JP2023158948A - Method for producing ppGpp-enriched organisms and organisms - Google Patents

Abstract

[Problem] To provide a ppGpp-enriched organism. [Solution] A method for producing a ppGpp-enriched organism includes a ppGpp synthase gene that includes a region encoding a ppGpp synthesis domain but does not include a region encoding a ppGpp degradation domain, and an inducer that controls transcription of the ppGpp synthase gene. An expression unit comprising a sex promoter and a region encoding a chloroplast transit peptide adjacent to the ppGpp synthetase gene is used to control an organism having chloroplasts using an inducer that activates the inducible promoter. Including nurturing in the presence of. [Selection diagram] Figure 1

Description

The present invention relates to a method for producing a ppGpp-enriched organism and an organism.

ppGpp (guanosine tetraphosphate) shown below is a nucleotide discovered as a signal substance during the nutrient starvation response of bacteria.

ppGpp and pppGpp (guanosine pentaphosphate) are synthesized and decomposed as shown in the following reaction formula (1). This reaction is catalyzed by ppGpp synthetase. For example, Non-Patent Document 1 describes Rel as a ppGpp synthase possessed by Bacillus subtilis.

Plants also have ppGpp synthase. For example, Non-Patent Document 2 describes that Arabidopsis thaliana has four types of ppGpp synthases.

Furthermore, when the amount of ppGpp increases or decreases within the chloroplasts of plants, the plant's resistance to nutrient starvation and the total weight of the plant change. As a method for increasing the amount of ppGpp in chloroplasts, there is a method of highly expressing a gene encoding ppGpp synthase RSH (RelA-SpoT Homolog).

For example, Non-Patent Document 3 describes a method for highly expressing RSH2 or RSH3 among the four types of ppGpp synthases that Arabidopsis has.

Patrick Pausch, Maha Abdelshahid, Wieland Steinchen, Heinrich Schafer, Fabio Lino Gratani, Sven-Andreas Freibert, Christiane Wolz, Kursad Turgay, Daniel N.Wilson, GertBange. Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel. Cell Reports, Volume 32, Issue 11 (2020) 108157. Sumire Ono, Sae Suzuki, Doshun Ito, Shota Tagawa, Takashi Shiina, Shinji Masuda. Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis. Plant Cell Physiol (2021 ) 61(12):2077-2086. Maekawa, M., Honoki, R., Ihara, Y. et al. Impact of the plastidial stringent response in plant growth and stress responses. Nature Plants 1, 15167 (2015).

The present invention aims to provide ppGpp-enriched organisms.

According to the first aspect of the present invention, a ppGpp synthetase gene that includes a region encoding a ppGpp synthesis domain and does not include a region encoding a ppGpp degradation domain; and an inducible promoter that controls transcription of the ppGpp synthase gene; An organism comprising an expression unit containing the ppGpp synthase gene and an adjacent region encoding a chloroplast transit peptide and having chloroplasts is grown in the presence of an inducer that activates the inducible promoter. A method of producing a ppGpp-enriched organism is provided.

According to a second aspect of the present invention, a ppGpp synthetase gene that includes a region encoding a ppGpp synthesis domain and does not include a region encoding a ppGpp degradation domain; and an inducible promoter that controls transcription of the ppGpp synthase gene; There is provided an organism having chloroplasts, comprising an expression unit comprising the ppGpp synthase gene and an adjacent region encoding a chloroplast transit peptide.

According to the present invention, ppGpp-enriched organisms are provided.

FIG. 1 is a schematic diagram showing an example of an expression unit. FIG. 2 is a schematic diagram showing domains conserved in RSH of Arabidopsis. FIG. 3 is an image showing the results of Western blotting before and after estrogen induction. FIG. 4 is a graph showing the relationship between estrogen concentration and ppGpp concentration on the second day after estrogen induction. FIG. 5 is a graph showing the relationship between estrogen concentration and ppGpp concentration on day 28 after estrogen induction.

Embodiments of the present invention will be described below. The embodiments described below are more specific implementations of any of the above aspects. The matters described below can be incorporated into each of the above aspects alone or in combination.

<Method for producing ppGpp-enriched organisms>
Hereinafter, a method for producing a ppGpp-enriched organism according to an embodiment of the present invention will be described.
First, an organism into which the expression unit has been introduced is prepared. The expression unit-introduced organism is an organism that is equipped with an expression unit and has chloroplasts. The expression unit-introduced organism can be obtained, for example, by introducing the expression unit into an organism having chloroplasts. The organism containing chloroplasts is, for example, a plant cell, a plant tissue, a plant organ, a plant individual, or a combination thereof. The plant is, for example, Arabidopsis. For example, existing gene introduction methods such as the Agrobacterium method can be used to introduce the expression unit.

FIG. 1 is a schematic diagram showing an example of an expression unit.
Expression unit 1 is a unit for expressing ppGpp synthase. As shown in FIG. 1, the expression unit 1 includes a ppGpp synthase gene 10, an inducible promoter 11, a region 12 encoding a chloroplast transit peptide, and a region 13 encoding a labeled protein.

The ppGpp synthase gene 10 is a gene encoding ppGpp synthase. The ppGpp synthase gene 10 includes a region encoding a ppGpp synthesis domain (hereinafter referred to as a first region) and does not include a region encoding a ppGpp degrading domain (hereinafter referred to as a second region). The second region is, for example, a region encoding a ppGpp hydrolysis domain.

Preferably, the ppGpp synthase gene 10 does not have a region encoding a domain that controls the activity of the ppGpp synthesis domain. The domain that controls the activity of the ppGpp synthesis domain is, for example, the TGS (ThrRS, GTPase, SpoT) control domain or the EF hand domain (EFh). In this case, ppGpp is likely to be efficiently accumulated within the organism.

Preferably, the ppGpp synthase gene 10 has a yjbM gene consisting of the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity with the yjbM gene consisting of the nucleotide sequence represented by SEQ ID NO: 1. It is a yjbM-like gene that contains the ppGpp synthesis domain coding region and does not contain the ppGpp degradation domain coding region.

The yjbM gene is a gene encoding the YjbM protein, which is a ppGpp synthase possessed by Bacillus subtilis. The yjbM gene has a first region but does not have a second region. Furthermore, the yjbM gene does not have a region encoding a domain that controls the activity of the ppGpp synthesis domain. Therefore, when a yjbM gene or a yjbM-like gene is used as the ppGpp synthase gene 10, ppGpp can be easily accumulated efficiently in an organism.

The table below shows the base sequence of the yjbM gene. In the base sequence shown below, the region from 45th to 164th is the first region.

The inducible promoter 11 is a promoter that controls transcription of the ppGpp synthase gene 10. Inducible promoter 11 is activated by an inducing substance.

Inducible promoter 11 is, for example, an estrogen-inducible promoter.

When an estrogen-inducible promoter is used as the inducible promoter 11, estrogen can be used as the inducer.

The region 12 (hereinafter referred to as the third region) encoding the chloroplast transit peptide is adjacent to the ppGpp synthase gene 10. Specifically, the third region is adjacent to the ppGpp synthase gene 10 such that the chloroplast transit peptide and the ppGpp synthase encoded by the ppGpp synthase gene 10 form a fusion protein. For example, the third region is adjacent to the ppGpp synthase gene 10 on the 5' end side of the ppGpp synthase gene 10.

The region 13 encoding a labeled protein (hereinafter referred to as the fourth region) encodes a labeled protein that labels the ppGpp synthase encoded by the ppGpp synthase gene 10. The fourth region is adjacent to the ppGpp synthase gene 10. Specifically, the fourth region is adjacent to the ppGpp synthase gene 10 such that the labeled protein and the ppGpp synthase encoded by the ppGpp synthase gene 10 form a fusion protein. The labeled protein is, for example, a FLAG (registered trademark) tag.

Next, the expression unit-introduced organism is grown in the presence of an inducer. According to this cultivation, first, a first fusion protein containing a chloroplast transit peptide encoded by the third region, a ppGpp synthetase encoded by the ppGpp synthase gene 10, and a labeled protein encoded by the fourth region is produced. Expression is induced. The first fusion protein then translocates to the chloroplast. Next, the chloroplast transit peptide possessed by the first fusion protein is cleaved. This produces a second fusion protein containing the ppGpp synthase and the labeled protein. Next, the second fusion protein catalyzes the ppGpp synthesis reaction. This reaction causes ppGpp to accumulate in chloroplasts. In this way, ppGpp-enriched organisms are obtained.

Cultivation is performed using, for example, a liquid medium or soil.

When using a liquid medium, the expression unit-introduced organism can be grown in the presence of the inducer by bringing the expression unit-introduced organism into contact with an inducer-containing liquid medium containing the inducer and the liquid medium.

The concentration of the inducer in the inducer-containing liquid medium is preferably within the range of 0.01 to 10 μM.

When soil is used, the expression unit-introduced organism can be grown in the presence of the inducing substance by supplying an inducing substance-containing liquid containing the inducing substance and liquid to the expression unit-introduced organism. The liquid is, for example, water.

Preferably, the concentration of the inducer in the inducer-containing liquid is within the range of 0.01 to 10 μM.

Preferably, the expression unit-introduced organism is grown in the presence of an inducer under an oligotrophic environment.

More preferably, the expression unit-introduced organism is grown in the presence of the inducer in an environment that is poor in phosphate or nitrogen sources. As described in the above-mentioned Non-Patent Document 3, Arabidopsis thaliana in which a large amount of ppGpp has been accumulated by overexpressing RSH3, a ppGPp synthase possessed by Arabidopsis, can be cultivated in an environment lacking in nitrogen sources. Can keep green leaves. Furthermore, Arabidopsis thaliana that has accumulated a large amount of ppGpp can maintain a low amount of anthocyanin even when cultured in an environment lacking in phosphate sources. Thus, ppGpp-enriched organisms have tolerance to oligotrophic environments. Therefore, when grown in an oligotrophic environment, ppGpp-enriched organisms have a superior appearance compared to organisms that accumulate a small amount of ppGpp.

The method for producing ppGpp-enriched organisms has been described above.

In addition, in the method described above, the fourth region is provided on the 3' end side of the ppGpp synthetase gene 10, but the fourth region may be provided between the third region and the ppGpp synthase gene 10. good. Further, another region may be interposed between the fourth region and the ppGpp synthase gene 10. Further, the fourth region may be omitted.

Further, another region may be interposed between the third region and the ppGpp synthase gene 10.

Furthermore, before growing the expression unit-introduced organism in the presence of the inducer, the expression unit-introduced organism may be grown in the absence of the inducer. For example, Arabidopsis thaliana that has accumulated a large amount of ppGpp has a lower photosynthetic ability than Arabidopsis thaliana that has accumulated a smaller amount of ppGpp, but has a higher amount of biomass. Therefore, when an expression unit-introduced organism is grown in the absence of an inducer and then an expression unit-introduced organism is grown in the presence of an inducer, a large amount of organic matter synthesized by photosynthesis is retained, and the amount of biomass is reduced. It is possible to obtain organisms enriched with ppGpp.

<Effect>
As mentioned above, Arabidopsis has four types of ppGpp synthases.
FIG. 2 is a schematic diagram showing domains conserved in these ppGpp synthases, ie, RSH1, RSH2, RSH3, and CRSH4. In FIG. 2, "cTP" indicates a chloroplast targeting peptide, i.e., a chloroplast transit peptide, "HD" indicates a (p)ppGpp hydrolysis domain, and "SYNTH" indicates a (p)ppGpp synthesis domain. "TGS" indicates the TGS regulatory domain, "ACT(RRM)" indicates the ACT regulatory domain (RNA recognition motif domain), "EFh" indicates the calcium-binding EF hand motif in the EF hand domain, and "synth G _376S ” indicates the ppGpp synthesis domain in which the 376th amino acid residue is substituted from glycine to serine, “HDc” indicates the degraded ppGpp hydrolysis domain, and “TM” indicates the putative transmembrane region. "RPP5-ID" indicates the RPP5 interaction domain. Note that "synth G ₃₇₆ S" is not considered to have any activity as a ppGpp synthesis domain. Furthermore, "HDc" is also considered not to have any activity as a ppGpp hydrolysis domain.

As shown in FIG. 2, ppGpp synthases RSH2 and RSH3 have not only a ppGpp synthesis domain but also a ppGpp hydrolysis domain. Therefore, RSH2 and RSH3 catalyze not only the synthesis reaction of ppGpp but also the decomposition reaction. Therefore, it is thought that the amount of ppGpp synthesized by RSH2 and RSH3 does not match the amount of ppGpp accumulated.

On the other hand, the above-mentioned expression unit introduced organism contains the ppGpp synthase gene 10. As described above, the ppGpp synthase gene 10 includes the first region and does not include the second region. Therefore, according to the method described above, the amount of ppGpp synthesized by the ppGpp synthase encoded by the ppGpp synthase gene 10 can match the amount of accumulated ppGpp. Furthermore, according to the method described above, it is possible to efficiently accumulate ppGpp. Furthermore, according to the method described above, it is possible to control the amount of ppGpp accumulated by controlling the expression level of ppGpp synthase.

Furthermore, as a prior art, there is an Arabidopsis mutant in which RSH3, a ppGpp synthase possessed by Arabidopsis, is overexpressed. In this Arabidopsis thaliana mutant, a large amount of ppGpp is accumulated compared to the wild type Arabidopsis thaliana. Moreover, the above-mentioned Arabidopsis thaliana mutant has a higher tolerance to environments with poor nitrogen sources than wild-type Arabidopsis thaliana. Furthermore, this Arabidopsis mutant has a higher amount of biomass than wild-type Arabidopsis.

However, since RSH3 is constantly expressed in the Arabidopsis mutant described above, RSH3 cannot be expressed at any timing. Therefore, it is neither possible to accumulate ppGpp at arbitrary timing nor to adjust the amount of accumulated ppGpp.

On the other hand, in the method for producing a ppGpp-enriched organism described above, transcription of the ppGpp synthase gene is controlled by an inducible promoter. Therefore, ppGpp synthetase can be expressed at any timing depending on the addition of the inducer. Therefore, it is possible to accumulate ppGpp at any timing and to adjust the amount of accumulated ppGpp.

Furthermore, ppGpp-enriched organisms have high tolerance to oligotrophic environments and have a large amount of biomass. Such organisms are therefore useful in agriculture, eg crop production.

Examples of the present invention will be described below.

<1. Preparation of transformants>
First, a plasmid containing the expression unit shown in FIG. 1 was prepared. Specifically, the yjbM gene and the region encoding the chloroplast transit peptide of the Arabidopsis recA gene (locus: At1g79050 in The Arabidopsis Information Resource (TAIR)) were amplified by PCR, and the resulting DNA fragments were transformed into a binary vector. It was cloned into the multiple cloning site of pER8. As the yjbM gene, the yjbM gene described in Reference 1 below was used. Furthermore, as pER8, pER8 described in Reference 2 below was used. pER8 has a multiple cloning site downstream of the estrogen-inducible promoter.

(Reference 1) Nanamiya H., Kasai K., Nozawa A., Yun CS., Nirisawa T., Murakami K., Natori Y., Kawamura F. and Tozawa, Y. (2008) Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis. Mol. Microbiol. 67: 291-304.
(Reference 2) Zuo J., Niu QW., Chua NH. (2000) An estrogen receptor-based transactivator XVE mediated highly inducible gene expression in transgenic plants. Plant J. 24: 265-273.
Next, the expression unit was introduced into Arabidopsis using the obtained plasmid.
By the above method, an organism into which the expression unit had been introduced, ie, a transformant, was obtained. Here, five transformants were obtained. The five transformants obtained are called transformants #1 to #5, respectively.

<2. Western blotting>
First, a part of the plant before estrogen induction was collected from each of the five transformants obtained by the method described above. Next, the five transformants and wild type Arabidopsis were grown for 2 days in an estrogen-containing liquid medium. Thereafter, part of the plant was collected from each of the five transformants and the wild type Arabidopsis.

Each collected plant was homogenized with 5 μL of TE buffer (pH 7.5) per mg. 20 μL of the obtained crushed material was taken into a new tube, 20 μL of 2x SDS sample buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue) was added and boiled for 5 minutes. This was used as a sample for electrophoresis.

Next, polyacrylamide electrophoresis (SDS-PAGE) was performed under the following conditions. The current was set at a constant current of 30 to 50 mA, and a migration buffer (Tris 3.03 g/L, glycine 14.31 g/L, SDS 1.0 g/L) was used as the buffer. As a marker, the trade name "Precision Plus Protein (registered trademark) 2-color standard #1610374" (manufactured by Bio-Rad) was used.

Next, a PDVF membrane (manufactured by Millipore) of the same size as the gel after electrophoresis and 10 pieces of filter paper were cut out. Next, the laminate obtained by stacking 5 filter papers, membrane, gel, and 5 filter papers in this order was placed in a semi-dry blotting device (manufactured by ATTO), and a constant current of 2 mA x 2 mA was applied to the membrane size [cm ² ]. Proteins were transferred to the membrane by allowing time to flow.

Next, the membrane after transfer was washed with methanol, and then immersed in blocking buffer (20 mM Tris/HCl pH 8.0, 88% NaCl, 3% skim milk) for blocking for 1 hour. Thereafter, the membrane was immersed in a solution prepared by adding 1/1000 amount of the primary antibody anti-FLAG M2 (manufactured by SIGMA) to a blocking buffer, and treated with the primary antibody overnight at 4°C.

The membrane was then washed three times with TBST buffer (20mM Tris/HCl pH 8.0, 88% NaCl, 0.05% Tween20). Next, washing was performed three times for 5 minutes using TBST buffer. Thereafter, the membrane was immersed in a solution prepared by adding 1/1000 volume of the secondary antibody anti-rabbit HRP (manufactured by Funakoshi) to TBST buffer, and treated with the secondary antibody at room temperature for 1 hour. Thereafter, washing was performed three times for 5 minutes using TBST buffer. This washed away the secondary antibody. Next, the signal was detected using ECL Plus (manufactured by GE Healthcare).

FIG. 3 is an image showing the results of Western blotting before and after estrogen induction. The "WT" column in FIG. 3 shows the results for wild-type Arabidopsis.

As shown in FIG. 3, a band was confirmed around about 27 kDa. This is the value obtained by subtracting the molecular size of the chloroplast-translocating peptide from the protein size of 33.3 kDa, which is predicted from the primary structure of the fusion protein of the chloroplast-translocating peptide, YjbM protein, and FLAG (registered trademark) tag. It is close to 25.7 kDa. From this, it is clear that the expressed YjbM protein is localized in chloroplasts. In this way, a transformant expressing YjbM protein by estrogen induction was obtained.

<3. Measurement of ppGpp concentration>
First, the transformant #1 described above was grown in an estrogen-containing liquid medium with an estrogen concentration of 0, 0.001, 0.01, 0.1, 1, or 10 μM. Next, part of the shoot part of the plant was collected 2 days and 28 days after estrogen induction.

Next, the ppGpp concentration in the collected shoot part was measured. The ppGpp concentration was measured using a method similar to that described in Reference 3 below. Specifically, first, the chute portion was crushed with liquid nitrogen. Thereafter, a sample solution was prepared using 3 mL of formic acid and 3 mL of ammonium acetate. Thereafter, 1 mL of chloroform was added to the sample solution, and solid phase extraction was performed. Next, the obtained sample was subjected to column treatment and then subjected to mass spectrometry using LC-MS/MS.

(Reference 3) Ihara, Y., Ohta, H. and Masuda, S. (2015) A highly sensitive quantification method for the accumulation of alarmone ppGpp in Arabidopsis thaliana using UPLC-ESI-qMS/MS. J. Plant Res. 128: 511-518.
FIG. 4 is a graph showing the relationship between estrogen concentration and ppGpp concentration on the second day after estrogen induction. FIG. 5 is a graph showing the relationship between estrogen concentration and ppGpp concentration on day 28 after estrogen induction.

As shown in FIGS. 4 and 5, estrogen treatment increased ppGpp concentration. Moreover, as shown in FIG. 5, the amount of ppGpp increased according to the estrogen concentration.

As is clear from the results of Western blotting and ppGpp concentration measurement described above, by expressing ppGpp synthase at an arbitrary timing, it was possible to induce accumulation of ppGpp in chloroplasts.

1... Expression unit 10... ppGpp synthase gene 11... Inducible promoter 12... Region 13... Region

Claims (5)

a ppGpp synthetase gene that includes a region encoding a ppGpp synthesis domain and does not include a region encoding a ppGpp degradation domain; an inducible promoter that controls transcription of the ppGpp synthase gene; and an inducible promoter adjacent to the ppGpp synthase gene. A ppGpp-enriched organism comprising an expression unit comprising a region encoding a chloroplast transit peptide, and growing an organism having chloroplasts in the presence of an inducer that activates the inducible promoter. manufacturing method. 2. The manufacturing method according to claim 1, further comprising growing the organism in the absence of the inducer before growing the organism in the presence of the inducer. 2. The manufacturing method according to claim 1, wherein the growth of the organism in the presence of the inducer is performed in an oligotrophic environment. The ppGpp synthase gene consists of a yjbM gene consisting of the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity with the yjbM gene consisting of the nucleotide sequence represented by SEQ ID NO: 1. , a yjbM-like gene having the region encoding the ppGpp synthesis domain and not having the region encoding the ppGpp degradation domain. a ppGpp synthetase gene that includes a region encoding a ppGpp synthesis domain and does not include a region encoding a ppGpp degradation domain; an inducible promoter that controls transcription of the ppGpp synthase gene; and an inducible promoter adjacent to the ppGpp synthase gene. An organism having chloroplasts, comprising an expression unit comprising a region encoding a chloroplast-translocating peptide.