New light-controlled repressor protein optolacl and use method thereof

Abstract

A new light-controlled repressor protein OptoLacl and a light-controlled gene expression system constructed on the basis of OptoLacl.

Description

Novel light-controlled repressor protein OptoLacI and its application method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application 202310707956.4 filed on June 14, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of protein engineering or synthetic biology in biotechnology, specifically to gene expression regulation, and more specifically to a novel light-controlled repressor protein OptoLacI and a light-controlled gene expression system constructed using OptoLacI, as well as the application of the light-controlled expression system in protein production and metabolic engineering. Background Art

[0004] In metabolic engineering practice, the introduction of exogenous pathways can lead to uneven distribution of material and energy flows, an imbalance in endogenous metabolic resources, and ultimately growth defects in production strains and limited product yields. Therefore, regulating metabolic pathways to improve metabolic imbalances is a common strategy in metabolic engineering practice. This includes enhancing precursor supply and substrate or product transport through gene overexpression, knocking out competing pathways, and overexpressing genes related to the target pathway. As the preferred host for the production of a wide range of compounds, Escherichia coli has broad applications in the fields of chemistry, food, pharmaceuticals, agriculture, and energy. In 1961, French scientists F. Jacob and J. Monod established the lactose operon model for Escherichia coli. The lac operon induction system based on this model is the most commonly used induction system in E. coli. In the absence of the lactose analog isopropyl β-d-1-thiogalactopyranoside (IPTG), the repressor protein LacI binds to the lac operator (LacO) upstream of the target gene, inhibiting transcription of the target gene. Upon IPTG binding to the repressor protein LacI, the LacI repressor dissociates from the lacO site, thereby initiating transcription of the target gene. In metabolic engineering, the addition of IPTG is the primary method for regulating metabolic pathways. However, in metabolic engineering and industrial protein production, the chemical inducer IPTG has drawbacks such as cytotoxicity, high cost, and irreversible induction, and it lacks precise temporal and spatial control. Consequently, researchers have been continuously searching for alternatives. With the continued development of optogenetic tools, light, with its low toxicity, easy availability, reversible control, and low cost, has become an ideal method for inducing gene expression. However, existing light-controlled gene expression systems face problems such as complex control components, complex operations, and imperfect bidirectional regulatory tools.

[0005] SUMMARY OF THE INVENTION

[0006] In response to the problems of cytotoxicity, high cost, and irreversible regulation in the existing chemical induction of Escherichia coli mentioned above, as well as the problems of complex control components, complex operation, and imperfect bidirectional regulation tools faced in the light-controlled Escherichia coli gene expression system, the present invention primarily provides a new light-controlled repressor protein OptoLacI, and develops two sets of light-controlled gene expression systems based on OptoLacI to achieve bidirectional induction by blue light and darkness. It has high temporal and spatial resolution and has excellent characteristics in protein expression and metabolic engineering.

[0007] In one aspect, 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.

[0008] In certain embodiments, the LOV domain is a LOV2 domain; preferably, the LOV domain is selected from the LOV2 domain (AsLOV2) of the oat phytochrome 1 gene, the photosensitive protein EL222 from Staphylococcus australis, the photosensitive domain LOV2 from Arabidopsis thaliana, or mutants thereof; preferably, the mutant is a cyclic rearrangement mutant or a mutant with altered response speed; preferably, the LOV domain is AsLOV2, for example, comprising the sequence shown in SEQ ID NO: 1; preferably, the LOV domain is cpLOV27, for example, comprising the sequence shown in SEQ ID NO: 2.

[0009] In certain embodiments, the insertion position 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, between positions 152 and 153; preferably, the insertion position is selected from between positions 335 and 336 of the LacI protein.

[0010] In certain embodiments, the insertion position is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, between positions 153 and 154; preferably, the insertion position is selected from between positions 311 and 312 of the LacI protein.

[0011] In certain embodiments, the LacI protein is wild-type; preferably, the wild-type LacI protein comprises the sequence shown in SEQ ID NO:26.

[0012] In certain embodiments, the LacI protein is a modified LacI protein, which comprises an amino acid substitution selected from the following compared to the wild-type LacI protein: (i) the amino acid at position 220 is substituted with F, and / or, (ii) the amino acid at position 84 is substituted with E, C, S, T, or I; preferably, the wild-type LacI protein comprises the sequence shown in SEQ ID NO: 26; preferably, the modified LacI protein comprises the sequence shown in SEQ ID NO: 27 or 28, or comprises 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.

[0013] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is optionally connected to the LacI protein via a peptide linker.

[0014] In certain embodiments, the light-controlled repressor protein comprises the sequence shown in any one of SEQ ID NOs: 11, 29, 6-8, 10, 12-14, 16 or comprises the sequence shown in SEQ ID NO: 3 or 17; preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO: 11 or 29; preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO: 3.

[0015] In one aspect, the present invention provides a nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein described above.

[0016] In one aspect, the present invention provides a vector comprising the nucleic acid construct described above.

[0017] In certain embodiments, the vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises the nucleotide sequence encoding the light-controlled repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a target gene or a cloning site for integrating the target gene; preferably, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes.

[0018] In certain embodiments, the second nucleic acid construct comprises a LacO operator selected from LacO1, LacOid, or a combination thereof.

[0019] In certain embodiments, the LacO operator gene contained in the second nucleic acid construct comprises at least one copy (e.g., 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, for example 3 to 5) of a LacO1 operator sequence; preferably, the LacO1 operator sequence comprises the sequence shown in SEQ ID NO: 24.

[0020] In certain embodiments, the LacO operator contained in the second nucleic acid construct further contains LacOid; preferably, the LacOid is located downstream of LacO1; preferably, the LacO operator contains LacO1 and LacOid from 5' to 3' direction.

[0021] In certain embodiments, the promoter comprised by the second nucleic acid construct is selected from the group consisting of T7 promoter of T7 phage, lac promoter, tac promoter, and lacUV5 promoter.

[0022] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein; preferably, the promoter is selected from the wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter.

[0023] In one aspect, 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.

[0024] In certain embodiments, the host cell is Escherichia coli, and the Escherichia coli has an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated into its genome; preferably, the endogenous LacI gene of the Escherichia coli is disrupted; preferably, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0025] In certain embodiments, the E. coli further comprises the vector comprising the first nucleic acid construct and the second nucleic acid construct as described above.

[0026] In one aspect, the present invention provides a system for regulating the expression of a gene of interest, comprising:

[0027] (1) a first nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein described above;

[0028] (2) A second nucleic acid construct comprising a promoter, a LacO operator, and a second nucleic acid construct of a target gene.

[0029] In certain embodiments, the second nucleic acid construct is as defined in any of the embodiments of any aspect above.

[0030] In certain embodiments, the second nucleic acid construct comprises a plurality of target genes arranged in tandem; preferably, an additional promoter is optionally inserted between the plurality of target genes.

[0031] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein; preferably, the promoter is selected from the wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter.

[0032] In certain embodiments, the system comprises at least two of the first nucleic acid constructs, one of which is integrated into the genome of the host cell, and the other and the second nucleic acid construct are present on a vector (e.g., an expression vector); wherein the at least two first nucleic acid constructs are identical to or different from each other; preferably, the vector is the vector comprising the first nucleic acid construct and the second nucleic acid construct as described above.

[0033] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are present on a vector (eg, an expression vector); preferably, the vector is the vector comprising the first nucleic acid construct and the second nucleic acid construct as described above.

[0034] In certain embodiments, the first nucleic acid construct is integrated into the genome of the host cell and the second nucleic acid construct is present on a vector (eg, an expression vector).

[0035] In certain embodiments, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli.

[0036] In one aspect, the present invention provides a kit comprising a host cell and an expression vector, wherein:

[0037] (1) The host cell has an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated into its genome; and the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises the exogenous nucleotide sequence encoding the light-controlled repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site for integrating a target gene; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli;

[0038] or,

[0039] (2) The host cell has an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated 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;

[0040] or,

[0041] (3) The host cell has not integrated the exogenous nucleotide sequence encoding the light-controlled repressor protein into its genome; and 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.

[0042] In certain embodiments, the second nucleic acid construct is as defined in any of the embodiments of any aspect above.

[0043] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein; preferably, the promoter is selected from the wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter.

[0044] In certain embodiments, (1) or (2) the host cell is Escherichia coli, and the Escherichia coli has an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated into its genome; preferably, the endogenous LacI gene of the Escherichia coli is destroyed; preferably, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0045] In one aspect, the present invention provides a method for regulating the expression of a gene of interest, comprising:

[0046] (1) providing the system described above in a host cell;

[0047] (2) Cultivating the host cell under conditions that allow expression of the target gene and inducing expression of the target gene.

[0048] In certain embodiments, step (1) comprises:

[0049] (i) providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated into its genome; preferably, the expression vector is the vector comprising the first nucleic acid construct and the second nucleic acid construct described above; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli;

[0050] or,

[0051] (ii) providing an expression vector comprising the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises the exogenous nucleotide sequence encoding the light-controlled repressor protein integrated into its genome; preferably, the host cell is a prokaryotic cell; preferably, the host cell is Escherichia coli;

[0052] or,

[0053] (iii) providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell does not contain the exogenous nucleotide sequence encoding the light-controlled repressor protein integrated into its genome; preferably, the expression vector is the vector comprising the first nucleic acid construct and the second nucleic acid construct as described above; preferably, the host cell is a prokaryotic cell, such as Escherichia coli.

[0054] In certain embodiments, (i) or (ii) the host cell is Escherichia coli, and the Escherichia coli has an exogenous nucleotide sequence encoding the light-controlled repressor protein described above integrated into its genome; preferably, the endogenous LacI gene of the Escherichia coli is disrupted; preferably, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence; preferably, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0055] In certain embodiments, the insertion position 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, and between positions 152 and 153; preferably, the insertion position is selected from between positions 335 and 336 of the LacI protein; the induction conditions described in step (2) include: culturing the host cell under dark conditions to induce the expression of the target gene; preferably, the method further includes culturing the recombinant host cell under blue light conditions to inhibit the expression of the target gene.

[0056] In certain embodiments, the insertion position in the light-controlled repressor protein is selected from the following amino acid positions of the LacI protein: between positions 311 and 312, and between positions 153 and 154; preferably, the insertion position is selected from between positions 311 and 312 of the LacI protein; the induction conditions described in step (2) include: culturing the host cells under blue light conditions to induce the expression of the target gene; preferably, the method further includes culturing the host cells under dark conditions to inhibit the expression of the target gene.

[0057] In one aspect, the present invention provides the light-controlled repressor protein, nucleic acid construct, vector, host cell, system, or kit described above, for use in regulating the expression of a target gene; preferably, the use includes regulating protein expression; preferably, the use includes regulating metabolic pathways and / or biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The invention is further described in the following non-limiting drawings:

[0059] Figure 1: Screening results of photoswitchable LacI mutants.

[0060] Figure 2: Schematic diagram of the light-controlled Escherichia coli genome structure.

[0061] Figure 3: Effect of the number of tandem LacO1 on the dark-inducible gene expression strain.

[0062] Figure 4: Effect of the number of tandem LacO1 on blue light-inducible gene expression strains.

[0063] Figure 5: Optimization results of the induction intensity of the dark-inducible gene expression system.

[0064] Figure 6: Effects of different promoters on the darkness-inducible gene expression system.

[0065] Figure 7: Optimization results of the induction multiple of the dark-inducible gene expression system.

[0066] Figure 8: Results of using the dark-inducible gene expression system for protein expression. Panel A shows the expression of alkaline protease (36.8 kD); Panel B shows the expression of PETase (29.0 kD); and Panel C shows the expression of GDH (29.4 kD). M represents protein marker (in kD). The numbers 0, 2, 4, 6, 9, and 12 represent the sample numbers of cell lysates obtained after dark induction for different times (0 h, 2 h, 4 h, 6 h, 9 h, and 12 h), respectively.

[0067] Figure 9: Schematic illustration of the application of the blue light-inducible gene expression system in protein expression. Panel A shows the expression results of FadA (41.6 kD); Panel B shows the expression results of MdhII (35.9 kD). M represents protein marker (unit: KD), L represents cell lysate sample after 9 hours of blue light induction, and L represents cell lysate sample after 9 hours of dark culture.

[0068] Figure 10: Schematic diagram of the expression cassettes of key genes for 1,3-propanediol biosynthesis.

[0069] Figure 11: Application of dark induction system in metabolic pathway flux control. No IPTG was added during the fermentation of S1-IPTG:BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA; S1-IPTG:BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA was added at OD 600 =0.1-0.6 when adding IPTG; S2LL:BL21_Dark_D v1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA Fermentation was always carried out under blue light; S2LD:BL21_Dark_D v1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA at OD 600 =0.1-0.6, switch to light-proof state.

[0070] Figure 12: Comparison of GFP expression using the light-controlled gene expression system and the IPTG-inducible system.

[0071] Detailed Description of the Invention

[0072] definition

[0073] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the following definitions and explanations of relevant terms are provided.

[0074] As used herein, the term "comprising" and its variations are used synonymously with the term "including" and its variations and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments and are also disclosed.

[0075] As used herein, the articles "a," "an," and "the" mean "at least one," unless the context in which the article is used clearly indicates otherwise.

[0076] As used herein, the terms "Lac repressor" or "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. Herein, when referring to the amino acid position of the LacI protein, the sequence shown in SEQ ID NO: 26 is used for description. For example, the expression "amino acid residue at position 335 of the LacI protein" refers to the amino acid residue at position 335 of the sequence shown in SEQ ID NO: 26 and its corresponding position. The corresponding position refers to the position in the sequence to be compared that is equivalent to the specific amino acid position in SEQ ID NO: 26 when the sequence to be compared is optimally aligned with SEQ ID NO: 26 (i.e., to obtain the highest percentage identity).

[0077] As used herein, the term "LOV domain" refers to a light-oxygen-voltage (LOV) domain, typically derived from a light-sensing protein from plants, algae, or bacteria. Preferably, the LOV domain is a LOV2 domain, preferably from oats, Staphylococcus amurensis, or Arabidopsis thaliana. In certain exemplary embodiments, the LOV domain has the amino acid sequence of SEQ ID NO: 1, 30, or 31, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0078] As used herein, the term "circular permutation" refers to the process of connecting the original N-terminus and C-terminus of a protein through a linker and introducing a new terminus by cleaving the original peptide bond of the protein. The new protein thus formed is called a circularly permuted mutant.

[0079] As used herein, the term "gene" is broadly used to refer to a DNA nucleic acid associated with a biological function. As used herein, a "target gene" refers to any nucleic acid sequence that may and / or will be meaningful in controlling its transcription level.

[0080] As used herein, the term "transcription" refers to the synthesis of RNA from a DNA template; the term "translation" refers to the synthesis of a polypeptide from an mRNA template. Transcription and translation are collectively referred to as "expression."

[0081] As used herein, the term "operably linked" refers to the functional connection of elements, wherein the elements are in a relationship that allows them to operate in an intended manner. For example, in the case of a promoter, the promoter is functionally linked to the gene of interest such that the promoter sequence is able to initiate transcription of the gene of interest.

[0082] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. One type of vector is an episomal vector, i.e., a nucleic acid capable of extrachromosomal replication. Another type of vector is an integrative vector, which is designed to recombine with the genetic material of the host cell. Vectors typically contain one or more restriction endonuclease recognition sites and / or sites for site-specific recombination, at which exogenous DNA fragments can be cleaved and ligated into the vector. The vector may contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0083] As used herein, the term "expression cassette" refers to a combination of a gene to which control elements are operably linked for expression. Control elements may include, for example, a promoter sequence, a start codon, a stop codon, and a terminator. The expression cassette may contain additional regulatory sequences and other sequences, such as enhancers, signal sequences, enhancers, introns, IRES sequences, and the like.

[0084] As used herein, the term "promoter" refers to an expression control element that allows RNA polymerase binding and initiation of transcription.

[0085] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells, fungal cells, insect cells, mammalian cells, and plant cells.

[0086] Light-operated repressor protein

[0087] The inventors have completed the present invention based on the following insights and findings after research, and specifically provide the following aspects.

[0088] The Lac repressor protein (LacI) regulates the transcription of genes required for lactose metabolism in Escherichia coli. Composed of 360 amino acid residues, LacI comprises two domains: the N-terminal domain (amino acids 1-59) contains a helix-turn-helix motif essential for DNA binding, while the C-terminal domain contains binding sites for chemical inducers and their analogs. The C-terminal 30 amino acids mediate tetramerization of the repressor protein. Two subunits of the LacI tetramer bind to the operator LacO1, while the other two bind to the operator LacO2 or LacO3, forming a loop between the operators and preventing RNA polymerase from binding to the promoter. Binding of LacI to the chemical inducer IPTG stabilizes the protein in a stable conformation with low affinity for the operator, resulting in reduced operator occupancy and a derepressed state of the repressor protein LacI, initiating transcription of downstream genes. The blue-light-responsive light-oxygen-voltage sensing domain 2 (LOV2) has a small molecular weight (100-140 amino acids) and its chromophore, flavin mononucleotide (FMN), is ubiquitous in various cell types. Therefore, LOV2 is a widely used light-controlled element. In various cell types, the LOV2 domain has been applied to reversibly and spatiotemporally manipulate cell signaling and behavior, including reversibly controlling intracellular protein degradation, protein-protein interactions, and protein localization. The LOV2 domain contains a Jα helix at its C-terminus. In the dark, the Jα helix forms a stable conformation within the β-sheet of the Per-Arnt-Sim (PAS) core domain. Upon illumination with blue light, covalent binding between the conserved cysteine ​​residue in the LOV2 domain and FMN causes a rotation of the conserved glutamine residue, ultimately leading to a conformational change in the Jα helix and its separation from the central β-sheet. Based on the repressor protein LacI and the light-controlled element LOV2, it is possible for us to mutate both and construct a new light-controlled repressor protein for precise regulation of gene expression.

[0089] In one aspect, the present invention provides a light-controlled 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.

[0090] In certain 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. References to amino acid positions of the LacI protein herein are made with reference to the sequence set forth in SEQ ID NO: 26.

[0091] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is directly linked to the LacI protein.

[0092] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is connected to the LacI protein via a peptide linker (eg, a peptide linker comprising one or several flexible amino acids).

[0093] LOV domain

[0094] Any LOV domain known in the art can be used in the present invention. In certain embodiments, the LOV domain is a LOV2 domain. LOV2 from different species all belong to the light-responsive domain and play the same role in different species. Furthermore, LOV2 from different species have highly similar three-dimensional structures and the corresponding allosteric mechanisms are the same. Therefore, LOV2 domains from different species are all suitable for use in the present invention.

[0095] In certain embodiments, the LOV domain is selected from the LOV2 domain of the oat phytochrome 1 gene (AsLOV2), the light-sensitive protein EL222 from Staphylococcus amurensis, the light-sensitive domain LOV2 from Arabidopsis thaliana, or mutants thereof.

[0096] In certain embodiments, the mutant is a cyclic rearrangement mutant. In this article, "cyclic rearrangement" has a meaning well known to those skilled in the art, which refers to connecting the original N-terminus and C-terminus of a protein by a linker and introducing a new terminus by cleaving the original peptide bond of the protein.

[0097] In certain embodiments, the mutant is a mutant with an altered response speed. Such mutants are known to those skilled in the art and typically include mutations selected from the following: V461I, V74I, L165I, wherein 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(2009)). Introducing mutations that alter LOV2's response speed does not change the mechanism and fact that it undergoes conformational changes in response to blue light, and thus these mutants are also applicable to the present invention.

[0098] In certain embodiments, the LOV domain is AsLOV2. In certain embodiments, the AsLOV2 comprises the amino acid sequence shown in SEQ ID NO:1.

[0099] In certain embodiments, the LOV domain is a circularly permuted mutant of AsLOV2. In certain embodiments, the circularly permuted mutant of AsLOV2 is cpLOV27, which comprises the amino acid sequence shown in SEQ ID NO:2.

[0100] In certain embodiments, the LOV domain is the light-sensitive protein EL222 from Staphylococcus aureus, for example comprising the amino acid sequence shown in SEQ ID NO:30.

[0101] In certain embodiments, the LOV domain is the photosensitive domain LOV2 from Arabidopsis thaliana, for example, comprising the amino acid sequence shown in SEQ ID NO:31.

[0102] LacI protein

[0103] In certain embodiments, the LacI protein is a wild-type LacI protein. In certain embodiments, the wild-type LacI protein comprises the sequence shown in SEQ ID NO:26.

[0104] In certain embodiments, the LacI protein is a modified LacI protein, which comprises an amino acid substitution selected from the following compared to the wild-type LacI protein: (i) the amino acid at position 220 is substituted with F, and / or, (ii) the amino acid at position 84 is substituted with E, C, S, T, or I.

[0105] In certain embodiments, the modified LacI protein comprises the sequence shown in SEQ ID NO: 27 or 28, or comprises the following sequence: the amino acid at position 84 is substituted with C, S, T, or I compared to SEQ ID NO: 27 or 28.

[0106] In certain embodiments, the modified LacI protein comprises the sequence shown in SEQ ID NO: 27 or 28.

[0107] Dark-responsive light-activated repressor proteins

[0108] In certain embodiments, the light-operated repressor protein of the present invention has darkness-responsive activity. As used herein, "dark-responsive activity" means that the light-operated repressor protein does not exert a repressive effect under dark conditions and allows expression of the target gene. The light-operated repressor protein preferably exerts a repressive effect under blue light (e.g., 430-495 nm, such as 450-480 nm) and inhibits expression of the target gene.

[0109] In certain embodiments, the insertion position in the dark-responsive light-operated repressor protein is selected from the following amino acid positions of the LacI protein: between positions 335 and 336 (such as Loop-2 (Q335-T336)), between positions 314 and 315 (such as Loop-1 (K314-G315)), between positions 315 and 316 (such as Loop-1 (G315-N316)), between positions 316 and 317 (such as Loop-1 (N316- Q317)), between positions 334 and 335 (such as Loop-2 (T334-Q335)), between positions 336 and 337 (such as Loop-2 (T336-A337)), between positions 337 and 338 (such as Loop-2 (A337-S338)), between positions 338 and 339 (such as Loop-2 (S338-P339)), between positions 152 and 153 (such as Loop-3 (D152-Q153)).

[0110] In certain embodiments, the insertion position in the darkness-responsive light-operated repressor protein is between positions 335 and 336 of the LacI protein (eg, Loop-2 (Q335-T336)).

[0111] In some embodiments, the light-operated repressor protein responsive to darkness comprises the amino acid sequence shown in any one of SEQ ID NOs: 11, 29, 6-8, 10, 12-14, 16. In some embodiments, the light-operated repressor protein responsive to darkness comprises the amino acid sequence shown in SEQ ID NO: 11 or 29.

[0112] Blue light-responsive light-activated repressor protein

[0113] In certain embodiments, the light-controlled repressor protein of the present invention has activity in response to blue light. As used herein, "activity in response to blue light" means that the light-controlled repressor protein does not exert a repressive effect under conditions of blue light (e.g., 430-495 nm, such as 450-480 nm) and allows expression of the target gene. The light-controlled repressor protein preferably exerts a repressive effect under dark conditions and inhibits expression of the target gene.

[0114] In certain embodiments, the insertion position in the blue light-responsive light-controlled repressor protein is selected from the following amino acid positions of the LacI protein: between positions 311 and 312 (such as Loop-1 (Q311-A312)), between positions 153 and 154 (such as Loop-3 (Q153-T154)).

[0115] In certain embodiments, the insertion position in the blue light-responsive light-controlled repressor protein is between positions 311 and 312 of the LacI protein (eg, Loop-1 (Q311-A312)).

[0116] In certain embodiments, the light-controlled repressor protein responsive to blue light comprises the amino acid sequence shown in SEQ ID NO: 3 or 17. In certain embodiments, the light-controlled repressor protein responsive to blue light comprises the amino acid sequence shown in SEQ ID NO: 3.

[0117] The light-controlled repressor protein of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology) or by chemical synthesis methods (such as the Fmoc solid phase method). The light-controlled repressor protein of the present invention is not limited by its production method.

[0118] Nucleic acid constructs and vectors

[0119] 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.

[0120] In another aspect, the present invention provides a vector comprising the nucleic acid construct as described above.

[0121] In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is a plasmid.

[0122] In certain 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 light-controlled repressor protein of the present invention, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a target gene.

[0123] In certain 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 light-controlled repressor protein of the present invention, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site for integrating the target gene (e.g., a multiple cloning site MCS).

[0124] In certain embodiments, the second nucleic acid construct comprises a LacO operator selected from LacO1, LacOid, or a combination thereof.

[0125] In certain embodiments, the LacO operator comprised in the second nucleic acid construct comprises at least one copy (e.g., 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, for example, 3 to 5) of a LacO1 operator sequence. In certain embodiments, the LacO operator comprised in the second nucleic acid construct comprises 3 to 5 copies of a LacO1 operator sequence. In certain embodiments, the LacO1 operator sequence comprises the sequence set forth in SEQ ID NO: 24.

[0126] In certain embodiments, the LacO operator comprised by the second nucleic acid construct comprises LacO1 and LacOid. In certain embodiments, the LacOid is located downstream of LacO1. In certain embodiments, the LacO operator comprises LacO1 and LacOid from the 5' to 3' direction.

[0127] In certain embodiments, the promoter included in the second nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter included in the second nucleic acid construct is selected from the T7 promoter of T7 phage, lac promoter, tac promoter, lacUV5 promoter.

[0128] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein of the present invention. In certain embodiments, the promoter comprised by the first nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter is selected from the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter.

[0129] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located on different expression cassettes of the same vector.

[0130] In certain embodiments, the vector is a pET-28a-based plasmid, wherein the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-controlled repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0131] host cells

[0132] In another aspect, the present invention provides a host cell comprising the nucleic acid construct or vector as described above.

[0133] In certain embodiments, the host cell is a prokaryotic cell.

[0134] In certain embodiments, the host cell is Escherichia coli.

[0135] In certain embodiments, the host cell is Escherichia coli, and the exogenous nucleotide sequence encoding the light-controlled repressor protein of the present invention is integrated into the genome of the Escherichia coli.

[0136] In certain embodiments, the endogenous LacI gene of the Escherichia coli is destroyed. In certain embodiments, the destruction includes: loss-of-function mutations (e.g., addition, deletion and / or substitution of one or more bases), deletion or substitution with an exogenous nucleotide sequence. "Loss-of-function mutation" refers to a mutation that causes the protein encoded and expressed by the mutant gene to lose its biological functional activity. 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 or substitution or addition of gene fragments), as long as the gene containing the loss-of-function mutation cannot produce or express a protein with biological functional activity.

[0137] In certain embodiments, both copies of the endogenous LacI gene of the E. coli are replaced with the exogenous nucleotide sequence.

[0138] In certain embodiments, one copy of the endogenous LacI gene of the E. coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0139] In certain embodiments, the integration of exogenous nucleotide sequences contained in the host cell and / or the disruption of endogenous genes can be achieved by any gene editing system well known to those skilled in the art. Exemplary gene editing systems include CRISPR / Cas, ZFN, TALEN, etc.

[0140] In certain embodiments, the Escherichia coli having an exogenous nucleotide sequence encoding the light-controlled repressor protein of the present invention integrated into its genome may further comprise the vector (eg, expression vector) comprising the first nucleic acid construct and the second nucleic acid construct as described above.

[0141] Systems for regulating target gene expression

[0142] In another aspect, the present invention provides a system for regulating the expression of a gene of interest, comprising:

[0143] (1) a first nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein of the present invention;

[0144] (2) A second nucleic acid construct comprising a promoter, a LacO operator, and a second nucleic acid construct of a target gene.

[0145] In certain embodiments, the second nucleic acid construct comprises a LacO operator selected from LacO1, LacOid, or a combination thereof.

[0146] In certain embodiments, the LacO operator comprised in the second nucleic acid construct comprises at least one copy (e.g., 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, for example, 3 to 5) of a LacO1 operator sequence. In certain embodiments, the LacO operator comprised in the second nucleic acid construct comprises 3 to 5 copies of a LacO1 operator sequence. In certain embodiments, the LacO1 operator sequence comprises the sequence set forth in SEQ ID NO: 24.

[0147] In certain embodiments, the LacO operator comprised by the second nucleic acid construct comprises LacO1 and LacOid. In certain embodiments, the LacOid is located downstream of LacO1. In certain embodiments, the LacO operator comprises LacO1 and LacOid from the 5' to 3' direction.

[0148] In certain embodiments, the promoter included in the second nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter included in the second nucleic acid construct is selected from the T7 promoter of T7 phage, lac promoter, tac promoter, lacUV5 promoter.

[0149] In certain embodiments, the second nucleic acid construct comprises a plurality of target genes arranged in tandem. In certain embodiments, an additional promoter is optionally inserted between the plurality of target genes.

[0150] In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled repressor protein of the present invention. In certain embodiments, the promoter comprised by the first nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter is selected from the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter.

[0151] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes of the same vector. In certain embodiments, the vector is a pET-28a-based plasmid, wherein the original gene sequence encoding LacI in pET-28a is replaced with a nucleotide sequence encoding the light-controlled repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0152] In certain embodiments, the light-controlled repressor protein is selected from the light-controlled repressor protein responsive to darkness of the present invention. In such embodiments, the system for regulating the expression of the gene of interest is a dark-inducible gene expression system, wherein the light-controlled repressor protein does not exert a repressive effect under dark conditions and allows the expression of the gene of interest, and preferably exerts a repressive effect under blue light conditions (e.g., 430-495 nm, such as 450-480 nm) and inhibits the expression of the gene of interest.

[0153] In certain embodiments, the light-controlled repressor protein is selected from the light-controlled repressor protein responsive to blue light of the present invention. In such embodiments, the system for regulating the expression of the target gene is a blue light-inducible gene expression system, and 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, and preferably exerts a repressive effect under dark conditions and inhibits the expression of the target gene.

[0154] Reagent test kit

[0155] On the other hand, 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-controlled repressor protein of the present invention integrated into its genome; and the expression vector comprises a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct comprises the exogenous nucleotide sequence encoding the light-controlled repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator gene, and a cloning site for integrating the target gene (e.g., a multiple cloning site MCS).

[0156] In certain embodiments, the host cell is a prokaryotic cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-controlled repressor protein integrated into its genome. In certain embodiments, the endogenous LacI gene of the Escherichia coli is destroyed. In certain embodiments, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence. In certain embodiments, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0157] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes. In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-operated repressor protein; preferably, the promoter is selected from the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In certain embodiments, the vector is a pET-28a-based plasmid, in which the original gene sequence encoding LacI in pET-28a is replaced with the nucleotide sequence encoding the light-operated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0158] On the other hand, 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-controlled repressor protein of the present invention integrated into its genome; and the expression vector comprises the second nucleic acid construct.

[0159] In certain embodiments, the host cell is a prokaryotic cell. In certain embodiments, preferably, the host cell is Escherichia coli.

[0160] In certain embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-operated repressor protein integrated into its genome. In certain embodiments, the endogenous LacI gene of the Escherichia coli is disrupted. In certain embodiments, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence. In certain embodiments, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0161] On the other hand, the present invention provides a kit comprising a host cell and an expression vector, wherein: the host cell has not integrated the exogenous nucleotide sequence encoding the light-controlled repressor protein into its genome; and the expression vector comprises the first nucleic acid construct and the second nucleic acid construct.

[0162] In certain embodiments, the host cell is a prokaryotic cell, such as Escherichia coli.

[0163] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes. In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-operated repressor protein; preferably, the promoter is selected from the wild-type LacI promoter, the LacUV5 promoter, the tac promoter, and the trc promoter. In certain embodiments, the vector is a pET-28a-based plasmid, in which the original gene sequence encoding LacI in pET-28a is replaced with the nucleotide sequence encoding the light-operated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0164] In certain embodiments, in the second nucleic acid construct of any of the above aspects, the LacO operator is selected from LacO1, LacOid, or a combination thereof.

[0165] In certain embodiments, in the second nucleic acid construct of any of the above aspects, the LacO operator comprises at least one copy (e.g., 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, for example, 3 to 5) of a LacO1 operator sequence. In certain embodiments, the LacO operator comprises 3 to 5 copies of a LacO1 operator sequence. In certain embodiments, the LacO1 operator sequence comprises the sequence set forth in SEQ ID NO: 24.

[0166] In certain embodiments, the second nucleic acid construct of any of the above aspects, wherein the LacO operator further comprises LacOid. In certain embodiments, the LacOid is located downstream of LacO1. In certain embodiments, the LacO operator comprises LacO1 and LacOid from the 5' to 3' direction.

[0167] In certain embodiments, the second nucleic acid construct described in any of the above aspects, wherein the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell; preferably selected from the T7 promoter of T7 phage, lac promoter, tac promoter, lacUV5 promoter.

[0168] In certain embodiments, the kit described in any one of the above aspects is used to regulate the expression of a target gene.

[0169] In certain embodiments, the light-controlled repressor protein is selected from the light-controlled repressor protein responsive to darkness of the present invention. In such embodiments, the kit is used for dark-induced gene expression, i.e., it does not exert a repressive effect under dark conditions and allows the expression of the target gene, and preferably exerts a repressive effect and inhibits the expression of the target gene under blue light (e.g., 430-495 nm, such as 450-480 nm).

[0170] In certain embodiments, the light-controlled repressor protein is selected from the blue light-responsive light-controlled repressor protein of the present invention. In such embodiments, the kit is used for blue light-induced gene expression, i.e., under blue light (e.g., 430-495 nm, such as 450-480 nm), it does not exert a repressive effect 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.

[0171] Method and application for regulating target gene expression

[0172] In another aspect, the present invention provides a light-controlled repressor protein, nucleic acid construct, vector, host cell, system, or kit of the present invention for use in regulating expression of a target gene. The regulation of target gene expression is performed in vitro. The use is for non-therapeutic purposes.

[0173] The target gene involved in the present invention preferably encodes a target protein. There are no restrictions on the target protein. The target protein can be a polypeptide that is not naturally present in the host cell, i.e., a heterologous protein, or can be natural to the host cell, i.e., a homologous protein of the host cell. The target protein involved in the present invention can be any peptide with biological activity, 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 certain embodiments, the target protein can be any protein suitable for the treatment or prevention of mammals (e.g., humans).

[0174] Furthermore, those skilled in the art are aware that microorganisms can produce important compounds through anabolism. Therefore, by regulating the anabolic pathways within microbial cells, the amount of metabolite synthesis can be altered (e.g., increased). Therefore, 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 certain embodiments, the metabolic pathways can be endogenous or exogenous to E. coli, including but not limited to pathways for the production of 3-HP and 1,3-PDO. Therefore, in certain embodiments, the target genes involved in the present invention also include genes involved in compound biosynthesis or microbial anabolic pathways, thereby achieving regulation of compound biosynthesis or microbial metabolic pathways.

[0175] In another aspect, the present invention provides a method for regulating the expression of a target gene, comprising:

[0176] (1) providing the system for regulating target gene expression of the present invention in a host cell;

[0177] (2) Cultivating the host cell under conditions that allow expression of the target gene and inducing expression of the target gene.

[0178] In certain embodiments, step (1) comprises: providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the light-controlled repressor protein of the present invention integrated into its genome.

[0179] In certain embodiments, the host cell is a prokaryotic cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-controlled repressor protein integrated into its genome. In certain embodiments, the endogenous LacI gene of the Escherichia coli is destroyed. In certain embodiments, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence. In certain embodiments, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0180] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes. In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-operated repressor protein; preferably, the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell, preferably selected from the wild-type LacI promoter, LacUV5 promoter, tac promoter, trc promoter. In certain embodiments, the vector is a pET-28a-based plasmid, in which the original gene sequence encoding LacI in pET-28a is replaced with the nucleotide sequence encoding the light-operated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0181] In certain embodiments, step (1) comprises: providing an expression vector comprising the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell comprises the exogenous nucleotide sequence encoding the light-controlled repressor protein integrated into its genome.

[0182] In certain embodiments, the host cell is a prokaryotic cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is an Escherichia coli having an exogenous nucleotide sequence encoding a light-controlled repressor protein integrated into its genome. In certain embodiments, the endogenous LacI gene of the Escherichia coli is destroyed. In certain embodiments, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence. In certain embodiments, one copy of the endogenous LacI gene of the Escherichia coli is replaced with the exogenous nucleotide sequence, and the other copy of the endogenous LacI gene is knocked out.

[0183] In certain embodiments, step (1) comprises: providing an expression vector comprising the first nucleic acid construct and the second nucleic acid construct in the system; introducing the expression vector into a host cell, wherein the host cell does not contain the exogenous nucleotide sequence encoding the light-controlled repressor protein integrated into its genome.

[0184] In certain embodiments, the host cell is a prokaryotic cell. In certain embodiments, the host cell is Escherichia coli.

[0185] In certain embodiments, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes. In certain embodiments, the first nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-operated repressor protein; preferably, the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell, preferably selected from the wild-type LacI promoter, LacUV5 promoter, tac promoter, trc promoter. In certain embodiments, the vector is a pET-28a-based plasmid, in which the original gene sequence encoding LacI in pET-28a is replaced with the nucleotide sequence encoding the light-operated repressor protein of the present invention, and the original lac operon in pET-28a is replaced with the LacO operator gene contained in the second nucleic acid construct.

[0186] In certain embodiments, the step of introducing the expression vector into the host cell described in any of the above embodiments can be performed by any means known in the art, such as transfection, transformation or transduction.

[0187] In certain embodiments, in the second nucleic acid construct described in any of the above embodiments, the LacO operator is selected from LacO1, LacOid, or a combination thereof.

[0188] In certain embodiments, in the second nucleic acid construct described in any of the above embodiments, the LacO operator comprises at least one copy (e.g., 1 to 8 copies, such as 1, 2, 3, 4, 5, 6, 7, 8, for example, 3 to 5 copies) of a LacO1 operator sequence. In certain embodiments, the LacO operator comprises 3 to 5 copies of a LacO1 operator sequence. In certain embodiments, the LacO1 operator sequence comprises the sequence set forth in SEQ ID NO: 24.

[0189] In certain embodiments, the second nucleic acid construct described in any of the above embodiments, wherein the LacO operator further comprises LacOid. In certain embodiments, the LacOid is located downstream of LacO1. In certain embodiments, the LacO operator comprises LacO1 and LacOid from the 5' to 3' direction.

[0190] In certain embodiments, the second nucleic acid construct described in any of the above embodiments, wherein the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell, is preferably selected from the T7 promoter of T7 phage, lac promoter, tac promoter, and lacUV5 promoter.

[0191] In certain embodiments, the light-controlled repressor protein is selected from the darkness-responsive light-controlled repressor protein of the present invention; and the induction conditions described in step (2) include: culturing the host cell under dark conditions to induce expression of the target gene. In certain embodiments, the method further includes culturing the recombinant host cell under blue light (e.g., 430-495 nm, such as 450-480 nm) to inhibit expression of the target gene.

[0192] In certain embodiments, the light-controlled repressor protein is selected from the blue light-responsive light-controlled repressor protein of the present invention; the induction conditions described in step (2) include: culturing the host cell under blue light (e.g., 430-495 nm, such as 450-480 nm) to induce expression of the target gene. In certain embodiments, the method further includes culturing the host cell under dark conditions to inhibit expression of the target gene.

[0193] In certain embodiments, the blue light described herein refers to light with a wavelength between 430-495 nm (eg, 450-480 nm). The blue light described herein can be emitted by a light source, including LED light panels, light strips, light tubes, and the like.

[0194] In certain embodiments, the expression vector comprising the first nucleic acid construct and the 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 the first nucleic acid construct, the second expression cassette comprises a promoter, a LacO operator gene, and a cloning site (e.g., a multiple cloning site MCS) for integrating a target gene, and integrating the target gene into the cloning site to provide a recombinant expression vector integrated with the target gene.

[0195] In certain embodiments, the expression vector comprising the 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 multiple cloning site MCS) for integrating a target gene, and integrating the target gene into the cloning site to provide a recombinant expression vector integrated with the target gene.

[0196] In certain embodiments, the method further comprises harvesting, isolating and / or purifying the expression product of the gene of interest (eg, protein of interest). Beneficial effects

[0197] The novel light-controlled repressor protein OptoLacI provided by the present invention not only meets the requirements of single-component regulation of gene expression, but also can achieve gene expression regulation with high temporal and spatial resolution. At the same time, it has two systems of blue light induction and darkness induction. OptoLacI and the operating sequence form a modular light-controlled system, which can be used immediately and has stronger system compatibility.

[0198] The present invention is based on a light-controlled gene expression system for Escherichia coli constructed based on a light-controlled repressor protein. The system includes tool strains of various induction strengths, and the induction system can adjust the expression intensity of the target gene by changing the frequency of blue light irradiation and the intensity of blue light. The blue light-induced gene expression system of the present invention exhibits extremely rigorous induction advantages, with very little expression leakage. The dark-induced gene expression system of the present invention has a good application effect in protein expression, and the induction effect is comparable to the IPTG induction effect. The dark-induced gene expression system of the present invention has an excellent application effect in metabolic regulation, has a more rigorous regulation of metabolic pathways, and the target product synthesized by dark induction has a higher yield than the IPTG induction system. The dark-induced gene expression system of the present invention and the blue light-induced gene expression system both have excellent spatial rigor. Example

[0199] The invention will now be described in the following non-limiting examples.

[0200] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0201] Example 1: Design of light-controlled repressor protein

[0202] To facilitate the screening of light-controlled repressor proteins, we used green fluorescent protein as a reporter gene. The restriction endonuclease sites at the 5' and 3' ends of green fluorescent protein were NheI and XhoI, respectively. The plasmid containing the GFP gene was amplified and double-digested with NheI and XhoI, respectively, on the plasmid containing the GFP gene and the pET-28a vector (purchased from Novagen) (Table 1). The pET-28a vector has the target transcription module (i.e., T7 promoter-lac operator-multiple cloning site) and contains the repressor gene lacI module; T4 DNA ligase was used for ligation (Table 2), and the ligation reaction system was transformed into competent cells DH5α and cultured overnight at 37°C. The next day, a single clone was picked for culture and sequencing. The correctly sequenced plasmid was named pZH37, in which the GFP gene was inserted into the multiple cloning site.

[0203] Table 1. Components of the double enzyme digestion system (50 μl)

[0204] Table 2 T4 DNA ligation system components (10 μl)

[0205] Compared with wild-type LacI, the mutation W220F was introduced into the repressor protein LacI. W220F The repression ability was enhanced, and the IPTG-induced GFP leakage expression level was reduced by 10 times. Therefore, in order to construct a rigorous light-controlled gene expression system, the LacI in the pZH37 plasmid was mutated to LacI W220F The mutated plasmid was named pZH49. In addition, the article reported the addition of the operator gene (LexA op ) repeat sequence can effectively increase the rigor of the light-controlled gene expression system, so the number of LacO1 genes in the lac operon part of the pZH37 plasmid was mutated to 3, and the mutated plasmid was named pZH39. At the same time, based on the pZH37 plasmid, the repressor gene lacI module was constructed as LacI W220F The mutant and lac operon portion of the plasmid pZH44 are 3*LacO1. Therefore, using GFP as a reporter gene, the fluorescence intensity of plasmids pZH37, pZH49, pZH39, and pZH44 was tested in BL21(DE3) cells, providing a basic template for constructing a rigorous light-controlled gene expression system. Plasmids pZH37, pZH49, pZH39, and pZH44 were transformed into BL21(DE3) competent cells, 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 incubated at 37°C in a shaker for 1 hour. An appropriate amount of the bacterial suspension was spread onto solid LB plates containing kanamycin resistance and cultured in a 37°C incubator overnight. The next day, single colonies were picked and the effects of different plasmids on the stringency of IPTG induction were tested in liquid LB.

[0206] Compared with wild-type LacI, the W220F mutation reduced IPTG-induced expression leakage and increased GFP expression, indicating that LacI W220F It is beneficial to reduce IPTG-induced leaky expression. When three LacO1 manipulation genes are connected in series, the expression level of GFP remains unchanged, and the IPTG-induced leaky expression level remains almost unchanged. W220F mutation and 3*LacO1 mutation, GFP expression level was not affected, and the leakage of IPTG-induced GFP expression was further reduced. Therefore, we also contained LacI W220Fand 3*LacO1 plasmid pZH44 as templates for photoswitchment.

[0207] The repressor protein LacI, derived from Escherichia coli strain K12, consists of 360 amino acids. LacI molecules bind to each other to form a tetramer. Amino acids 1-59 specifically bind to the operator gene, while amino acids 340-360 mediate tetramer formation. Based on the principles of light-controlled element conformational changes and the structure of the repressor protein LacI (PDB ID: 1LBG), a rational design was used to insert the light-controlled element LOV2 into solvent-exposed loops, including loops 1, 2, and 3. The amino acid sequences of the light-controlled element AsLOV2 (SEQ ID NO: 1) or its mutant cpLOV27 (SEQ ID NO: 2) were obtained by gene synthesis. The LOV2 gene was constructed into the LacI candidate loop site in pZH44 using the Gibson assembly method (Table 3). The Gibson assembly product was transformed into DH5α competent cells, 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 incubated in a 37°C constant temperature shaker for 1 hour. An appropriate amount of bacterial liquid was spread on a solid LB plate containing kanamycin resistance and cultured in a 37°C constant temperature incubator overnight.

[0208] Table 3 Gibson reaction components (10 μl)

[0209] The next day, single clones were picked and placed in LB medium containing kanamycin, cultured at 37°C in a constant temperature shaker for 8-9 hours, plasmids were extracted, and sent to a sequencing company for sequencing. The plasmids after successful sequencing were named pZH36, pZH46, pZH47, pZH48, pZH50, pZH51, pZH52, pZH53, pZH54, pZH55, pZH56, pZH57, pZH58, pZH62, pZH63, pZH64, and pZH65 (Table 4).

[0210] Table 4. Correspondence between LOV2 and cpLOV27 insertion positions and plasmid names

[0211] Example 2: Screening of light-controlled repressor proteins

[0212] In order to screen LacI mutants with optogenetic activity, pZH44 was used as a control plasmid. 200 ng of the control plasmid and the test plasmid (Table 4) were transformed into BL21 (DE3) competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, and ice-bathed for 2 minutes. 900 μl of LB medium was added to the competent cells and incubated at 37°C for 1 hour. An appropriate amount of bacterial solution was spread on a solid LB plate containing kanamycin resistance and cultured in a 37°C constant temperature incubator overnight. The next day, a single colony was picked and transferred to LB medium containing kanamycin, cultured at 37°C for 12 hours, and the bacterial solution was transferred at a 1% inoculum size. The strains containing the control plasmid and the test plasmid were transferred to two 24-well plates. The 24-well plates were placed in a blue light (90 μmol / m 2 The cells were cultured in the dark for 12 h. The control strain was induced with 1 mM IPTG for the same duration in the dark. The green fluorescence intensity of the different strains was then measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 530 nm, as well as the differences in GFP fluorescence values, to determine the light-responsiveness of the LacI mutants.

[0213] Strains containing LacI mutant plasmids pZH47, pZH36, pZH48, pZH50, pZH51, pZH55, pZH56, pZH57, and pZH62 showed dark-induced GFP expression activity. The LacI mutant corresponding to pZH36 exhibited superior induction characteristics, with the highest dark-induced fold increase and lower leaky 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 vector containing the target transcription module and the light-controlled repressor protein gene is called a light-controlled vector.

[0214] Example 3: Construction of light-controlled Escherichia coli strains

[0215] pZH36 corresponding to the mutant LacI M9 (OptoLacI D ) has dark-responsive activity, while the mutant LacI corresponding to pZH58 M1 (OptoLacI L ) has activity in response to blue light. Based on OptoLacI D 、OptoLacI L We constructed light-controlled E. coli strains. First, we used BL21 (DE3) as the starting strain. The BL21 (DE3) genome contains two copies of wild-type LacI. We selected 500bp of base sequences upstream and downstream of the wild-type LacI as homology arms and aligned the homology arms with OptoLacI. DThe genes were co-constructed into the Donor plasmids, named pZH76 and pZH78. On the plasmid containing the Cas9 protein gene, a plasmid containing two N20s was constructed, and the plasmid was named pZH83. D The wild-type LacI site was integrated into the genome of BL21 (DE3), and two OptoLacI sites were integrated into the upstream and downstream LacI sites of the genome. D The strain was named BL21_Dark_D V1 , while the upstream LacI of the genome was knocked out and the downstream LacI was replaced by OptoLacI D The strain was named BL21_Dark_S V1 The same method was used to construct the Donor plasmids pZH77 and pZH79, the targeting plasmid was pZH84, and the blue light response mutant OptoLacI was used to generate the target plasmid. L , constructing a blue light-responsive E. coli strain and inserting OptoLacI L The wild-type LacI site was integrated into the genome of BL21 (DE3), and two OptoLacI sites were integrated into the upstream and downstream LacI sites of the genome. L The strain was named BL21_Light_D, and the upstream LacI of the genome was knocked out and the downstream LacI was replaced by OptoLacI L The obtained strain was named BL21_Light_S (Figure 2). The strain with the light-controlled repressor protein gene integrated into its genome is called a light-controlled strain.

[0216] Example 4: Performance test of light-controlled Escherichia coli with dark-induced gene expression

[0217] In order to test the performance of the strain for dark-induced gene expression and optimize the effect of different numbers of LacO1 in series on the plasmid on its light-controlled performance, we constructed plasmids with different numbers of LacO1 in series (Table 5). The repressor gene modules of each plasmid were mutant LacI. M9 These plasmids were transformed into dark-induced light-controlled strains, cultured in a blue light environment, and then transferred to 24-well plates at a 1% inoculum to induce GFP expression under dark conditions.

[0218] Table 5 Correspondence between dark induction system plasmids and different numbers of LacO1

[0219] The results are shown in Figure 3. In the dark-induced light-controlled strain BL21_Dark_S, as the number of LacO1 tandems on the plasmid increased, the stringency of dark-induced GFP expression showed an increasing trend, among which 3*LacO1 and 4*LacO1 had the same induction multiple (Figure 3A). V1 In the experiments, the stringency of dark-induced GFP expression increased with the number of LacO1 tandems on the plasmid, with 3*LacO1 and 4*LacO1 showing the same induction fold ( Figure 3B ).

[0220] Example 5: Performance test of light-controlled Escherichia coli with blue light-induced gene expression

[0221] In order to test the performance of strains that can induce gene expression by blue light and optimize the effect of different numbers of LacO1 in series on the plasmid, we constructed plasmids with different numbers of LacO1 in series (Table 6). The repressor gene modules of each plasmid were mutant LacI. M1 These plasmids were transformed into blue light-induced light-controlled strains, cultured in a dark environment, and then transferred to 24-well plates at a 1% inoculation rate to induce GFP expression under blue light conditions.

[0222] Table 6 Correspondence between blue light inducible system plasmids and different numbers of LacO1

[0223] The results are shown in Figure 4. In the blue-light-induced light-controlled strain BL21_Light_S, the stringency of blue-light-induced GFP expression showed an increasing trend as the number of LacO1 tandems on the plasmid increased, with 3*LacO1 and 4*LacO1 having the same induction fold (Figure 4A). In the blue-light-induced light-controlled strain BL21_Light_D, the stringency of blue-light-induced GFP expression showed an increasing trend as the number of LacO1 tandems on the plasmid increased, with 3*LacO1 and 4*LacO1 having the same induction fold (Figure 4B).

[0224] Example 6: Optimization of the induction intensity of the dark-induced gene expression system

[0225] In metabolic engineering and industrial production, the dark-induced system has higher application potential. However, the current dark-induced gene expression system has the problem of low induction intensity. Therefore, we optimized the induction intensity of the dark-induced gene expression system. By saturation mutation of the key amino acid sites of LacI, we screened mutants with enhanced induction intensity. After mutating it into different mutants, the induction intensity increased by 2.9 to 6.9 times. When the 84th lysine of this site was mutated to glutamic acid, its induction intensity was higher than that of LacI. W220FThe increase was about 6.5 times (Figure 5). Therefore, based on this, we obtained the second generation of light-controlled repressor protein OptoLacI DV2 (SEQ ID NO: 29), BL21_Dark_S V1 With BL21_Dark_D V1 The site on the genome was mutated to obtain the second version of the dark-induced gene expression strain BL21_Dark_S V2 With BL21_Dark_D V2 .

[0226] Example 7: Effects of different promoters on the dark-induced gene expression system

[0227] In order to test the performance of the strain for dark-induced gene expression and characterize the effect of different promoters on the plasmid on its light-controlled performance, we constructed plasmids containing different promoters (T7, tac, lac, lacUV5) on the plasmid, and the repressor gene module of each plasmid was mutant LacI. M9-K84E These plasmids were transformed into the dark-induced light-controlled strain BL21_Dark_D V1 The cells were cultured in a blue light environment and then transferred to a 24-well plate at a 1% inoculum to induce GFP expression in the dark. The results are shown in Figure 6. V1 In the experiment, for plasmids carrying different promoters, the dark-induced GFP expression levels were similar, but the rigor of dark-induced GFP expression was different. This experimental result proves that T7, tac, lac, and lacUV5 promoters are all suitable for dark-inducible gene expression systems.

[0228] Example 8: Optimization of the induction factor of the dark-induced gene expression system

[0229] In BL21_Dark_D V2 On this basis, by optimizing the number of LacO1 tandems (1-8), we continued to optimize the induction multiple of the dark-induced gene expression system, gradually increased the number of LacO1 tandems on the plasmid pZH36, and transformed it into BL21_Dark_D V2The strain was cultured under blue light and then transferred to a 24-well plate at a 1% inoculum size to induce GFP expression in the dark. The results showed that the optimal induction multiple was achieved when 4*LacO1 (pML308) was added in series on the plasmid (Figure 7A). A series of plasmids were also constructed based on the LacO1 mutant LacOid (SEQ ID NO: 25) on the plasmid pZH36 to test their dark-induced effects. The results showed that the tandem use of LacO1 and LacOid still had a good dark-induced effect, and the overall expression level was increased (Figure 7B).

[0230] Example 9: Application of Dark-Inducible Gene Expression System in Protein Expression

[0231] In metabolic engineering practice, the light-controlled system for dark-induced protein production has significant advantages over the light-controlled system for blue-light-induced protein production. Therefore, we explored the application of the dark-induced gene expression system in protein expression. Taking glucose dehydrogenase (GDH, accession number: P40288), PET degrading enzyme PETase (accession number: A0A0K8P0E4) and alkaline protease (alkalineprotease) (accession number: P00780) as examples, the expression of different genes by the light-controlled system for dark-induced production was tested (Table 7). The plasmids corresponding to the above-mentioned target proteins are pML333, pML351, and pML353, respectively. Among them, the number of LacO1 in the target transcription module is 4, and the light-controlled repressor protein module is OptoLacI DV2 .

[0232] Table 7 Summary of application information of dark-inducible gene expression system in protein expression

[0233] Take 200ng of plasmid and transform it into BL21_Dark_D V1 or BL21_Dark_D V2 Competent cells were placed in an ice bath for 30 min, heat-shocked at 42°C for 90 s, and then placed in an ice bath for 2 min. 900 μl of LB medium was added to the competent cells and incubated at 37°C for 1 h in a light-controlled shaker with a blue light intensity of 90 μmol / m 2 / s, take an appropriate amount of bacterial solution and spread it on a solid LB plate containing kanamycin resistance, and culture it overnight in a 37℃ blue light incubator (blue light intensity is 80μmol / m2 / s). The next day, pick a single clone and transfer it to LB medium containing kanamycin, and culture it in a 37℃ light-controlled shaker for 12 hours. Transfer the bacterial solution to a 24-well plate with a 1% inoculum volume, and place the 24-well plate in a blue light incubator (80μmol / m2 / s). 2The cells were cultured at OD600 = 0.1-0.8, and the 24-well plate was completely covered with tin foil. Induction was performed in the dark, and samples were taken at 0, 2, 4, 6, 9, and 12 hours. Samples at different time points were centrifuged at 12,000 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 culture solution, and the cells were boiled at 100°C for 20 minutes. A 2 μl sample was collected and subjected to 12% SDS-PAGE analysis. The light-controlled expression of dark-induced genes was determined by SDS-PAGE.

[0234] The research results are shown in Figure 8, where Figure A is GDH, Figure B is PETase, and Figure C is alkalineprotease. As the dark induction time increases, the target protein band gradually thickens, and the target protein band is obvious and clear. There is very little expression leakage before dark induction (0h), indicating that the dark-inducible gene expression system has a good application effect in protein expression.

[0235] Example 10: Application of blue light-induced gene expression system in protein expression

[0236] In order to characterize the induction characteristics of the blue light-induced gene expression system, we also explored the application of the blue light-induced gene expression system in protein expression. Taking acetyl-CoA C-acyltransferase (FadA) (accession number: A4XSM9) and malate dehydrogenase (MdhII) (accession number: D9PVI7) as examples, the expression of different genes by the light-controlled system induced by blue light was tested (Table 8). The plasmids corresponding to the above-mentioned target proteins are pML359 and pML361, respectively. Among them, the number of LacO1 in the target transcription module is 1, and the light-controlled repressor protein module is LacI. M1 .

[0237] Table 8 Summary of application information of blue light-induced gene expression system in protein expression

[0238] Take 200 ng of plasmid and transform it into BL21_Light_D competent cells, ice bath for 30 min, heat shock at 42 °C for 90 s, ice bath for 2 min, add 900 μl LB medium to the competent cells, incubate at 37 °C in a light-controlled shaker for 1 h, and the blue light intensity is 80 μmol / m 2 / s, take an appropriate amount of bacterial solution and spread it on a solid LB plate containing kanamycin resistance, and culture it in a 37℃ incubator overnight. The next day, pick a single clone and transfer it to LB medium containing kanamycin, and culture it in a 37℃ shaking incubator for 12 hours. Transfer the bacterial solution to a 24-well plate with a 1% inoculum volume, cover the 24-well plate with tin foil, and culture the 24-well plate to OD600 = 0.1-0.8, and place it in a blue light (80μmol / m2 The samples were induced for 9 hours ( / s) and then sampled. Samples at different time points were centrifuged at 12,000 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 solution, and the cells were boiled at 100°C for 20 minutes. 2 μl of the sample was subjected to 12% SDS-PAGE analysis to determine the light-controlled effect of darkness-induced gene expression.

[0239] The research results are shown in Figure 9, where Figure A is FadA and Figure B is MdhII. The target protein bands in the blue light-induced group are obvious and clear, while no obvious protein bands are found in the corresponding positions in the dark control group, indicating that the blue light-induced gene expression system has very little expression leakage in protein expression and has good application effects.

[0240] Example 11: Application of Dark-Inducible Gene Expression System in 1,3-Propanediol Production

[0241] In metabolic engineering practice, resolving the conflicting demands on culture media for host cell growth and target product production is a major challenge. Compared to light-controlled systems for blue-light-induced protein production, light-controlled systems for dark-induced protein production are more rigorous and can effectively control metabolic flux allocation by manipulating the expression of key enzymes. Therefore, we explored the application of dark-induced gene expression systems for metabolic flux control. Using 1,3-propanediol as an example, we tested the ability of dark-induced light-controlled systems to control metabolic flux in the 1,3-propanediol biosynthesis pathway.

[0242] First, glycerol dehydratase (dhaB1234, gene sequence shown in SEQ ID NO: 20) and glycerol dehydratase reactivator (gdrAB, gene sequence shown in SEQ ID NO: 21, SEQ ID NO: 22) were cloned from the Klebsiella pneumoniae genome, and alcohol dehydrogenase (yqhD, gene sequence shown in SEQ ID NO: 23) was cloned from the Escherichia coli MG1655 genome. These were cloned into the expression vector pCDFDuet (purchased from Novagen) to construct the recombinant plasmid pCDFDuet-dhaB1234-gdrB-yqhD-gdrA (as shown in Figure 10, the expression cassette of the key gene for 1,3-propanediol biosynthesis). The pCDFDuet-1 vector contains a T7 promoter and a lac operator sequence.

[0243] Transformed into BL21(DE3) and BL21_Dark_D v1 The 1,3-propanediol production strains BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA and BL21_Dark_D were constructed.v1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA. Specifically, 200 ng of plasmid was transformed into BL21_Dark_D V1 Or BL21 (DE3) competent cells, ice bath for 30 minutes, heat shock at 42 ° C for 90 seconds, ice bath for 2 minutes, add 900 μl LB medium to the competent cells. The host cell is BL21_Dark_D V1 The cells were incubated at 37°C in a light-controlled shaker for 1 h with a blue light intensity of 90 μmol / m 2 / s; host cells are BL21 (DE3) and directly recovered in the dark for 1 hour. Take an appropriate amount of bacterial solution and spread it on a solid LB plate containing kanamycin resistance. The host cells are BL21_Dark_D V1 The host cells were cultured in a 37°C blue light incubator (blue light intensity was 80 μmol / m2 / s), and the host cells were cultured in a 37°C incubator overnight. V1 / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA, pick a single clone and transfer it to LB medium containing kanamycin, and culture it in a light-controlled shaker at 37℃ for 12-18h as a seed solution. Transfer the bacterial solution to a 24-well plate at a 1% inoculum volume, and place the 24-well plate under blue light (80μmol / m 2 / s), and cultured to OD 600 =0.1~0.6, one group used tin foil to cover the 24-well plate completely and induce in the dark, while the other group was still cultured under blue light, and samples were taken after fermentation for 48h. For BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA, single clones were picked and transferred to LB medium containing kanamycin, and cultured at 37℃ on a shaker for 12-18h as seed liquid. The bacterial liquid was transferred to a 24-well plate at a 1% inoculum volume, and the 24-well plate was placed in a blue light (80μmol / m 2 / s), and cultured to OD 600 = 0.1-0.6. One group was treated with IPTG to induce 1,3-PDO synthesis, while the other group was not treated with IPTG. Samples were taken after 48 hours of fermentation. 800 μL of the fermentation broth was centrifuged at 12,000 rpm, and the supernatant was collected for 1,3-PDO production analysis using a HPLC method reported in the literature.

[0244] Table 8 Fermentation comparison test design

[0245] The results are shown in Figure 11. Compared with the ordinary BL21 (DE3) using IPTG to control the metabolic pathway flux of 1,3-propylene glycol, there is no significant difference between the induced group and the uninduced group. The dark induction expression system is more rigorous, and the induced group has a significantly higher 1,3-propylene glycol yield than the uninduced group. In addition, the BL21_Dark_D v1 The 1,3-propanediol production in the BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA group was significantly higher than that in the BL21(DE3) / pCDFDuet-dhaB1234-gdrB-yqhD-gdrA group, indicating that the darkness-inducible gene expression system has a good application effect in metabolic flux control.

[0246] The above results show that: (1) By inserting light-controlled elements (LOV2 and its mutants) into the target protein sequence, light-controlled protein function can be achieved. (2) The Escherichia coli light-controlled gene expression system developed based on the light-controlled repressor protein OptoLacI can be applied to protein production. The inducer is dark conditions or blue light irradiation. The induction cost is low and the induction operation is fast, which has great advantages in protein production. (3) The Escherichia coli light-controlled gene expression system developed based on the light-controlled repressor protein OptoLacI can be applied to the production of dynamically regulated chemicals. (4) Light-controlled expression can be achieved by providing a light-controlled vector in a light-controlled strain, providing a common expression vector in a light-controlled strain, and providing a light-controlled vector in a common strain.

[0247] Example 12: Comparison of the OptoLacI system and the IPTG induction system

[0248] The dark-induced gene expression plasmid pML308 was transformed into the light-controlled strain BL21_Dark_D V1 and BL21_Dark_D V2 The single clone was picked and cultured overnight under blue light conditions. The next day, the inoculation volume was transferred to a 24-well plate at 1% and placed in a dark environment. The inoculation volume was 37°C for 12 hours, and the GFP fluorescence intensity was detected. The blue light-induced gene expression plasmid pZH251 was transformed into the light-controlled strain BL21_Light_D, and the single clone was picked and cultured overnight under dark conditions. The next day, the inoculation volume was 1% and transferred to a 24-well plate at 1% and placed in a blue light environment. The inoculation volume was 37°C for 9 hours, and the GFP fluorescence intensity was detected. At the same time, pZH37 was transformed into BL21 (DE3), and the single clone was picked and cultured until OD 600 =0.5, 1 mM IPTG was added, induced at 37°C for 12 h, and the fluorescence intensity of GFP was detected.

[0249] The results, as shown in Figure 12, show that while the expression intensities of the dark-inducible and blue-light-inducible gene expression systems were lower than those induced by IPTG, the blue-light-inducible gene expression system achieved an induction multiple of 75 times, while the IPTG-inducible system achieved an induction multiple of only 16 times. This indicates that the blue-light-inducible system has more rigorous induction characteristics and can avoid unnecessary expression leakage. Furthermore, the dark-inducible and blue-light-inducible gene expression systems of the present invention offer greater adjustability, allowing protein expression and leakage expression to be controlled by adjusting the blue light pulse pattern and blue light intensity, a feature not found in the IPTG system.

[0250] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative and non-exhaustive, and is not intended to limit the disclosed embodiments. Those skilled in the art will appreciate that various modifications and variations of the details may be made in light of all the teachings disclosed herein, and that such modifications are within the scope of the present invention. The present invention is defined in its entirety by the appended claims and any equivalents thereof.

[0251] Sequence information

[0252] SEQ ID NO: 1 (AsLOV2)

[0253] SEQ ID NO:2 (cpLOV27)

[0254] SEQ ID NO:3(LacI M1 )

[0255] SEQ ID NO:4(LacI M2 )

[0256] SEQ ID NO:5(LacI M3 )

[0257] SEQ ID NO:6(LacI M4 )

[0258] SEQ ID NO:7(LacI M5 )

[0259] SEQ ID NO:8(LacI M6 )

[0260] SEQ ID NO:9(LacI M7 )

[0261] SEQ ID NO: 10 (LacI M8 )

[0262] SEQ ID NO:11(LacI M9 )

[0263] SEQ ID NO: 12 (LacI M10 )

[0264] SEQ ID NO: 13 (LacI M11 )

[0265] SEQ ID NO: 14 (LacI M12 )

[0266] SEQ ID NO: 15 (LacI M13 )

[0267] SEQ ID NO: 16 (LacI M14 )

[0268] SEQ ID NO: 17 (LacI M15 )

[0269] SEQ ID NO: 18 (LacI M16 )

[0270] SEQ ID NO: 19 (LacI M17 )

[0271] SEQ ID NO: 20 (glycerol dehydratase dhaB1234)

[0272] SEQ ID NO: 21 (glycerol dehydratase reactivating enzyme gdrA)

[0273] SEQ ID NO: 22 (glycerol dehydratase reactivating enzyme gdrB)

[0274] SEQ ID NO: 23 (alcohol dehydrogenase yqhD)

[0275] SEQ ID NO: 24 (LacO1 operator sequence)

[0276] SEQ ID NO: 25 (LacOid operator sequence)

[0277] SEQ ID NO:26 (LacI WT Manipulator protein)

[0278] SEQ ID NO:27 (LacI W220F Manipulator protein)

[0279] SEQ ID NO:28 (LacI W220F+K84E Manipulator protein)

[0280] SEQ ID NO:29 (OptoLacI DV2 )

[0281] SEQ ID NO: 30 (Photosensitive protein EL222 from Staphylococcus aureus)

[0282] SEQ ID NO: 31 (photosensitive domain LOV2 from Arabidopsis thaliana)

Claims

1. A light-controlled 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.

2. The light-controlled repressor protein according to claim 1, wherein The LOV domain is a LOV2 domain; Preferably, the LOV domain is selected from the LOV2 domain (AsLOV2) of the oat phytochrome 1 gene, the photosensitive protein EL222 from Staphylococcus australis, the photosensitive domain LOV2 from Arabidopsis thaliana, or mutants thereof; Preferably, the mutant is a cycle rearrangement mutant or a mutant with altered response speed; Preferably, the LOV domain is AsLOV2, for example comprising the sequence shown in SEQ ID NO: 1; Preferably, the LOV domain is cpLOV27, for example comprising the sequence shown in SEQ ID NO:

2.

3. The light-controlled repressor protein according to claim 1 or 2, wherein The insertion position 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, between positions 152 and 153; Preferably, the insertion position is between positions 335 and 336 of the LacI protein.

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

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

26.

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

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 is optionally connected to the LacI protein via a peptide linker.

8. The light-controlled repressor protein according to any one of claims 1 to 7, comprising a sequence as shown in any one of SEQ ID NOs: 11, 29, 6-8, 10, 12-14, 16 or a sequence as shown in SEQ ID NO: 3 or 17; Preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO: 11 or 29; Preferably, the light-controlled repressor protein comprises the sequence shown in SEQ ID NO:

3.

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

10. A vector comprising the nucleic acid construct according to claim 9.

11. The vector of claim 10, comprising a first nucleic acid construct and a second nucleic acid construct, wherein: The first nucleic acid construct comprises the nucleotide sequence encoding the light-controlled repressor protein, and the second nucleic acid construct comprises a promoter, a LacO operator, and a target gene or a cloning site for integrating a target gene; Preferably, the first nucleic acid construct and the second nucleic acid construct are located in different expression cassettes.

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

13. The vector according to claim 11 or 12, wherein The LacO operator gene contained in the second nucleic acid construct comprises at least one copy (e.g., 1 to 8 copies, such as 1, 2, 3, 4, 5, 6, 7, 8, such as 3 to 5 copies) of the LacO1 operator sequence; Preferably, the LacO1 operator sequence comprises the sequence shown in SEQ ID NO:

24.

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

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

16. The vector according to any one of claims 11 to 15, wherein The first nucleic acid construct further 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 wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter.

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

18. The host cell according to claim 17, wherein The host cell is Escherichia coli, and the Escherichia coli has an exogenous nucleotide sequence encoding the light-controlled repressor protein according to any one of claims 1 to 8 integrated into its genome; Preferably, the endogenous LacI gene of the Escherichia coli is disrupted; Preferably, both copies of the endogenous LacI gene of the Escherichia coli are replaced with the exogenous nucleotide sequence; Preferably, one copy of the endogenous LacI gene of the Escherichia coli is replaced by 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 11-16.

20. A system for regulating the expression of a target gene, comprising: (1) a first nucleic acid construct comprising a nucleotide sequence encoding the 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, and a second nucleic acid construct of a target gene.

21. The system of claim 20, wherein: The second nucleic acid construct is as defined in any one of claims 11-15.

22. The system of claim 20 or 21, wherein: The second nucleic acid construct comprises a plurality of target genes arranged in tandem; Preferably, additional promoters are optionally inserted between the multiple target genes.

23. The system of any one of claims 20-22, wherein: The first nucleic acid construct further 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 wild-type LacI promoter, LacUV5 promoter, tac promoter, and trc promoter.

24. The system of any one of claims 20 to 23, wherein: (1) The system comprises at least two of the first nucleic acid constructs, one of which is integrated into the genome of the host cell, and the other and the second nucleic acid construct are present on a vector (e.g., an expression vector); wherein the at least two first nucleic acid constructs are identical to or different from each other; preferably, the vector is 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 (eg, an expression vector); preferably, the vector is defined in any one of claims 11 to 16; or, (3) the first nucleic acid construct is integrated into the genome of the 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.

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

26. A method for regulating the expression of a target gene, comprising: (1) providing the system according to any one of claims 20 to 24 in a host cell; (2) Cultivating the host cell under conditions that allow expression of the target gene and inducing expression of the target gene.

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

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

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

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