to an expression regulatory element that enables the binding and initiation of RNA polymerase. As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, and includes, but is not limited to, prokaryotic cells, fungal cells, insect cells, mammalian cells, and plant cells.

[0028] Light-regulated repressor proteins Based on the inventors' research, the present invention has been completed in accordance with the following insights and discoveries, and in particular the following embodiments are provided. Lac repressor protein (LacI) is a protein that regulates the transcription of genes necessary for lactose metabolism in E. coli. LacI consists of 360 amino acid residues and its structure includes an N-terminal domain and a C-terminal domain. The N-terminal domain (amino acids 1-59) contains a helical-turn-helical motif essential for DNA binding, and the C-terminal domain contains a binding site for chemical derivatives and their analogs, where the 30 amino acid residues at the C-terminus mediate the tetramerization of the repressor protein. Two subunits of the LacI tetramer bind to the operator gene LacO1, and the remaining two subunits bind to the operator gene LacO2 or LacO3, forming a loop between the operator genes and inhibiting RNA polymerase from binding to the promoter. When LacI is bound to the chemical derivative IPTG, a stable protein structure with low affinity to the operator gene is maintained, reducing the occupancy rate of the operator gene and derepressing the repressor protein LacI, thereby initiating the transcription of downstream genes. The blue light-responsive photooxygen voltage-sensing domain 2 (LOV2) has a low molecular weight (100-140 amino acids), and its chromophore, flavin mononucleotide (FMN), is universally present in various cells, making LOV2 a widely used photoregulated element. In various cells, the LOV2 domain is applied to the reversible spatiotemporal manipulation of cell signaling and behavior, including the reversible regulation of intracellular proteolysis, protein-protein interactions, and protein localization. The C-terminus of the LOV2 domain contains a Jα helix. Under dark conditions, this Jα helix forms a stable structure between itself and the β-sheet of the Per-Arnt-Sim (PAS) core domain. Under blue light irradiation, conserved cysteine ​​residues within the LOV2 domain covalently bond with FMN residues, causing rotation of conserved glutamine residues, which ultimately leads to a structural change in the Jα helix and its separation from the central β-sheet. Based on the repressor protein LacI and the light-regulating element LOV2, it is possible to construct novel light-regulating repressor proteins for precise regulation of gene expression by mutating both of them.

[0029] In one embodiment, the present invention provides a light-regulated repressor protein comprising a LacI protein and a LOV domain inserted between adjacent amino acids in the loop 1, loop 2, or loop 3 region of the LacI protein. In some embodiments, the loop 1 region of the LacI protein refers to amino acid positions 311-317, the loop 2 region refers to amino acid positions 333-340, and the loop 3 region refers to amino acid positions 152-156. In this specification, when referring to amino acid positions of the LacI protein, it refers to the sequence described in SEQ ID NO: 26. In some embodiments, the N-terminus and / or C-terminus of the LOV domain are directly ligated to the LacI protein. In some embodiments, the N-terminus and / or C-terminus of the LOV domain are linked to the LacI protein via a peptide linker (e.g., a peptide linker comprising one or more flexible amino acids).

[0030] LOV domain Any LOV domain known in the art can be used in the present invention. In some embodiments, the LOV domain is an LOV2 domain. LOV2 domains from different species all belong to the photoresponsive domain and play the same role in different species; furthermore, the three-dimensional structures of LOV2 domains from different species are highly similar, and the corresponding allosteric mechanisms are the same. Therefore, LOV2 domains from different species are all applicable to the present invention.

[0031] In some embodiments, the LOV domain is selected from the LOV2 domain of Avena sativa phototropin 1 (AsLOV2), the photosensitive protein EL222 from Erythrobacter litralis, the photosensitive domain LOV2 from Arabidopsis thaliana, or a variant thereof. In some embodiments, the mutant is a circular permutation mutant. In this specification, “circular permutation” has a meaning familiar to those skilled in the art and refers to the introduction of a new term by linking the original N-terminus and C-terminus of a protein via a linker and cleaving the original peptide bond of the protein. In some embodiments, the mutants are those with altered response rates. Such mutants are well known to those skilled in the art and typically include mutations selected from V461I, V74I, and L165I, where the amino acid positions refer to positions in AsLOV2, and the corresponding positions in other LOV domains can be determined by sequence alignment (see, for example, Zoltowski, BD, B. Vaccaro, and BR Crane, Mechanism-based tuning of a LOV domain photoreceptor. Nature Chemical Biology, 5(11): 827-834 (2)). Since introducing mutations that alter response rates into LOV2 does not change the mechanism and facts of its allosteric response to blue light, these mutants are also applicable to the present invention.

[0032] In some embodiments, the LOV domain is AsLOV2. In some embodiments, AsLOV2 contains the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the LOV domain is a circular permutation variant of AsLOV2. In some embodiments, the circular permutation variant of AsLOV2 is cpLOV27, which contains the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the LOV domain is, for example, the photosensitive protein EL222 derived from Erythrobacter litralis, which includes the amino acid sequence described in SEQ ID NO: 30. In some embodiments, the LOV domain is, for example, the photosensitive domain LOV2 derived from Arabidopsis thaliana, which includes the amino acid sequence described in Sequence ID No. 31.

[0033] LacI protein In some embodiments, the LacI protein is a wild-type LacI protein. In some embodiments, the wild-type LacI protein contains the sequence described in SEQ ID NO: 26. In some embodiments, the LacI protein is a modified LacI protein that, compared to the wild-type LacI protein, includes: (i) a substitution of the amino acid at position 220 with F, and / or (ii) a substitution of the amino acid at position 84 with E, C, S, T, or I. In some embodiments, the modified LacI protein includes the sequence described in SEQ ID NO: 27 or 28, or a sequence in which the amino acid at position 84 is substituted with C, S, T, or I compared to SEQ ID NO: 27 or 28. In some embodiments, the modified LacI protein comprises the sequence described in SEQ ID NO: 27 or 28.

[0034] Dark-responsive, light-regulated repressor proteins In some embodiments, the light-controlled repressor proteins of the present invention have dark-responsive activity. As used herein, "dark-responsive activity" means that the light-controlled repressor protein does not exert a repressive effect under dark conditions and allows the expression of the target gene. Preferably, the light-controlled repressor protein exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) and inhibits the expression of the target gene.

[0035] In some embodiments, the insertion sites in dark-responsive, light-regulated repressor proteins are the following amino acid positions in the LacI protein: between positions 335 and 336 (e.g., loop-2 (Q335-T336)), between positions 314 and 315 (e.g., loop-1 (K314-G315)), between positions 315 and 316 (e.g., loop-1 (G315-N316)), and between positions 316 and 317 (e.g., loop-1 (N3 The following are selected: 16-Q317), between 334th and 335th place (e.g., loop-2(T334-Q335)), between 336th and 337th place (e.g., loop-2(T336-A337)), between 337th and 338th place (e.g., loop-2(A337-S338)), between 338th and 339th place (e.g., loop-2(S338-P339)), and between 152nd and 153rd place (e.g., loop-3(D152-Q153)). In some embodiments, the insertion site in the dark-responsive, light-regulated repressor protein is between positions 335 and 336 of the LacI protein (e.g., loop-2 (Q335-T336)). In some embodiments, the dark-responsive, light-regulated repressor protein comprises the amino acid sequence described in any one of SEQ ID NOs: 11, 29, 6-8, 10, 12-14, and 16. In some embodiments, the dark-responsive, light-regulated repressor protein comprises the amino acid sequence described in SEQ ID NO: 11 or 29.

[0036] Blue light-responsive photoregulatory repressor protein In some embodiments, the light-controlled repressor proteins of the present invention have blue light-responsive activity. In this specification, "blue light-responsive activity" refers to the fact that the light-controlled repressor protein does not exert a repressive effect under blue light conditions (e.g., 430-495 nm, such as 450-480 nm), and allows the expression of the target gene. It is desirable that the light-controlled repressor protein exerts a repressive effect and inhibits the expression of the target gene under dark conditions. In some embodiments, the insertion site in the blue light-responsive photoregulatory repressor protein is selected from the following amino acid positions of the LacI protein: between positions 311 and 312 (e.g., loop-1 (Q311-A312)) or between positions 153 and 154 (e.g., loop-3 (Q153-T154)).

[0037] In some embodiments, the insertion site in the blue light-responsive photoregulatory repressor protein is located between positions 311 and 312 of the LacI protein (e.g., loop-1 (Q311-A312)). In some embodiments, the blue light-responsive photoregulatory repressor protein comprises the amino acid sequence described in SEQ ID NO: 3 or 17. The light-controlled repressor proteins of the present invention can be prepared by various methods well known in the art, such as genetic engineering methods (recombinant technology) or chemical synthesis methods (e.g., Fmoc solid-phase method). The light-controlled repressor proteins of the present invention are not limited by the method of their production.

[0038] nucleic acid constructs and vectors In another aspect, the present invention provides a nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein of the present invention. In another embodiment, the present invention provides a vector comprising the nucleic acid construct described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding the photoregulated repressor protein of the present invention, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a target gene.

[0039] In some embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises a nucleotide sequence encoding the photoregulated repressor protein of the present invention, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site (e.g., a multi-cloning site MCS) for incorporating the target gene. In some embodiments, the LacO operator gene included in the second nucleic acid construct is selected from LacO1, LacOid, or a combination thereof.

[0040] In some embodiments, the LacO operator gene included in the second nucleic acid construct contains at least one copy (e.g., 1 to 8 copies such as 1, 2, 3, 4, 5, 6, 7, 8 copies, or 3 to 5 copies) of the LacO1 operator sequence. In some embodiments, the LacO operator gene included in the second nucleic acid construct contains 3 to 5 copies of the LacO1 operator sequence. In some embodiments, the LacO1 operator sequence includes the sequence described in SEQ ID NO: 24. In some embodiments, the LacO operator gene included in the second nucleic acid construct comprises LacO1 and LacOid. In some embodiments, LacOid is located downstream of LacO1. In some embodiments, the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction. In some embodiments, the promoter included in the second nucleic acid construct is recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e., it can be any promoter as long as the host cell's RNA polymerase is used. In some embodiments, the promoter included in the second nucleic acid construct is selected from the group consisting of the T7 promoter of the T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter.

[0041] In some embodiments, the first nucleic acid construct includes a promoter operably ligated to a nucleotide sequence encoding the light-controlled repressor protein of the present invention. In some embodiments, the promoter included in the first nucleic acid construct can be any promoter, as long as it is recognized by an RNA polymerase encoded by an RNAP gene contained in the host chromosome, i.e., as long as the RNA polymerase of the host cell is used. In some embodiments, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In some embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes within the same vector. In some embodiments, the vector is a pET-28a-based plasmid, where the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with a LacO operon contained in a second nucleic acid construct.

[0042] host cell In another embodiment, the present invention provides a host cell comprising a nucleic acid construct or vector as described above. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is Escherichia coli, and the Escherichia coli has an exogenous nucleotide sequence that encodes the light-controlled repressor protein of the present invention, which is incorporated into its genome.

[0043] In some embodiments, the endogenous LacI gene of Escherichia coli is disrupted. In some embodiments, disruption includes: loss-of-function mutations (e.g., addition, deletion, and / or substitution of one or more bases), deletions, or substitutions by foreign nucleotide sequences. A “loss-of-function mutation” refers to a mutation that causes the protein encoded and expressed by the mutated gene to lose its biological function. Loss-of-function mutations include, but are not limited to, missense mutations, nonsense mutations, frameshift mutations, base deletions, base substitutions, base additions, and any combination thereof (e.g., deletion, substitution, or addition of a gene segment), provided that the gene containing the loss-of-function mutation is unable to produce or express a protein with biological function.

[0044] In some embodiments, both copies of the endogenous LacI gene in E. coli are replaced with exogenous nucleotide sequences. In some embodiments, one copy of the endogenous LacI gene in E. coli is replaced with an exogenous nucleotide sequence, while the other copy of the endogenous LacI gene is knocked out. In some embodiments, the integration of exogenous nucleotide sequences contained within host cells and / or the disruption of endogenous genes can be achieved using any gene editing system well known to those skilled in the art. Typical gene editing systems include CRISPR / Cas, ZFN, TALEN, and the like. In some embodiments, Escherichia coli having an exogenous nucleotide sequence encoding the light-controlled repressor protein of the present invention, incorporated into its genome, may further comprise a vector (e.g., an expression vector) containing the first nucleic acid construct and the second nucleic acid construct as described above.

[0045] Systems that regulate target gene expression In another embodiment, the present invention is a system for regulating target gene expression, (1) A first nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein of the present invention. (2) A second nucleic acid construct comprising a promoter, a LacO operator gene, and a target gene. The system includes providing a system. In some embodiments, the LacO operator gene included in the second nucleic acid construct is selected from LacO1, LacOid, or a combination thereof. In some embodiments, the LacO operator gene included in the second nucleic acid construct contains at least one copy (e.g., 1 to 8 copies such as 1, 2, 3, 4, 5, 6, 7, 8 copies, or 3 to 5 copies) of the LacO1 operator sequence. In some embodiments, the LacO operator gene included in the second nucleic acid construct contains 3 to 5 copies of the LacO1 operator sequence. In some embodiments, the LacO1 operator sequence includes the sequence described in SEQ ID NO: 24.

[0046] In some embodiments, the LacO operator gene included in the second nucleic acid construct comprises LacO1 and LacOid. In some embodiments, LacOid is located downstream of LacO1. In some embodiments, the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction.

[0047] In some embodiments, the promoter included in the second nucleic acid construct is recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e., it can be any promoter as long as the host cell's RNA polymerase is used. In some embodiments, the promoter included in the second nucleic acid construct is selected from the group consisting of the T7 promoter of the T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter. In some embodiments, the second nucleic acid construct includes a plurality of target genes arranged in tandem. In some embodiments, additional promoters are optionally inserted between the plurality of target genes. In some embodiments, the first nucleic acid construct further includes a promoter operably ligated to a nucleotide sequence encoding the light-controlled repressor protein of the present invention. In some embodiments, the promoter included in the first nucleic acid construct can be any promoter, as long as it is recognized by an RNA polymerase encoded by an RNAP gene contained in the host chromosome, i.e., as long as the RNA polymerase of the host cell is used. In some embodiments, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter.

[0048] In some embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes within the same vector. In some embodiments, the vector is a pET-28a-based plasmid, where the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with a LacO operon contained in the second nucleic acid construct. In some embodiments, the light-regulated repressor protein is selected from the dark-responsive light-regulated repressor proteins of the present invention. In such embodiments, the system that regulates the expression of the target gene is a dark-inducible gene expression system, where the light-regulated repressor protein does not exert a repressive effect under dark conditions, allows the expression of the target gene, and preferably exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) to inhibit the expression of the target gene. In some embodiments, the light-controlled repressor protein is selected from the blue light-responsive light-controlled repressor proteins of the present invention. In such embodiments, the system that regulates the expression of the target gene is a blue light-inducible gene expression system, where the light-controlled repressor protein does not exert a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) and allows the expression of the target gene, and preferably exerts a repressive effect under dark conditions to inhibit the expression of the target gene.

[0049] kit In another embodiment, the present invention provides a kit comprising a host cell and an expression vector, wherein the host cell has an exogenous nucleotide sequence encoding the light-regulated repressor protein of the present invention, incorporated within its genome; the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises an exogenous nucleotide sequence encoding the light-regulated repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site (e.g., a multi-cloning site MCS) for incorporating a target gene.

[0050] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence that encodes a light-regulated repressor protein integrated into its genome. In some embodiments, the endogenous LacI gene of Escherichia coli is disrupted. In some embodiments, both copies of the endogenous LacI gene of Escherichia coli are replaced with an exogenous nucleotide sequence. In some embodiments, one copy of the endogenous LacI gene of Escherichia coli is replaced with an exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0051] In some embodiments, the first and second nucleic acid constructs are located on different expression cassettes. In some embodiments, the first nucleic acid construct comprises a promoter operably ligated to a nucleotide sequence encoding a light-regulated repressor protein; preferably, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In some embodiments, the vector is a pET-28a-based plasmid, where the original LacI gene sequence in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with a LacO operon contained in the second nucleic acid construct.

[0052] In another embodiment, the present invention provides a kit comprising a host cell and an expression vector, wherein the host cell has an exogenous nucleotide sequence encoding the photoregulated repressor protein of the present invention, which is incorporated into its genome, and the expression vector comprises a second nucleic acid construct. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is preferably Escherichia coli. In some embodiments, the host cell is *E. coli* having an exogenous nucleotide sequence that encodes a light-regulated repressor protein, integrated into its genome. In some embodiments, the endogenous LacI gene of *E. coli* is disrupted. In some embodiments, both copies of the endogenous LacI gene of *E. coli* are replaced with the exogenous nucleotide sequence. In some embodiments, one copy of the endogenous LacI gene of *E. coli* is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out. In another embodiment, the present invention provides a kit comprising a host cell and an expression vector, wherein the host cell does not contain an exogenous nucleotide sequence encoding a photoregulated repressor protein incorporated into its genome; and the expression vector comprises a first nucleic acid construct and a second nucleic acid construct.

[0053] In some embodiments, the host cell is a prokaryotic cell such as E. coli. In some embodiments, the first and second nucleic acid constructs are located on different expression cassettes. In some embodiments, the first nucleic acid construct further comprises a promoter operably ligated to a nucleotide sequence encoding a light-regulated repressor protein; preferably, the promoter is selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In some embodiments, the vector is a pET-28a-based plasmid, where the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operator in pET-28a is replaced with a LacO operator gene contained in the second nucleic acid construct.

[0054] In some embodiments, in the second nucleic acid construct according to any of the above aspects, the LacO operator gene is selected from LacO1, LacOid, or a combination thereof. In some embodiments, in the second nucleic acid construct according to any of the above embodiments, the LacO operator gene includes at least one copy (e.g., 1 to 8 copies such as 1, 2, 3, 4, 5, 6, 7, 8 copies, or 3 to 5 copies) of the LacO1 operator sequence. In some embodiments, the LacO operator gene includes 3 to 5 copies of the LacO1 operator sequence. In some embodiments, the LacO1 operator sequence includes the sequence described in SEQ ID NO: 24.

[0055] In some embodiments, in the second nucleic acid construct according to any of the above embodiments, the LacO operator gene further comprises LacOid. In some embodiments, LacOid is located downstream of LacO1. In some embodiments, the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction. In some embodiments, in the second nucleic acid construct described in any of the above embodiments, the promoter is any promoter as long as it is recognized by an RNA polymerase encoded by an RNAP gene contained in the host chromosome, i.e., a host cell RNA polymerase is used; preferably selected from the T7 promoter of the T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter. In some embodiments, the kit described in any of the above embodiments is used to regulate the expression of a target gene.

[0056] In some embodiments, the light-controlled repressor protein is selected from the dark-responsive light-controlled repressor proteins of the present invention. In such embodiments, the kit is used for dark-induced gene expression, that is, it does not exert a repressive effect under dark conditions and allows the expression of the target gene, and preferably exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) to inhibit the expression of the target gene. In some embodiments, the light-controlled repressor protein is selected from the blue light-responsive light-controlled repressor proteins of the present invention. In such embodiments, the kit is used for blue light-induced gene expression, i.e., it does not exert a repressive effect under blue light (e.g., 430-495 nm such as 450-480 nm) and allows the expression of the target gene, and preferably exerts a repressive effect and inhibits the expression of the target gene under dark conditions.

[0057] Methods and uses for modulating target gene expression In other embodiments, the present invention provides the use of the photoregulated repressor proteins, nucleic acid constructs, vectors, host cells, systems, or kits of the present invention for regulating the expression of target genes. The regulation of target gene expression is performed in vitro. This use is for non-therapeutic purposes. The target gene in the present invention preferably encodes a target protein. The target protein is not limited. The target protein may be a polypeptide not naturally present in the host cell, i.e., a heterologous protein, or it may be naturally present in the host cell, i.e., a homologous protein of the host cell. The target protein in the present invention may be any bioactive peptide, including but not limited to enzymes, regulatory proteins, receptors, hormones, cytokines, membrane proteins, transport proteins, antigens, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, antibodies, or derivatives thereof. In some embodiments, the target protein may be any protein suitable for therapeutic or prophylactic purposes in mammals (e.g., humans).

[0058] Furthermore, it is well known to those skilled in the art that microorganisms can produce important compounds through anabolic pathways, and therefore, by regulating anabolic pathways within microbial cells, it is possible to alter (e.g., enhance) the synthetic output of metabolites. Thus, the light-controlled repressor proteins, nucleic acid constructs, vectors, host cells, systems, or kits of the present invention can also be used to regulate microbial metabolic pathways. In some embodiments, the metabolic pathways are endogenous or exogenous in E. coli and include, but are not limited to, the 3-HP and 1,3-PDO production pathways. Therefore, in some embodiments, the target genes related to the present invention also include genes involved in compound biosynthesis or microbial anabolic pathways, thereby achieving regulation of compound biosynthesis or microbial metabolic pathways.

[0059] In another embodiment, the present invention provides a method for regulating the expression of a target gene, (1) To provide a system of the present invention for regulating the expression of a target gene in a host cell; (2) Inducing the expression of the target gene by culturing host cells under conditions that enable the expression of the target gene. This provides a method that includes [something].

[0060] In some embodiments, step (1) comprises: providing an expression vector comprising a first nucleic acid construct and a second nucleic acid construct of the system; and introducing the expression vector into a host cell, the host cell having an exogenous nucleotide sequence encoding the photoregulated repressor protein of the present invention, which is incorporated into its genome. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-regulated repressor protein integrated into its genome. In some embodiments, the endogenous LacI gene of Escherichia coli is disrupted. In some embodiments, both copies of the endogenous LacI gene of Escherichia coli are replaced with an exogenous nucleotide sequence. In some embodiments, one copy of the endogenous LacI gene of Escherichia coli is replaced with an exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0061] In some embodiments, the first and second nucleic acid constructs are located on different expression cassettes. In some embodiments, the first nucleic acid construct further comprises a promoter operably linked to a nucleotide sequence encoding a light-regulated repressor protein; preferably, the promoter is any promoter recognized by an RNA polymerase encoded by an RNAP gene contained in the host chromosome, i.e., any promoter as long as the host cell's RNA polymerase is used, preferably selected from the group consisting of the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In some embodiments, the vector is a pET-28a-based plasmid, where the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with a LacO operon contained in the second nucleic acid construct.

[0062] In some embodiments, step (1) comprises: providing an expression vector comprising a second nucleic acid construct of the system; and introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding a photoregulatory repressor protein, which is incorporated into its genome. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-regulated repressor protein integrated into its genome. In some embodiments, the endogenous LacI gene of Escherichia coli is disrupted. In some embodiments, both copies of the endogenous LacI gene of Escherichia coli are replaced with an exogenous nucleotide sequence. In some embodiments, one copy of the endogenous LacI gene of Escherichia coli is replaced with an exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0063] In some embodiments, step (1) comprises: providing an expression vector comprising a first nucleic acid construct and a second nucleic acid construct of the system; and introducing the expression vector into a host cell, wherein the host cell does not contain exogenous nucleotide sequences encoding a photoregulated repressor protein that have been incorporated into its genome. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is Escherichia coli. In some embodiments, the first and second nucleic acid constructs are located on different expression cassettes. In some embodiments, the first nucleic acid construct further comprises a promoter operably ligated to a nucleotide sequence encoding a light-regulated repressor protein; preferably, the promoter is any promoter recognized by RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e., any promoter as long as the host cell's RNA polymerase is used, preferably selected from the group consisting of the wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter. In some embodiments, the vector is a pET-28a-based plasmid, where the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-regulated repressor protein of the present invention, and the original lac operator in pET-28a is replaced with a LacO operator gene contained in the second nucleic acid construct.

[0064] In some embodiments, the step of introducing the expression vector described in any of the above embodiments into host cells can be carried out by any means well known in the art, such as transfection, transformation, or transduction. In some embodiments, in the second nucleic acid construct described in any of the embodiments above, the LacO operator gene is selected from LacO1, LacOid, or a combination thereof. In some embodiments, in the second nucleic acid construct described in any of the embodiments above, the LacO operator gene includes at least one copy (e.g., 1 to 8 copies such as 1, 2, 3, 4, 5, 6, 7, 8 copies, or 3 to 5 copies) of the LacO1 operator sequence. In some embodiments, the LacO operator gene includes 3 to 5 copies of the LacO1 operator sequence. In some embodiments, the LacO1 operator sequence includes the sequence described in SEQ ID NO: 24.

[0065] In some embodiments, in the second nucleic acid construct described in any of the embodiments above, the LacO operator gene further comprises LacOid. In some embodiments, LacOid is located downstream of LacO1. In some embodiments, the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction. In some embodiments, in the second nucleic acid construct described in any of the embodiments above, the promoter is any promoter as long as it is recognized by an RNA polymerase encoded by an RNAP gene contained in the host chromosome, i.e., a host cell RNA polymerase is used; preferably selected from the group consisting of the T7 promoter of the T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter.

[0066] In some embodiments, the light-regulated repressor protein is selected from the dark-responsive light-regulated repressor proteins of the present invention; the induction conditions in step (2) include: culturing host cells in the dark to induce the expression of the target gene. In some embodiments, the method further includes culturing recombinant host cells under blue light (e.g., 430–495 nm, such as 450–480 nm) to inhibit the expression of the target gene. In some embodiments, the light-controlled repressor protein is selected from the blue light-responsive light-controlled repressor proteins of the present invention; the induction conditions in step (2) include culturing host cells under blue light (e.g., 430–495 nm, such as 450–480 nm) to induce the expression of the target gene. In some embodiments, the method further includes culturing host cells under dark conditions to inhibit the expression of the target gene. In some embodiments, the blue light referred to herein refers to light with a wavelength of 430–495 nm (e.g., 450–480 nm). The blue light referred to herein may be emitted from light sources such as LED panels, light strips, and lamps.

[0067] In some embodiments, an expression vector comprising a first nucleic acid construct and a second nucleic acid construct can be obtained by the following steps: providing a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid construct and the second expression cassette comprises a promoter, a LacO operator gene, and a cloning site (e.g., a multi-cloning site MCS) for incorporating a target gene; and providing a recombinant expression vector into which the target gene is incorporated by incorporating the target gene into the cloning site. In some embodiments, an expression vector comprising a second nucleic acid construct can be obtained by the following steps: providing a vector comprising a promoter, a LacO operator gene, and a cloning site (e.g., a multi-cloning site MCS) for incorporating a target gene; and providing a recombinant expression vector into which the target gene is incorporated by incorporating the target gene into the cloning site. In some embodiments, the method further includes collecting, separating, and / or purifying the expression product of the target gene (e.g., the target protein).

[0068] Beneficial effects The novel light-controlled repressor protein OptoLacI provided by this invention not only satisfies the requirements for single-component regulation of gene expression but also achieves high temporal and spatial resolution of gene expression regulation. It also combines two systems: a blue light-inducible system and a dark-inducible system. OptoLacI and the operator sequence form a modular light-controlled system that is ready for immediate use and exhibits broad system compatibility. This invention provides a light-controlled E. coli gene expression system based on a light-controlled repressor protein. This system includes tool strains with various induction intensities. This induction system allows for the adjustment of target gene expression intensity by changing the frequency and intensity of blue light irradiation. The blue light-inducible gene expression system of this invention exhibits the advantage of extremely precise induction with minimal expression leakage. The dark-light-inducible gene expression system of this invention shows excellent application effects in protein expression and has an induction effect comparable to IPTG induction. The dark-light-inducible gene expression system of this invention shows excellent application effects in metabolic regulation, providing more precise control of metabolic pathways and achieving improved yield of target products of dark-inducible synthesis compared to the IPTG induction system. Both the dark-light-inducible and blue light-inducible gene expression systems of this invention exhibit excellent spatial precision. [Examples]

[0069] The present invention will be described in conjunction with the following non-limiting embodiments. Those skilled in the art will understand that the examples are for illustrative purposes only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the test methods in the examples are conventional. Where no specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed. All reagents or equipment whose manufacturers are not specified are commercially available conventional products.

[0070] (Example 1) Design of light-controlled repressor proteins To facilitate the screening of light-regulated repressor proteins, green fluorescent protein (GFP) was used as a reporter gene. The restriction endonuclease sites at the 5' and 3' ends of green fluorescent protein are NheI and XhoI, respectively. Plasmids containing the GFP gene were amplified, and the plasmid containing the GFP gene and the pET-28a vector (purchased from Novagen) were double-digested with NheI and XhoI (Table 1). Here, the pET-28a vector had a target transcription module (i.e., T7 promoter-lac operator-multicloning site) and contained the repressor gene lacI module. Ligation was performed using T4 DNA ligase (Table 2). The ligation reaction system was transformed into competent DH5α cells and cultured overnight at 37°C. The following day, single clones were collected, cultured, and sequenced. The correctly sequenced plasmid was named pZH37, and the GFP gene was inserted into the multicloning site.

[0071] [Table 1]

[0072] [Table 2]

[0073] Compared to wild-type LacI, when the mutant W220F was introduced into the repressor protein LacI, the resulting LacI W220F The inhibitory ability was enhanced, and the IPTG-induced GFP leak expression level was reduced to one-tenth. Therefore, in order to construct a strictly photoregulated gene expression system, LacI of the pZH37 plasmid was modified. W220F The mutant plasmid was mutated and named pZH49. Furthermore, the operator gene (LexA opIt has been reported that increasing the number of repeats in the lac operon can effectively enhance the precision of light-regulated gene expression systems. Therefore, the number of tandem repeats in the LacO1 gene in the lac operon of the pZH37 plasmid was mutated to 3, and the resulting plasmid was named pZH39. On the other hand, based on plasmid pZH37, plasmid pZH44 was constructed, which has the LacIW220F mutant as the repressor gene lacI module and 3*LacO1 as the lac operon portion. Using GFP as a reporter gene, the fluorescence intensity of plasmids pZH37, pZH49, and pZH44 in BL21(DE3) cells was tested, thereby providing a basic template for constructing a strictly light-regulated gene expression system. Plasmids pZH37, pZH49, and pZH44 were used to transform BL21(DE3) competent cells. After incubation in an ice bath for 30 minutes, the cells were subjected to heat shock at 42°C for 90 seconds, incubated in an ice bath for 2 minutes, and 900 μl of LB medium was added to the competent cells. The cells were incubated in a shaker at 37°C for 1 hour. Appropriate amounts of bacterial culture were spread onto kanamycin-containing solid LB plates and incubated overnight at 37°C. The following day, single colonies were collected, and the effect of different plasmids on the strictness of IPTG induction in liquid LB was tested.

[0074] Compared to wild-type LacI, the W220F mutant reduces IPTG-induced expression leak while simultaneously increasing GFP expression, which is related to LacI W220F This has shown to be beneficial in reducing IPTG-induced leak. However, when the three operator gene LacO1 tandem repeat was introduced, GFP expression levels remained unchanged, and IPTG-induced leak also remained largely unchanged. W220F When the 3*LacO1 mutation was introduced simultaneously, GFP expression levels were unaffected, but IPTG-induced GFP leak was further reduced. Therefore, LacI W220F Plasmid pZH44, which contains both and 3*LacO1, was used as a template for photo-controlled modification.

[0075] The repressor protein LacI, derived from E. coli strain K12, consists of 360 amino acids. LacI molecules bind to each other to form tetramers. Amino acid residues 1-59 specifically bind to the operator gene, while amino acids 340-360 mediate tetramer formation. Based on the principle of structural change of the light-regulating element and the structure of the repressor protein LacI (PDB ID: 1LBG), the light-regulating element LOV2 was rationally inserted into the solvent exposure loops, including loops 1, 2, and 3. The amino acid sequences of the light-regulating element AsLOV2 (SEQ ID NO: 1) or its mutant cpLOV27 (SEQ ID NO: 2) were obtained by gene synthesis. The LOV2 gene was constructed in the LacI candidate loop region of pZH44 using the Gibson assembly method (Table 3). The Gibson assembly product transformed DH5α-competent cells, incubated in an ice bath for 30 minutes, then subjected to heat shock treatment at 42°C for 90 seconds, and further incubated in an ice bath for 2 minutes. 900 μl of LB medium was added to competent cells, and the cells were incubated in a shaker at 37°C for 1 hour. Subsequently, an appropriate amount of bacterial culture was spread onto a kanamycin-containing solid LB plate and incubated overnight at 37°C.

[0076] [Table 3] The following day, single clones were collected and cultured in kanamycin-containing LB medium at 37°C for 8-9 hours. Plasmids were extracted and sent to a sequencing company for sequencing. Plasmids that were successfully sequenced were named pZH36, pZH46, pZH47, pZH48, pZH50, pZH51, pZH52, pZH53, pZH54, pZH55, pZH56, pZH57, pZH58, pZH62, pZH63, pZH64, and pZH65 (Table 4).

[0077] [Table 4]

[0078] (Example 2) Screening of light-regulated repressor proteins To screen for LacI mutants with optogenetic activity, pZH44 was used as a control plasmid. BL21(DE3) competent cells were transformed with 200 ng each of the control and test plasmids (Table 4), incubated on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, followed by incubation on ice for 2 minutes. 900 μl of LB medium was added to the competent cells and incubated at 37°C for 1 hour using a shaker. Subsequently, an appropriate amount of bacterial culture was spread onto a kanamycin-containing solid LB plate and incubated overnight at 37°C. The following day, single colonies were collected and cultured in kanamycin-containing LB medium at 37°C for 12 hours using a shaker. The bacterial culture was then transferred with a 1% inoculation. The strains containing the control and test plasmids were each transferred to two 24-well plates, and the 24-well plates were heated under blue light (90 μmol / m²). 2 The cells were incubated for 12 hours each in the excitation wavelength ( / second) and in the dark. 1 mM IPTG was added to the control strain and induced in the dark for the same duration. Next, the green fluorescence intensity of the different strains was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 530 nm, and the photoresponse activity of the LacI mutants was evaluated by comparing the differences in GFP fluorescence values.

[0079] Strains containing LacI mutant plasmids pZH47, pZH36, pZH48, pZH50, pZH51, pZH55, pZH56, pZH57, and pZH62 exhibited dark-induced GFP expression activity. The LacI mutant corresponding to pZH36 showed superior induction characteristics, exhibiting the highest dark-induced increase and lower leak expression. Strains containing plasmids pZH63 and pZH58 exhibited blue light-induced activity, with the mutant corresponding to pZH58 showing more pronounced blue light-induced activity (Figure 1). The resulting vectors, containing the target transcription module and the light-regulated repressor protein gene, are called light-regulated vectors.

[0080] (Example 3) Construction of Light-Controlled Escherichia coli Strains Mutant LacI Corresponding to pZH36 M9 (OptoLacI D ) showed dark-responsive activity, while the mutant LacI M1 (OptoLacI L ) showed blue-light-responsive activity. Based on OptoLacI D and OptoLacI L , light-controlled Escherichia coli strains were constructed. First, BL21(DE3) containing 2 copies of wild-type LacI in its genome was used as the starting strain; after selecting 500 bp nucleotide sequences upstream and downstream of the wild-type LacI genome as homologous arms, the homologous arms were co-constructed with OptoLacI D in a donor plasmid, and the resulting plasmids were named pZH76 and pZH78. Based on a plasmid containing the Cas9 protein gene, a plasmid containing two N20 sequences was constructed, and the resulting plasmid was named pZH83. Using the CRISPR-Cas9 system, OptoLacI D was integrated into the wild-type LacI locus on the BL21(DE3) genome. The strain with both the upstream and downstream LacI loci on the genome replaced by 2 copies of OptoLacI D was named BL21_Dark_D v1 , and the strain with the upstream LacI knocked out and the downstream LacI replaced by OptoLacI D was named BL21_Dark_S V1 . Using the same method, donor plasmids pZH77 and pZH79 were constructed, the target plasmid was pZH84, and based on the blue-light-responsive mutant OptoLacI L , blue-light-responsive Escherichia coli strains were constructed. OptoLacI L was integrated into the wild-type LacI locus on the BL21(DE3) genome. The strain with both the upstream and downstream LacI loci replaced by 2 copies of OptoLacI L was named BL21_Light_D, and the strain with the upstream LacI knocked out and the downstream LacI replaced by OptoLacI LThe strain in which the gene was replaced was named BL21_Light_S (Figure 2). The strain obtained as a result of the integration of the light-regulating repressor protein gene into the genome was called a light-regulating strain.

[0081] (Example 4) Performance evaluation of light-controlled E. coli with dark-induced gene expression To evaluate the performance of strains exhibiting dark-induced gene expression and to optimize the effect of different numbers of LacO1 tandem copies on light control performance, plasmids containing different numbers of LacO1 tandem copies were constructed (Table 5). The repressor gene module of each plasmid was mutant LacI M9 These plasmids were transformed into dark-inducible light-controlled strains and cultured under blue light. Subsequently, they were transferred to 24-well plates at a 1% inoculation, and GFP expression was induced under dark conditions.

[0082] [Table 5]

[0083] The results are shown in Figure 3. In the dark-inducible light-controlled strain BL21_Dark_S, the strictness of dark-inducible GFP expression tended to increase with increasing LacO1 tandem copy number on the plasmid, and the induction multipliers for 3*LacO1 and 4*LacO1 were similar (Figure 3A). Dark-inducible light-controlled strain BL21_Dark_D v1 The accuracy of dark-induced GFP expression tended to increase with increasing LacO1 tandem copy number on the plasmid, and the induction multipliers for 3*LacO1 and 4*LacO1 were similar (Figure 3B).

[0084] (Example 5) Performance evaluation of light-controlled E. coli with blue light-induced gene expression. To evaluate the performance of strains exhibiting blue light-induced gene expression and to optimize the effect of different numbers of LacO1 tandem copies on light control performance, plasmids containing different numbers of LacO1 tandem copies were constructed (Table 6). The repressor gene module of each plasmid was mutant LacI M1 These plasmids were transformed into blue light-inducible photoregulatory strains, cultured in the dark, and then transferred to 24-well plates at a 1% inoculation. GFP expression was induced under blue light conditions.

[0085] [Table 6] The results are shown in Figure 4. In the blue light-inducible photoregulatory strain BL21_Light_S, the precision of blue light-inducible GFP expression tended to increase with increasing LacO1 tandem copy number on the plasmid, and the induction factors for 3*LacO1 and 4*LacO1 were similar (Figure 4A). In the blue light-inducible photoregulatory strain BL21_Light_D, the precision of blue light-inducible GFP expression tended to increase with increasing LacO1 tandem copy number on the plasmid, and the induction factors for 3*LacO1 and 4*LacO1 were similar (Figure 4B).

[0086] (Example 6) Optimization of induction intensity in dark-induced gene expression systems Dark-induced gene expression systems have greater potential applications in metabolic engineering and industrial production. However, current dark-induced gene expression systems suffer from low induction intensity. Therefore, we optimized the induction intensity of dark-induced gene expression systems. Saturation mutations were performed at key amino acid sites of LacI, and mutants with enhanced induction intensity were screened. After mutating these mutants into different mutants, the induction intensity increased from 2.9 to 6.9 times. When lysine at position 84 was mutated to glutamate, LacI W220F Compared to the previous method, the induction intensity was improved by approximately 6.5 times (Figure 5). Therefore, based on these results, the second-generation photoregulated repressor protein OptoLacI DV2(Sequence code 29) was obtained. BL21_Dark_S V1 and BL21_Dark_D v1 By mutating this region on the genome, a second version of the dark-induced gene expression strain, BL21_Dark_S, was created. V2 and BL21_Dark_D V2 I obtained it.

[0087] (Example 7) The effect of different promoters in dark-induced gene expression systems To evaluate the performance of strains capable of dark-induced gene expression and to characterize the effect of different promoters on light-controlled performance, plasmids containing different promoters (T7, tac, lac, lacUV5) were constructed. The repressor gene module of each plasmid was mutant LacI M9-K84E These plasmids were used in the dark-inducible light-controlled strain BL21_Dark_D. v1 The cells were transformed and cultured under blue light. Subsequently, the plasmid was transferred to a 24-well plate at a 1% inoculation, and GFP expression was induced under dark conditions. The results are shown in Figure 6. Dark-inducible light-controlled strain BL21_Dark_D v1 In this study, the dark-inducible GFP expression levels were similar for plasmids with different promoters, but the precision of dark-inducible GFP expression differed. These results indicate that the T7, tac, lac, and lacUV5 promoters are all suitable for dark-inducible gene expression systems.

[0088] (Example 8) Optimization of induction multipliers in dark-induced gene expression systems BL21_Dark_D V2 Based on this, the induction multiplier of the dark-induced gene expression system was further optimized by adjusting the LacO1 tandem copy number (1-8). The LacO1 tandem copy number was stepwise increased on plasmid pZH36, and then BL21_Dark_D V2The strains were transformed. After culturing the strains under blue light, they were transferred to 24-well plates with a 1% inoculation, and GFP expression was induced under dark conditions. The results showed that the optimal induction factor was achieved when 4*LacO1 (pML308) were present in tandem on the plasmid (Figure 7A). Simultaneously, a series of plasmids were constructed on plasmid pZH36 based on the LacO1 mutant LacOid (SEQ ID NO: 25), and their dark-induced effect was tested. The results showed that using LacO1 and LacOid in tandem resulted in a good dark-induced effect and improved the overall expression level (Figure 7B).

[0089] (Example 9) Application of dark-induced gene expression systems to protein expression In the practice of metabolic engineering, light-controlled systems for dark-induced protein production have significant advantages compared to light-controlled systems for blue light-induced protein production. Therefore, the inventors investigated the application of dark-induced gene expression systems to protein expression. Using glucose dehydrogenase (GDH, accession number: P40288), PETase (accession number: A0A0K8P0E4), and alkalin protease (accession number: P00780) as examples, the expression of different genes by light-controlled systems for dark-induced production was tested (Table 7). The plasmids corresponding to the target proteins were pML333, pML351, and pML353, respectively, with a LacO1 copy number of 4 in the target transcription module, and the light-controlled repressor protein module was OptoLacI DV2 That was the case.

[0090] [Table 7]

[0091] 200 ng of plasmid was collected and placed in BL21_Dark_D v1 Or BL21_Dark_D V2Competent cells were transformed, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then incubated on ice for 2 minutes. 900 μl of LB medium was added to the competent cells, and blue light intensity of 90 μmol / m² was applied. 2 The culture was incubated at 37°C for 1 hour in a light-controlled shaker at 0 / second. An appropriate amount of bacterial culture was spread onto a kanamycin-containing solid LB plate and placed in a blue light incubator (blue light intensity of 80 μmol / m²). 2 The cells were incubated overnight at 37°C at 80 μmol / m³ / second. The following day, a single colony was collected and incubated for 12 hours on a light-controlled shaker at 37°C in kanamycin-containing LB medium. The bacterial suspension was transferred to a 24-well plate with a 1% inoculation, and the 24-well plate was heated under blue light (80 μmol / m³ / second). 2 Set to / seconds, OD 600 The cells were incubated until the pH was 0.1-0.8. The 24-well plates were completely covered with aluminum foil for dark induction. Samples were taken at 0, 2, 4, 6, 9, and 12 hours. Samples from different time points were centrifuged at 12,000 rpm for 3 minutes to remove the LB medium, and the cells were resuspended in PBS. An appropriate amount of SDS-PAGE loading buffer was added to the resuspended bacterial suspension, and after boiling at 100°C for 20 minutes, 2 μl samples were taken and 12% SDS-PAGE analysis was performed. The photoregulatory effect of dark-induced gene expression was evaluated by SDS-PAGE.

[0092] The results are shown in Figure 8, with panel A representing GDH, panel B representing PETase, and panel C representing alkaline protease. As the dark induction time increased, the bands for the target proteins gradually became darker, clearer, and sharper. Minimal expression leakage was observed before dark induction (0 hours), indicating that the dark-inducible gene expression system has excellent applicability in protein expression.

[0093] (Example 10) Application of blue light-induced gene expression systems to protein expression To characterize the inductive properties of the blue light-induced gene expression system, the inventors also investigated its application to protein expression. Using acetyl-CoA C-acyltransferase (FadA) (accession number: A4XSM9) and malate dehydrogenase (MdhII) (accession number: D9PVI7) as examples, the expression of different genes by a photoregulatory system for blue light-induced production was tested (Table 8). The plasmids corresponding to the target proteins were pML359 and pML361, respectively, the LacO1 copy number in the target transcription module was 1, and the photoregulatory repressor protein module was LacI M1 That was the case.

[0094] [Table 8] BL21_Light_D competent cells were transformed with 200 ng of plasmid, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, then incubated on ice for 2 minutes; 900 μl of LB medium was added to the competent cells, and blue light intensity of 80 μmol / m² was used. 2 The culture was incubated at 37°C for 1 hour in a light-controlled shaker at 0 / second. An appropriate amount of bacterial culture was spread onto a kanamycin-containing solid LB plate and incubated overnight at 37°C. The next day, a single colony was taken and incubated in kanamycin-containing LB medium on a shaker for 12 hours at 37°C. The bacterial culture was transferred to a 24-well plate with a 1% inoculation. The 24-well plate was completely covered with aluminum foil and OD 600 The plate was incubated until it reached 0.1-0.8. Then, the plate was exposed to blue light (80 μmol / m³). 2The cells were placed at 12000 rpm for 9 hours before sampling. Samples from different time points were centrifuged at 12000 rpm for 3 minutes, the LB medium was removed, and the cells were resuspended in PBS. An appropriate amount of SDS-PAGE loading buffer was added to the resuspended bacterial suspension, and the mixture was boiled at 100°C for 20 minutes. 2 μl samples were taken and 12% SDS-PAGE analysis was performed to evaluate the light-controlled effect of dark-induced gene expression by SDS-PAGE. The results are shown in Figure 9, with panel A representing FadA and panel B representing MdhII. In the blue light-induced group, the target protein band was clear and distinct, while in the dark-controlled group, no clear protein band was observed at the corresponding position. This indicates that the blue light-induced gene expression system exhibited minimal expression leakage and good application performance.

[0095] (Example 11) Application of dark-induced gene expression systems in 1,3-propanediol production In the practice of metabolic engineering, resolving the conflict between host cell growth and the demand for culture substrates in target product synthesis is a significant challenge. Compared to light-controlled systems for blue light-induced protein production, light-controlled systems for dark-induced protein production are more rigorous and effectively control metabolic flux allocation by regulating the expression of key enzymes. Therefore, we investigated the application of dark-induced gene expression systems to metabolic flux regulation. Using 1,3-propanediol as an example, we tested the regulation of the metabolic flux of the 1,3-propanediol biosynthesis pathway by light-controlled systems for dark-induced production.

[0096] First, glycerol dehydratase (dhaB1234, having the gene sequence described in SEQ ID NO: 20) and glycerol dehydratase reactivating enzyme (gdrAB, having the gene sequences described in SEQ ID NOs: 21 and 22) were cloned from the genome of Klebsiella pneumoniae, and alcohol dehydrogenase (yqhD, having the gene sequence described in SEQ ID NO: 23) was cloned from the genome of Escherichia coli MG1655. These genes were then cloned into the expression vector pCDFDuet (purchased from Novagen), and a recombinant plasmid pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (shown in Figure 10, an expression cassette for the major genes in 1,3-propanediol biosynthesis) was constructed. The pCDFDuet-1 vector contained the T7 promoter and lac operon sequence.

[0097] BL21(DE3) and BL21_Dark_D v1 Through transformation, the 1,3-propanediol-producing strains BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA and BL21_Dark_D were produced. v1 The plasmids / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA were constructed. Specifically, 200 ng of plasmid was collected and used in BL21_Dark_D v1 Alternatively, BL21(DE3) competent cells were transformed, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and incubated on ice for 2 minutes. 900 μl of LB medium was added to the competent cells. BL21_Dark_D v1 Host cells containing blue light at a blue light intensity of 90 μmol / m² 2 The host cells containing BL21(DE3) were incubated at 37°C for 1 hour in a light-controlled shaker; and then directly recovered in the dark for 1 hour. An appropriate amount of bacterial culture was spread onto a kanamycin-containing solid LB plate. BL21_Dark_D v1 Host cells containing this substance were placed in a blue light incubator (80 μmol / m³). 2Host cells, including BL21(DE3), were incubated overnight at 37°C (at 0 / second). Fermentation comparison experiments were performed according to Table 8. BL21_Dark_D V1 For / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA, a single colony was collected and cultured in kanamycin-containing LB medium at 37°C on a light-controlled shaker for 12-18 hours to obtain a seed culture. The bacterial culture was transferred to a 24-well plate with a 1% inoculation. The 24-well plate was then heated under blue light (80 μmol / m³). 2 Set to / seconds, OD 600 The cultures were incubated until the concentration reached 0.1-0.6. One group's 24-well plate was completely covered with aluminum foil and incubated in the dark, while the other group's 24-well plate was incubated under blue light. Samples were taken 48 hours after fermentation. For BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA, a single colony was taken and incubated in kanamycin-containing LB medium on a shaker at 37°C for 12-18 hours to obtain a seed culture. The bacterial culture was transferred to a 24-well plate with a 1% inoculation. The 24-well plate was incubated under blue light (80 μmol / m³). 2 Set to / seconds, OD 600 The mixture was incubated until the pH was between 0.1 and 0.6. IPTG was added to one group's 24-well plate to induce 1,3-PDO synthesis, while IPTG was not added to the other group's 24-well plate. Samples were taken 48 hours after fermentation. 800 μL of fermentation broth was taken and centrifuged at 12,000 rpm. The supernatant was collected, and the yield of 1,3-PDO was determined using the HPLC method reported in the literature.

[0098] [Table 9]

[0099] The results are shown in Figure 11. While the difference between the induced and uninduced groups in the metabolic pathway flux of 1,3-propanediol in the BL21(DE3) strain under conventional IPTG control was not significant, the dark-induced expression system showed superior rigor. The induced group showed a significant increase in 1,3-propanediol production compared to the uninduced group, and BL21_Dark_D v1 1,3-propanediol production in the / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA induction group was significantly higher than in the BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA group. This indicates that dark-induced gene expression systems have a good application effect in regulating metabolic flux.

[0100] The results above demonstrate the following: (1) Photoregulation of protein function can be achieved by inserting a photoregulatory element (LOV2 and its variants) into the target protein sequence; (2) The photoregulatory E. coli gene expression system developed based on the photoregulatory repressor protein OptoLacI can be applied to protein production using dark conditions or blue light irradiation as inducers, has low induction costs and rapid induction procedures, and offers significant advantages in protein production; (3) The photoregulatory E. coli gene expression system developed based on the photoregulatory repressor protein OptoLacI can be applied to dynamically regulate the production of chemical substances; (4) Photoregulatory expression can be achieved by providing a photoregulatory vector to a photoregulatory strain, providing a common expression vector to a photoregulatory strain, and providing a photoregulatory vector to a common strain.

[0101] (Example 12) Comparison of OptoLacI and IPTG induction systems The light-controlled strain BL21_Dark_D was expressed using the dark-inducible gene expression plasmid pML308. V1 and BL21_Dark_D V2Each cell was transformed. A single clone was collected and cultured overnight under blue light. The next day, the cells were transferred to a 24-well plate with a 1% inoculation and cultured in the dark at 37°C for 12 hours. GFP fluorescence intensity was measured. After transforming the light-controlled strain BL21_Light_D with the plasmid pZH251, which induces gene expression under blue light, a single clone was collected and cultured overnight in the dark. The next day, the cells were inoculated into a 24-well plate with a 1% inoculation and cultured under blue light at 37°C for 9 hours. GFP fluorescence intensity was measured. Meanwhile, BL21(DE3) was transformed with pZH37, and a single clone was collected and cultured under blue light. 600 After culturing until the GFP level reached 0.5, 1 mM IPTG was added, and the cells were induced at 37°C for 12 hours. Subsequently, the GFP fluorescence intensity was measured.

[0102] The results are shown in Figure 12. The expression intensities of the dark-inducible gene expression system and the blue light-inducible gene expression system were lower than those of the IPTG-inducible system. However, in terms of induction factor, the blue light-inducible gene expression system showed an induction factor of 75 times, while IPTG induction showed an induction factor of only 16 times. This indicates that the blue light-inducible system has more precise induction characteristics and can avoid unwanted expression leaks. Furthermore, the dark-inducible gene expression system and the blue light-inducible gene expression system of the present invention are more adjustable, and protein expression levels and leak expression can be controlled by adjusting the blue light pulse mode and intensity, a feature that the IPTG system lacked. As described above, various embodiments of the present invention have been explained. The above description is illustrative and not exhaustive. It is not limited to the disclosed embodiments. Those skilled in the art will understand that various modifications and variations can be made in detail based on all the disclosed teachings, and all such modifications fall within the scope of the protection of the present invention. The entire scope of the present invention is given by the appended claims and their equivalents.

[0103] Sequence information Sequence ID 1 (AsLOV2) LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA Sequence ID 2 (cpLOV27) REGVMLIKKTAENIDEAAKELGGGSGGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAE Sequence ID 3 (LacI M1 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTP INSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQ LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA AVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID 4 (LacI M2 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPI NSIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIPPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQA LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA VKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ sequence number 5 (LacI M3 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPI NSIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIPPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAV LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA KGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ sequence number 6 (LacI M4 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPIN SIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLRLAGWHKYLTRNQIQPIAREGDFSAMSGFQQTMQMLNEGIPPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVK LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA GNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ sequence number 7(LacI M5 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPIN SIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIPPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKG LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA NQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ sequence number 8 (LacI M6 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINS IIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIPPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGN LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA QLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ sequence number 9 (LacI M7 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDG TRLGVEHLVALGHQQIALAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPN LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA TQTASPRALADSLQLARQVSRLESGQ sequence number 10 (LacI M8 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT RLGVEHLVALGHQQIALAGPLSSVSARLLRAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA QTASPRALADSLQLARQVSRLESGQ sequence number 11 (LacI M9 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT RLGVEHLVALGHQQIALAGPLSSVSARLLRAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQ LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA TASPRALADSLQLARQVSRLESGQ sequence number 12 (LacI M10 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTR LGVEHLVALGHQQIALAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQT LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA ASPRALADSLQLARQVSRLESGQ sequence number 13 (LacI M11 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTR LGVEHLVALGHQQIALAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA SPRALADSLQLARQVSRLESGQ sequence number 14(LacI M12 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRL GVEHLVALGHQQIALAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTAS LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA PRALADSLMQLARQVSRLESGQ sequence number 15 (LacI M13 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRL GVEHLVALGHQQIALAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASP LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA RALADSLMQLARQVSRLESGQ sequence number 16 (LacI M14 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDREGVMLIKKTAENIDEAAKELGGGSGGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAE QTPINSIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAERGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSMLQLARQVSRLESGQ sequence number 17(LacI M15 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQ REGVMLIKKTAENIDEAAKELGGGSGGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAE TPINSIIFSHEDGTRLGVEHLVALGHQQIALAGPLSSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSMLQLARQVSRLESGQ sequence number 18 (LacI M16 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQT REGVMLIKKTAENIDEAAKELGGGSGGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID 19 (LacI M17 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTP REGVMLIKKTAENIDEAAKELGGGSGGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAE INSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLLAGWHKYLTRNQIQPIAEREGDFSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID No. 20 (Glycerol dehydratase dhaB1234) * Sequence ID No. 21 (Glycerol dehydratase reactivating enzyme gdrA) * Sequence ID No. 22 (Glycerol dehydratase reactivating enzyme gdrB) MSLSPPGVRLFYDPRGHHAGAINELCWGLEEQGVPCQTITYDGGGDAAALGALARSSPLRVGIGLSASGEIALTHAQLPADAPLATGHVTDSDDHLRTLGANAGQLVKVLPLSERN* Sequence ID 23 (Alcohol dehydrogenase yqhD) MNNFNLHTPTRILFGKGAIAGLREQIPHDARVLITYGGGSVKKTGVLDQVLDALKGMDVLEFGGIEPNPAYETLMNAVKLVREQKVTFLLAVGGGSVLDGTKFIAAAANYPENIDPWHILQTGGKEIKSAIPMGCVLTLPATGSESNAGAVISRKTTGDKQAFHSAHVQPVFAVLDPVYTYTLPPRQVANGVVD AFVHTVEQYVTKPVDAKIQDRFAEGILLTLIEDGPKALKEPENYDVRANVMWAATQALNGLIGAGVPQDWATHMLGHELTAMHGLDHAQTLAIVLPALWNEKRDTKRAKLLQYAERVWNITEGSDDERIDAAIAATRNFFEQLGVPTHLSDYGLDGSSIPALLKKLEEHGMTQLGENHDITLDVSRRIYEAAR* Sequence ID 24 (LacO1 operator sequence) GGAATTGTGAGCGGATAACAATTCC Sequence ID 25 (LacOid operator sequence) AATTgtgagcgcTCAcaatt Sequence ID 26 (LacI WT Operator protein) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAI K SRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD W SAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID 27 (LacIW220F Operator protein) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAI K SRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD F SAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID 28 (LacI W220F+K84E Operator protein) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAI E SRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD F SAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ Sequence ID 29 (OptoLacI DV2 ) VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAI E SRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD F SAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQ LERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAA TASPRALADSLMQLARQVSRLESGQ Sequence ID 30 (EL222, a light-sensitive protein derived from Erythrobacter litralis) MLDMGQDRPIDGSGAPGADDTRVEVQPPAQWVLDLIEASPIASVVSDPRLADNPLIAINQ AFTDLTGYSEEECVGRNCRFLAGSGTEPWLTDKIRQGVREHKPVLVEILNYKKDGTPFRN AVLVAPIYDDDDELLYFLGSQVEVDDDQPNMGMARRERAAEEMLKTLSPRQLEVTTLVASG LRNKEVAARLGLSEKTVKMHRGLVMEKLNLKTSADLVRIAVEAGI Sequence ID 31 (LOV2, a photosensitive domain derived from Arabidopsis thaliana) GSPEFIEKNFVISDPRLPDNPIIFASDSFLELTEYSREEILGRNCRFLQGPETDQATVQKIRDAIRDQREITVQLINYTKSGKKFWNLFHLQPMRDQKGELQYFIGVQLDGSDHV

Claims

1. A light-regulated repressor protein comprising a LacI protein and an LOV domain inserted between adjacent amino acids in the loop 1, loop 2, or loop 3 region of the LacI protein.

2. The LOV domain is the LOV2 domain; Preferably, the LOV domain is selected from the LOV2 domain of Avena sativa phototropin 1 (AsLOV2), the photosensitive protein EL222 derived from Erythrobacter litoralis, the photosensitive domain LOV2 derived from Arabidopsis thaliana, or a variant thereof; Preferably, the mutant is a circular permutation mutant or a mutant with altered response rate; Preferably, the LOV domain is AsLOV2 containing, for example, the sequence described in Sequence ID No. 1; Preferably, the LOV domain is cpLOV27 containing, for example, the sequence described in SEQ ID NO: 2, the light-controlled repressor protein according to claim 1.

3. The insertion site is selected from the following amino acid positions of the LacI protein: between positions 335 and 336, between positions 314 and 315, between positions 315 and 316, between positions 316 and 317, between positions 334 and 335, between positions 336 and 337, between positions 337 and 338, between positions 338 and 339, or between positions 152 and 153; Preferably, the insertion site is between positions 335 and 336 of the LacI protein, the light-controlled repressor protein according to claim 1 or 2.

4. The insertion site is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, or between positions 153 and 154; Preferably, the insertion site is between positions 311 and 312 of the LacI protein, the light-controlled repressor protein according to claim 1 or 2.

5. The LacI protein is wild-type LacI protein; Preferably, the light-regulated repressor protein according to any one of claims 1 to 4, wherein the wild-type LacI protein comprises the sequence described in SEQ ID NO:

26.

6. The LacI protein is a modified LacI protein, and compared to the wild-type LacI protein, it comprises: (i) a substitution of the amino acid at position 220 with F, and / or (ii) an amino acid substitution selected from the substitution of the amino acid at position 84 with E, C, S, T, or I; Preferably, the modified LacI protein comprises the sequence described in SEQ ID NO: 27 or 28, or a sequence in which the amino acid at position 84 is substituted with C, S, T, or I compared to SEQ ID NO: 27 or 28, according to any one of claims 1 to 4.

7. The light-controlled repressor protein according to any one of claims 1 to 6, wherein the N-terminus and / or C-terminus of the LOV domain may be linked to the LacI protein via a peptide linker.

8. The sequence includes one of the sequences described in sequence numbers 11, 29, 6-8, 10, 12-14, and 16, or the sequence described in sequence number 3 or 17. Preferably, the sequence includes the sequence described in Sequence ID No. 11 or 29; Preferably, a light-controlled repressor protein according to any one of claims 1 to 7, comprising the sequence described in SEQ ID NO:

3.

9. A nucleic acid construct comprising a nucleotide sequence encoding a light-controlled repressor protein according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid construct described in claim 9.

11. The present invention comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises the nucleotide sequence encoding the photoregulated repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a target gene or a cloning site for incorporating the target gene; Preferably, the vector according to claim 10, wherein the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes.

12. The LacO operator gene contained in the second nucleic acid construct is selected from LacO1, LacOid, or a combination thereof. The vector according to claim 11.

13. The LacO operator gene contained in the second nucleic acid construct comprises at least one copy (for example, 1 to 8 copies such as 1, 2, 3, 4, 5, 6, 7, 8 copies, or for example, 3 to 5 copies) of the LacO1 operator sequence; Preferably, the vector according to claim 11 or 12, wherein the LacO1 operator sequence includes the sequence described in sequence number 24.

14. The LacO operator gene contained in the second nucleic acid construct further comprises LacOid; Preferably, the LacOid is located downstream of LacO1; Preferably, the vector according to claim 13, wherein the LacO operator gene comprises LacO1 and LacOid in the 5' to 3' direction.

15. The vector according to any one of claims 11 to 14, wherein the promoter included in the second nucleic acid construct is selected from the T7 promoter of T7 phage, the lac promoter, the tac promoter, and the lacUV5 promoter.

16. The first nucleic acid construct comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein; Preferably, the promoter is selected from the group consisting of a wild-type LacI promoter, a LacUV5 promoter, a tac promoter, and a trc promoter, as described in any one of claims 11 to 15.

17. comprising the nucleic acid construct according to claim 9, or the vector according to any one of claims 10 to 16; Preferably, it is a prokaryotic cell; Preferably, a host cell, which is E. coli.

18. Escherichia coli having an exogenous nucleotide sequence that encodes a light-controlled repressor protein according to any one of claims 1 to 8, which is incorporated into its genome; Preferably, the endogenous LacI gene of the E. coli is disrupted; Preferably, both copies of the endogenous LacI gene in the E. coli are substituted with the exogenous nucleotide sequence; Preferably, the host cell according to claim 17, wherein one copy of the endogenous LacI gene of Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

19. The host cell according to claim 18, wherein the Escherichia coli further comprises the vector according to any one of claims 11 to 16.

20. A system that regulates the expression of target genes, (1) A first nucleic acid construct comprising a nucleotide sequence encoding a light-controlled repressor protein according to any one of claims 1 to 8; (2) A second nucleic acid construct comprising a promoter, a LacO operator gene, and the target gene. A system that includes this.

21. The system according to claim 20, wherein the second nucleic acid construct is defined in any one of claims 11 to 15.

22. The second nucleic acid construct comprises a plurality of target genes arranged in tandem; Preferably, the system according to claim 20 or 21, wherein additional promoters may be inserted between the plurality of target genes.

23. The first nucleic acid construct further comprises a promoter operably ligated to the nucleotide sequence encoding the light-controlled repressor protein; Preferably, the promoter is selected from the group consisting of a wild-type LacI promoter, a LacUV5 promoter, a tac promoter, and a trc promoter, according to any one of claims 20 to 22.

24. (1) The system comprises at least two of the first nucleic acid constructs, one of which is incorporated into the genome of a host cell and the other which is present together with the second nucleic acid construct on a vector (e.g., an expression vector); at least two of the first nucleic acid constructs are either identical or different from each other; preferably, the vector is as defined in any one of claims 11 to 16; Or, (2) The first nucleic acid construct and the second nucleic acid construct are present on a vector (e.g., an expression vector); preferably, the vector is defined in any one of claims 11 to 16; Or, (3) The first nucleic acid construct is incorporated into the genome of a host cell, and the second nucleic acid construct is present on a vector (e.g., an expression vector); Preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli, according to any one of claims 20 to 23.

25. A kit comprising host cells and an expression vector, (1) The host cell has an exogenous nucleotide sequence encoded in its genome that encodes a light-regulated repressor protein according to any one of claims 1 to 8; the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises an exogenous nucleotide sequence encoding the light-regulated repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site for incorporating a target gene; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli, for example, as defined in claim 18; Or, (2) The host cell does not contain an exogenous nucleotide sequence encoding the light-regulated repressor protein incorporated into its genome; the expression vector comprises the first nucleic acid construct and the second nucleic acid construct; preferably, the host cell is a prokaryotic cell such as Escherichia coli; Or, (3) The host cell has an exogenous nucleotide sequence that encodes a light-regulated repressor protein according to any one of claims 1 to 8, incorporated into its genome, and the expression vector comprises the second nucleic acid construct; preferably the host cell is a prokaryotic cell; preferably the host cell is Escherichia coli, as defined in claim 18, for example. Preferably, the kit wherein the second nucleic acid construct is as defined in any one of claims 11 to 15.

26. A method for regulating the expression of a target gene, (1) Providing the system according to any one of claims 20 to 24 to a host cell; (2) Culture host cells under conditions that enable the expression of the target gene, and induce the expression of the target gene. Methods that include...

27. Step (1) is: (i) to provide an expression vector comprising the first nucleic acid construct and the second nucleic acid construct of the system; and to introduce the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding a photoregulatory repressor protein according to any one of claims 1 to 8, incorporated into its genome; preferably the expression vector is as defined in any one of claims 11 to 16; preferably the host cell is a prokaryotic cell; preferably the host cell is Escherichia coli, for example, as defined in claim 18. Or, (ii) to provide an expression vector comprising the first nucleic acid construct and the second nucleic acid construct of the system; and introducing the expression vector into a host cell, wherein the host cell does not contain an exogenous nucleotide sequence encoding the light-regulated repressor protein incorporated into its genome; preferably the expression vector is defined in any one of claims 11 to 16; preferably the host cell is a prokaryotic cell such as Escherichia coli, Or, (iii) The method of claim 26, comprising: (iii) providing an expression vector comprising the second nucleic acid construct of the system; and introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the photoregulatory repressor protein, incorporated into its genome; preferably the host cell is a prokaryotic cell; preferably the host cell is Escherichia coli, as defined in claim 18, for example.

28. The light-controlled repressor protein is defined in claim 3, and the induction conditions in step (2) include: culturing the host cells under dark conditions to induce the expression of the target gene; Preferably, the method according to claim 26 or 27, further comprising culturing recombinant host cells under blue light conditions to inhibit the expression of the target gene.

29. The light-controlled repressor protein is defined in claim 4, and the induction conditions in step (2) include: culturing the host cells under blue light conditions to induce the expression of the target gene; Preferably, the method according to claim 26 or 27, further comprising culturing the host cells under dark conditions to inhibit the expression of the target gene.

30. The use of a light-regulated repressor protein according to any one of claims 1 to 8, a nucleic acid construct according to claim 9, a vector according to any one of claims 10 to 16, a host cell according to any one of claims 17 to 19, a system according to any one of claims 20 to 24, or a kit according to claim 25 for regulating the expression of a target gene, Preferably, this includes regulating protein expression; Preferably, use involves regulation of metabolic pathways and / or biosynthesis.

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