JPH08214882A - Acetohydroxy acid synthase gene of photosynthetic bacteria - Google Patents

Abstract

PURPOSE: To obtain the subject new gene coding for two subunits of an acetohydroxy acid synthase originated from Rhodobacter capsulatus belonging to photosynthetic bacterial strain and giving an amino acid synthase to use CO2 as a carbon source as well as an energy source. CONSTITUTION: This new DNA useful e.g. for the production of enzymes for the synthesis of amino acids using CO2 as a carbon source as well as an energy source codes for a large subunit of an acetohydroxy acid synthase having an amino acid sequence of formula I and a small subunit of an acetohydroxy acid synthase having an amino acid sequence of formula II and is originated from Rhodobacter capsulatus (ATCC 11166) which is a photosynthetic bacterial strain. The DNA can be produced by extracting the whole DNA of Rhodobacter capsulatus, selecting a pair of primers composed of the DNA part of an acetohydroxy acid synthase and cloning the synthesized product by PCR method using the primers.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an acetohydroxy acid synthase (EC 4.1.3.) Derived from the photosynthetic bacterium Rhodobacter capsulatus.
18) relating to DNA.

[0002]

2. Description of the Prior Art An enzyme acetohydroxy acid synthase having catalytic activity for the first step from α-ketobutyric acid to L-isoleucine or the first step from pyruvate to L-valine is L-isoleucine, L-valine. It is an enzyme involved in the initial stage of the biosynthesis system and is the rate-limiting enzyme for these biosynthesis. Therefore, the gene encoding acetohydroxy acid synthase (AHAS gene)
It is expected that the productivity of these amino acids by microorganisms can be improved by utilizing

Acetohydroxy acid synthase forms a tetramer consisting of two large subunits and two small subunits. Regarding the gene encoding acetohydroxy acid synthase (AHAS gene),
In microorganisms, a gene derived from Escherichia coli [Nucleic. Acids Res. 13, p. 3995 (1985), Pr
oc.Natl.Acad.Sci.USA.78, p.922 (1981), J. Bacteriol.
147, p.797 (1981)], Bacillus subtilis
ubtilis) -derived gene [GenBank, AccessionL03181],
Brevibacterium flavu
m) -derived gene [DNA Sequence-J. DNA Sequencing and
Mapping, vol.3, pp.303-310) and the like have been isolated and their nucleotide sequences have been determined.

By the way, photosynthetic bacteria having the ability to fix CO ₂ are expected to be used for amino acid production using CO ₂ as a carbon source and an energy source. Regarding the nucleotide sequence of the AHAS gene derived from photosynthetic bacteria, As far as the inventor knows, there are no reports.

[0005]

SUMMARY OF THE INVENTION The present invention has been made for the purpose.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, by utilizing a gene recombination technique, a large subunit of acetohydroxy acid synthase was obtained from photosynthetic bacteria. The inventors have found that the DNA encoding the small subunit can be isolated, and completed the present invention.

That is, the gist of the present invention is the photosynthetic bacterium Rhodobacter capsulatus.
DNA encoding a large subunit of acetohydroxy acid synthase, a DNA encoding a small subunit of acetohydroxy acid synthase from photosynthetic bacterium Rhodobacter capsulatus, and a large subunit of acetohydroxy acid synthase DNA having a coding DNA and a DNA coding for a small subunit of acetohydroxy acid synthase.

The "DNA encoding the large subunit of acetohydroxy acid synthase" of the present invention means α-
Ketobutyric acid to L-isoleucine or pyruvate to L
-Means a DNA encoding a large subunit constituting acetohydroxy acid synthase, an enzyme having the catalytic activity of the first step leading to valine, and further including a 5'untranslated region, 3'untranslated region, promoter and the like DN
A may be included. In addition, "DNA encoding a small subunit of acetohydroxy acid synthase"
The term "means a DNA encoding a small subunit constituting acetohydroxy acid synthase, and may further include a DNA containing a 5'untranslated region, a 3'untranslated region, a promoter and the like.

Hereinafter, the DNA encoding the large subunit of acetohydroxy acid synthase or the DNA containing the same may be abbreviated as "AHAS large subunit gene". Further, the DNA encoding the small subunit of acetohydroxy acid synthase or the DNA containing the same may be abbreviated as "AHAS small subunit gene". Furthermore, the AHAS large subunit gene, the AHAS small subunit gene, and the AHAS large subunit gene and AHA
DNA having both S small subunit gene
May be collectively referred to simply as "AHAS gene".

The base sequence of the AHAS large subunit gene of the present invention includes a base sequence capable of encoding the amino acid sequence shown in SEQ ID NO: 1. AHA of the present invention
The nucleotide sequence of the S large subunit gene is not limited to this sequence, and α-ketobutyric acid or pyruvate when the acetohydroxy acid synthase is constituted together with the small subunit of acetohydroxy acid synthase in the above amino acid sequence. Any of the DNA encoding the large subunit of acetohydroxy acid synthase having a substitution, deletion, insertion or transfer of an amino acid residue that does not substantially impair the activity of synthesizing acetohydroxy acid from an acid is the AHAS large of the present invention. It is included in the subunit gene. More specific examples of the base sequence of the AHAS large subunit gene of the present invention include the base sequence shown in SEQ ID NO: 2 and the base sequence substantially the same as this. Here, “substantially the same” means that the AHAS large subunit encoded by the DNA having the base sequence has substantially the same enzymatic properties as the AHAS large subunit encoded by the base sequence shown in SEQ ID NO: 2. There is something.

The base sequence of the AHAS small subunit gene of the present invention includes a base sequence capable of encoding the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence of the AHAS small subunit gene of the present invention is
Not limited to this sequence, among the amino acid sequence, the activity of synthesizing acetohydroxy acid from α-ketobutyric acid or pyruvic acid when constituting acetohydroxy acid synthase with the large subunit of acetohydroxy acid synthase Any DNA encoding the small subunit of acetohydroxy acid synthase having a substitution, deletion, insertion or transfer of amino acid residues that does not substantially harm is included in the AHAS large subunit gene of the present invention. . More specifically, the base sequence of the AHAS small subunit gene of the present invention includes the base sequence shown in SEQ ID NO: 2 and the base sequence substantially the same as this. Here, “substantially the same” means AHAS encoded by a DNA having the nucleotide sequence.
It means that the small subunit has substantially the same enzymatic properties as the AHAS small subunit encoded by the nucleotide sequence shown in SEQ ID NO: 2.

An example of the method for obtaining the gene of the present invention will be described below.

<Isolation of AHAS Gene> AHA of the present invention
Although the S gene can be synthesized after its nucleotide sequence is determined, it is usually a photosynthetic bacterium, for example, Rhodobacter capsultas.
atus) (ATCC11166) and the like on the chromosome,
The chromosomal DNA can be separated and obtained by the method described below from among the cleaved fragments generated by cleaving it with an appropriate restriction enzyme.

(1) Preparation of Chromosome Library First, the above photosynthetic bacterium, for example, Rhodobacter capsulatus, is cultured according to a known method [see, for example, J.Bac.156, p.507, (1983)]. ,
Cells are collected from the culture and chromosomal DNA is extracted from the cells. The chromosomal DNA is, for example, J. Bac.156, p.507, (1
It can be easily extracted from the cells by the method described in 983).

This chromosomal DNA is cleaved with an appropriate restriction enzyme such as BamHI, the resulting DNA fragment is inserted into an appropriate cloning vector such as pUC118 (Takara Shuzo), and the obtained recombinant vector is used. An appropriate host, for example, Escherichia coli JM109 (Takara Shuzo) is transformed to obtain a transformant.

(2) Selection of clones Recombinant plasmid DNA was extracted from the obtained transformants, and the AHAS gene derived from Escherichia coli [Nuclei
c. Acids Res. 13, p. 3995 (1985), Proc.Natl.Acad.Sci.
USA.78, p.922 (1981), J.Bacteriol.147, p.797 (198
1)] Rhodobacter capsulatus (ATCC 11166) by Southern hybridization using a common region sequence as a probe
It is possible to select a transformant carrying a recombinant plasmid in which the AHAS gene of the origin is inserted [DNA Sequ
ence-J. DNA Sequencing and Mapping, vol.3, pp.303-3
See 10].

As one of the AHAS genes of the present invention thus obtained, the above-mentioned Rhodobacter capsulatus (A
The chromosomal DNA of TCC11166) is a restriction enzyme BamH
The size obtained by cutting out with I is about 4.0 kb
DNA fragments can be mentioned.

Table 1 shows the number of recognition sites and the size of the fragment when the DNA fragment containing the AHAS gene of about 4.0 kb was digested with various restriction enzymes.

[0019]

[Table 1]

In the present specification, "the number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid containing the same is completely decomposed in the presence of a restriction enzyme, and the decomposed product is subjected to 1% agarose according to a method known per se. It was subjected to gel electrophoresis and 4% polyacrylamide gel electrophoresis, and the value determined from the number of degradable fragments was adopted.

In addition, the "size of the cleaved fragment" and the size of the plasmid are such that, when agarose gel electrophoresis is used, Escherichia coli lambda phage (λ ph
based on the standard line drawn by the swimming distance when a mixture of DNA fragments of known molecular weight obtained by cleaving the DNA of age) with the restriction enzyme HindIII is swept on the same agarose gel, and polyacrylamide gel electrophoresis. In the case of using the induction, when a DNA fragment mixture of known molecular weight obtained by digesting the DNA of Escherichia coli Phi-X174 phage (φX174 phase) with the restriction enzyme HaeIII was run on the same polyacrylamide gel. The size of the cleaved DNA fragment or each DNA fragment of the plasmid can be calculated based on the standard line drawn by the swimming distance. In the determination of the size of each DNA fragment, regarding the size of the fragment of 1 kb or more,
By using the results obtained by 1% agarose gel electrophoresis and by using the results obtained by 4% acrylamide gel electrophoresis for fragment sizes of about 0.1 kb to less than 1 kb, accurate measurements can be obtained. It will be possible.

On the other hand, the above Rhodobacter capsulatus (ATCC11166)
AHA having a size of about 4.0 kb obtained by directly cutting the chromosomal DNA of Escherichia coli with a restriction enzyme BamHI
The base sequence of the S fragment is plasmid pUC18 or pU
Dideoxynucleotide enzymatic method using C19 etc. [dide
Oxy chain termination method; Sanger, F et al., Proc.
Nat. Acad. Sci. USA, 74,5463, (1977)].

The size determined above is about 4.
The sequence in the 0 kb DNA fragment is represented by SEQ ID NO: in the Sequence Listing below.
1 and 2 are shown. From the open reading frame present in this sequence, the gene coding for acetohydroxy acid synthase (AHAS gene) is the amino acid sequence from amino acid number 1 to amino acid number 578 (AHAS large subunit) and amino acid number 1 to It was revealed that it encodes an amino acid sequence up to amino acid number 186 (AHAS small subunit). The AHAS gene is not only isolated from natural bacterial chromosomal DNA, such as photosynthetic bacterial chromosomal DNA, but also a commonly used DNA synthesizer, such as Beckman System-1 Plus.
It may be a compound synthesized using s).

As described above, the DNA fragment of the present invention obtained from a bacterial chromosome such as Rhodobacter capsulatus (ATCC11166) has a base sequence of the acetohydroxy acid synthase unless it substantially impairs its function. Some bases may be replaced with other bases, may be deleted, new bases may be inserted, or a part of the base sequence may be transferred. Further, it may be a nucleotide sequence that hybridizes to those nucleotide sequences, and any of these derivatives is included in the DNA fragment containing the gene encoding the acetohydroxy acid synthase of the present invention.

While the present invention has been described above, it will be described more specifically with reference to the following examples. It should be understood, however, that the examples are to be considered as an aid in gaining a specific appreciation of the invention and are not to be construed as limiting the scope of the invention in any way.

[0026]

【Example】

[Example 1] Rhodobacter capsulatus (ATCC
Cloning of AHAS partial fragment derived from 11166)

(A) Rhodobacter capsulatus (A
Extraction of total DNA of TCC11166) Rhodobacter capsulatus (ATCC11166)
Using a platinum loop, PYE medium 1L [composition: peptone
3g, yeast extract 3g, malic acid 0.6g, butyric acid
0.2 g, sodium bicarbonate 0.1 g], and photoanaerobically cultivated at 30 ° C. until the late logarithmic growth phase to collect the cells [J. Bac 156, p. 507, (1983)].

The obtained bacterial cells were adjusted to a concentration of 10 mg / ml with 10 μg / ml lysozyme and NaCl-20 m.
M Tris buffer (pH 8.0) and 1 mM EDTA
Aqueous solution containing 2Na (concentration of each component is final concentration)
Suspended in water. Next, proteinase K was added to this suspension so that the final concentration was 100 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour.
Incubated for hours. Further, sodium dodecyl sulfate was added so that the final concentration was 0.5%, and the mixture was kept at 50 ° C. for 6 hours for lysis. An equal amount of phenol / chloroform mixed solution was added to the lysate, and the mixture was gently shaken at room temperature for 10 minutes, and then the whole was centrifuged (3,800 xg, 15 minutes, 1 minute).
5 to 20 ° C.), and the supernatant fraction was collected. Sodium acetate was added to this supernatant so that the concentration was 0.3 M, and then twice the amount of ethanol was slowly added. The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried. 10 in the obtained DNA
mM Tris buffer (pH 7.5), 1 mM EDTA
・ Add 5 ml of an aqueous solution containing 2Na, leave it at 4 ° C overnight, and use it for PCR (polymerase chain reaction) template DN.
Used as A.

(B) Selection of PCR Primer The nucleotide sequence of the AHAS gene has been determined in some bacteria such as those derived from Escherichia coli [Nuclei
c. Acids Res. 13, p. 3995 (1985), Proc.Natl.Acad.Sci.
USA.78, p.922 (1981), J.Bacteriol.147, p.797 (198
1)]. The region conserved among these various microorganisms, particularly the region conserved not only in the amino acid sequence but also in the DNA base sequence, was examined, and a pair of primers having the following base sequences were selected, and applied biotechnology 394 DNA / made by Systems (Applied Biosystems)
It was synthesized using an RNA synthesizer.

(A-1) ATHGGIWMIG AYGCITTYCARGA 23 (SEQ ID NO: 3) (b-1) CCRTGCATIC CIARCATICC 20 (SEQ ID NO: 4) (where R is A or G, Y is C or T, M is A or C) , W is A or T, H is A or C or T, where A is adenine, G is guanine, C is cytosine, T is thymine, and I is hypoxanthine (deoxyinosine as a nucleotide).

(C) PCR reaction In the PCR, a DNA thermal cycler manufactured by Perkin Elmer Cetus Co., Ltd. was used, and as a reaction reagent, a recombinant tack DNA polymerase polymerase Takara tack (Reco
mbinant TaqDNA Polymerase TaKaRa Taq) (Takara Shuzo Co., Ltd.) was used under the following conditions.

[0032]

[Reaction Solution]: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA MgCl2 10 μl (DNA content 1 μM or less) 1 μl each of the primers prepared in the above (B) (final concentration 0) 0.25 μM) Glycerol 10 μl Dimethylsulfoxide 5 μl Recombinant tack DNA / Polymerase 0.5 μl Sterile distilled water 46.5 μl

The above mixture was mixed, and using 100 μl of the reaction solution, 25 cycles of the following PCR cycle were performed. [PCR cycle]: Denaturation process: 94 ° C. 60 seconds Annealing process: 55 ° C. 120 seconds Extension process: 72 ° C. 180 seconds

(D) Detection of reaction product 10 μl of the reaction solution obtained in the above item (C) was electrophoresed on a 2% agarose gel to detect a DNA fragment of about 0.5 kb.

(E) Cloning of amplified fragment 3 μl of the reaction solution obtained in the above (C) was mixed with 1 μl of the PCR product cloning vector pGEM-T vector (commercially available from PROMEGA), and T4 DNA ligase buffer (10 ×) was mixed. 1 μl of each component of T4 DNA ligase 1 unit was added, and the volume was adjusted to 10 μl with sterile distilled water.
The reaction was carried out at 5 ° C. for 3 hours to combine the vector with the PCR product to obtain a recombinant plasmid mixture.

Using the obtained plasmid mixture, the calcium chloride method [Journal of Molecular Biology, Vol. 53,
Page 159 (1970)] was used to transform Escherichia coli JM109 (Takara Shuzo), and ampicillin 5
It was smeared on a medium containing 0 mg [trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g dissolved in 1 L of distilled water].

The strain grown on this medium was subjected to liquid culture by a conventional method, recombinant plasmid DNA was extracted from the culture, and the plasmid DNA was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, the length of the plasmid pGEM-T was 3.
In addition to 0 kb DNA fragment, insert D of about 0.5 kb in length
NA fragment was observed.

Example 2 Determination of Nucleotide Sequence of Amplified Fragment Regarding the amplified fragment having a size of about 0.5 kb obtained in the item (E) of Example 1, the nucleotide sequence of the amplified fragment was determined by the dideoxynucleotide termination method. Law) [Sanger,
F. et al., Proceeding of National A
cademy of Science, Volume 74, page 5463 (19)
77)].

As a result of the nucleotide sequence determination, the inserted fragment showed a high homology with a part of the E. coli-derived AHAS gene (a region sandwiched by the primers used for PCR),
It was confirmed that a part of the AHAS gene derived from Rhodobacter capsulatus was cloned.

Example 3 Cloning of DNA Fragment Containing AHAS Gene Derived from Rhodobacter capsulatus 90 μl of the total DNA solution of Rhodobacter capsulatus obtained in (A) of Example 1 above was treated with the restriction enzyme Sau3.
Using AI 5 units, reaction was carried out at 37 ° C. for 10 minutes for partial decomposition. This degraded DNA and λFixII / XhoI
Partial Fill-In Vector Kit (Part
ial Fill-In Vector Kit) and Klenow fragment (Toyobo Co., Ltd.) were used to prepare a chromosomal DNA library according to the protocol.

Next, the λDNA library phage solution (2 to 5 × 10 4 pfu; diluted with SM buffer) and the culture solution of Escherichia coli P2392 (manufactured by Toyobo Co., Ltd.) were mixed in equivalent amounts and incubated at 37 ° C. for 15 minutes. 3 to 4 ml of λ medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, MgS) kept warm at 50 ° C.
Dissolve 2g of O4 / 7H2O and 5g of agar in 1L of distilled water)
Λ plate (trypton 10 g, NaCl 5
g, MgSO4 · 7H2O 2 g and agar 10 g were dissolved uniformly in 1 L of distilled water), and cultured at 37 ° C. for 12 to 16 hours.

The plaques formed on this medium were adsorbed on a nitrocellulose filter, and the filter was dissolved in distilled water containing 20 g of NaOH and 87.5 g of NaCl to 1 L, and tris (hydroxymethyl) amino. 121.2 g of methane and NaCl 3
0 g was dissolved in distilled water, and the solution was adjusted to pH 7.0 with 6N HCl to make 1 L, and the solution was placed on filter paper soaked for 5 minutes each, and treated. After air-drying this filter,
Dry heat treatment was carried out at 80 ° C. for 2 hours to fix the DNA on the filter.

AHAS obtained by the PCR method shown in Example 1
Plaque hybridization was performed using the partial gene fragment as a probe and the filter prepared above. At this time, the probe was labeled using a random labeling kit (Takara Shuzo). Plaque hybridization can be performed using standard methods (Molecu
lar Cloning), published by Cold Spring Harbor Laboratory (1
989)]. As a result, positive plaques can be selected, and phage DNA can be selected from the plaques.
Was extracted, and an insert fragment having a size of 4.0 kb that was cut out from the restriction enzyme BamHI was confirmed. This insert fragment was inserted into the BamHI site of plasmid pUC19,
It was used for confirmation of the entire AHAS gene region.

The number of recognition sites and the size of the cleaved fragment when the size of the DNA fragment of 4.0 kb was cleaved with various restriction enzymes were as shown in Table 1 above. The restriction enzyme cleavage point map is shown in FIG.

[Example 4] Confirmation of the entire region of AHAS gene In order to identify the site of the AHAS gene existing on the DNA fragment of 4.0 kb obtained in Example 3, the following operation was performed. The sequence was determined.

DNA fragment solution 14 having a size of 4.0 kb
μl using the restriction enzyme Sau3AI 5units,
Partial decomposition was carried out by reacting at 37 ° C for 1 to 5 minutes. On the other hand, the cloning vector pUC118 (commercially available from Takara Shuzo) was cleaved with the restriction enzyme BamHI, and the digested ends were dephosphorylated. This vector is mixed with the above-described partially degraded DNA fragment, and 50 mM Tris buffer (pH 7.6), 10 mM
Dithiothreitol, 1 mM ATP, 10 mM Mg
Each component of Cl2 and T4 DNA ligase 1 unit (the concentration of each component is the final concentration) was added and reacted at 16 ° C for 10 hours to ligate the vector and the partially degraded DNA fragment to obtain a recombinant plasmid mixture.

On the other hand, the size obtained in the embodiment is 4.0.
14 μl of the kb DNA fragment solution was added to the restriction enzyme TaqI.
Using 50 units, it was reacted for 5 to 8 minutes for partial decomposition. The cloning vector pUC118 is a restriction enzyme Ac
It was cleaved with cI, dephosphorylated and ligated with the partially digested DNA fragment in the same manner as above to obtain a recombinant plasmid mixture.

Using each of the plasmid mixtures obtained above, the calcium chloride method [Journal of Molecular Biology] was used.
, 53, p. 159 (1970)] to transform Escherichia coli JM109 (manufactured by Takara Shuzo), and a medium containing 50 μg / ml of ampicillin [Trypton 10].
g, yeast extract 5 g, NaCl 5 g and agar 16 g were dissolved in distilled water 1 L].

Each of the growing strains on this medium is liquid-cultured by a conventional method, plasmid DNA is extracted from the culture solution, and the nucleotide sequence thereof is determined by the dideoxynucleotide method (dideoxy chain terminator method) using pUC118 (Takara Shuzo).
[Sanger F. et al. , Proceeding of National Academy of Science
National Academy of Science), Vol. 74, page 54
63 (1977)].

Due to the presence of the open reading frame in the base sequence, the AHAS gene has a base sequence shown in SEQ ID NO: 2 and a 1734 base pair encoding 578 amino acids having the base sequence shown in SEQ ID NO: 1 in the late sequence listing. It was revealed that it is composed of 558 base pairs encoding 186 amino acids.

[0051]

INDUSTRIAL APPLICABILITY By breeding and improving a photosynthetic bacterium using the AHAS gene provided by the present invention, CO ₂ can be used for the production of amino acids as a carbon source and an energy source.

[0052]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1734 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Rhodobacter capsulatus Strain name: ATCC11166 Sequence characteristics Symbol: CDS Presence position: 1-1734 Method for determining characteristics: E sequence ATG ACC GGC GCG GAA ATG GTG GTG CAG GCG CTG AAG GAT CAG GGG GTC 48 Met Thr Gly Ala Glu Met Val Val Gln Ala Leu Lys Asp Gln Gly Val 1 5 10 15 GAC ACG ATC TTC GGA TAC CCG GGC GGC GCC GTG CTT CCG ATC TAT GAC 96 Asp Thr Ile Phe Gly Tyr Pro Gly Gly Ala Val Leu Pro Ile Tyr Asp 20 25 30 GCG ATC TTT CAG CAA AAC GAC ATC CGC CAC ATC CTG GTG CGG CAT GAA 144 Ala Ile Phe Gln Gln Asn Asp Ile Arg His Ile Leu Val Arg His Glu 35 40 45 CAG GCC GCG GTG CAC ATG GCC GAG GGC TAT GCC CGC TCG ACC GGC AAG 192 Gln Ala Ala Val His Met Ala Glu Gly Tyr Ala Arg Ser Thr Gly Lys 50 55 60 CCG GGC GTG GTG CTG GTG ACC TCG GGC CCG GGG GCg ACG AAT gCG GTG 240 Pro Gl y Val Val Leu Val Thr Ser Gly Pro Gly Ala Thr Asn Ala Val 65 70 75 80 ACC GGG CTG ACC GAT GCC TTG ATG GAC AGC ATT CCG CTG GTC GTC TTC 288 Thr Gly Leu Thr Asp Ala Leu Met Asp Ser Ile Pro Leu Val Val Phe 85 90 95 TCG GGT CAG GTG CCC ACC TTC ATG ATC GGC ACC GAC GGT TTT CAG GAA 336 Ser Gly Gln Val Pro Thr Phe Met Ile Gly Thr Asp Gly Phe Gln Glu 100 105 110 GCC GAC ACG ATC GGC ATC ACC CGC CCC TGC ACC AAG CAC AAC TGG CTG 384 Ala Asp Thr Ile Gly Ile Thr Arg Pro Cys Thr Lys His Asn Trp Leu 115 120 125 GTG AAG GAC CCG GCG AAA CTG TCG GAA ACC ATC CAT CAG GCC TTC CAT 432 Val Lys Asp Pro Ala Lys Leu Ser Glu Thr Ile His Gln Ala Phe His 130 135 140 GTC GCC ACC TCG GGG CGG CCC GGC CCG GTc TTG ATC GAC CTG CCC AAG 480 Val Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Ile Asp Leu Pro Lys 145 150 155 160 GAC GTG CAA TTC GCC ACC GGC CAA TAC ACC GGG CCG AAG CTC GCC CGC 528 Asp Val Gln Phe Ala Thr Gly Gln Tyr Thr Gly Pro Lys Leu Ala Arg 165 170 175 GTC GAT CAC TAT CAG CCC AAG GTC AAG GGC GAC ATC GAT GCG ATC ACC 5 76 Val Asp His Tyr Gln Pro Lys Val Lys Gly Asp Ile Asp Ala Ile Thr 180 185 190 AAG CTC GTC GAG CTG ATC GAA AAG GCG GAA CGG CCG ATC TTT TAC ACC 624 Lys Leu Val Glu Leu Ile Glu Lys Ala Glu Arg Pro Ile Phe Tyr Thr 195 200 205 GGC GGC GGG GTG ATC AAC TCA GGC CCC GGG GCG TCG CAA CTC TTG CGC 672 Gly Gly Gly Val Ile Asn Ser Gly Pro Gly Ala Ser Gln Leu Leu Arg 210 215 220 GAG CTG GTC GAT GCG ACG GGC TTC CCG ATC ACC TCG ACG CTG ATG GGG 720 Glu Leu Val Asp Ala Thr Gly Phe Pro Ile Thr Ser Thr Leu Met Gly 225 230 235 240 CTG GGC GCC TAT CCG GCC TCG GGC AAG GGC TGG CTC GGC ATG CTC GGC 768 Leu Gly Ala Tyr Pro Ala Ser Gly Lys Gly Trp Leu Gly Met Leu Gly 245 250 255 ATG CAC GGG CTT TAC GAG GCC AAT CTG GCG ATG CAC GGC TGC GAT CTG 816 Met His Gly Leu Tyr Glu Ala Asn Leu Ala Met His Gly Cys Asp Leu 260 265 270 ATG ATC AAC CTC GGG GCG CGG TTC GAT GAC CGC ATC ACC GGG CGC GTC 864 Met Ile Asn Leu Gly Ala Arg Phe Asp Asp Arg Ile Thr Gly Arg Val 275 280 285 GCC GAT TTC AGC CCC CAT TCG ACC AAG GCG CAT GTC GAT CAT GTC GATATC GAC GCC 912 Ala Asp Phe Ser Pro His Ser Thr Lys Ala His Val Asp Ile Asp Ala 290 295 300 TCG TCC ATC AAC AAG ATC ATC ACT GTC GAT CTG CCG ATC GTC GGC GAT 960 Ser Ser Ile Asn Lys Ile Ile Thr Val Asp Leu Pro Ile Val Gly Asp 305 310 315 320 ATC GGC CAT GTG CTG GAA GAC CTG CTC AAG GTC TGG AAA GCG CGC GGG 1008 Ile Gly His Val Leu Glu Asp Leu Leu Lys Val Trp Lys Ala Arg Gly 325 330 335 CGC AAG GTC AAC AAG GAA GGC CTG GCG AAC TGG TGG GGC CGG ATC GAC 1056 Arg Lys Val Asn Lys Glu Gly Leu Ala Asn Trp Trp Gly Arg Ile Asp 340 345 350 GAG TGG AAG AAG GTG CGC TGC CTT GCC TAC ACC AAC TCC GAC GCG ATC 1104 Trp Lys Lys Val Arg Cys Leu Ala Tyr Thr Asn Ser Asp Ala Ile 355 360 365 ATC AAA CCG CAA TAT GCG CTG GAG CGG CTG GAC GCC CTG ACC AGG GGG 1152 Ile Lys Pro Gln Tyr Ala Leu Glu Arg Leu Asp Ala Leu Thr Arg Gly 370 375 380 CGC AAC CGC TAC ATC ACC ACC GAA GTC GGC CAG CAT CAG ATG TGG GCG 1200 Arg Asn Arg Tyr Ile Thr Thr Glu Val Gly Gln His Gln Met Trp Ala 385 390 395 400 GCG CAA TAT CTG GGC TTC GAG GCG CCC AAT CGC TGG ATG ACC TCG GGC 1248 Ala Gln Tyr Leu Gly Phe Glu Ala Pro Asn Arg Trp Met Thr Ser Gly 405 410 415 GGT TTG GGG ACG ATG GGC TAT GGT TTC CCG GCC TCG ATC GGG GTG CAG 1296 Gly Leu Gly Thr Met Gly Tyr Gly Phe Pro Ala Ser Ile Gly Val Gln 420 425 430 ATC GCG CAT CCG GAC GCG CTC GTC ATC AAC ATC GCG GGC GAG GCC AGC 1344 Ile Ala His Pro Asp Ala Leu Val Ile Asn Ile Ala Gly Glu Ala Ser 435 440 445 TGG CTG ATG AAC ATG CAG GAA ATG GGC ACG GCG ACG CAG TTC CGC ACC 1392 Trp Leu Met Asn Met Gln Glu Met Gly Thr Ala Thr Gln Phe Arg Thr 450 455 460 CCG GTG AAG CAG TTC ATC CTG AAC AAC GAG CGG CTG GGC ATG GTG CGG 1440 Pro Val Lys Gln Phe Ile Leu Asn Asn Glu Arg Leu Gly Met Val Arg 465 470 475 480 CAG TGG CAG GAT CTG CTG CAT GGC GGG CGC TAT TCG CAA AGC TGG TCG 1488 Gln Trp Gln Asp Leu Leu His Gly Gly Arg Tyr Ser Gln Ser Trp Ser 485 490 495 GAA AGC CTG CCC GAT TTC GTG AAG CTG GCG GAA AGC TTC GGC TGG AAG 1536 Glu Ser Leu Pro Asp Phe Val Lys Leu Ala Glu Ser Phe Gly Trp Lys 500 505 510 GGG ATC ATC TGC CGC GAT CCG AAG GAG CTG GAC GAT GCG ATC ATG GAG 1584 Gly Ile Ile Cys Arg Asp Pro Lys Glu Leu Asp Asp Ala Ile Met Glu 515 520 525 ATG ATC GAC TAC GAT GGT CCG GTG CTT TTT GAT TGT CTG GTC GAG AAG Met Ile Asp Tyr Asp Gly Pro Val Leu Phe Asp Cys Leu Val Glu Lys 530 535 540 CAC GAG AAC TGC TTC CCG ATG ATC CCG TCC GGC AAG CCG CAT AAC GAA 1680 His Glu Asn Cys Phe Pro Met Ile Pro Ser Gly Lys Pro His Asn Glu 545 550 555 560 ATG CTG CTC TCG GCC GAT GCG GCG GCC TTT GCG GGC GGC GCG GCG CTG 1728 Met Leu Leu Ser Ala Asp Ala Ala Ala Phe Ala Gly Gly Ala Ala Leu 565 570 575 GTG TGA 1734 Val 578

SEQ ID NO: 2 Sequence length: 558 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Rhodobacter capshratus Strain name: ATCC11166 Sequence features Characteristic symbol: CDS Location: 1-558 Method for determining characteristic: E sequence ATG GCC GCT CTG AAC ATC AAG AAA GGC TCC ACG AGC CAC TCC GCC TAT 48 Met Ala Ala Leu Asn Ile Lys Lys Gly Ser Thr Ser His Ser Ala Tyr 1 5 10 15 GAC CTG CGC GAC TTC CAT TCG GAT ATC GAA CGC AGC CAC ACG CTC GCC 96 Asp Leu Arg Asp Phe His Ser Asp Ile Glu Arg Ser His Thr Leu Ala 20 25 30 GTG ATC GTC GAG AAC GAA CCG GGG GTT CTG GCC CGG GTG ATC GGG CTG 144 Val Ile Val Glu Asn Glu Pro Gly Val Leu Ala Arg Val Ile Gly Leu 35 40 45 TTT TCC GGG CGC GGC TAC AAC ATC GAC AGC CTG ACC GTG GCG GAA ATC 192 Phe Ser Gly Arg Gly Tyr Asn Ile Asp Ser Leu Thr Val Ala Glu Ile 50 55 60 GAC CAC AAG GGG CAC CGG AGC CGC ATC ACC ATC GTC ACC CGC GGC AC C 240 Asp His Lys Gly His Arg Ser Arg Ile Thr Ile Val Thr Arg Gly Thr 65 70 75 GAA GCG GTG ATC GAA CAG ATC CGC GCG CAG CTG GGC CGG ATC GTG CCG 288 Glu Ala Val Ile Glu Gln Ile Arg Ala Gln Leu Gly Arg Ile Val Pro 80 85 90 95 GTG CAC GAG GTC CAC GAC CTG ACC ACC GAG GCG AAG GCG GTG GAG CGG 336 Val His Glu Val His Asp Leu Thr Thr Glu Ala Lys Ala Val Glu Arg 100 105 110 GAA CTG GCC TTG TTC AAG GTC ACG GCG ACG GGG GAA AAG CGG GTG GAA 384 Glu Leu Ala Leu Phe Lys Val Thr Ala Thr Gly Glu Lys Arg Val Glu 115 120 125 TCT TTG CGT CTG GCC GAT ATC TTC CGC GCC TCG GTG GTG GAC AGC ACG 432 Ser Leu Arg Leu Ala Asp Ile Phe Arg Ala Ser Val Val Asp Ser Thr 130 135 140 CTG GAA AGC TTC ATT TTC GAG ATC ACC GGC ACC TCG GAA AAG ATC GAT 480 Leu Glu Ser Phe Ile Phe Glu Ile Thr Gly Thr Ser Glu Lys Ile Asp 145 150 155 GCC TTT GCC GAA TTG ATG CGG CCG CTC GGT CTG GTC GAT GTG GCG CGC 528 Ala Phe Ala Glu Leu Met Arg Pro Leu Gly Leu Val Asp Val Ala Arg 160 165 170 175 ACC GGC GTG GCC GCG CTG GCG CGG GGC GTC TGA 558 Thr Gly Val Ala Ala Leu Ala Arg Gly Val 180 185

SEQ ID NO: 3 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA ATHGGNWMNG AYGCNTTYCARGA 23

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA CCRTGCATNC CNARCATNCC 20

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ritsuko Imai 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Toyo Kaijuku Building 8th floor Foundation for Global Environmental Technology Research (72) Inventor Yasushi Uchida 1 2-8-11 Nishishinbashi, Minato-ku, Tokyo 7th Toyo Kaijuku Building 8th floor, Research Institute for Global Environmental Technology (72) Inventor Hideaki Yukawa 2-8-11 Nishishinbashi, Minato-ku, Tokyo Toyo Kaiji Building 8th floor, Research Institute for Global Environmental Technology

Claims (7)

[Claims] 1. A DN encoding a large subunit of acetohydroxy acid synthase derived from the photosynthetic bacterium Rhodobacter capsulatus.
A. 2. Having the amino acid sequence shown in SEQ ID NO: 1,
Substitution, deletion, or insertion of amino acid residues that do not substantially impair the activity of synthesizing acetohydroxy acid from α-ketobutyric acid or pyruvate when configuring acetohydroxy acid synthase with the small subunit of acetohydroxy acid synthase The DNA according to claim 1, which encodes a large subunit of acetohydroxy acid synthase which may be possessed. 3. The DN according to claim 2, which has the base sequence shown in SEQ ID NO: 1 or a base sequence substantially the same as this.
A. 4. A DN encoding a small subunit of acetohydroxy acid synthase derived from the photosynthetic bacterium Rhodobacter capsulatus.
A. 5. Having the amino acid sequence shown in SEQ ID NO: 2,
Substitution, deletion, or insertion of amino acid residues that do not substantially impair the activity of synthesizing acetohydroxy acid from α-ketobutyric acid or pyruvate when configuring acetohydroxy acid synthase with the large subunit of acetohydroxy acid synthase The DNA according to claim 4, which encodes a small subunit of acetohydroxy acid synthase which may be possessed. 6. The DN according to claim 5, which has the base sequence shown in SEQ ID NO: 2 or a base sequence substantially identical thereto.
A. 7. A DNA encoding the large subunit of acetohydroxy acid synthase according to claim 1,
A DNA comprising the DNA encoding the small subunit of acetohydroxy acid synthase according to claim 4.
A.