Genetic switches

Abstract

To provide a mutant sequence of a gene switch sensor having higher functionalization.SOLUTION: The present invention discloses a composition containing a polypeptide for inducible control of target gene expression in an organism. A polynucleotide encoding the polypeptide includes a gene switch expression sequence, and a target gene sequence whose expression is induced by binding of a complex, in which a transcriptional regulator encoded by the gene switch expression sequence is bound to an inducer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to mutated sequences of artificial transcription factors and gene switches utilizing them. More specifically, this disclosure relates to gene switches obtained by an efficient selection method that can dramatically improve the performance of conventional early-type gene switch sensors. [Background technology]

[0002] In recent years, gene switch sensors (metabolite sensors) that respond to compounds such as metabolites have attracted attention, and their development has been particularly focused on bacteria such as Escherichia coli. However, in yeast, a eukaryote, the transcriptional control region is longer than in bacteria, and its functional region is unclear, making it difficult to develop high-performance gene switch sensors.

[0003] In developing gene switch sensors, it is essential to select mutants that are in an activated state under conditions where gene expression should be ON, and / or in a repressed state under conditions where gene expression should be OFF (ON selection / OFF selection). It is important to select under appropriate conditions for both of these states. While conventional fluorescence activating cell sorting allows for the selection conditions to be changed, only one condition can be examined at a time. Furthermore, changing the selection conditions is difficult when using drug-based selection techniques. Summary of the Invention [Means for solving the problem]

[0004] The present inventors have obtained mutants of artificial transcription factors and identified useful mutations. The present disclosure provides such mutants and / or gene switches.

[0005] Thus, the present disclosure provides: (Item 1) A composition comprising a polypeptide for inducibly controlling expression of a target gene in an organism, wherein a polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a target gene sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulator encoded by the gene switch expression sequence with an inducer. (Item 2A) (DAPG-ON) The composition described in the preceding item, wherein the gene switch comprises an amino acid mutation at one or more positions selected from 5, 6, 20, 70, 86, 109, 117, 143, and 187 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item 2) (DAPG-ON) The composition described in the preceding item, wherein the gene switch comprises one or more mutations selected from P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item 2B) (DAPG-ON) Inversion The composition according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item 3) (Camphor-OFF) The composition of any one of the preceding items, wherein the gene switch comprises a mutation that disrupts the function of the nuclear localization signal. (Item 4) (Camphor-OFF) The composition according to any one of the preceding items, wherein the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal. (Item 5A) (Camphor-OFF) The composition according to any one of the preceding items, wherein the frameshift mutation of the nuclear localization signal comprises a mutation in PKKKRKV in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item 5) (Camphor-OFF) The composition according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises a mutation from PKKKRKV to RKERSKI in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item 6A) (HSL-ON) The composition according to any one of the preceding items, wherein the gene switch comprises amino acid mutations at one or more positions selected from 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item 6) (HSL-ON) The composition according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item 7A) (Borneol-ON) The composition according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item 7) (Borneol-ON) The composition according to any one of the preceding items, wherein the gene switch comprises a Y40C mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item 8A) (Tet-ON) The composition according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 8 in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item 8) (Tet-ON) The composition according to any one of the preceding items, wherein the gene switch comprises a K8N mutation in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item 9A) (DAPG-OFF) The composition according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at one or more positions selected from 157 and 41 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item 9) (DAPG-OFF) The composition according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from R157H and E41G in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item 10) (DAPG-ON) mutation site A nucleic acid molecule in which one or more bases selected from positions 13, 16, 349 to 351, 349 to 351, 256, 257, 427, 325, 59, 209, and 559 of the base sequence represented by SEQ ID NO: 5 or a variant thereof have been mutated. (Item 11) (DAPG-ON) Specific mutation A nucleic acid molecule according to the above item, comprising, in the base sequence represented by SEQ ID NO: 5 or a variant thereof, one or more mutations selected from C13T, T16C, A350G, mutations of AA at positions 350 and 351 to CG, mutation of CAA at positions 349 to 351 to ATT, mutation of CAA at positions 349 to 351 to GGT, A257C, mutation of AA at positions 256 and 257 to GC, mutation of AA at positions 256 and 257 to TC, mutation of AA at positions 256 and 257 to GG, G427A, T325C, A59G, A209G, and A559G. (Item 12) (DAPG-ON) inversion mutation A nucleic acid molecule according to any one of the preceding items, comprising, in the base sequence represented by SEQ ID NO: 5 or a variant thereof, one or more mutations selected from A350G, mutations of AA at positions 350 and 351 to CG, mutations of CAA at positions 349 to 351 to ATT, mutations of CAA at positions 349 to 351 to GGT, A209G, and A559G. (Item 13) The nucleic acid molecule according to any one of the preceding items, wherein the polypeptide encoded by the nucleic acid molecule functions as a polypeptide for inducible control of target gene expression in an organism. (Item A1) A polypeptide used for inducible control of target gene expression in an organism, wherein a polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a target gene sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer. (Item A2A) (DAPG-ON) The polypeptide described in the preceding item, wherein the gene switch comprises amino acid mutations at one or more positions selected from 5, 6, 20, 70, 86, 109, 117, 143, and 187 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item A2) (DAPG-ON) The polypeptide according to the preceding item, wherein the gene switch comprises one or more mutations selected from P5S, S6P, K20R, E70G, K86T, K86A, K86S, K86G, F109L, Q117R, Q117P, Q117G, Q117N, E143K, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item A2B) (DAPG-ON) Inversion The polypeptide according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item A3) (Camphor-OFF) The polypeptide according to any one of the preceding items, wherein the gene switch comprises a mutation that disrupts the function of the nuclear localization signal. (Item A4) (Camphor-OFF) The polypeptide according to any one of the preceding items, wherein the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal. (Item A5A) (Camphor-OFF) The polypeptide according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises an amino acid mutation in PKKKRKV in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item A5) (Camphor-OFF) The polypeptide according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises a mutation from PKKKRKV to RKERSKI in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item A6A) (HSL-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises amino acid mutations at one or more positions selected from 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item A6) (HSL-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item A7A) (Borneol-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item A7) (Borneol-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises a Y40C mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item A8A) (Tet-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 8 in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item A8) (Tet-ON) The polypeptide according to any one of the preceding items, wherein the gene switch comprises a K8N mutation in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item A9A) (DAPG-OFF) The polypeptide according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at one or more positions selected from 157 and 41 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item A9) (DAPG-OFF) The polypeptide according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from R157H and E41G in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item B1) 1. Use of a polypeptide for producing a composition for inducible control of target gene expression in an organism, wherein a polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a target gene sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer. (Item B2A) (DAPG-ON) The use described in the above item, wherein the gene switch comprises amino acid mutations at one or more positions selected from 5, 6, 20, 70, 86, 109, 117, 143, and 187 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item B2) (DAPG-ON) The use according to the preceding item, wherein the gene switch comprises one or more mutations selected from P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item B2B) (DAPG-ON) Inversion The use according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item B3) (Camphor-OFF) The use according to any one of the preceding items, wherein the gene switch comprises a mutation that disrupts the function of the nuclear localization signal. (Item B4) (Camphor-OFF) The use according to any one of the preceding items, wherein the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal. (Item B5A) (Camphor-OFF) The use according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises a mutation in PKKKRKV in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item B5) (Camphor-OFF) The use according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises a mutation from PKKKRKV to RKERSKI in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item B6A) (HSL-ON) The use according to any one of the preceding items, wherein the gene switch comprises amino acid mutations at one or more positions selected from 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item B6) (HSL-ON) The use according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item B7A) (Borneol-ON) The use according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item B7) (Borneol-ON) The use according to any one of the preceding items, wherein the gene switch comprises a Y40C mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item B8A) (Tet-ON) The use according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 8 in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item B8) (Tet-ON) The use according to any one of the preceding items, wherein the gene switch comprises a K8N mutation in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item B9A) (DAPG-OFF) The use according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at one or more positions selected from 157 and 41 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item B9) (DAPG-OFF) The use according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from R157H and E41G in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item C1) A method for inducibly controlling the expression of a target gene in an organism using a composition comprising a polypeptide for inducibly controlling the expression of a target gene in an organism, wherein a polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a target gene sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer. (Item C2A) (DAPG-ON) The method according to the preceding item, wherein the gene switch comprises amino acid mutations at one or more positions selected from 5, 6, 20, 70, 86, 109, 117, 143, and 187 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item C2) (DAPG-ON) The method according to the preceding item, wherein the gene switch comprises one or more mutations selected from P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item C2B) (DAPG-ON) Inversion The method according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item C3) (Camphor-OFF) The method according to any one of the preceding items, wherein the gene switch comprises a mutation that disrupts the function of a nuclear localization signal. (Item C4) (Camphor-OFF) The method according to any one of the preceding items, wherein the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal. (Item C5A) (Camphor-OFF) The method according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises an amino acid mutation in PKKKRKV in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item C5) (Camphor-OFF) The method according to any one of the preceding items, wherein the frameshift mutation in the nuclear localization signal comprises a mutation from PKKKRKV to RKERSKI in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item C6A) (HSL-ON) The method according to any one of the preceding items, wherein the gene switch comprises amino acid mutations at one or more positions selected from 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item C6) (HSL-ON) The method according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Item C7A) (Borneol-ON) The method according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item C7) (Borneol-ON) The method according to any one of the preceding items, wherein the gene switch comprises a Y40C mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Item C8A) (Tet-ON) The method according to any one of the preceding items, wherein the gene switch comprises an amino acid mutation at position 8 in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item C8) (Tet-ON) The method according to any one of the preceding items, wherein the gene switch comprises a K8N mutation in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Item C9A) (DAPG-OFF) The method according to any one of the preceding items, wherein the gene switch comprises amino acid mutations at one or more positions selected from 157 and 41 in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. (Item C9) (DAPG-OFF) The method according to any one of the preceding items, wherein the gene switch comprises one or more mutations selected from R157H and E41G in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.

[0006] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] Note that features and significant actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings. [Effects of the Invention]

[0008] This disclosure provides mutation sequences for highly functional gene switches and sensors. Useful mutations in transcription factors (proteins) are important because they are expected to function similarly not only in yeast but also in other organisms, including bacteria. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the S / N ratio of an improved Tet-ON gene switch in one embodiment of the present disclosure. [Figure 2] FIG. 2 shows the mutation sites and S / N ratios of various gene switches developed in one embodiment of the present disclosure. [Figure 3] FIG. 3 is a schematic diagram showing flux control from FPP to β-carotene using the gene switch developed in one embodiment of the present disclosure. [Figure 4] FIG. 4 shows the nucleotide and amino acid (AA, shown in brackets) mutations found in the rPhlTA expression cassette of the DAPG-ON switch evolved in one embodiment of the present disclosure. [Figure 5] FIG. 5 shows the nucleotide and amino acid (AA, shown in brackets) mutations found in the LuxTA expression cassette of the HSL-ON switch evolved in one embodiment of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of a gene switch in Pichia yeast developed in one embodiment of the present disclosure. [Figure 7] FIG. 7 is a graph showing the relationship between changes in phlO copy number and changes in expression level in a Pichia yeast gene switch according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0011] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0012] As used herein, "about" means ±10% of the preceding numerical value.

[0013] As used herein, the terms "gene switch" and "gene switch sensor" are used interchangeably and refer to a factor (e.g., a molecule, a complex thereof, or a fusion thereof) containing a transcriptional regulatory domain and having a site for interaction (e.g., binding) with a substance (activator) that can activate a target gene by interacting (e.g., binding) with the nucleic acid constituting the gene. The activity of such a factor can be regulated by the interaction (e.g., binding or dissociation) of an inducer, thereby changing its function. When an activator interacts (e.g., binds) with a gene switch, the degree or state of interaction (e.g., binding) of the gene switch with the target sequence changes, resulting in the repression or induction of expression of the target gene. For example, a "gene switch" as used herein is a molecule containing a transcriptional activation domain and having a site for binding with a substance that can activate the molecule by binding, and the binding of the substance results in binding or dissociation from the target sequence.

[0014] As used herein, "gene switch expression sequence" refers to a nucleic acid sequence that encodes a gene switch when the gene switch is a protein or polypeptide.

[0015] As used herein, the term "activator of a gene switch" refers to a substance, such as a compound, that activates or changes the function of a gene switch by interacting with (e.g., binding to) the gene switch, thereby directly or indirectly inducing the regulation of expression of one or more genes. The activator may be different for each gene switch. Examples of combinations of gene switches and activators include rtetTA and Dox, CamTA and D-Camphor, PhlTA and DAPG, and LuxTA and HSL.

[0016] As used herein, the term "target sequence of a gene switch" refers to a nucleic acid sequence located 5' upstream of a gene encoding a protein of interest that controls the transcription of the target gene. Preferably, the nucleic acid sequence has promoter activity and may be a promoter sequence or an artificial promoter in which an operator sequence is fused with a core promoter. Preferably, an enhancer can act indirectly or directly on the target sequence of the gene switch. The action of the gene switch on the target sequence can be regulated by the addition of an inducer, thereby controlling the expression of the target gene; if the gene switch does not act on the target sequence, the target gene will not be expressed. For example, the gene switch rtetTA acts on its target sequence, the tetO promoter (an artificial promoter containing tetO), by the addition of its activator Dox, and promotes the transcription of the target gene located downstream of the promoter.

[0017] As used herein, the term "promoter" refers to a region on DNA that controls the initiation of gene transcription and directly regulates the level of transcription, and refers to a nucleic acid sequence that initiates transcription upon binding of RNA polymerase. The promoter can be appropriately selected depending on the species of host cell used. When yeast is used as a host, any promoter can be used, as long as it can be expressed in host cells such as yeast. Examples of promoters include the GAL1 promoter, GAL10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, TDH promoter, DAS1, and AOX1 promoter.

[0018] As used herein, the term "transcriptional regulatory factor" refers to a protein that acts by acting on a regulatory DNA element such as a promoter. Transcriptional regulatory factors are broadly classified into transcriptional repressors (repressors) and transcriptional activators (activators). Transcriptional repressors act on regulatory DNA elements to suppress gene transcription and reduce the amount of gene expression. Transcriptional activators act on regulatory DNA elements to promote gene transcription and increase the amount of gene expression. In one embodiment of the present disclosure, known transcriptional repressors and transcriptional activators can be used. The expression of a gene sequence located downstream of a regulatory DNA element on which a transcriptional regulatory factor acts is suppressed or promoted by the action of the transcriptional regulatory factor. Gene expression refers to a series of processes in which genetic information is transcribed into mRNA and further translated into the amino acid sequence of the protein encoded by the gene. If expression is promoted, the protein encoded by the gene is produced and its amount increases, whereas if expression is suppressed, the protein encoded by the gene is not produced and its amount decreases.

[0019] As used herein, a "candidate transcriptional regulatory factor" is a factor that can be a candidate for a transcriptional regulatory factor.

[0020] As used herein, the term "inducer" or "inducing agent" refers to a factor that can activate, inactivate, or change the function of a transcriptional regulatory factor by binding to the transcriptional regulatory factor, thereby directly or indirectly regulating the expression of one or more genes.

[0021] As used herein, a "DNA library" refers to a nucleic acid library containing nucleic acid sequences isolated from nature or synthetic nucleic acid sequences. Sources of nucleic acid sequences isolated from nature include, but are not limited to, genomic and cDNA sequences derived from eukaryotic cells, prokaryotic cells, or viruses. Libraries in which any sequence (e.g., signal, tag, etc.) is added to a sequence isolated from nature are also included in the DNA library of the present disclosure.

[0022] As used herein, the term "host cell" refers to a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. A host cell can include not only the particular subject cell but also the progeny of such a cell. A host cell can be any cell, including prokaryotic or eukaryotic cells, that has the appropriate traits to receive and produce a heterologous nucleic acid or protein. Examples of host cells include prokaryotic and eukaryotic cells, bacterial cells, mycobacterial cells, fungal cells, yeast cells, plant cells, insect cells, non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas.

[0023] As used herein, the term "expression vector" refers to vector DNA that delivers an exogenous gene to a host cell and is capable of expressing the target gene in the host cell. The vector DNA is not particularly limited as long as it is replicable in the host, and can be appropriately selected depending on the type of host and intended use. The vector DNA may be obtained by extracting naturally occurring DNA, or it may be vector DNA that lacks portions of DNA other than those necessary for replication. Representative examples of vector DNA include vector DNA derived from plasmids, bacteriophages, and viruses. Examples of plasmid DNA include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, and yeast-derived plasmids. Examples of bacteriophage DNA include λ phage. Examples of virus-derived vector DNA include vectors derived from animal viruses such as retroviruses, vaccinia viruses, adenoviruses, papovaviruses, SV40, fowlpox viruses, and pseudorabies viruses, as well as vectors derived from insect viruses such as baculoviruses. Other examples include vector DNA derived from transposons, insertion elements, and yeast chromosomal elements. Alternatively, vector DNAs created by combining these elements can be used, such as vector DNAs (cosmids, phagemids, etc.) created by combining genetic elements of plasmids and bacteriophages. The vector DNA must incorporate a gene of interest so that it can be expressed, and its components include at least the gene of interest and a regulatory DNA element, such as a promoter. In addition to these elements, if desired, gene sequences carrying information related to replication and control can be combined and incorporated into the vector DNA using known techniques. Examples of such gene sequences include cis elements such as ribosome binding sequences, terminators, signal sequences, and enhancers, splicing signals, and selection markers (dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, etc.). One or more gene sequences selected from these can be incorporated into the vector DNA.

[0024] As used herein, the term "fusion gene" refers to a new gene that is generated by artificially or naturally linking multiple genes, and includes genes that are generated as a result of recombination such as chromosomal translocation, insertion, and inversion. Fusion genes may also encode fusion proteins. The number of genes linked in a fusion gene is not particularly limited, as long as the fusion gene can produce a functional protein. The orientation of each gene in a fusion gene is also not particularly limited, and may be all forward (the original transcription direction), all reverse (the opposite of the original transcription direction), or a combination of forward and reverse. Furthermore, in a fusion gene, each gene may be linked at any position. As used herein, "fusion genes" includes not only genes in which different genes are linked, but also genes in which the same genes are linked.

[0025] As used herein, "operably linked" means that the polypeptide encoded by the nucleic acid is linked to an element such as a promoter so that the polypeptide is expressed in a state that exhibits the biological activity of the polypeptide under the control of the element.

[0026] As used herein, the term "random mutation" refers to randomly introduced mutations in a nucleic acid sequence, and the number of mutations may be one or more. The mutations in random mutations may be artificially introduced or may be naturally occurring mutations.

[0027] As used herein, the term "marker gene" refers to a gene encoding any protein that is translated within a cell, functions as a marker, and enables the identification of cell types that satisfy specific conditions. There are no particular limitations on the protein as long as it can be translated within a cell and function as a marker, and it includes, for example, proteins that exhibit fluorescence, luminescence, or color development, or proteins that can be visualized and quantified by supporting fluorescence, luminescence, or color development. Fluorescent proteins include blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, HyPer, and mUkG (Umikinoko Green); yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; and KusabiraOrange. (e.g., hmKO2) and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, and mPlum, but are not limited to these.

[0028] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0029] In one aspect, the present disclosure provides a composition comprising a polypeptide for inducibly controlling expression of a gene of interest in an organism, wherein the polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a gene of interest sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulator encoded by the gene switch expression sequence with an inducer. Such a polypeptide or composition functions as a gene switch or gene switch molecule.

[0030] In one embodiment, the organism may be any prokaryotic or eukaryotic organism, such as an animal, plant, fungus, or protist. Preferably, the eukaryotic organism is a fungus, and more preferably, the eukaryotic organism is a yeast. Suitable yeasts in the present disclosure include, for example, yeasts belonging to the genus Saccharomyces, yeasts belonging to Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), and methanol-utilizing yeasts (Ogataea polymorpha, Hansenula polymorpha, Candida boidinii, etc.).

[0031] In one embodiment, the gene switch expression sequence can be any sequence, and for example, when the gene switch is a protein or polypeptide, it can be a nucleic acid sequence encoding the gene switch. The gene switch expression sequence is a factor containing a transcription activation domain having a site at which a substance (activator) that can activate a molecule by interacting with (e.g., binding to) the nucleic acid that constitutes a gene of interest interacts with (e.g., binds to) the nucleic acid. In one embodiment, the gene switch expression sequence can include a sequence that expresses a transcription regulator such as CamTA, PhlTA, LuxTA, or rtetTA, or an artificial transcription regulator that is a modified version of such a transcription regulator.

[0032] In one embodiment, the transcriptional regulatory factor is any factor that acts on a regulatory DNA element such as a promoter, and includes, for example, a transcriptional repressor (repressor) that acts on a regulatory DNA element to suppress gene transcription and reduce the amount of gene expression, and a transcriptional activator that acts on a regulatory DNA element to promote gene transcription and increase the amount of gene expression. In one embodiment, the transcriptional regulatory factor may include CamTA, PhlTA, LuxTA, rtetTA, or an artificial transcriptional regulatory factor obtained by modifying such a transcriptional regulatory factor. In one embodiment, the transcriptional regulatory factor may be fused with a transcriptional activation domain (e.g., VP16, 48), other activation domain sequences, a transcriptional repressor, a nuclear localization signal (NLS), or the like.

[0033] In one embodiment, the inducer is any factor that can activate, inactivate, or change the function of a transcriptional regulator by binding to the transcriptional regulator. In one embodiment, the inducer may be, for example, DAPG, D-Camphor, HSL, or Dox.

[0034] In one embodiment, the complex in which the transcriptional regulatory factor and the inducer are bound may be bound in any manner as long as the function of the complex is to directly or indirectly regulate the expression of one or more genes.

[0035] In one embodiment, the operator sequence can be located adjacent to the promoter sequence, either upstream or downstream, and can be one or multiple operator sequences connected in tandem, such as about 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 operator sequences.

[0036] In one embodiment, the polypeptides (gene switches) contained in the composition of the present disclosure may have a common structure, for example, in that they contain an artificial transcription activation domain (VP16 x 3 = VP48) or a nuclear localization signal (NLS). In another embodiment, the artificial promoters may have a common structure in that they contain a core promoter (derived from GAL1) (or the AOX1 or DAS1 core promoter when using Pichia yeast). In one embodiment, the transcription factors (artificial transcription factors) or their binding sequences (artificial promoters) of the present disclosure may each contain a corresponding bacterial repressor and operator.

[0037] In one embodiment, the polypeptides (gene switches) included in the composition of the present disclosure may be: (DAPG-ON) an amino acid sequence represented by SEQ ID NO: 1 or a variant thereof containing one or more mutations selected from positions 5, 6, 20, 70, 86, 109, 117, 143, and 187; (HSL-ON) an amino acid sequence represented by SEQ ID NO: 3 or a variant thereof containing one or more mutations selected from positions 116, 201, 140, and 33; (Borneol-ON) an amino acid sequence represented by SEQ ID NO: 2 or a variant thereof containing a mutation at position 40; (Tet-ON) an amino acid sequence represented by SEQ ID NO: 4 or a variant thereof containing a mutation at position 8; (Camphor-OFF) an amino acid sequence represented by SEQ ID NO: 2 or a variant thereof containing a mutation in PKKKRKV; or (DAPG-OFF) an amino acid sequence represented by SEQ ID NO: 1 or a variant thereof containing one or more mutations selected from positions 157 and 41.

[0038] Specifically, for example, (DAPG-ON) those having the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof, which contain one or more mutations selected from P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A; (HSL-ON) those having the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof, which contain one or more mutations selected from S116Y, W201R, H140N, and T33A; (Borne ol-ON) The amino acid sequence represented by SEQ ID NO: 2 or a variant thereof, which contains a Y40C mutation; (Tet-ON) The amino acid sequence represented by SEQ ID NO: 4 or a variant thereof, which contains a K8N mutation; (Camphor-OFF) The amino acid sequence represented by SEQ ID NO: 2 or a variant thereof, which contains a PKKKRKV to PKRKERSKI mutation; (DAPG-OFF) The amino acid sequence represented by SEQ ID NO: 1 or a variant thereof, which contains one or more mutations selected from R157H and E41G.

[0039] The nucleic acids or proteins used in the present disclosure may include sequences or variants thereof in which one or more amino acids or nucleotides have been mutated (e.g., substituted, deleted, and / or added) in the target amino acid or base sequence. Here, "one or more" in the full-length amino acid sequence of the chimeric protein generally refers to 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less, or 1 amino acid). Furthermore, in the amino acid sequence of a domain, "one or more" generally refers to 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (e.g., 3 amino acids or less, 2 amino acids or less, or 1 amino acid). To maintain the biological activity of the present disclosure, it is desirable that the mutated amino acid residue be mutated to another amino acid whose amino acid side chain properties are conserved. For example, the nature of the amino acid side chains can include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the characters in parentheses represent the single-letter amino acid symbols). These are also referred to herein as "conservative substitutions." When mutations are anticipated in the polypeptides of the present disclosure, non-conservative substitutions are preferred, and it is understood that conservative substitutions may be made at amino acid positions where mutations are not anticipated.It is known that proteins having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids can maintain their biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M.J. & Smith, M., Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Therefore, in one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values ​​or less. Proteins with deletions or other modifications can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. Site-directed mutagenesis can be performed using, for example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.). Mutant antibodies with deletions or other modifications can be selected to have activity similar to that of the wild-type antibody by performing various characterization techniques such as FACS analysis or ELISA.

[0040] As used herein, the phrase "mutation of one or more amino acids in an amino acid sequence (e.g., insertion, substitution, and / or deletion, or addition to one or both termini)" refers to a modification that involves substitution of a number of amino acids, such as those that may occur naturally, by well-known techniques such as site-directed mutagenesis, or by natural mutation. The modified amino acid sequence may be one in which, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted, or deleted, or added to one or both termini. The modified amino acid sequence may preferably have one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of the present disclosure. In one embodiment, the modified amino acid sequence includes only a portion of the sequence, such as one in which the first Met has been deleted, or one in which the N-terminus or C-terminus has been deleted, in the amino acid sequences or nucleic acid sequences of SEQ ID NOs: 1 to 5.

[0041] In one embodiment of the present disclosure, in addition to the mutations specified in the polypeptide or composition of the present disclosure, one or more additional mutations may be contained, as long as they do not substantially affect the function (such as a gene switch) of the present disclosure.

[0042] In one embodiment of the present disclosure, a gene switch can be applied to the mass production of useful proteins. For example, protein production involves forcing host cells, such as E. coli, to express a target protein obtained from a heterologous organism. To produce a protein that is toxic to the host cell, the host cells are grown to a sufficient number and then induced for forced expression at an appropriate time. Therefore, a gene switch according to one embodiment of the present disclosure has a sufficiently low basal expression level when not induced (i.e., low leakage expression) and sufficient gene expression when expression is induced (ON) (i.e., a large ON / OFF expression level ratio).

[0043] In still another embodiment, the gene switch of the present disclosure can regulate the expression of downstream genes with high sensitivity even when the concentration of the inducer is low.

[0044] In another embodiment, the gene switch can be used as a tool for metabolic engineering. In metabolic engineering, multiple enzyme genes are simultaneously expressed in a single host cell to construct a biosynthetic pathway for a target substance. To achieve optimal results in the constructed artificial biosynthetic pathway, such as maximizing the yield of the final product per biomass and minimizing by-products, it is necessary to precisely and independently regulate the expression levels of each gene. Therefore, in one embodiment of the present disclosure, a gene switch can be provided that has the desired ON / OFF switching properties and can continuously regulate the expression levels of each gene switch when regulating the expression of multiple genes simultaneously in a single cell, without an inducer of one gene switch causing the malfunction of another gene switch.

[0045] In another embodiment, the gene switch sensor of the present disclosure can easily and quickly measure the production amount (concentration) of a target metabolite using GFP or the like, without using analytical techniques such as HPLC or GCMS. When the gene switch sensor of the present disclosure is used as a metabolic sensor, not only can a highly sensitive gene switch sensor be used to measure compounds produced in low amounts, but also a less sensitive gene switch sensor can be used to measure the production amount of a strain whose production amount has increased, and the method of the present disclosure allows for easy sensitivity adjustment.

[0046] In one embodiment of the present disclosure, when using a transcription factor in a host including not only yeast but also prokaryotes such as E. coli and other eukaryotes, the same effect can be obtained by introducing the mutations identified in the artificial transcription factor obtained in the present disclosure. Both cases of introducing the mutations into a natural transcription factor and cases of introducing the mutations into an artificial transcription factor are conceivable.

[0047] In one embodiment of the present disclosure, improved versions of various gene switch sensors (e.g., tetracycline-responsive, homoserine lactone (HSL)-responsive, 2,4-diacetylphloroglucinol (DAPG)-responsive, etc.) can be easily produced. Furthermore, gene sensors that showed almost no response in the initial sensors can be easily improved to a level where a clear response is observed. Reverse switches can also be easily constructed. Furthermore, by using tandem operator sequences, even more dramatic improvements in response performance have been achieved. (artificial transcription factor) In one aspect of the present disclosure, there is provided a composition comprising a polypeptide for inducible control of expression of a gene of interest in an organism, wherein a polynucleotide encoding the polypeptide comprises a gene switch expression sequence and a gene of interest sequence whose expression is induced by binding of a complex formed by binding a transcriptional regulator encoded by the gene switch expression sequence with an inducer. (DAPG-ON gene switch) In one embodiment of the present disclosure, a DAPG-ON type gene switch can be provided. In the case of an artificial transcription factor (PhlTA) using a 2,4-diacetylphloroglucinol (DAPG)-responsive transcription factor (PhlF), only an OFF type switch (DAPG-OFF) is known. However, in one embodiment of the present disclosure, a DAPG-ON type gene switch can be provided in which the responsiveness is reversed by adding the Q117R, K86T, or E143K mutation to PhlF in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof. In one embodiment, the responsiveness of this DAPG-ON type gene switch can be reversed simply by introducing Q117R, Q117P, Q117G, or Q117N. In one embodiment, the responsiveness of the DAPG-ON type gene switch can be more significantly reversed by further adding K86T and E143K. In one embodiment, the sensitivity to DAPG can be improved by adding the F109L, K86S, K86A, or K86G mutation in addition to these mutations. In another embodiment, the increase in OFF (in the absence of DAPG) expression (increased leakage) associated with increased sensitivity can be suppressed by mutation of S5P or P6S. Furthermore, in one embodiment, the addition of mutations of P5S, S6P, or D90G can improve responsiveness. For example, when increasing sensitivity by introducing a mutation such as F109L, leakage expression during OFF can be suppressed. Regarding F109, even when 50 variants were screened from a site-saturation library (NNK) of the F109 residue, only F109L was found as a sensitivity-enhancing mutation, suggesting that mutations other than L do not increase sensitivity.

[0048] In another embodiment, a DAPG-ON switch with inverted responsiveness can be provided by mutations of K20R, E70G, and / or T187A. In one embodiment, inversion of responsiveness cannot be achieved without the absence of either E70G or T187A.

[0049] Furthermore, in one embodiment, tandem use of operator sequences can dramatically improve responsiveness (500-fold or more with 6 copies of phlO).

[0050] In another aspect of the present disclosure, the DAPG-ON gene switch can be provided as encoded by a nucleic acid molecule containing one or more mutations selected from the following in the nucleotide sequence represented by SEQ ID NO: 5 or a variant thereof: C13T, T16C, A350G, AA to CG mutations at positions 350 and 351, CAA to ATT mutations at positions 349 and 351, CAA to GGT mutations at positions 349 and 351, A257C, AA to GC mutations at positions 256 and 257, AA to TC mutations at positions 256 and 257, AA to GG mutations at positions 256 and 257, G427A, T325C, A59G, A209G, and A559G. In this case, the polypeptide encoded by the nucleic acid molecule can function as a polypeptide for inducible control of target gene expression in Pichia yeast. (DAPG-OFF gene switch) In one embodiment of the present disclosure, a DAPG-OFF gene switch can be provided. In one embodiment of the present disclosure, an artificial transcription factor (PhlTA) using a 2,4-diacetylphloroglucinol (DAPG)-responsive transcription factor (PhlF) can be used, for example, by adding one or more mutations selected from R157H and E41G to PhlF in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof (mutations G470A (corresponding to R157H) and A122G (corresponding to E41G) in the nucleic acid sequence represented by SEQ ID NO: 5), thereby providing a gene switch with a higher S / N ratio than conventional OFF-type switches (DAPG-OFF). (HSL-ON type gene switch) In one embodiment of the present disclosure, an HSL-ON type gene switch can be provided. In one embodiment of the present disclosure, a gene switch (HSL-ON) can be provided that utilizes an artificial transcription factor (LuxTA) that uses a homoserine lactone (HSL)-responsive transcription factor (LuxR). Adding the S116Y mutation to LuxR can improve sensitivity to HSL compared to the previously known S116A mutation. Furthermore, in one embodiment, adding W201R, T33A, and / or H140N mutations in addition to S116Y can further improve sensitivity to HSL. Therefore, in one embodiment of the present disclosure, the gene switch preferably contains one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. (Borneol-ON gene switch) In one embodiment of the present disclosure, a Borneol-ON type gene switch can be provided, in which the gene switch preferably contains a Y40C mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. (Tet-ON gene switch) In one embodiment of the present disclosure, a Tet-ON gene switch can be provided. In one embodiment of the present disclosure, a gene switch (Tet-ON) can be provided that utilizes an artificial transcription factor (rTetTA) that uses a doxycycline (Dox)-responsive transcription factor (TetR). By adding the R8K mutation, a higher S / N ratio (approximately 600 times) can be achieved than the previously known G72V mutation (approximately 500 times). Therefore, in one embodiment of the present disclosure, the gene switch preferably contains the R8K mutation in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof. (Camphor-OFF type gene switch) In one embodiment of the present disclosure, a camphor-OFF gene switch can be provided. In one embodiment of the present disclosure, a gene switch (Camphor-OFF) can be provided that utilizes an artificial transcription factor (CamTA) that employs a D-camphor-responsive transcription factor (CamR). Functional expression in yeast can be achieved by disrupting the NLS function (e.g., by frameshift or insertion of a stop codon) of the nuclear localization signal (NLS) located at the C-terminus, thereby avoiding toxicity. In one embodiment of the present disclosure, a frameshift mutation that disrupts NLS function can be exemplified by a PKKKRKV to PKRKERSKI mutation in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof. In this case, the gene sequence can include the A695del mutation. In another embodiment, a switch with improved sensitivity not only to camphor but also to borneol and β-pinene can be provided by adding a Y40C (or Q87R, or R20K) mutation to CamR. (Gene switch in Pichia yeast) In one embodiment of the present disclosure, an artificial promoter fusing an operator sequence (phlO) with a core promoter (e.g., the AOX1 or DAS1 core promoter derived from Pichia yeast) can be used as a gene switch in Pichia pastoris (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris) or methanol-assimilating yeast (e.g., Ogataea polymorpha, Hansenula polymorpha, Candida boidinii). This allows expression of a PhlF mutant (PhlTA mutant) created in budding yeast to function as a DAPG-ON promoter in methanol-assimilating yeast, including Pichia yeast. In one embodiment, up to 48 copies of the operator sequence (phlO) can be tandemly arranged to create an extremely powerful artificial inducible promoter that functions in methanol-assimilating yeast.

[0051] In one embodiment of the present disclosure, a DNA library can be prepared using various known methods, and is not particularly limited as long as random mutations are introduced into the gene switch expression sequence and / or the promoter to which the transcriptional regulatory factor encoded by the gene switch expression sequence binds. In one embodiment of the present disclosure, such a DNA library can be introduced into host cells by introducing an expression vector into the host cells. An "expression vector" refers to vector DNA that carries an exogenous gene into a host cell and is capable of expressing a target gene in the host cell. The vector DNA is not particularly limited as long as it is replicable in the host, and can be selected appropriately depending on the type of host and intended use. The vector DNA may be obtained by extracting naturally occurring DNA, or it may be vector DNA that lacks portions of DNA other than those necessary for replication. Representative examples of vector DNA include vector DNA derived from plasmids, bacteriophages, and viruses. Examples of plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis, and yeast. Examples of bacteriophage DNA include λ phage. Examples of virus-derived vector DNA include vectors derived from animal viruses such as retroviruses, vaccinia viruses, adenoviruses, papovaviruses, SV40, fowlpox viruses, and pseudorabies viruses, as well as vectors derived from insect viruses such as baculoviruses. Other examples include vector DNA derived from transposons, insertion elements, and yeast chromosomal elements. Alternatively, examples include vector DNA created by combining these, such as vector DNA (cosmids, phagemids, etc.) created by combining genetic elements of plasmids and bacteriophages. A gene of interest must be incorporated into the vector DNA so that it can be expressed, and its components must consist of at least the gene of interest and a regulatory DNA element, such as a promoter.In addition to these elements, if desired, gene sequences carrying information related to replication and regulation can be combined and incorporated into vector DNA using known techniques. Examples of such gene sequences include cis elements such as ribosome binding sequences, terminators, signal sequences, and enhancers, splicing signals, and selection markers (dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, etc.). One or more gene sequences selected from these can be incorporated into vector DNA.

[0052] A known genetic engineering technique can be used to incorporate a gene of interest into vector DNA. For example, a gene of interest can be digested at specific sites with an appropriate restriction enzyme, then mixed with similarly digested vector DNA, and religated with a ligase. Alternatively, a desired vector DNA can be obtained by ligating an appropriate linker to the gene of interest and inserting it into the multicloning site of a vector suitable for the purpose.

[0053] The method for introducing an expression vector into a host cell is not particularly limited as long as it is a method that can introduce vector DNA into the host cell and cause expression of a target gene in the host cell, and any known method appropriately selected depending on the species of the host cell may be used, such as the lithium acetate method, electroporation, calcium phosphate method, and lipofection.

[0054] In one embodiment of the present disclosure, it is not necessary to include the expression sequence and the gene switch in the same expression vector, and this can also be achieved by co-transforming an expression vector containing the expression sequence of the fusion reporter gene and an expression vector containing the gene switch into the same cell.

[0055] The cells used in the present disclosure are not particularly limited, and various types of cells, such as yeast and Escherichia coli, can be used. In one embodiment of the present disclosure, cell death or cell extinction refers to a state in which cellular functions, such as proliferation, have been lost. For example, when cells such as yeast are cultured on a solid medium, this refers to a state in which the ability to grow from a single cell and form a cell colony of a certain size or larger that can be visually counted has been lost. In other embodiments, cell death or cell extinction may also include active cell death (apoptosis), which actively removes unnecessary or damaged cells caused by physiological or pathological factors, and passive cell death (necrosis), which is a response to external factors.

[0056] In one embodiment of the present disclosure, budding yeast, fission yeast, etc. can be used as the yeast. Examples of budding yeast that can be used include the Saccharomyces genus (e.g., Saccharomyces cerevisiae), Zygosaccharomyces genus (e.g., Zygosaccharomyces rouxii), Pichia yeast (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), and methanol-utilizing yeast (Ogataea polymorpha, Hansenula polymorpha, Candida boidinii, Ogataea minuta, Ogataea angusta, Pichia methanolica, Ogataea parapolymorpha, etc.). Examples of fission yeast that can be used include the Zygosaccharomyces genus (e.g., Zygosaccharomyces rouxii). Among these, particularly preferred yeasts are Saccharomyces cerevisiae or Pichia yeasts (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris). In one embodiment, a methanol-assimilating yeast is defined as a yeast cell that can be cultured using methanol as a sole carbon source. However, the term "methanol-assimilating yeast cell" also encompasses yeast cells that were originally methanol-assimilating yeast cells but have lost their ability to assimilate methanol due to artificial modification or mutation.

[0057] Pichia yeast has the ability to utilize methanol and is widely used industrially, primarily for protein production. In this yeast, the AOX1 promoter is widely used as a methanol-inducible system. This AOX1 promoter has very strong expression levels, and it is said that 5% of the total mRNA in cells grown under methanol is derived from AOX1p, and its expression can be strictly controlled by methanol.

[0058] In one embodiment of the present disclosure, a novel inducible promoter comparable to the AOX1 promoter can be provided by using rPhlTA, an artificial transcriptional activator, and DAPG as an inducer.

[0059] Gene switches can be used as inducible promoters, and there is a high demand for high-performance artificial gene switches that overcome the drawbacks of natural inducible promoters, which are difficult to use. Furthermore, gene switches allow for easy indirect quantitative evaluation of products and regulation of gene expression, leading to a growing demand for their use in recent years. If this method could be used to easily create high-performance gene switch sensors in yeast, which is also useful as an industrial microorganism, it would dramatically reduce the time required for strain construction and enable advanced metabolic and gene network control. Therefore, industrial applications are expected, particularly in the fields of metabolic engineering and synthetic biology. Furthermore, the gene switch disclosed herein can be used for mass production of useful proteins, expression control of toxic proteins, and simple concentration measurement of metabolic compounds.

[0060] In one embodiment of the present disclosure, an inducible promoter can be used for inducible expression (secretion or intracellular production) of a protein.

[0061] In one embodiment of the present disclosure, Tet-ON, DAPG-ON, and HSL-ON, which have excellent S / N ratios, can be used to control the expression of metabolic enzymes. For example, they can be used for enzymes that branch metabolic pathways. For example, in the production of squalene, a type of terpenoid, turning off downstream ergosterol pathway enzymes that consume squalene only at the timing of high squalene production can increase squalene production. Alternatively, in other embodiments, useful proteins that exhibit high cytotoxicity, such as antibody proteins, can be used as candidates. Unlike metabolic engineering, which involves constructing a chemical conversion process, antibody protein expression is directly proportional to production. Therefore, expression levels must be maximized. However, if the S / N ratio of the expression induction system is low, expression leakage occurs, making it impossible to obtain a yeast strain due to cytotoxicity caused by protein overexpression. DAPG-ON, Tet-ON, and HSL-ON switches, due to their high S / N ratios, are expected to enable high production of toxic proteins.

[0062] In one embodiment of the present disclosure, a gene switch can be used to switch on / off the expression of genes that are particularly toxic to cells. Overexpression of antibody proteins, which are expected to be used as pharmaceuticals, or heterologous enzyme genes responsible for the synthesis of plant metabolites in yeast often results in cytotoxicity. However, even genes that are toxic during the cell growth phase can sometimes have their toxicity alleviated if their expression is induced after growth is complete. Therefore, by using a gene switch to turn off the expression of a toxic gene during the growth phase and turn it on after growth is complete, cytotoxicity caused by overexpression of a heterologous gene can be avoided.

[0063] In one embodiment of the present disclosure, gene switches have also attracted attention in the field of biocomputing. A "genetic circuit," consisting of a combination of multiple gene switches, can be interpreted as an analogy to an "electronic circuit," which processes external information provided to a cell. Indeed, in the field of synthetic biology, numerous gene circuits with the same topology as logic circuits implemented in actual computers have been designed and constructed with the aim of constructing gene circuits and endowing cells with new functions. However, unlike electronic circuits, the ON / OFF switching of gene switches is analog, limiting the number of gene circuit combinations possible, limiting their scalability. Genetic switches with a superior OFF / ON ratio may potentially address these weaknesses of gene circuits.

[0064] In one embodiment of the present disclosure, the disclosed method can be used to create new high-performance gene switches or metabolic sensors in various biological species, primarily yeast, other eukaryotes, and prokaryotic cells, including Escherichia coli. The created switch sensors can be licensed or sold as part of kits, or contracted for development. The created genetic switches can also be used as inducible promoters for new protein production, which are safe, inexpensive, and easy to use (e.g., no explosion-proof equipment required, no carbon source limitations, etc.), to precisely control gene expression in metabolic pathways, or to develop high-performance (high-yielding) substance-producing strains for practical processes or research purposes (including in-house and contract development). Furthermore, the developed metabolic sensors can be used for simple, high-throughput quantification of target products and by-products, instead of HPLC or GC / MS, thereby accelerating the development of high-performance (high-yielding) substance-producing strains.

[0065] In one embodiment of the present disclosure, a gene switch using a cheaper and safer compound as an inducer can be developed and licensed or sold as part of a kit. Transcription factors from various biological species are screened using the physical properties of the inducer as an index, and a gene switch prototype is created using this. Random genetic mutations are introduced into the components of the prototype. OFF / ON selection is then performed on the gene switch library thus created. Specifically, yeast strains containing the gene switch library are cultured in the presence of 100 nM 5FdU and then transferred to a medium containing the inducer to induce gene expression. ON selection is performed by transferring the culture medium to a medium containing 1-3 mM Zeocin and the inducer. By adjusting the inducer concentration according to the application, gene switch mutants that respond to lower concentrations of the inducer can be isolated (enhanced sensitivity). The developed gene switch can be used to control the expression of metabolic enzymes that are key to substance production or to induce the production of proteins that place a heavy burden on the cell. In the contract development of switch sensors, a similar evolutionary molecular engineering cycle is carried out for a specified pair of inducer and transcription factor. For inducer-transcription factor pairs, those that respond to compounds important in metabolic engineering, such as the target product or its intermediates, are selected, and metabolic sensors that can easily monitor their concentrations are developed. The metabolic sensors obtained in this way are used to speed up strain development.

[0066] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and can be found in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology," Wiley, and annual updates; Sninsky, JJ et al. (1999); "PCR Applications: Protocols for Functional Genomics," Academic Press; and "Experimental Methods for Gene Transfer and Expression Analysis," a special edition of Experimental Medicine, Yodosha, 1997, all of which are incorporated herein by reference in their entirety.

[0067] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other examples include Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0068] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves.

[0069] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0070] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0071] Example 1: Evaluation of S / N ratio of Tet-ON gene switch Previous studies have shown that the Tet-ON gene switch showed only a three-fold induction upon Dox addition, but this was due to nonspecific binding of rtTetTA to tetO and the lack of p tetO7 This is thought to be due to the fortuitous appearance of a promoter upstream of tetO. To reduce nonspecific binding to tetO, the entire region of the rTetTA expression cassette was randomly mutated by error-prone PCR (epPCR), and the resulting PCR product was then transformed into p tetO7 The resulting transformants (~10 5 We performed OFF selection on the pool of cells (10 unique clones) by culturing them in 5FdU-containing liquid medium without Dox to eliminate mutants with leaky (i.e., Dox-independent) TBG expression. Next, we performed ON selection under various conditions to eliminate non-functional variants and thereby enrich for favorable switches. Specifically, the pool of OFF-selected cells was aliquoted into 14 test tubes containing various combinations of Dox (0.01–10 μg / mL) and Zeocin (1 or 2 mM), and the samples were shaken overnight.

[0072] Each of the 14 ON-selected cell mixtures was analyzed using flow cytometry (Figure 1A). Functional switches were enriched to different degrees under various selection conditions. In all cases, significant enrichment of desirable (Dox-inducible) mutants was observed at all nonzero concentrations of Dox tested (Figure 1A). In general, the output signal under induction conditions (Dox+) was higher for pools from runs 8–14, in which higher selection pressure (2 mM Zeocin) was applied under ON conditions, than for their corresponding pools under lower selection pressure (runs 1–7; 1 mM Zeocin). However, pools selected under ON with 2 mM Zeocin were contaminated with a high proportion of "always ON" variants that exhibited high fluorescence signals even in the absence of Dox. We found that ON selection with 2 mM Zeocin also exerted a toxic effect on positive (desirable) clones with high output upon Dox induction, thereby reducing the proportion of switching mutants compared to non-switching mutants (superactivators) with the "always ON" phenotype. In both cases, low concentrations of Dox, especially below 0.1 μg / mL, resulted in the gradual accumulation of leaky variants in the pools. In this particular case, the most efficient enrichment of functional switches was achieved in runs 9 and 11 (pools #9 and #11). Runs 8 and 11 were used for subsequent screening to isolate variants with both the highest induction output and the highest stringency.

[0073] From the two selected pools, 93 clones were randomly selected and subjected to fluorescent screening in a 96-well plate (Figure 1B). A total of 48% (45 / 93) of the tested clones showed more than 5-fold induction with Dox (10 μg / mL), indicating that the best rTetTA variant (rTetTA K8N、L131L ) showed 8- and 11-fold induction in the presence of 0.3 and 10 μg / mL Dox, respectively ( Fig. 1C ).

[0074] As shown in Figure 1A, random mutations were introduced into the expression cassette encoding the Tet-ON switch rTetTA, and the resulting library was subjected to parallel OFF / ON selection under different selection conditions. To screen for conditions that enrich for switch variants, the resulting cell populations were assessed for Dox-dependent fluorescence shifts. The green and blue regions represent variants with undesired behavior, i.e., leaky expression (10 in the absence of Dox). 4 cells with GFP fluorescence or higher) and inactive variants (10 in the presence of 10 μg / mL Dox 4 Figure 1B shows the relative abundance (%) of switching variants (cells with GFP fluorescence less than 100%). The relative abundance (%) of switching variants was obtained by subtracting both of these two values ​​from 100%. The dashed line indicates the histogram obtained from a yeast strain harboring a plasmid without rTetTA. Figure 1B shows the results of fluorescent screening of Tet-ON variants in a 96-well plate. Figure 1C shows the transfer function of selected variants. Error bars represent the mean ± SD of three independent experiments. 50% effective concentration (EC 50 ) values ​​were calculated from dose-response curves fitted to the Hill equation by the least squares method. The top panel shows the effect of the wild-type and evolved Tet-ON switch (rTetTA K8N、L13L Fluorescence images of cell pellets from a yeast strain carrying ) incubated with or without 10 μg / mL Dox are shown. Example 2: Improvement of gene switch Following the protocol for developing the Tet-ON system, any transcription factor can be used as a component of a eukaryotic transcriptional switch. However, the performance of the resulting switch is unpredictable due to differences in stability, DNA-binding affinity, and how their function changes when fused with other proteins or domains to generate sTAs. Furthermore, the switching behavior of sTAs is highly dependent on various factors, including expression level, promoter location and copy number, and strain type. In practice, new systems must be re-evolved to ensure appropriate performance under each condition.

[0075] To improve negatively regulated eukaryotic gene switches, we reconstructed two yeast transcriptional switches: the 2,4-diacetylphlorogucinol (DAPG)-repressible (DAPG-OFF) switch and the D-Camphor-repressible (Camphor-OFF) switch (Figure 2A). Specifically, we fused the DAPG- and D-Camphor-responsive bacterial repressors (PhlF and CamR) with a VP48 activation domain and a nuclear localization signal (NLS) to generate sTAs (PhlTA and CamTA, respectively) with protein sequences identical to the published sequences. Furthermore, we replaced seven copies of tetO with single operators for PhlF and CamR (phlO and camO, respectively). GAL1-c and synthetic promoters (p phlO1 and p camO1 ) was constructed. Although our method was as consistent as possible with published methods, the initial construct did not function as reported. This was likely due to slight differences in the cis-regulatory elements (promoter / terminator) used to drive sTA expression. Furthermore, we observed high toxicity with the PhlTA expression plasmid. When the plasmid was transformed into yeast, only a few viable colonies insensitive to DAPG were obtained (Figure 2B). These isolates were thought to be mutants with spontaneous down-tuning mutations in the PhlTA expression plasmid that likely alleviate the toxic effects of overexpressing VP16. The CamTA expression plasmid exhibited lower toxicity in yeast, but the reconstituted Camphor-OFF switch showed significant leaky expression in the presence of D-camphor (Figure 2C).

[0076] We mutated the entire PhlTA and CamTA expression cassettes using epPCR, and then performed ON / OFF selection on the resulting libraries. This selection rapidly isolated functional DAPG-OFF mutants (1-2E and 1-6H) that showed a 4-fold reduction in expression upon DAPG addition (Figure 2B), as well as a Camphor-OFF switch mutant (1-8D) that showed a 2-fold improvement in responsiveness to D-Camphor (S / N ratio) (Figure 2C). Notably, no obvious toxicity was observed in yeast for the newly isolated mutants. Five of the best-performing Camphor-OFF switches shared a frameshift mutation (A695del) (from amino acid PKKKRKV to RKERSKI) that abolished the entire NLS. Meanwhile, four of the best-performing DAPG-OFF switches retained an intact NLS but carried the same mutation in the PhlTA expression cassette. These observations suggest that partial impairment of NLS function (and possibly nuclear localization) is necessary for optimal CamTA activity to ensure no or less leaky TBG expression in the presence of D-camphor. In control experiments, no transcriptional activation by CamTA lacking the NLS was detected.

[0077] Figure 2 shows a schematic diagram of the method for creating and evolving yeast transcriptional switches (evaluated by signal-to-noise ratio, sensitivity, and behavioral type). Figure 2A shows the yeast gene switch developed in this disclosure. Plasmid expression of the identified sTA mutants resulted in the evolution of p phlO1 , p camO1 , or p luxO1Dose-dependent activation of genes under the control of β-glucan was observed. Figures 2B–2F show the transfer functions of the parent and mutant DAPG-OFF switch (Figure 2B), Camphor-OFF switch (Figure 2C), DAPG-ON switch (Figure 2D), and HSL-ON switch (Figure 2F). TBG-derived GFP fluorescence was plotted as a function of each inducer concentration. Error bars represent the mean ± SD of three independent experiments. The concentrations of inducers added during OFF / ON selection are indicated by arrows and dashed lines. EC 50 Values ​​were calculated from dose-response curves fitted to the Hill equation by least-squares and expressed in micromolar units. Figures 2E and 2G show the structural mapping of mutations in PhlF and LuxR that invert / sensitize PhlTA and sensitize LuxTA, respectively. The structures of PhlF and LuxR were modeled using the Swiss-Model server based on the crystal structures of the TetR family transcriptional regulator SCO0332 (PDB: 2ZB9) and the quorum sensor protein TraR (PDB: 1L3L), respectively. DNA structures were derived from the corresponding reference crystal structures. (Example 3: Conversion of DAPG-OFF system to DAPG-ON system) Mutations can lead to the emergence of transcription factors with novel switching behaviors. Various bacterial repressors are known to reverse their ligand response through several mutations. Therefore, we investigated whether mutagenesis of the gene encoding the aforementioned DAPG-OFF switch could convert the construct into a DAPG-ON switch. To this end, we subjected the same PhlTA library used to isolate the DAPG-OFF switch to OFF selection in the absence of DAPG, followed by ON selection in the presence of this compound (5 μM). Thirty variants were randomly selected from the surviving pool and subjected to OFF / ON screening. As a result, DAPG-ON variants (1-11E and 1-11G) were isolated, which exhibited an 8-fold DAPG-dependent increase in fluorescence (Figure 2D, Figure 4A). Mutational analysis of these variants revealed the presence of three novel mutations. One of them (Q117R) was essential for the inversion of the function of PhlTA (rPhlTA), and the other two mutations (E143K and / or K86T) were essential for improving the responsiveness (both sensitivity and fold change) of the inverted switch (Figure 2E). Another cycle of mutagenesis, selection, and screening using a lower concentration of DAPG (0.5 μM) for ON selection reduced the EC by up to 8-fold. 50 We obtained three second-generation variants (2-1E, 2-4E, and 2-7E) that exhibited enhanced sensitivity to DAPG (Figure 2D). All of these second-generation variants shared a novel mutation (F109L) that caused increased sensitivity to DAPG (Figure 2E). PhlF has previously been modified in E. coli for sensitivity and selectivity to DAPG, but the mutations that exhibited the reversed PhlF (rPhlF) function shown in this example have not been reported previously. Similarly, mutations that increase the sensitivity of rPhlF have not been reported previously.

[0078] Figure 4 shows the nucleotide and amino acid (AA) mutations found in the rPhlTA expression cassette of the evolved DAPG-ON switch. Mutations found in the first-generation (Figure 4A) and second-generation (Figure 4B) rPhlTA expression cassettes are indicated by red lines, except for the second-generation mutant, whose mutation was also found in the parent (1-11E) mutant. Fold change was calculated from the data shown in Figure 2D as the mean ± SD ratio of fluorescence intensity in the presence and absence of 10 μM DAPG. Example 4: Other Novel Switches To date, prokaryotic transcriptional activators have not been utilized as transcriptional activators in yeast. Therefore, we attempted to produce sTA using Vibrio fischeri LuxR, a 3-oxo-hexanoylhomoserine lactone (HSL)-inducible bacterial transcriptional activator, as the inducible component. Simple plasmid expression of LuxTA (a fusion of LuxR, VP48, and NLS, Figure 2A) resulted in p luxO1 Under the control of the GAL1 core promoter fused to the LuxR-binding box (luxO), HSL-dependent expression of TBG was not observed (Figure 2F). Using this nonfunctional parent as starting material, we performed two rounds of mutagenesis and selection with decreasing concentrations of inducer (round 1, 10 μM HSL; round 2, 1 μM HSL) and successfully identified functional HSL-ON variants. Thus, for the first time, we rapidly enabled yeast to sense and respond to bacterial signals.

[0079] The best-performing variant (Figure 2F), which showed a 6-fold enhancement of TBG expression upon the addition of 3 μM HSL, had two nonsynonymous mutations (S116Y and W201R) (Figure 2G, Figure 5). In the yeast HSL-ON switch, both mutations were sufficient to sensitize LuxTA to HSL alone, albeit to different degrees. However, each mutation behaved quite differently in E. coli. As seen in yeast, the S116Y mutation, located in the ligand-binding region of the LuxR structure, inhibited the LuxR / p binding in E. coli. luxIt increased the HSL sensitivity of the transcription factor. On the other hand, another mutation (W201R), located on the surface of LuxR, known to interact with E. coli RNA polymerase, significantly reduced expression from the lux promoter in E. coli. The W201R substitution appears to negatively affect LuxR's function as an RNA polymerase recruiter, which may explain why this mutation was previously overlooked in screens for LuxR mutations that sensitize proteins for HSL activation. In yeast HSL-ON switches, the role of LuxR is to facilitate HSL-induced DNA binding. Therefore, this sensitizing mutation could be identified only in yeast genetic switches.

[0080] Figure 5 shows the nucleotide and amino acid (AA) mutations found in the LuxTA expression cassettes of the evolved HSL-ON switches. Mutations found in the first-generation (Figure 5A) and second-generation (Figure 5B) LuxTA expression cassettes are indicated by red lines, except for the second-generation mutants in which the mutation was also found in the parent (1-4A) mutant. Fold change was calculated from the data shown in Figure 2F as the mean ± SD ratio of fluorescence intensity in the presence and absence of 100 μM HSL (3 μM for 2-4F). Example 5: Integration of yeast switch mutants into an AND-gated β-carotene biosynthetic pathway We have developed a series of gene switches in yeast with improved signal-to-noise ratios, and we attempted to apply these structures to pathway flux control. 2-1E , HSL-ON 2-4F , and Tet-ON 1-11F ) were integrated into different chromosomes of yeast (Figure 3A). The resulting strains were transformed with plasmids (p tetO7 , p phlO6 , and p luxO5) was transformed. The potential promoter sequence contained in the vector sequence more than 500 sequences upstream of synP was deleted, and the number of repeats of phlO and luxO was increased. GFP fluorescence of the resulting cells was induced only in the presence of their cognate inducers, and was observed at 10 2 These synthetic promoters were induced by the above factors (Fig. 3B). All of these synthetic promoters were stringent, i.e., they showed low basal TBG expression in the absence of inducers.

[0081] We attempted to exploit the orthogonal regulatory potential of these switches by using them as regulators of the β-carotene biosynthetic pathway, and to synthesize β-carotene in a nine-step process using S. cerevisiae Bts1p in combination with the CrtYB and CrtI proteins of Xanthophyllomyces dendrorous (Fig. 3C).

[0082] When yeast was transformed with a plasmid expressing all three genes (BTS1, crtYB, and crtI) under the control of a constitutive promoter, the yeast produced β-carotene, regardless of the presence or absence of an inducer (Figure 3D). phlO6 and p tetO7 Since all three of these genes had to be expressed, β-carotene production was expected only in the presence of both DAPG and Dox (AND-gated). tetO7 Leaky expression from the promoter was found to be non-negligible, likely due to the strong catalytic activity of CrtYB, resulting in significant mispigmentation in the absence of Dox. This leaky expression could be attributed to the gene switch used to control crtYB. tetO7 From p luxO5The β-carotene biosynthesis pathway was then placed under AND gate control by the combination of HSL and DAPG. In this strain, the expected AND gate behavior was observed, in that β-carotene biosynthesis was observed only in the presence of both DAPG and HSL. A similar strategy was employed to construct a strain in which the β-carotene biosynthesis pathway was placed under AND gate control.

[0083] Figure 3 shows the flux control from FPP to β-carotene using the newly developed gene switch. Figure 3A shows the yeast strain used for AND gate control of carotenoid biosynthesis. 2-1E , HSL-ON 2-4F , and Tet-ON K8N,L131L Three plasmids expressing sTA for use in the switch were chromosomally integrated. phlO6 , p tetO7 , and p luxO5 ) were expressed only in the presence of the corresponding inducers (DAPG, Dox, and HSL, respectively). Figure 3B shows the flow cytometric results of orthogonal GFP expression control using Dox, DAPG, and HSL. Figure 3C shows the synthetic pathway to β-carotene. FPP stands for farnesyl diphosphate, and GGPP stands for geranylgeranyl diphosphate. Figure 3D shows a schematic diagram of steady-state β-carotene biosynthesis and AND-gated β-carotene biosynthesis. The constitutive promoter or synP(p) was used in the combinations shown on the left. phlO6 , p tetO7 , and p luxO5 The cell pellets of yeast strains expressing BTS1, crtYB, and crtI under the control of β-actin (β-actin) are shown. These strains were inoculated into liquid media containing different combinations of inducers (Dox, DAPG, and HSL) and cultured at 30°C for 24 hours. The inducer concentrations were DAPG (3 μM), HSL (3 μM), and Dox (10 μg / mL). Example 6: Development of a gene switch in Pichia yeast In this example, a gene switch was developed using the yeast Pichia pastoris. Specifically, as shown in Figure 6A, an artificial transcription activator (rPhlTA) was generated by fusing a DAPG-responsive bacterial repressor (PhlF) with a VP48 activation domain and a nuclear localization signal (NLS), and cloned between the GAP promoter and the AOX1 terminator. Furthermore, a single or tandem array of 18 PhlF operators (phlO) was fused to the AOX1 core promoter, driving a synthetic promoter (p phlO1および p phlO18 ) was constructed and cloned upstream of the EGFP gene. The AOX1 terminator was placed downstream of EGFP. The rPhlTA expression cassette and artificial promoter cassette were then integrated into the T38473 and ARG4 loci of Komagataella phaffii CBS 7435 (Fig. 6A).

[0084] As a result, as shown in Figure 6B, rPhlTA was inserted into the ARG4 locus and the artificial promoter p phlO1 or p phlO18 When an EGFP cassette containing the α- and β-actin-containing ...

[0085] As shown in Figure 6C, rPhlTA was inserted into the T38473 locus and the artificial promoter p phlO1 or p phlO18 When an EGFP cassette containing the α-terminal fragment was integrated into the T38473 locus, GFP expression at 0 μM DAPG was significantly increased, and the signal-to-noise ratio was reduced by approximately 7 and 300 times (approximately 1.6 kbp or 1 kbp).

[0086] The whole (approximately 1.6 kbp) or part (1 kbp) of the ARG4 gene was transformed with the artificial promoter p phlO1 or p phlO18When cloned upstream of p, only the GFP expression level at 0 μM DAPG was reduced, as shown in Figure 6D. phlO1 When phlO was used, the S / N ratio was restored by 300-fold. This indicates that inserting a sequence of 1 kbp or more upstream of the artificial promoter is necessary to achieve a high S / N ratio regardless of the genomic locus to which it is inserted. As shown in Figure 6E, a similar effect was observed when the copy number of phlO was increased to 48, extending the entire phlO tandem repeat to approximately 1.6 kbp, resulting in a S / N ratio of over 3000-fold.

[0087] Gene switches for yeast, including Pichia pastoris, can reverse the regulatory pattern not only by using artificial transcriptional activators but also by fusing a transcriptional repression motif, such as Mxi1 (a transcriptional repressor), in place of VP16.

[0088] In gene switches for Pichia yeast, using AOX1core or DAS1core as an artificial promoter is more efficient at controlling gene expression than using GAL1core. In Pichia yeast, artificial promoters using GAL1core have extremely low activity, but by changing to core promoters such as AOX1 or DAS1 (strong methanol-inducible promoters) derived from Pichia yeast or GAP, the ON (gene expression induction) value becomes significantly larger.

[0089] As mentioned above, while a high signal-to-noise ratio was achieved when the copy number of phlO was increased to 48, high responsiveness was also observed at 1, 6, 12, 18, and 24 copies. Although the maximum expression level during ON (gene induction) did not change significantly, we found that leaky expression significantly decreased with increasing copy number of phlO (Figures 6B, 6E, and 7). This suggests that the length of the DNA upstream of the promoter may affect the reduction in leaky expression. It is thought that some upstream transcription is responsible for leaky expression, and that this effect is reduced as the distance from the upstream site increases, resulting in a decrease in leaky expression. Regarding leaky expression caused by upstream sequences, it is possible to develop artificial promoters that are almost completely free of leaky expression by modifying the upstream sequence or increasing the distance from the upstream site.

[0090] Furthermore, placing ARG4 upstream of the operator sequence instead of the tandem copies of phlO (48 copies) also results in extremely low leaky expression. This suggests that ARG4 functions as an insulator. ARG4 also functions as an insulator when shortened to approximately 1000 bp. Even with phlO1, a signal-to-noise ratio of approximately 600-fold can be achieved (Fig. 6C, Fig. 6D). Increasing the operator copy number and lengthening the sequence also reduces leaky expression in the same way, suggesting that the distance from upstream is an important factor.

[0091] (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0092] The gene switch disclosed herein can be used for mass production of useful proteins, expression control of toxic proteins, and simple concentration measurement of metabolic compounds. This can significantly increase the productivity of biopharmaceuticals and industrial enzymes, dramatically reduce the time required for strain construction, and enable advanced metabolic and gene network control. Therefore, industrial applications are expected, particularly in the fields of metabolic engineering and synthetic biology. [Sequence List Free Text]

[0093] SEQ ID NO: 1: Amino acid sequence of DAPG-responsive transcription factor (PhlF) SEQ ID NO: 2: Amino acid sequence of D-camphor-responsive or Borneol-responsive transcription factor (CamR) SEQ ID NO: 3: Amino acid sequence of HSL-responsive transcription factor (LuxR) SEQ ID NO: 4: Amino acid sequence of Dox-responsive transcription factor (TetR) SEQ ID NO: 5: Nucleic acid sequence of transcription factor (PhlF)

Claims

1. A composition comprising a polynucleotide for inducible control of target gene expression in an organism, wherein the polynucleotide comprises a gene switch expression sequence and the target gene sequence, the expression of which is induced by binding of a complex formed by binding of a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer, The composition, wherein the gene switch comprises one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO:

1.

2. A composition comprising a polynucleotide for inducible control of target gene expression in an organism, wherein the polynucleotide comprises a gene switch expression sequence and the target gene sequence, the expression of which is induced by binding of a complex formed by binding of a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer, A composition, wherein the gene switch comprises a Y40C mutation in the amino acid sequence represented by SEQ ID NO:

2.

3. A composition comprising a polynucleotide for inducible control of target gene expression in an organism, wherein the polynucleotide comprises a gene switch expression sequence and the target gene sequence, the expression of which is induced by binding of a complex formed by binding of a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer, The composition, wherein the gene switch comprises a K8N mutation in the amino acid sequence represented by SEQ ID NO:

4.

4. A composition comprising a polynucleotide for inducible control of target gene expression in an organism, wherein the polynucleotide comprises a gene switch expression sequence and the target gene sequence, the expression of which is induced by binding of a complex formed by binding of a transcriptional regulatory factor encoded by the gene switch expression sequence with an inducer, The composition, wherein the gene switch comprises one or more mutations selected from R157H and E41G in the amino acid sequence represented by SEQ ID NO:

1.

5. The composition of any one of claims 1 to 4, wherein the gene switch further comprises a nuclear localization signal.

6. The composition of claim 5 , wherein the gene switch comprises a mutation that disrupts the function of the nuclear localization signal.

7. The composition of claim 6 , wherein the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal.

8. A nucleic acid molecule in which one or more bases selected from positions 13, 16, 349 to 351, 349 to 351, 256, 257, 427, 325, 59, 209, and 559 in the base sequence represented by SEQ ID NO: 5 have been mutated, and which contains one or more mutations selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1, and encodes a polypeptide having the ability to inducibly regulate the expression of a gene of interest in an organism.

9. The nucleic acid molecule of claim 8, comprising one or more mutations selected from C13T, T16C, A350G, mutations of AA at positions 350 and 351 to CG, mutations of CAA at positions 349 to 351 to GGT, A257C, mutations of AA at positions 256 and 257 to GC, mutations of AA at positions 256 and 257 to TC, mutations of AA at positions 256 and 257 to GG, G427A, T325C, A59G, A209G, and A559G in the base sequence represented by SEQ ID NO:

5.

10. The nucleic acid molecule according to claim 8 or 9, comprising one or more mutations selected from A350G, mutations of AA at positions 350 and 351 to CG, mutations of CAA at positions 349 to 351 to GGT, A209G, and A559G in the base sequence represented by SEQ ID NO: 5.

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