Novel shuttle vector

Abstract

The present invention addresses the problem of providing a novel vector using a replication initiation point derived from Thermus thermophilus. The problem is solved by providing a shuttle vector that includes a specific first replication initiation point derived from Thermus thermophilus and a second replication initiation point derived from a biological species other than the Thermus thermophilus.

Description

New Shuttle Vector

[0001] The present invention relates to a shuttle vector comprising a replication origin derived from Thermus thermophilus and a replication origin derived from another biological species.

[0002] Thermus thermophilus, a model thermophilic microorganism, is known for its high ability to take up foreign DNA, and this property has led to the establishment of a genetic manipulation system for it. For example, various genetic manipulation methods are applicable, such as the development of shuttle vectors for E. coli based on plasmids present in Thermus thermophilus, and the use of high recombination efficiency with homologous sequences for gene insertion and deletion in the chromosome.

[0003] Up until now, the development of plasmid vector systems has mainly used pTT8 derived from Thermus thermophilus strain HB8 as the origin of replication (Non-Patent Document 1). Recently, plasmids that are presumed to have a replication origin different from that of pTT8 have been developed (Non-Patent Document 2), but the plasmids that are widely and commonly used have almost exclusively been pTT8-type plasmids.

[0004] Koyama Y et al., A plasmid vector for an extreme thermophile, Thermus thermophilus., FEMS Microbiol Lett., 1990, Oct;60(1-2):97-101. doi: 10.1016 / 0378-1097(90)90352-q.Verdu et al., A Modular Vector Toolkit with a Tailored Set of Thermosensors To Regulate Gene Expression in Thermus thermophilus., ACS Omega., 2019, Aug 27;4(11):14626-14632. doi: 10.1021 / acsomega.9b02107. eCollection 2019 Sep 10.

[0005] In view of the above, an object of the present invention is to provide a novel vector using a replication origin derived from Thermus thermophilus.

[0006] As a result of extensive research aimed at solving the above problems, the present inventors discovered the following findings for the first time, and based on these findings, completed the present invention. ・A large number of types of plasmids different from the pTT8 type were discovered among Thermus thermophilus newly isolated from the natural environment. ・The replication origin in the plasmid was successfully identified, and a vector having the replication origin was constructed. ・A shuttle vector between Thermus thermophilus and Escherichia coli, having the replication origin, was constructed. ・The shuttle vector was capable of stably replicating in both hosts. ・The shuttle vector had superior stability to conventional shuttle vectors using pTT8.

[0007] That is, one aspect of the present invention relates to the following. [1] A shuttle vector comprising a first replication origin derived from Thermus thermophilus, and a second replication origin derived from a biological species other than Thermus thermophilus, wherein the first replication origin is any of the following polynucleotides: (a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (a2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in which one or more bases have been deleted, substituted or added, and which functions as a replication origin; (a3) ​​a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 1, and which functions as a replication origin; (b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which one or more bases have been deleted, substituted or added, and which functions as a replication origin; (b3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 2, and which functions as a replication origin; (c1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3; (c2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3, in which one or several bases have been deleted, substituted or added, and which functions as an origin of replication; (c3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 3, and which functions as an origin of replication; (d1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4, in which one or several bases have been deleted, substituted or added, and which functions as an origin of replication; (d3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 4, and which functions as an origin of replication; (e1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;(e2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5, and which functions as an origin of replication; (e3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 5, and which functions as an origin of replication; (f1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 27; (f2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 27, and which functions as an origin of replication; (f3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 27, and which functions as an origin of replication; (g1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 28; (g2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 28, and which functions as an origin of replication; (g3) A polynucleotide consisting of a nucleotide sequence having 80% or more homology to the nucleotide sequence shown in SEQ ID NO: 28 and functioning as a replication origin. [2] The shuttle vector according to [1], wherein the first replication origin is any of (a1), (b1), (c1), (d1), (e1), (f1), or (g1). [3] The shuttle vector according to [1] or [2], wherein the second replication origin is derived from Escherichia coli. [4] The shuttle vector according to [3], wherein the second replication origin is ColE1, pMB, p15a, pSC101, or pRSF. [5] The shuttle vector according to any one of [1] to [4], which is pIOK1Hg (SEQ ID NO: 6), pIOK2Hg (SEQ ID NO: 7), pIOK3Hg (SEQ ID NO: 8), pIOK4Hg (SEQ ID NO: 9), pIOK5Hg (SEQ ID NO: 10), pIOK6Hg (SEQ ID NO: 11), pIOK7Hg (SEQ ID NO: 12), pIOK8Hg (SEQ ID NO: 13), pIOK9Hg (SEQ ID NO: 14), pIOK10Hg (SEQ ID NO: 15), pFK1Hg (SEQ ID NO: 29), pFK2Hg (SEQ ID NO: 30), pSG1Hg (SEQ ID NO: 31), or pSG2Hg (SEQ ID NO: 32).[6] The shuttle vector according to any one of [1] to [5], further comprising at least one sequence selected from the group consisting of a promoter sequence, a multicloning site, and a drug resistance gene. [7] A transformant comprising the shuttle vector according to any one of [1] to [6]. [8] A method for producing a protein, comprising the step of culturing the transformant according to [7].

[0008] According to the present invention, a novel vector using a replication origin derived from Thermus thermophilus can be provided, and also, a shuttle vector capable of stable replication can be provided.

[0009] Figure 1-1 shows the results of measuring the plasmid copy number of the shuttle vector of the present invention in T. thermophilus using real-time PCR. Figure 1-2 shows the results of measuring the plasmid copy number of the shuttle vector of the present invention in T. thermophilus using real-time PCR. Figure 2-1 shows the results of evaluating the maintenance stability of the shuttle vector of the present invention in T. thermophilus. Figure 2-2 shows the results of evaluating the maintenance stability of the shuttle vector of the present invention in T. thermophilus. Figure 3-1 shows the results of comparing the maintenance stability of the shuttle vector of the present invention with a shuttle vector derived from pTT8. Figure 3-2 shows the results of comparing the maintenance stability of the shuttle vector of the present invention with a shuttle vector derived from pTT8. Figure 4 shows colony formation in a plasmid compatibility test.

[0010] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0011] (1. Definitions) As used herein, "Thermus thermophilus" refers to a type of Gram-negative aerobic bacterium classified in the genus Thermus. Its optimum growth temperature is 75°C, and it is classified as a thermophile. Furthermore, as used herein, "thermophiles" refer to microorganisms whose optimum growth temperature is 50°C or higher.

[0012] As used herein, the term "origin of replication" refers to a specific sequence on DNA (e.g., a plasmid or vector) at which replication of that DNA begins. The "origin of replication" includes a series of sequences necessary for replication and may also include replication genes and other sequences involved in replication. The "origin of replication" can also be referred to as the "replication region."

[0013] As used herein, the term "shuttle vector" refers to a vector that is capable of replicating in two or more different species.

[0014] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and refers to a polymer of nucleotides.

[0015] As used herein, "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence" and refers to a sequence of deoxyribonucleotides (abbreviated A, G, C, and T).

[0016] (2. Shuttle Vector) In one embodiment of the present invention, a shuttle vector is provided which comprises a first origin of replication derived from Thermus thermophilus and a second origin of replication derived from a biological species other than Thermus thermophilus.

[0017] The present inventors have conducted extensive research with the aim of providing a novel vector using a replication origin derived from Thermus thermophilus. Specifically, the present inventors discovered many plasmids different from the pTT8 type (pTthSNM1-1d, pTthSNM1-1e, pTthSNM1-7e, pTthSNM1-7f, and pTthSNM3-3d) among Thermus thermophilus strains newly isolated from natural environments, and have continued to investigate the possibility of using these plasmids as novel vectors.

[0018] However, it has been common knowledge in the art that it is extremely difficult to identify the region essential for replication in a plasmid from genome information alone. Furthermore, due to the high GC content unique to Thermus thermophilus, it has been necessary to experimentally verify the stability and replication ability of the plasmid when combined with a heterologous gene fragment from E. coli or another strain.

[0019] Under these circumstances, in the present invention, an origin of replication was deduced based on the gene sequence of a novel plasmid contained in Thermus thermophilus, and a vector having said origin of replication was constructed. Furthermore, a shuttle vector between Thermus thermophilus and Escherichia coli having said origin of replication was constructed, and its ability to replicate continuously in both hosts was confirmed (multiple host exchanges), and its replication stability was evaluated under conditions in which drugs or the like were not added, thereby confirming that it is a shuttle vector that replicates stably. It is surprising that the novel vector containing the origin of replication derived from Thermus thermophilus according to the present invention is capable of replication while maintaining high stability.

[0020] (Thermus thermophilus-derived first origin of replication) The shuttle vector of the present invention contains a Thermus thermophilus-derived first origin of replication.

[0021] In one embodiment of the present invention, the first replication origin is any of the following polynucleotides: (a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (a2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, in which one or several bases have been deleted, substituted or added, and which functions as a replication origin; (a3) ​​a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 1, and which functions as a replication origin; (b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2, in which one or several bases have been deleted, substituted or added, and which functions as a replication origin; (b3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 2, and which functions as a replication origin; (c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3; (c2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 3, and which functions as an origin of replication; (c3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 3, and which functions as an origin of replication; (d1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 4, and which functions as an origin of replication; (d3) a polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 4, and which functions as an origin of replication; (e1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5; (e2) a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 5, and which functions as an origin of replication; (e3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology to the nucleotide sequence shown in SEQ ID NO: 5 and having a function as a replication origin;(f1) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 27; (f2) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 27 in which one or more bases have been deleted, substituted or added, and which functions as an origin of replication; (f3) A polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 27, and which functions as an origin of replication; (g1) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 28; (g2) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 28 in which one or more bases have been deleted, substituted or added, and which functions as an origin of replication; (g3) A polynucleotide consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 28, and which functions as an origin of replication.

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[0029] (a1) is a polynucleotide derived from pTthSNM1-1d (GenBank ID: AP025599), and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1.

[0030] (a2) is a polynucleotide derived from pTthSNM1-1d, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 1, and which functions as an origin of replication.

[0031] In the polynucleotide (a2), "several" means, for example, 2 to 50, 2 to 40, 2 to 30, or 2 to 20, preferably 2 to 10, more preferably 2 to 8, even more preferably 2 to 6, and particularly preferably 2 to 4.

[0032] Whether or not the polynucleotide (a2) functions as an origin of replication can be determined by any method known in the art, for example, by constructing a plasmid containing a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in which one or several nucleotides have been deleted, substituted, or added, introducing the plasmid into Thermus thermophilus, culturing the Thermus thermophilus, and then determining whether or not the plasmid can be replicated in the Thermus thermophilus.

[0033] (a3) is a polynucleotide derived from pTthSNM1-1d, which consists of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 1 and which functions as an origin of replication.

[0034] In the polynucleotide (a3), the homology to the nucleotide sequence shown in SEQ ID NO: 1 is preferably 80% or more, and more preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In this specification, "homology" can also be rephrased as "identity."

[0035] Whether or not the polynucleotide of (a3) ​​functions as an origin of replication can be determined by the same method as in (a2) above.

[0036] (b1) is a polynucleotide derived from pTthSNM1-1e (GenBank ID: AP025607) and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:2.

[0037] (b2) is a polynucleotide derived from pTthSNM1-1e, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2, and which functions as an origin of replication.

[0038] In the polynucleotide of (b2), the term "several" refers to the same as described in (a2) above.

[0039] Whether or not the polynucleotide of (b2) has the function of a replication origin can be determined by the same method as in (a2) above.

[0040] (b3) is a polynucleotide derived from pTthSNM1-1e, which consists of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 2, and which functions as an origin of replication.

[0041] In the polynucleotide of (b3), the homology with the base sequence shown in SEQ ID NO: 2 is as described in (a3) ​​above.

[0042] Whether or not the polynucleotide of (b3) functions as an origin of replication can be determined by the same method as in (a2) above.

[0043] (c1) is a polynucleotide derived from pTthSNM1-7e (GenBank ID: AP025608), and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:3.

[0044] (c2) is a polynucleotide derived from pTthSNM1-7e, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 3, and which functions as an origin of replication.

[0045] In the polynucleotide of (c2), the term "several" refers to the same as described in (a2) above.

[0046] Whether or not the polynucleotide of (c2) functions as an origin of replication can be determined by the same method as in (a2) above.

[0047] (c3) is a polynucleotide derived from pTthSNM1-7e, which consists of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 3, and which functions as an origin of replication.

[0048] In the polynucleotide of (c3), the homology with the base sequence shown in SEQ ID NO: 3 is as described in (a3) ​​above.

[0049] Whether or not the polynucleotide of (c3) functions as an origin of replication can be determined by the same method as in (a2) above.

[0050] (d1) is a polynucleotide derived from pTthSNM1-7f (GenBank ID: AP025600) and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:4.

[0051] (d2) is a polynucleotide derived from pTthSNM1-7f, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 4, and which functions as an origin of replication.

[0052] In the polynucleotide (d2), the term "several" refers to the same as in (a2) above.

[0053] Whether or not the polynucleotide (d2) functions as an origin of replication can be determined by the same method as in (a2) above.

[0054] (d3) is a polynucleotide derived from pTthSNM1-7f, consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 4, and having the function of an origin of replication.

[0055] In the polynucleotide of (d3), the homology with the base sequence shown in SEQ ID NO: 4 is as described in (a3) ​​above.

[0056] Whether or not the polynucleotide (d3) functions as an origin of replication can be determined by the same method as in (a2) above.

[0057] (e1) is a polynucleotide derived from pTthSNM3-3d (GenBank ID: AP025612) and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:5.

[0058] (e2) is a polynucleotide derived from pTthSNM3-3d, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 5, and which functions as an origin of replication.

[0059] In the polynucleotide of (e2), the term "several" refers to the same as described in (a2) above.

[0060] Whether or not the polynucleotide of (e2) has the function of a replication origin can be determined by the same method as in (a2) above.

[0061] (e3) is a polynucleotide derived from pTthSNM3-3d, consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 5, and having the function of an origin of replication.

[0062] In the polynucleotide of (e3), the homology with the base sequence shown in SEQ ID NO: 5 is as described in (a3) ​​above.

[0063] Whether or not the polynucleotide of (e3) has the function of a replication origin can be determined by the same method as in (a2) above.

[0064] (f1) is a polynucleotide derived from pTthSNM1-1f (GenBank ID: AP025601.1) and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27.

[0065] (f2) is a polynucleotide derived from pTthSNM1-1f, which consists of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 27, and which functions as an origin of replication.

[0066] In the polynucleotide of (f2), the term "several" refers to the same as described in (a2) above.

[0067] Whether or not the polynucleotide of (f2) has the function of a replication origin can be determined by the same method as in (a2) above.

[0068] (f3) is a polynucleotide derived from pTthSNM1-1f, consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 27, and having the function of an origin of replication.

[0069] In the polynucleotide of (f3), the homology with the base sequence shown in SEQ ID NO: 5 is as described in (a3) ​​above.

[0070] Whether or not the polynucleotide (f3) functions as an origin of replication can be determined by the same method as in (a2) above.

[0071] (g1) is a polynucleotide derived from pTthSNM1-1g (GenBank ID: AP025602.1) and is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28.

[0072] (g2) is a polynucleotide derived from pTthSNM1-1g, and is a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 28, and which functions as an origin of replication.

[0073] In the polynucleotide (g2), the term "several" refers to the same as in (a2) above.

[0074] Whether or not the polynucleotide of (g2) functions as a replication origin can be determined by the same method as in (a2) above.

[0075] (g3) is a polynucleotide derived from pTthSNM1-1g, consisting of a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 28, and having the function of an origin of replication.

[0076] In the polynucleotide of (g3), the homology with the base sequence shown in SEQ ID NO: 5 is as described in (a3) ​​above.

[0077] Whether or not the polynucleotide of (g3) functions as a replication origin can be determined by the same method as in (a2) above.

[0078] (Second origin of replication derived from a biological species other than Thermus thermophilus) The shuttle vector of the present invention comprises a second origin of replication derived from a biological species other than Thermus thermophilus.

[0079] The second origin of replication is not particularly limited as long as it is derived from a biological species other than Thermus thermophilus, and examples thereof include origins of replication derived from Escherichia coli, yeast, actinomycetes, Bacillus subtilis, etc. From the viewpoint of convenience in genetic manipulation, an origin of replication derived from Escherichia coli is preferably used.

[0080] The replication origin derived from E. coli is not particularly limited, but examples thereof include replication origins such as ColE1, pMB, p15a, pSC101, and pRSF.

[0081] The yeast-derived replication origin is not particularly limited, but examples thereof include the 2 μm replication origin.

[0082] The actinomycete-derived replication origin is not particularly limited, but examples thereof include replication origins such as SCP2, SLP1.2, and pIJ101.

[0083] (Other Features) The shuttle vector of the present invention preferably further comprises at least one sequence selected from the group consisting of a promoter sequence, a multicloning site, and a drug resistance gene. The promoter sequence, multicloning site, and drug resistance gene can be selected appropriately based on the type of vector used, the purpose of the experiment, other conditions, etc.

[0084] Any promoter (promoter sequence) known to those skilled in the art can be used, and examples include, but are not limited to, the lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, lacI promoter, lacZ promoter, T3 promoter, T7 promoter, SV40 early promoter, SV40 late promoter, and retrovirus LTR promoter.

[0085] The multicloning site is not particularly limited, and examples thereof include the multicloning sites of pUC18 vector, pCOLD vector, etc. The number of multicloning sites may be one or more.

[0086] The drug resistance gene is not particularly limited, but examples thereof include the kanamycin resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, gentamicin resistance gene, neomycin resistance gene, apramycin resistance gene, bleomycin resistance gene, zeocin resistance gene, etc. In consideration of suitability as a selection marker, the kanamycin resistance gene, neomycin resistance gene, hygromycin resistance gene, bleomycin resistance gene, and zeocin resistance gene are preferred.

[0087] In one embodiment of the present invention, the shuttle vector of the present invention may further comprise one or more other replication origins in addition to the first and second replication origins. The other replication origins are preferably derived from a biological species different from that of the first and second replication origins.

[0088] (One aspect of shuttle vector) In one embodiment of the present invention, the shuttle vector of the present invention is preferably pIOK1Hg (SEQ ID NO: 6), pIOK2Hg (SEQ ID NO: 7), pIOK3Hg (SEQ ID NO: 8), pIOK4Hg (SEQ ID NO: 9), pIOK5Hg (SEQ ID NO: 10), pIOK6Hg (SEQ ID NO: 11), pIOK7Hg (SEQ ID NO: 12), pIOK8Hg (SEQ ID NO: 13), pIOK9Hg (SEQ ID NO: 14), pIOK10Hg (SEQ ID NO: 15), pFK1Hg (SEQ ID NO: 29), pFK2Hg (SEQ ID NO: 30), pSG1Hg (SEQ ID NO: 31) or pSG2Hg (SEQ ID NO: 32). In one embodiment of the present invention, the shuttle vector of the present invention is preferably pIOK1Hg (SEQ ID NO: 6), pIOK2Hg (SEQ ID NO: 7), pIOK3Hg (SEQ ID NO: 8), pIOK4Hg (SEQ ID NO: 9), pIOK5Hg (SEQ ID NO: 10), pIOK6Hg (SEQ ID NO: 11), pIOK7Hg (SEQ ID NO: 12), pIOK8Hg (SEQ ID NO: 13), pIOK9Hg (SEQ ID NO: 14), pIOK10Hg (SEQ ID NO: 15), pFK1Hg (SEQ ID NO: 29) or pFK2Hg (SEQ ID NO: 30).

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[0127] In one embodiment of the present invention, there is provided a shuttle vector comprising a first replication origin derived from Thermus thermophilus and a second replication origin derived from a biological species other than Thermus thermophilus, wherein the first replication origin is derived from pTthSNM1-1d, pTthSNM1-1e, pTthSNM1-7e, pTthSNM1-7f, pTthSNM3-3d, pTthSNM1-1f, or pTthSNM1-1g.

[0128] In one embodiment of the present invention, there is provided a foreign gene expression vector comprising an origin of replication derived from Thermus thermophilus. In one embodiment of the present invention, the origin of replication is derived from pTthSNM1-1d, pTthSNM1-1e, pTthSNM1-7e, pTthSNM1-7f, pTthSNM3-3d, pTthSNM1-1f, or pTthSNM1-1g. In one embodiment of the present invention, the origin of replication is any one of the polynucleotides (a1) to (g3), preferably (a1), (b1), (c1), (d1), (e1), (f1), or (g1). In one embodiment of the present invention, the origin of replication is preferably any one of the polynucleotides (a1) to (f3).

[0129] (Method of Constructing Shuttle Vector) The shuttle vector of the present invention can be constructed using any method known in the art. For example, when a plasmid derived from Thermus thermophilus is used as the base vector, it can be constructed by inserting a second origin of replication derived from a biological species other than Thermus thermophilus into the plasmid derived from Thermus thermophilus. Furthermore, when a plasmid derived from a biological species other than Thermus thermophilus is used as the base vector, it can be constructed by inserting a first origin of replication derived from Thermus thermophilus into the plasmid derived from the biological species other than Thermus thermophilus. The method of inserting an origin of replication into a plasmid can be any method known in the art.

[0130] The orientation of the replication origin in the shuttle vector of the present invention is not particularly limited. For example, when two replication origins are present, the two replication origins may be oriented in the same direction or in opposite directions.

[0131] (3. Transformant) In one embodiment of the present invention, a transformant containing the shuttle vector is provided. The transformant is produced by introducing the shuttle vector into a host.

[0132] The host is not particularly limited, but preferred are Thermus thermophilus (for example, HB27 strain, HB8 strain, etc.), Escherichia coli (for example, JM109 strain, DH5 alpha strain, etc.), and the like.

[0133] The transformation method is not particularly limited, and conventionally known methods such as the calcium chloride method, calcium phosphate method, liposome method, DEAE-dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, transduction, and infection can be suitably used. Such methods are described in many standard laboratory manuals, such as "Basic Methods in Molecular Biology" by Davis et al. (1986) and "Molecular Cloning." Furthermore, in the case of Thermus thermophilus, natural transformation is generally used, and transformation can be carried out, for example, by the method described in Non-Patent Document 1.

[0134] (4. Method for Producing Protein) In one embodiment of the present invention, there is provided a method for producing a protein, which includes a step of culturing the transformant. The protein in this production method is not particularly limited, and any desired protein can be produced.

[0135] The transformant can be cultured by any method known in the art, and the culture conditions can be appropriately determined by those skilled in the art.

[0136] In one embodiment of the present invention, this production method is carried out, for example, by introducing a gene encoding a desired protein (e.g., an exogenous protein) into a shuttle vector of the present invention, transforming the cell with the shuttle vector, and then culturing the resulting transformant under certain conditions.

[0137] In one embodiment of the present invention, the production method preferably includes a step of purifying the target protein from the transformant or culture medium containing the target protein obtained in the culturing step.

[0138] The step of purifying the protein is carried out, for example, by preparing a cell extract from the transformant by a well-known method, or by recovering the culture medium and then purifying the protein from the cell extract or culture medium by a well-known method.

[0139] The purification method is not particularly limited, but examples include ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.

[0140] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0141] (Experimental materials) <Bacterial strains> Escherichia coli JM109 (hereinafter sometimes referred to as "E. coli") Thermus thermophilus HB27 (hereinafter sometimes referred to as "T. thermophilus")

[0142] <Culture medium>

[0143]

[0144] For the cultivation of T. thermophilus, the above medium was supplemented with 0.4 mM MgSO4 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.35 mM CaCl2 (Fujifilm Wako Pure Chemical Industries, Ltd.), and the agar medium was supplemented with 15 g / L agar (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0145] <Antibiotics> Hygromycin B (Fujifilm Wako Pure Chemical Industries, Ltd.) 100 μg / mL Hygromycin B was added to the medium to the above concentration.

[0146] <DNA polymerase> KOD FX Neo (Toyobo)

[0147] <Real-time PCR reagent> KAPA SYBR Fast qPCR Kit (Nippon Genetics)

[0148] (method)

[0149] <Culturing of Escherichia coli> Static or shaking culture was carried out at 37°C.

[0150] <Cultivation of T. thermophilus> Static (agar plate) or shaking culture (liquid medium) was performed at 70°C.

[0151] <Transformation of E. coli> Transformation of E. coli was performed using the following procedure. 1. Thaw competent cells (stored at -80°C) on ice for 5 minutes. 2. Add 1 μl of DNA solution to 100 μl of competent cells. 3. Leave on ice for 10 minutes. 4. Heat shock at 42°C for 45 seconds. 5. Leave on ice for 2 minutes. 6. Add 500 μl of SOC medium and incubate at 37°C for 1 hour.

[0152] <Transformation of T. thermophilus> Transformation of T. thermophilus was carried out using the following procedure. 1. Cultivate the cells until stationary phase. 2. Add 1 μl of DNA solution to 100 μl of cell culture. 3. Cultivate with shaking at 70°C for 3 hours.

[0153] <Plasmid extraction> Plasmid extraction was carried out using FastGene Plasmid Mini Kits (Nihon Genetics) according to the protocol described in the kit.

[0154] <Gibson Assembly>

[0155]

[0156] A reagent was prepared with the above composition and incubated at 50°C for 1 hour.

[0157] <PCR> PCR was carried out under the conditions described below.

[0158] <Electrophoresis> Electrophoresis was carried out using a 1% agarose gel at 135 V for 15 minutes.

[0159] <Gel extraction> Gel extraction was performed using the FastGene Gel / PCR Extraction Kit (Nippon Genetics) according to the protocol described in the kit. Elution was performed with 30 μl of 10 mM Tris-HCl, pH 8.5.

[0160] <Plasmid extraction> Gel extraction was performed using the FastGene Plasmid mini Kit (Nippon Genetics) according to the protocol described in the kit. Elution was performed with 50 μl of 10 mM Tris-HCl, pH 8.5.

[0161] <Genome extraction> Genome extraction was performed using the Extrap Soil DNA Kit Plus Ver. 2 (Funakoshi) according to the protocol described in the kit. Elution was performed with 100 μl of 10 mM Tris-HCl pH 8.5.

[0162] <Real-time PCR> Samples were prepared using the KAPA SYBR Fast qPCR kit (Nihon Genetics) according to the protocol described in the kit. Measurements were performed using a QuantStudio® 3 Real-time PCR System (Thermo Fisher Scientific).

[0163] (Search for new ori) First, to find plasmids possessing new replication genes (Rep proteins) among the plasmids held in our laboratory, we used BLAST searches to predict the replication genes of each plasmid. We then classified the plasmids based on the sequence homology of the predicted replication genes, and found that many of the plasmids fell into three major groups. Since one of these groups had not yet been used as a plasmid vector, we decided to construct a new shuttle vector based on the plasmids belonging to this group (pTthSNM1-1d, pTthSNM1-1e, pTthSNM1-7e, pTthSNM1-7f, pTthSNM3-3d, pTthSNM1-1f, and pTthSNM1-1g).

[0164] (Construction of New Shuttle Vectors) First, the sequences between the putative replication gene and the preceding and following ORFs of each T. thermophilus plasmid were amplified by PCR. For pTthSNM1-1d and pTthSNM3-3d, two downstream protein genes of unknown function were also amplified. For pTthSNM1-1f, 400 bp upstream and 200 bp downstream of the putative replication gene were also amplified. For pTthSNM1-1g, 200 bp upstream and 200 bp downstream of the putative replication gene were also amplified. Next, the PCR-amplified DNA fragments were inserted into the E. coli vector pHSG298Hg using Gibson Assembly in two orientations for each fragment. pHSG298Hg is a pUC-type plasmid with a ColE1-type replication mechanism and a thermostable hygromycin resistance gene as a selectable marker. The constructed shuttle vectors (hereinafter sometimes referred to as "plasmids") are shown in Tables 24 and 25.

[0165]

[0166]

[0167] The base sequence of pHSG298Hg (SEQ ID NO: 26) is shown below.

[0168]

[0169] (Vector Preparation) Using vector pHSG298Hg, in which the kanamycin resistance gene of commercially available vector pHSG298 (manufactured by Takara Bio) was replaced with a heat-stable hygromycin resistance gene, an on was inserted downstream of the hygromycin resistance gene. A linear full-length plasmid of pHSG298Hg was prepared by inverse PCR using primers of SEQ ID NOs: 24 and 25. KOD one (manufactured by Takara Bio) was used for vector PCR.

[0170] After the reaction, the reaction solution was cooled to room temperature, and 0.5 μl (equivalent to 10 U) of Dpn I (NEB) was added, followed by reaction at 37° C. for 2 hours.

[0171] <PCR materials> DNA template 0.2 μl Fw primer 0.5 μl Rv primer 0.5 μl KOD One 8 μl dH2O 10.3 μl 20 μl

[0172] <PCR conditions> 98℃ 2 minutes, 98℃ 10 seconds, 57℃ 5 seconds, 68℃ 20 seconds *30 cycles, 68℃ 10 seconds

[0173] <Primer>

[0174]

[0175] (Preparation of Ori Fragment) Ori fragment was prepared under the following conditions.

[0176] <PCR materials> DNA template 0.2 μl Fw primer 0.5 μl Rv primer 0.5 μl KOD FENeo 0.4 μl S*Buffer for FXNeo 10 μl 2 mM dNTPS 4 μl dH2O 4.4 μl 20 μl

[0177] <PCR conditions> 98℃ 2 minutes, 98℃ 10 seconds, 57℃ 30 seconds, 68℃ 90 seconds *30 cycles, 68℃ 10 seconds

[0178] <Primer>

[0179]

[0180]

[0181] The vector and Ori fragment were separated and purified by agarose gel electrophoresis. A portion of the 30 μl eluate was subjected to Gibson assembly as described above. After incubation at 50°C for 1 hour, a portion of the reaction mixture (0.5 μl) was added to 100 μl of JM109 competent cells for transformation. Plasmids were prepared from colonies that appeared on LB / Hg medium, and sequences were confirmed by the Sanger method.

[0182] (Confirmation of the shuttle ability of the constructed plasmid) The constructed plasmid was confirmed to be capable of autonomous replication in both E. coli and T. thermophilus hosts. First, the constructed plasmid was used to transform E. coli JM109. The E. coli JM109 transformant grew at 37°C on LB plates containing hygromycin, confirming that the plasmid was autonomously replicating in E. coli JM109. Next, the plasmid was extracted from the E. coli JM109 transformant, and the extracted plasmid was used to transform T. thermophilus HB27. The T. thermophilus HB27 transformant grew at 70°C on LB plates containing hygromycin, confirming that the plasmid was autonomously replicating in T. thermophilus HB27. Next, the plasmid was extracted from the T. thermophilus HB27 transformant, and the extracted plasmid was used to transform E. coli JM109. This transformant also acquired hygromycin resistance, confirming that the plasmid was autonomously replicating within the bacterial cells. This process of host replication was repeated five times.

[0183] These experiments confirmed that the constructed plasmid replicated autonomously in both E. coli and T. thermophilus hosts, and that the amplified plasmid could be used to transform both E. coli and T. thermophilus hosts.

[0184] (Determination of plasmid copy number) The plasmid copy number of the constructed plasmid in T. thermophilus was measured using real-time PCR. Here, the plasmid copy number refers to the relative copy number (i.e., the number of plasmids per chromosome). First, the genome was extracted from the transformant. Real-time PCR was performed using the extracted genome as a template. The target sequence of the chromosome was a part of the DNA polymerase I gene, and the target sequence of the plasmid was a part of the hygromycin resistance gene. The results are shown in Figures 1-1 and 1-2. Figures 1-1 and 1-2 show that the copy number of each plasmid was as follows:・IOK1: 1.06 (standard deviation 0.04) ・IOK2: 1.35 (standard deviation 0.04) ・IOK3: 0.89 (standard deviation 0.07) ・IOK4: 1.15 (standard deviation 0.13) ・IOK5: 1.05 (standard deviation 0.05) ・IOK6: 1.84 (standard deviation 0.07)・IOK7: 0.86 (standard deviation 0.18) ・IOK8: 0.73 (standard deviation 0.17) ・IOK9: 1.72 (standard deviation 0.07) ・IOK10: 1.41 (standard deviation 0.07) ・FK1: 1.38 (standard deviation 0.12) ・FK2: 1.05 (standard deviation 0.10)・SG1: 0.05 (standard deviation 0.06) ・SG2: 0.46 (standard deviation 0.17)

[0185] (Measurement of Plasmid Maintenance Stability) The maintenance stability of the constructed plasmids in T. thermophilus was evaluated. Maintenance stability is an indicator of whether a plasmid can be stably maintained in the absence of selective pressure such as antibiotics. Transformants were subcultured in antibiotic-free liquid medium for one week. The medium was replaced and samples were collected every 24 hours. The sampled cells were plated on agar medium containing or not containing hygromycin. After overnight static culture, the number of colonies on the agar medium was counted. Plasmid maintenance stability was evaluated by dividing the number of colonies on the agar medium containing hygromycin by the number of colonies on the agar medium without hygromycin. The results are shown in Figures 2-1 and 2-2. Figure 2-1 indicates that pIOK1 to pIOK10 have plasmid maintenance stability. Figure 2-2 indicates that pFK1Hg and pFK2Hg have plasmid maintenance stability. On the other hand, pSG1 and pSG2 had relatively low plasmid maintenance stability.

[0186] (Comparison with pTT8-Derived Shuttle Vector) The constructed plasmid (pIOK9) was compared with the pTT8-derived shuttle vector for maintenance stability. Transformants were subcultured in antibiotic-free liquid medium for one week. Medium was replaced and samples were collected every 24 hours. The sampled cells were plated on agar plates containing or not containing hygromycin. After overnight static culture, the number of colonies on the agar plates was counted. Plasmid maintenance stability was evaluated by dividing the number of colonies on the agar plates containing hygromycin by the number of colonies on the agar plates without hygromycin. The results are shown in Figure 3. pSN2 was constructed using the forward primer for IOK1 (SEQ ID NO: 22) and the reverse primer for IOK2 (SEQ ID NO: 23) and pTT8 (GenBank ID: AP024986 (NZ_AP024986.1)) as the template. Figures 3-1 and 3-2 show that pIOK9 has superior plasmid retention stability compared to pTT8-derived shuttle vectors. Furthermore, pFK2 has similar plasmid retention stability to pIOK4 and is more stable than the pTT8-type shuttle vector pSN2.

[0187] (Plasmid Compatibility Test) The newly constructed pFK and pSG vectors were tested for compatibility with shuttle vectors with other replication mechanisms. Plasmid compatibility refers to the ability of plasmids to stably coexist within the same host. In this example, plasmid compatibility was tested using pFK2, pSG2, pIOK6, and pSN2 (pTT8 type). To easily test compatibility, the antibiotic resistance gene of each shuttle vector was replaced with a kanamycin (Km) resistance gene to construct plasmids pFK2Km, pSG2Km, pIOK6Km, and pSN2Km. Two plasmids with different antibiotic resistance genes (HgR and KmR) were simultaneously introduced into T. thermophilus HB27, and compatibility was tested by the acquisition of two antibiotic resistances (HgR and KmR). All six combinations of these four plasmids resulted in colony formation on TR / Hg / Km agar medium (Figure 4). The transformants were cultured overnight in TR / Hg / Km liquid medium, and the plasmids were extracted from the cells. When the plasmids were introduced into E. coli DH5α, colonies were formed on both LB / Hg and LB / Km agar plates. The transformants were cultured overnight in LB / Hg or LB / Km liquid medium, and the plasmids were extracted and sequenced. The sequence of the plasmid extracted from E. coli matched the sequences of the two plasmids initially introduced into T. thermophilus HB27. These results confirmed that all combinations of plasmids coexist stably within the same host.

[0188] The present invention provides a shuttle vector capable of stable replication, and is therefore extremely useful as a basic technology for genetic engineering. This application is based on Japanese Patent Application No. 2024-048786 (filing date: March 25, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A shuttle vector comprising a first replication origin derived from Thermus thermophilus, and a second replication origin derived from a biological species other than Thermus thermophilus, wherein the first replication origin is any of the following polynucleotides: (a1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (a2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in which one or more bases have been deleted, substituted or added, and which functions as an origin of replication; (a3) ​​a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 1, and which functions as an origin of replication; (b1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which one or more bases have been deleted, substituted or added, and which functions as an origin of replication; (b3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 2, and which functions as an origin of replication; (c1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3; (c2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3, in which one or several bases have been deleted, substituted or added, and which functions as an origin of replication; (c3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 3, and which functions as an origin of replication; (d1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4; (d2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4, in which one or several bases have been deleted, substituted or added, and which functions as an origin of replication; (d3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 4, and which functions as an origin of replication; (e1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5; (e2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5, in which one or several bases have been deleted, substituted or added, and which functions as an origin of replication;(e3) A polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 5 and functioning as an origin of replication; (f1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27; (f2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27 in which one or more bases have been deleted, substituted or added and functioning as an origin of replication; (f3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 27 and functioning as an origin of replication; (g1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28; (g2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28 in which one or more bases have been deleted, substituted or added and functioning as an origin of replication; (g3) a polynucleotide consisting of a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 28 and functioning as an origin of replication.

2. The shuttle vector of claim 1, wherein the first replication origin is any of (a1), (b1), (c1), (d1), (e1), (f1) or (g1).

3. The shuttle vector of claim 1, wherein the second replication origin is derived from Escherichia coli.

4. The shuttle vector of claim 3, wherein the second origin of replication is a ColE1, pMB, p15a, pSC101, or pRSF origin of replication.

5. The shuttle vector of claim 1, which is pIOK1Hg (SEQ ID NO: 6), pIOK2Hg (SEQ ID NO: 7), pIOK3Hg (SEQ ID NO: 8), pIOK4Hg (SEQ ID NO: 9), pIOK5Hg (SEQ ID NO: 10), pIOK6Hg (SEQ ID NO: 11), pIOK7Hg (SEQ ID NO: 12), pIOK8Hg (SEQ ID NO: 13), pIOK9Hg (SEQ ID NO: 14), pIOK10Hg (SEQ ID NO: 15), pFK1Hg (SEQ ID NO: 29), pFK2Hg (SEQ ID NO: 30), pSG1Hg (SEQ ID NO: 31) or pSG2Hg (SEQ ID NO: 32).

6. The shuttle vector of claim 1, further comprising at least one sequence selected from the group consisting of a promoter sequence, a multicloning site, and a drug resistance gene.

7. A transformant comprising the shuttle vector according to any one of claims 1 to 6.

8. A method for producing a protein, comprising the step of culturing the transformant described in claim 7.

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