WO2022118972A1 - 高発現高制御性遺伝子スイッチ - Google Patents

高発現高制御性遺伝子スイッチ Download PDF

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WO2022118972A1
WO2022118972A1 PCT/JP2021/044573 JP2021044573W WO2022118972A1 WO 2022118972 A1 WO2022118972 A1 WO 2022118972A1 JP 2021044573 W JP2021044573 W JP 2021044573W WO 2022118972 A1 WO2022118972 A1 WO 2022118972A1
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gene
acid sequence
seq
mutation
variant
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French (fr)
Japanese (ja)
Inventor
将大 冨永
純 石井
洋一郎 伊藤
健太 能崎
昭彦 近藤
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Kobe University NUC
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Kobe University NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present disclosure relates to mutant sequences of artificial transcription factors, gene switches using them, and gene switch systems using them. More specifically, the present disclosure provides a highly expressed and highly regulatory gene switch, and a gene switch system utilizing the same.
  • the present inventors provide a novel gene switch and a gene switch system using the novel gene switch. More specifically, the present disclosure provides a highly expressed and highly regulatory gene switch, and a gene switch system utilizing the same. Such highly expressed and highly regulated gene switches are used in Pichia pastoris (Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), and methanol-utilizing yeast (Ogatanea ph). It is a variant of and has useful variants.
  • the present disclosure provides: (Item 1) A composition for inducible control of target gene expression in Pikia yeast or methanol-utilizing yeast, which comprises a nucleic acid sequence of the target gene and a gene switch expression sequence operably linked to the nucleic acid sequence.
  • a composition comprising a gene construct, wherein a complex of a transcriptional regulator and an inducing factor encoded by the gene switch expression sequence binds to a regulatory DNA element to control the expression of the gene of interest.
  • the transcription factor is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and The composition according to the above item, which comprises one or more mutations selected from T187A.
  • the transcription factor 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.
  • the mutation that disrupts the function of the nuclear localization signal includes a frameshift mutation of the nuclear localization signal.
  • the frameshift mutation of 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.
  • the transcriptional regulator comprises a mutation of K8N in the amino acid sequence represented by SEQ ID NO: 5 or a variant thereof.
  • the transcriptional regulator 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.
  • nucleic acid molecule of any one of the above items comprising mutations to GGT, A209G, and one or more mutations selected from A559G.
  • nucleic acid molecule according to any one of the above items wherein the polypeptide encoded by the nucleic acid molecule functions as a polypeptide for inducible control of expression of a target gene in an organism.
  • (Item H1) The composition according to any one of the above items for detecting the production amount of a gene product encoded by the target gene, the inducing factor thereof, or a metabolite thereof.
  • (Item A1) A method of producing the target protein A step of providing a gene construct comprising a target nucleic acid sequence encoding the target protein and a gene switch expression sequence operably linked to the nucleic acid sequence, the transcriptional regulatory factor encoded by the gene switch expression sequence. The step in which the expression of the nucleic acid sequence of interest is controlled by binding to the regulatory DNA element by the complex in which the nucleic acid and the inducer are bound. A step of introducing the gene construct into a host, wherein the host comprises the inducer.
  • the host comprises Pichia yeast or methanol assimilating yeast.
  • the transcription factor is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and The method according to any one of the above items, comprising one or more mutations selected from T187A.
  • the transcription factor 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.
  • the method described in paragraph 1. The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation that disrupts the function of a nuclear localization signal.
  • the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal.
  • (Item A7) The method according to any one of the above items, wherein the frameshift mutation of 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 A8) 13. the method of.
  • (Item A9) The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation in Y40C in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • (Item A10) The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation in K8N in the amino acid sequence represented by SEQ ID NO: 5 or a variant thereof.
  • (Item A11) The method according to any one of the above items, wherein the transcriptional regulator 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.
  • the Pichia yeast is selected from Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • (Item C1) A protein produced by the method according to any one of the above items.
  • (Item E1) A processed product and / or a metabolite obtained by processing and / or metabolizing a protein produced by the method according to any one of the above items.
  • (Item B1) A method for producing a processed product and / or a metabolite of a protein of interest.
  • a step of providing a gene construct comprising a target nucleic acid sequence encoding the target protein and a gene switch expression sequence operably linked to the nucleic acid sequence, the transcriptional regulatory factor encoded by the gene switch expression sequence.
  • the step in which the expression of the nucleic acid sequence of interest is controlled by binding to the regulatory DNA element by the complex in which the nucleic acid and the inducer are bound.
  • a step in which the production of the target protein is controlled by placing the host under conditions in which the transcriptional regulatory factor and the inducing factor bind to each other.
  • a method comprising the steps of recovering the protein of interest and performing the desired processing and / or metabolic treatment to obtain a processed product and / or a metabolite.
  • the host comprises Pichia yeast or methanol assimilating yeast.
  • the transcription factor is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and The method according to any one of the above items, comprising one or more mutations selected from T187A.
  • the transcription factor 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.
  • the method described in paragraph 1. The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation that disrupts the function of a nuclear localization signal.
  • the mutation that disrupts the function of the nuclear localization signal comprises a frameshift mutation of the nuclear localization signal.
  • (Item B7) The method according to any one of the above items, wherein the frameshift mutation of 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 B8) 13. the method of.
  • (Item B9) The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation in Y40C in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • (Item B10) The method according to any one of the above items, wherein the transcriptional regulator comprises a mutation in K8N in the amino acid sequence represented by SEQ ID NO: 5 or a variant thereof.
  • (Item B11) The method according to any one of the above items, wherein the transcriptional regulator 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.
  • the Pichia yeast is selected from Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • (Item D1) A processed product and / or a metabolite produced by the method according to any one of the above items.
  • (Item F1) A gene construct containing a nucleic acid sequence of interest encoding a protein of interest and a gene switch expression sequence operably linked to the nucleic acid sequence, with transcriptional regulators and inducers encoded by the gene switch expression sequence.
  • the transcription factor is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and The genetic construct according to the above item, comprising one or more mutations selected from T187A.
  • the transcription factor 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.
  • the frameshift mutation of 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 F7 13 Gene construct.
  • (Item F8) The gene construct according to any one of the above items, wherein the transcriptional regulator comprises a mutation in Y40C in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • (Item F9) The gene construct according to any one of the above items, wherein the transcriptional regulator comprises a mutation in K8N in the amino acid sequence represented by SEQ ID NO: 5 or a variant thereof.
  • (Item F10) The gene construct according to any one of the above items, wherein the transcriptional regulator 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 F11) The genetic construct according to any one of the above items, wherein the Pichia yeast is selected from Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • (Item G1) A cell containing the gene construct according to any one of the above items.
  • compositions containing a polypeptide for inducible control of target gene expression in Pikia yeast or methanol-utilizing yeast wherein the polynucleotide encoding the polypeptide is a gene switch expression sequence and the gene switch expression sequence.
  • a composition comprising a transcriptional regulatory factor encoded by the gene and a gene sequence of interest whose expression is induced by the binding of a complex to which an inducing factor is bound.
  • the gene switch is selected from the 5-position, 6-position, 20-position, 70-position, 86-position, 109-position, 117-position, 143-position, and 187-position in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the composition according to the above item which comprises a mutation of an amino acid at one or more positions.
  • the gene switch is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A.
  • the composition according to the above item, comprising one or more mutations selected from. (Item 2B) (DAPG-ON) Inversion One or more of the gene switch selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • composition according to any one of the above items which comprises a mutation.
  • (Item 3) (Camphor-OFF) The composition according to any one of the above items, wherein the gene switch comprises a mutation in which the function of a nuclear localization signal is disrupted.
  • (Item 4) (Camphor-OFF) The composition according to any one of the above items, wherein the mutation that disrupts the function of the nuclear localization signal includes a frameshift mutation of the nuclear localization signal.
  • 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 7) (Borneolu-ON) The composition according to any one of the above items, wherein the gene switch comprises a mutation in Y40C 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 above items, wherein the gene switch comprises a mutation in the amino acid 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 above items, wherein the gene switch comprises a mutation of K8N in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • (Item 9A) (DAPG-OFF) 13. Composition.
  • the Pichia yeast is selected from Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • (Item 12) (DAPG-ON) Specific mutations Mutations of AA at positions C13T, T16C, A350G, positions 350 and 351 to CG at positions 349 to CG in the base sequence represented by SEQ ID NO: 5 or a variant thereof. Mutations of CAA at positions 351 to ATT, mutations of CAA at positions 349 to 351 to GGT, mutations of A257C and AA at positions 256 and 257 to GC, and mutations of AA at positions 256 and 257 to TC.
  • the nucleic acid molecule of the above item comprising mutations of AA at positions 256 and 257 to GG, one or more mutations selected from G427A, T325C, A59G, A209G, and A559G.
  • (Item 13) (DAPG-ON) Inverted Mutation
  • mutations of AA at positions A350G, 350 and 351 to CG, and ATT of CAA at positions 349 to 351 The nucleic acid molecule according to any one of the above items, which comprises a mutation to GGT at positions 349 to 351 and one or more mutations selected from A209G, and A559G.
  • nucleic acid molecule according to any one of the above items, wherein the polypeptide encoded by the nucleic acid molecule functions as a polypeptide for inducible control of expression of a target gene in an organism.
  • (Item A1) A polynucleotide used for inducible control of target gene expression in Pikia yeast or methanol-utilizing yeast, wherein the polynucleotide encoding the polypeptide is composed of a gene switch expression sequence and the gene switch expression sequence.
  • a polypeptide comprising a transcriptional regulatory factor encoded and a gene sequence of interest whose expression is induced by binding of a complex to which an inducing factor is bound.
  • the gene switch is selected from the 5-position, 6-position, 20-position, 70-position, 86-position, 109-position, 117-position, 143-position, and 187-position in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the polypeptide according to the above item which comprises a mutation of an amino acid at one or more positions.
  • the gene switch is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A.
  • the polypeptide according to the above item, comprising one or more mutations selected from. (Item A2B) (DAPG-ON) Inversion One or more of the gene switch selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the polypeptide according to any one of the above items which comprises a mutation.
  • (Item A3) (Camphor-OFF)
  • (Item A4) (Camphor-OFF)
  • (Item A6A) HSL-ON
  • the gene switch comprises a mutation in an amino acid at one or more positions selected from positions 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof.
  • the polypeptide according to any one of the above. (Item A6) (HSL-ON)
  • 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.
  • Polypeptide polypeptide.
  • (Item A7A) (Borneolu-ON)
  • (Item A7) (Borneolu-ON)
  • (Item A8A) (Tet-ON)
  • (Item B1) The use of a polypeptide to produce a composition for inducible control of gene expression of interest in Pikia yeast or methanol-utilizing yeast, wherein the polynucleotide encoding the polypeptide is a gene switch expression sequence and. Use comprising a transcriptional regulatory factor encoded by the gene switch expression sequence and a gene sequence of interest whose expression is induced by binding of a complex to which an inducing factor is bound.
  • the gene switch is selected from the 5-position, 6-position, 20-position, 70-position, 86-position, 109-position, 117-position, 143-position, and 187-position in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the gene switch is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A. Uses according to the above item, comprising one or more mutations selected from. (Item B2B) (DAPG-ON) Inversion One or more of the gene switch selected from Q117R, Q117P, Q117G, Q117N, E70G, and T187A in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the frameshift mutation of 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.
  • the gene switch comprises a mutation in an amino acid at one or more positions selected from positions 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. Use as described in any one of the items.
  • (Item B6) HSL-ON
  • 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) (Borneolu-ON)
  • the gene switch comprises a mutation in the amino acid at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.
  • (Item B7) (Borneolu-ON) The use according to any one of the above items, wherein the gene switch comprises a mutation in Y40C 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 above items, wherein the gene switch comprises a mutation in the amino acid 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 above items, wherein the gene switch comprises a mutation in K8N in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • (Item B9A) (DAPG-OFF) 13. Use of.
  • (Item B9) (DAPG-OFF) The use according to any one of the above 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.
  • the Pichia yeast is selected from Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris.
  • (Item C1) A method for inducibly controlling the expression of a target gene in Pikia yeast or methanol-utilizing yeast using a composition containing a polypeptide for inducible control of target gene expression in Pikia yeast or methanol-utilizing yeast.
  • the gene of interest whose expression is induced by the binding of the polynucleotide encoding the polypeptide to the gene switch expression sequence and the complex in which the transcriptional regulatory factor and the inducing factor encoded by the gene switch expression sequence are bound. Methods, including sequences. (Item C2A) (DAPG-ON)
  • the gene switch is selected from the 5-position, 6-position, 20-position, 70-position, 86-position, 109-position, 117-position, 143-position, and 187-position in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the method according to the above item which comprises mutation of an amino acid at one or more positions.
  • the gene switch is P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K, F109L, K20R, E70G, and T187A.
  • the method according to the above item comprising one or more mutations selected from.
  • (Item C2B) (DAPG-ON) Inversion One or more of the gene switch 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 above items, wherein the gene switch comprises a mutation in which the function of a nuclear localization signal is disrupted.
  • (Item C4) (Camphor-OFF) The method according to any one of the above items, wherein the mutation that disrupts the function of the nuclear localization signal includes a frameshift mutation of the nuclear localization signal.
  • the frameshift mutation of the nuclear localization signal comprises a mutation of an amino acid in PKKKRKV in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.
  • the frameshift mutation of 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.
  • the gene switch comprises a mutation in an amino acid at one or more positions selected from positions 116, 201, 140, and 33 in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof. The method described in any one of the above.
  • (Item C6) HSL-ON
  • 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.
  • Method. (Item C7A) (Borneolu-ON) The method according to any one of the above items, wherein the gene switch comprises a mutation in the amino acid at position 40 in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.
  • (Item C7) (Borneolu-ON) The method according to any one of the above items, wherein the gene switch comprises a mutation in Y40C 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 above items, wherein the gene switch comprises a mutation in the amino acid 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 above 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) 13. the method of.
  • the gene switch of the present disclosure has high expression and high controllability.
  • the gene switch of the present disclosure is strictly controlled by a compound (inducing substance) other than methanol, and realizes a high degree of controllability not found in conventional gene switches.
  • the present disclosure relates to Pichia pastoris (Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), methanol-utilizing yeast (Ogataea polynemori, high-function yeast, etc.)
  • a mutant sequence of a gene switch sensor can be provided.
  • Useful mutations on transcription factors (proteins) are important deliverables as they are expected to function similarly not only in yeast, but also in other species, including bacteria.
  • FIG. 1 shows the improved S / N ratio of the Tet-ON type gene switch in one embodiment of the present disclosure.
  • FIG. 2 shows mutation sites of various gene switches developed in one embodiment of the present disclosure and their S / N ratios.
  • FIG. 3 is a schematic diagram showing flux control from FPP to ⁇ -carotene using the gene switch developed in one embodiment of the present disclosure.
  • FIG. 4 shows nucleotide and amino acid (AA, shown in parentheses) mutations found in the rPhlTA expression cassette of the DAPG-ON switch evolved in one embodiment of the present disclosure.
  • FIG. 5 shows mutations in nucleotides and amino acids (AA, shown in parentheses) found in the LuxTA expression cassette of the HSL-ON switch evolved in one embodiment of the present disclosure.
  • FIG. 6 is a schematic diagram of a gene switch in Pichia yeast developed in one embodiment of the present disclosure.
  • FIG. 7 is a graph showing the relationship between the change in the number of copies of phlO and the change in the expression level in the gene switch of Pichia yeast in one embodiment of the present disclosure.
  • FIG. 8 shows that mutations in P5S and / or S6P in rPhlTA suppress the expression of leaks in the absence of DAPG.
  • FIG. 9 is a diagram showing the effect of the length of the AOX1 core promoter in the artificial promoter on the responsiveness of the DAPG-ON system.
  • FIG. 10 is a diagram showing the effect of the length of the DAS1 core promoter in the artificial promoter on the responsiveness of the DAPG-ON system.
  • FIG. 11 is a diagram showing a gene expression system (KpTet-ON) for Dox-induced Pichia yeast developed in one embodiment of the present disclosure.
  • FIG. 12 is a diagram showing a gene expression system (KpHSL-ON) for HSL-induced Pichia yeast developed in one embodiment of the present disclosure.
  • FIG. 13 is a diagram showing a Pichia yeast DAPG-OFF system developed in one embodiment of the present disclosure.
  • FIG. 14 is a diagram showing the secretory production of Nanobody antibody using the DAPG-ON / OFF switch developed in one embodiment of the present disclosure.
  • FIG. 15 is a diagram showing the secretory production of Nanobody antibody using the DAPG-OFF switch developed in one embodiment of the present disclosure.
  • FIG. 16 is a diagram showing 5L scale fermentation production of small molecule antibodies using Fed-batch culture and DAPG-OFF system or GAP promoter system.
  • a “gene switch” or “gene switch sensor” is a factor that is interchangeably used and contains a transcriptional regulatory domain (eg, a molecule, a complex thereof, or a fusion thereof, etc.).
  • a transcriptional regulatory domain eg, a molecule, a complex thereof, or a fusion thereof, etc.
  • the "gene switch” may be referred to as a "transcription factor” and can be used interchangeably.
  • the activity of such factors is regulated by the interaction (eg, binding or dissociation) of the inducing substance (activator, or inducing factor), and its function can be changed.
  • the interaction (eg, binding) of an inducing substance (activator, or inducing factor) with a gene switch changes the degree or state of its interaction (eg, binding) with the target sequence of the gene switch.
  • the expression of the gene of interest is suppressed or induced.
  • the "gene switch" in the present specification is a molecule containing a transcriptional activation domain, and a site to which a substance capable of activating the molecule by binding (for example, an activating substance or an inducing factor) binds. Examples thereof include molecules having the above, and binding or dissociation to the target sequence due to the binding of the substance.
  • the “gene switch system” refers to the entire system using the gene switch as described above, that is, a transcription factor and its binding sequence (expression control sequence, regulatory DNA element, etc.), and further downstream thereof. Refers to the whole including the promoter of.
  • the term "highly controllable" of a gene switch means that gene expression downstream of a promoter controlled by the function of a gene switch (transcriptional regulator) can be turned on and off strictly. It means that the expression level ratio (S / N ratio) between on and off of expression is high. That is, when the maximum expression level (expression level in the on state) is high, the leakage expression (expression level in the off state) is low, and / or the response sensitivity is high, it can be said to be a highly regulatory gene switch.
  • the "gene switch expression sequence” means a nucleic acid sequence encoding the gene switch when the gene switch (transcriptional regulator) is a protein or polypeptide.
  • a “gene switch activator” activates a gene switch or alters its function by interacting with (eg, binding to) a gene switch (transcriptional regulator), resulting in.
  • a substance that directly or indirectly induces the regulation of the expression of one or more genes for example, such a compound.
  • the "activating substance” may be referred to as an "inducing factor” or an “inducing substance” and can be used interchangeably.
  • the activator can also be different for each gene switch (transcription factor). Examples of the combination of the gene switch (transcription factor) and the activating substance include rtetTA and Dox, CamTA and D-Camphor, PhlTA and DAPG, LuxTA and HSL, and the like.
  • a "gene switch target sequence” is a nucleic acid sequence located 5'upstream of a gene encoding a gene of interest, and refers to a nucleic acid sequence or regulatory DNA element that controls transcription of the target gene. It may preferably be an artificial promotor having a promotor activity and having a promotor arrangement or a fusion of an operator arrangement and a core promotor.
  • the enhancer can also act indirectly or directly on the target sequence of the gene switch (transcription factor).
  • the action of a gene switch (transcriptional regulator) on a target sequence can be regulated by the addition of an inducer, thereby controlling the expression of the target gene, unless the gene switch (transcriptional regulator) acts on the target sequence.
  • the target gene is not expressed.
  • the gene switch rtetTA acts on its target sequence teO promoter (artificial promoter containing teto) by the addition of its activator Dox, and promotes transcription of the target gene located downstream of the promoter.
  • the term "promotor” refers to a region on DNA that controls the initiation of transcription of a gene and directly regulates the degree of transcription thereof, and refers to a nucleic acid sequence that initiates transcription by binding to RNA polymerase. ..
  • the promoter can be appropriately selected and used depending on the type of host cell used. When yeast is used as a host, the promoter is not particularly limited as long as it can be expressed in a host cell such as yeast, and any of them may be used.
  • examples of the promoter include a GAL1 promoter, a GAL10 promoter, a heat shock protein promoter, an MF ⁇ 1 promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a TDH promoter, a DAS1 promoter, and an AOX1 promoter.
  • transcriptional regulator refers to a protein that acts on a regulatory DNA element such as a promoter.
  • Transcription regulators are broadly divided into transcriptional repressors (repressors) and transcriptional activators (activators).
  • Transcription suppressors can act on regulatory DNA elements to suppress gene transcription and reduce gene expression.
  • Transcriptional activators can act on regulatory DNA elements to promote gene transcription and increase gene expression.
  • known factors can be used as the transcriptional repressor and the transcriptional activator. The expression of a gene sequence located downstream of a regulatory DNA element on which a transcriptional regulator acts is suppressed or promoted by the action of the transcriptional regulator.
  • Gene expression means a series of processes in which gene information is transcribed into mRNA and further translated as an amino acid sequence of a protein encoded by the gene. If expression is promoted, a gene-encoded protein is produced and its amount increases, and if expression is suppressed, a gene-encoded protein is not produced and its amount decreases.
  • the "candidate transcription factor” is a factor that can be a candidate for a transcription factor.
  • inducing factor As used herein, the term “inducing factor”, “inducing substance” or “inducing agent” is used to activate, inactivate, or change the function of a transcriptional regulator by binding to the transcriptional regulator. As a result, a factor capable of regulating the direct or indirect expression of one or more genes.
  • a "DNA library” is a nucleic acid library containing a nucleic acid sequence isolated from nature or a synthetic nucleic acid sequence. Sources of naturally isolated nucleic acid sequences include, but are not limited to, eukaryotic cells, prokaryotic cells, or virally derived genomic or cDNA sequences. A library in which an arbitrary sequence (eg, signal, tag, etc.) is added to a sequence isolated from nature is also included in the DNA library of the present disclosure.
  • an arbitrary sequence eg, signal, tag, etc.
  • the term "host cell” refers to a cell into which a heterologous (eg, exogenous) nucleic acid or protein has been introduced.
  • the host cell can include not only a specific target cell but also a progeny of the cell.
  • Host cells include prokaryotic or eukaryotic cells and are any cells that have the appropriate traits to receive and produce heterologous nucleic acids or proteins. Includes, for example, 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 hybridoma or quadroma. Is done.
  • the term "expression vector” refers to a vector DNA that carries an external 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 can be replicated in the host, and can be appropriately selected depending on the type of host and the purpose of use.
  • the vector DNA may be a vector DNA obtained by extracting a naturally occurring DNA, or a vector DNA in which a part of the DNA other than the part necessary for replication is missing.
  • Representative vector DNAs include, for example, plasmids, bacteriophages, and virus-derived vector DNAs.
  • plasmid DNA a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, or the like can be used.
  • bacteriophage DNA include ⁇ phage and the like.
  • virus-derived vector DNA include vectors derived from animal viruses such as retrovirus, vaccinia virus, adenovirus, papovavirus, SV40, chicken pox virus, and pseudomad dog disease virus, and vectors derived from insect viruses such as baculovirus. be able to.
  • vector DNA derived from a transposon, an insertion element, a yeast chromosomal element, and the like can be mentioned.
  • a vector DNA prepared by combining these for example, a vector DNA prepared by combining genetic elements of a plasmid and a bacteriophage (such as cosmid and phagemid) can be mentioned. It is necessary to incorporate the target gene into the vector DNA so that the target gene is expressed, and at least the target gene and a regulatory DNA element, for example, a promoter are included in the vector DNA. In addition to these elements, if desired, gene sequences carrying information on replication and regulation can be combined and incorporated into vector DNA by known techniques.
  • Such gene sequences include, for example, ribosome binding sequences, terminators, signal sequences, cis-elements such as enhancers, splicing signals, and selectable markers (dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, etc.). can. One or more gene sequences selected from these can be integrated into the vector DNA.
  • fusion gene refers to a new gene that is generated by artificially or naturally linking multiple genes, and is the result of recombination such as chromosomal translocation, insertion, or inversion. Things are also included.
  • the fusion gene may also encode a fusion protein.
  • the number of genes linked in the fusion gene is not particularly limited as long as it can give rise to a protein that is functional as a fusion gene.
  • the orientation of each gene in the fusion gene is also not particularly limited, and all are forward (original transcription orientation), all are reverse (opposite to the original transcription orientation), and any combination of forward and reverse. May be good. Further, in the fusion gene, each gene may be linked at any position.
  • the "fusion gene” in the present specification includes not only a gene in which different genes are linked but also a gene in which the same gene is linked.
  • operably linked means that a polypeptide encoded by a nucleic acid is linked to an element such that it is expressed in a state that exhibits the biological activity of the polypeptide under the control of an element such as a promoter. It means that it has been done.
  • an "operably linked” sequence is an expression control sequence or regulatory DNA element flanking the gene of interest, and expression that acts trans or at a distance to control the gene of interest. Includes either regulatory sequences or regulatory DNA elements.
  • random mutation refers to the random inclusion of mutations in a nucleic acid sequence and such mutations, where the number of mutations may be one or two or more. There may be. Mutations in random mutations may be artificially introduced or may be naturally occurring mutations.
  • the term "marker gene” refers to a gene encoding an arbitrary protein that is translated intracellularly, functions as a marker, and enables discrimination of a cell type that meets specific conditions.
  • the protein is not particularly limited as long as it is a protein that can be translated intracellularly and functions as a marker.
  • a protein that exhibits fluorescence, luminescence, or coloration, or by assisting fluorescence, luminescence, or coloration, etc. Contains proteins that can be visualized and quantified.
  • the fluorescent protein examples include blue fluorescent proteins such as Siris and EBFP; cyanofluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (eg, h Green fluorescent protein such as EGFP, GFP2, HyPer, mUkG (Umikinoko Green); Yellow fluorescent protein such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBana, etc.
  • blue fluorescent proteins such as Siris and EBFP
  • cyanofluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP
  • TurboGFP AcGFP
  • TagGFP Azami-Green
  • Yellow fluorescent protein such as TagYFP, EYFP, Venus, YFP, PhiYFP,
  • Orange fluorescent protein such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawbury, etc .; Red fluorescent protein such as TurboFP602, mRFP1, JRed, KillerRed, , MPlum and other near-infrared fluorescent proteins, but are not limited thereto.
  • the "gene” refers to a factor that defines a genetic trait, and the “gene” may refer to a "polynucleotide", an “oligonucleotide”, and a “nucleic acid”.
  • the present disclosure describes Pichia pastoris (Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), Komagataella pseudopastoris, etc.
  • a transcriptional regulatory factor encoded by the gene switch expression sequence comprising a gene construct comprising the nucleic acid sequence of the gene of interest and a gene switch expression sequence operably linked to the nucleic acid sequence.
  • a composition in which the expression of the target gene is regulated by binding to the regulatory DNA element by the complex in which the gene and the inducer are bound. Such compositions function in gene switch systems.
  • Pichia pastoris (Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris) is known as a methanol-utilizing yeast, has excellent ability to secrete and produce proteins from the host, and produces proteins such as enzymes. It is used.
  • the gene of a foreign protein can be highly expressed by a gene expression system using the AOX1 promoter, which is a strong methanol-inducible promoter.
  • the promoter for expressing the target gene in Pichia yeast is not particularly limited as long as it can function in Pichia yeast in order to initiate the transcription of the gene, and the AOX1 promoter is preferably used.
  • an inducible promoter of Pichia yeast such as DAS1, DAS2, AOX2, HGT1, FDH1, FLD, CTA1, PMP20, FLD1, CAT1, CUP1, ICL1 and a constitutive expression type GAP can be used in the same manner. Can be done.
  • Pichia yeast (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris), methanol-utilizing yeast (Ogataeapolymorpha, Hansenula polymorpha, Candida boidinii, Ogataea minuta, Ogataea angusta, Pichia methanolica, etc.) Can be used.
  • particularly preferable yeasts are Pichia yeasts (Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris).
  • a methanol-utilizing yeast is defined as a yeast cell that can be cultivated using methanol as the sole carbon source. Originally, it was a methanol-utilizing yeast cell, but it was artificially modified. Alternatively, yeast cells that have lost their methanol assimilation performance due to mutation are also included in the methanol-utilizing yeast cells.
  • Pichia yeast has the property of assimilating methanol and is widely used industrially mainly in protein production.
  • the AOX1 promoter is widely used as a methanol induction system.
  • the expression level of this AOX1 promoter is very strong, and it is said that 5% of the total mRNA amount is derived from AOX1p in cells grown under methanol, and its expression can be strictly controlled by methanol.
  • rPhlTA which is an artificial transcription activator
  • DAPG DAPG
  • the gene switch expression sequence is an arbitrary sequence, eg, if the gene switch (transcriptional regulator) is a protein or polypeptide, it can be a nucleic acid sequence encoding the gene switch (transcriptional regulator). ..
  • the gene switch expression sequence is a site where a substance (activator or inducing factor) that can activate the molecule by interacting (for example, binding) with a nucleic acid constituting the target gene interacts (for example, binds). It is a factor containing a transcriptional activation domain having.
  • the gene switch expression sequence can include, for example, a sequence expressing a transcriptional regulator such as PhlTA, CamTA, LuxTA, rtetTA, or an artificial transcriptional regulator modified from such a transcriptional regulator.
  • the transcriptional regulator is an arbitrary factor that acts on and acts on a regulatory DNA element such as a promoter, eg, acts on the regulatory DNA element to suppress gene transcription and reduce gene expression. Includes transcriptional repressors (repressors) and transcriptional activators that act on regulatory DNA elements to promote gene transcription and increase gene expression.
  • the transcriptional regulator can include PhlTA, CamTA, LuxTA, rtetTA and artificial transcriptional regulators modified from such transcriptional regulators.
  • the transcriptional regulator can be fused with a transcriptional activation domain (VP16, 48, etc.), other activation domain sequences, transcriptional repressors, nuclear localization signals (NLS), and the like.
  • the inducing factor is any factor that, by binding to a transcriptional regulator, can activate, inactivate, or alter its function.
  • the inducing factor can be, for example, DAPG, D-Camphor, HSL, Dox.
  • any complex in which a transcriptional regulator and an inducing factor is bound is capable of regulating the direct or indirect expression of one or more genes as a function of the complex. It may be bonded in such an manner.
  • the operator sequence can be located close to upstream or downstream of the promoter sequence, and the operator sequence may be one or tandem to connect a plurality. In the case of tandemization, for example, about 2, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about. Operator sequences such as 45, about 50, about 55, about 60, and about 65 can be connected and used.
  • the artificial promoter may comprise a common structure in that it comprises a core promoter (derived from GAL1) (core promoters of AOX1 and DAS1 when Pichia yeast is used).
  • the transcription factor (artificial transcription factor) of the present disclosure or the binding sequence thereof (artificial promoter) can include a repressor and an operator derived from the corresponding bacteria, respectively.
  • the gene switches (transcriptional regulators) of the present disclosure are highly expressed (eg, S / N ratios of at least about 200, about 300, about 400, about 500, about 600, about 700, about 800, About 900, about 1000, about 1200, about 1500, about 2000, about 2500, about 3000, about 3500, or about 4000) and / or high controllability (eg, semi-response concentration as response sensitivity ⁇ EC50).
  • the high expression and high controllability property is a high level not found in conventional gene switches.
  • the gene switch (transcriptional regulator) of the present disclosure is a 5-position, 6-position, 20-position, 70-position, in the amino acid sequence represented by (DAPG-ON) SEQ ID NO: 1 or a variant thereof. Containing one or more mutations selected from positions 86, 109, 117, 143, and 187; at position 116 in the amino acid sequence represented by (HSL-ON) SEQ ID NO: 3 or a variant thereof. , 201, 140, and 33; one containing the mutation at position 40 in the amino acid sequence represented by (Borneol-ON) SEQ ID NO: 2 or a variant thereof.
  • the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof may contain one or more mutations selected from positions 157 and 41.
  • (DAPG-ON) SEQ ID NO: 1 or a variant thereof P5S, S6P, Q117R, Q117P, Q117G, Q117N, K86T, K86A, K86S, K86G, E143K. , F109L, K20R, E70G, and one containing one or more mutations selected from T187A; (HSL-ON) in the amino acid sequence represented by SEQ ID NO: 3 or a variant thereof, S116Y, W201R, H140N, and T33A.
  • the nucleic acid or protein used in the present disclosure is a sequence or a variant thereof in which one or more amino acids or nucleotides are mutated (eg, substituted, deleted and / or added) in the amino acid or base sequence of interest.
  • "one or more" in the full-length amino acid sequence of the chimeric protein is usually 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 1 amino acid).
  • "one or more” is usually 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (for example, 3 amino acids or less, 2 amino acids or less, 1).
  • the mutated amino acid residue be mutated to another amino acid that preserves the properties of the amino acid side chain.
  • the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and 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 group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side chains Amino acids (H, F, Y, W) having an amino acid (H, F, Y, W) can be mentioned (all in parentheses represent one-letter notation of amino acids (A, I, L, M, F, P, W, Y,
  • non-conservative substitutions are preferred, and at amino acid positions where mutations are not expected, conservative substitutions are used. It is understood that it may be done. It is known that a protein having an amino acid sequence modified by deletion, addition and / or substitution with another amino acid of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81,5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487- 6500, Wang, A.
  • the "several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be less than or equal to any one of them.
  • the deleted protein can be produced, for example, by a site-specific mutagenesis method, a random mutagenesis method, biopanning using an antibody phage library, or the like.
  • a site-specific mutagenesis method for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) Can be used. It is possible to select an antibody having the same activity as the wild type from the mutant type antibody into which a deletion or the like has been introduced by performing various characterizations such as FACS analysis and ELISA.
  • mutation of one or more amino acids in an amino acid sequence refers to site-specific mutagenesis. It means that the modification has been made by a well-known technical method such as a law, or by a natural mutation, by substitution of a plurality of amino acids to the extent that it can occur naturally.
  • the modified amino acid sequence may include, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, particularly preferably 1 to 2 amino acids inserted, substituted, or missing. It can be lost, or added to one or both ends.
  • the modified amino acid sequence is preferably an amino acid sequence in which the amino acid sequence has one or more (preferably one or several or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of the present disclosure. There may be. Further, in one embodiment, the variant is, for example, a sequence in which the first Met is deleted in each amino acid sequence or nucleic acid sequence of SEQ ID NOs: 1 to 5, or the N-terminal or C-terminal thereof is deleted. Includes only a portion of.
  • the present disclosure may further contain a plurality of additional mutations.
  • the gene switch system of the present disclosure can be applied to mass production of useful proteins, in which, for example, host cells such as Escherichia coli are forced to express a target protein obtained from a heterologous organism.
  • the technique is used.
  • the gene switch according to one embodiment of the present disclosure has a sufficiently low basal expression level at the time of non-induction (that is, a small leakage expression), and sufficient gene expression when the expression is induced (ON). This is done (that is, the expression level ratio at the time of ON / OFF is large).
  • the gene switch (transcriptional regulator) of the present disclosure can regulate the expression of a downstream gene with high sensitivity even when the concentration of the inducing factor is low.
  • the gene switch system of the present disclosure can be used as a means of metabolic engineering.
  • metabolic engineering multiple enzyme genes are simultaneously expressed in one host cell to construct a biosynthetic pathway for a certain target substance.
  • the expression levels of individual genes should be detailed and independently. It needs to be adjusted. Therefore, in one embodiment of the present disclosure, when the expression of a plurality of genes is regulated simultaneously in one cell, the inducer of one gene switch (transcriptional regulator) has the desired ON / OFF switching characteristics.
  • each gene switch transcriptional regulator
  • / or the gene switch system using it without malfunctioning another gene switch (transcriptional regulator) and / or the gene switch system using it. It is possible to provide a gene switch (transcriptional regulator) capable of continuously regulating the gene and / or a gene switch system utilizing the gene switch.
  • the production amount (concentration) of the target metabolite can be measured easily and at high speed by GFP or the like without using an analysis method such as HPLC or GCMS. Can be done.
  • the gene switch system of the present disclosure is used as a metabolite sensor, not only the low-production compound is measured by using the highly sensitive gene switch sensor, but also the desensitized gene switch system is used. It is also possible to measure the production amount of the strain whose production amount has increased, and according to the method of the present disclosure, the sensitivity can be easily adjusted.
  • the mutation identified by the artificial transcription factor obtained in the present disclosure is used.
  • the same effect can be obtained. Both the case of adding a mutation to a natural transcription factor and the case of adding a mutation to an artificial transcription factor can be considered.
  • various gene switches transcriptional regulators
  • gene switch systems utilizing them tetracycline responsive, homoserine lactone (HSL) responsive, 2,4-diacetylphloroglucinol ( An improved version of DAPG) responsive type, etc.
  • HSL homoserine lactone
  • DAPG 2,4-diacetylphloroglucinol
  • the gene sensor which hardly responded to the initial sensor, can be easily improved to the level where the response can be properly observed.
  • a reversible switch can be easily constructed.
  • tandemizing the operator arrangement a further dramatic improvement in response performance was achieved.
  • Pichia pastoris Pichia pastoris (Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris) and methanol-utilizing yeast (Ogataea polynyl gene for gene-inducing yeast (Ogataea polyn))
  • the composition comprises a gene construct comprising the nucleic acid sequence of the gene of interest and a gene switch expression sequence operably linked to the nucleic acid sequence, and is a transcriptional regulator and induction encoded by the gene switch expression sequence.
  • a composition is provided in which the expression of the target gene is regulated by binding the complex to which the factor is bound to the regulatory DNA element.
  • DAPG-ON type gene switch In one embodiment of the present disclosure, a DAPG-ON type gene switch (transcriptional regulator) and / or a gene switch system utilizing the DAPG-ON type gene switch (transcriptional regulator) can be provided.
  • a DAPG-ON type gene switch transcriptional regulator
  • a gene switch system utilizing the DAPG-ON type gene switch transcriptional regulator
  • an artificial transcription factor PhlTA
  • DAPG 2,4-diacetylfluoroglucinol
  • PhlF 2,4-diacetylfluoroglucinol
  • DAPG-OFF only an OFF-type switch (DAPG-OFF) is known.
  • a DAPG-ON type gene switch whose responsiveness is reversed by adding a mutation of Q117R, K86T, E143K to PhlF in the amino acid sequence represented by SEQ ID NO: 1 or a variant thereof.
  • the DAPG-ON type gene switch is responsive only by introducing Q117R, Q117P, Q117G, or Q117N.
  • the DAPG-ON type gene switch can be more significantly reversed in its responsiveness with the addition of K86T and E143K.
  • the sensitivity to DAPG can also be improved by further adding the F109L, K86S, K86A, or K86G mutations in addition to these mutations.
  • the increase in OFF (absence of DAPG) expression (increase in leakage) associated with increased sensitivity can be suppressed by mutations in S5P or P6S.
  • the responsiveness can be improved by adding a mutation of P5S or S6P, and for example, when the F109L mutation or the like is introduced to increase the sensitivity, the occurrence of leakage at the time of OFF can be suppressed. can.
  • F109 even if 50 variants are screened from the site-saturation library (NNK) of F109 residues, only F109L is found as a sensitizing mutation, so mutations other than L do not sensitize. be able to.
  • DAPG-ON type switch in which the responsiveness is reversed even with a mutation of K20R, E70G, and / or T187A.
  • the inversion of responsiveness cannot be obtained without either E70G or T187A.
  • the responsiveness can be dramatically improved by tandemizing the operator sequence (500 times or more with phlO ⁇ 6 copies).
  • the DAPG-ON type gene switch (transcriptional regulator) is AA at positions C13T, T16C, A350G, 350 and 351 in the base sequence represented by SEQ ID NO: 5 or a variant thereof. Mutations to CG, mutations of CAA at positions 349 to 351 to ATT, mutations of CAA at positions 349 to 351 to GGT, mutations of A257 and AA to GC at positions 256 and 351 and Encoded by a nucleic acid molecule containing one or more mutations selected from mutations of AA at position 257 to TC, mutations of AA at positions 256 and 257 to GG, G427A, T325C, A59G, A209G, and A559G. It can also be provided as.
  • the polypeptide encoded by the nucleic acid molecule can also function as a polypeptide for inducible control of target gene expression in Pichia yeast.
  • DAPG-OFF type gene switch In one embodiment of the present disclosure, a DAPG-OFF type gene switch (transcriptional regulator) and / or a gene switch system utilizing the DAPG-OFF type gene switch (transcriptional regulator) can be provided.
  • an artificial transcription factor (PhlTA) with a 2,4-diacetylfluoroglusinol (DAPG) responsive transcription factor (PhlF) is used, for example, the amino acid represented by SEQ ID NO: 1.
  • HSL-ON type gene switch In one embodiment of the present disclosure, an HSL-ON type gene switch (transcription factor) and / or a gene switch system utilizing the same can be provided. In one embodiment of the present disclosure, it is possible to provide a gene switch system (HSL-ON) using an artificial transcription factor (LuxTA) using a homoserine lactone (HSL) -responsive transcription factor (LuxR), and the LuxR can be provided.
  • a gene switch system HSL-ON
  • LuxTA artificial transcription factor
  • HSL homoserine lactone
  • LuxR homoserine lactone
  • the sensitivity to HSL can be improved as compared with the conventionally known S116A mutation.
  • the sensitivity to HSL can be further improved by adding the W201R, T33A, and / or H140N mutations in addition to S116Y.
  • said gene switch comprises one or more mutations selected from S116Y, W201R, H140N, and T33A in the amino acid sequence represented by SEQ ID NO:
  • a Borneol-ON type gene switch (transcription factor) and / or a gene switch system utilizing the Borneol-ON type gene switch (transcriptional regulator) can be provided.
  • the gene switch contains a mutation of Y40C in the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof.
  • Tet-ON type gene switch In one embodiment of the present disclosure, a Tet-ON type gene switch (transcription factor) and / or a gene switch system utilizing the same can be provided.
  • a gene switch system Tet-ON using an artificial transcription factor (rTetTA) using a doxicyclin (Dox) responsive transcription factor (TetR) can be provided to generate an R8K mutation.
  • rTetTA artificial transcription factor
  • Dox doxicyclin responsive transcription factor
  • the gene switch comprises a mutation in R8K in the amino acid sequence represented by SEQ ID NO: 4 or a variant thereof.
  • a Camphor-OFF type gene switch (transcription factor) and / or a gene switch system utilizing the same can be provided.
  • a gene switch system (Camphor-OFF) using an artificial transcription factor (CamTA) using a D-camphor-responsive transcription factor (CamR) can be provided and placed at the C-terminal.
  • CamTA artificial transcription factor
  • CamR D-camphor-responsive transcription factor
  • frameshift mutations that disrupt such NLS function include mutations from PKKKRKV to PKRKERSKI in the amino acid sequence represented by SEQ ID NO: 2 or variants thereof.
  • the gene sequence can include a mutation of A695del.
  • a Y40C (or Q87R, or R20K) mutation to CamR, it is possible to provide a switch having improved sensitivity not only to camphor but also to borneol and ⁇ -pinene.
  • Pichia pastoris Pichia pastoris: Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris
  • methanol-utilizing yeast Ogataeaophila
  • an artificial promoter fused with a core promoter such as the core promoter of Pichia yeast-derived AOX1 or DAS1
  • a core promoter such as the core promoter of Pichia yeast-derived AOX1 or DAS1
  • PhlF mutant PhlTA mutant
  • tandemizing the operator sequence (phrO) up to 48 copies can create a highly potent artificial inducible promoter that functions in methanol-utilizing yeast.
  • a DNA library can be made using a variety of known methods and to a promoter to which a gene switch expression sequence and / or a transcriptional regulator encoded by the gene switch expression sequence binds. It is not particularly limited as long as it has a random mutation introduced.
  • such a DNA library can be introduced into a host cell by introducing an expression vector into the host cell.
  • the "expression vector” is a vector DNA that carries an external gene to a host cell and can express the target gene in the host cell.
  • the vector DNA is not particularly limited as long as it can be replicated in the host, and can be appropriately selected depending on the type of host and the purpose of use.
  • the vector DNA may be a vector DNA obtained by extracting a naturally occurring DNA, or a vector DNA in which a part of the DNA other than the part necessary for replication is missing.
  • Representative vector DNAs include, for example, plasmids, bacteriophages, and virus-derived vector DNAs.
  • plasmid DNA a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, or the like can be used.
  • the bacteriophage DNA include ⁇ phage and the like.
  • virus-derived vector DNA examples include vectors derived from animal viruses such as retrovirus, vaccinia virus, adenovirus, papovavirus, SV40, chicken pox virus, and pseudomad dog disease virus, and vectors derived from insect viruses such as baculovirus. be able to.
  • vector DNA derived from a transposon, an insertion element, a yeast chromosomal element, and the like can be mentioned.
  • a vector DNA prepared by combining these for example, a vector DNA prepared by combining genetic elements of a plasmid and a bacteriophage (such as cosmid and phagemid) can be mentioned.
  • target gene it is necessary to incorporate the target gene into the vector DNA so that the target gene is expressed, and at least the target gene and a regulatory DNA element, for example, a promoter are included in the vector DNA.
  • a regulatory DNA element for example, a promoter
  • gene sequences carrying information on replication and regulation can be combined and incorporated into vector DNA by known techniques.
  • Such gene sequences include, for example, ribosome binding sequences, terminators, signal sequences, cis-elements such as enhancers, splicing signals, and selectable markers (dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, etc.). can.
  • One or more gene sequences selected from these can be integrated into the vector DNA.
  • a known genetic engineering technique can be applied to the method of incorporating the target gene into the vector DNA.
  • a method can be used in which the gene of interest is treated with an appropriate restriction enzyme, cleaved at a specific site, then mixed with the similarly treated vector DNA, and recombined with ligase.
  • the desired vector DNA can also be obtained by ligating an appropriate linker to the target gene and inserting it into the multicloning site of the vector suitable for the target.
  • the method for introducing the expression vector into the host cell is not particularly limited as long as it is an introduction method capable of introducing the vector DNA into the host cell to express the target gene in the host cell, and is known as appropriately selected depending on the species of the host cell. Any of the methods may be used. For example, a lithium acetate method, an electroporation method, a calcium phosphate method, a lipofection method and the like can be mentioned.
  • the expression sequence and the gene switch in the same expression vector, and the expression vector containing the expression sequence of the fusion reporter gene and the gene switch (transcriptional regulator). It can also be carried out by co-transforming an expression vector containing the above into the same cell.
  • the gene switch system can be used as an inducible promoter, and there is a high need for a high-performance artificial gene switch system that overcomes the drawbacks of the natural inducible promoter, which has characteristics that are difficult to use.
  • the gene switch system can perform simple indirect quantitative evaluation of products and regulation of gene expression, the need for its use has been increasing in recent years.
  • yeast which is also useful as an industrial microorganism, if a high-performance gene switch sensor and / or a gene switch system using it can be easily produced by this method, the time for strain construction can be overwhelmingly shortened. It can perform advanced metabolism and gene network control. Therefore, industrial application is expected especially in the fields of metabolic engineering and synthetic biology.
  • the gene switch of the present disclosure can be used for mass production of useful proteins, control of expression of proteins showing toxicity, simple concentration measurement of metabolic compounds, and the like.
  • an inducible promoter can be utilized for inducible expression (secretion or intracellular production) of a protein.
  • Tet-ON, DAPG-ON, and HSL-ON having an excellent S / N ratio can be assumed to be used for controlling the expression of metabolic enzymes.
  • it may be used for an enzyme that branches a metabolic pathway.
  • the production amount of squalene can be increased by turning off the enzyme in the downstream ergosterol pathway that consumes squalene only at the timing of high production of squalene.
  • useful proteins that are highly toxic to cells, such as antibody proteins can be mentioned as candidates.
  • the expression level of antibody proteins is directly proportional to the production amount. Therefore, it is necessary to increase the expression level to the limit, but if the S / N ratio of the expression induction system is low, expression leakage occurs, and it is impossible to obtain a yeast strain due to cytotoxicity due to overexpression of protein. be. Due to its high signal-to-noise ratio, DAPG-ON, Tet-ON, and HSL-ON switches are expected to produce high toxic proteins.
  • the gene switch system can be used to turn ON / OFF the expression of genes that are particularly toxic to cells.
  • Intrayeast expression of antibody proteins expected to be used as pharmaceuticals or heterologous enzyme genes responsible for the synthesis of plant metabolites often show cytotoxicity when overexpressed. However, even if a gene is toxic during the cell proliferation phase, its toxicity may be alleviated if the expression is induced after the proliferation is completed. Therefore, by using a gene switch system to turn off the expression of the toxic gene during the growth phase and turn it on after the growth is completed, cytotoxicity due to overexpression of the heterologous gene can be avoided.
  • the gene switch system is also attracting attention in the field of biocomputing.
  • a "gene circuit" composed of a combination of a plurality of gene switch systems can be interpreted as an analogy of an "electronic circuit” that processes external information given to a cell.
  • ON / OFF switching of gene switches is analog, so the number of combinations of gene circuits is limited to a few, limiting their expandability. There is a possibility that such a weak point of the gene circuit can be solved by a gene switch having an excellent ratio of OFF and ON.
  • the present disclosure is for creating a high-performance new gene switch system or metabolite sensor in various species such as yeast as a main component, other eukaryotes, and prokaryotes containing Escherichia coli. Method can be used. You can also sell the switch sensor you create as part of a license or kit, or develop a switch sensor on a contract basis.
  • the created gene switch system can be used as a promoter for inducing new protein production that is safe, inexpensive, and easy to use (for example, no explosion-proof equipment is required, carbon sources are not limited, etc.), and gene expression in metabolic pathways is strictly expressed.
  • the developed metabolite sensor is used to easily quantify the production volume of target products and by-products instead of HPLC and GCMS, accelerating the development speed of high-performance (high-production) substance production strains. You can also let it. Therefore, in one embodiment of the present disclosure, it is possible to provide a composition for detecting the production amount of a gene product, an inducing factor, or a metabolite thereof encoded by a gene of interest.
  • a gene switch using a cheaper and safer compound as an inducer, and / or a gene switch system using the same, has been developed and licensed or used as a part of a kit. Can be sold. Transcription factors possessed by various species will be screened using the physical properties of the inducer as an index, and a gene switch (transcriptional regulator) using this and / or a prototype of a gene switch system using it will be prototyped. Random gene mutations are introduced into the components of the prototype prototype. OFF / ON selection is performed on the gene switch library thus prepared.
  • gene expression is induced by culturing a yeast strain carrying a gene switch library in the presence of 100 nM of 5 FdU and then transferring it to a medium containing an inducer. ON selection is performed by transferring the culture medium to a medium containing 1 to 3 mM Zeocin and an inducer.
  • the gene switch (transcription factor) mutant that responds to the lower concentration inducer is also isolated (higher sensitivity).
  • the developed gene switch (transcriptional regulator) and / or the gene switch system using it is used to control the expression of metabolic enzymes that are the key to substance production and to induce the production of proteins that have a heavy load on cells.
  • Pichia yeast can be used for the production of valuable proteins because it can be cultivated at high density and, above all, the amount of protein secreted and expressed is large. In particular, even in the expression of human-derived recombinant proteins, disulfide formation and post-translational modification, which are more likely to fold correctly than Escherichia coli and the like, are likely to occur and are often used.
  • DAPG-ON gene switch transcriptional regulator
  • / or the gene switch system using it in Pichia yeast, from the existing antibody production process using methanol to a safe antibody production process free of methanol. And can be migrated.
  • a gene construct comprising a nucleic acid sequence of interest encoding a protein of interest and a gene switch expression sequence operably linked to the nucleic acid sequence, encoded by the gene switch expression sequence. It is possible to provide a gene construct in which the expression of the nucleic acid sequence of interest is regulated by binding to the regulatory DNA element by the complex in which the transcriptional regulatory factor and the inducing factor are bound. By using such a construct, the production of a desired protein can be controlled.
  • the construct of the present disclosure can be produced and modified by using a technique well known in the art, and the production method thereof is not particularly limited.
  • the gene construct of the present disclosure can be introduced into a cell using a technique well known in the art, and a vector DNA containing the gene construct of the present disclosure is introduced into a host cell to express the target gene in the host cell.
  • the method is not particularly limited as long as it can be obtained, and a method suitable for the host cell can be preferably adopted.
  • Examples of the protein encoded by the nucleic acid sequence that can be included in the gene construct of the present disclosure include any protein described in the column of (Production: Primary product).
  • the cells thus obtained are arranged together with a predetermined inducing factor under conditions under which the expression of a desired protein can be controlled, so that the expression of the protein can be turned on and off and the expression level can be controlled.
  • the present disclosure provides cells containing the genetic constructs of the present disclosure.
  • the cells into which the construct of the present disclosure is introduced may be any cell including eukaryotes and prokaryotes, and may be, for example, yeast (Pikia yeast, sprouting yeast), Escherichia coli, bacillus, lactic acid bacterium, and the like. Examples include Escherichia coli, filamentous fungi, and microalgae.
  • a method for producing a protein of interest which comprises a gene construct comprising a nucleic acid of interest encoding the protein of interest and a gene switch expression sequence operably linked to the nucleic acid of interest.
  • the expression of the target nucleic acid sequence is controlled by binding to the regulatory DNA element by the complex in which the transcriptional regulatory factor and the inducing factor encoded by the gene switch expression sequence are bound.
  • the step of providing a gene construct can provide a gene construct comprising a nucleic acid sequence of interest encoding the protein of interest and a gene switch expression sequence operably linked to said nucleic acid sequence.
  • the target nucleic acid sequence and the gene switch expression sequence do not have to be contained in the same expression vector, and the expression vector containing the target nucleic acid sequence and the expression vector containing the gene switch expression sequence are transformed into the same cell. You can also do it.
  • the expression of the target protein from the target nucleic acid sequence can be controlled.
  • the gene switch of the present disclosure can control the on / off of its production at a desired timing, and can control its production amount. , It is possible to mass-produce useful proteins and antibody proteins and control the expression of toxic proteins.
  • Examples of the target protein that can be produced by using the gene switch of the present disclosure or the gene switch system include enzymes for synthesizing useful substances such as amino acids and polylactic acid, and target compounds (for example, PET). Examples thereof include an enzyme for degrading a persistent compound), an enzyme for synthesizing an antibacterial substance such as an antibiotic, and an antibody protein that specifically interacts with a target protein.
  • a method for producing a processed product and / or a metabolite of a protein of interest wherein the nucleic acid sequence encoding the protein of interest and a gene switch expression sequence operably linked to the nucleic acid sequence are expressed.
  • a step of controlling the production of the target protein by setting the conditions and a step of recovering the target protein and performing a desired processing and / or metabolic treatment to obtain a processed product and / or a metabolite. The method is provided.
  • a gene switch or gene switch system of the present disclosure is used to produce a protein of interest, and the protein is metabolized, fermented, or modified to produce useful production. Goods can be obtained and the present disclosure may also include such secondary products in the present disclosure.
  • the gene switch of the present disclosure, or a protein produced or recovered using the gene switch system can be further modified, sugar chains can be added or removed, and the gene switch of the present disclosure, Alternatively, the protein produced or recovered using a gene switch system can be physically or chemically modified.
  • the expression of the metabolite is controlled by using the induced expression by the gene switch system of the present disclosure, the switching between the growth phase and the fermentation phase, and the avoidance of toxicity are controlled to increase the fermentation productivity of the metabolite. You can also.
  • the gene switch of the present disclosure or a method for processing, modifying, or metabolizing a target protein that can be produced by using the gene switch system, includes removal of an expression tag, immobilization on a carrier, and the like.
  • Post-translational modifications such as glycosylation, acetylation, succinylation, and phosphorylation, incubation with substances that are raw materials for useful substances, and disruption of microorganisms that produce the target protein can be mentioned.
  • the processed product and / or metabolite of the target protein that can be produced by using the gene switch of the present disclosure or the gene switch system includes low molecular weight organic compounds such as amino acids and organic acids, polylactic acid, polyhydroxybutyric acid and the like. Biopolymers, artificial seasonings using extracts of microorganisms high in target compounds, supplements, biosensors using immobilized enzyme proteins, and the like can be mentioned.
  • the gene switch or the gene switch system of the present disclosure by applying the gene switch or the gene switch system of the present disclosure, it is possible to develop a fed batch production technique suitable for high-mix production in the production of biopharmacy by microorganisms.
  • fed-batch culture also enables mass production of Nanobodies.
  • by initiating expression induction by the gene switch or the gene switch system of the present disclosure it is possible to increase the antibody production amount.
  • the gene switch, or gene switch system of the present disclosure can also be applied to develop a transcription expression system suitable for continuous production or batch production, or to search for useful genes necessary for the development of high-performance microorganisms. It can be carried out.
  • Pichia yeast and a methanol-induced promoter are generally used, but the present invention is not limited to this, and other organisms can also be used.
  • the gene switches, or gene switch systems of the present disclosure are capable of developing safe and / or hyperpotent, inducible or homeostatic expression promoters. By applying the gene switch or gene switch system of the present disclosure, it is possible to design or improve an artificial activator or repressor that can be freely designed.
  • the gene switch or gene switch system of the present disclosure can also be used for evolutionary engineering because it can design artificial (amino acids, nucleic acids, etc.) sequences that can be freely designed.
  • the gene switches, or gene switch systems of the present disclosure can also be utilized to assess the productivity of comprehensive (systematic) low molecular weight biomaterials (eg, antibodies), protein transport, quality control, and protease development folding. It can also be applied to or combined with technologies such as improvement of sugar chains and addition of sugar chains.
  • the gene switch or gene switch system of the present disclosure it is possible to construct a high molecular weight antibody-producing strain by accumulating useful factors in Pichia yeast, for example, pair screening useful factor, useful trans factor, etc. It can be applied to gene-deficient useful factors, transport system useful factors, non-yeast-derived useful factors, etc., and in one embodiment, further the 2 g / L Nanobody production already achieved by batch culture in a test tube is further increased. Can be enhanced.
  • higher production is possible with the jar fermenter, and the gene switch or the gene switch system of the present disclosure can also be applied to such a production system.
  • the gene switch of the present disclosure or the gene switch system and the gene products and products obtained thereby, are highly functional biomaterials (eg, antibodies, etc.) by the so-called DBTL (design, build, test, learn) cycle. It is possible to rapidly create high-producing strains of metabolites, other various substances such as proteins, lipids and sugars, organella, etc.).
  • DBTL design, build, test, learn
  • the arrangement, structure, and modified cell design of biological substances are assumed.
  • biological substances for example, antibodies
  • Gene products and processed products are available.
  • the gene switch of the present disclosure or the gene switch system and the gene products and processed products obtained thereby, it is possible to improve the biopharmaceutical fed batch production technique by microorganisms, and the fed batch suitable for high-mix production. Production technology can also be improved.
  • Another example is the production of biopharmacy by microorganisms, which can be improved by combining the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained thereby.
  • the gene switch of the present disclosure can be used for rapid development using microorganisms and robotics ⁇ digital.
  • the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained by the gene switch system can be used in an ultra-high throughput experimental system utilizing robotics.
  • the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained thereby can be directly or indirectly used in the design support system utilizing the information analysis / measurement data.
  • a biological substance for example, an antibody module can be obtained and improved in performance, and the high-performance biological substance can be obtained.
  • the gene switch or gene switch system of the present disclosure and the gene products and processed products obtained by the gene switch can be used for the development of the acquisition technique of (for example, an antibody fragment / antibody-like molecule), and a biological substance (for example, an antibody) can be used. )
  • the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained thereby can be directly or indirectly utilized for specificity to a target that is difficult to obtain, high stability and high expression, and the like.
  • the gene switch or gene of the present disclosure is also used for assembling modules of biological substances (for example, antibodies): high functionality and optimization, and creation of next-generation multivalent multivalent antibodies (high functionality and multifunction).
  • the switch system and the gene products and processed products obtained by the switch system can be utilized.
  • the gene switch of the present disclosure may be used for producing a high-quality antibody drug conjugate (ADC) or site-specific cross-linking with a unique enzyme. It can also be applied to development.
  • ADC antibody drug conjugate
  • site-specific cross-linking with a unique enzyme. It can also be applied to development.
  • the gene switch or gene switch system of the present disclosure and the gene products and processed products obtained thereby can produce high-quality next-generation antibodies for practical use, and the next generation in Pikia yeast can be produced.
  • the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained thereby can be used for high quality and high production of the antibody.
  • the gene switch of the present disclosure can be used for rapid optimization of production strains / induction / culture conditions, and also for reduction of HCP / glycosylation. Can be done.
  • the gene switch or gene switch system of the present disclosure and the gene products and processed products obtained thereby can also be used for productivity verification in various cells, for example, CHO cells.
  • the gene switch or gene switch system of the present disclosure and the gene products and processed products obtained thereby can be incorporated or applied. ..
  • the gene switch of the present disclosure can impart the function of a biological substance (for example, an antibody), improve the quality and produce it.
  • a biological substance for example, an antibody
  • the gene switch of the present disclosure, or the gene switch system and the gene product obtained by the gene switch can be used to rapidly optimize the hereditary type, induction / culture conditions, and culture process of Pikia yeast for each candidate antibody.
  • Processed products can be applied, culture process optimization by online automatic control / prediction model construction, analysis / identification and reduction of HCP / sugar chains, high quality of Pikia yeast production system, etc. can be performed, other than antibodies.
  • a similar process can be used for biological materials in the yeast.
  • the gene switch of the present disclosure can evaluate the productivity and performance of biological substances (for example, antibodies) in CHO cells, and can be used for clearing cells (cleared cells). Performance verification and GMP production with CHO cells, etc. can also be performed.
  • biological substances for example, antibodies
  • Performance verification and GMP production with CHO cells, etc. can also be performed.
  • the system to be implemented is automatic DNA synthesis and automatic purification of biological substances (antibodies, etc.) with the robotics ⁇ digital infrastructure in mind.
  • Microfluidic systems Large-scale sequence analysis can be performed in combination as needed.
  • the acquisition, functionalization and maturation of biological material modules can also be improved or improved by utilizing the gene switches of the present disclosure, or gene switch systems and the gene products and processed products obtained thereby.
  • Humanization, improvement of affinity / structural stability, cross-linking of site-specific drugs, modification of sugar chain addition mechanism, improvement of pharmacokinetics, etc. can also be obtained with the gene switch or gene switch system of the present disclosure and thereby. It can be achieved by applying the following gene products and processed products.
  • the gene switch of the present disclosure can also be applied to multispecificity: combination optimization and production of biomaterials (eg, antibodies) in, for example, Pichia yeast.
  • biomaterials eg, antibodies
  • the gene switch of the present disclosure, or the gene switch system and the gene products and processed products obtained by the gene switch system can be applied to rapid and massively parallel breeding and production, which is a specialty of microorganisms.
  • an antibody using CHO cells can be applied.
  • Production can be exemplified.
  • productivity and performance comparison in CHO cells can be performed, and the gene switch or gene switch system of the present disclosure and the gene products and processed products obtained thereby can be utilized as needed.
  • the gene switch of the present disclosure or the gene switch system and the gene products and processed products obtained by the gene switch system can be applied to the transplantation to IgG.
  • the gene switch of the present disclosure or the gene switch system and the gene products and processed products obtained by the gene switch system, can be applied to the optimization of bispecific antibodies and the like.
  • gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) can also be used, and others, for example, Gait. , M. J. (1985). Oligonicleotide Synthesis: A Practical Approach, IRL Press; Gait, M. Gait. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.M. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.M. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z.
  • Example 1 Development of gene switch in Pichia yeast
  • a gene switch was developed using Pichia yeast.
  • a DAPG-responsive bacterial repressor (PhlF) is fused with a VP48 activation domain and a nuclear localization signal (NLS) to generate an artificial transcriptional activator (rPhlTA).
  • NLS nuclear localization signal
  • rPhlTA artificial transcriptional activator
  • GAP promoter and AOX1 terminator cloned.
  • phlO PhlF operator
  • 18 tandem bicycles are connected to AOX1.
  • the S / N ratios were about 600 and 3000 times, respectively.
  • This is comparable to the performance of the AOX1 promoter, which is known to be most capable of inducing expression in conventional Pichia yeast, has no restrictions on medium components (mainly sugar sources), and is extremely compared to conventional low-performance artificial promoters. It was found to show a high signal-to-noise ratio.
  • Gene switches for yeast can reverse the mode of control by fusing not only artificial transcriptional activators but also transcriptional repressive motifs such as Mxi1 (transcriptional repressor) instead of VP16. ..
  • gene expression can be controlled more efficiently by using AOX1 core or DAS1 core as an artificial promoter than by using GAL1 core.
  • the artificial promoter using GAL1core has extremely low activity, and when it is turned on (gene expression induction) by changing to a core promoter such as AOX1 or DAS1 (strong methanol-inducing promoter) or GAP derived from Pichia yeast. The value will be extremely large.
  • ARG4 is placed upstream of the operator sequence instead of the phlO tandem copy (48 copies), the leakage expression is similarly extremely small. It is considered that ARG4 is functioning as an insulator. Even if ARG4 is cut down to about 1000bp, it functions as an insulator. Even with phlO1, an S / N ratio of about 600 times can be achieved (FIGS. 6C and 6D). Even if the number of copies of the operator is increased and the sequence is lengthened, the leakage occurrence is similarly reduced, so it is considered that the length of the distance from the upstream has an effect.
  • the resulting transformants ( ⁇ 105 unique clones) were cultured in Dox-free 5FdU -containing liquid medium to remove leaking (ie, Dox-independent) mutants with TBG expression. Then, OFF selection was performed. Next, ON selection was performed under various conditions to remove non-functional variants and thereby concentrate good switches. Specifically, the pool of OFF-selected cells was dispensed into 14 tubes containing various combinations of Dox (0.01-10 ⁇ g / mL) and zeocin (1 or 2 mM), and a sample was dispensed. It was shaken in the evening.
  • 93 clones were randomly selected from the two selected pools and subjected to fluorescence screening on 96-well plates (FIG. 1B).
  • a total of 48% (45/93) of the clones tested showed more than 5-fold induction with Dox (10 ⁇ g / mL), with the highest rTetTA mutants (rTetTA K8N, L131L ) at 0.3 and 10 ⁇ g / mL.
  • rTetTA K8N, L131L the highest rTetTA mutants
  • Fig. 1C In the presence of Dox, 8-fold and 11-fold induction was shown, respectively.
  • a random mutation was introduced into the expression cassette encoding rTetTA of the Tet-ON switch, and the obtained library was subjected to a parallel operation of OFF / ON selection under different selection conditions.
  • the resulting cell population was evaluated for Dox-dependent fluorescence shift.
  • the regions shown in green and blue show undesired behavior: variants with leaking expression (cells with 104 or more GFP fluorescence in the absence of Dox) and inactive variants (in the presence of 10 ⁇ g / mL Dox). Each relative amount ( %) of (cells with GFP fluorescence less than 104) is shown.
  • the relative quantity (%) of the switching variant was obtained by subtracting both of these two numbers from 100%.
  • the dashed line shows a histogram obtained from a yeast strain having a plasmid that does not contain rTetTA.
  • FIG. 1B shows the results of fluorescence screening on a 96-well plate of the Tet-ON variant.
  • FIG. 1C shows the transfer function of the selected mutant. Error bars represent the mean ⁇ SD of three independent experiments.
  • the 50% effective concentration (EC 50 ) value was calculated from a dose-response curve adapted to the Hill equation by the least squares method.
  • the upper panel shows fluorescence images of yeast strain cell pellets with wild-type and evolved Tet-ON switches (rTetTA K8N, L13L ) incubated with and without 10 ⁇ g / mL Dox.
  • Example 3 Improvement of gene switch
  • Any transcription factor can be used as a component of the eukaryotic transcription switch according to the protocol for developing the Tet-ON system.
  • different transcription factors are obtained because they differ in stability, DNA binding affinity, and how their function changes when fused with other proteins or domains to produce sTA.
  • Switch performance is unpredictable.
  • the switching behavior of sTA is highly dependent on various factors (expression level, promoter position and number of copies, and strain type). In reality, new systems need to be re-evolved to ensure proper performance under each condition.
  • DAPG 2,4-Diacetylfluorologsinol
  • DAPG-OFF D-Camphor-repressible
  • Camphor-OFF D-Camphor-repressible switch
  • DAPG and CamR D-Camphor responsive bacterial repressors
  • NLS nuclear localization signal
  • FIG. 2 shows a schematic diagram showing the production and evolution method of the yeast transfer switch (evaluated by S / N ratio, sensitivity, and behavior type).
  • FIG. 2A shows the yeast gene switch developed in the present disclosure. The plasmid expression of the identified sTA mutant expressed a dose-dependent activation gene placed under the control of pphrO1 , pcamO1 , or pluxO1 in the host cell.
  • 2B-2F show parent and variant transfer of DAPG-OFF switch (FIG. 2B), Camphor-OFF switch (FIG. 2C), DAPG-ON switch (FIG. 2D), and HSL-ON switch (FIG. 2F). Shown the function. GFP fluorescence from TBG was plotted as a function of each inducer concentration.
  • the error bars shown represent the mean ⁇ SD of the three independent experiments.
  • the concentration of the inducer added during the OFF / ON selection is indicated by an arrow and a broken line.
  • the EC50 value was calculated from a dose-response curve adapted to the Hill equation by the least squares method and expressed in micromolar units.
  • 2E and 2G show structural mappings 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 transcription factor SCO0332 (PDB: 2ZB9) and the quorum sensor protein TraR (PDB: 1L3L), respectively.
  • the DNA structure is taken from the corresponding reference crystal structure.
  • Example 4 Conversion from DAPG-OFF system to DAPG-ON system
  • Mutations allow the emergence of transcription factors with novel switching behavior. Repressors of various bacteria are known to reverse their ligand response by causing several mutations. Therefore, it was confirmed whether or not the construct can be converted into the DAPG-ON switch by inducing a mutation in the gene encoding the DAPG-OFF switch described above.
  • the same PhlTA library used to isolate the DAPG-OFF switch was subjected to OFF selection in the absence of DAPG and then ON selection in the presence of this compound (5 ⁇ M).
  • DAPG-ON variants (1-11E, 1-11G) were isolated and showed an 8-fold increase in DAPG-dependent fluorescence.
  • FIG. 2D, FIG. 4A Mutation analysis of this variant revealed the existence of three new mutations. One of them (Q117R) is essential for the functional reversal of PhlTA (rPhlTA) and the other two mutations (E143K and / or K86T) are the responsiveness of the inverted switch (both sensitivity and multiple change). It was essential for the improvement of (Fig. 2E).
  • FIG. 4 shows nucleotide and amino acid (AA, shown in parentheses) mutations found in the rPhlTA expression cassette of the evolved DAPG-ON switch. Found in first-generation (FIG. 4A) and second-generation (FIG. 4B) rPhlTA expression cassettes, with the exception of the second-generation mutant, which was also found in the parental (1-11E) mutant. The mutations that have been made are shown by the red line. Fold change was calculated from the data shown in FIG. 2D as the average ⁇ SD ratio of fluorescence intensity in the presence and absence of 10 ⁇ M DAPG.
  • coli RNA polymerase significantly reduced expression from the lux promoter in E. coli.
  • the W201R substitution is thought to have a negative effect on the function of LuxR as a recruiter for RNA polymerase, in order to find the LuxR mutation that this mutation has so far sensitized to proteins for activation by HSL. It could be the reason why it was overlooked in the screening.
  • yeast HSL-ON switches the role of LuxR is to promote HSL-induced DNA binding. Therefore, it is considered that this sensitizing mutation can be identified only by the yeast gene switch.
  • FIG. 5 shows mutations in nucleotides and amino acids (AA, shown in parentheses) found in the LuxTA expression cassette of the evolved HSL-ON switch. Except for the 2nd generation mutant in which the mutation was also found in the parent (1-4A) mutant, it was found in the 1st generation (Fig. 5A) and 2nd generation (Fig. 5B) LuxTA expression cassettes. The mutations made are shown by the red line. Fold change was calculated from the data shown in FIG. 2F as the average ⁇ SD ratio of fluorescence intensity in the presence and absence of 100 ⁇ M HSL (3 ⁇ M for 2-4F).
  • yeast switch mutant Integration of yeast switch mutant into AND gate ⁇ -carotene biosynthetic pathway
  • yeast switch mutant Since yeast has developed a series of gene switches with improved signal-to-noise ratio, we tried to apply these structures to pathway flux control.
  • Three isolated sTAs (DAPG-ON 2-1E , HSL-ON 2-4F , and Tet-ON 1-11F ) were integrated into different yeast chromosomes (FIG. 3A).
  • the resulting strain was transformed with plasmids (p tetO7 , pflO6 , and pluxO5 ) in which the gfp gene was cloned downstream of each synP.
  • the potential promoter sequence contained in the vector sequence 500 or more upstream of synP was deleted, and the number of repetitions of phlO and luxO was increased.
  • the GFP fluorescence of the resulting cells was induced only in the presence of their cognate inducers and by more than 102 factors (FIG. 3B). All of these synthetic promoters were stringent, i.e., showed low basal TBG expression in the absence of inducers.
  • the yeast When the yeast was transformed under the control of a promoter constructing a plasmid expressing all three genes (BTS1, crtYB, and crtI), the yeast produced ⁇ -carotene with or without an inducer (Fig. 3D).
  • the promoters of BTS1 and crtYB were replaced with pflO6 and ptetO7 , respectively. Since all three of these genes needed to be expressed, ⁇ -carotene production was expected only in the presence of both DAPG and Dox (AND-gate control). However, leaked expression by the ptetO7 promoter was found to be non-negligible, probably due to the strong catalytic activity of CrtYB, resulting in significant false pigmentation in the absence of Dox.
  • This leakage expression is alleviated by converting the gene switch used to control crtYB from ptetO7 to plusO5 , and the ⁇ -carotene biosynthetic pathway is placed under AND gate control by a combination of HSL and DAPG. rice field. In this strain, the expected AND gate behavior was observed, with ⁇ -carotene biosynthesis only seen in the presence of both DAPG and HSL. A similar strategy was adopted in the construction of strains in which the ⁇ -carotene biosynthetic pathway was placed under the AND gate.
  • FIG. 3 shows flux control from FPP to ⁇ -carotene using a newly developed gene switch.
  • FIG. 3A shows a yeast strain used for AND gate control of carotenoid biosynthesis. Three plasmids expressing sTA for use with the DAPG-ON 2-1E , HSL-ON 2-4F , and Tet-ON K8N, L131L switches were chromosomally integrated. The genes downstream of each synP ( pflO6 , ptetO7 , and plusO5 ) were expressed only in the presence of the corresponding inducers (DAPG, Dox, and HSL, respectively).
  • FIG. 3B shows the results of flow cytometric measurement of orthogonal GFP expression control using Dox, DAPG, and HSL.
  • FIG. 3C shows the synthetic route to ⁇ -carotene.
  • FPP stands for farnesyl diphosphate
  • GGPP stands for geranylgeranyl diphosphate.
  • FIG. 3D shows a schematic diagram of steady ⁇ -carotene biosynthesis and ⁇ -carotene biosynthesis by AND gate control.
  • Cell pellets of yeast strains (FIG. 3D right panel) expressing (BTS1, crtYB, and crtI) under the control of a constitutive promoter or synP ( pflO6 , ptetO7 , and luxO5 ) in the combination shown in the left panel of FIG. 3D. Is shown.
  • 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 ⁇ M), HSL (3 ⁇ M), and Dox (10 ⁇ g / mL).
  • Pichia yeast strains are transformed with a plasmid cloned downstream of the DAPG-ON switch with genes encoding industrially useful proteins such as low molecular weight antibodies and enzymes.
  • the antibody gene is placed downstream of the core promoter of AOX1 fused upstream with phlO and introduced into the genome of Pichia yeast.
  • the gene translated into the amino acid sequence of PhlTA is placed downstream of the GAP promoter and introduced into the Pichia yeast genome.
  • the obtained transformants are cultured in 3 to 5 ⁇ M DAPG-containing medium for 48 hours. The supernatant obtained by centrifuging the culture medium is used in the downstream antibody production process.
  • a culture supernatant containing the gene at a predetermined concentration is obtained by culturing using a test tube (SDS-PAGE).
  • a test tube SDS-PAGE
  • the antibody gene a single antibody gene derived from human, camel or mouse, a fusion gene obtained by tandem fusion of two or three different antibody genes, or a Nanobody of an antibody gene is used.
  • Example 8 Effect of P5S and S6P mutation of rPhlF
  • the effects of P5S and S6P mutations in the artificial transcription factor rPhlTA on responsiveness were investigated (FIG. 8).
  • the absence of P5S / S6P mutations the absence of DAPG, that is, the expression of leakage during OFF was significantly increased, and it was concluded that these mutations are mutations that suppress the expression of leakage during OFF.
  • Example 9 Confirmation of influence on expression by artificial promoter
  • the DAPG responsiveness did not change at all when the core promoter length was in the range of 177 to 237 bp (FIG. 9).
  • the core promoter was changed from the methanol-inducible AOX1 promoter to the methanol-inducible DAS1 promoter and 18 copies of phlO were fused, when the DAS1 core promoter was used up to the position of -124 or -159, the core promoter was used up to the position of -124 or -159.
  • the length of the AOX1 core promoter and the change to the DAS1 core promoter do not significantly affect the performance, that is, the construction of the artificial promoter is not limited to the AOX1 core promoter.
  • Example 10 Construction of Doxycycline (Dox) induction system for Pichia yeast
  • Example 12 Construction of DAPG-OFF switch for Pichia yeast
  • PhlTA dissociates from PhlO of the operator DNA in response to DAPG
  • it functions as a "DAPG-OFF switch” in which gene expression is turned off in response to DAPG
  • FIG. 13a When the switching function was investigated in Pichia yeast, it was slightly switched in the PhlTA mutant having the E41G mutation (Fig. 13b). Responsiveness was improved by modifying the terminator to regulate the expression level of the E41G mutant of PhlTA (Fig. 13c).
  • F109L mutation that increases the sensitivity of rPhlTA constituting the DAPG-ON system was added, it was found that the DAPG-OFF system could also be increased in sensitivity (FIG. 13d).
  • Example 14 Confirmation of expression of antibodies ALX-0171 and ALX-0081 by DAPG-OFF switch
  • DAPG-OFF switch Comparison with methanol induction system and constitutive expression system
  • Secretory production of two Nanobody antibodies developed by Abrynx, ALX-0171 and ALX-0081, using the DAPG-OFF switch was examined in in vitro culture (2 mL scale) (FIG. 15).
  • both antibodies were able to achieve secretory production exceeding the AOX1 promoter, which is a methanol induction system, and the GAP promoter, which is a composition expression system. It was also possible to suppress secretory production by adding DAPG.
  • Pichia yeast strains are transformed with a plasmid cloned from the amylase gene downstream of the DAPG-ON switch. Specifically, the amylase gene is placed downstream of the core promoter of AOX1 fused upstream with phlO and introduced into the genome of Pichia yeast. The gene translated into the amino acid sequence of PhlTA is placed downstream of the GAP promoter and introduced into the Pichia yeast genome. The obtained transformants are cultured in 3 to 5 ⁇ M DAPG-containing medium for 48 hours. The supernatant obtained by centrifuging the culture medium is used in the downstream processing process.
  • a culture supernatant containing the gene at a predetermined concentration is obtained by culturing using a test tube (SDS-PAGE).
  • a test tube SDS-PAGE
  • amylase gene a gene derived from the genus Aspergillus or the genus Bacillus is used.
  • Example 16 Decomposition of starch by amylase
  • the culture supernatant containing amylase can be used directly or after purification.
  • affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like can be used as a method for purifying the enzyme.
  • Culture supernatants or purified enzymes containing amylase can metabolize starch and break it down into glucose, maltose, and oligosaccharides. Mix an appropriate amount of culture supernatant or purified enzyme with starch and incubate under predetermined conditions.
  • the amylase enzyme activity of a protein can be measured by analyzing the solution after the enzyme reaction by an iodine starch reaction, HPLC or the like.
  • the gene switch of the present disclosure can be used for mass production of useful proteins, expression control of proteins showing toxicity, simple concentration measurement of metabolic compounds, and the like.
  • the productivity of biopharmacy and industrial enzymes can be greatly increased, the time for strain construction can be overwhelmingly shortened, and advanced metabolism and gene network control can be performed. Therefore, industrial application is expected especially in the fields of metabolic engineering and synthetic biology.
  • 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 HSL-responsive transcription factor (LuxR) )
  • SEQ ID NO: 4 Dox-responsive transcription factor (TetR) amino acid sequence
  • SEQ ID NO: 5 Transcription factor (PhlF) nucleic acid sequence

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JP2002522079A (ja) * 1998-08-13 2002-07-23 シンジェンタ リミテッド 遺伝子スイッチ
WO2018106892A1 (en) * 2016-12-07 2018-06-14 The Johns Hopkins University Orthogonal transcriptional switches derived from tet repressor homologs for saccharomyces cerevisiae
WO2019187911A1 (ja) * 2018-03-26 2019-10-03 国立大学法人神戸大学 新規細胞及びそれを用いた目的タンパク質の製造方法
JP2020501533A (ja) * 2016-11-24 2020-01-23 ケンブリッジ エンタープライズ リミテド 制御可能な転写

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JP2002522079A (ja) * 1998-08-13 2002-07-23 シンジェンタ リミテッド 遺伝子スイッチ
JP2020501533A (ja) * 2016-11-24 2020-01-23 ケンブリッジ エンタープライズ リミテド 制御可能な転写
WO2018106892A1 (en) * 2016-12-07 2018-06-14 The Johns Hopkins University Orthogonal transcriptional switches derived from tet repressor homologs for saccharomyces cerevisiae
WO2019187911A1 (ja) * 2018-03-26 2019-10-03 国立大学法人神戸大学 新規細胞及びそれを用いた目的タンパク質の製造方法

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