JPH09248194A - Production of hydrogen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing hydrogen in higher efficiency by subjecting hydrogen-productive photosynthetic bacteria to gene recombination operation. SOLUTION: Hydrogen-productive photosynthetic bacteria capable of expressing protein, having a structural gene group coding protein aggregates capable of forming photosynthetic organs and a promoter controlling transcription of the structural gene group onto a mRNA, is subjected to gene recombination operation, and hydrogen is produced by using the resultant photosynthetic bacteria. In this method for producing hydrogen, the gene recombination operation is conducted in such a way that a fragment provided with an enzyme- binding sequence portion bindable to mRNA synthase to be bound to the promoter is inserted into the bacteria and a vector free from the structural gene group is transduced into the original bacteria in advance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a structural gene group encoding a protein assembly (PUF) forming a photosynthetic organ, and the structural gene group is a messenger RNA (mRN).
A) A method for producing hydrogen in which a protein-expressing hydrogen-producing photosynthetic bacterium comprising a promoter for controlling transcription to A) is genetically modified to produce hydrogen using the hydrogen-producing photosynthetic bacterium. .

[0002]

2. Description of the Related Art Conventionally, various kinds of hydrogen-producing photosynthetic bacteria have been known, and many attempts have been made to utilize hydrogen gas obtained by culturing the hydrogen-producing photosynthetic bacteria. . In such a case, the hydrogen-producing photosynthetic bacterium may be cultivated as a wild strain as it is, but it may also be cultivated by performing a gene recombination operation and imparting various traits to the hydrogen-producing photosynthetic bacterium. It's starting.

[0003]

By the way, in the case of culturing hydrogen-producing photosynthetic bacteria to produce hydrogen,
The hydrogen-producing photosynthetic bacteria are housed in a predetermined culture container under anaerobic conditions, and the method of collecting hydrogen gas that is metabolized by irradiating the hydrogen-producing photosynthetic bacteria in the container with light is taken into the culture container. When the hydrogen-producing photosynthetic bacteria are contained, the bacteria living on the surface side of the culture container generate a large amount of hydrogen, while the bacteria living on the inner surface side of the culture container do not contribute much to hydrogen production. From the perspective that it may be, various attempts have been made in various fields to effectively utilize all bacteria for hydrogen production. However, there are many aspects in which it is not enough to simply study the shape of the container and the light irradiation conditions, and a drastic solution is desired.

Therefore, in view of the above situation, it is an object of the present invention to provide a technique capable of more efficiently producing hydrogen when carrying out a genetic recombination operation on a hydrogen-producing photosynthetic bacterium.

[0005]

[Means for Solving the Problems] When hydrogen-producing photosynthetic bacteria are housed in a culture vessel, bacteria that live on the surface side of the culture vessel generate a large amount of hydrogen, while they live on the inner surface side of the culture vessel. As the previous prediction that the bacteria may not contribute to the production of hydrogen so much, the protein aggregates forming the photosynthetic organs of the hydrogen-producing photosynthetic bacteria reach the inner surface of the culture vessel. One of the factors is that it inhibits The present inventors believe that if this factor is removed, all bacteria can be effectively utilized for hydrogen production, and a method for controlling expression of a protein assembly by a separately invented gene recombination operation (hereinafter When the expression of the protein assembly forming the photosynthetic organ of the hydrogen-producing photosynthetic bacterium is suppressed by the promoter competition method) in order to increase the translucency of the hydrogen-producing photosynthetic bacterium, it occurs in the container. We obtained new knowledge that the amount of hydrogen can be increased.

The outline of the promoter competition law is as follows. An mRNA synthase that binds to the promoter can be bound into a protein-expressing cell that comprises a structural gene group that encodes a protein assembly and a promoter that controls transcription of the structural gene group into mRNA. Having an enzyme-binding sequence containing a gene sequence of
A vector not containing the structural gene group is introduced. By doing so, the mRNA synthase can also bind to the vector, so that the frequency with which the mRNA synthase binds to the promoter to synthesize the protein is suppressed. This is because the mRNA synthase binds to the promoter of the structural gene group and synthesizes the mRNA of the structural gene group. The mRNA is then translated into protein. At this time, a messenger RN that binds to the promoter
If a vector having an enzyme-binding sequence portion containing a gene sequence (enzyme-binding sequence) capable of binding to A (mRNA) synthase and containing no structural gene group has been introduced into cells, It is thought that the mRNA synthesizing sequence can bind to the mRNA synthase. As a result, the frequency with which the mRNA synthase binds to the promoter in the cell decreases. Further, when the mRNA synthesizing enzyme binds to the enzyme-binding sequence portion of the vector,
The mRNA synthase is not involved in the expression of proteins encoded by the structural genes. In other words,
When the mRNA synthase binds to the promoter, it expresses the protein, and when it binds to the enzyme binding sequence part, it does not express the protein. Therefore, the vector is not usually introduced. In the cell,
Compared to the binding of mRNA synthase to the promoter,
MRNA synthase in the cells introduced with the vector,
The frequency of binding to the promoter is reduced, and the frequency of expression of the protein encoded by the structural gene group is reduced in the cells. Therefore, it is considered that the expression of the protein assembly can be controlled.

[Structure 1] The present invention is based on the above new finding, and the characteristic means of the present invention for achieving the above-mentioned object is a structural gene group encoding a protein assembly (PUF) forming a photosynthetic organ. And a promoter capable of controlling the transcription of the structural gene group into messenger RNA (mRNA), and carrying out a gene recombination operation on the protein-expressing hydrogen-producing photosynthetic bacterium. In the hydrogen production method of producing hydrogen using
Messenger RNA (mR that binds to the promoter
NA) is to insert an enzyme-binding fragment having an enzyme-binding sequence part capable of binding to a synthase and to introduce a vector not containing the structural gene group into the hydrogen-producing photosynthetic bacterium. The actions and effects are as follows.

[Effects] That is, according to the promoter competition method, the hydrogen-producing photosynthetic bacterium can be modified so that the protein aggregates forming the photosynthetic organs are less likely to be expressed. Further, the transparency of the hydrogen-producing photosynthetic bacterium is increased. Therefore, it is considered that the hydrogen-producing photosynthetic bacteria in the culture vessel receive sufficient light as a whole and work to produce hydrogen, which can contribute to the production of a large amount of hydrogen. By the way, since the expression level of the protein aggregates forming the photosynthetic organs is decreased in order to increase the translucency, the hydrogen-producing ability of the hydrogen-producing photosynthetic bacteria decreases when taken individually. It is considered that However, based on the above new findings, it is considered that there is a proportional relationship between the translucency and the expression level of the protein aggregate,
When the bacterial cell concentration is constant, a small number of bacterial cells are irradiated with light in excess of the required minimum amount that can maintain a sufficient hydrogen production amount, and other bacterial cells are exposed to light below the required minimum amount. Rather than irradiation, increase the number of cells that receive the minimum required light that can maintain a sufficient hydrogen production amount, and reduce the number of cells that receive only the light that does not reach the minimum required amount. If the light irradiation is performed as described above, it is considered that the utilization efficiency of the irradiated light energy is improved, so that the hydrogen-producing photosynthetic bacteria in the culture vessel have a minimum necessary light amount capable of maintaining the hydrogen production amount. As long as the expression level of the corresponding protein assembly can be secured, it is considered that there is a certain transmissivity that maximizes the hydrogen production amount between the transmissivity and the hydrogen production amount. If you adjust the amount appropriately,
It is thought that hydrogen production can be increased.

[0009] Therefore, in order to modify the bacterial cells, the "gene recombination method" in which the DNA base sequence is recombined so that the mRNA synthase is less likely to bind to the promoter, or the antisense RNA of the protein to be controlled is used. Introducing a coding DNA into a cell to bind the antisense RNA to the mRNA synthesized by the mRNA synthase,
When the ribosome binds to the mRNA and translates the mRNA into a protein, it is relatively easy to perform without complicated control such as “antisense RNA method” that makes it difficult to decipher the base sequence of the mRNA. By maximizing the advantage of the promoter competition method that the expression amount of protein aggregates can be controlled, it is possible to contribute to the increase of hydrogen production amount.

[Structure 2] As the hydrogen-producing photosynthetic bacterium, Rhodobacter sphaeroides (Rhod)
Observer sphaeroides), Rhodobacter
capsulatus), Rhodopseudomonas virid
It is preferable if it is one selected from is), the enzyme-binding sequence part may be a base sequence equivalent to the promoter of the hydrogen-producing photosynthetic bacterium, and the enzyme-binding sequence part may be the hydrogen atom. The promoter of the productive photosynthetic bacterium may have a modified nucleotide sequence, or the vector may contain a plurality of enzyme-binding sequence portions.

[Function and Effect 2] That is, various kinds of hydrogen-producing photosynthetic bacteria can be mentioned. Among them, Rhodobacter sphaeroides (Rhodobacter)
sphaeroides), Rhodobacter capsula
tus), Rhodo Pseudomonas Viridis (Rhod)
If one of the two types is used, it has high hydrogen productivity and is easy to handle, and if the enzyme-binding sequence part has a nucleotide sequence equivalent to the promoter of the hydrogen-producing photosynthetic bacterium, the promoter competition is promoted. There is an advantage that the method can be performed easily. Furthermore, if the enzyme-binding sequence part is a modified nucleotide sequence of the promoter of the hydrogen-producing photosynthetic bacterium, the degree of control of the expression level of the protein assembly by the promoter competition method can be variously changed, The degree of control of the expression level of the assembly, the enzyme-binding sequence part is easier to control so as not to significantly suppress the expression of the protein assembly than in the case of a base sequence equivalent to the promoter of the hydrogen-producing photosynthetic bacterium, It is possible to prevent the expression of protein aggregates from being suppressed too much by the promoter competition method.
Further, for example, when the vector contains a plurality of enzyme-binding sequence portions, it is easy to secure a large amount of suppression of the expression of the protein aggregate when the amount of the expression of the protein assembly is insufficient. . In the case where the vector is a vector containing a plurality of enzyme-binding sequence portions consisting of a modified nucleotide sequence of the promoter of the hydrogen-producing photosynthetic bacterium, the amount of suppression of the expression of the protein assembly can be arbitrarily set. Can be controlled to an appropriate value. As a result, when hydrogen is produced using the promoter competition method, the amount of suppression of protein aggregate expression is controlled to an appropriate value so that the hydrogen productivity of the hydrogen-producing photosynthetic bacterium used can be maximized. As a result, it has become possible to maximize the hydrogen production capacity for each hydrogen-producing photosynthetic bacterium.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The photosynthetic Rhodobacter sphaeroides RV strain (hereinafter abbreviated as RV strain) (Life Research Institute No. 7254) using the method for controlling the expression of a protein assembly of the present invention will be described below. An example of expressing a protein LH1-RC complex (hereinafter abbreviated as LH1-RC) will be described with reference to the drawings.

Photosynthetic bacterium Rhodobacter sphaeroides RV NCIB8253 strain (Rhodobacter
sphaeroides RV / NCIB8253 strain)
The gene sequence of (hereinafter, referred to as 8253 strain) is described in the literature [Molecular Microbiology (1991) 5 (11) p2649-2661,
"DNA sequencing and complementation / deletion analy
sis of the bch A-puf operon region of Rhodobactor s
phaeroides: in vitro mapping of the oxygen regurat
ed pufpromoter. ", CN Hunter et al.], and it is considered that a similar nucleotide sequence exists in the DNA of the RV strain. Therefore, the gene region considered to include the puff promoter of the 8253 strain was reported. Of a pair of oligonucleotides 1 and 2 which serve as a primer for synthesizing the RV strain by polymerase chain reaction (PCR) (see FIG. 5 and Chemical formula 1), and using the oligonucleotides 1 and 2 as primers, All D
PCR was performed using NA as a template. (See Experimental item 1-1)

[0014]

Embedded image Oligonucleotide 1; 5′-CAACATCGTCATGTTACCGCG-3 ′ Oligonucleotide 2; 5′-ACGGAGTGCAATTCCTGGCGC-3 ′

The PCR product is electrophoresed on an agarose gel (b.) In a TBE buffer solution (see a.) (Enzymes, chemicals, etc.) (hereinafter referred to simply as (a.)). After staining with ethidium bromide and then isolating, a DNA fragment of about 1200 to 1300 base pairs (bp) was obtained (see Experimental item 1-2). That is, this DNA
The fragment can be expected to have a nucleotide sequence corresponding to the puff promoter of the 8253 strain in the RV strain.
The DNA fragment was cloned in Escherichia coli (c.) And then subjected to a cleavage test with a restriction enzyme (Experiment 1-
3)), and it was found to contain a portion of the base sequence that is almost the same as the base sequence of the puff promoter of the 8253 strain (hereinafter, this base sequence is referred to as a promoter sequence portion). In other words, this DNA fragment is
It is considered to contain a promoter sequence involved in expressing 1-RC.

The above DNA fragment was cut out with a restriction enzyme EcoRI and labeled with a label (k.) For use as a probe for Southern hybridization. Southern hybridization is carried out using the probe, and a DNA library having the homologous sequence to the probe is selected from the DNA library of the RV strain (see Experiment 2-1) and inserted into the plasmid vector pBR322 to obtain a plasmid vector. pRVB2 (see FIG. 3) (hereinafter, simply referred to as pRVB2) was obtained (see Experimental item 2-2).

Next, in the RV strain, a pair of oligonucleotides 3 and 4 (see FIG. 6 and Chemical formula 2), which serve as primers for the region of the base sequence containing the promoter sequence portion, were synthesized, and PCR was performed using the vector pRVB2 as a template. Synthesis was carried out to obtain a DNA fragment (promoter fragment 5) (see Experimental item 3-1). Wide promoter range vector pKT230 having a kanamycin resistance gene
(Hereinafter referred to simply as pKT230) (e.) To obtain a promoter fragment-containing vector 6 (Experimental Item 3-
2).

[0018]

Embedded image Oligonucleotide 3 5′-CGGATCCCCAACCCTCTTTCATCG
CTGCCCTC-3 'Oligonucleotide 45'-CGGAATTCGGCGTCCGAACAGGGC
GTTGCAA-3 '

When the region corresponding to the promoter sequence in this promoter fragment-containing vector 6 was examined, it was found that the sequence was as shown in Chemical formula 3.

[0020]

Embedded image 1 10 20 5'-GGATCCCAAC CCTCTTTCAT 30 40 CGCTGCCTCT TTCCCGGGTG 50 60 CGGCGATCCG GCGCGCTACC 70 80 GGAACGCCCG TTATGGGATA 90 100 TGCAGGTGCC ACACGTGGTT 110 120 ACTGCAGGAA GTTTGCAACG 130 140 CCCTGTTCGA CGCCCTGTTC 150 CACGCCGAAT TC-3 '

40 of the promoter sequences shown in Chemical formula 3
DNA fragments mut1, mut2, mut3, mut containing a modified promoter sequence in which the base sequence at position -45 is replaced with the base sequences (1) to (4) in Chemical formula 4
4 (see Chemical Formula 5) is a DNA synthesizer (Cyclone Plus DNA Syn
thesizer manufactured by Japan Millipore)
Modified promoter fragment-containing vectors 31 and 3 in which NA fragments mut1, mut2, mut3 and mut4 are inserted
2, 33, 34 were obtained by the Kunkel method (m ₁ ) (see Experimental section 4-3).

[0022]

Embedded image

[0023]

[Chemical formula 5] 40 45 …… TTTCCCGGGT GCGGCG ATCCGGCGCG …… mut1 …… TTTCCCGGGT GCAGCG ATCCGGCGCG …… mut2 …… TTTCCCGGGT GCAACG ATCCGGCGCG …… mut3 …… TTTCCCGGGT CGCCGC ATCCGGCGCG …… mut4 GGCGCGGGATCG …… CGTCCGGGAT

The obtained plasmid vectors 31, 32,
E. coli S17-1 having conjugative transfer ability
(Hereinafter referred to simply as S17-1 strain) (f.) Was introduced by the calcium chloride method. Conjugated transfer is performed using the transformed E. coli S17-1 strain (S17-1 modified strain) and the RV strain carrying the modified promoter fragment-containing vectors 31, 32, 33, 34 (see Experimental item 5-1), Colonies of the modified vector-introduced RV strain into which the above vector was introduced, which were generated on the aSy agar medium containing kanamycin (gh), were selected, and the aSy liquid medium containing ganamycin (g.
h. ) (See Experimental Section 5-2). By comparing the absorption spectra (875 nm) derived from LH1-RC between the modified vector-introduced RV strain and the RV strain into which the promoter fragment-containing vector has not been introduced, RV
It was shown that the expression of LH1-RC in the strain was suppressed and the translucency of the cells was improved. In addition, the translucency of each cell is lower than that of the RV strain (hereinafter referred to as the RV-pp strain) into which a promoter sequence part whose unchanged translucency is introduced is used.
It can be seen that the amount of suppression of 1-RC expression is small and that the respective amounts of suppression of LH1-RC expression are different. Furthermore, these plasmid vectors 31, 32,
RV strain carrying 33, 34 (hereinafter, RV-mut1
Strain, RV-mut2 strain, RV-mut3 strain, RV-mu
t4 strain) was examined for the above-mentioned RV.
It was found that it showed a higher growth property than the -pp strain and showed a growth property relatively close to that of the RV strain.

[Experimental Item] <1> Identification of promoter involved in expression of LH1-RC in RV strain 1-1 PCR synthesis of nucleotide sequence of promoter 1-1 Base of paffoperon of strain 8253 reported in the above-mentioned paper When the starting base of puff B was 1 in the sequence, oligonucleotide 1 corresponding to the 20th base from the −1203 base and oligonucleotide 2 corresponding to the 20th base from the + 68th base were synthesized (see FIG. 5). The oligonucleotide 1 was used as a forward primer and the oligonucleotide 2 was used as a reverse primer, and a PCR synthesis reaction was performed using the total DNA extracted from the RV strain as a template to synthesize a nucleotide sequence. The reaction conditions are shown below.

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 97 ° C 30 seconds: Single-stranded DNA generation by thermal denaturation 50 30: Annealing of primer to single-stranded DNA 72 60: Extension of complementary strand by polymerase reaction ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━ 25 cycles

<2> Synthesis of vector pRVB2 (FIG. 3)
2-1 Southern hybridization After the DNA fragment was treated with a restriction enzyme, the DNA fragment was labeled with a (k.) Probe to obtain the total DN extracted from the RV strain.
Southern hybridization was performed on 2 μg of A.

First, 2 μg of total DNA extracted from RV strain
Then, 40 units of the restriction enzyme BamHI was added, and the mixture was reacted at 37 ° C. for 4 hours and electrophoresed on a 0.8% agarose gel (b.) In 1 × TBE buffer. Southern hybridization was performed on this gel using the above-mentioned probe, and from the gel portion corresponding to the detected band, D
The NA fragment was recovered.

2-2 Introduction of DNA Fragment into Vector Recovered DNA fragment A3 was cleaved with restriction enzyme BamHI using T4 DNA ligase to generate pBR322.
(D.) To obtain vector pRVB2.

2-3 Properties of pRVB2 As shown in FIG. 3, pRVB2 has a partial gene base sequence determined by subcloning, and as shown in FIG. 3, a promoter partial sequence involved in LH1-RC expression by the RV strain and LH1- It was found to have a protein-coding partial sequence that expresses RC (open arrow portion in the figure).

<3> pKT2 of promoter fragment C1
Introduction into 30 3-1 PCR Synthesis of Promoter Fragment As shown in FIGS. 5 and 6, when the starting base of puff B in the puff operon of the 8253 strain is set to 1, it corresponds to 30 bases from the −756th base, and Oligonucleotide 3 having a BamHI recognition sequence added to the 5 terminus, and -66
Corresponds to the 30th base from the second base, and EcoR at its 5 end
Oligonucleotide 4 added to the I recognition sequence was synthesized. The oligonucleotide 3 was used as a forward primer and the oligonucleotide 4 was used as a reverse primer, and PCR was performed using the pRVB2 as a template to synthesize a nucleotide sequence. As a result, a promoter fragment 5 containing the base sequence of the promoter is obtained. The reaction conditions are shown below.

(Reaction solution) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 10x buffer solution (l.): 10 μl dATP: 2 μl dCTP: 2 μl dGTP: 2 μl dTTP: 2 μl AmpliTaq DNA: 0.5 μl Oligonucleotide 3: 0.5 μl Oligonucleotide 4: 0.5 μl pRV2: 0.1 μl MilliQ water: total volume is 100 μl ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

(Conditions) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 95 ° C. 2 minutes: 1 ────── ─────────────────────────── 95 ° C 1 min: Single-stranded DNA generation by thermal denaturation 60 ° C 1 min: Single-stranded primer Annealing to DNA 72 ° C. 2 minutes: Extension of complementary strand by polymerase reaction 2 ───────────────────────────────── 72 ℃ 1 minute: ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 1 ── ～ 2 ── 30 cycles between

3-2 Synthesis of promoter fragment-containing vector To the PCR product containing promoter fragment 5, 20 units each of restriction enzyme BamHI and restriction enzyme EcoRI were added, and 10x buffer solution (l.) Was added to the total reaction solution. The reaction was carried out at 37 ° C. for 2 hours in a solution added with 1/10 of the amount. The reaction product was electrophoresed in 0.8% low melting point agarose gel (i.) In TBE buffer (a.). PC on the low melting point agarose gel (i.)
The R product was stained with ethidium bromide, a band of about 110 to 120 base pairs (bp) was confirmed under UV irradiation, a gel fragment corresponding to the band was cut out, dissolved by heating, and further with phenol / chloroform. Extract and
DNA was recovered from the gel fragment by ethanol precipitation to obtain 5 μg of the enzyme-treated fragment.

On the other hand, pKT2 having kanamycin resistance
4 μg of 30 (e.) Was treated in the same manner to cut into two fragments, a BamHI site at one end and an Ec at the other end.
A long fragment of pKT230 having an oRI site was separated using a low melting point agarose gel (i.), extracted with phenol / chloroform, and then precipitated with ethanol to obtain about 4 μg.

About 5 μg of the pKT230 long fragment was added to 1 μg of the enzyme-treated fragment and ligated with DNA ligase (j.) To synthesize promoter fragment-containing vector 6. This promoter fragment-containing vector 6 was used for comparison experiments and also for the synthesis of the following modified promoter sequence portion (see FIG. 4).

<4> Synthesis of Modified Promoter Sequence 4-1 Cloning Mutk is used as a kit for synthesizing a modified promoter sequence, in which the Kunkel method is applied to perform substitution, deletion, insertion, etc. of bases.
An-K (Takara Shuzo Co., Ltd.) was used. (N.). Vector pUC118 (hereinafter simply referred to as pUC118)
(P.) And the promoter fragment-containing vector 6 grown for convenience of handling were treated with BamHI and EcoRI, respectively, to obtain a long fragment of pUC118 and a short fragment of the promoter fragment-containing vector 6. These were ligated using ligase to obtain a promoter fragment-containing vector 7. This promoter fragment-containing vector 7
E. FIG. E. coli JM109 strain (hereinafter simply referred to as JM109 strain) was transformed with 50 μg / m of ampicillin.
1, X-gal 20 μg / ml, IPTG 0.2 m
The M-containing plate was applied. Normal pUC11
Since 8 has a lacZα gene, when this gene works normally, it shows a colony blue color of the JM109 strain. However, when the above-mentioned gene is inserted, the lacZα gene is destroyed, so that the JM109 strain forms a white colony. Therefore, white colonies were selected and proliferated (inoculated into LB medium containing 50 μg / ml of ampicillin, 3
By culturing overnight at 7 ° C.), separation (alkaline lysis method), and extraction, the JM109 modified strain having the promoter fragment-containing vector 7 can be obtained.

4-2 Preparation of Single-Stranded DNA Promoter fragment-containing vector 7 was prepared from the above JM109 modified strain by using E. coli CJ236 strain (hereinafter, simply C
(Abbreviated as J236 strain) (n.) Was transformed by the calcium salt method and cultured to form colonies to obtain a CJ236 modified strain. The colonies were transferred to 2 ml of 2xYT medium (streptomycin 8 μg / ml and ampicillin 150).
Pre-incubate with μg / ml) overnight. 1 ml of this culture
And inoculate 100 ml of the 2xYT medium,
Infect with phage M13K07. CJ236 modified strains infected with phage were cultured in the presence of kanamycin,
The culture was centrifuged to collect the supernatant and
/ PEG6000 solution (25 ml) was added, stirred, and allowed to stand at room temperature for 10 minutes, and the precipitate was collected by centrifugation (8,000 g, 10 minutes), dissolved in Tris-EDTA (5 ml), extracted with phenol, and then subjected to the alkaline SDS method. Extract to obtain single-stranded DNA body 8. The single-stranded DNA body 8 has a base sequence partially substituted with dU.

4-3 Synthesis of Complementary Strand Using the 4-3-1 DNA synthesizer, the above-mentioned DNA fragments mut1 to mut4 (see Chemical Formula 5) containing the modified promoter sequence were synthesized. The synthesized DNA fragments mut1 to mut4 were recovered by ethanol precipitation and phosphorylated at the 5 ′ end (1 of each of the synthesized DNA fragments mut1 to mut4).
5x Kination buffer for 0 pmol
(N.) 2 μl, T4 Polynucleotide
Add 10 U of Kinase (n.) And sterilized water, and add 10μ
In the reaction solution designated as 1, left still at 37 ° C. for 15 minutes and 70 ° C. for 10 minutes). 4-3-2 To 0.2 pmol of the synthesized single-stranded DNA body 8, Ann was added.
1 μl of ealing buffer (n.) and sterilized water were added to make 10 μl of reaction solution 1 μl and phosphorylated DN
When 1 μl of A fragment mut1 to mut4 is mixed and left standing at 65 ° C. for 15 minutes and 37 ° C. for 15 minutes, the single-stranded DNA is obtained.
The DNA fragments mut1 to mut4 hybridize to the body 8. 4-3-3 Subsequently, 25 μl of Extension buffer
(N.), 60 U of E. coli DNA ligase (n.), 1 U of T4 DNA polymerase was added, and
Let stand at ℃ for 2 hours. Finally, 3 μl of 0.2 M EDT
A, pH 8.0 was added and the mixture was allowed to stand at 65 ° C. for 5 minutes, and a plasmid 11 in which a complementary strand was formed in the single-stranded DNA body 8 was obtained.
~ 14 can be formed. 4-3-4 10 μl of the solution of the plasmids 11 to 14 was added to E. co
li BMH 71-18 mutS COMPETE
100 μl of nt Cells (hereinafter simply referred to as BMH71-18 strain) (n.) were mixed, and the mixture was mixed at 0 ° C. for 30 minutes.
For 45 minutes at 42 ° C and 2 minutes at 0 ° C for transformation, add 900 μl of 37 ° C SOC medium, and incubate at 37 ° C for 1 minute.
After shaking culture for a while, LB plate (ampicillin 150
(contains μg / ml) (n.) and cultures overnight at 37 ° C. to form colonies to obtain a modified BMH71-18 strain. In this state, the plasmids 11 to 14 in the BMH71-18 modified strain returned to the original state in the dU of the single-stranded DNA body 8 and transformed into modified promoter sequence-containing vectors 21 to 24 having a modified promoter sequence part. It is thought that it has become.

4-3-5 Plasmid Extraction of Mutant Strains The modified promoter sequence-containing vectors 21 to 24 were extracted from the formed colonies of BMH71-18 modified strains by the alkaline SDS method, and their nucleotide sequences were confirmed.

<5> Cloning of Modified Promoter Sequence-Containing Vector 5-1 Modified Promoter Sequence-Containing Vector Introduced RV Strain The modified promoter sequence-containing vectors 21 to 24 and pKT230 are treated with restriction enzymes BamHI and EcoRI, respectively, A short fragment of the digested product of the modified promoter sequence-containing vectors 21 to 24, and the pKT
The long fragment of 230 was ligated to obtain modified promoter sequence-containing vectors 31 to 34. The modified promoter sequence-containing vectors 31 to 34 and pKT230
Is transformed into the S17-1 strain in the same manner as the above <3> -2, and the modified S17-1 modified strain of the S17-1 strain is further applied on the same medium as the RV strain, Culture (3
The cells were conjugatively transferred at 0 ° C. under anaerobic and light conditions for 7 days, and transferred into the RV strain. The obtained culture was treated with aSy agar medium containing 10 μg / ml of kanamycin (g.
h. ), And by selecting the resulting colonies, modified promoter sequence-containing vectors 31 to 34 or p
RV-mut1 strain into which KT230 has been introduced to RV-m
ut4 strain and RV-PKT strain were obtained.

5-2 Cultivation of vector-introduced RV strains RV-mut1 to RV-mut4 strains and RV-
PKT strain (vector-introduced RV strain) was added to kanamycin 10
What was cultured in an aSy liquid medium (gh) containing μg / ml and grown to OD ₆₆₀ = 3 or more was
After inoculating 25% in L medium and culturing in Rubin with a volume of 200 ml at 37 ° C. under anaerobic / light conditions (1,500 lux) for one week, each of the vector-introduced RV strains produces hydrogen by photosynthesis. I was able to confirm that. Further, when the hydrogen production capacity of the vector-introduced RV strain was measured, it was as shown in FIG.

<Discussion> In other words, since the RV strain is producing hydrogen, it is clear that it expresses at least LH1-RC and, for example, the above-mentioned RV-mu.
Comparing the absorption spectrum of the culture solution of the t1 strain with the absorption spectrum of the culture solution obtained by similarly culturing the wild RV strain (see FIG. 8), a signal near 875 nm (near 850 nm) peculiar to LH1-RC was obtained. It was found that the shoulder) of the large peak of LH1-RC was reduced, and the expression of LH1-RC could be suppressed. In other words, RV
It was found that the expression of LH1-RC by the strain was suppressed and the translucency of the cells was improved.

Further, comparing the hydrogen producing capacities of the vector-introduced RV strains, the RV-mut1 strain to RV-
It was found that the mut4 strain exhibits an extremely high hydrogen-producing ability as compared with the RV-PKT strain, and in culturing the hydrogen-producing photosynthetic bacterium, the hydrogen-producing ability individually possessed by the cultured cells was reduced. Even so, it was proved that improving the light utilization efficiency of the entire culture container leads to the improvement of hydrogen production.

[Other Embodiments] In the above embodiment, the RV strain was transformed according to Experimental Items <1> to <5>, but the present invention is not limited to this.

For example, the base sequence equivalent to the promoter is not limited to the promoter fragment 5,
Various base sequences can be used depending on the method of selecting the primer. Also, in the experimental item <1>, 825
The nucleotide sequences of the puff promoters of the 3 strains are LH- of the RV strain.
MR capable of binding to the promoter of RV strain even if the nucleotide sequence of the promoter involved in RC is slightly different
If it can be predicted that NA synthase can be bound, it can be used in the following experimental section.
Nucleotide sequences to which vRNA synthetase can bind are collectively referred to as an enzyme-binding sequence.

Furthermore, although pKT230 was used as a vector, a plasmid which can be expressed in cells of the RV strain (for example, a wide host range vector such as pRK415 and pUI501) can be used as a vector.

In order to introduce the trait into the RV strain, electroporation may be used instead of transfecting E. coli by the calcium chloride method and then conjugative transfer.

Furthermore, the present invention can be applied to any cells other than the RV strain, even if there is a host vector system. In these cases, the nucleotide sequence of the promoter controlling the protein to be suppressed is clarified. The enzyme-bonded sequence of the promoter may be synthesized, and the operation corresponding to the above experimental item may be performed.

When a vector capable of introducing a plurality of DNA fragments is selected as a vector to be introduced into cells, a plurality of enzyme-binding sequence fragments containing an enzyme-binding sequence can be inserted into the vector. For example, by inserting a modified promoter sequence fragment containing a modified promoter sequence part having a low activity of suppressing protein expression by only one into the vector, it is possible to finely adjust the protein expression suppression amount. Become. Further, by connecting a plurality of the modified promoter sequence fragments or a plurality of promoter sequence fragments,
It is considered that the same effect can be obtained by synthesizing a new enzyme-binding sequence fragment containing a gene sequence having a plurality of sites capable of binding to a synthetic enzyme and inserting it into a vector. Furthermore, if hydrogen-producing photosynthetic bacteria that have undergone various genetic recombination operations are combined with genetic recombination operations related to the promoter competition method, it is necessary to reduce the activity of the hydrogen-producing photosynthetic bacteria due to the genetic recombination operation. It is convenient because the promoter competition method can be performed while suppressing it to a minimum.

<Enzymes, chemicals, etc.> a. TBE buffer solution: 5x Composition: Tris 54 g Boric acid 27.5 g 0.5 M EDTA 20 ml was dissolved in water to adjust the pH to 8.0 to make 1 liter.

B. Agarose gel: See Kem GTG Agaros
e (Takara Shuzo Co., Ltd.)

C. For cloning, E. coli TA one shot and vector p included in a cloning kit (TA Cloning system manufactured by Invitrogen)
I am using CRII.

D. Vector pBR322 (manufactured by Toyobo Co., Ltd.)

E. Vector pKT230: Literature "specifi
c-purpose plasmid cloning vectors II. Broad host r
ange, highcopy number, RSF1010-derived vectors, an
da host-vector system for genecloning in Pseudomo
nas. "M. Bagdasarian et al., Gene 16 (1981).

F. E. coli S17-1: Reference "Broad Host
Range Mobilization System for in vitro Genetic Eng
ineering: Transposon Mutagenesis in Gram Negative B
acteria. "R. Simon et al., Bio / technoloigy, Novembe
r (1983).

G. aSy liquid medium, (aSy agar medium) Composition: ammonium sulfate 1.25 g sodium succinate 9.8 g yeast extract 1.0 g basal medium (h.) 1 liter (pH 6.8) (aSy agar medium has the above composition) 1.5% Bacto agar added)

H. Basal medium composition: dipotassium hydrogen phosphate 0.75 g potassium dihydrogen phosphate 0.85 g boric acid 2.8 mg sodium molybdate dihydrate 0.75 mg zinc sulfate heptahydrate 0.24 mg manganese sulfate Tetrahydrate 2.1 mg Copper (II) nitrate trihydrate 0.04 mg Calcium chloride dihydrate 0.75 mg Ethylenediaminetetraacetic acid 2 mg Magnesium sulfate heptahydrate 0.2 mg Iron sulfate (II ) 10 mg vitamins (vitamin B1 biotin p-aminobenzoic acid nicotinic acid nicotinamide) What was made into 1 liter by dissolving a trace amount in distilled water (pH 6.8).

I. Low melting point agarose gel: NuSieve GTG
Agarose (Takara Shuzo Co., Ltd.)

J. DNA ligase: T4 DNA ligase (Takara Shuzo Co., Ltd.)

K. A DIG DNA labeling kit (Boehringer Mannheim) was used for labeling.

L. Buffer 10x Composition: Tris-HCl 50 mM Magnesium chloride 10 mM Sodium chloride 100 mM Dithiothreitol 1 mM

M. (Kunkel, TA (1985)
Proc. Natl. Acad. sci. USA. ,
82,488. )

N. Mutan-K (Takara Shuzo Co., Ltd.) contains the following solutions, plasmids and phages. E. FIG. coli CJ236 strain M. 13K07 phage Annealing buffer Kination buffer Extension buffer T4 Polyclinotide Kinase T4 DNA polymerase E. coli BMH71-18 mutS Comp
etent Cells. LB plate

P. Vector pUC118 (Takara Shuzo Co., Ltd.)

In addition, nx (n is a numerical value, for example, 1
x, 5x, 10x) indicates that the composition has a concentration suitable for use by diluting it n times, and conversely, it is a composition obtained by concentrating a solution having an appropriate concentration n times. It shows something.

Of course, for other enzymes, drugs, etc.,
The present invention is not limited to the above embodiment.

[Brief description of drawings]

FIG. 1 is a process diagram showing a strain creating method for carrying out the method for suppressing the expression of a protein assembly of the present invention.

FIG. 2 is a process diagram showing a strain creating method for carrying out the method for suppressing the expression of a protein assembly of the present invention.

FIG. 3: Restriction map of pRVB2

FIG. 4 Restriction enzyme map of promoter fragment-containing vector

FIG. 5 is a diagram showing a correspondence relationship between oligonucleotides 1 and 2 and a puff operon.

FIG. 6 is a diagram showing a correspondence relationship between oligonucleotides 3 and 4 and puff operons.

FIG. 7 is a graph showing the growth of microorganisms.

FIG. 8 is a graph showing an absorption spectrum of a culture solution of a microorganism.

Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12R 1:01) (C12N 1/21 C12R 1:01) (72) Inventor Masato Miyake 1-chome, Tsukuba-shi, Ibaraki 1-3 Industrial Technology Institute of Biotechnology Industrial Technology (72) Inventor Yasuo Asada 1-3-1 Higashi Tsukuba City, Ibaraki Prefecture Industrial Technology Institute of Biotechnology Industrial Research (72) Inventor Shobayashi Nishi, Minato-ku, Tokyo 2-8-11 Shimbashi 7th Toyo Kaijuku Building 8th floor, Research Institute for Global Environmental Industry, CO2 fixation etc. (72) Inventor Kyoko Nagamine 2-8-11 Nishishinbashi, Minato-ku, Tokyo 7th Toyo 8th floor of Maritime Building Research Institute for Global Environment and Industrial Technology CO2 fixation project room (72) Inventor Tadaaki Kawasugi 2-8-11 Nishishinbashi, Minato-ku, Tokyo 7th Toyo Kaijuku Building 8th floor Earth Departure from the project room (72) of CO2 fixation, etc. Akito Nakata Eiju 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Toyo Kaijuku Building 8th Floor, Research Institute for Global Environmental Science and Technology, CO2 immobilization project room (72) Inventor Rudmila Gee Basilieva Tokyo 2-8-11 Nishishinbashi, Minato-ku 7th Toyo Kaijuku Building 8th floor, Research Institute for Global Environmental Technology CO2 fixation etc. (72) Inventor Satoshi Nishikata 2-8-11 Nishishinbashi, Minato-ku, Tokyo 7th Toyo Kaijuku Building 8th floor, Research Institute for Global Environmental Industry, CO2 fixation etc.

Claims (5)

[Claims] 1. A protein assembly forming a photosynthetic organ (P
UF) -encoding structural genes and a promoter that controls the transcription of the structural genes into messenger RNA (mRNA). A method for producing hydrogen using the hydrogen-producing photosynthetic bacterium, comprising: a messenger RNA (mR) that binds to the promoter.
NA) A hydrogen production method in which an enzyme-binding fragment having an enzyme-binding sequence portion capable of binding to a synthase is inserted and a vector not containing the structural gene group is introduced into the hydrogen-producing photosynthetic bacterium. 2. The hydrogen-producing photosynthetic bacterium is Rhodobacter sp.
haeroides), Rhodobacter capsulatu
s), Rhodopseudomonas viridis (Rhodop)
The method for producing hydrogen according to claim 1, wherein the hydrogen production method is one selected from the group consisting of Seudomonas viridis). 3. The method for producing hydrogen according to claim 1, wherein the enzyme-binding sequence portion has a base sequence equivalent to the promoter of the hydrogen-producing photosynthetic bacterium. 4. The method for producing hydrogen according to claim 1, wherein the enzyme-bonded sequence portion is a nucleotide sequence obtained by modifying the promoter of the hydrogen-producing photosynthetic bacterium. 5. The method for producing hydrogen according to claim 1, 2 or 4, wherein the vector contains a plurality of enzyme-binding sequence portions.