Transformant and method for detecting presence or absence of hypophosphorous acid using the transformant

Abstract

To provide a transformant exhibiting a containment effect independent of phosphorous acid.SOLUTION: The transformant is deficient in functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein, and has a gene encoding a hypophosphite-specific transporter protein introduced thereinto.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a transformant and a method for detecting the presence or absence of hypophosphorous acid using the transformant. [Background technology]

[0002] In recent years, genetically modified organisms applicable to a wide variety of uses have been produced, and the produced genetically modified organisms are expected to be used for purposes such as oral vaccines or improving the natural environment.

[0003] On the other hand, there are several conditions that must be met before genetically modified organisms can actually be used. One of these conditions is that genetically modified organisms must be able to grow only in a limited location and cannot grow anywhere else (in other words, genetically modified organisms with a high containment effect). If such genetically modified organisms are created, even if they leak into the natural environment, they will not be able to grow there, and contamination of the natural environment by the genetically modified organisms can be prevented.

[0004] Various methods have been developed for producing such genetically modified organisms. One method is to confer auxotrophy on an organism for a compound that does not exist in nature (synthetic auxotrophy). Patent Document 1 is a specific example that discloses such a method. Patent Document 1 discloses transformants of Escherichia coli, blue-green algae, and the like, which have been developed by the present inventors and whose metabolism has been modified so that they grow dependent on phosphorous acid, which is hardly present in the environment, thereby exhibiting the world's highest level of containment effect. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 168163 Brochure Summary of the Invention [Problem to be solved by the invention]

[0006] However, in recent years, phosphorous acid may be released into the environment as wastewater from metal plating and as agricultural fertilizer, raising concerns that the presence of anthropogenic phosphorous acid in the environment may affect the containment effect. Therefore, there is a need to develop transformants that exhibit a containment effect independent of phosphorous acid.

[0007] An object of one aspect of the present invention is to provide a transformant that exhibits a phosphite-independent containment effect, and a method for detecting the presence or absence of hypophosphite using the transformant. [Means for solving the problem]

[0008] In order to solve the above problems, one aspect of the present invention includes the following configurations.

[0009] [1] A transformant that lacks the functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein, and that has a gene encoding a hypophosphate-specific transporter protein introduced into it, resulting in the transformant being unable to utilize phosphate and phosphorous acid for growth, but being able to utilize hypophosphate for growth.

[0010] [2] The transformant according to [1], into which a gene encoding an HtxA protein has been introduced, the hypophosphite-specific transporter protein being an HtxBCDE protein, and the HtxB protein of the HtxBCDE protein is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (1) to (3): (1) a polynucleotide consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 3; (2) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity; or (3) A polynucleotide having a sequence identity of 90% or more with a polynucleotide consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encoding a protein having hypophosphorous acid transport activity but no phosphate or phosphite transport activity.

[0011] [3] A gene encoding the HtxA protein has been introduced, The transformant according to [1], wherein the hypophosphite-specific transporter protein is a FocA protein, which is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (4) to (6): (4) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 24 or 25; (5) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having the base sequence represented by SEQ ID NO: 24 or 25, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity; or (6) A polynucleotide having a sequence identity of 90% or more with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 24 or 25, and encoding a protein having hypophosphate transport activity but not phosphate or phosphate transport activity.

[0012] [4] The transformant according to any one of [1] to [3], further comprising a gene encoding a phosphite dehydrogenase protein introduced therein.

[0013] [5] The transformant according to any one of [1] to [4], wherein the transformant is a transformant of a prokaryote.

[0014] [6] A transformant according to any one of [1] to [5], which can utilize phosphate for growth and has a growth advantage in a medium containing hypophosphorous acid as a phosphorus source over a competing strain that cannot utilize hypophosphorous acid for growth.

[0015] [7] A method for detecting the presence or absence of hypophosphorous acid, comprising: a culturing step of culturing a transformant according to any one of [1] to [6] in a medium to be detected; and a detecting step of detecting the presence or absence of growth of the transformant in the culturing step. [Effects of the Invention]

[0016] According to one aspect of the present invention, a phosphite-independent containment effect can be achieved for transformants. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a diagram illustrating the structure of a transformant according to an embodiment of the present invention. [Figure 2] FIG. 1 is a diagram for conveniently explaining the structures of membrane-bound HtxBCDE and HtxBCD proteins. [Figure 3] FIG. 1 shows the three-dimensional structure of the HtxB protein. [Figure 4] FIG. 1 shows the results of a specificity test of mutant HtxBCD transporters (Example 1). [Figure 5] 501 and 502 are diagrams showing the results of growth measurement of the hypophosphorous acid-encapsulating strain (Example 2). [Figure 6] FIG. 1 shows the results of evaluation of the specificity of FocA protein for inorganic phosphorus compounds (Example 4). [Figure 7] FIG. 1 shows the hypophosphite dependency of hypophosphite-dependent strains utilizing the FocA protein prepared in Example 5. [Figure 8] FIG. 1 shows the results of an evaluation of the specificity of a FocA protein homologue from the marine cyanobacterium Synechococcus sp. PCC 7002 for inorganic phosphorus compounds (Example 7). [Figure 9] FIG. 1 shows the results of verifying the growth advantage of hypophosphate-dependent strains in hypophosphate medium (Example 8). DETAILED DESCRIPTION OF THE INVENTION

[0018] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

[0019] [1. Basic principle] While phosphoric acid exists in large amounts in nature, phosphorous acid and hypophosphorous acid are absent or present in very small amounts, if at all. Furthermore, as mentioned above, anthropogenic phosphorous acid may be present in the environment. In this case, if a transformant is created that cannot utilize phosphoric acid and phosphorous acid for growth but can utilize hypophosphorous acid for growth, the transformant will be unable to grow even if it leaks into an environment where anthropogenic phosphorous acid is present. The present inventors came up with the idea that the transformant would exhibit a containment effect that is independent of phosphorous acid, and aimed to create the transformant.

[0020] The transformant according to this embodiment will be described with reference to Fig. 1. Fig. 1 is a diagram illustrating the structure of the transformant according to this embodiment. As shown in Fig. 1, a phosphate transporter protein and a phosphate ester transporter protein are basically present in an organism.

[0021] Phosphate transporter proteins are proteins that transport phosphate and reduced phosphorus compounds into cells, while phosphate ester transporter proteins are proteins that transport phosphate esters into cells.

[0022] When phosphate is supplied to the cell from nature, the transformant is able to grow dependent on phosphate in nature. On the other hand, when phosphate ester is supplied to the cell from nature, the transformant converts the phosphate ester into a phosphorus source using its own metabolic system and is able to grow dependent on the phosphorus source in nature. Therefore, the transformant according to this embodiment lacks the functions of both the phosphate transporter protein and the phosphate ester transporter protein.

[0023] However, if the functions of both the phosphate transporter protein and the phosphate ester transporter protein are deleted, not only phosphate and phosphate ester but also reduced phosphorus compounds (e.g., phosphorous acid, hypophosphorous acid) are not naturally supplied to the cells, and in this case, the transformant cannot grow depending on not only phosphate and phosphate ester but also reduced phosphorus compounds.

[0024] The present inventors have solved the above-mentioned problems by discovering that the hypophosphate transporter protein encoded by the HtxBCDE gene (or HtxBCD gene) derived from Pseudomonas stutzeri WM88 has the function of transporting reduced phosphorus compounds but not phosphate.

[0025] Specifically, the present inventors created a transformant that utilizes only reduced phosphorus compounds by deleting the function of the host's phosphate transport system and introducing an HtxBCDE gene (or an HtxBCD gene) into the host. When a transformant expresses an HtxBCDE protein (or an HtxBCD protein), the transformant can take up only reduced phosphorus compounds into the cell. The reduced phosphorus compounds are converted to phosphate by an intracellular metabolic system, and the transformant can grow using the phosphate. Furthermore, when the HtxA gene encoding hypophosphite dioxygenase is expressed simultaneously with the HtxBCDE gene, hypophosphorous acid taken up into the cell by the HtxBCDE protein is oxidized by the HtxA protein to produce phosphite within the cell, and the phosphite is oxidized by the PtxD protein to produce phosphate within the cell, allowing the transformant to grow using the phosphate.

[0026] Based on the above-mentioned principle, the inventors considered that if the HtxBCDE protein (or HtxBCD protein) could be modified into a protein that takes up hypophosphorous acid into cells but does not take up phosphate or phosphorous acid into cells, i.e., a hypophosphorous acid-specific transporter (hereinafter sometimes referred to as hpt-HtxBCDE), it would be possible to create a transformant according to this embodiment that exhibits a containment effect independent of phosphate.

[0027] Here, the details of the HtxBCDE protein and the HtxBCD protein will be explained using Figure 2. Figure 2 is a diagram for conveniently explaining the structure of the membrane-bound HtxBCDE protein and the HtxBCD protein. The HtxBCDE protein and the HtxBCD protein are membrane-bound protein complexes and are substrate-binding protein-dependent ABC transporters. The HtxBCDE protein and the HtxBCD protein are composed of an HtxB protein, an HtxC protein, an HtxD protein, and optionally an HtxE protein (not shown).

[0028] HtxB is a protein localized in the periplasm outside the cell membrane. HtxC is a protein that binds to the cell membrane. HtxD is a protein that exists in the cytoplasm and has ATPase activity. As shown in Figure 2, of these proteins, HtxB binds to substrates and is involved in selecting the substrates to transport into the cell.

[0029] Therefore, the present inventors focused on the HtxB protein and attempted to modify it to hpt-HtxBCDE. Figure 3 shows the three-dimensional structure of the HtxB protein. The amino acid sequence of the HtxB protein is shown in SEQ ID NO: 20.

[0030] In the amino acid sequence of the HtxB protein having the three-dimensional structure shown in FIG. 3, tryptophan at position 52 (W52) and aspartic acid at position 206 (D206) are known to be important amino acids for substrate binding.

[0031] Therefore, the present inventors hypothesized and tested that an HtxBCDE protein containing an HtxB protein mutated by substituting W52 or D206 with another amino acid could become hpt-HtxBCDE.

[0032] Specifically, the present inventors attempted to generate transformants in E. coli by performing three steps: (i) deleting the functions of a phosphate transporter protein and a phosphate ester transporter protein; (ii) introducing a hypophosphite dioxygenase protein (HtxA protein) that converts hypophosphite to phosphorous acid and a phosphite dehydrogenase protein (PtxD protein) that converts phosphorous acid to phosphate; and (iii) expressing an HtxBCDE protein (or HtxBCD protein) in which W52 or D206 in HtxB protein is substituted. As a result, it was found that when W52 in HtxB protein was substituted with alanine (W52A), D206 in HtxB protein was substituted with alanine (D206A), or D206 in HtxB protein was substituted with asparagine (D206N) in step (iii), the resulting transformants were able to grow using hypophosphite but were unable to grow using phosphate or phosphorous acid. It was also demonstrated that the transformant exhibited a phosphite-independent containment effect. The amino acid sequences of the W52A substitution mutant HtxB protein, the D206A substitution mutant HtxB protein, and the D206N substitution mutant HtxB protein are shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.

[0033] It has been reported that the introduction of substitution mutations W52A, D206A, or D206N into the HtxB protein results in a loss of substrate binding ability in in vitro tests. Considering this in vitro study, it is predicted that the introduction of substitution mutations W52A, D206A, or D206N into the HtxB protein in vivo will also result in the inability of the HtxB protein to bind to substrates, and as a result, the HtxBCDE protein will not take up either hypophosphite or phosphite into cells.

[0034] However, surprisingly, it was found that when a substitution mutation of W52A, D206A, or D206N is introduced into the HtxB protein, the HtxBCDE protein takes up hypophosphorous acid into cells, but does not take up phosphorous acid into cells, as described above. Therefore, the technical concept of the present embodiment, which is based on this finding, overturns conventional knowledge, is not predictable from conventional knowledge, and has been independently completed by the present inventors.

[0035] Furthermore, although the aforementioned hpt-HtxBCDE exhibits high hypophosphate specificity, its application to a limited host is limited due to its nature as it is derived from Pseudomonas stutzeri WM88. Therefore, the discovery of a more versatile hypophosphate-specific transporter was desired. Therefore, the present inventors conducted extensive research to identify a more versatile hypophosphate-specific transporter that could replace hpt-HtxBCDE. The present inventors then focused on the formate transporter FocA protein of Escherichia coli as a candidate for a new hypophosphate-specific transporter and verified its hypophosphate specificity.

[0036] Although FocA protein is known to incorporate hypophosphite as a formate analog, little is known about its specificity for other inorganic phosphorus compounds in vivo. Therefore, the present inventors evaluated the specificity of FocA protein for inorganic phosphorus compounds in Escherichia coli cells in vivo and found that a transformant expressing FocA protein can grow using hypophosphite but cannot grow using phosphate or phosphorous acid. Furthermore, the transformant was demonstrated to exhibit a containment effect independent of phosphite.

[0037] This embodiment will be described in further detail below.

[0038] 2. Transformants The transformant according to this embodiment is characterized in that it is unable to utilize phosphate and phosphorous acid for growth, but can utilize hypophosphorous acid for growth, because it lacks the functions of the gene encoding a phosphate transporter protein and the gene encoding a phosphate ester transporter protein, and has a gene encoding a hypophosphorous acid-specific transporter protein introduced into it.

[0039] Hosts for the transformant of this embodiment include, for example, prokaryotes such as Escherichia coli, lactic acid bacteria, and photosynthetic bacteria, eukaryotic microorganisms such as yeast, and plants, but of course, the present invention is not limited to these hosts. The transformant of this embodiment can be produced by manipulating a small number of genes. Therefore, any organism (e.g., a microorganism) can be used as a host. The transformant of this embodiment is preferably a prokaryotic transformant, and more preferably an Escherichia coli transformant.

[0040] As used herein, the term "phosphate transporter protein" refers to a protein that has the activity of taking up both phosphate and reduced phosphorus compounds into cells, while the term "phosphate ester transporter protein" refers to a protein that has the activity of taking up phosphate esters into cells.

[0041] In this case, a transformant lacking the functions of the gene encoding the phosphate transporter protein and the gene encoding the phosphate transporter protein may be prepared by artificially mutating the host. Alternatively, a transformant may be prepared using a host that does not originally have the functions of the gene encoding the phosphate transporter protein and the gene encoding the phosphate transporter protein (for example, a host that does not have in its genome the gene encoding the phosphate transporter protein and the gene encoding the phosphate transporter protein, or a host in which the phosphate transporter protein and the phosphate transporter protein are not expressed).

[0042] Different biological species have different types of phosphate transporter proteins and phosphate ester transporter proteins present in their cells. Therefore, the types of phosphate transporter proteins and phosphate ester transporter proteins that are functionally deficient in the transformant of this embodiment are not particularly limited. The genes encoding the phosphate transporter proteins and the genes encoding the phosphate ester transporter proteins whose functions are to be deficient may be appropriately determined depending on the host.

[0043] For example, when the host of the transformant is Escherichia coli, examples of the phosphate transporter protein and phosphate ester transporter protein include the proteins disclosed in JP 2018-033386 A.

[0044] The phosphate transporter protein may be at least one protein selected from the group consisting of PitA protein, PitB protein, PstSCAB protein, and PhnCDE protein. Whether or not a protein has phosphate transport activity can be confirmed by expressing a gene encoding a desired protein into an organism lacking the functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein, and then growing the microorganism in a medium containing various phosphorus sources. If the microorganism grows in a medium containing phosphate, it can be determined that the protein has phosphate transport activity.

[0045] The phosphate transporter protein may be at least one protein selected from the group consisting of UhpT protein, UgpB protein, and GlpT protein. Whether or not a protein has phosphate transport activity can be confirmed by expressing a gene encoding a desired protein into an organism lacking the function of a gene encoding a phosphate transporter protein and a gene encoding a phosphate transporter protein, and then growing the microorganism in a medium containing various phosphorus sources. If the microorganism grows in a medium containing phosphate, the protein can be determined to have phosphate transport activity.

[0046] The transformant according to this embodiment is a transformant into which a gene encoding a hypophosphite-specific transporter protein has been introduced, as well as a gene encoding a hypophosphite dioxygenase protein that converts hypophosphite to phosphorous acid.

[0047] As used herein, the term "hypophosphate-specific transporter protein" refers to a protein that has the activity of transporting hypophosphate into cells but does not have the activity of transporting phosphate and phosphate into cells. Examples of the hypophosphate-specific transporter protein include HtxBCDE protein (or HtxBCD protein) in which W52 or D206 in HtxB protein has been mutated by substitution, FocA protein, etc. The genes encoding the HtxBCDE protein and HtxBCD protein may be derived from Pseudomonas stutzeri WM88.

[0048] Furthermore, in the transformant of this embodiment, the hypophosphite-specific transporter protein has a hypophosphite-specific binding protein, which is one of the components of the hypophosphite-specific transporter protein, and has hypophosphite-binding specificity, i.e., the ability to bind to hypophosphite but not to phosphate or phosphite. A transformant having this configuration takes up hypophosphite into cells but not to phosphate or phosphite. Therefore, the transformant exhibits a containment effect that is independent of phosphate and phosphite.

[0049] Furthermore, the transformant according to this embodiment has a growth advantage over competing strains (contaminating bacteria) in a medium containing hypophosphorous acid as a phosphorus source. For example, when a competing strain is added to the transformant according to this embodiment and cultured using hypophosphorous acid and phosphoric acid as phosphorus sources, the transformant according to this embodiment grows more favorably than the competing strain in a medium containing hypophosphorous acid as a phosphorus source, while the competing strain grows more favorably in a medium containing phosphoric acid as a phosphorus source. The growth advantage of the transformant according to this embodiment is evaluated as described in Example 8 below. In Example 8 below, Pseudomonas putida MY11-41 (which can utilize phosphoric acid for growth but cannot utilize phosphoric acid or hypophosphorous acid for growth) was used as the competing strain, and 1 / 100 of the competing strain was added to the transformant according to this embodiment in an amount equivalent to 1 / 100 of the amount used in the transformant according to this embodiment. The transformant according to this embodiment grew more favorably than the competing strain in a medium containing hypophosphorous acid as a phosphorus source. As a result, the transformant according to this embodiment grew more favorably than the competing strain in a medium containing hypophosphorous acid as a phosphorus source.

[0050] Hypophosphorous acid is a phosphorus source that cannot be utilized by general microorganisms. Therefore, by using hypophosphorous acid as the phosphorus source in the medium and using the transformant according to this embodiment, contamination of the growth system of the transformant with external microorganisms can be prevented, and only the transformant can grow preferentially.

[0051] The hypophosphite-specific transporter protein used in the transformant according to this embodiment will be specifically described below.

[0052] First, the hypophosphate-specific transporter protein used in the transformant of this embodiment is composed of HtxB protein, HtxC protein, and HtxD protein, all of which have hypophosphate-binding specificity, and may optionally be composed of HtxE protein, which constitutes an additional cell membrane-binding protein. That is, the hypophosphate-specific transporter protein is an HtxBCDE protein (or an HtxBCD protein) obtained by introducing a W52A, D206A, or D206N substitution mutation into an HtxB protein. Specifically, the hypophosphate-specific transporter protein is an HtxBCDE protein (or an HtxBCD protein) in which the HtxB protein consists of an amino acid sequence represented by any one of SEQ ID NOs: 21 to 23.

[0053] More specifically, the transformant according to this embodiment has a gene encoding an HtxA protein introduced therein, and the hypophosphite-specific transporter protein is an HtxBCDE protein, of which the HtxB protein is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (1) to (3): (1) a polynucleotide consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 3; (2) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity; or (3) A polynucleotide having a sequence identity of 90% or more with a polynucleotide consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encoding a protein having hypophosphorous acid transport activity but no phosphate or phosphite transport activity.

[0054] Furthermore, the hypophosphate-specific transporter protein used in the transformant of this embodiment is FocA protein. FocA protein is a formate transporter in Escherichia coli. FocA protein is known to take up hypophosphate as a formate analog, but little knowledge has been gained regarding its specificity for other inorganic phosphorus compounds in vivo. The present inventors evaluated the specificity of FocA protein for inorganic phosphorus compounds in Escherichia coli cells and found that FocA protein is a hypophosphate-specific transporter protein that has the activity of taking up hypophosphate into cells but does not have the activity of taking up phosphate or phosphorus acid into cells. Homologous genes of FocA protein exist in a wide range of biological species, from prokaryotes to eukaryotes, improving its versatility across different biological species.

[0055] Furthermore, in the transformant of this embodiment, the gene encoding the FocA protein may be a gene encoding the FocA protein of E. coli, or a gene encoding a FocA protein homologue of the marine cyanobacterium Synechococcus sp. PCC 7002. Furthermore, the hypophosphate-specific transporter protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 26 or 27. The protein consisting of the amino acid sequence shown in SEQ ID NO: 26 corresponds to the FocA protein of E. coli, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 27 corresponds to the FocA protein homologue of the marine cyanobacterium Synechococcus sp. PCC 7002.

[0056] More specifically, the transformant according to this embodiment is characterized in that a gene encoding an HtxA protein is introduced into the transformant, and the hypophosphite-specific transporter protein is a FocA protein, which is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (4) to (6): (4) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 24 or 25; (5) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having the base sequence represented by SEQ ID NO: 24 or 25, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity; or (6) A polynucleotide having a sequence identity of 90% or more with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 24 or 25, and encoding a protein having hypophosphate transport activity but not phosphate or phosphate transport activity.

[0057] The nucleotide sequence shown in SEQ ID NO: 24 is a gene encoding the FocA protein of Escherichia coli. Furthermore, the nucleotide sequence shown in SEQ ID NO: 25 is a gene encoding a FocA protein homolog of the marine cyanobacterium Synechococcus sp. PCC 7002. Using the BLAST program, the FocA protein homolog of the marine cyanobacterium Synechococcus sp. PCC 7002 shows approximately 28% amino acid identity with the FocA protein of Escherichia coli.

[0058] It is known that the FocA protein is expressed in Escherichia coli under anaerobic conditions. The endogenous FocA protein of Escherichia coli is unable to utilize hypophosphorous acid for aerobic growth. Therefore, to achieve the ability to utilize hypophosphorous acid for growth but not phosphate or phosphate for growth under aerobic conditions, it is necessary to introduce a gene consisting of any one of the polynucleotides (4) to (6) to forcibly express the FocA protein. The present inventors have confirmed that Escherichia coli that do not forcibly express the FocA protein (endogenous FocA protein) are unable to utilize hypophosphorous acid for growth (see Example 4 below, "Control" in Figure 6).

[0059] In the transformant of this embodiment, the HtxA protein may be a protein consisting of the protein shown in (7) or (8) below, a protein comprising at least a portion of the protein shown in (7) or (8) below, a protein consisting of a protein encoded by a gene consisting of the polynucleotide shown in (9) or (10) below, or a protein comprising at least a portion of a protein encoded by a gene consisting of the polynucleotide shown in (9) or (10) below: (7) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18; (8) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18 in which one or several amino acids are deleted, substituted, or added, and having hypophosphite dioxygenase activity; (9) A polynucleotide consisting of the base sequence represented by SEQ ID NO: 4; or (10) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having the base sequence represented by SEQ ID NO: 4 and encodes a protein having hypophosphite dioxygenase activity.

[0060] Furthermore, whether a protein does not have phosphate and phosphite transport activity but does have hypophosphite transport activity can be confirmed by introducing a gene encoding a desired protein into an organism lacking the functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein in an expressible manner, and then growing the microorganism in a medium containing various phosphorus sources. If the microorganism does not grow in a medium containing phosphate or a medium containing phosphite, but grows in a medium containing hypophosphite, it can be determined that the protein does not have phosphate and phosphite transport activity but does have hypophosphite transport activity.

[0061] With respect to the polynucleotide of (3) or (6) above, the higher the sequence identity, the more preferable, and may be, for example, 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. However, when the polynucleotide of (3) above has a sequence identity of 90% or more with the polynucleotide represented by SEQ ID NO: 1, at least the sequence (codon) corresponding to alanine (A) at position 52 of the HtxB protein is identical to that of the polynucleotide represented by SEQ ID NO: 1; when the polynucleotide has a sequence identity of 90% or more with the polynucleotide represented by SEQ ID NO: 2, at least the sequence (codon) corresponding to alanine (A) at position 206 of the HtxB protein is identical to that of the polynucleotide represented by SEQ ID NO: 2; and when the polynucleotide has a sequence identity of 90% or more with the polynucleotide represented by SEQ ID NO: 3, at least the sequence (codon) corresponding to asparagine (N) at position 206 of the HtxB protein is identical to that of the polynucleotide represented by SEQ ID NO: 3. The sequence identity of polynucleotides can be calculated, for example, using GENETYX-WIN (trade name, manufactured by Genetyx Co., Ltd.) according to the product manual.

[0062] The transformant of this embodiment may further have a gene encoding a phosphite dehydrogenase protein introduced therein. With this configuration, hypophosphorous acid taken up into the cell can be efficiently converted into phosphate, allowing the transformant of this embodiment to grow more efficiently depending on hypophosphorous acid.

[0063] The gene encoding the phosphite dehydrogenase protein can be a gene derived from Pseudomonas stutzeri WM88 (eg, the PtxD gene).

[0064] More specifically, the phosphite dehydrogenase protein may be a protein consisting of the protein shown in (11) or (12) below, a protein comprising at least a portion of the protein shown in (11) or (12) below, a protein consisting of a protein encoded by a gene consisting of the polynucleotide shown in (13) or (14) below, or a protein comprising at least a portion of a protein encoded by a gene consisting of the polynucleotide shown in (13) or (14) below: (11) A protein consisting of the amino acid sequence represented by SEQ ID NO: 19; (12) A protein consisting of the amino acid sequence represented by SEQ ID NO: 19 in which one or several amino acids are deleted, substituted, or added, and having phosphite dehydrogenase activity; (13) A polynucleotide consisting of the base sequence represented by SEQ ID NO: 5; or (14) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having the base sequence represented by SEQ ID NO: 5 and encodes a protein having phosphite dehydrogenase activity.

[0065] Whether or not a protein has phosphite dehydrogenase activity can be determined by determining whether the protein is NADP + Dependent or NADP + Dependently oxidizes phosphorous acid to HPO4 2- Specifically, the desired protein and HPO3 2- and NAD + or NADP + After mixing, HPO4 2- is produced, the protein can be determined to have phosphite dehydrogenase activity.

[0066] In the transformant of this embodiment, genes encoding PtxD and HtxA may be introduced so that PtxD and HtxA are expressed as a single polypeptide chain PtxD-HtxA by translational fusion. In this case, the single polypeptide chain PtxD-HtxA may be a polypeptide chain PtxD-5xGly-HtxA in which a 5xGly sequence is introduced between PtxD and HtxA for the purpose of increasing protein mobility.

[0067] More specifically, one polypeptide chain PtxD-5xGly-HtxA may be a protein consisting of the protein shown in (15) or (16) below, a protein comprising at least a portion of the protein shown in (15) or (16) below, a protein consisting of a protein encoded by a gene consisting of the polynucleotide shown in (17) or (18) below, or a protein comprising at least a portion of a protein encoded by a gene consisting of the polynucleotide shown in (17) or (18) below: (15) A protein consisting of the amino acid sequence represented by SEQ ID NO: 17; (16) A protein consisting of the amino acid sequence represented by SEQ ID NO: 17 in which one or several amino acids are deleted, substituted, or added, and having phosphite dehydrogenase activity and hypophosphite dioxygenase activity; (17) A polynucleotide consisting of the base sequence represented by SEQ ID NO: 16; or (18) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide consisting of the base sequence represented by SEQ ID NO: 16 and encodes a protein having phosphite dehydrogenase activity and hypophosphite dioxygenase activity.

[0068] As used herein, "an amino acid in which one or several amino acids have been deleted, substituted or added" is not particularly limited to the position at which the deletion, substitution or addition occurs.

[0069] Furthermore, the number of amino acids intended by "one or several amino acids" is not particularly limited, but may be 50 or less, 40 or less, 30 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 amino acid.

[0070] The amino acid substitution is preferably a conservative substitution. Conservative substitution refers to the substitution of a specific amino acid with another amino acid having similar chemical properties and / or structure to the amino acid. Examples of chemical properties include hydrophobicity (hydrophobicity and hydrophilicity) and charge (neutrality, acidity, and basicity). Examples of structures include aromatic rings, aliphatic hydrocarbon groups, and carboxyl groups present in side chains or as functional groups in side chains.

[0071] Examples of conservative substitutions include, for example, substitutions of serine with threonine, lysine with arginine, and phenylalanine with tryptophan amino, although the present invention is not limited to these substitutions.

[0072] As used herein, the term "stringent conditions" refers to conditions under which a double-stranded polynucleotide specific to a base sequence is formed, but a non-specific double-stranded polynucleotide is not formed. In other words, these conditions can be defined as conditions under which highly homologous nucleic acids hybridize with each other, for example, at a temperature 15°C lower, preferably 10°C lower, and more preferably 5°C lower than the melting temperature (Tm value) of a perfectly matched hybrid.

[0073] For example, hybridization can be carried out for 16 to 24 hours in a buffer solution consisting of 0.25 M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1x Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C, followed by two 15-minute washes in a buffer solution consisting of 20 mM Na2HPO4, pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C.

[0074] Another example is a hybridization solution containing 25% formamide, or more stringent conditions, such as 50% formamide, 4x SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, and 20 μg / mL denatured salmon sperm DNA, which is prehybridized overnight at 42°C. The labeled probe is then added and incubated overnight at 42°C for hybridization. Subsequent washes can be performed with a solution and temperature conditions of approximately 1x SSC, 0.1% SDS, and 37°C. More stringent conditions include 0.5x SSC, 0.1% SDS, and 42°C, while even more stringent conditions include 0.2x SSC, 0.1% SDS, and 65°C. Thus, the more stringent the hybridization wash conditions, the more specific the hybridization. However, the above combinations of SSC, SDS, and temperature conditions are merely examples, and a person skilled in the art would be able to achieve similar stringency by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.), as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

[0075] 3. Method for detecting the presence or absence of hypophosphorous acid The method for detecting the presence or absence of hypophosphorous acid according to this embodiment is characterized by having a culture step in which the transformant according to this embodiment is cultured in a medium to be detected, and a detection step in which the presence or absence of growth of the transformant in the culture step is detected.

[0076] In the detection method according to this embodiment, if the transformant grows during the culture process, it can be determined that hypophosphorous acid is contained in the culture medium to be detected, and if the transformant does not grow during the culture process, it can be determined that hypophosphorous acid is not contained in the culture medium to be detected.

[0077] The components and form of the medium to be detected are not particularly limited. For example, the medium to be detected may be in a liquid or solid form.

[0078] When the medium to be detected is liquid, the presence or absence of growth of the transformant in the culture step can be detected, for example, by measuring the turbidity (e.g., OD600) of the medium in the detection step. On the other hand, when the medium to be detected is solid, the presence or absence of growth of the transformant in the culture step can be detected, for example, by checking the presence or absence of colonies of the transformant formed on the solid medium in the detection step.

[0079] The reduced phosphorus compound detected by the detection method according to this embodiment is hypophosphorous acid, which means that the detection method according to this embodiment can specifically detect hypophosphorous acid. [Example]

[0080] An embodiment of the present invention will now be described.

[0081] (Examples relating to HtxBCDE proteins) <Culture medium used> LB (yeast extract-tryptone) medium was used as the natural medium, and MOPS synthetic medium was used as the minimal medium.

[0082] LB-G3Pi: LB medium supplemented with glycerol 3-phosphate (G3Pi) to a final concentration of 0.5 mM. MOPS-P0: MOPS synthetic medium without phosphorus source; MOPS-Pi: MOPS-0 medium supplemented with a stock solution of phosphoric acid (Pi) to a final concentration of 1.0 mM. MOPS-Pt: MOPS-0 medium supplemented with a stock solution of phosphorous acid (Pt) to a final concentration of 1.0 mM. MOPS-HPt: MOPS-0 medium supplemented with stock solution of hypophosphorous acid (HPt) to a final concentration of 1.0 mM MOPS-G3Pi: MOPS-0 medium supplemented with a stock solution of glycerol 3-phosphate (G3Pi) to a final concentration of 1.0 mM.

[0083] <Parent strain (E. coli strain) used> MT2012: E. coli contains four phosphate transporter proteins: PitA, PitB, PstSCAB, and PhnCDE. MT2012 is a wild-type strain, MG1655, in which the genes encoding these four phosphate transporter proteins and the gene encoding the phoA protein have been disrupted (ΔpitA, ΔpitB, ΔphnC, ΔpstSCABphoU, ΔphoA) (Motomura, K. et al., "Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation." FEMS microbiology letters 320, 25-32, 2011.).

[0084] RN1006: RN1006 is a strain in which seven genes, including those encoding phosphate transporter proteins (pitA, pitB, and phnC), those encoding phosphate ester transporter proteins (glpT, ugpB, and uhpT), and the gene encoding alkaline phosphatase protein (phoA), have been deleted from the wild-type strain MG1655.

[0085] RN1008: RN1008 is a strain in which (i) the genes encoding phosphite and hypophosphite transporter proteins (htxABCDE) and the gene encoding phosphite dehydrogenase protein (ptxD) were introduced into RN1006, and (ii) pstSCAB, a gene encoding a phosphate transporter protein, was further deleted.

[0086] <Plasmid construction> The primers used for constructing the plasmids are shown in Table 1.

[0087] [Table 1]

[0088] ptxD-htxA / pTWV229 Linearized DNA was obtained by inverse PCR using primers Ptac4071-2_fw and ptxD dTAG-5G_rv with the PtxD expression plasmid Ptac4071-ptxD / pTWV229 (Hirota et al. A novel biocontainment strategy makes bacterial growth and survival dependent on phosphite. Sci. Rep. 7: 44748). Next, PCR was performed using primers 5G-htxA_fw and htxA_rv with the htxBCDE / pSTV28 (Motomura, K. et al. Synthetic phosphorus metabolic pathway for biosafety and contamination management of cyanobacterial cultivation. ACS Synth. Biol. 7, 9, 2189-2198, 2018) as a template to obtain an amplified htxA DNA fragment. The linearized DNA of the amplified Ptac4071-ptxD / pTWV229 was then ligated to the amplified htxA DNA fragment using the In Fusion Cloning Kit (Takara Bio) to obtain ptxD-htxA / pTWV229. In ptxD-htxA / pTWV229, the PtxD and HtxA translation regions (SEQ ID NO: 16) were designed to express PtxD and HtxA as a single polypeptide chain through translational fusion, with a 5xGly sequence inserted between them to enhance protein mobility. The amino acid sequence of the protein translated from the PtxD and HtxA translation regions is shown in SEQ ID NO: 17.

[0089] htxBCDE(HtxB W52A) / pSTV28 htxBCDE(HtxB W52A) / pSTV28 is a plasmid that expresses the HtxBCDE protein, which is an HtxB protein with a W52A substitution mutation introduced. This plasmid was constructed as follows.

[0090] A PCR reaction solution was prepared by mixing PrimeSTAR Max Premix, template (htxBCDE / pSTV28), primer pair HtxB_W52A_fw and HtxB_W52A_rv, and sterilized water in the composition shown in Table 2. The reaction was carried out under the conditions shown in Table 3. The resulting PCR product was transformed into the cloning host DH5α, and the plasmid was extracted from the resulting transformant to obtain htxBCDE(HtxB W52A) / pSTV28.

[0091] [Table 2]

[0092] [Table 3]

[0093] htxBCDE(HtxB D206A) / pSTV28 htxBCDE(HtxB D206A) / pSTV28 is a plasmid that expresses the HtxBCDE protein into which a substitution mutation of D206A has been introduced in the HtxB protein.

[0094] This plasmid was obtained in the same manner as htxBCDE(HtxB W52A) / pSTV28, except that HtxB_D206A_fw and HtxB_D206A_rv were used as the primer pair.

[0095] htxBCDE(HtxB D206N) / pSTV28 htxBCDE(HtxB D206N) / pSTV28 is a plasmid that expresses the HtxBCDE protein into which the D206N substitution mutation has been introduced in the HtxB protein.

[0096] This plasmid was obtained in the same manner as htxBCDE(HtxB W52A) / pSTV28, except that HtxB_D206N_fw and HtxB_D206N_rv were used as the primer pair.

[0097] htxBCDE(HtxB W52A,D206A) / pSTV28 htxBCDE(HtxB W52A, D206A) / pSTV28 is a plasmid that expresses the HtxBCDE protein into which substitution mutations W52A and D206A have been introduced into the HtxB protein.

[0098] This plasmid was obtained in the same manner as htxBCDE(HtxB W52A) / pSTV28, except that the template (htxBCDE(HtxB W52A) / pSTV28) and the primer pair HtxB_D206A_fw and HtxB_D206A_rv were used.

[0099] htxBCDE(HtxB W52A,D206N) / pSTV28 htxBCDE(HtxB W52A, D206N) / pSTV28 is a plasmid that expresses the HtxBCDE protein into which substitution mutations W52A and D206N have been introduced into the HtxB protein.

[0100] This plasmid was obtained in the same manner as htxBCDE(HtxB W52A,D206A) / pSTV28, except that HtxB_D206N_fw and HtxB_D206N_rv were used as a primer pair.

[0101] Example 1: Specificity test of mutant HtxBCD transporters First, ptxD-htxA / pTWV229 was introduced into the inorganic phosphate transporter specificity test strain MT2012 by the calcium chloride method to obtain a transformant. Next, each of the following plasmids (a) to (f) was introduced into the transformant to prepare six test strains.

[0102] (a) htxBCDE / pSTV28, (b) htxBCDE(HtxB W52A) / pSTV28, (c) htxBCDE(HtxB D206A) / pSTV28, (d) htxBCDE(HtxB D206N) / pSTV28, (e) htxBCDE(HtxB W52A,D206A) / pSTV28, (f)htxBCDE(HtxB W52A,D206N) / pSTV28.

[0103] The six test strains were used to examine the uptake ability of the HtxBCD transporter by spot assay. Specifically, the following procedure was performed for each of the six test strains. First, the test strain was inoculated into 5 mL of LB-G3Pi and pre-cultured in a constant temperature shaking incubator. Next, 1 mL of the pre-culture solution was sampled and washed twice with the same amount of MOPS-0, and the OD600 was measured using a spectrophotometer. After diluting with MOPS-0 to an OD600 of 0.1, the OD was measured at 1.0 x 10 -6 The culture was diluted 10-fold from 0.01 to 0.01. Finally, 10 μL of each dilution was spotted onto MOPS-Pi, MOPS-Pt, MOPS-HPt, MOPS-G3Pi, and MOPS-P0 agar plates and cultured in an incubator at 37°C. The same procedure was also performed on strain RH781, which expresses PstSCAB instead of HtxBCDE as a transport protein control.

[0104] The results are shown in Figure 4. In Figure 4, "HtxBCDE" is the test strain into which the plasmid (a) was introduced. "HtxBCDE (W52A)" is the test strain into which the plasmid (b) was introduced. "HtxBCDE (D206A)" is the test strain into which the plasmid (c) was introduced. "HtxBCDE (D206N)" is the test strain into which the plasmid (d) was introduced. "HtxB*CDE (W52A, D206A)" is the test strain into which the plasmid (e) was introduced. "HtxB*CDE (W52A, D206N)" is the test strain into which the plasmid (f) was introduced. "PstSCAB" is RH781.

[0105] As shown in Figure 4, HtxBCDE(W52A), HtxBCDE(D206A), and HtxBCDE(D206N) could not grow on MOPS-Pi or MOPS-Pt but could grow on MOPS-HPt. These test strains were unable to utilize phosphate and phosphite for growth, but were able to utilize hypophosphate for growth. Thus, HtxBCDE proteins (single mutants) containing the W52A, D206A, or D206N substitution mutations in the HtxB protein were found to be hypophosphate-specific transporter proteins. Furthermore, the growth patterns of the spots suggested that the HtxBCDE proteins containing the W52A or D206A substitution mutations were particularly efficient at uptake of hypophosphate.

[0106] Furthermore, HtxB*CDE(W52A,D206A) and HtxB*CDE(W52A,D206N) did not grow in MOPS-Pi, MOPS-Pt, or MOPS-HPt media. HtxBCDE proteins (double mutants) containing substitutions of W52A and D206A or W52A and D206N in the HtxB protein completely lost their transport function and could not uptake either hypophosphite or phosphite.

[0107] Example 2: Preparation of hypophosphorous acid-containing strains and growth measurement RN1006 was used as the parent strain. First, RN1006 was co-transformed with ptxD-htxA / pTWV229 and either htxBCDE(W52A) / pSTV28 or htxBCDE(D206A) / pSTV28 by electroporation, and selected on LB agar medium containing the appropriate antibiotic. Next, the pstSCAB-phoU gene was disrupted with pst,pho::Km P1 λ phage. Of the resulting transformants, the one containing htxBCDE(W52A) / pSTV28 was designated AM101, and the one containing htxBCDE(D206A) / pSTV28 was designated AM102.

[0108] The growth of hypophosphite-encapsulated strains AM101 and AM102 was examined by measuring the turbidity of the liquid culture medium. First, AM101 and AM102 were precultured in MOPS-HPt medium in the same manner as described in Example 1 (Specificity Test of Mutant HtxBCD Transporters). Then, 1 mL of the AM101 and AM102 preculture was sampled and washed three times with the same volume of MOPS-0. OD600 was measured using a spectrophotometer and diluted with MOPS-0 to an OD600 of 1. Then, 1% (50 μL) of the cells were inoculated into 5 mL of MOPS-HPt liquid medium and placed in a thermostatic shaking incubator. Shaking culture was performed at 160 rpm and 37°C. The turbidity during shaking culture was automatically measured using a turbidimeter to examine growth. The growth of the phosphorous acid- and hypophosphite-encapsulated strain RN1008 was also examined using the same procedure.

[0109] 501 and 502 in Figure 5 show the results of measuring the growth of hypophosphorous acid-encapsulated strains. As shown in 501 and 502 in Figure 5, when comparing the growth of AM101 and AM102 with that of RN1008 in MOPS-HPt, it was found that AM101 and AM102 had a longer lag phase than RN1008. Furthermore, it was found that the final OD600 values ​​were similar among RN1008, AM101, and AM102.

[0110] Example 3: Escape assay of hypophosphite-encapsulated strains The hypophosphate-encapsulated strains AM101 and AM102 were evaluated by escape assay for the possibility of the emergence of mutants that escape hypophosphate encapsulation. AM101 and AM102 were precultured in MOPS-HPt medium as described in Example 1 (Specificity Test of Mutant HtxBCD Transporters). Then, 1 mL of the AM101 and AM102 preculture was sampled and inoculated into 200 mL of MOPS-HPt (permissive medium). The culture was placed in a constant-temperature shaker and cultured at 160 rpm and 37°C for 24 hours. The culture was washed three times with 30 mL of sterile water and then suspended in 1 mL of sterile water. The bacterial cell suspension was plated on a square dish containing LB agar medium (nonpermissive medium) and cultured at 37°C for 21 days, and the appearance of colonies was observed. To determine the total number of bacteria used in this assay, 100 μL of the 24-hour culture was diluted, spread on MOPS-HPt agar medium, and cultured in an incubator at 37°C. The number of colonies formed (CFU) was counted, and the total number of bacteria in the preculture was calculated from this value.

[0111] As a result, AM101 and AM102 were cultured in non-permissive medium for 21 days, but no colonies were observed for either strain. The detection limit in this experimental system was calculated using the following formula. [Detection limit (escapees / CFU)] = 1 / ([Number of colonies formed (CFU)] x [Dilution factor (-)] ÷ [Volume of culture solution spread (mL)] x [Volume of culture solution used (mL)]) The detection limit in this experiment was 1.5 × 10 -11 Below (AM101), 3.3×10 -11 These values ​​are below the detection limit (1.0 × 10) set by the NIH (National Institute of Health) that must be met for the establishment of biological containment technology. -8 It was found that the following criteria were fully met (Torres, et al. Essays Biochem. 2016).

[0112] (Examples relating to FocA protein) <Isolation and cloning of the FocA gene from E. coli> The primers used are as follows: Primer A: 5'-CCTAGGAGCATCACCGTGAAAGCTGACAACCCTTTTG-3' (SEQ ID NO: 28); Primer B: 5'-CAGGTCGACTCTAGATTAATGGTGGTCGTTTTCACG-3' (SEQ ID NO: 29); Primer C: 5′-TCTAGAGTCGACCTGCAGGC-3′ (SEQ ID NO: 30); Primer D: 5'-GGTGATGCTCCTAGGATCCCCG-3' (SEQ ID NO: 31).

[0113] The FocA gene was amplified by PCR using the genomic DNA of the E. coli MG1655 strain as a template with primers A and B. The resulting FocA gene was cloned into linearized E. coli vector pSTV28 by PCR using primers C and D to obtain the FocA expression plasmid FocA / pSTV28.

[0114] <Isolation and cloning of the FocA homologue gene from the marine cyanobacterium Synechococcus sp. PCC 7002> In addition to Primer C and Primer D, the primers used were as follows: Primer E: 5'-CCTAGGAGCATCACCATGGACTACGTAATCCCCAAAG-3' (SEQ ID NO: 32); Primer F: 5'-CAGGTCGACTCTAGATTACCGCAACACAGGATTTTTC-3' (SEQ ID NO: 33).

[0115] Using the amino acid sequence of the FocA protein derived from Escherichia coli strain MG1655 as a query, we used the BLAST program to search for bacterial species containing FocA protein homologs. As a result, we found a gene (referred to as the 7002-FocA gene) encoding a protein showing approximately 28% amino acid identity to the genome of the marine cyanobacterium Synechococcus sp. PCC 7002. The 7002-FocA gene was PCR-amplified using primers E and F and the genomic DNA of PCC 7002 strain as a template. The resulting 7002-FocA gene was cloned into the linearized E. coli vector pSTV28 by PCR using primers C and D to obtain the 7002-FocA expression plasmid 7002FocA / pSTV28.

[0116] Example 4: Evaluation of specificity of FocA protein for inorganic phosphorus compounds First, ptxD-htxA / pTWV229 was introduced into the inorganic phosphate transporter specificity test strain MT2012 by the calcium chloride method to obtain a transformant. Next, the pSTV28, htxBCDE / pSTV28, and FocA / pSTV28 plasmids were introduced into the transformant to generate three test strains.

[0117] The following procedure was performed on each of the three test strains. First, the test strain was inoculated into LB medium and cultured overnight at 37°C, after which the cells were washed with MOPS-P0. The washed cell suspension was inoculated into MOPS-Pi, MOPS-Pt, MOPS-HPt, and MOPS-G3Pi, respectively, and cultured at 37°C for 8 days. The substrate specificity of the FocA protein was evaluated based on growth in each medium. In the above procedure, 50 μg / mL carbenicillin and 30 μg / mL chloramphenicol were added to all media, including the preculture, to prevent loss of the plasmid.

[0118] The results are shown in Figure 6. In Figure 6, "Control" is a test strain into which pSTV28 was introduced. "HtxBCDE" is a test strain into which htxBCDE / pSTV28 was introduced. "FocA" is a test strain into which FocA / pSTV28 was introduced.

[0119] As shown in Figure 6, the test strain into which FocA / pSTV28 was introduced, i.e., the FocA protein-expressing strain, did not grow in a medium containing only phosphate as the sole phosphorus source (MOPS-Pi) or a medium containing only phosphorous acid as the sole phosphorus source (MOPS-Pt), but grew only in a medium containing only hypophosphorous acid as the sole phosphorus source (MOPS-HPt). This indicates that the FocA protein is a hypophosphorous acid-specific transporter protein that specifically takes up hypophosphorous acid.

[0120] Example 5: Construction of hypophosphite-dependent strains using FocA protein Adoption of hypophosphate dependence in the host organism requires (i) disruption of all endogenous phosphate transporters, (ii) expression of the hypophosphate oxidase HtxA and phosphite oxidase PtxD proteins, and (iii) expression of a hypophosphate-specific transporter.

[0121] Therefore, first, E. coli strain A' in which all endogenous phosphate transporter genes on the chromosome were disrupted was prepared using RN1006 according to the following procedure.

[0122] E. coli strain A': We generated E. coli strain A' by disrupting all endogenous phosphate transporter genes on the chromosome of RN1006 using genome editing to delete pstSCAB-phoU without inserting a selectable marker. Prior to genome editing, a plasmid (pSIMcpf1-pitA) containing a phosphate transporter (pitA) gene was introduced into RN1006 to support phosphorus uptake when all phosphate transporter genes on the chromosome were disrupted. pSIMcpf1-pitA is a temperature-sensitive plasmid. Therefore, pSIMcpf1-pitA replicates when E. coli strain A' is cultured at 30°C, but does not replicate when E. coli strain A' is cultured at 37°C.

[0123] Transformant A was generated by introducing ptxD-htxA / pTWV229 and FocA / pSTV28 into E. coli strain A', which had all of the endogenous phosphate transporter genes on the chromosome disrupted as described above. Transformant A was then cultured at 37°C, and the temperature-sensitive plasmid (pSIMcpf1-pitA) was removed from transformant A to generate a hypophosphite-dependent E. coli strain. The resulting hypophosphite-dependent E. coli strain was designated NM1012.

[0124] NM1012 was inoculated into MOPS-Pi, MOPS-Pt, MOPS-HPt, and LB media and cultured at 37°C for 7 days to confirm its hypophosphate dependence. The results are shown in Figure 7. As shown in Figure 7, it was confirmed that the hypophosphate-dependent E. coli strain NM1012 grows only in media containing hypophosphate.

[0125] Example 6: Evaluation of the containment effect of hypophosphite-dependent strains using FocA protein The hypophosphite-dependent E. coli strain NM1012 was inoculated into MOPS-glucose synthetic medium (containing 0.5 mM IPTG, 50 μg / mL carbenicillin, and 30 μg / mL chloramphenicol) containing hypophosphite (final concentration 5.0 mM) and cultured overnight at 37°C. The cells were then inoculated at 1% into 100 mL of medium of the same composition. After 24 hours of culture at 37°C, the cells were washed by centrifugation and resuspension in sterile water. The washed cells were then suspended in 2 mL of sterile water. 1 mL of this suspension was spread onto LB agar plates (Nunc™ Square BioAssay Dishes, 245 mm x 245 mm x 25 mm, Thermo Fisher Scientific) containing 50 μg / mL carbenicillin and 30 μg / mL chloramphenicol. The plates were then cultured at 37°C for 3 weeks and observed for the presence of colonies.

[0126] To measure the total bacterial count, 100 μL was collected from the culture medium cultured at 37°C for 24 hours, diluted 106-fold with sterile water, and then spread onto three MOPS-glucose synthetic agar plates (containing 0.5 mM IPTG, 50 μg / mL carbenicillin, and 30 μg / mL chloramphenicol) containing hypophosphorous acid (final concentration 5.0 mM) and cultured at 37°C for three days. The total bacterial count used to evaluate the containment effect was calculated from the average number of colonies obtained on the three agar plates.

[0127] As a result, NM1012 was cultured at 37°C for 3 weeks, but no colonies of NM1012 mutants capable of utilizing phosphorus sources other than hypophosphorous acid were observed. From the results of two experiments, the frequency of emergence of escape mutants that escaped from containment was 6.6 × 10 -12 / CFU was calculated.

[0128] Example 7: Evaluation of the specificity of the FocA protein homologue of the marine cyanobacterium Synechococcus sp. PCC 7002 for inorganic phosphorus compounds First, ptxD-htxA / pTWV229 and 7002-FocA / pSTV28 were introduced into the inorganic phosphate transporter specificity test strain MT2012 to prepare a test strain.

[0129] The prepared test strain was inoculated into LB medium and cultured overnight at 37°C, after which the cells were washed with MOPS-P0. The washed cell suspension was inoculated into MOPS-Pi, MOPS-Pt, MOPS-HPt, and MOPS-G3Pi, respectively, and cultured at 37°C for 8 days. The substrate specificity of the 7002-FocA protein was evaluated based on growth in each medium. To prevent loss of the plasmid, 50 μg / mL carbenicillin and 30 μg / mL chloramphenicol were added to all media, including the preculture, during the above procedure.

[0130] The results are shown in Figure 8. As shown in Figure 8, the test strain into which 7002-FocA / pSTV28 was introduced, i.e., the 7002-FocA protein-expressing strain, did not grow in a medium containing phosphate as the sole phosphorus source (MOPS-Pi) or a medium containing phosphorous acid as the sole phosphorus source (MOPS-Pt), but grew only in a medium containing hypophosphorous acid as the sole phosphorus source (MOPS-HPt). This indicates that the 7002-FocA protein is a hypophosphate-specific transporter protein that specifically takes up hypophosphate.

[0131] Example 8: Verification of the growth advantage of hypophosphate-dependent strains in hypophosphate medium Hypophosphorous acid is a phosphorus source that cannot be utilized by general microorganisms. Therefore, using hypophosphorous acid as the phosphorus source in the medium is expected to prevent contamination by external microorganisms and allow only microorganisms capable of utilizing hypophosphorous acid to grow preferentially. Therefore, we evaluated the growth advantage of hypophosphorous acid-dependent Escherichia coli strains in hypophosphorous acid-containing medium.

[0132] The hypophosphite-confined strain AM101 was mixed with its competitor, Pseudomonas putida MY11-41 (hereafter simply referred to as MY11-41), and competitive culture was performed. Prior to competitive culture, AM101 was pre-cultured in MOPS-HPt medium containing appropriate antibiotics, and MY11-41 was pre-cultured in MOPS-Pi medium. MY11-41 can utilize phosphate for growth, but cannot utilize phosphorous acid or hypophosphite for growth.

[0133] MY11-41 is a Pseudomonas putida MY11 (wild-type) strain that was treated with the mutagen NTG (N-methyl-N'-nitro-N-nitrosoguanidine) to accumulate high amounts of polyphosphate, but it does not have the ability to utilize phosphite or hypophosphite (Reference: Morohoshi, T., Yamashita, T., Kato, J., Ikeda, T., Takiguchi, N., Ohtake, H., and Kuroda, A.: A method for screening polyphosphate-accumulating mutants which remove phosphate efficiently from synthetic wastewater, J. Biosci. Bioeng., 95, 637-640 (2003)).

[0134] After washing the preculture solutions of AM101 and MY11-41 three times with sterile water, both AM101 and MY11-41 were inoculated into 50 mL of MOPS-glucose medium containing 0.4 mM phosphate as a phosphorus source (hereinafter referred to as phosphate medium) or 50 mL of MOPS-glucose medium containing 0.4 mM hypophosphorous acid as a phosphorus source (hereinafter referred to as hypophosphorous acid medium). The inoculation amount of AM101 and MY11-41 was 10 mL per medium. 6 cfu / mL, 10 4The target addition was a concentration of 100:1 cfu / mL. The exact number of cells in the phosphate medium was determined by plating AM101 and MY11-41 on agar media containing the same components as those used in the pre-culture.

[0135] After the inoculation, the phosphate medium and hypophosphate medium were cultured at 37°C and 160 rpm for 48 hours with shaking. After the culture, 160 or 560 μL samples were collected over time from the phosphate medium culture medium and the hypophosphate medium culture medium, respectively. The collected samples were then spread on agar media containing the same components as those used in the pre-culture and cultured in an incubator at 37°C. The number of colonies formed (CFU) was counted, and the number of AM101 and MY11-41 present in the phosphate medium culture medium and the hypophosphate medium culture medium at each collection time was measured. The abundance ratios of AM101 and MY11-41 present in the phosphate medium culture medium and the hypophosphate medium culture medium were calculated from the measured number of AM101 and MY11-41 present.

[0136] The results are shown in Figure 9. The initial cell count of the inoculated cells was 1.65 x 10 for AM101 and MY11-41 in the phosphate medium. 6 cfu / mL, 2.70 × 10 4 cfu / mL for AM101 and MY11-41, respectively. 6 cfu / mL, 3.95 × 10 4 cfu / mL.

[0137] The number of bacteria 24 hours after the start of cultivation was 1.48 × 10 for AM101 in the case of phosphate medium. 6 cfu / mL, MY11-41 was 6.27 × 10 8 cfu / mL were present. In other words, AM101 could not grow on phosphate medium, and MY11-41 grew predominantly.

[0138] In the case of hypophosphite medium, AM101 is 7.41 x 10 8 cfu / mL, MY11-41 strain was 8.00 × 10 4cfu / mL. In other words, MY11-41 could not grow on hypophosphite medium, and only AM101 grew predominantly. As a result, the dominance rate of AM101 reached 99.99% after 24 hours and remained so until the end of the culture. [Industrial Applicability]

[0139] The present invention can be widely used in fields requiring transformants (for example, fields for producing oral vaccines and fields aimed at improving the natural environment).

Claims

1. A transformant that is unable to utilize phosphate and phosphorous for growth but can utilize hypophosphorous acid for growth because it lacks the functions of a gene encoding a phosphate transporter protein and a gene encoding a phosphate ester transporter protein and has a gene encoding a hypophosphorous acid-specific transporter protein introduced into it.

2. A gene encoding the HtxA protein has been introduced, The transformant according to claim 1, wherein the hypophosphite-specific transporter protein is an HtxBCDE protein, and the HtxB protein of the HtxBCDE protein is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (1) to (3): (1) a polynucleotide consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 3; (2) A polynucleotide that hybridizes under stringent conditions with DNA having a base sequence complementary to a polynucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity; or (3) A polynucleotide that has a sequence identity of 90% or more with a polynucleotide consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 3, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity.

3. A gene encoding the HtxA protein has been introduced, The transformant according to claim 1, wherein the hypophosphite-specific transporter protein is a FocA protein, which is a mutant protein encoded by a gene consisting of any one of the following polynucleotides (4) to (6): (4) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 24 or 25; (5) A polynucleotide that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to a polynucleotide consisting of the base sequence represented by SEQ ID NO: 24 or 25, and encodes a protein that does not have phosphate and phosphate transport activity but has hypophosphate transport activity; or (6) A polynucleotide that has a sequence identity of 90% or more to a polynucleotide consisting of the base sequence represented by SEQ ID NO: 24 or 25, and encodes a protein that does not have phosphate or phosphite transport activity but has hypophosphite transport activity.

4. 4. The transformant according to claim 1, further comprising a gene encoding a phosphite dehydrogenase protein introduced thereinto.

5. The transformant according to any one of claims 1 to 3, wherein the transformant is a transformant of a prokaryote.

6. The transformant according to any one of claims 1 to 3, which has a growth advantage in a medium containing hypophosphorous acid as a phosphorus source over a competing strain that can utilize phosphate for growth and cannot utilize hypophosphorous acid for growth.

7. a culturing step of culturing the transformant according to any one of claims 1 to 3 in a medium to be detected; a detection step of detecting whether or not the transformant has grown in the culture step.

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