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Method for constructing recombinant bacterium for producing non-native protein, and utilization of same UPDATE 実績あり

外国特許コード F190009893
整理番号 07783-EP
掲載日 2019年8月26日
出願国 欧州特許庁(EPO)
出願番号 11795797
公報番号 2584037
公報番号 2584037
出願日 平成23年6月16日(2011.6.16)
公報発行日 平成25年4月24日(2013.4.24)
公報発行日 平成28年8月10日(2016.8.10)
国際出願番号 JP2011063778
国際公開番号 WO2011158895
国際出願日 平成23年6月16日(2011.6.16)
国際公開日 平成23年12月22日(2011.12.22)
優先権データ
  • 特願2010-137635 (2010.6.16) JP
  • 特願2011-047663 (2011.3.4) JP
  • 2011JP63778 (2011.6.16) WO
発明の名称 (英語) Method for constructing recombinant bacterium for producing non-native protein, and utilization of same UPDATE 実績あり
発明の概要(英語) The present invention provides a novel method of producing a recombinant bacterium for production of a non-natural protein, including: (1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon; (2) expressing an aminoacyl-tRNA synthetase in the bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an ±-hydroxy acid; (3) (i) introducing a DNA construct into the bacterium, which DNA construct is for expressing, in the absence of a release factor for terminating translation at UAG codon, a function of at least one gene selected from the group consisting of genes each of which loses its function when a gene that codes for the release factor is defective and/or introducing an alteration into said at least one gene in a chromosome of the bacterium, which alteration is for expressing the function of said at least one gene in the absence of the release factor; and (4) causing the gene that codes for the release factor in the bacterium to be defective.
従来技術、競合技術の概要(英語) Background Art
There has been a protein in which an amino acid residue at a desired position is substituted with a non-natural amino acid and/or an α-hydroxy acid, and such protein is called a non-natural protein (or super protein). The non-natural protein has a new biological activity, catalytic activity, structure and function, which are not found in natural proteins. Because of these features, non-natural protein synthesis is an indispensable basic technology for overcoming limitations of conventional protein engineering.
In recent years, it has become possible to synthesize a non-natural protein by using Escherichia coli, yeast, insect cells, and mammalian cells. Usually, a non-natural amino acid is introduced at an amber codon (UAG codon) (which is one of stop codons), because it is not desirable that a non-natural amino acid be introduced at a non-specific position in such a synthesizing system.
It is known that the introduction of a non-natural amino acid at an amber codon competes with release factor, which terminates translation at a UAG codon. Non-Patent Literatures 1 and 2 disclose that weakening RF-1 activity (which is a release factor for Escherichia coli) improves introduction efficiency for a non-natural amino acid. Furthermore, Non-Patent Literature 3 discloses that decreasing the amount of RF-1 in cell-free protein synthesis system enables efficient introduction of a non-natural amino acid at an amber codon.
Furthermore, it is considered that introduction efficiency can be further improved by causing a prfA gene coding for RF-1 to be defective. As one method for causing the prfA gene to be defective, Non-Patent Literature 4 discloses a method including replacing amber codons of all genes on a genome with ocher codons or opal codons. Non-Patent Literatures 5 and 6 report Escherichia. coli in which a prfA gene is caused to be defective by replacing all amber codons with ocher codons.
Citation List
Patent Literature
Patent Literature 1
Specification of the United States Patent Application No. 2009/0317910
Non-Patent Literatures
Non-Patent Literature 1
Ryden, S.M., and Isaksson, L.A., Molecular and General Genetics, 193, 38-45 (1984)
Non-Patent Literature 2
Kleina, L.G., et al., Journal of Molecular Biology, 213, 705-717 (1990)
Non-Patent Literature 3
Short III, G.F., et al., Biochemistry, 38, 8808-8819 (1999)
Non-Patent Literature 4
Forster, A.C. et al., Genome Research, 17, 1-6 (2007)
Non-Patent Literature 5
Wang, H.H., et al., Nature, 460, 894-898 (2009)
Non-Patent Literature 6
"Synthetic Biology Projects", [online], [browsing date: April 30, 2009], Internet <URL:http://arep.med.harvard.edu/SBP/>
特許請求の範囲(英語) [claim1]
1. A method of producing a recombinant bacterium for production of a non-natural protein, comprising the steps of:
(1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon;
(2) expressing an aminoacyl-tRNA synthetase in a bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an α-hydroxy acid;
(3) subjecting a bacterium to a process for causing, in the absence of a release factor for terminating translation at UAG codon, the translation of at least one gene into a protein that functions normally, wherein said at least one gene is selected from the group consisting of the genes coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli and the genes accD, acpS, cspR, dapB, divIC, dnaA, fint, folD, ftsA, may, mrpD, murE, murG, plsX, ppnK, racE, resB, resC, rnpA, rplX, rpmGB, rpmH, rpsG, secA, secY, topA, trmD, yacM, ydiC, yloQ, ypuH and ysxC of Bacillus subtilis; and
(4) causing the gene that codes for the release factor in a bacterium to be defective, the bacteria in the steps (1) through (4) being identical, and
the process being (i) a process of introducing, into the bacterium, a DNA construct for causing, in the absence of the release factor, the translation of said at least one gene into a protein that functions normally and/or (ii) a process of introducing, into said at least one gene in a chromosome of the bacterium, an alteration for causing, in the absence of the release factor, the translation of said at least one gene into a protein that functions normally,
wherein said at least one gene is at least one gene, the defect of which alone is lethal, among all genes whose translation is terminated at UAG codon.

[claim2]
2. A method of producing a recombinant bacterium for production of a non-natural protein, comprising the steps of:
(5) expressing, in a bacterium which expresses tRNA that recognizes UAG codon, an aminoacyl-tRNA synthetase that acylates the tRNA with a non-natural amino acid or an α-hydroxy acid;
(6) subjecting a bacterium to a process for causing, in the absence of a release factor for terminating translation at UAG codon, the translation of at least one gene into a protein that functions normally, wherein said at least one gene is selected from the group consisting of the genes coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli and the genes accD, acpS, cspR, dapB, divIC, dnaA, fmt, folD, ftsA, map, mreD, murE, murG, plsX, ppnK, racE, resB, resC, rnpA, rplX, rpmGB, rpmH, rpsG, secA, secY, topA, trmD, yacM, ydiC, yloQ, ypuH and ysxC of Bacillus subtilis; and
(7) causing the gene that codes for the release factor in the bacterium to be defective, the process being (i) a process of introducing, into the bacterium, a DNA construct for causing, in the absence of the release factor, the translation of said at least one gene into a protein that functions normally and/or (ii) a process of introducing, into said at least one gene in a chromosome of the bacterium, an alteration for causing, in the absence of the release factor, the translation of said at least one gene into a protein that functions normally,
wherein said at least one gene is at least one gene, the defect of which alone is lethal, among all genes whose translation is terminated at UAG codon.

[claim3]
3. The method according to claim 1 or 2, wherein, in the bacterium, another tRNA which recognizes UAG codon but is not acylated by the aminoacyl-tRNA synthetase is expressed, said method further comprising the step of causing a gene that codes for said another tRNA to be defective.

[claim4]
4. The method according to claim 1 or 2, wherein the non-natural protein is a non-natural protein that includes the non-natural amino acid or the α-hydroxy acid.

[claim5]
5. The method according to any one of claims 1 through 4, wherein the bacterium is Escherichia coli.

[claim6]
6. The method according to any one of claims 1 through 5, wherein said at least one gene is any six genes selected from the group consisting of coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli.

[claim7]
7. The method according to any one of claims 1 through 5, wherein said at least one gene is coaD, hda, mreC and hemA genes of Escherichia coli.

[claim8]
8. The method according to any one of claims 1 through 7, wherein said at least one gene further includes another gene, defect of which causes a reduction in growth rate of the bacterium.

[claim9]
9. The method according to claim 8, wherein said another gene defect of which causes a reduction in growth rate of the bacterium is a sucB gene of Escherichia coli.

[claim10]
10. The method according to any one of claims 1 through 5, wherein:
the bacterium is Escherichia coli; and
the DNA construct causes the translation of at least one gene selected from the group consisting of coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli into a protein that functions normally.

[claim11]
11. The method according to claim 10, wherein the DNA construct further expresses a sucB gene of Escherichia coli.

[claim12]
12. The method according to any one of claims 1 through 11, wherein the DNA construct is selected from a bacterial artificial chromosome, a plasmid, and linear DNA, each of which is recruited in trans and/or in cis to the chromosome of the bacterium.

[claim13]
13. A DNA construct for producing a recombinant bacterium for production of a non-natural protein, which DNA construct causes, in the presence of (i) tRNA that recognizes UAG codon and (ii) an aminoacyl-tRNA synthetase which acylates the tRNA with a non-natural amino acid or an α-hydroxy acid but in the absence of a release factor for terminating translation at UAG codon, the translation of said of at least one gene into a protein that functions normally, wherein said at least one gene is selected from the group consisting of the genes coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli and the genes accD, acpS, cspR, dapB, divIC, dnaA, fmt, folD, ftsA, map, mrpD, murE, murG, plsX, ppnK, racE, resB, resC, rnpA, rplX, rpmGB, rpmH, rpsG, secA, secY, topA, trmD, yacM, ydiC, yloQ, ypuH and ysxC of Bacillus subtilis,
wherein said at least one gene is at least one gene, the defect of which alone is lethal, among all genes whose translation is terminated at UAG codon.

[claim14]
14. A recombinant bacterium for production of a non-natural protein,
which recombinant bacterium expresses tRNA that recognizes UAG codon;
which recombinant bacterium expresses an aminoacyl-tRNA synthetase which acylates the tRNA with a non-natural amino acid or an α-hydroxy acid;
(i) into which recombinant bacterium a DNA construct has been introduced, which DNA construct is for causing, in the absence of a release factor for terminating translation at UAG codon, the translation of at least one gene into a protein that functions normally, wherein said at least one gene is selected from the group consisting of the genes coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli and the genes accD, acpS, cspR, dapB, divIC, dnaA, fmt, folD, ftsA, map, mrpD, murE, murG, plsX, ppnK, racE, resB, resC, rnpA, rplX, rpmGB, rprnH, rpsG, secA, secY, topA, trmD, yacM, ydiC, yloQ, ypuH and ysxC of Bacillus subtilis; and/or (ii) in which recombinant bacterium, an alteration has been introduced into said at least one gene in a chromosome of the recombinant bacterium, which alteration is for causing the translation, in the absence of the release factor, of said at least one gene into a protein that functions normally; and
in which recombinant bacterium the gene that codes for the release factor is defective, wherein said at least one gene is at least one gene, the defect of which alone is lethal, among all genes whose translation is terminated at UAG codon.

[claim15]
15. The recombinant bacterium according to claim 14, which is derived from Escherichia coli.

[claim16]
16. A method of producing a non-natural protein with use of a recombinant bacterium, comprising expressing, in a recombinant bacterium set forth in claim 15 or in an extract of the recombinant bacterium,
(a) an aminoacyl-tRNA synthetase capable of activating a non-natural amino acid or an α-hydroxy acid,
(b) tRNA which recognizes UAG codon and is capable of being attached to the non-natural amino acid or the α-hydroxy acid in the presence of the aminoacyl-tRNA synthetase, and
(c) a gene that codes for a desired protein, which gene has at least one nonsense mutation occurred randomly or in a desired position.

[claim17]
17. The method according to claim 16, wherein the recombinant bacterium is derived from Escherichia coli.

[claim18]
18. An extract of a recombinant bacterium for use in production of a non-natural protein,
which recombinant bacterium expresses tRNA that recognizes UAG codon;
which recombinant bacterium expresses an aminoacyl-tRNA synthetase which acylates the tRNA with a non-natural amino acid or an α-hydroxy acid;
(i) into which recombinant bacterium a DNA construct has been introduced, which DNA construct is for causing, in the absence of a release factor for terminating translation at UAG codon, the translation of at least one gene into a protein that functions normally, wherein said at least one gene is selected from the group consisting of the genes coaD, murF, hda, mreC, hemA, lpxK and lolA of Escherichia coli and the genes accD, acpS, cspR, dapB, divIC, dnaA, fmt, folD, ftsA, map, mrpD, murE, murG, plsX, ppnK, racE, resB, resC, rnpA, rplX, rprnGB, rpmH, rpsG, secA, secY, topA, trmD, yacM, ydiC, yloQ, ypuH and ysxC of Bacillus subtilis; and/or (ii) in which recombinant bacterium, an alteration has been introduced into said at least one gene in a chromosome of the recombinant bacterium, which alteration is for causing the translation, in the absence of the release factor, of said at least one gene into a protein that functions normally; and
in which recombinant bacterium the gene that codes for the release factor is defective, wherein said at least one gene is at least one gene, the defect of which alone is lethal, among all genes whose translation is terminated at UAG codon.

[claim19]
19. The extract according to claim 18, wherein the recombinant bacterium is derived from Escherichia coli.
  • 出願人(英語)
  • RIKEN
  • 発明者(英語)
  • YOKOYAMA, Shigeyuki
  • MUKAI, Takahito
  • SAKAMOTO, Kensaku
  • MATSUMOTO, Akiko
国際特許分類(IPC)
指定国 Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

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