JP2000300256A - Production of isoprenoid compounds by micro-organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain isoprenoid compounds that is useful for treatment of heart diseases, osteoporosis and the like in high yield by culturing a specific transformant in a medium. SOLUTION: A DNA including one or more DNAs selected from (A) a DNA encoding a protein having the activity of catalyzing the reaction forming 1- deoxy-D-xylose 5-phosphate from pyruvic acid and glyceraldehyde-5-phosphate, (B) a DNA encoding farnesyl pyrophophate synthetase, (C) a DNA encoding a protein having the amino acid sequence of the formula I or the like, (D) a DNA encoding the protein having the amino acid sequence of formula II or the like and (E) a DNA encoding the protein having the activity of catalyzing the reaction forming 2-C-methyl-D-erythritol-4-phosphate from 1-deoxy-D-xylose-5- phosphate are incorporated into a vector and the resultant vector is transduced into the host cells. Then, the resultant transformant is culture in a medium to form the objective isoprenoid compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an isoprenoid compound using a transformant derived from a prokaryote, and a method for searching for an antibacterial or herbicidal active substance involved in a non-mevalonic acid pathway.

[0002]

2. Description of the Related Art Isoprenoids are a general term for compounds having an isoprene unit having 5 carbon atoms as a basic skeleton, and are biosynthesized by polymerization of isopentenyl pyrophosphate (IPP). There are a wide variety of isoprenoid compounds in nature, many of which are useful to humans. For example, ubiquinone plays an important role in vivo as an essential component of the electron transport system, is used as a medicament effective for heart disease, and is increasing in demand in Europe and the United States as a health food.

[0003] Vitamin K is an important vitamin involved in the blood coagulation system, and is used as a hemostatic agent. Recently, its involvement in bone metabolism has been suggested, and its application to the treatment of osteoporosis is expected. Menaquinone is licensed as a pharmaceutical. In addition, ubiquinone and vitamin K have an inhibitory effect on the adhesion of shellfish, and are expected to be applied to paints for preventing adhesion of shellfish.

Further, a carotenoid having a carbon number of 4
A compound having an isoprene skeleton of 0 has an antioxidant effect, and some compounds such as β-carotene, astaxanthin, and cryptoxanthin are expected to have cancer prevention and immunostimulatory activities. As described above, isoprenoid compounds contain many useful substances, and if these inexpensive production methods are established, it is thought that there will be great social and medical benefits.

[0005] Production of isoprenoid compounds by fermentation has been studied for some time, and attempts have been made to examine culture conditions, breed strains by mutagenesis, and to improve production by genetic engineering techniques. However, its effect is limited to individual compound species, and no effective method is known for isoprenoid compounds in general. Isopentenyl pyrophosphate (IPP), the basic skeleton unit of isoprenoid compounds, has been proven to be biosynthesized from acetyl-CoA via mevalonic acid (mevalonic acid pathway) in eukaryotes such as animals and yeasts. .

In the mevalonate pathway, 3-hydroxy-3-
Methylglutaryl-CoA (HMG-CoA) reductase is considered to be rate-limiting [Mol. Biol. Cell, 5 , 655 (199
4)], attempts have been made to increase the productivity of carotenoids by increasing the expression of HMG-CoA reductase in yeast [Misawa et al.
7)].

[0007] In prokaryotes, there is no evidence demonstrating the existence of the mevalonate pathway, and another pathway, namely, 1-deoxy-D-xylulose 5-phosphate formed by the condensation of pyruvate and glyceraldehyde 3-phosphate. A non-mevalonate pathway in which IPP is biosynthesized via is found in many prokaryotes [Biochem. J., 295 , 517 (1993)].
^From the experiment using ¹³ C-labeled substrate, 1-deoxy-D-
It has been suggested that xylulose 5-phosphate is converted to IPP via 2-C-methyl-D-erythritol 4-phosphate [Tetrahedron Lett. 38 , 4769 (199
7)].

In Escherichia coli, the enzyme 1-deoxy-, which condenses pyruvate and glyceraldehyde 3-phosphate to biosynthesize 1-deoxy-D-xylulose 5-phosphate.
A gene encoding D-xylulose 5-phosphate synthase (DXS) has been identified [Proc. Natl. Acad. Sc.
i. USA., 94 , 12857 (1997)]. The gene contains four Os, including ispA, which encodes farnesyl pyrophosphate synthase.
It is included in the RF operon.

Further, in Escherichia coli, 1-deoxy-
It is known that there is an activity to convert D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate [Tetrahedron Lett. 39 , 4509 (1998)].
There is no description or suggestion about manipulating these genes contained in the operon to improve the productivity of isoprenoid compounds at present. Although knowledge about the non-mevalonate pathway in prokaryotes is slowly accumulating, the enzymes involved and many of the genes encoding them are still unknown.

In photosynthetic bacteria, chorismate is converted to 4-
There is known a method for efficiently producing ubiquinone-10 by introducing a gene for the enzyme ubiC that converts to hydroxybenzoate (ubiC gene) and a gene for p-hydroxybenzoate transferase (ubiA) (Japanese Unexamined Patent Publication No. Hei 8- 107789), there is no example of improving the productivity of isoprenoid compounds by manipulating the enzyme gene of the non-mevalonate pathway.
Furthermore, there is no knowledge about how a prokaryote is affected by inhibiting a reaction on the non-mevalonic acid pathway by mutation, drug treatment, or the like.

[0011]

An object of the present invention is to produce isoprenoid compounds useful for medicines, health foods, shellfish adhesion preventive paints, etc. for the purpose of heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation and the like. A DNA containing one or more DNAs involved in synthesis is incorporated into a vector, the resulting recombinant DNA is introduced into a prokaryotic host cell, and the resulting transformant is cultured in a medium, and A method for producing an isoprenoid compound, comprising producing and accumulating an isoprenoid compound and collecting the isoprenoid compound from the culture, and a method for producing a DNA encoding a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound. The resulting recombinant DNA is introduced into a host cell, and the resulting transformant is introduced into a medium. Feeding the protein, accumulating and producing the protein in a culture, and collecting the protein from the culture, to provide a method for producing the protein, the protein, and a DNA encoding the protein. . Another object of the present invention is to provide a method for searching for an antibacterial and herbicidally active substance, which is characterized by searching for a substance that inhibits an enzymatic reaction on a non-mevalonate pathway.

[0012]

Means for Solving the Problems The present inventors have searched for DNA that can improve the productivity of isoprenoids by prokaryotes, and improved the productivity of isoprenoids by introducing the obtained DNA into prokaryotes. The inventors have found that the present invention can be performed, and have completed the present invention.

That is, the first invention of the present application provides the following (a), (b), (c), (d), (e) and (f).
A DNA containing one or more DNAs selected from the following is incorporated into a vector, the obtained recombinant DNA is introduced into prokaryotic host cells, and the obtained transformant is cultured in a medium.
A method for producing an isoprenoid compound, comprising producing and accumulating an isoprenoid compound in a culture, and collecting the isoprenoid compound from the culture.

(A) is a DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate; (b) farnesyl DNA encoding pyrophosphate synthase, (c) is a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 3, or one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein. Encoding a protein comprising an amino acid sequence represented by the following sequence and having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound; (d) a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4; Has one or several amino acids deleted, substituted or Consists pressurized amino acid sequence, and DNA encoding a protein having an activity that can improve the biosynthesis efficiency of isoprenoid compounds,
(E) is from 1-deoxy-D-xylulose 5-phosphate to 2-C
DNA encoding a protein having an activity of catalyzing a reaction that produces -methyl-D-erythritol 4-phosphate, wherein (f) is selected from (a), (b), (c), (d) and (e) And DNA encoding a protein that hybridizes under stringent conditions with the DNA to be obtained and has substantially the same activity as the protein encoded by the selected DNA.

In the present specification, deletion, substitution or addition of an amino acid can be carried out by a site-directed mutagenesis method which is a well-known technique prior to the filing of the application. It means a number, for example, 1 to 5 amino acids that can be deleted, substituted or added by a specific mutagenesis method.

One or several such amino acids are deleted,
The protein consisting of the substituted or added amino acid sequence can be obtained by molecular cloning: A Laboratory
Manual (Molecular Cloning, A laboratory manua
l), 2nd edition (Sambrook, Frits
ch), edited by Maniatis, Cold Spring Harbor Laboratory Press (Cold Spring Ha)
rbor Laboratory Press), 1989 (hereinafter abbreviated as Molecular Cloning Second Edition)], Current Pr.
otocols in Molecular Biology, John Wiley & Sons (1
987-1997), NucleicAcids Research, 10 , 6487 (198
2), Proc. Natl. Acad. Sci., USA, 79 , 6409 (1982), Ge
ne, 34 , 315 (1985), Nucleic Acids Research, 13 , 44
31 (1985), Proc. Natl. Acad. Sci USA, 82 , 488 (198
It can be prepared according to the method described in 5) and the like.

In the above, as a DNA encoding a protein that catalyzes a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate, for example, SEQ ID NO: 1, 26 or 28
Encoding a protein having the amino acid sequence described in 1.
A or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequences of these proteins, and 1-deoxy-D-xylulose 5 comprising pyruvic acid and glyceraldehyde 3-phosphate. -A DNA encoding a protein having an activity of catalyzing a reaction to generate phosphate, and the like.

Specific examples include a DNA having the nucleotide sequence of SEQ ID NO: 6, a DNA having the nucleotide sequence of SEQ ID NO: 27 or 29, and the like. As the DNA encoding farnesyl pyrophosphate synthase, for example, DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 or deletion or substitution or addition of one or several amino acids in the amino acid sequence of the protein And DNA encoding a protein having a farnesyl pyrophosphate synthase activity. As a specific example, a DNA having the base sequence of SEQ ID NO: 7 and the like can be mentioned.

Specific examples of the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 3 include a DNA having the base sequence of SEQ ID NO: 8, and the like. Specific examples of the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 4 include a DNA having the base sequence of SEQ ID NO: 9, and the like.

As a DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, for example, SEQ ID NO: 5 or DNA encoding a protein having the amino acid sequence described in No. 30; an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein; and 1-deoxy-D-xylulose 5 And DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from -phosphate.

As a specific example of the DNA, SEQ ID NO: 1
DNA having the base sequence described in 0 or 31 can be mentioned. The term "DNA that hybridizes under stringent conditions" refers to the use of a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like, using the DNA or a fragment of the DNA as a probe. Means a DNA obtained by colony or plaque-derived DNA or a filter on which a fragment of the DNA is immobilized.
After performing hybridization at 65 ° C. in the presence of １．０1.0 M NaCl, the SSC solution of about 0.1 to 2 times (the composition of the 1 × concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate). ) Can be used to identify DNAs that can be identified by washing the filter at 65 ° C.

Hybridization can be carried out according to the method described in Molecular Cloning, Second Edition and the like. As a hybridizable DNA, specifically, a DNA having at least 70% or more homology with a nucleotide sequence selected from SEQ ID NOs: 1, 2, 3, 4 and 5, and preferably a DN having at least 90% homology
A can be given.

Examples of the isoprenoid compound include ubiquinone, vitamin K ₂ , carotenoid and the like. The second invention of the present application provides the following (a) and (b)
And a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound selected from (c).

(A) is a protein having the amino acid sequence of SEQ ID NO: 3, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein; (b) Is a protein having the amino acid sequence of SEQ ID NO: 4, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and (c) is a protein having the amino acid sequence of SEQ ID NO: 5. It is a protein having an amino acid sequence or a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein.

According to a third aspect of the present invention, there is provided a transformant obtained by incorporating a DNA encoding the protein of the second aspect into a vector, introducing the obtained recombinant DNA into a host cell, Is cultured in a culture medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture, thereby producing a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound. Is the way. In the above, a microorganism belonging to the genus Escherichia , a microorganism belonging to the genus Rhodobacter or E
Microorganisms belonging to the genus rwinia can be mentioned.

The fourth invention of the present application provides the following (a):
DNA encoding a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound selected from (b), (c) and (d). (A) is a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 3, (b) is a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4, and (c) is a DNA encoding the protein having the amino acid sequence of SEQ ID NO: 5. DNA encoding a protein having
(D) is a DNA having the nucleotide sequence of SEQ ID NO: 8,
(E) is a DNA having the nucleotide sequence of SEQ ID NO: 9,
(F) is a DNA having the nucleotide sequence of SEQ ID NO: 10,
(G) is a DNA that hybridizes with the DNA according to any one of (a) to (f) under stringent conditions.

The fifth invention of the present application relates to a method of producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde triphosphate, followed by 2-C-methyl-D-phosphate.
A substance that inhibits a reaction of a protein having an activity of an enzyme selected from enzymes present on a non-mevalonate pathway for biosynthesizing isopentenyl pyrophosphate via biosynthesis of erythritol 4-phosphate. This is a method for searching for a substance having antibacterial activity, which is characterized by searching.

The sixth invention of the present application relates to a method of producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde triphosphate, followed by 2-C-methyl-D-phosphate.
A substance that inhibits a reaction of a protein having an activity of an enzyme selected from enzymes present on a non-mevalonate pathway for biosynthesizing isopentenyl pyrophosphate via biosynthesis of erythritol 4-phosphate. This is a method for searching for a substance having herbicidal activity, characterized by searching.

In the above inventions 5 and 6, examples of the protein include the following proteins (a) and (b). (A) Pyruvic acid and glyceraldehyde 3-
A protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from phosphoric acid, (b)
Is from 2-deoxy-D-xylulose 5-phosphate to 2-
It is a protein having an activity of catalyzing a reaction producing C-methyl-D-erythritol 4-phosphate.

In the above, as a protein which catalyzes a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, for example, a protein having the amino acid sequence of SEQ ID NO: 1 ,
Or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and which is composed of pyruvic acid and glyceraldehyde 3-phosphate and 1-deoxy-D-xylulose 5-phosphate.
A protein having an activity of catalyzing a reaction for producing phosphoric acid can be given.

As a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, for example, an amino acid sequence represented by SEQ ID NO: 5 is used. Protein,
Alternatively, the protein comprises an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein, and comprises 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol A protein having an activity of catalyzing a reaction for producing 4-phosphate can be given. A seventh invention of the present invention is a substance having an antibacterial activity obtained by the search method of the fifth invention. However, known substances obtained by the search method are not included in the present invention.

The present inventors have developed 2-C-methyl-D-erythritol 4-phosphate which is a product of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction and the reaction assumed in this enzyme reaction. Intermediate and fosmidomycin [3- (N-formyl-N-hydroxyamino) propylphosphonic ac
Focusing on structural similarity to id], based on the assumption that fosmidomycin has 1-deoxy-D-xylulose 5-phosphate reductoisomerase inhibitory activity and also has antibacterial activity, An experiment was conducted on the search method of the fifth invention shown in Example 10 to find that fosmidmycin was a substance having the inhibitory activity and also having an antibacterial activity. However, the known fosmidmycin is excluded from the present invention.

An eighth aspect of the present invention is a substance having a herbicidal activity obtained by the search method of the sixth aspect. As described above, known substances obtained by the search method are not included in the present invention. Hereinafter, the present invention will be described in detail.

[0034]

DETAILED DESCRIPTION OF THE INVENTION Acquisition of DNA encoding a protein involved in biosynthesis of isoprenoid compound (1) Acquisition of DNA encoding a protein involved in biosynthesis of isoprenoid compound utilizing the nucleotide sequence of DNA encoding DXS (DXS gene) Nucleotide sequence information of the chromosome and DXS gene of Escherichia coli [Proc. Natl. Acad. Sci. USA., 94 , 12857 (199
7)], the DNA region containing the DXS gene from Escherichia coli or the DNA region of the gene near the DXS gene was cloned by PCR (Science, 230 , 1350 (1985)).
Can be obtained.

As base sequence information including the DXS gene,
For example, the base sequence described in SEQ ID NO: 11 can be mentioned. As a method for obtaining the DNA region containing the DXS gene, the following method can be specifically mentioned.

Escherichia coli, for example, E. coli XL1-Blue strain (available from Toyobo) is transformed into a medium suitable for Escherichia coli, for example, LB liquid medium (10 g of bactotripton (Difco), 5 g of yeast extract (Difco), Culture medium adjusted to pH 7.2 containing 1 g of NaCl in 1 liter of water] according to a conventional method. After the culture, cells are obtained from the culture by centrifugation.

Chromosomal DNA is obtained from the obtained cells by a known method (eg, Molecular Cloning, 2nd edition).
Is isolated. Using the nucleotide sequence information set forth in SEQ ID NO: 11, a sense primer and an antisense primer containing the DXS gene or containing the nucleotide sequence corresponding to the DNA region of the gene adjacent to the DXS gene were converted to DN.
A: Synthesize using a synthesizer.

After amplification by the PCR method, an appropriate restriction enzyme site, for example, a restriction enzyme site such as Bam HI or Eco RI is provided at the 5 'end of each of the sense primer and the antisense primer so that the amplified DNA fragment can be introduced into a plasmid. It is preferable to add them.

The combination of the sense primer and the antisense primer includes, for example, SEQ ID NOs: 12 and 1
3, SEQ ID NOs: 14 and 15, SEQ ID NOs: 12 and 1
6, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 1
3, DNAs having the nucleotide sequence of the combination of SEQ ID NOs: 22 and 23, and the like.

Using chromosomal DNA as a template, these primers, TaKaRa LA-PCR ^™ Kit Ver.2 (Takara Shuzo) or E
PCR is performed using DNA Thermal Cycler (PerkinElmer Japan) using xpand ^™ High-Fidelity PCR System (Boehringer Mannheim) or the like.

As the conditions for PCR, the primer
kb or less for 30 seconds at 94 ° C.
One cycle consists of a reaction step consisting of 5 ° C. for 30 seconds to 1 minute and 72 ° C. for 2 minutes. In the case of a DNA fragment exceeding 2 kb, one reaction step consisting of 98 ° C. for 20 seconds and 68 ° C. for 3 minutes is performed. After 30 cycles, 7
Conditions for reacting at 2 ° C. for 7 minutes can be given.

The amplified DNA fragment is cleaved at the same site as the restriction enzyme site provided by the above primer with an appropriate vector capable of being amplified in Escherichia coli, and then subjected to agarose electrophoresis, sucrose density gradient ultracentrifugation or the like. The DNA fragment is fractionated and collected.

Using the recovered DNA fragment, a conventional method, for example,
Molecular Cloning Second Edition, Current Protocol
s in Molecular Biology, Supplement 1-38, John Wil
ey & Sons (1987-1997), DNA Cloning 1: Core Techniqu
es, A PracticalApproach, Second Edition, Oxford Un
iversity Press (1995) etc., or a commercially available kit such as SuperScript Plasmid System for
A cloning vector was prepared using cDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies) or ZAP-cDNA Synthesis Kit (manufactured by Stratagene), and Escherichia coli, for example, E. coli DH5α strain ( (Available from Toyobo)
Is transformed.

As a cloning vector for transforming Escherichia coli, any phage vector or plasmid vector can be used as long as it can autonomously replicate in Escherichia coli K12 strain. You may. In particular,
ZAP Express (Strategies, 5 , 58, manufactured by Stratagene)
(1992)), pBluescript II SK (+) (Nucleic Acids Rese
arch, 17 , 9494 (1989)], Lambda ZAP II (Stratagene), λgt10, λgt11 [DNA Cloning, APractica
l Approach, 1 , 49 (1985)], λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U
(Manufactured by Pharmacia), pcD2 [H. Okayamaand P. Berg; M
ol. Cell. Biol., 3 , 280 (1983)], pMW218 (manufactured by Wako Pure Chemical Industries), pUC118 (manufactured by Takara Shuzo), pEG400 [J. Bac., 172 ,
2392 (1990)] and pQE-30 (manufactured by QIAGEN).

From the obtained transformant, the desired DN
A containing plasmid can be prepared by a conventional method, for example, Molecular Cloning, Second Edition, Current Protocols in Molecul.
ar Biology, Supplement 1-38, John Wiley & Sons (1
987-1997), DNA Cloning 1: Core Techniques, A Practi
cal Approach, Second Edition, Oxford University Pr
It can be obtained by the method described in ess (1995) and the like.

According to the method, DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate, farnesyl pyrophosphate synthase , A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 3, a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4, etc., and a plasmid containing one or more of these DNAs be able to.

As the plasmid, for example, the above DNA
Plasmid pADO-1 containing the DNA having the nucleotide sequence of SEQ ID NO: 6, or plasmid pDXS-1 or pQEDXS-1, plasmid pISP-1 containing the DNA having the nucleotide sequence of SEQ ID NO: 7, and SEQ ID NO: 8 Plasmid pX containing DNA having the nucleotide sequence of
SE-1 and plasmid pTFE-1 containing a DNA having the nucleotide sequence of SEQ ID NO: 9 and the like.

Using a base sequence of a DNA fragment derived from Escherichia coli inserted into these plasmids, a homolog of the DNA is obtained from another prokaryote, for example, a microorganism belonging to the genus Rhodobacter by the same method as described above. Can be.

(2) Obtaining a DNA encoding a protein having an activity capable of complementing a methyl erythritol auxotroph mutant of Escherichia coli (a methyl erythritol auxotrophic complement gene) Obtaining an Escherichia coli methyl erythritol auxotroph The E. coli W3110 strain (ATCC14948) is cultured according to a conventional method.

After culturing, cells are obtained from the resulting culture by centrifugation. The cells are placed in a suitable buffer, for example,
After washing with a 0.05 M trismaleic acid buffer (pH 6.0) or the like, the cells are suspended in the same buffer so that the cell concentration becomes 10 ⁴ to 10 ¹⁰ cells / ml.

Using the suspension, a mutation treatment is carried out by a conventional method. As a conventional method, for example, a method in which NTG is added to the suspension so as to have a final concentration of 600 mg / l, and the suspension is kept at room temperature for 20 minutes to perform mutation treatment can be mentioned. The mutated suspension is cultured in a minimal agar medium supplemented with 0.05-0.5% methylerythritol.

Examples of the minimal agar medium include a medium obtained by adding agar to an M9 medium (Molecular Cloning, 2nd edition). Methylerythritol can be chemically synthesized according to the method described in Tetrahedron Letters, 38 , 35, 6184 (1997).

After culturing, the colonies that grew and formed were removed from the minimal agar medium and methyl erythritol in 0.05 to 0.5.
%, The strain showing the requirement for methylerythritol, that is, a strain that can grow on a minimal agar medium containing methylerythritol but cannot grow on a minimal agar medium is selected as a target mutant strain. The ME7 strain can be mentioned as a methylerythritol-requiring mutant strain obtained by the operation.

Acquisition of Methylerythritol-Required Complementary Gene Escherichia coli, for example, E. coli W3110 strain (ATCC 14948) is inoculated into a culture medium, for example, LB liquid medium, and cultured to a logarithmic growth phase according to a conventional method. After the culture, the obtained culture is centrifuged to collect the cells.

A chromosomal DNA is isolated and purified from the obtained cells according to a conventional method (for example, the method described in Molecular Cloning, Second Edition). Chromosomal DN obtained by isolating and purifying chromosomal DNA obtained by the method described in (1) above
A can also be used. An appropriate amount of the chromosomal DNA is partially digested with an appropriate restriction enzyme, for example, Sau 3AI, and the digested DNA fragment obtained is subjected to a conventional method, for example, sucrose density gradient ultracentrifugation (26,000 rpm, 26,000 rpm).
(0 ° C., 20 hr).

The size obtained by the fractionation is 4 to 6 k
A chromosomal genomic library is prepared by ligating the DNA fragment b with a vector digested with an appropriate restriction enzyme, for example, pMW118 (Nippon Gene). Using the prepared chromosome library, the methylerythritol-required mutant strain isolated above, for example, the ME7 strain, is transformed according to a conventional method (for example, the method described in Molecular Cloning, Second Edition).

The transformant is applied to a minimal agar medium containing a drug corresponding to the drug resistance gene of the vector, for example, M9 agar medium containing 100 μg / l of ampicillin, and cultured at 37 ° C. overnight. By this method, a transformant in which the requirement for methylerythritol has been restored can be selected.

From the obtained transformant, a plasmid is extracted by a conventional method. As a plasmid capable of restoring the requirement for methylerythritol, for example, pMEW
73, pQEDXR can be given. The nucleotide sequence of the DNA introduced into the plasmid is determined.

As the base sequence determined by the method,
A sequence including the base sequence of the yaeM gene shown in SEQ ID NO: 10 and the like can be mentioned. Utilizing the obtained nucleotide sequence information of the yaeM gene,
A homolog of the yaeM gene can be obtained by the same method as described above.

II. Production of a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound In order to express the DNA obtained as described above in a host cell, first, the DNA fragment of interest is converted into a restriction enzyme Or a DNA degrading enzyme, into a DNA fragment of an appropriate length containing the gene, inserted into the expression vector downstream of the promoter, and then inserted the expression vector into a host cell suitable for the expression vector. Introduce inside.

As the host cell, any cell that can express the target gene can be used. For example, Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus,
Bacteria belonging to the genus Microbacterium, Kluyveromyces, Saccharomyces, Schizosaccharomyces,
Examples include yeast, animal cells, insect cells, and the like belonging to the genus Trichosporon, Siwaniomyces and the like.

As the expression vector, those which can replicate autonomously in the above-mentioned host cells or can be integrated into a chromosome, and which contain a promoter at a position where the above-mentioned DNA of interest can be transcribed are used. When a bacterium or the like is used as a host cell, an expression vector for expressing the DNA is capable of autonomous replication in the bacterium, and at the same time, is a recombinant vector comprising a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence. It is preferably a vector. A gene that controls the promoter may be included.

Examples of expression vectors include pBTrp2,
pBTac1, pBTac2 (both commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invit
rogen), pGEMEX-1 (Promega), pQE-8 (QIAGE
N), pQE-30 (QIAGEN), pKYP10 (JP-A-58-11)
0600), pKYP200 [Agricultural Biological Chemistr
y, 48 , 669 (1984)], pLSA1 (Agric. Biol. Chem., 53 ,
277 (1989)), pGEL1 (Proc. Natl. Acad. Sci. USA,
82 , 4306 (1985)), pBluescriptII SK +, pBluescriptI
ISK (-) (Stratagene), pTrS30 (FERMBP-5407), pT
rS32 (FERM BP-5408), pGEX (Pharmacia), pET-3
(Novagen), pTerm2 (US4686191, US4939094, US51
60735), pSupex, pUB110, pTP5, pC194, pUC18 (gene,
33 , 103 (1985)), pUC19 (Gene, 33 , 103 (1985)), p
STV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118
(Manufactured by Takara Shuzo), pPA1 (JP-A-63-233798), pEG400
[J. Bacteriol., 172 , 2392 (1990)], pQE-30 (QIAGEN
And the like).

The promoter may be any promoter as long as it can be expressed in a host cell. For example, tr
p promoter (P trp ), lac promoter (P lac ),
P _L promoter, P _R promoter, promoter derived from the P _SE such as promoter, E. coli, phage and the like, SPO1
Promoters, SPO2 promoters, penP promoters and the like can be mentioned. In addition, a promoter (P trp x2) in which two P trps are connected in series, a promoter artificially designed and modified such as a tac promoter, a letI promoter, and a lacT7 promoter can also be used.

The ribosome binding sequence is not particularly limited as long as it can be expressed in a host cell, and a suitable distance (for example, 6 to 18 bases) is provided between the Shine-Dalgarno sequence and the initiation codon. It is preferable to use a plasmid adjusted to ()). Target DN
Although the expression of A does not necessarily require a transcription termination sequence,
Preferably, it is desirable to arrange a transcription termination sequence immediately below the structural gene.

As host cells,EscherichiaGenus,Coryn
ebacteriumGenus,BrevibacteriumGenus,BacillusGenus,Microb
acteriumGenus,SerratiaGenus,PseudomonasGenus,Agrobacteri
umGenus,AlicyclobacillusGenus,AnabaenaGenus,Anacystis
Genus,ArthrobacterGenus,AzobacterGenus,ChromatiumGenus,Erw
iniaGenus,MethylobacteriumGenus,PhormidiumGenus,Rhodobac
terGenus,RhodopseudomonasGenus,RhodospirillumGenus,Scene
desmunGenus,StreptomycesGenus,SynnecoccusGenus,Zymomonas
Microorganisms belonging to the genus and the like can be given, preferably,
EscherichiaGenus,CorynebacteriumGenus,Brevibacterium
Genus,BacillusGenus,PseudomonasGenus,AgrobacteriumGenus,Al
icyclobacillusGenus,AnabaenaGenus,AnacystisGenus,Arthrob
acterGenus,AzobacterGenus,ChromatiumGenus,ErwiniaGenus,Met
hylobacteriumGenus,PhormidiumGenus,RhodobacterGenus,Rhod
opseudomonasGenus,RhodospirillumGenus,ScenedesmunGenus,S
treptomycesGenus,SynnecoccusGenus,ZymomonasBelongs to the genus
Microorganisms and the like can be mentioned.

Specific examples of the microorganism include, for example, Escher
ichia coli XL1-Blue, Escherichiacoli XL2 -Blue, Esc
herichia coli DH1, Escherichia coli DH5α, Escheri
chiacoli MC1000 , Escherichia coli KY3276, Escheric
hia coli W1485, Escherichia coli JM109, Escherichi
a coli HB101, Escherichia coli No.49, Escherichia
coli W3110, Escherichia coli NY49, Escherichia col
i MP347, Escherichia coli NM522, Bacillussubtili
s , Bacillusamyloliquefacines , Brevibacteriumammmon
iagenes , Brevibacterium immariophilum ATCC14068, Br
evibacteriumsac charolyticum ATCC14066, Brevibacter
iumflavum ATCC14067, Brevibacteriumlactofermentum
ATCC13869, Corynebacterium glutamicum ATCC13032, C
orynebacterium glutamicum ATCC14297, Corynebacteri
um acetoacidophilum ATCC13870, Microbacteriumammon
iaphilum ATCC15354, Serratiaficaria , Serratiafonti
cola , Serratialiquefaciens , Serratiamarcescens , Ps
eudomonas sp.D -0110, Agrobacterium radiobacter , A
grobacterium rhizogenes , Agrobacterium rubi , Anaba
enacylindrica , Anabaenadoliolum , Anabaenaflos-aqua
e , Arthrobacteraurescens , Arthrobactercitreus , Art
hrobacterglobformis , Arthrobacter hydrocarboglutami
cus , Arthrobactermysorens , Arthrobacternicotiana
e , Arthrobacter paraffineus , Arthrobacter protophorm
iae , Arthrobacterroseoparaffinus , Arthrobactersulf
ureus , Arthrobacterureafaciens , Chromatiumbuderi ,
Chromatiumtepidum , Chromatiumvinosum , Chromatiumwa
rmingii , Chromatium fluviatile , Erwinia uredovora ,
Erwinia carotovora , Erwinia ananas , Erwinia herbic
ola , Erwinia punctata , Erwinia terreus , Methylobac
teriumrhodesianum , Methylobacteriumextorquens , Pho
rmidium sp.ATCC29409, Rhodobacter capsulatus , Rho
dobactersphaeroides , Rhodopseudomonasblastica , Rho
dopseudomonasmarina , Rhodopseudomonaspalustris , Rh
odospirillumrubrum , Rhodospirillumsalexigens , Rhod
ospirillumsalinarum , Streptomycesambofaciens , Stre
ptomycesaureofaciens , Streptomycesaureus , Streptom
ycesfungicidicus , Streptomycesgriseochromogenes , S
treptomycesgriseus , Streptomyceslividans , Streptom
ycesolivogriseus , Streptomycesrameus , Streptomyces
tanashiensis , Streptomycesvinaceus , Zymomonas mobi
lis etc. can be given.

As a method for introducing a recombinant vector, any method for introducing DNA into the above host cells can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)),
Protoplast method (JP-A-63-2483942), or Gene,
17 , 107 (1982) and Molecular & General Genetics, 168 ,
111 (1979).

When yeast is used as a host cell, as an expression vector, for example, YEp13 (ATCC3711)
5), YEp24 (ATCC37051), YCp50 (ATCC3741)
9), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast. For example, PHO5 promoter, PGK promoter, GAP promoter, A
DH promoter, gal1 promoter, gal10
Promoter, heat shock protein promoter, M
Promoters such as the Fα1 promoter and CUP1 promoter can be mentioned.

As host cells, Saccharomyces cerevisae , Schizosaccharomyces pombe , Kluyveromyces lactis , Trichosporonplulu
s ), Schwanniomycesa
lluvius ) and the like.

As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, electroporation [Methods.
nzymol., 194 , 182 (1990), spheroplast method (Pro
Natl. Acad. Sci. USA, 75 , 1929 (1978)], lithium acetate method [J. Bacteriol., 153 , 163 (1983)], Proc. Nat.
l. Acad. Sci. USA, 75 , 1929 (1978).

When animal cells are used as host cells, expression vectors such as pcDNAI and pcD
M8 (commercially available from Funakoshi), pAGE107 [JP
3-22979; Cytotechnology, 3 , 133, (1990)], pAS
3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329 ,
840, (1987)], pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103
[J. Biochem., 101 , 1307 (1987)], pAGE210 and the like.

As the promoter, any promoter that can be expressed in animal cells can be used. For example, IE (imme) of cytomegalovirus (human CMV) can be used.
diateearly) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter,
SRα promoter and the like can be mentioned. Further, an enhancer of the IE gene of human CMV may be used together with the promoter. Namalba cells, as host cells,
HBT5637 (JP-A-63-299), COS1 cells, COS7 cells, CHO cells and the like can be mentioned.

As a method for introducing a recombinant vector into animal cells, any method capable of introducing DNA into animal cells can be used, for example, an electroporation method [Cytotechnology, 3 , 133 (1990)], a calcium phosphate method. (Japanese Patent Application Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci., USA, 84 , 7413 (1987)], virolo
gy, 52 , 456 (1973). Acquisition and culturing of transformants are described in JP-A-2-227.
075 or JP-A-2-257891.

When an insect cell is used as a host, for example, a baculovirus expression vectors a laboratory manual (Baculovirus Expr.
ession Vectors, A Laboratory Manual), current
Protocols in Molecular Biology Supplement 1-38 (1987-1997), Bio / Technology, 6 , 47
(1988) and the like, the protein can be expressed.

That is, after a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in an insect cell culture supernatant, the recombinant virus is further infected into insect cells to express the protein. Can be done. Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pB139
luBacIII (both manufactured by Invitrogen) and the like.

Examples of the baculovirus include, for example, Autographa californica nuclea polyhedrosis virus, which is a virus that infects night insects.
(Autographa californica nuclear polyhedrosis viru
s) and the like can be used. Spodop as an insect cell
terafrugiperda ovarian cells Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Co.
mpany), New York (1992)], Trich
Oplusiani ovary cells such as High5 (manufactured by Invitrogen) can be used.

Examples of the method for co-transferring the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc.
Natl. Acad. Sci. USA, 84 , 7413 (1987)]. As a method for expressing the gene, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning Second Edition, etc., in addition to direct expression.

When expressed by yeast, animal cells or insect cells, a protein having a sugar or a sugar chain added thereto can be obtained. Recombinant D incorporating the above DNA
By culturing the transformant having NA in a medium, producing and accumulating a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound in the culture, and collecting the protein from the culture, A protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound can be produced.

A method for culturing a transformant for producing a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound of the present invention in a medium can be carried out according to a usual method used for culturing a host. . When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism. ,
Either a natural medium or a synthetic medium may be used as long as the transformant can be cultured efficiently.

The carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose,
Sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.

Examples of the nitrogen source include ammonium salts of various inorganic acids and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn corn. Chip liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.

As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
The culturing temperature is preferably 15 to 40 ° C., and the culturing time is usually 16
Hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0084] If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-
the β-D- thiogalactopyranoside (IPTG) or the like, tr
When culturing a microorganism transformed with an expression vector using the p promoter, indoleacrylic acid (IA
A) may be added to the medium.

As a medium for culturing a transformant obtained by using animal cells as host cells, R.R.
PMI1640 medium [The Journal of the American Me
dical Association, 199 , 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], DMEM medium [Virology, 8 , 396 (1959)], 199 medium [Proceed
ing of the Society for the Biological Medicine, 73 ,
1 (1950)] or a medium obtained by adding fetal bovine serum or the like to such a medium.

The cultivation is usually carried out at pH 6-8, 30-40 ° C.
This is performed for 1 to 7 days under conditions such as the presence of 5% CO ₂ . Also,
If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

As a medium for culturing a transformant obtained by using an insect cell as a host cell, generally used T
NM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400, ExCell405 (all from JRH Biosciences), Grace's Insect Medium
[Grace, TCC, Nature, 195 , 788 (1962)] and the like can be used.

The cultivation is usually carried out at pH 6 to 7, at 25 to 30 ° C. for 1 to 5 days. If necessary, an antibiotic such as gentamicin may be added to the medium during the culture. From a culture of the transformant of the present invention, a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound of the present invention can be isolated and purified by a conventional enzyme isolation and purification method. Good.

For example, when the protein of the present invention is expressed in a dissolved state in the cells, the cells are recovered by centrifugation after suspension of the culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, and the like. The cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Diethylaminoethyl (DEAE)-Sepharose, DIAION
Anion exchange chromatography using a resin such as HPA-75 (Mitsubishi Kasei), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), butyl sepharose, phenyl sepharose, etc. Purified standard using hydrophobic chromatography method using resin, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc. alone or in combination Can be obtained.

When the protein is expressed by forming an insoluble form in the cells, the cells are similarly recovered, crushed, and centrifuged to obtain a precipitate fraction obtained by a conventional method. After recovering the protein, the insoluble form of the protein is solubilized with a protein denaturant. After diluting the lysate into a solution containing no protein denaturing agent or a solution in which the concentration of the protein denaturing agent is so low that the protein is not denatured, or dialyzed to form the protein into a normal three-dimensional structure, A purified sample can be obtained by the same isolation and purification method.

When the protein of the present invention or a derivative such as a modified sugar thereof is secreted extracellularly, the protein or a derivative such as a sugar chain adduct thereof can be recovered in the culture supernatant. That is, the culture is treated by a method such as centrifugation as described above to obtain a soluble fraction, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to. Examples of the protein obtained in this manner include a protein having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOS: 1 to 5.

The protein expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Also, Kuwawa Trading (US Advanced ChemTech), Perkin Elmer Javan (US Perkin-Elmer), Pharmacia Biotech (Sweden Pharmacia Biotech), Aloka (U.S. Protein Technology Instrument), Kurabo Industries (US Synthecell-US) Vega), Nippon Perceptive Limited (U.S. PerSeptive), Shimadzu Corporation, or other peptide synthesizers.

III. Production of isoprenoid compounds II. The transformant obtained in the above step II. The isoprenoid compound can be produced by culturing according to the method described in the above, producing and accumulating the isoprenoid compound in the culture, and collecting the isoprenoid compound from the culture.

[0094] By this culture, isoprenoid compounds such as ubiquinone, vitamin K ₂ and carotenoid can be produced. As a specific example, for example, Escheric
Production of ubiquinone-8 or menaquinone-8 using a microorganism belonging to the genus hia as a transformant, production of ubiquinone-10 using a microorganism belonging to the genus Rhodobacter as a transformant, Arth
robacter genus in the production of vitamin K ₂ in which the microorganism is a transformant belonging, production of astaxanthin that a microorganism belonging to the genus Agrobacterium and transformant, lycopene a microorganism belonging to the genus Erwinia were the transformant, beta-carotene, zeaxanthin And the like.

After completion of the culture, the isoprenoid compound is extracted by adding a suitable solvent to the culture solution, and the precipitate is removed by centrifugation or the like, followed by performing various chromatography to isolate and purify the isoprenoid compound. it can.

IV. Search for a substance that inhibits the enzyme activity on the non-mevalonate pathway (1) Measurement of the enzyme activity on the non-mevalonate pathway The enzyme activity on the non-mevalonate pathway is measured according to the usual enzyme activity measurement method. be able to. That is, the pH of the buffer used in the reaction solution for activity measurement may be within a pH range that does not inhibit the activity of the target enzyme, and is preferably in a range including the optimal pH.

For example, in the case of 1-deoxy-D-xylulose 5-phosphate reductoisomerase,
10, preferably 6-9. As the buffer, any buffer can be used as long as it does not inhibit the enzyme activity and can achieve the above pH. As the buffer,
Tris-HCl buffer, phosphate buffer, borate buffer, HEP
An ES buffer, a MOPS buffer, a bicarbonate buffer, or the like can be used. 1-deoxy-D-xylulose 5
-In phosphate reductoisomerase, for example, Tris-HCl buffer is preferably used.

The buffer may be used at any concentration as long as it does not inhibit the enzyme activity, but is preferably from 1 mM to 1M. When a coenzyme is required for the target enzyme, the coenzyme is added to the reaction solution. For example,
In 1-deoxy-D-xylulose 5-phosphate reductoisomerase, NADPH, NADH or other electron donors can be used.
ADPH can be raised.

The concentration of the coenzyme to be added can be any concentration as long as it does not inhibit the reaction, but is preferably 0.01 mM to 100 mM, more preferably 0.1 mM.
M to 10 mM concentration. Metal ions may be added to the reaction solution as needed. Any metal ions can be added as long as they do not inhibit the reaction, but preferably include Co ²⁺ , Mg ²⁺ , and Mn ²⁺ .

Metal ions can be added as metal salts, for example, chlorides, sulfates, carbonates, phosphates and the like. The concentration of the metal ion to be added can be any concentration as long as it does not inhibit the reaction, but is preferably 0 mM to 100 mM, more preferably 0.1 mM to 10 mM.

A substrate for the target enzyme is added to the reaction solution. For example, in 1-deoxy-D-xylulose 5-phosphate reductoisomerase,
Add D-xylulose 5-phosphate. The concentration of the substrate can be any concentration as long as it does not hinder the reaction, but the concentration in the reaction solution is preferably 0.01 mM to
0.2M.

The concentration of the enzyme used in the reaction is not particularly limited, but the reaction is usually carried out in a concentration range of 0.01 mg / ml to 100 mg / ml. The enzyme used does not necessarily need to be purified to a single substance, and may be a sample mixed with other artificial proteins as long as it does not interfere with the reaction. In the search in (2) below, a cell extract containing the enzyme activity or a cell having the enzyme activity can also be used.

The reaction temperature may be within a temperature range that does not inhibit the activity of the target enzyme, and is preferably in a range including the optimum temperature. That is, the reaction temperature is from 10 ° C to 60 ° C,
The temperature is more preferably from 30 ° C to 40 ° C. The activity can be detected by a method capable of measuring the substrate or the reaction product by reducing the substrate accompanying the reaction or increasing the reaction product.

As the method, there can be mentioned, for example, a method of separating and quantifying a target substance by high performance liquid chromatography (HPLC) if necessary. When NADH or NADPH increases or decreases with the progress of the reaction, the activity can be directly measured by measuring the absorbance at 340 nm of the reaction solution. For example, 1-deoxy-
In the case of D-xylulose 5-phosphate reductoisomerase, the activity can be detected by measuring the decrease in absorbance at 340 nm with a spectrophotometer to determine the NADPH that decreases with the progress of the reaction.

(2) Search for a substance that inhibits the enzyme activity on the non-mevalonate pathway The search for a substance that inhibits the enzyme activity on the non-mevalonate pathway is performed by adding the target substance to the enzyme activity measurement system described in (1) above. In addition, the reaction can be carried out in the same manner, and a search can be made for a substance that suppresses the amount of reduction of the substrate or a substance that suppresses the amount of production of the reaction product as compared with the case of no addition.

As a search method, a method of tracking the amount of decrease in the substrate or the amount of increase in the reaction product over time, or measuring the amount of decrease in the substrate or the amount of increase in the reaction product after reacting for a certain period of time. And the like. In the method of tracking the amount of decrease in the substrate or the amount of increase in the reaction product over time, it is preferable to measure the amount of decrease in the substrate or the amount of increase in the reaction product at intervals of about 15 seconds to 20 minutes during the reaction. , More preferably at intervals of 1 to 3 minutes.

In a method for measuring the amount of decrease in the substrate or the amount of increase in the reaction product after reacting for a certain period of time,
The reaction time is preferably from 10 minutes to 1 day, more preferably from 30 minutes to 2 hours. A substance that inhibits the enzymatic activity on the non-mevalonate pathway inhibits the growth of microorganisms and plants having the non-mevalonate pathway. The present inventors have found for the first time that the substance inhibits the growth of the microorganism and the plant.

Since the non-mevalonate pathway is present in microorganisms and plants but not in animals or humans, the above-described search method inhibits enzyme activity on the non-mevalonate pathway which does not affect humans and animals. The substance can be obtained.
The substance can be an effective antimicrobial or herbicide. Examples of the present invention will be described below, but the present invention is not limited to these examples. The genetic recombination experiments described in the Examples were performed using the method described in Molecular Cloning, 2nd Edition (hereinafter, referred to as ordinary method) unless otherwise specified.

[0109]

EXAMPLES Example 1 Obtaining a DNA Encoding a Protein Involved in the Biosynthesis of an Isoprenoid Compound (1) Obtaining a DNA Encoding a Protein Involved in the Biosynthesis of an Isoprenoid Compound Using the Base Sequence of the DXS Gene of Escherichia coli One platinum loop of E. coli XL1-Blue strain (purchased from Toyobo)
The cells were inoculated into 0 ml of LB liquid medium and cultured at 37 ° C. overnight.

After culturing, cells were obtained from the resulting culture by centrifugation. From the cells, the chromosome DN
A was isolated and purified. Bam HI and Eco RI at the 5 'end having a combination of the nucleotide sequences of SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 12 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 13.
Sense and antisense primers having restriction enzyme cleavage sites, respectively, SEQ ID NOS: 22 and 23
Sense primers and antisense primers each having a Bam HI restriction enzyme cleavage site at the 5 'end having the following nucleotide sequence combination were synthesized using a DNA synthesizer.

Using chromosomal DNA as a template, these primers and TaKaRa LA-PCR ^™ Kit Ver.2 (Takara Shuzo), Exp
PCR was performed with DNA Thermal Cycler (Perkin Elmer Japan) using and and ^TM High-Fidelity PCR System (Boehringer Mannheim) or Taq DNA polymerase (Boelinnger).

[0112] The PCR was carried out using a DNA fragment of 2 kb or less as 94
The reaction step consisting of 30 ° C. for 30 seconds, 55 ° C. for 30 seconds to 1 minute, and 72 ° C. for 2 minutes is defined as one cycle, and the DNA fragment exceeding 2 kb is subjected to a reaction step consisting of 98 ° C. for 20 seconds and 68 ° C. for 3 minutes. As one cycle, after 30 cycles, the reaction was carried out at 72 ° C. for 7 minutes.

[0113] Among the amplified DNA fragments by PCR, DNA fragments amplified using sense and antisense primers having Bam HI and Eco RI restriction enzyme cleavage sites, respectively at the 5 'end restriction enzymes Bam HI and Eco Digested with RI
The DNA fragment amplified using the sense primer and the antisense primer each having a Bam HI restriction enzyme cleavage site was digested with the restriction enzyme Bam HI.

[0114] After digestion, these restriction enzymes treated DNA fragment was subjected to agarose gel electrophoresis to obtain Bam HI- Eco RI-treated DNA fragments and Bam HI-treated DNA fragment. Broad host range vector pE with lac promoter
G400 [J. Bac., 172 , 2392 (1990)] and restriction enzyme B
After digestion with am HI and Eco RI, agarose gel electrophoresis was carried out, and Bam HI- Eco RI treated pEG40.
Zero fragments were obtained.

PUC118 (Takara Shuzo) was replaced with restriction enzyme B
After digestion with am HI, Ba subjected to agarose gel electrophoresis
An mHI-treated pUC118 fragment was obtained. After mixing with Bam HI- Eco RI processing pEG400 fragments for Bam HI- Eco RI-treated DNA fragments each obtained above, ethanol precipitated, and dissolved the resulting DNA precipitate 5μl of distilled water, the ligation reaction , To obtain recombinant DNAs.

Using the recombinant DNA, E. coli (purchased from Toyobo) DH5α strain was transformed by a conventional method, and the transformant was spread on an LB agar medium containing 100 μg / ml of spectinomycin. Cultured overnight at ℃. Several growing colonies of spectinomycin resistant transformants were cultured with shaking in 10 ml of LB liquid medium containing 100 μg / ml of spectinomycin at 37 ° C. for 16 hours.

The cells were obtained by centrifuging the obtained culture solution. A plasmid was isolated from the cells according to a conventional method. The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted therein.

DN having the nucleotide sequence of SEQ ID NO: 6
A, a plasmid containing the DNA having the nucleotide sequence of SEQ ID NO: 7, the DNA having the nucleotide sequence of SEQ ID NO: 8 and the plasmid containing the DNA having the nucleotide sequence of SEQ ID NO: 9
O-1, a plasmid containing the DNA having the nucleotide sequence of SEQ ID NO: 6 is pDXS-1, a plasmid containing the DNA having the nucleotide sequence of SEQ ID NO: 7 is pISP-1, and a DNA having the nucleotide sequence of SEQ ID NO: 8 Plasmid containing pXSE-1 having the nucleotide sequence of SEQ ID NO: 9
The plasmid containing NA was named pTFE-1.

Further, the Bam HI processing D obtained above
After mixing NA fragment and Bam HI treated pUC118 fragment, subjected to ethanol precipitation, the resulting DNA precipitate was dissolved in 5μl of distilled water, it was obtained recombinant DNA by performing a ligation reaction. Thereafter, E. coli was transformed in the same manner as described above, and a plasmid was isolated from the E. coli.

In the same manner as described above, the plasmid isolated by the above method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined, thereby confirming that the plasmid had the DNA fragment of interest inserted therein. The plasmid the Ba
After mHI treatment, the target DNA fragment was recovered in the same manner as described above, and subcloned into an expression vector pQE30 (Qiagen) by a conventional method. The plasmid having the nucleotide sequence of SEQ ID NO: 6 obtained by the subcloning was named pQEDXS-1.

(2) Acquisition of Methylerythritol-Requiring Complementary Gene Acquisition of Escherichia coli Methylerythritol-Requiring Mutant E. coli W3110 strain (ATCC14948) was inoculated into an LB liquid medium and cultured until logarithmic growth. After the culture, cells were obtained from the resulting culture by centrifugation. The cells were added to 0.0
After washing with 5M Tris-maleate buffer solution (pH 6.0), the cells were suspended in the same buffer solution so that the cell concentration was 10 ⁹ cells / ml.

The suspension was added with NTG to a final concentration of 600 mg / g.
1 and kept at room temperature for 20 minutes to perform mutation treatment. The obtained mutant-treated cells were spread on an M9 minimal agar medium [Molecular Cloning 2nd Edition] plate containing 0.1% methylerythritol and cultured. Methylerythritol is available from Tetrahedron Letters, 38 , 35, 61.
84 (1997).

M9 containing 0.1% of methylerythritol
A colony that grew on the minimal agar medium was replicated on an M9 minimal agar medium and an M9 minimal agar medium containing 0.1% of methyl erythritol, showing a requirement for methyl erythritol, ie, 0.1% of methyl erythritol.
A strain which can grow on the M9 minimal agar medium containing but cannot grow on the M9 minimal agar medium was selected as the target mutant strain. The methyl erythritol-requiring mutant strain ME7 obtained by the selection was used in the following experiments.

Acquisition of Methylerythritol-Required Complementary Gene E. coli W3110 strain (ATCC 14948) was inoculated into an LB liquid medium, cultured until the logarithmic growth phase, and then centrifuged to collect cells. Chromosomal DNA was isolated and purified from the obtained cells according to a conventional method.

The chromosomal DNA (200 μg) was digested with restriction enzyme Sa.
u 3AI, and the resulting digested DNA fragment was subjected to sucrose density gradient ultracentrifugation (26,000 rpm).
m, 20 ° C., 20 hr). Vector magnitude obtained by fractionation is a DNA fragment of 4～6Kb, was digested with restriction enzymes Bam HI pMW11
8 (manufactured by Nippon Gene Co., Ltd.) to prepare a chromosome genomic library.

Using the prepared chromosome library, the ME7 strain isolated above was transformed according to a conventional method. The obtained transformant was spread on an LB agar medium containing 100 μg / l of ampicillin, and cultured at 37 ° C. overnight. Plasmids were extracted from a plurality of colonies grown in the culture, and the nucleotide sequence was determined.

The clone whose nucleotide sequence was determined was SEQ ID NO: 1.
It had a sequence containing the base sequence shown in FIG. These plasmids were named pMEW41 and pMEW73. Plasmids extracted from one of the clones having this sequence were cloned into pMEW
Named 73. pMEW73 was double-digested with the Hin dIII and Sac I, Hin having the nucleotide sequence shown in SEQ ID NO: 10 obtained dIII- Sac I treatment the DNA fragment broad host range vector pEG400 [J. Bac., 172, 2392 ( 1990)] to construct pEGYM1.

[0128] The Hin dIII- Sac I Hin d processing DNA fragment of the vector pUC19 [Gene, 33, 103 (1985)]
It was prepared pUCYM-1 linked to III-Sac I site. Genb
From the chromosomal base sequence information of Escherichia coli based on the database of ank, it was found that the DNA fragment inserted into the vector contained the yaeM gene.

A recombinant vector capable of sufficiently expressing the yaeM gene was obtained by PCR [Science, 230 , 1350 (1985)].
Was constructed by the following method. A sense primer having the sequence shown in SEQ ID NO: 20 and an antisense primer having the sequence shown in SEQ ID NO: 21 were synthesized using a DNA synthesizer.

A restriction enzyme site of Bam HI was added to each of the 5 'ends of the sense primer and the antisense primer. Using the chromosomal DNA as a template and these primers and Taq DNA polymerase (manufactured by Boelinnger), PCR was performed using a DNA Thermal Cycler (manufactured by PerkinElmer Japan) to amplify the yaeM gene.

The PCR was performed at 94 ° C. for 30 seconds and at 55 ° C. for 3 seconds.
After 1 cycle and 30 cycles of a reaction step consisting of 0 seconds and 72 ° C. for 2 minutes, the reaction was carried out at 72 ° C. for 7 minutes. After digesting the amplified DNA fragment and pUC118 (manufactured by Takara Shuzo) with the restriction enzyme Bam HI,
The fragments were purified by agarose gel electrophoresis.

After mixing these purified fragments, ethanol precipitation was performed. The obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain a recombinant DNA. The recombinant DNA is yaeM
After confirming that the gene was a DNA sequence, it was subcloned into an expression vector pQE30 (Qiagen). The resulting recombinant DNA was pQE
Named YM1.

After transforming ME7 strain using pQEYM1 according to a conventional method, the transformant was transformed with ampicillin 100
It was spread on an LB agar medium containing μg / ml and cultured at 37 ° C. overnight. It was confirmed that the transformed strain formed a colony at a growth rate comparable to that of the wild-type strain.
It was found that the mutation of ME7 strain was complemented by the aeM gene.

Example 2 Production of Ubiquinone-8 (CoQ8) by Recombinant E. coli (1) Plasmids pADO-1 and pADO obtained in Example 1
DXS-1, pXSE-1 or p as control
EG400 was introduced into E. coli DH5α strain,
Transformants E. coli DH5α / pADO-1 and E. coli DH5α / pD resistant to spectinomycin at a concentration of 0 μg / ml
XS-1, E. coli DH5α / pXSE-1 and E. coli DH5α / pE
G400 was acquired respectively.

[0135] Thiamin (thiamine) and vitamin B _6, respectively 100 mg / l, p-hydroxybenzoic acid 50mg
/ L, L containing 100 μg / ml of spectinomycin
These transformants were inoculated into a test tube containing 10 ml of B medium, and cultured with shaking at 30 ° C. for 72 hours. After completion of the culture, each culture was concentrated 10-fold.

To 300 μl of each concentrated solution, 300 μl of 2-butanol and 300 μl of glass beads were added, and the mixture was added to a multi-beads shocker MB-200 (manufactured by Yasui Kikai).
The solvent was extracted from the isoprenoid compound while crushing the cells for minutes, and then the 2-butanol layer was collected by centrifugation. The amount of CoQ8 produced by the transformant was calculated by quantitatively analyzing CoQ8 in the butanol layer by high performance liquid chromatography (LC-10A, manufactured by Shimadzu Corporation).

The column is Develosil ODS-HG-5 (Nomura Chemical)
And using a solution of methanol: n-hexane = 8: 2 as a mobile phase, and analyzing at a flow rate of 1 ml / min and a measurement wavelength of 275 nm. The results are shown in Table 1.

[0138]

[Table 1]

The amount of CoQ8 produced was determined by the control strain DH5
Compared to α / pEG400, DH5α / pADO-1, DH5α / pDXS-1 and DH5α / pXSE-1 were significantly higher. In particular, the highest productivity was obtained with DH5α / pADO-1 into which all the DNAs obtained in Example 1 were introduced.

[0140] (2) M9 medium in test tubes containing 10 ml, E obtained above (1). Coli DH5α / pDXS -1 also <br/> is inoculated respectively E. Coli DH5α / pEG400, 30 Shaking culture was performed at 72 ° C for 72 hours. After completion of the culture, the amount of CoQ8 produced by the transformant was calculated in the same manner as in the above (1). The results are shown in Table 2.

[0141]

[Table 2]

The production amount of CoQ8 was determined by the control strain DH5
DH5α / pDXS-1 was significantly higher than α / pEG400. (3) the pEG400 as a plasmid pEGYM1 or control obtained in Production Example 1 of CoQ8 by recombinant E. coli E. Coli was introduced into DH5α strain, 100 [mu] g / ml concentration of spectrum transformant E showing resistance to spectinomycin. coli DH5α / pEGYM1 and E. co
li DH5α / pEG400 each was obtained.

Glucose 1%, Vitamin B ₁ 100mg
/ L, vitamin B ₆ 100mg / l, was inoculated these transformants LB medium supplemented p- hydroxy benzoic acid 50 mg / l in test tubes containing 10 ml, and 72 hours by shaking culture at 30 ° C.. After completion of the culture, the amount of CoQ8 produced by the transformant was calculated in the same manner as in the above (1).
The results are shown in Table 3.

[0144]

[Table 3]

The amount of CoQ8 produced was determined based on the control strain DH5a.
DH5a / pEGYM1 was significantly higher than / pEG400.

Example 3 Production of Menaquinone-8 (MK-8) by Recombinant Escherichia coli (1) A TB medium [bactotripton (Difco), 12 g, supplemented with 100 μg / ml spectinomycin)
Yeast extract (manufactured by Difco) 24 g, glycerol 5 g
Is dissolved in 900 ml of water, and KH ₂ PO ₄ is
₂ HPO ₄ Medium] which was prepared by adding 100ml of an aqueous solution containing 0.72M to test tube containing 10 ml, Example 2
Obtained in (1), E. Coli DH5α / pADO-1 or E. Co
li was inoculated DH5 [alpha / PEG 400 were each 72 hours shaking culture at 30 ° C..

After completion of the culture, MK-8 was quantified in the same manner as in the method of quantifying CoQ8 in Example 2 (1), and the amount of MK-8 produced by the transformant was calculated. The results are shown in Table 4.

[0148]

[Table 4]

The amount of MK-8 produced was determined by the control strain DH5
Compared to α / pEG400, DH5α / pADO-1 was significantly higher. (2) E. coli DH5α / pDXS- obtained in Example 2 (1)
1 or E. coli DH5α / pEG400 was cultured in the same manner as in (1) above, and the amount of MK-8 produced by the transformant was calculated. The results are shown in Table 5.

[0150]

[Table 5]

The production of MK-8 was controlled by the control strain DH5.
Compared to α / pEG400, DH5α / pDXS-1 was significantly higher.

Example 4 CoQ by Erwinia carotovora
8. Production of plasmid pDXS-1 obtained in Example 1 or pEG400 as a control in Erwinia carotovora IFO
The transformants IFO-3380 / pDXS-1 and IFO-3380 / pEG400 exhibiting resistance to spectinomycin at a concentration of 100 μg / ml were obtained.

These transformants were inoculated into test tubes containing 10 ml of LB medium supplemented with 100 μg / ml of spectinomycin, and cultured with shaking at 30 ° C. for 72 hours. After completion of the culture, the amount of CoQ8 produced by the transformant was calculated in the same manner as in Example 2 (1). The results are shown in Table 6.

[0154]

[Table 6]

The amount of CoQ8 produced was determined by the control strain IFO-
IFO-3380 / pDXS-1 was significantly higher than 3380 / pEG400.

Example 5 Production of Ubiquinone and Carotenoid by Erwinia uredovora Plasmids pUCYM-1, pQEDXS-1, pQE obtained in Example 1
YM-1 or pUC19 and pQE30 as a control were subjected to Erwinia uredovora DSM-30 by electroporation.
Transformant E. uredovora DSM-30080 / pU which has been introduced into strain 080 and has resistance to ampicillin at a concentration of 100 μg / ml.
CYM-1, E. uredovora DSM-30080 / pQEDXS-1, E. uredov
ora DSM-30080 / pQEYM-1, E. uredovora DSM-30080 / pU
C19 and E. uredovora DSM-30080 / pQE30 were obtained.

[0157] Ampicillin 100 [mu] g / ml, 1% glucose, vitamin B ₁ 100 mg / l, vitamin B ₆ 1
00 mg / l, p-hydroxybenzoic acid 50 mg / l
These transformants were inoculated into a test tube containing 10 ml of the added LB medium, and cultured with shaking at 30 ° C. for 72 hours.

After completion of the culture, the amount of CoQ8 produced by the transformant was calculated in the same manner as in Example 2 (1).
The production amount of the carotenoid pigment was calculated by measuring the absorbance at 450 nm of the 2-butanol layer obtained by the same method as in Example 2 (1) using a spectrophotometer. The results are shown in Table 7.

[0159]

[Table 7]

Both the production amount of CoQ8 and the production amount of carotenoid pigment were significantly higher in DSM-30080 / pUCYM-1 than in control strain DSM-30080 / pUC19. Similarly, both the production amount of CoQ8 and the production amount of carotenoid pigment were compared with the control strain DSM-30080 / pQE30,
-30080 / pQEYM-1 and DSM-30080 / pQEDXS-1 were significantly higher.

Example 6 Photosynthetic bacterium Rhodobacter sphaer
Obtaining DNA encoding the protein involved in the biosynthesis of isoprenoid compounds from oides (1) R. Acquisition of DXS gene from sphaeroides Using the DXS gene sequence found in Escherichia coli, DXS homologs conserved in other species were searched from genbank.
As a result, Haemophilus influenzae (P45205), Rhodobac
ter capsulatus (P26242), Bacillussubtilis (P54523),
Synechocystis sp.PCC6803 (P73067) and Mycobacteri
Homolog was found in un tuberculosis (O07184) and the like.
These sequences were compared to select a highly conserved amino acid sequence. A nucleotide sequence corresponding to the conserved amino acid sequence is designed in consideration of the codon usage of R. sphaeroides , and a DNA fragment having the nucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 33 as a sense primer is sequenced as an antisense primer. The DNA fragment having No. 34 was synthesized using a DNA synthesizer.

R. sphaeroides KY4113 strain (FERM-P4675)
Using the chromosomal DNA as a template, the above primer and Expa
Using the nd ^™ High-Fidelity PCR System (Boehringer Mannheim), PCR was performed with DNA Thermal Cycler (Perkin Elmer Japan). PCR is 94
A reaction step consisting of 40 ° C. for 40 seconds, 60 ° C. for 40 seconds, and 72 ° C. for 1 minute was defined as one cycle, and after 30 cycles, the reaction was carried out at 72 ° C. for 7 minutes to obtain a target DNA fragment. . This DNA fragment is converted to DIG DNA Lab
D using ling Kit (Boehringer Mannheim)
IG labeled.

To obtain the full length DXS gene of R. sphaeroides , a genomic library of KY4113 strain was prepared. The KY4113 strain was cultured overnight in an LB medium to extract chromosomal DNA. After partial digestion with the restriction enzyme Sau 3AI, a DNA fragment of 4 to 6 kb was purified by sucrose density gradient ultracentrifugation. The DNA fragment and Bam HI digested vector pUC19 Ligation Pack (Nippon Gene)
And transformed into Escherichia coli DH5α. The transformant was spread on an LB plate containing 100 μg / ml of ampicillin to obtain about 10,000 colonies.

When screening was performed by colony hybridization using the DIG-labeled DNA fragment obtained as described above as a probe, two types of DNA fragments were detected. When each was sequenced, an ORF highly homologous to the known DXS gene of another species was found from each DNA fragment. The amino acid sequence shown in SEQ ID NO: 26 was named DXS1, and the amino acid sequence shown in SEQ ID NO: 27 was named DXS2.

(2) Confirmation of complementation using Escherichia coli DXS gene-deficient strain Acquisition of Escherichia coli DXS gene-deficient strain The E. coli W3110 strain (ATCC14948) was inoculated into an LB liquid medium and cultured until the exponential growth phase. After the culture, cells were obtained from the resulting culture by centrifugation. The cells were added to 0.0
After washing with 5M Tris-maleate buffer solution (pH 6.0), the cells were suspended in the same buffer solution so that the cell concentration was 10 ⁹ cells / ml.

The suspension was added with NTG to a final concentration of 600 mg / g.
1 and kept at room temperature for 20 minutes to perform mutation treatment. The obtained mutant-treated cells were spread on an M9 minimal agar medium [molecular cloning second edition] plate containing 0.1% of 1-deoxyxylulose and cultured.
1-Deoxyxylulose is available from JCS Perkin Trans
Chemical synthesis was carried out according to the method described in I , 2131-2137 (1982).

A colony grown on an M9 minimal agar medium containing 0.1% of 1-deoxyxylulose was replicated on an M9 minimal agar medium and an M9 minimal agar medium containing 0.1% of 1-deoxyxylulose, A strain showing a requirement for 1-deoxyxylulose, which can grow on the M9 minimal agar medium containing 0.1% of 1-deoxyxylulose but cannot grow on the M9 minimal agar medium, was selected as the target mutant.

The mutant strain selected and obtained by the method is
It was named E1 strain. When pDXS-1 was introduced into the ME1 strain, it complemented the requirement for 1-deoxyxylulose. Therefore, the ME1 strain was determined to be a strain deficient in the DXS gene.

(3) Complementation test of DXS1 and DXS2 DXS1 consisting of SEQ ID NO: 27 derived from KY4113 strain
And the DNA fragment encoding DXS2 consisting of SEQ ID NO: 29 were each isolated from the vector pUC
A plasmid ligated downstream of the 19 lac promoter was constructed.

Each of these constructed plasmids was ME
When introduced into one strain, DXS1 and DXS2 both complemented the 1-deoxyxylulose requirement of ME1 strain. From the above, R. sphaeroides has two DXS1 and DXS2 DXS genes that have an activity of catalyzing a reaction to generate 1-deoxy-D-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate. It turned out to have.

(4) Acquisition of R. sphaeroides- derived Methylerythritol -Required Complementary Gene The E. coli methylerythritol-required mutant ME7 obtained in Example 1 (2) was replaced with methylerythritol 0.
After inoculating the cells in an LB liquid medium containing 1% and culturing until the logarithmic growth phase, the cells were collected by centrifugation.

The cells were cultured in 1 mM containing 10% glycerol.
The medium was washed twice with an aqueous HEPES solution to remove the medium components as much as possible. The washed cells were mixed with the R. cerevisiae prepared in Example 6 (1) .
A plasmid extracted from the genomic library of sphaeroides KY4113 strain was introduced by electroporation according to a conventional method.

After the introduction, the cells were spread on an LB agar medium containing 100 μg / l of ampicillin, and cultured at 37 ° C. overnight. The growing colonies were picked, inoculated and cultured in an LB liquid medium, and a plasmid was extracted from the cultured cells.

When the extracted plasmid was re-introduced into the ME7 strain, it grew on a medium not containing methylerythritol , so that the extracted plasmid contained R. sphaeroides.
It was confirmed that a DNA fragment complementary to the requirement of methyl erythritol from the origin was contained. As a result of determining the base sequence of this DNA fragment, Escherichia coli ya consisting of SEQ ID NO: 31 was obtained.
A DNA sequence encoding an amino acid sequence highly homologous to eM was found.

Example 7 Production of Ubiquinone-10 (CoQ10) by Recombinant Photosynthetic Bacteria DNA Fragment D of SEQ ID NO: 27 Obtained in Example 6
DNA fragment DXS2 consisting of XS1 and SEQ ID NO: 29
Upstream, ligated the glnB promoter derived from KY4113 strain,
Plasmids created by insertion into the broad host range vector pEG400 were named pRSDX-1 and pRSDX-2. Also, yaeM and DX
A plasmid in which S1 was ligated in tandem and ligated downstream of the glnB promoter was constructed and named pRSYMDX1. These plasmids, R by electroporation (Bio-Rad). sphaeroides KY4113 strain.

After the introduction, the cells were spread on an LB agar medium containing spectinomycin at a concentration of 100 μg / ml, and cultured at 30 ° C. for 3 days. 100 μg / ml of growing colonies
After inoculating in an LB medium containing spectinomycin at a concentration and culturing overnight, the cultured cells were collected by centrifugation.

Plasmid extraction from the obtained cells (Qiagen
(Manufactured by the company), and it was confirmed that each of the introduced plasmids was retained. The transformant obtained in this manner was KY41
13 / pRSDX-1, KY4113 / pRSDX-2, KY4113 / pRSYMDX1, and K
It was named Y4113 / pEG400. Seed medium [glucose 2%, peptone 1%, yeast extract 1%, NaCl 0.5% (pH
7.2, adjusted with NaOH)] into a test tube containing 5 ml of each transformant, and inoculated with a platinum loop and cultured at 30 ° C. for 24 hours.

0.5 ml of the obtained culture was added to a ubiquinone-10 production medium (molasses 4%, glucose 2.7%, corn steep liquor 4%, ammonium sulfate 0.8%,
1% potassium phosphate 0.05%, 2 potassium phosphate 0.0
A medium containing 5%, magnesium sulfate heptahydrate 0.025%, ferrous sulfate heptahydrate 3 mg / l, thiamine 8 mg / l, nicotinic acid 8 mg / l, and trace element 1 ml / l was adjusted to pH 9. After the adjustment, 1 ml of calcium carbonate was added and sterilized in an autoclave, and the mixture was added to a test tube containing 5 ml, followed by shaking culture at 30 ° C. for 5 days. After completion of the culture, the amount of CoQ10 produced by the transformant was calculated in the same manner as in the method of quantifying CoQ8 in Example 2 (1).
Table 8 shows the results.

[0179]

[Table 8]

The amount of CoQ10 produced was determined based on the control strain KY41.
It was significantly higher in KY4113 / pRSDX-1, KY4113 / pRSDX-2 and KY4113 / pRSYMDX1 compared to 13 / pEG400.

Example 8 Measurement of Activity of Enzyme Encoded by yaeM Gene (1) Higher Expression of yaeM Gene A recombinant plasmid capable of sufficiently expressing the yaeM gene was subjected to PCR (Science, 230 , 1350 (1985)). And constructed by the following method. A sense primer having the sequence shown in SEQ ID NO: 24 and an antisense primer having the sequence shown in SEQ ID NO: 25 were synthesized using a DNA synthesizer.

A Bam HI restriction enzyme site was added to each of the 5 'ends of the sense primer and the antisense primer. Using the chromosomal DNA as a template and these primers and Taq DNA polymerase (manufactured by Boehringer), the yaeM gene was amplified by performing PCR using a DNA Thermal Cycler (manufactured by PerkinElmer Japan).

The PCR was performed at 94 ° C. for 30 seconds and at 55 ° C. for 3 seconds.
After 1 cycle and 30 cycles of a reaction step consisting of 0 seconds and 72 ° C. for 2 minutes, the reaction was carried out at 72 ° C. for 7 minutes. After digesting the amplified DNA fragment and pUC118 (manufactured by Takara Shuzo) with the restriction enzyme Bam HI,
The fragments were purified by agarose gel electrophoresis.

After mixing these purified fragments, ethanol precipitation was performed, and the obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain a recombinant DNA. The recombinant DNA is yaeM
The gene was confirmed by determining the DNA sequence.

A plasmid was extracted from the recombinant, digested with a restriction enzyme Bam HI, and subjected to agarose gel electrophoresis to obtain a Bam HI-treated yaeM gene-containing DNA fragment. Restriction enzyme Bam to pQE30 (manufactured by QIAGEN)
After digestion with HI, agarose gel electrophoresis was performed and Bam H
An I-treated pQE30 fragment was obtained.

The Bam HI processing yaeM obtained above
After mixing the gene-containing DNA fragment with the Bam HI digested pQE30 fragment, ethanol precipitation was performed, and the obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain a recombinant DNA. The recombinant D
With NA, E. After transformation according to a conventional method to CoIi JM109 strain, the transformant was spread on LB agar medium containing ampicillin 100 [mu] g / ml, and cultured overnight at 37 ° C..

[0187] A plasmid was isolated from the Escherichia coli in the same manner as described above. In the same manner as described above, the plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined, thereby confirming that the plasmid had the DNA fragment of interest inserted therein. This plasmid was named pQEDXR.

(2) Measurement of the activity of the yaeM gene product Purification of the yaeM gene product The pQEDXR prepared in (1) was purified from E. coli having pREP4 by a conventional method.
E.coli M15 strain (QIAGEN) and 200 μg of ampicillin
/ ml, M15 / pREP4 + pQE resistant to kanamycin 25 μg / ml
DXR strain was obtained.

The M15 / pREP4 + pQEDXR strain was transformed with ampicillin 200
The cells were cultured at 37 ° C. in 100 ml of an LB liquid medium containing μg / ml and kanamycin 25 μg / ml, and when the turbidity at 660 nm reached 0.8, isopropylthiogalactoside was added to a final concentration of 0.2 mM. . 3 more
After culturing at 7 ° C. for 5 hours, centrifugation (3,000 rpm,
10 minutes) to remove the culture supernatant. 10 cells
The suspension was suspended in 6 ml of 0 mM Tris-HCl buffer (pH 8.0), and crushed while cooling with ice using an ultrasonic crusher (SONIFIER, manufactured by BRANSON). The obtained cell lysate is centrifuged (1
(20,000 rpm, 20 minutes, 4 ° C.), and the supernatant was collected. The cell extract centrifuged supernatant is transferred to a Ni-NTA resin column.
(Manufactured by QIAGEN), 20 ml of washing buffer [100 m
M Tris-HCl (pH 8.0), 50 mM imidazole,
0.5% Tween 20]. Then, 10 ml of an elution buffer [100 mM Tris-HCl (pH 8.0), 200 mM imidazole] was passed through the column, and the eluate was fractionated by 1 ml. The protein content of each fraction was measured (using a protein quantification kit from BioRad), and the fraction containing the protein was defined as a purified protein fraction.

Substrate 1-deoxy-D-xylulose 5-
Preparation of phosphoric acid 1-deoxy-D-xylulose 5-phosphate as a reaction substrate was prepared as follows. Detection of 1-deoxy-D-xylulose 5-phosphate was performed by HPLC [column: Senshup.
ak NH2-1251-N (4.6 x 250mm, manufactured by Senshu), moving bed: 1
00 mM KH ₂ PO ₄ (pH 3.5)]
m was measured by measuring the absorbance.

The plasmid pQDXS-1 which highly expresses the dxs gene of Escherichia coli was introduced into the E. coli M15 / pREP4 strain in the same manner as described above to obtain the M15 / pREP4 + pQDXS-1 strain. Example 8
The cells were cultured in the same manner as in (2), and the dxs enzyme protein was purified using a Ni-NTA resin column.

The purified dxs protein was added to a 20 ml reaction solution [100 mM Tris-HCl (pH 7.5), 10 mM sodium pyruvate, 30 mM DL-glyceraldehyde-3-phosphate, 1.5 mM thiamine pyrophosphate, 10 mM
MMgCl ₂ , 1 mM DL-dithiothreitol] and kept at 37 ° C.

After reacting for 12 hours, the reaction mixture was washed with water for 300 hours.
After the solution was diluted to 1 ml and passed through an activated carbon column (2.2 × 8 cm), the solution was passed through a Dowex 1-X8 (Cl − type, 3.5 × 25 cm) and eluted with 1% saline. After concentration of the eluted fraction,
The solution was passed through adex G-10 (1.8 × 100 cm) and eluted with water. The fraction containing 1-deoxy-D-xylulose 5-phosphate was freeze-dried to obtain about 50 mg of a white powder. NMR analysis (A-500, manufactured by JEOL Ltd.) confirmed that the powder was 1-deoxy-D-xylulose 5-phosphate.

Measurement of enzyme activity of yaeM gene product 100 mM Tris-HCl (pH 7.5), 1 mM MmCl
2, 0.3 mM NADPH and y obtained in Example 8 (2)
To 1 ml of the reaction solution containing the aeM gene product was added 0.3 mM (final concentration) of 1-deoxy-D-xylulose 5-phosphate synthesized as described above, and the mixture was incubated at 37 ° C. Increase or decrease of NADPH during incubation by 340 n
When the absorbance of M was measured by a method of measuring with a spectrophotometer (UV-160, manufactured by Shimadzu Corporation), it was found that NADPH decreased with time.

To confirm the structure of the above reaction product, the reaction was carried out on a larger scale as follows, and the product was isolated. After similarly incubating 200 ml of a reaction solution having the same composition as above except that the concentration of 1-deoxy-D-xylulose 5-phosphate was 0.15 mM at 37 ° C. for 30 minutes, the whole amount was passed through an activated carbon column, After diluting the passing solution with water to 1 L, Dowex 1-X8 (Cl − type, 3.5 × 20)
cm).

Elution was carried out with 400 ml of 1% saline, and Sephadex
 G-10 (1.8x100cm), eluted with water
Was. The reaction product can be isolated by freeze-drying the eluted fraction.
Was. The amount of the reaction product isolated from the HR-FABMS analysis
Child formula is C_FiveH₁₂O₇P [m / z 215.0276 (M-
H)^-, △ -4.5 mmu].¹H and ¹³
The following chemical shift was obtained from the CNMR analysis.

¹ H NMR (D ₂ O, 500 MHz): δ 4.03 (ddd, J = 11.
5, 6.5, 2.5 Hz, 1H), 3.84 (ddd, J = 11.5, 8.0, 6.5 H
z, 1H), 3.78 (dd, J = 80, 2.5 Hz, 1H), 3.60 (d, J = 12.0
Hz, 1H), 3.50 (d, J = 12.0 Hz, 1H), 1.15 (s, ³ H); ¹³ C
NMR (D ₂ O, 125 MHz): δ 75.1 (C-2), 74.8 (C-3), 67.4 (C-
1), 65.9 (C-4), 19.4 (2-Me) This reaction product was treated with alkaline phosphatase (Takara Shuzo).
The chemical shift obtained by ¹ H and ¹³ C NMR analysis of the compound obtained by the treatment described in Tetrahedron Letter,
38 , 6184 (1997). It completely matched the chemical shift obtained by NMR analysis of 2-C-methyl-D-erythritol synthesized by the method described in (1997).

The optical rotation of the former is [α] _D ²¹ = + 6.0.
_{(C = 0.050, H 2 O} ) at, have been reported [Tetrah
edron Letter, 38 , 6184 (1997)] 2-C-methyl-D
-Optical rotation of erythritol [α] _D ²⁵ = + 7.0 (c =
0.13, consistent with H ₂ O).

From these results, it was revealed that the reaction product of the yaeM gene product was 2-C-methyl-D-erythritol 4-phosphate. That is, the yaeM gene product is 1-deoxy-during the consumption of NADPH.
D-xylulose 5-phosphate to 2-C-methyl-D-
It was found to have activity to produce erythritol 4-phosphate. Based on this catalytic activity, this enzyme was named 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

1-Deoxy-D-xylulose 5-
Properties of Phosphate Reductoisomerase Using the 1 ml reaction system described in Example 8 (2),
The enzymatic properties of deoxy-D-xylulose 5-phosphate reductoisomerase were examined. Here, 1u is defined as the activity of oxidizing 1 mmol of NADPH per minute. N
When ADPH was replaced by NADH, the activity was 1/100.
It fell below.

When 1-deoxy-D-xylulose was used in place of 1-deoxy-D-xylulose 5-phosphate, no reaction occurred. From SDS-PAGE analysis,
This enzyme was found to be composed of a 42 kDa polypeptide. Table 9 shows the effect of metal addition to the reaction system.

[0202]

[Table 9]

1-Deoxy-D- in the presence of MnCl ₂
The Km for xylulose 5-phosphate and NADPH were 249 μM and 7.4 μM, respectively. FIG. 1 shows the effect of the reaction temperature, and FIG. 2 shows the effect of the reaction pH.

Example 9 Preparation and Properties of YaeM-Deficient Mutant (1) Preparation of yaeM-Defective Mutant In order to examine whether 1-deoxy-D-xylulose 5-phosphate reductoisomerase is essential for cell growth, The defective mutant was prepared as follows. A kanamycin resistance gene cassette for insertion into the yaeM gene was created as follows.

The plasmid pME obtained in Example 1 (2)
W41 was digested with restriction enzyme Bal I and then subjected to agarose gel electrophoresis to obtain a Bai I I-treated DNA fragment. Tn5
After digestion with restriction enzymes Hind III and Sam I, DN
The ends of the fragments were blunt-ended using A blunting kit (Takara Shuzo). The obtained blunted DNA fragment was prepared using B
After mixing with aii I treatment pMEW41DNA fragment, ethanol precipitated, and the resulting DNA precipitate was dissolved in 5μl of distilled water, it was obtained recombinant DNA by performing a ligation reaction.

Using the recombinant DNA, E. coli JM109
After transforming the strain (purchased from Takara Shuzo) according to a conventional method, the transformant was spread on an LB agar medium containing 100 μg / ml of ampicillin and 15 μg / ml of kanamycin, and then incubated at 37 ° C.
Overnight. For several colonies of ampicillin-resistant transformants that have grown,
The cells were shake-cultured in 10 ml of an LB liquid medium containing g / ml and 15 μg / ml of kanamycin at 37 ° C. for 16 hours. The cells were obtained by centrifuging the obtained culture solution.

A plasmid was isolated from the cells according to a conventional method. The plasmid isolated by this method was cleaved with various restriction enzymes to examine the structure, and it was confirmed that the plasmid had the target DNA fragment inserted therein. This plasmid was named pMEW41Km. Using pMEW41Km, the yaeM gene on the chromosome was disrupted by homologous recombination. A schematic diagram of the recombination is shown in FIG.

PMEW41Km was replaced with restriction enzymes Hind III and Sa
After digestion with cI, agarose gel electrophoresis was performed to purify the linear fragment. Using this fragment, Escherichia coli FS1576 was transformed according to a conventional method. The FS1576 strain is available from the National Institute of Genetics under the name ME9019. The transformant was spread on an LB agar medium containing 15 μg / ml of kanamycin and 1 g / l of 2-C-methyl-D-erythritol, and
Cultured at 7 ° C overnight.

Some of the grown kanamycin-resistant colonies were cultured with shaking in 10 ml of an LB liquid medium containing 15 μg / ml of kanamycin and 1 g / l of 2-C-methyl-D-erythritol at 37 ° C. for 16 hours. The cells were obtained by centrifuging the obtained culture solution. Chromosomal DNA was isolated from the cells according to a conventional method.

[0210] limit the chromosomal DNA enzyme Sma I or P
Digested with stI . The chromosome of FS1576 strain was treated in the same manner. According to a conventional method, these restriction enzyme-treated DN
After A was subjected to agarose gel electrophoresis, it was subjected to Southern hybridization analysis using the kanamycin resistance gene and the yaeM gene as probes. As a result, the chromosome of the kanamycin-resistant colony had the structure shown in FIG. 3, and it was confirmed that the yaeM gene was disrupted and disrupted by the kanamycin-resistant gene as intended.

(2) Properties of the yaeM-deficient mutant strain The yaeM-deficient strain and the parent strain FS1576 strain prepared by the above-described procedure were transformed with an LB agar medium and 2-C-
It was spread on an LB agar medium containing 1 g / l of methyl-D-erythritol and cultured at 37 ° C. Table 10 shows the degree of growth two days later.

[0212]

[Table 10]

The yaeM-deficient mutant was 2-C-methyl-D
Since the cells could not grow on a medium to which erythritol was not added, it became clear that this gene was essential for cell growth in the absence of 2-C-methyl-D-erythritol.

Example 10 Effect of 1-deoxy-D-xylulose 5-phosphate reductoisomerase inhibitor on the growth of bacteria 2-product which is a product of 1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction Since fosmidmycin is structurally similar to C-methyl-D-erythritol 4-phosphate and the reaction intermediate assumed in this enzyme reaction,
The following experiment was performed based on the presumption that fosmidmycin may inhibit the enzyme.

Fosmidomycin was added to the 1-deoxy-D-xylulose 5-phosphate reductoisomerase activity measurement system shown in Example 8 and the effect on the enzyme activity was examined. Fosmidomycin [Chem. Pharm. Bull., 30 ,
111-118 (1982)].

The reaction system shown in Example 8 (2)
Various concentrations of fosmidomycin were added to a system scaled down to ml (each concentration was the same), incubated at 37 ° C, and the increase / decrease of NADPH was measured using a benchmark microplate reader (Bio-Rad).

As shown in FIG. 4, fosmidomycin was found to inhibit 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Escherichia coli W3110 strain was added to LB agar medium, fosmidomycin at 3.13 mg /
LB agar medium and fosmidmycin 3.13
mg / l and 2-C-methyl-D-erythritol 0.2
Each was spread on LB agar medium containing 5 g / l, and
And cultured.

Two days after the culture, the bacteria were able to grow on the LB agar medium, the two kinds of medium on the LB agar medium containing fosmidomycin and 0.25 g / l of 2-C-methyl-D-erythritol. However, the bacteria could not grow on the LB agar medium to which only fosmidmycin was added.

From these results, it was clarified that fosmimycin inhibits the growth of bacteria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase. From the above, yaeM (1
-Deoxy-D-xylulose 5-phosphate reductoisomerase) can be an effective antibacterial or herbicide.

[0220]

Industrial Applicability According to the present invention, it is involved in the biosynthesis of isoprenoid compounds useful for medicines, health foods, and shellfish adhesion preventive paints for the purpose of heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation, etc. A DNA containing one or more DNAs is incorporated into a vector, the obtained recombinant DNA is introduced into a prokaryotic host cell, and the obtained transformant is cultured in a medium to produce an isoprenoid compound in the culture. A method for producing an isoprenoid compound, comprising accumulating and collecting an isoprenoid compound from the culture, a DNA comprising one or more DNAs encoding a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound Was integrated into a vector, and the resulting recombinant D
Introducing the NA into a host cell, culturing the resulting transformant in a medium, producing and accumulating the protein in a culture, and collecting the protein from the culture to produce the protein. Method and the protein, and 1-deoxy-D
Antibacterial and / or herbicidal activity by searching for a novel enzyme protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from xylulose 5-phosphate and a substance inhibiting the enzyme A method for searching for a compound having activity can be provided.

[0221]

[Sequence List] SEQUENCE LISTING <110> KYOWA HAKKO KOGYO CO., LTD. <120> A METHOD OF PRODUCING AN ISOPRENOID COMPOUND USING MICROORGANISM <130> H11-0421N2 <140> <141> <150> H10-103101 <151> 1998-04-14 <150> H10-221910 <151> 1998-08-05 <150> H11-035739 <151> 1999-02-15 <160> 34 <170> PatentIn Ver. 2.0 <210> 1 <211 > 620 <212> PRT <213> Escherichia coli <400> 1 Met Ser Phe Asp Ile Ala Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser 1 5 10 15 Thr Gln Glu Leu Arg Leu Leu Pro Lys Glu Ser Leu Pro Lys Leu Cys 20 25 30 Asp Glu Leu Arg Arg Tyr Leu Leu Asp Ser Val Ser Arg Ser Ser Gly 35 40 45 His Phe Ala Ser Gly Leu Gly Thr Val Glu Leu Thr Val Ala Leu His 50 55 60 Tyr Val Tyr Asn Thr Pro Phe Asp Gln Leu Ile Trp Asp Val Gly His 65 70 75 80 Gln Ala Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Lys Ile Gly 85 90 95 Thr Ile Arg Gln Lys Gly Gly Leu His Pro Phe Pro Trp Arg Gly Glu 100 105 110 Ser Glu Tyr Asp Val Leu Ser Val Gly His Ser Ser Thr Ser Ile Ser 115 120 125 Ala Gly Ile Gly Ile Ala Val Ala Ala Glu Lys G lu Gly Lys Asn Arg 130 135 140 Arg Thr Val Cys Val Ile Gly Asp Gly Ala Ile Thr Ala Gly Met Ala 145 150 155 160 Phe Glu Ala Met Asn His Ala Gly Asp Ile Arg Pro Asp Met Leu Val 165 170 175 Ile Leu Asn Asp Asn Glu Met Ser Ile Ser Glu Asn Val Gly Ala Leu 180 185 190 Asn Asn His Leu Ala Gln Leu Leu Ser Gly Lys Leu Tyr Ser Ser Leu 195 200 205 Arg Glu Gly Gly Lys Lys Val Phe Ser Gly Val Pro Pro Ile Lys Glu 210 215 220 Leu Leu Lys Arg Thr Glu Glu His Ile Lys Gly Met Val Val Pro Gly 225 230 235 240 Thr Leu Phe Glu Glu Leu Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly 245 250 255 His Asp Val Leu Gly Leu Ile Thr Thr Leu Lys Asn Met Arg Asp Leu 260 265 270 270 Lys Gly Pro Gln Phe Leu His Ile Met Thr Lys Lys Gly Arg Gly Tyr 275 280 285 Glu Pro Ala Glu Lys Asp Pro Ile Thr Phe His Ala Val Pro Lys Phe 290 295 300 Asp Pro Ser Ser Gly Cys Leu Pro Lys Ser Ser Gly Gly Leu Pro Ser 305 310 315 320 Tyr Ser Lys Ile Phe Gly Asp Trp Leu Cys Glu Thr Ala Ala Lys Asp 325 330 335 Asn Lys Leu Met Ala Ile Thr Pro Ala Met Arg G lu Gly Ser Gly Met 340 345 350 Val Glu Phe Ser Arg Lys Phe Pro Asp Arg Tyr Phe Asp Val Ala Ile 355 360 365 Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Ile Gly Gly 370 375 380 Tyr Lys Pro Ile Val Ala Ile Tyr Ser Thr Phe Leu Gln Arg Ala Tyr 385 390 395 400 Asp Gln Val Leu His Asp Val Ala Ile Gln Lys Leu Pro Val Leu Phe 405 410 415 Ala Ile Asp Arg Ala Gly Ile Val Gly Ala Asp Gly Gln Thr His Gln 420 425 430 Gly Ala Phe Asp Leu Ser Tyr Leu Arg Cys Ile Pro Glu Met Val Ile 435 440 445 Met Thr Pro Ser Asp Glu Asn Glu Cys Arg Gln Met Leu Tyr Thr Gly 450 455 460 Tyr His Tyr Asn Asp Gly Pro Ser Ala Val Arg Tyr Pro Arg Gly Asn 465 470 475 480 480 Ala Val Gly Val Glu Leu Thr Pro Leu Glu Lys Leu Pro Ile Gly Lys 485 490 495 Gly Ile Val Lys Arg Arg Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly 500 505 510 Thr Leu Met Pro Glu Ala Ala Lys Val Ala Glu Ser Leu Asn Ala Thr 515 520 525 Leu Val Asp Met Arg Phe Val Lys Pro Leu Asp Glu Ala Leu Ile Leu 530 535 540 Glu Met Ala Ala Ser His Glu Ala Leu Val Thr Val G lu Glu Asn Ala 545 550 555 560 Ile Met Gly Gly Ala Gly Ser Gly Val Asn Glu Val Leu Met Ala His 565 570 575 Arg Lys Pro Val Pro Val Leu Asn Ile Gly Leu Pro Asp Phe Phe Ile 580 585 590 Pro Gln Gly Thr Gln Glu Glu Met Arg Ala Glu Leu Gly Leu Asp Ala 595 600 605 Ala Gly Met Glu Ala Lys Ile Lys Ala Trp Leu Ala 610 615 620 <210> 2 <211> 299 <212> PRT <213> Escherichia coli <400> 2 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40 45 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn 50 55 60 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser 65 70 75 80 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg 85 90 95 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu 100 105 110 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala 115 120 125 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg 165 170 175 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu 180 185 190 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu 195 200 205 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp 210 215 220 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys 290 295 <210 > 3 <211> 80 <212> PRT <213> Escherichia coli <400> 3 Met Pro Lys Lys Asn Glu Ala Pro Ala Ser Phe Glu Lys Ala Leu Ser 1 5 10 15 Glu Leu Glu Gln Ile Val Thr Arg Leu Glu Ser Gly Asp Leu Pro Leu 20 25 30 Glu Glu Ala Leu Asn Glu Phe Glu Arg Gly Val Gln Leu Ala Arg Gln 35 40 45 Gly Gln Ala Lys Leu Gln Gln Ala Glu Gln Arg Val Gln Ile Leu Leu 50 55 60 Ser Asp Asn Glu Asp Ala Ser Leu Thr Pro Phe Thr Pro Asp Asn Glu 65 70 75 80 <210> 4 <211> 348 <212> PRT <213> Escherichia coli <400> 4 Val Thr Gly Val Asn Glu Cys Ser Arg Ser Thr Cys Asn Leu Lys Tyr 1 5 10 15 Asp Glu Tyr Ser Arg Ser Gly Ser Met Gln Tyr Asn Pro Leu Gly Lys 20 25 30 Thr Asp Leu Arg Val Ser Arg Leu Cys Leu Gly Cys Met Thr Phe Gly 35 40 45 Glu Pro Asp Arg Gly Asn His Ala Trp Thr Leu Pro Glu Glu Ser Ser 50 55 60 Arg Pro Ile Ile Lys Arg Ala Leu Glu Gly Gly Ile Asn Phe Phe Asp 65 70 75 80 Thr Ala Asn Ser Tyr Ser Asp Gly Ser Ser Glu Glu Ile Val Gly Arg 85 90 95 Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp Val Val Val Ala Thr Lys 100 105 110 Val Phe His Arg Val Gly Asp Leu Pro Glu Gly Leu Ser Arg Ala Gln 115 120 125 Ile Leu Arg Ser Ile Asp Asp Ser Leu Arg Arg Leu Gly Met Asp Tyr 130 135 140 Val Asp Ile Leu Gln Ile His Arg Trp Asp Tyr Asn Thr Pro Ile Glu 145 150 155 160 Glu Thr Leu Glu Ala Leu Asn Asp Val Val Lys Ala Gly Lys Ala Arg 165 170 175 Tyr Ile Gly Ala Ser Ser Met His Ala Ser Gln Phe Ala Gln Ala Leu 180 185 190 Glu Leu Gln Lys Gln His Gly Trp Ala Gln Phe Val Ser Met Gln Asp 195 200 205 His Tyr Asn Leu Ile Tyr Arg Glu Glu Glu Arg Glu Met Leu Pro Leu 210 215 220 Cys Tyr Gln Glu Gly Val Ala Val Ile Pro Trp Ser Pro Leu Ala Arg 225 230 235 240 Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr Ala Arg Leu Val Ser 245 250 255 Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu Ser Asp Glu Asn Asp Ala 260 265 270 270 Gln Ile Ala Glu Arg Leu Thr Gly Val Ser Glu Glu Leu Gly Ala Thr 275 280 285 Arg Ala Gln Val Ala Leu Ala Trp Leu Leu Ser Lys Pro Gly Ile Ala 290 295 300 Ala Pro Ile Ile Gly Thr Ser Arg Glu Glu Gln Leu Asp Glu Leu Leu 305 310 315 320 Asn Ala Val Asp Ile Thr Leu Lys Pro Glu Gln Ile Ala Glu Leu Glu 325 330 335 Thr Pro Tyr Lys Pro His Pro Val Val Gly Phe Lys 340 345 <210> 5 <211> 398 <212> PRT <213> Escherichia coli < 400> 5 Met Lys Gln Leu Thr Ile Leu Gly Ser Thr Gly Ser Ile Gly Cys Ser 1 5 10 15 Thr Leu Asp Val Val Arg His Asn Pro Glu His Phe Arg Val Val Ala 20 25 30 Leu Val Ala Gly Lys Asn Val Thr Arg Met Val Glu Gln Cys Leu Glu 35 40 45 Phe Ser Pro Arg Tyr Ala Val Met Asp Asp Glu Ala Ser Ala Lys Leu 50 55 60 Leu Lys Thr Met Leu Gln Gln Gln Gly Ser Arg Thr Glu Val Leu Ser 65 70 75 80 Gly Gln Gln Ala Ala Cys Asp Met Ala Ala Leu Glu Asp Val Asp Gln 85 90 95 Val Met Ala Ala Ile Val Gly Ala Ala Gly Leu Leu Pro Thr Leu Ala 100 105 110 Ala Ile Arg Ala Gly Lys Thr Ile Leu Leu Ala Asn Lys Glu Ser Leu 115 120 125 Val Thr Cys Gly Arg Leu Phe Met Asp Ala Val Lys Gln Ser Lys Ala 130 135 140 Gln Leu Leu Pro Val Asp Ser Glu His Asn Ala Ile Phe Gln Ser Leu 145 150 155 160 Pro Gln Pro Ile Gln His Asn Leu Gly Tyr Ala Asp Leu Glu Gln Asn 165 170 175 Gly Val Val Ser Ile Leu Leu Thr Gly Ser Gly Gly Pro Phe Arg Glu 180 185 190 Thr Pro Leu Arg Asp Leu Ala Thr Met Thr Pro Asp Gln Ala Cys Arg 195 200 205 His Pro Asn Trp Ser Met Gly Arg Ly s Ile Ser Val Asp Ser Ala Thr 210 215 220 Met Met Asn Lys Gly Leu Glu Tyr Ile Glu Ala Arg Trp Leu Phe Asn 225 230 235 240 Ala Ser Ala Ser Gln Met Glu Val Leu Ile His Pro Gln Ser Val Ile 245 250 255 His Ser Met Val Arg Tyr Gln Asp Gly Ser Val Leu Ala Gln Leu Gly 260 265 270 Glu Pro Asp Met Val Arg Gln Leu Pro Thr Pro Trp Ala Trp Pro Asn 275 280 285 Arg Val Asn Ser Gly Val Lys Pro Leu Asp Phe Cys Lys Leu Ser Ala 290 295 300 Leu Thr Phe Ala Ala Pro Asp Tyr Asp Arg Tyr Pro Cys Leu Lys Leu 305 310 315 320 Ala Met Glu Ala Phe Glu Gln Gly Gln Ala Ala Thr Thr Ala Leu Asn 325 330 335 Ala Ala Asn Glu Ile Thr Val Ala Ala Phe Leu Ala Gln Gln Ile Arg 340 345 350 Phe Thr Asp Ile Ala Ala Leu Asn Leu Ser Val Leu Glu Lys Met Asp 355 360 365 Met Arg Glu Pro Gln Cys Val Asp Asp Val Leu Ser Val Asp Ala Asn 370 375 380 Ala Arg Glu Val Ala Arg Lys Glu Val Met Arg Leu Ala Ser 385 390 395 <210> 6 <211> 1860 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1) .. (1860) <400> 6 atg agt ttt gat att gcc aaa tac ccg acc ctg gca ctg gtc gac tcc 48 Met Ser Phe Asp Ile Ala Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser 1 5 10 15 acc cag gag tta cga ctg ttg ccg aaa gag agt tta ccg aaa ctc tgc 96 Thr Gln Glu Leu Arg Leu Leu Pro Lys Glu Ser Leu Pro Lys Leu Cys 20 25 30 gac gaa ctg cgc cgc tat tta ctc gac agc gtg agc cgt tcc agc ggg 144 Asp Glu Leu Arg Arg Tyr Leu Leu Asp Ser Val Ser Arg Ser Ser Gly 35 40 45 cac ttc gcc tcc ggg ctg ggc acg gtc gaa ctg acc gtg gcg ctg cac 192 His Phe Ala Ser Gly Leu Gly Thr Val Glu Leu Thr Val Ala Leu His 50 55 60 tat gtc tac aac acc ccg ttt gac caa ttg att tgg gat gtg ggg cat 240 Tyr Val Tyr Asn Thr Pro Phe Asp Gln Leu Ile Trp Asp Val Gly His 65 70 75 80 cag gct tat ccg cat aaa att ttg acc gga cgc cgc gac aaa atc ggc 288 Gln Ala Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Lys Ile Gly 85 90 95 acc atc cgt cag aaa ggc ggt ctg cac ccg ttc ccg tgg cgc ggc gaa 336 Thr Ile Arg Gln Lys Gly Gly Leu His Pro Phe Pro Trp Arg Gly Glu 100 105 110 agc gaa tat gac gta tta agc gtc ggg cat tca tca acc tcc atc agt 384 Ser Glu Tyr Asp Val Leu Ser Val Gly His Ser Ser Thr Ser Ile Ser 115 120 125 gcc gga att ggt att gcg gtt gct gcc gaa aaa gaa ggc aaa aat cgc 432 Ala Gly Ile Gly Ile Ala Val Ala Ala Glu Lys Glu Gly Lys Asn Arg 130 135 140 cgc acc gtc tgt gtc att ggc gat ggc gcg att acc gca ggc atg gcg 480 Arg Thr Val Cys Val Ile Gly Asp Gly Ala Ile Thr Ala Gly Met Ala 145 150 155 160 ttt gaa gcg atg aat cac gcg ggc gat atc cgt cct gat atg ctg gtg 528 Phe Glu Ala Met Asn His Ala Gly Asp Ile Arg Pro Asp Met Leu Val 165 170 175 att ctc aac gac aat gaa atg tcg att tcc gcg ctc 576 Ile Leu Asn Asp Asn Glu Met Ser Ile Ser Glu Asn Val Gly Ala Leu 180 185 190 aac aac cat ctg gca cag ctg ctt tcc ggt aag ctt tac tct tca ctg 624 Asn Asn His Leu Ala Gln Leu Leu Ser Gly Lys Leu Tyr Ser Ser Leu 195 200 205 cgc gaa ggc ggg aaa aaa gtt ttc tct ggc gtg ccg cca att aaa gag 672 Arg Glu Gly Gly Lys Lys Val Phe Ser Gly Val Pro Pro Ile Lys Glu 210 215 220 ctg ctc aaa cgc acc gagaa cat att aaa ggc atg gta gtg cct ggc 720 Leu Leu Lys Arg Thr Glu Glu His Ile Lys Gly Met Val Val Pro Gly 225 230 235 240 acg ttg ttt gaa gag ctg ggc ttt aac tac atc ggc ccg gtg gac ggt Phe Glu Glu Leu Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly 245 250 255 cac gat gtg ctg ggg ctt atc acc acg cta aag aac atg cgc gac ctg 816 His Asp Val Leu Gly Leu Ile Thr Thr Leu Lys Asn Met Arg Asp Leu 260 265 270 aaa ggc ccg cag ttc ctg cat atc atg acc aaa aaa ggt cgt ggt tat 864 Lys Gly Pro Gln Phe Leu His Ile Met Thr Lys Lys Gly Arg Gly Tyr 275 280 285 285 gaa ccg gca gaa aaa gac ccg atc act gcc gtg cct aaa ttt 912 Glu Pro Ala Glu Lys Asp Pro Ile Thr Phe His Ala Val Pro Lys Phe 290 295 300 gat ccc tcc agc ggt tgt ttg ccg aaa agt agc ggc ggt ttg ccg agc 960 Asp Pro Ser Ser Gly Cysu Lys Ser Ser Gly Gly Leu Pro Ser 305 310 315 320 tat tca aaa atc ttt ggc gac tgg ttg tgc gaa acg gca gcg aaa gac 1008 Tyr Ser Lys Ile Phe Gly Asp Trp Leu Cys Glu Thr Ala Ala Lys Asp 325 330 335 aac aag ctg atg gcg att act ccg gcg atg cgt gaa ggt tcc ggc atg 1056 Asn Lys Leu Met Ala Ile Thr Pro Ala Met Arg Glu Gly Ser Gly Met 340 345 350 gtc gag ttt tca cgt aaa ttc ccg gat cgc tactt gc 1104 Val Glu Phe Ser Arg Lys Phe Pro Asp Arg Tyr Phe Asp Val Ala Ile 355 360 365 gcc gag caa cac gcg gtg acc ttt gct gcg ggt ctg gcg att ggt ggg 1152 Ala Glu Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Ile Gly Gly 370 375 380 tac aaa ccc att gtc gcg att tac tcc act ttc ctg caa cgc gcc tat 1200 Tyr Lys Pro Ile Val Ala Ile Tyr Ser Thr Phe Leu Gln Arg Ala Tyr 385 390 395 400 400 gat cag gtg ctg cat gac gcg att caa aag ctt ccg gtc ctg ttc 1248 Asp Gln Val Leu His Asp Val Ala Ile Gln Lys Leu Pro Val Leu Phe 405 410 415 gcc atc gac cgc gcg ggc att gtt ggt gct gac ggt caa acc cat cag 1296 Ala Ile Asp Ala Gly Ile Val Gly Ala Asp Gly Gln Thr His Gln 420 425 430 ggt gct ttt gat ctc tct tac ctg cgc tgc ata ccg gaa atg gtc att 1344 Gly Ala Phe Asp Leu Ser Tyr Leu Arg Cys Ile Pro Glu Met Val Ile 435 440 445 atg acc ccg agc gat gaa aac gaa tgt cgc cag atg ctc tat acc ggc 1392 Met Thr Pro Ser Asp Glu Asn Glu Cys Arg Gln Met Leu Tyr Thr Gly 450 455 460 tat cac tat aac gat ggc ccg tca gc tac ccg cgt ggc aac 1440 Tyr His Tyr Asn Asp Gly Pro Ser Ala Val Arg Tyr Pro Arg Gly Asn 465 470 475 480 gcg gtc ggc gtg gaa ctg acg ccg ctg gaa aaa cta cca att ggc aaa 1488 Ala Val Thal Pro Leu Glu Lys Leu Pro Ile Gly Lys 485 490 495 ggc att gtg aag cgt cgt ggc gag aaa ctg gcg atc ctt aac ttt ggt 1536 Gly Ile Val Lys Arg Arg Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly 500 505 510 ac atg cca gaa gcg gcg aaa gtc gcc gaa tcg ctg aac gcc acg 1584 Thr Leu Met Pro Glu Ala Ala Lys Val Ala Glu Ser Leu Asn Ala Thr 515 520 525 ctg gtc gat atg cgt ttt gtg aaa ccg ctt gat ga 1632 Leu Val Asp Met Arg Phe Val Lys Pro Leu Asp Glu Ala Leu Ile Leu 530 535 540 gaa atg gcc gcc agc cat gaa gcg ctg gtc acc gta gaa gaa aac gcc 1680 Glu Met Ala Ala Ser His Glu Ala Leu Val Thr V al Glu Glu Asn Ala 545 550 555 560 att atg ggc ggc gca ggc agc ggc gtg aac gaa gtg ctg atg gcc cat 1728 Ile Met Gly Gly Ala Gly Ser Gly Val Asn Glu Val Leu Met Ala His 565 570 575 cgt ccc gtg ctg aac att ggc ctg ccg gac ttc ttt att 1776 Arg Lys Pro Val Pro Val Leu Asn Ile Gly Leu Pro Asp Phe Phe Ile 580 585 590 ccg caa gga act cag gaa gaa atg cgc gcc gaa ctc gccc cg gcc ccc gccc Gly Thr Gln Glu Glu Met Arg Ala Glu Leu Gly Leu Asp Ala 595 600 605 gct ggt atg gaa gcc aaa atc aag gcc tgg ctg gca 1860 Ala Gly Met Glu Ala Lys Ile Lys Ala Trp Leu Ala 610 615 620 620 <210> 7 < 211> 897 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1) .. (897) <400> 7 atg gac ttt ccg cag caa ctc gaa gcc tgc gtt aag cag gcc aac cag 48 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 gcg ctg agc cgt ttt atc gcc cca ctg ccc ttt cag aac act ccc gtg 96 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 gtc gaa acc atg cag tat ggc gca tta tta ggt ggt aag cgc ctg cga 144 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40 45 cct ttc ctg gtt tat gcc acc ggt cat atg ttc ggc gtt agc aca aac 192 Pro Phe Leu Val Tyr Ala Thrly His Met Phe Gly Val Ser Thr Asn 50 55 60 acg ctg gac gca ccc gct gcc gcc gtt gag tgt atc cac gct tac tca 240 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser 65 70 75 80 tta att cat gat gat tta ccg gca atg gat gat gac gat ctg cgt cgc 288 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg 85 90 95 ggt ttg cca acc tgc cat gtg aag ttt ggc gaa gca ac gc atg 336 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu 100 105 110 gct ggc gac gct tta caa acg ctg gcg ttc tcg att tta agc gat gcc 384 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ileu Ser Asp Ala 115 120 125 gat atg ccg gaa gtg tcg gac cgc gac aga att tcg atg att tct gaa 432 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 ctg gcg agc gcc agt ggt attcc gga atg tgc g gt ggt cag gca tta 480 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 gat tta gac gcg gaa ggc aaa cac gta cct ctg gac gcg ctt gag cgt 528 Asp Leu Asp Ala Glu His Val Pro Leu Asp Ala Leu Glu Arg 165 170 175 att cat cgt cat aaa acc ggc gca ttg att cgc gcc gcc gtt cgc ctt 576 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu 180 185 190 ggt gca tta agc gcc gga gat aaa gga cgt cgt gct ctg ccg gta ctc 624 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu 195 200 205 gac aag tat gca gag agc atc ggc ctt gcc ttc cag gtt ca 672 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp 210 215 220 atc ctg gat gtg gtg gga gat act gca acg ttg gga aaa cgc cag ggt 720 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 gcc gac cag caa ctt ggt aaa agt acc tac cct gca ctt ctg ggt ctt 768 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 gag caa gcc cgg aag aaa gcc c gg gat ctg atc gac gat gcc cgt cag 816 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 tcg ctg aaa caa ctg gct gaa cag tca ctc gat acc tcg gca ctg gag Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 gcg cta gcg gac tac atc atc cag cgt aat aaa 897 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys 290 295

<210> 8 <211> 240 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1) .. (240) <400> 8 atg ccg aag aaa aat gag gcg ccc gcc agc ttt gaa aag gcg ctg agc 48 Met Pro Lys Lys Asn Glu Ala Pro Ala Ser Phe Glu Lys Ala Leu Ser 1 5 10 15 gag ctg gaa cag att gta acc cgt ctg gaa agt ggc gac ctg ccg ctg 96Glu Ile Val Thr Arg Leu Glu Ser Gly Asp Leu Pro Leu 20 25 30 gaa gag gcg ctg aac gag ttc gaa cgc ggc gtg cag ctg gca cgt cag 144 Glu Glu Alu Leu Asn Glu Phe Glu Arg Gly Val Gln Leu Ala Arg Gln 35 45 ggg cag gcc aaa tta caa caa gcc gaa cag cgc gta caa att ctg ctg 192 Gly Gln Ala Lys Leu Gln Gln Ala Glu Gln Arg Val Gln Ile Leu Leu 50 55 60 tct gac aat gaa gac gcc tct cta acc cct tttca gac aat gag 240 Ser Asp Asn Glu Asp Ala Ser Leu Thr Pro Phe Thr Pro Asp Asn Glu 65 70 75 80 <210> 9 <211> 1044 <212> DNA <213> Escherichia coli <220> <221> CDS <222 > (1) .. (1044) <400> 9 gtg act ggg gtg aac gaa tgc agc cgc agc aca tgc aac ttg aag tat 48 Val Thr Gly Val Asn G lu Cys Ser Arg Ser Thr Cys Asn Leu Lys Tyr 1 5 10 15 gac gag tat agc agg agt ggc agc atg caa tac aac ccc tta gga aaa 96 Asp Glu Tyr Ser Arg Ser Gly Ser Met Gln Tyr Asn Pro Leu Gly Lys 20 25 30 acc gac ctt cgc gtt tcc cga ctt tgc ctc ggc tgt atg acc ttt ggc 144 Thr Asp Leu Arg Val Ser Arg Leu Cys Leu Gly Cys Met Thr Phe Gly 35 40 45 gag cca gat cgc ggt aat cac gca tgg aca ctg ccg ga gaa agc agc 192 Glu Pro Asp Arg Gly Asn His Ala Trp Thr Leu Pro Glu Glu Ser Ser 50 55 60 cgt ccc ata att aaa cgt gca ctg gaa ggc ggc ata aat ttc ttt gat 240 Arg Pro Ile Ile Lys Arg Ala Leu Glu Gly Ile Asn Phe Phe Asp 65 70 75 80 acc gcc aac agt tat tct gac ggc agc agc gaa gag atc gtc ggt cgc 288 Thr Ala Asn Ser Tyr Ser Asp Gly Ser Ser Glu Glu Ile Val Gly Arg 85 90 95 gca ctg cgg gat ttc gcc cgt cgt gaa gac gtg gtc gtt gcg acc aaa 336 Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp Val Val Val Ala Thr Lys 100 105 110 gtg ttc cat cgc gtt ggt gat tta ccg gaa gga tta tcc cgt gcg ca Phe His Arg Val Gly Asp Leu Pro Glu Gly Leu Ser Arg Ala Gln 115 120 125 att ttg cgc tct atc gac gac agc ctg cga cgt ctc ggc atg gat tat 432 Ile Leu Arg Ser Ile Asp Asp Ser Leu Arg Arg Leu Gly Met Asp Tyr 130 135 140 gtc gat atc ctg caa att cat cgc tgg gat tac aac acg ccg atc gaa 480 Val Asp Ile Leu Gln Ile His Arg Trp Asp Tyr Asn Thr Pro Ile Glu 145 150 155 160 gag acg ctg gaa gcc ctc aac gac gtg gta aaa gcc ggg cgt 528 Glu Thr Leu Glu Ala Leu Asn Asp Val Val Lys Ala Gly Lys Ala Arg 165 170 175 tat atc ggc gcg tca tca atg cac gct tcg cag ttt gct cag gca ctg 576 Tyr Ile Gly Ala Ser Ser Met His Ala Ser Gln Phe Ala Gln Ala Leu 180 185 190 gaa ctc caa aaa cag cac ggc tgg gcg cag ttt gtc agt atg cag gat 624 Glu Leu Gln Lys Gln His Gly Trp Ala Gln Phe Val Ser Met Gln Asp 195 200 205 cac tac aat ctg atttat gaa gaa gag cgc gag atg cta cca ctg 672 His Tyr Asn Leu Ile Tyr Arg Glu Glu Glu Arg Glu Met Leu Pro Leu 210 215 220 tgt tat cag gag ggc gtg gcg gta att cca tgg agc ccg ctg gca Agg 720 CyGly Val Ala Val Ile Pro Trp Ser Pro Leu Ala Arg 225 230 235 240 ggc cgt ctg acg cgt ccg tgg gga gaa act acc gca cga ctg gtg tct 768 Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr Ala Arg Leg Val Ser 245 250 255 gat gag gtg ggg aaa aat ctc tat aaa gaa agc gat gaa aat gac gcg 816 Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu Ser Asp Glu Asn Asp Ala 260 265 270 270 cag atc gca gag cgg tta aca ggc gac ctg ggg gcg aca 864 Gln Ile Ala Glu Arg Leu Thr Gly Val Ser Glu Glu Leu Gly Ala Thr 275 280 285 cga gca caa gtt gcg ctg gcc tgg ttg ttg agt aaa ccg ggc att gcc 912 Arg Ala Gln Val Ala Leu Ala Leu Ser Lys Pro Gly Ile Ala 290 295 300 gca ccg att atc gga act tcg cgc gaa gaa cag ctt gat gag cta ttg 960 Ala Pro Ile Ile Gly Thr Ser Arg Glu Glu Gln Leu Asp Glu Leu Leu 305 310 315 320 aac gc gt gat atc act ttg aag ccg gaa cag att gcc gaa ctg gaa 1008 Asn Ala Val Asp Ile Thr Leu Lys Pro Glu Gln Ile Ala Glu Leu Glu 325 330 335 acg ccg tat aaa ccg cat cct gtc gta gga ttt yr 1044 Pro s Pro His Pro Val Val Gly Phe Lys 340 345 <210> 10 <211> 1194 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1) .. (1194) <400> 10 atg aag caa ctc acc att ctg ggc tcg acc ggc tcg att ggt tgc agc 48 Met Lys Gln Leu Thr Ile Leu Gly Ser Thr Gly Ser Ile Gly Cys Ser 1 5 10 15 acg ctg gac gtg gtg cgc cat aat ccc cgacactt gta gtt gcg 96 Thr Leu Asp Val Val Arg His Asn Pro Glu His Phe Arg Val Val Ala 20 25 30 ctg gtg gca ggc aaa aat gtc act cgc atg gta gaa cag tgc ctg gaa 144 Leu Val Ala Gly Lys Asn Val Thr Arg Met Val Glu Gln Cys Leu Glu 35 40 45 ttc tct ccc cgc tat gcc gta atg gac gat gaa gcg agt gcg aaa ctt 192 Phe Ser Pro Arg Tyr Ala Val Met Asp Asp Glu Ala Ser Ala Lys Leu 50 55 60 ctt aaa acg atg cta cag caa cag ggt agc cgc acc gaa gtc tta agt 240 Leu Lys Thr Met Leu Gln Gln Gln Gly Ser Arg Thr Glu Val Leu Ser 65 70 75 80 ggg caa caa gcc gct tgc gat atg gca gcg ctt gag gat gtt gat cag 288 Gly Gln Gln Ala Ala Cys Asp Met Ala Ala Leu Glu Asp Val Asp Gln 85 90 95 gtg atg gca g cc att gtt ggc gct gct ggg ctg tta cct acg ctt gct 336 Val Met Ala Ala Ile Val Gly Ala Ala Gly Leu Leu Pro Thr Leu Ala 100 105 110 gcg atc cgc gcg ggt aaa acc att ttg ctg gcc aat aaa gaa tca Ala Ile Arg Ala Gly Lys Thr Ile Leu Leu Ala Asn Lys Glu Ser Leu 115 120 125 gtt acc tgc gga cgt ctg ttt atg gac gcc gta aag cag agc aaa gcg 432 Val Thr Cys Gly Arg Leu Phe Met Asp Ala Val Lys Gln Ser Lys Ala 130 135 140 caa ttg tta ccg gtc gat agc gaa cat aac gcc att ttt cag agt tta 480 Gln Leu Leu Pro Val Asp Ser Glu His Asn Ala Ile Phe Gln Ser Leu 145 150 155 160 ccg caa cct atc cag cat aat ctg gga tac gct gac ctt gag caa aat 528 Pro Gln Pro Ile Gln His Asn Leu Gly Tyr Ala Asp Leu Glu Gln Asn 165 170 175 ggc gtg gtg tcc att tta ctt acc ggg tct ggt ggc cct ttc cgt gag 576 Gly Val Val Ile Leu Leu Thr Gly Ser Gly Gly Pro Phe Arg Glu 180 185 190 acg cca ttg cgc gat ttg gca aca atg acg ccg gat caa gcc tgc cgt 624 Thr Pro Leu Arg Asp Leu Ala ThrMet Thr Pro Asp Gln Ala Cys Arg 195 200 205 c at ccg aac tgg tcg atg ggg cgt aaa att tct gtc gat tcg gct acc 672 His Pro Asn Trp Ser Met Gly Arg Lys Ile Ser Val Asp Ser Ala Thr 210 215 220 atg atg aac aaa ggt ctg gaa tac att gaa gcg cgt tggg ttt aac 720 Met Met Asn Lys Gly Leu Glu Tyr Ile Glu Ala Arg Trp Leu Phe Asn 225 230 235 240 gcc agc gcc agc cag atg gaa gtg ctg att cac ccg cag tca gtg att 768 Ala Ser Ala Ser Gln Met Glu Val His Pro Gln Ser Val Ile 245 250 255 cac tca atg gtg cgc tat cag gac ggc agt gtt ctg gcg cag ctg ggg 816 His Ser Met Val Arg Tyr Gln Asp Gly Ser Val Leu Ala Gln Leu Gly 260 265 270 270 gaa ccg gat atg gta cgc caa ttg ccc aca cca tgg gca tgg ccg aat 864 Glu Pro Asp Met Val Arg Gln Leu Pro Thr Pro Trp Ala Trp Pro Asn 275 280 285 cgc gtg aac tct ggc gtg aag ccg ctc gat ttt tgc aaa cta ag g Asn Ser Gly Val Lys Pro Leu Asp Phe Cys Lys Leu Ser Ala 290 295 300 ttg aca ttt gcc gca ccg gat tat gat cgt tat cca tgc ctg aaa ctg 960 Leu Thr Phe Ala Ala Pro Asp Tyr Asp Arg Tyr Pro Cys Leu Lys Leu305 310 315 320 gcg atg gag gcg ttc gaa caa ggc cag gca gcg acg aca gca ttg aat 1008 Ala Met Glu Ala Phe Glu Gln Gly Gln Ala Ala Thr Thr Ala Leu Asn 325 330 335 gcc gca aac gaa atc acc gtt gtt ctt gcg caa caa atc cgc 1056 Ala Ala Asn Glu Ile Thr Val Ala Ala Phe Leu Ala Gln Gln Ile Arg 340 345 350 ttt acg gat atc gct gcg ttg aat tta tcc gta ctg gaa aaa atg gat 1104 Phe Thr Asp Ile Ala Asn Leu Ser Val Leu Glu Lys Met Asp 355 360 365 atg cgc gaa cca caa tgt gtg gac gat gtg tta tct gtt gat gcg aac 1152 Met Arg Glu Pro Gln Cys Val Asp Asp Val Leu Ser Val Asp Ala Asn 370 375 380 gcg cgt gaa gtc gcc aga aaa gag gtg atg cgt ctc gca agc 1194 Ala Arg Glu Val Ala Arg Lys Glu Val Met Arg Leu Ala Ser 385 390 395 <210> 11 <211> 4390 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (208) .. (447) <220> <221> CDS <222> (450) .. (1346) <220> <221> CDS <222> (1374) .. (3233) ) <220> <221> CDS <222> (3344) .. (4390) <400> 11 atggcggcaa tggttcgttg gcaagcctta agcgacttgt atagggaaaa atacagcagc 60 ccacacctgc ggctgcatcc aggcgcggaa gtataccact aacatcgctt tgctgtgcac 120 atcaccttac cattgcgcgt tatttgctat ttgccctgag tccgttacca tgacggggcg 180 aaaaatattg agagtcagac attcatt atg ccg aag aaa aat gag gcg ccc gcc 234 Met Pro Lys Lys Asn Glu Ala Pro Ala 1 5 agc ttt gaa aag gcg ctg agc gag ctg gaa cag att gta acc cgt ctg 282 Ser Phe Glu Lys Ala Leu Ser Glu Leu Glu Gln Ile Val Thr Arg Leu 10 15 20 25 gaa agt ggc gac ctg ccg ctg gaa gag gcg ctg aac gag ttc gaa cgc 330 Glu Ser Gly Asp Leu Pro Leu Pro Leu Glu Ala Leu Asn Glu Phe Glu Arg 30 35 40 ggc gtg cag ctg gca cgt cag ggg cag gcc aaa tta caa caa gcc gaa 378 Gly Val Gln Leu Ala Arg Gln Gly Gln Ala Lys Leu Gln Gln Ala Glu 45 50 55 cag cgta caa att ctg ctg tct gac aat gaa gac gcc tct cta acc 426 Gln Arg Val Gln Ile Leu Leu Ser Asp Asn Glu Asp Ala Ser Leu Thr 60 65 70 cct ttt aca ccg gac aat gag ta atg gac ttt ccg cag caa ctc gaa 473 Pro Phe Thr Pro Asp Asn Glu Met Asp Phe Pro Gln Gln Leu Glu 75 80 15 gcc tgc gtt aag cag gcc aac cag gcg ctg agc c gt ttt atc gcc cca 521 Ala Cys Val Lys Gln Ala Asn Gln Ala Leu Ser Arg Phe Ile Ala Pro 10 15 20 ctg ccc ttt cag aac act ccc gtg gtc gaa acc atg cag tat ggc gca 569 Leu Pro Phe Gln Asn Thr Pro Val Val Glu Thr Met Gln Tyr Gly Ala 25 30 35 40 tta tta ggt ggt aag cgc ctg cga cct ttc ctg gtt tat gcc acc ggt 617 Leu Leu Gly Gly Lys Arg Leu Arg Pro Phe Leu Val Tyr Ala Thr Gly 45 50 55 cat atg ttc ggc gtt agc aca aac acg ctg gac gca ccc gct gcc gcc 665 His Met Phe Gly Val Ser Thr Asn Thr Leu Asp Ala Pro Ala Ala Ala 60 65 70 gtt gag tgt atc cac gct tac tca tta att cat gat gat tta ccg gca 713 Val Glu Cys Ile His Ala Tyr Ser Leu Ile His Asp Asp Leu Pro Ala 75 80 85 atg gat gat gac gat ctg cgt cgc ggt ttg cca acc tgc cat gtg aag 761 Met Asp Asp Asp Asp Leu Arg Arg Gly Leu Pro Thr Cys His Val Lys 90 95 100 ttt ggc gaa gca aac gcg att ctc gct ggc gac gct tta caa acg ctg 809 Phe Gly Glu Ala Asn Ala Ile Leu Ala Gly Asp Ala Leu Gln Thr Leu 105 110 115 120 gcg ttc tcg att tta ag gcc gat atg ccg gaa gtg tcg gac cgc 857 Ala Phe Ser Ile Leu Ser Asp Ala Asp Met Pro Glu Val Ser Asp Arg 125 130 135 gac aga att tcg atg att tct gaa ctg gcg agc gcc agt ggt att gcc 905 Asp Arg Ile Ser Met Ile Ser Glu Ala Ser Ala Ser Gly Ile Ala 140 145 150 gga atg tgc ggt ggt cag gca tta gat tta gac gcg gaa ggc aaa cac 953 Gly Met Cys Gly Gly Gln Ala Leu Asp Leu Asp Ala Glu Gly Lys His 155 160 165 gta cct ctg gcg ctt gag cgt att cat cgt cat aaa acc ggc gca 1001 Val Pro Leu Asp Ala Leu Glu Arg Ile His Arg His Lys Thr Gly Ala 170 175 180 ttg att cgc gcc gcc gtt cgc ctt ggt gca tta agc gcc gga gat aaa 1049 Leu Ile Arg Ala Ala Val Arg Leu Gly Ala Leu Ser Ala Gly Asp Lys 185 190 195 200 gga cgt cgt gct ctg ccg gta ctc gac aag tat gca gag agc atc ggc 1097 Gly Arg Arg Ala Leu Pro Val Leu Asp Lys Tyr Alu Glu Ile Gly 205 210 215 ctt gcc ttc cag gtt cag gat gac atc ctg gat gtg gtg gga gat act 1145 Leu Ala Phe Gln Val Gln Asp Asp Ile Leu Asp Val Val Gly Asp Thr 220 225 230 gca acg ttg gga aaa cgc cag ggt gcc gac cag caa ctt ggt aaa agt 1193 Ala Thr Leu Gly Lys Arg Gln Gly Ala Asp Gln Gln Leu Gly Lys Ser 235 240 245 acc tac cct gca ctt ctg ggt ctt gag caa gcc cgg aag aaa gcc cgg 1241 Thr Tyr Pro Ala Leu Leu Gly Leu Glu Gln Ala Arg Lys Lys Ala Arg 250 255 260 gat ctg atc gac gat gcc cgt cag tcg ctg aaa caa ctg gct gaa cag 1289 Asp Leu Ile Asp Asp Ala Arg Gln Ser Leu Lys Gln Leu Ala Glu G275 265 280 tca ctc gat acc tcg gca ctg gaa gcg cta gcg gac tac atc atc cag 1337 Ser Leu Asp Thr Ser Ala Leu Glu Ala Leu Ala Asp Tyr Ile Ile Gln 285 290 295 cgt aat aaa taaacaataa gtatta gat gcctg at gtagtcc atg Arg Asn Lys Met Ser Phe Asp Ile Ala 15 5 aaa tac ccg acc ctg gca ctg gtc gac tcc acc cag gag tta cga ctg 1439 Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser Thr Gln Glu Leu Arg Leu 10 15 20 ttg ccg aaa gag agt tta ccg aaa ctc tgc gac gaa ctg cgc cgc tat 1487 Leu Pro Lys Glu Ser Leu Pro Lys Leu Cys Asp Glu Leu Arg Arg Tyr 25 30 35 tta ctc gac agc gtg agc cgt tcc agc ggg cacttc tcc ggg ctg 1535 Leu Leu Asp Ser Val Ser Arg Ser Ser Gly His Phe Ala Ser Gly Leu 40 45 50 ggc acg gtc gaa ctg acc gtg gcg ctg cac tat gtc tac aac acc ccg 1583 Gly Thr Val Glu Leu Thr Val Ala Leu His Tyr Val Tyr Asn Thr Pro 55 60 65 70 ttt gac caa ttg att tgg gat gtg ggg cat cag gct tat ccg cat aaa 1631 Phe Asp Gln Leu Ile Trp Asp Val Gly His Gln Ala Tyr Pro His Lys 75 80 85 att ttg acc gga cgc cgc gac aaa atc ggc acc atc cgt cag aaa ggc 1679 Ile Leu Thr Gly Arg Arg Asp Lys Ile Gly Thr Ile Arg Gln Lys Gly 90 95 100 ggt ctg cac ccg ttc ccg tgg cgc ggc gaa agc gata g Leu His Pro Phe Pro Trp Arg Gly Glu Ser Glu Tyr Asp Val Leu 105 110 115 agc gtc ggg cat tca tca acc tcc atc agt gcc gga att ggt att gcg 1775 Ser Val Gly His Ser Ser Thr Ser Ile Ser Ala Gly Ile Gly Ile Ala 120 125 130 gtt gct gcc gaa aaa gaa ggc aaa aat cgc cgc acc gtc tgt gtc att 1823 Val Ala Ala Glu Lys Glu Gly Lys Asn Arg Arg Thr Val Cys Val Ile 135 140 145 150 ggc gat ggc gcg att acc gca ggc gcg t tt gaa gcg atg aat cac 1871 Gly Asp Gly Ala Ile Thr Ala Gly Met Ala Phe Glu Ala Met Asn His 155 160 165 gcg ggc gat atc cgt cct gat atg ctg gtg att ctc aac gac aat gaa 1919 Ala Gly Asp Met Leu Val Ile Leu Asn Asp Asn Glu 170 175 180 atg tcg att tcc gaa aat gtc ggc gcg ctc aac aac cat ctg gca cag 1967 Met Ser Ile Ser Glu Asn Val Gly Ala Leu Asn Asn His Leu Ala Gln 185 190 195 ctg tcc ggt aag ctt tac tct tca ctg cgc gaa ggc ggg aaa aaa 2015 Leu Leu Ser Gly Lys Leu Tyr Ser Ser Leu Arg Glu Gly Gly Lys Lys 200 205 210 gtt ttc tct ggc gtg ccg cca att aaa gag cc 2063 Val Phe Ser Gly Val Pro Pro Ile Lys Glu Leu Leu Lys Arg Thr Glu 215 220 225 230 gaa cat att aaa ggc atg gta gtg cct ggc acg ttg ttt gaa gag ctg 2111 Glu His Ile Lys Gly Met Val Val Pro Gly Thr Leu Phe Glu Glu Leu 235 240 245 ggc ttt aac tac atc ggc ccg gtg gac ggt cac gat gtg ctg ggg ctt 2159 Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly His Asp Val Leu Gly Leu 250 255 260 atc acc acg cta aag aa catg cgc gac ctg aaa ggc ccg cag ttc ctg 2207 Ile Thr Thr Leu Lys Asn Met Arg Asp Leu Lys Gly Pro Gln Phe Leu 265 270 275 cat atc atg acc aaa aaa ggt cgt ggt tat gaa ccg gca gaa aaa gac 2 Met Thr Lys Lys Gly Arg Gly Tyr Glu Pro Ala Glu Lys Asp 280 285 290 ccg atc act ttc cac gcc gtg cct aaa ttt gat ccc tcc agc ggt tgt 2303 Pro Ile Thr Phe His Ala Val Pro Lys Phe Asp Pro Ser Ser Gly Cys 295 300 305 310 ttg ccg aaa agt agc ggc ggt ttg ccg agc tat tca aaa atc ttt ggc 2351 Leu Pro Lys Ser Ser Gly Gly Leu Pro Ser Tyr Ser Lys Ile Phe Gly 315 320 325 gac tgg ttg tgc gaa acg gca gc aac aag ctg atg gcg att 2399 Asp Trp Leu Cys Glu Thr Ala Ala Lys Asp Asn Lys Leu Met Ala Ile 330 335 340 act ccg gcg atg cgt gaa ggt tcc ggc atg gtc gag ttt tca cgt aaa 2447 Thr Pro Gla Get Arg Ser Gly Met Val Glu Phe Ser Arg Lys 345 350 355 ttc ccg gat cgc tac ttc gac gtg gca att gcc gag caa cac gcg gtg 2495 Phe Pro Asp Arg Tyr Phe Asp Val Ala Ile Ala Glu Gln His Ala Val 360 365 370 acc ttt gct gcg ggt ctg gcg att ggt ggg tac aaa ccc att gtc gcg 2543 Thr Phe Ala Ala Gly Leu Ala Ile Gly Gly Tyr Lys Pro Ile Val Ala 375 380 385 390 att tac tcc act ttc ctg caa cgc gg gat tat g cat gac 2591 Ile Tyr Ser Thr Phe Leu Gln Arg Ala Tyr Asp Gln Val Leu His Asp 395 400 405 gtg gcg att caa aag ctt ccg gtc ctg ttc gcc atc gac cgc gcg ggc 2639 Val Ala Ile Gln Lys Leu Pro Val Leu Phe Ile Asp Arg Ala Gly 410 415 420 att gtt ggt gct gac ggt caa acc cat cag ggt gct ttt gat ctc tct 2687 Ile Val Gly Ala Asp Gly Gln Thr His Gln Gly Ala Phe Asp Leu Ser 425 430 435 tac ctg cgc tgc ata ccg gaa atg gtc att atg acc ccg agc gat gaa 2735 Tyr Leu Arg Cys Ile Pro Glu Met Val Ile Met Thr Pro Ser Asp Glu 440 445 450 aac gaa tgt cgc cag atg ctc tat acc ggc tat cac tat aac gat Gc cs 2783 Asn Arg Gln Met Leu Tyr Thr Gly Tyr His Tyr Asn Asp Gly 455 460 465 470 ccg tca gcg gtg cgc tac ccg cgt ggc aac gcg gtc ggc gtg gaa ctg 2831 Pro Ser Ala Val Arg Tyr Pro Arg Gly Asn Ala Val Glu Leu 475 480 485 acg ccg ctg gaa aaa cta cca att ggc aaa ggc att gtg aag cgt cgt 2879 Thr Pro Leu Glu Lys Leu Pro Ile Gly Lys Gly Ile Val Lys Arg Arg 490 495 500 ggc gag aaa ctg gc atc ggt acg ctg atg cca gaa gcg 2927 Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly Thr Leu Met Pro Glu Ala 505 510 515 gcg aaa gtc gcc gaa tcg ctg aac gcc acg ctg gtc gat atg Cgt Ttt A975 Ala Leu Asn Ala Thr Leu Val Asp Met Arg Phe 520 525 530 gtg aaa ccg ctt gat gaa gcg tta att ctg gaa atg gcc gcc agc cat 3023 Val Lys Pro Leu Asp Glu Ala Leu Ile Leu Glu Met Ala Ala Ser His 535 540 540 gaa gcg ctg gtc acc gta gaa gaa aac gcc att atg ggc ggc gca ggc 3071 Glu Ala Leu Val Thr Val Glu Glu Asn Ala Ile Met Gly Gly Ala Gly 555 560 560 565 agc ggc gtg aac gag gc gc gat gcc cgt ccc gtg 3119 Ser Gly Val Asn Glu Val Leu Met Ala His Arg Lys Pro Val Pro Val 570 575 580 ctg aac att ggc ctg ccg gac ttc ttt att ccg caa gga act cag gaa 3167 Leu Asn Ile Gly Leu Pro Asp Phe Phe Ile Pro Gln Gly Thr Gln Glu 585 590 595 gaa atg cgc gcc gaa ctc ggc ctc gat gcc gct ggt atg gaa gcc aaa 3215 Glu Met Arg Ala Glu Leu Gly Leu Asp Ala Ala Gly Met Glu Ala Lys 600 605 gcc atc ctg gca taatccctac tccactcctg ctatgcttaa 3263 Ile Lys Ala Trp Leu Ala 615 620 gaaattattc atagactcta aataattcga gttgcaggaa ggcggcaaac gagtgaagcc 3323 ccaggagctt acataagtaa gt g ag gc gc ag gc gc ag gc gc gc gc gc gc gc gc gc ag aac ttg aag tat gac gag tat agc agg agt ggc agc atg caa tac 3424 Cys Asn Leu Lys Tyr Asp Glu Tyr Ser Arg Ser Gly Ser Met Gln Tyr 15 20 25 aac ccc tta gga aaa acc gac ctt cgc gtt tcc cgactt tgc ggc 3472 Asn Pro Leu Gly Lys Thr Asp Leu Arg Val Ser Arg Leu Cys Leu Gly 30 35 40 tgt atg acc ttt ggc gag cca gat cgc ggt aat cac gca tgg aca ctg 3520 Cys Met Thr Phe Gly Glu Pro Asp Arg Gly Asn His Ala Trp Thr Leu 45 50 55 ccg gaa gaa agc agc cgt ccc ata att aaa cgt gca ctg gaa ggc ggc 3568 Pro Glu Glu Ser Ser Arg Pro Ile I le Lys Arg Ala Leu Glu Gly Gly 60 65 70 75 ata aat ttc ttt gat acc gcc aac agt tat tct gac ggc agc agc gaa 3616 Ile Asn Phe Phe Asp Thr Ala Asn Ser Tyr Ser Asp Gly Ser Ser Glu 80 85 90 gag atc gtc ggt cgc gca ctg cgg gat ttc gcc cgt cgt gaa gac gtg 3664 Glu Ile Val Gly Arg Ala Leu Arg Asp Phe Ala Arg Arg Glu Asp Val 95 100 105 gtc gtt gcg acc aaa gtg ttc cat cgc gtt ggt gat gatta 3712 Val Val Ala Thr Lys Val Phe His Arg Val Gly Asp Leu Pro Glu Gly 110 115 120 tta tcc cgt gcg caa att ttg cgc tct atc gac gac agc ctg cga cgt 3760 Leu Ser Arg Ala Gln Ile Leu Arg Ser Ile Asp Asp Ser Leu Arg Arg 125 130 135 ctc ggc atg gat tat gtc gat atc ctg caa att cat cgc tgg gat tac 3808 Leu Gly Met Asp Tyr Val Asp Ile Leu Gln Ile His Arg Trp Asp Tyr 140 145 150 155 aac acg ccg atc gaga g ac ctg gaa gcc ctc aac gac gtg gta aaa 3856 Asn Thr Pro Ile Glu Glu Thr Leu Glu Ala Leu Asn Asp Val Val Lys 160 165 170 gcc ggg aaa gcg cgt tat atc ggc gcg tca tca atg cac gct tcg cag 3904 Ala Gly A rg Tyr Ile Gly Ala Ser Ser Met His Ala Ser Gln 175 180 185 ttt gct cag gca ctg gaa ctc caa aaa cag cac ggc tgg gcg cag ttt 3952 Phe Ala Gln Ala Leu Glu Leu Gln Lys Gln His Gly Trp Ala Gln Phe 190 195 200 gtc agt atg cag gat cac tac aat ctg att tat cgt gaa gaa gag cgc 4000 Val Ser Met Gln Asp His Tyr Asn Leu Ile Tyr Arg Glu Glu Glu Arg 205 210 215 gag atg cta cca ctg tgt tat cag gag ggc gg att cca tgg 4048 Glu Met Leu Pro Leu Cys Tyr Gln Glu Gly Val Ala Val Ile Pro Trp 220 225 230 235 agc ccg ctg gca agg ggc cgt ctg acg cgt ccg tgg gga gaa act acc 4096 Ser Pro Leu Ala Arg Gly Arg Leu Thr Arg Pro Trp Gly Glu Thr Thr 240 245 250 gca cga ctg gtg tct gat gag gtg ggg aaa aat ctc tat aaa gaa agc 4144 Ala Arg Leu Val Ser Asp Glu Val Gly Lys Asn Leu Tyr Lys Glu Ser 255 260 265 gat gaa aat gac gcg cag atc gca gag cgg tta aca ggc gtc agt gaa 4192 Asp Glu Asn Asp Ala Gln Ile Ala Glu Arg Leu Thr Gly Val Ser Glu 270 275 280 gaa ctg ggg gcg aca cga gca caa gtt gcg ctg gccg gtt gtt 40 Glu Leu Gly Ala Thr Arg Ala Gln Val Ala Leu Ala Trp Leu Leu Ser 285 290 295 aaa ccg ggc att gcc gca ccg att atc gga act tcg cgc gaa gaa cag 4288 Lys Pro Gly Ile Ala Ala Pro Ile Ile Gly Thr Ser Ar Glu Glu Gln 300 305 310 315 ctt gat gag cta ttg aac gcg gtg gat atc act ttg aag ccg gaa cag 4336 Leu Asp Glu Leu Leu Asn Ala Val Asp Ile Thr Leu Lys Pro Glu Gln 320 325 330 att gcc gaa ctgcc ac tat aaa ccg cat cct gtc gta gga ttt 4384 Ile Ala Glu Leu Glu Thr Pro Tyr Lys Pro His Pro Val Val Gly Phe 335 340 345 aaa taa 4390 Lys <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 12 ccggatccat ggcggcaatg gttcgttggc aag 33 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 13 ccgaattctt atttaaatcc tacgacagga tgcg 34 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 14 ccggatccat g agttttgat attgccaaat acc 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 15 ccgaattctt atgccagcca ggccttgatt ttg 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 16 ccgaattctt actcattgtc cggtgtaaaa ggg 33 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic DNA <400> 17 ccggatccat ggactttccg cagcaactcg aag 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 18 ccgaattctt atttattacg ctggatgatg tag 33 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 19 ccggatccta atccctactc cactcctgct atg 33 < 210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 20 gggggatcca agcaactcac cattc tgggc 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 21 gggggatccg cttgcgagac gcatcacctc 30 <210> 22 <211> 32 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 22 gggggatcca gttttgatat tgccaaatac cc 32 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Synthetic DNA <400> 23 gggggatcct gccagccagg ccttgatttt gg 32

<210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 24 gggggatccg agcaactcac cattctgggc 30 <210> 25 <211> 30 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 25 gggggatccg cttgcgagac gcatcacctc 30 <210> 26 <211> 637 <212> PRT <213> Rhodobacter sphaeroides <400> 26 Met Thr Asp Arg Pro Cys Thr Pro Thr Leu Asp Arg Val Thr Leu Pro 1 5 10 15 Val Asp Met Lys Gly Leu Thr Asp Arg Glu Leu Arg Ser Leu Ala Asp 20 25 30 Glu Leu Arg Ala Glu Thr Ile Ser Ala Val Ser Val Thr Gly Gly His 35 40 45 Leu Gly Ala Gly Leu Gly Val Val Glu Leu Thr Val Ala Leu His Ala 50 55 60 Val Phe Asp Ala Pro Arg Asp Lys Ile Ile Trp Asp Val Gly His Gln 65 70 75 80 Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Arg Ile Arg Thr 85 90 95 Leu Arg Gln Gly Gly Gly Leu Ser Gly Phe Thr Lys Arg Ser Glu Ser 100 105 110 Pro Tyr Asp Cys Phe Gly Ala Gly His Ser Ser Thr Ser Ile Ser Ala 115 120 12 5 Ala Val Gly Phe Ala Ala Ala Arg Glu Met Gly Gly Asp Thr Gly Asp 130 135 140 Ala Val Ala Val Ile Gly Asp Gly Ser Met Ser Ala Gly Met Ala Phe 145 150 155 160 Glu Ala Leu Asn His Gly Gly His Leu Lys Asn Arg Val Ile Val Ile 165 170 175 Leu Asn Asp Asn Glu Met Ser Ile Ala Pro Pro Val Gly Ala Leu Ser 180 185 190 Ser Tyr Leu Ser Arg Leu Tyr Ala Gly Ala Pro Phe Gln Asp Phe Lys 195 200 205 Ala Ala Ala Lys Gly Ala Leu Gly Leu Leu Pro Glu Pro Phe Gln Glu 210 215 220 Gly Ala Arg Arg Ala Lys Glu Met Leu Lys Ser Val Thr Val Gly Gly 225 230 235 240 Thr Leu Phe Glu Glu Leu Gly Phe Ser Tyr Val Gly Pro Ile Asp Gly 245 250 255 His Asp Leu Asp Gln Leu Leu Pro Val Leu Arg Thr Val Lys Gln Arg 260 265 270 Ala His Ala Pro Val Leu Ile His Val Ile Thr Lys Lys Gly Arg Gly 275 280 285 Tyr Ala Pro Ala Glu Ala Ala Arg Asp Arg Gly His Ala Thr Asn Lys 290 295 300 Phe Asn Val Leu Thr Gly Ala Gln Val Lys Pro Val Ser Asn Ala Pro 305 310 315 320 Ser Tyr Thr Lys Val Phe Ala Gln Ser Leu Ile Lys Glu Ala Glu Val 325 330 Three 35 Asp Glu Arg Ile Cys Ala Val Thr Ala Ala Met Pro Asp Gly Thr Gly 340 345 350 Leu Asn Leu Phe Gly Glu Arg Phe Pro Lys Arg Thr Phe Asp Val Gly 355 360 365 Ile Ala Glu Gln His Ala Val Thr Phe Ser Ala Ala Leu Ala Ala Gly 370 375 380 Gly Met Arg Pro Phe Cys Ala Ile Tyr Ser Thr Phe Leu Gln Arg Gly 385 390 395 400 Tyr Asp Gln Ile Val His Asp Val Ala Ile Gln Arg Leu Pro Val Arg 405 410 415 Phe Ala Ile Asp Arg Ala Gly Leu Val Gly Ala Asp Gly Ala Thr His 420 425 430 Ala Gly Ser Phe Asp Val Ala Phe Leu Ser Asn Leu Pro Gly Ile Val 435 440 445 Val Met Ala Ala Ala Asp Glu Ala Glu Leu Val His Met Val Ala Thr 450 455 460 Ala Ala Ala His Asp Glu Gly Pro Ile Ala Phe Arg Tyr Pro Arg Gly 465 470 475 480 Asp Gly Val Gly Val Glu Met Pro Val Lys Gly Val Pro Leu Gln Ile 485 490 495 495 Gly Arg Gly Arg Val Val Arg Glu Gly Thr Arg Ile Ala Leu Leu Ser 500 505 510 510 Phe Gly Thr Arg Leu Ala Glu Val Gln Val Ala Ala Glu Ala Leu Arg 515 520 525 Ala Arg Gly Ile Ser Pro Thr Val Ala Asp Ala Arg Phe Ala Lys Pro 530 535 540 L eu Asp Arg Asp Leu Ile Leu Gln Leu Ala Ala His His Glu Ala Leu 545 550 555 560 Ile Thr Ile Glu Glu Gly Ala Ile Gly Gly Phe Gly Ser His Val Ala 565 570 575 Gln Leu Leu Ala Glu Ala Gly Val Phe Asp Arg Gly Phe Arg Tyr Arg 580 585 590 Ser Met Val Leu Pro Asp Thr Phe Ile Asp His Asn Ser Ala Glu Val 595 600 605 Met Tyr Ala Thr Ala Gly Leu Asn Ala Ala Asp Ile Glu Arg Lys Ala 610 615 620 620 Leu Glu Thr Leu Gly Val Glu Val Leu Ala Arg Arg Ala 625 630 635 <210> 27 <211> 1911 <212> DNA <213> Rhodobacter sphaeroides <220> <221> CDS <222> (1) .. (1911) <400> 27 atg acc gac aga ccc tgc acg ccg acg ctc gac cgg gtg acg ctc ccg 48 Met Thr Asp Arg Pro Cys Thr Pro Thr Leu Asp Arg Val Thr Leu Pro 1 5 10 15 gtg gac atg aag ggc ctc acg gac cgt gag ctg cgc tcg ctg gcc gac 96 Val Asp Met Lys Gly Leu Thr Asp Arg Glu Leu Arg Ser Leu Ala Asp 20 25 30 gag ctg cgg gcc gaa acg atc tcg gcc gtg tcg gtg acg ggc ggg cat 144 Glu Leu Arg Ala Glu Thr Ile Ser Ala Val Ser Val Thr Gly Gly His 35 40 45 ctg ggc gca ggc ctc ggc gtg gtg gag ttg acg gtt gcg ctg cat gcg 192 Leu Gly Ala Gly Leu Gly Val Val Glu Leu Thr Val Ala Leu His Ala 50 55 60 gtc ttc gat gcg ccg cgc gac aag atc atc tgg gac gtg ggc cac cap Apa Phe Pro Arg Asp Lys Ile Ile Trp Asp Val Gly His Gln 65 70 75 80 tgc tac ccc cac aag atc ctg acc ggg cgg cgc gac cgc atc cgc aca 288 Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Arg Ile Arg Thr 85 90 95 ctg cgg cag ggc ggg ggt ctc tcg ggc ttc acc aag cgc tcc gag agc 336 Leu Arg Gln Gly Gly Gly Leu Ser Gly Phe Thr Lys Arg Ser Glu Ser 100 105 110 ccc tac gac tgt ttc ggc gcg gc acc tcc tcg atc tcg gcc 384 Pro Tyr Asp Cys Phe Gly Ala Gly His Ser Ser Thr Ser Ile Ser Ala 115 120 125 gcg gtg ggc ttt gcc gcg gcg cgc gag atg ggc ggc gac acg ggc gac 432 Ala Val Gly Phe Ala Ala Ag Met Gly Gly Asp Thr Gly Asp 130 135 140 gcg gtg gcg gtg atc ggc gat ggc tcg atg tcg gcc ggc atg gcc ttc 480 Ala Val Ala Val Ile Gly Asp Gly Ser Met Ser Ala Gly Met Ala Phe 145 150 155 160 ggg aac cac gg c ggg cac ctg aag aac cgg gtg atc gtg atc 528 Glu Ala Leu Asn His Gly Gly His Leu Lys Asn Arg Val Ile Val Ile 165 170 175 ctg aac gac aat gag atg agc atc gcg ccg ccg gtg ggg gcg ctg Asp Asn Glu Met Ser Ile Ala Pro Pro Val Gly Ala Leu Ser 180 185 190 tccg tat ctc tcg cgg ctc tat gcg ggc gcg ccg ttc cag gac ttc aag 624 Ser Tyr Leu Ser Arg Leu Tyr Ala Gly Ala Pro Phe Gln Asp Phe 195 200 205 gcg gcc gcc aag gga gcg ctc ggg ctt ctg ccc gaa ccg ttc cag gag 672 Ala Ala Ala Lys Gly Ala Leu Gly Leu Leu Pro Glu Pro Phe Gln Glu 210 215 220 ggc gcg cgc cgc gag agag agag agag gtc acc gtc ggc ggc 720 Gly Ala Arg Arg Ala Lys Glu Met Leu Lys Ser Val Thr Val Gly Gly 225 230 235 240 acg ctc ttc gag gag ctg ggt ttc tcc tat gtc ggc ccg atc gac ggg 768 Thr Leu Phe Glu Glu Phe Ser Tyr Val Gly Pro Ile Asp Gly 245 250 255 cac gat ctc gac cag ctt ctg ccg gtg ctg cgg acc gtc aag cag cgg 816 His Asp Leu Asp Gln Leu Leu Pro Val Leu Arg Thr Val Lys Gln Arg 260 265 270 270 gcg cat gc g ccg gtg ctg atc cat gtc atc acc aag aag ggc agg ggc 864 Ala His Ala Pro Val Leu Ile His Val Ile Thr Lys Lys Gly Arg Gly 275 280 285 tat gct ccg gcc gag gcc gcg cgc gac cgc ggc cat gcc ac gag ac 912 Tyr Ala Pro Ala Glu Ala Ala Arg Asp Arg Gly His Ala Thr Asn Lys 290 295 300 ttc aac gtc ctg acc ggc gcg cag gtg aag ccg gtc tcg aac gcc ccc 960 Phe Asn Val Leu Thr Gly Ala Gln Val Lys Pro Val Asn Ala Pro 305 310 315 320 tcc tac acc aag gtc ttc gcc cag agc ctc atc aag gag gcc gag gtc 1008 Ser Tyr Thr Lys Val Phe Ala Gln Ser Leu Ile Lys Glu Ala Glu Val 325 330 335 gac gag cgg atc tgc gc gt acg gcc gcc atg ccg gac ggg acg ggg 1056 Asp Glu Arg Ile Cys Ala Val Thr Ala Ala Met Pro Asp Gly Thr Gly 340 345 350 ctc aac ctc ttc ggc gag cgg ttt ccg aag cgc acc ttc gac gtg Asc Leu Leu Gly Glu Arg Phe Pro Lys Arg Thr Phe Asp Val Gly 355 360 365 atc gcg gaa cag cat gcg gtg acc ttc tcg gcg gcg ctt gcg gca ggc 1152 Ile Ala Glu Gln His Ala Val Thr Phe Ser Ala Ala Leu Ala Ala Gly 370 75 380 ggc atg cgg ccc ttc tgc gcg atc tat tcc acc ttc ctc cag cgc ggc 1200 Gly Met Arg Pro Phe Cys Ala Ile Tyr Ser Thr Phe Leu Gln Arg Gly 385 390 395 400 tac gac cag atc gtg g cat gc gc cgc ctg ccg gtg cgc 1248 Tyr Asp Gln Ile Val His Asp Val Ala Ile Gln Arg Leu Pro Val Arg 405 410 415 ttc gcc atc gat cgc gcg ggc ctc gtg ggg gcg gac ggc gcc acc cat 1296 Phe Ala Ile Asp Val Gly Ala Asp Gly Ala Thr His 420 425 430 gcg ggc tcg ttc gac gtg gcc ttc ctg tcg aac ctg ccc ggc atc gtg 1344 Ala Gly Ser Phe Asp Val Ala Phe Leu Ser Asn Leu Pro Gly Ile Val 435 at 440 g 445 gcc gcc gac gag gcc gag ctc gtc cat atg gtg gcc acc 1392 Val Met Ala Ala Ala Asp Glu Ala Glu Leu Val His Met Val Ala Thr 450 455 460 gcc gcc gcc cat gac gaa ggg ccc atc gcc ttc cgc tac ccg cgc gg Ala Ala Ala His Asp Glu Gly Pro Ile Ala Phe Arg Tyr Pro Arg Gly 465 470 475 480 gac ggc gtg ggg gtc gag atg ccg gtg aag ggc gtg ccg ctc cag atc 1488 Asp Gly Val Gly Val Glu Met Pro Val Lys l Pro Leu Gln Ile 485 490 495 ggc cgc ggc cgt gtg gtg cgc gag ggc acg cga atc gcg ctt ttg tcc 1536 Gly Arg Gly Arg Val Val Arg Glu Gly Thr Arg Ile Ala Leu Leu Ser 500 505 510 ttc ggc acc gag gtg cag gtg gcc gcc gag gcg ctg cgt 1584 Phe Gly Thr Arg Leu Ala Glu Val Gln Val Ala Ala Glu Ala Leu Arg 515 520 525 gcg cgc ggg atc tct ccc acg gtt gcg gat gcg cgc Ttt gcaaag ccg Ile Ser Pro Thr Val Ala Asp Ala Arg Phe Ala Lys Pro 530 535 540 ctc gac cgg gat ctg atc ctg cag ctc gcg gcc cat cac gag gcg ctt 1680 Leu Asp Arg Asp Leu Ile Leu Gln Leu Ala Ala His His Glu Ala Leu 545 550 555 560 atc acc atc gag gag ggc gcc atc ggc ggt ttc ggc agc cat gtg gcg 1728 Ile Thr Ile Glu Glu Gly Ala Ile Gly Gly Phe Gly Ser His Val Ala 565 570 575 cag ctt ctg gcc gag gcc ggg cgt ggc ttc cgg tat cgc 1776 Gln Leu Leu Ala Glu Ala Gly Val Phe Asp Arg Gly Phe Arg Tyr Arg 580 585 590 tcg atg gtg ctg ccc gac acg ttc atc gac cac aac agc gcg gag gtg 1824 Ser Met Val Phe Ile Asp His Asn Ser Ala Glu Val 595 600 605 atg tat gcc acc gcc ggg ctg aat gcg gcc gac ata gag cgg aag gcg 1872 Met Tyr Ala Thr Ala Gly Leu Asn Ala Ala Asp Ile Glu Arg Lys Ala 610 615 620 ctg g acg ctg ggg gtg gag gtc ctc gcc cgc cgc gcc 1911 Leu Glu Thr Leu Gly Val Glu Val Leu Ala Arg Arg Ala 625 630 635 <210> 28 <211> 648 <212> PRT <213> Rhodobacter sphaeroides <400> 28 Met Thr Asn Pro Thr Pro Arg Pro Glu Thr Pro Leu Leu Asp Arg Val 1 5 10 15 Cys Cys Pro Ala Asp Met Lys Ala Leu Ser Asp Ala Glu Leu Glu Arg 20 25 30 Leu Ala Asp Glu Val Arg Ser Glu Val Ile Ser Val Val Ala Glu Thr 35 40 45 Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala 50 55 60 Leu His Ala Val Phe Asn Thr Pro Thr Asp Lys Leu Val Trp Asp Val 65 70 75 80 Gly His Gln Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Glu Gln 85 90 95 Met Arg Thr Leu Arg Gln Lys Gly Gly Leu Ser Gly Phe Thr Lys Arg 100 105 110 Ser Glu Ser Ala Tyr Asp Pro Phe Gly Ala Ala His Ser Ser Thr Thr Ser 115 120 125 Ile Ser Ala Ala Leu Gly Phe Ala Met Gly Arg Glu Leu Gly Gln Pro 130 135 140 Val Gly Asp Thr Ile Ala Val Ile Gly Asp Gly Ser Ile Thr Ala Gly 145 150 155 160 Met Ala Tyr Glu Ala Leu Asn His Ala Gly His Leu Asn Lys Arg Leu 165 170 175 Phe Val Ile Leu Asn Asp Asn Asp Met Ser Ile Ala Pro Pro Val Gly 180 185 190 Ala Leu Ala Arg Tyr Leu Val Asn Leu Ser Ser Lys Ala Pro Phe Ala 195 200 205 Thr Leu Arg Ala Ala Ala Asp Gly Leu Glu Ala Ser Leu Pro Gly Pro 210 215 220 Leu Arg Asp Gly Ala Arg Arg Ala Arg Gln Leu Val Thr Gly Met Pro 225 230 235 240 Gly Gly Gly Thr Leu Phe Glu Glu Leu Gly Phe Thr Tyr Val Gly Pro 245 250 255 Ile Asp Gly His Asp Met Glu Ala Leu Leu Gln Thr Leu Arg Ala Ala 260 265 270 Arg Ala Arg Thr Thr Gly Pro Val Leu Ile His Val Val Thr Lys Lys 275 280 285 Gly Lys Gly Tyr Ala Pro Ala Glu Asn Ala Pro Asp Lys Tyr His Gly 290 295 300 Val Asn Lys Phe Asp Pro Val Thr Gly Glu Gln Lys Lys Ser Val Ala 305 310 315 320 Asn Ala Pro Asn Tyr Thr Lys Val Phe Gly Ser Thr Leu Thr Glu Glu 325 330 335 Ala Ala Arg Asp Pro Arg Ile Val Ala Ile Thr Ala Ala Met Pro Ser 340 345 350 350 Gly Thr Gly Val Asp Ile Met Gln Lys Arg Phe Pro Asn Arg Val Phe 355 360 365 Asp Val Gly Ile Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu 370 375 380 Ala Gly Ala Gly Met Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Leu 385 390 395 400 Gln Arg Gly Tyr Asp Gln Ile Ala His Asp Val Ala Leu Gln Asn Leu 405 410 415 Pro Val Arg Phe Val Ile Asp Arg Ala Gly Leu Val Gly Ala Asp Gly 420 425 430 Ala Thr His Ala Gly Ala Phe Asp Val Gly Phe Leu Thr Ser Leu Pro 435 440 445 Asn Met Thr Val Met Ala Ala Ala Asp Glu Ala Glu Leu Ile His Met 450 455 460 Ile Ala Thr Ala Val Ala Phe Asp Glu Gly Pro Ile Ala Phe Arg Phe 465 470 475 480 480 Pro Arg Gly Glu Gly Val Gly Val Glu Met Pro Glu Arg Gly Thr Val 485 490 495 Leu Glu Pro Gly Arg Gly Arg Val Val Arg Glu Gly Thr Asp Val Ala 500 505 510 Ile Leu Ser Phe Gly Ala His Leu His Glu Ala Leu Gln Ala Ala Lys 515 520 525 Leu Leu Glu Ala Glu Gly Val Ser Val Thr Val Ala Asp Ala Arg Phe 530 535 540 Ser Arg Pro Leu Asp Thr Gly Leu Ile Asp Gln Leu Val Arg His His 545 550 555 560 Ala Ala Leu Val Thr Val Glu Gln Gly Ala Met Gly Gly Phe Gly Ala 565 570 575 His Val Met His Tyr Leu Ala Asn Ser Gly Gly Phe Asp Gly Gly Leu 580 585 590 Ala Leu Arg Val Met Thr Leu Pro Asp Arg Phe Ile Glu Gln Ala Ser 595 600 605 Pro Glu Asp Met Tyr Ala Asp Ala Gly Leu Arg Ala Glu Asp Ile Ala 610 615 620 620 Ala Thr Ala Arg Gly Ala Leu Ala Arg Gly Arg Val Met Pro Leu Arg 625 630 635 640 Gln Thr Ala Lys Pro Arg Ala Val 645

<210> 29 <211> 1944 <212> DNA <213> Rhodobacter sphaeroides <220> <221> CDS <222> (1) .. (1944) <400> 29 atg acc aat ccc acc ccg cga ccc gaa acc ccg ctt ttg gat cgc gtc 48 Met Thr Asn Pro Thr Pro Arg Pro Glu Thr Pro Leu Leu Asp Arg Val 1 5 10 15 tgc tgc ccg gcc gac atg aag gcg ctg agt gac gcc ga ctg gag cgg 96 Cys Cys Pro Ala Asp Met Lys Ala Leu Ser Asp Ala Glu Leu Glu Arg 20 25 30 ctg gcc gac gaa gtg cgt tcc gag gtg att tcg gtc gtt gcc gag acg 144 Leu Ala Asp Glu Val Arg Ser Glu Val Ile Ser Val Val Ala Glu Thr 35 40 45 gga gga cat ctg ggg tcc tcg ctg ggg gtg gtc gag ctg acc gtc gcg 192 Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala 50 55 60 ctg cat gca gtc ttc aac acg ccc acc gac aag ctc gt tgg gac gtg 240 Leu His Ala Val Phe Asn Thr Pro Thr Asp Lys Leu Val Trp Asp Val 65 70 75 80 ggc cac cag tgc tac ccc cac aag atc ctc acc ggc cgg cgc gag cag 288 Gly His Gln Cys Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Glu Gln 85 90 95 atg cgc acc ctg cgc cag aag gg c ggc ctc tcg ggc ttc acc aag cgc 336 Met Arg Thr Leu Arg Gln Lys Gly Gly Leu Ser Gly Phe Thr Lys Arg 100 105 110 tcg gaa tcc gcc tac gac ccg ttc ggc gcg gcc cat tcc tcg acc tcg A 384 Ser Tyr Asp Pro Phe Gly Ala Ala His Ser Ser Thr Ser 115 115 125 atc tcg gcc gcg ctc ggc ttt gcc atg ggc cgc gag ctg ggc caa ccc 432 Ile Ser Ala Ala Leu Gly Phe Ala Met Gly Arg Glu Leu Gly Gln Pro 130 135 140 gtg ggc gac acg atc gcc gtg atc ggc gac ggc tcg atc acc gcg ggc 480 Val Gly Asp Thr Ile Ala Val Ile Gly Asp Gly Ser Ile Thr Ala Gly 145 150 155 160 atg gcc tac gag gcg ctg aac cac gcg gg aac aag cgc ctg 528 Met Ala Tyr Glu Ala Leu Asn His Ala Gly His Leu Asn Lys Arg Leu 165 170 175 ttc gtg atc ctg aac gac aat gac atg agc atc gcg ccg ccc gtg ggg 576 Phe Val Ile Asu Asn Asn Ser Ile Ala Pro Pro Val Gly 180 185 190 gct ctg gcg cgc tat ctc gtg aat ctc tcc tcg aag gcg ccc ttc gcc 624 Ala Leu Ala Arg Tyr Leu Val Asn Leu Ser Ser Lys Ala Pro Phe Ala 195 200 205 acg ctg cgc gc c gcc gac ggg ctc gag gcc tcg ctg ccg ggg ccg 672 Thr Leu Arg Ala Ala Ala Asp Gly Leu Glu Ala Ser Leu Pro Gly Pro 210 215 220 ctc cgc gac ggg gcg cgc cgg gcg cgc cag ctc ggg acc 720 Arg Asp Gly Ala Arg Arg Ala Arg Gln Leu Val Thr Gly Met Pro 225 230 235 240 ggc ggg ggc acg ctc ttc gag gag ctg ggc ttc acc tat gtg ggt ccc 768 Gly Gly Gly Thr Leu Phe Glu Glu Leu Gly Phe Thr Gly Pro 245 250 255 atc gac ggc cac gac atg gag gcg ctg ctc cag acg ctg cgc gcg gcg 816 Ile Asp Gly His Asp Met Glu Ala Leu Leu Gln Thr Leu Arg Ala Ala 260 265 270 270 cgg gcc cgg acc acg ggg cc g atc cat gtg gtc acg aag aag 864 Arg Ala Arg Thr Thr Gly Pro Val Leu Ile His Val Val Thr Lys Lys 275 280 285 ggc aag ggc tac gcc cct gcc gag aat gcc ccc gac aag tat cac ggg 912 Gly Lys Gly Tyr Ala Pro Ala Glu Asn Ala Pro Asp Lys Tyr His Gly 290 295 300 gtg aac aag ttc gac ccc gtc acg ggc gag cag aag aag tcg gtc gcc 960 Val Asn Lys Phe Asp Pro Val Thr Gly Glu Gln Lys Lys Ser Val Ala 305 310 315 320 a ac gcg ccg aac tac acc aag gtc ttc ggc tcc acc ctg acc gag gag 1008 Asn Ala Pro Asn Tyr Thr Lys Val Phe Gly Ser Thr Leu Thr Glu Glu 325 330 335 gcc gcg cgc gat ccg cgc atc gtg gcc atc atg gg gcc ccc tcg 1056 Ala Ala Arg Asp Pro Arg Ile Val Ala Ile Thr Ala Ala Met Pro Ser 340 345 350 ggc acc ggc gtc gac atc atg cag aag cgt ttc ccg aac cgc gtc ttc 1104 Gly Thr Gly Val Asp Ile Met Gln Lys Ar Pro Asn Arg Val Phe 355 360 365 gac gtg ggc atc gcc gag cag cat gcc gtg acc ttc gcg gcg ggc ctt 1152 Asp Val Gly Ile Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu 370 375 380 380 gcc ggg gcc ggg atag ccc ttc tgc gcg atc tat tcc tcg ttc ctg 1200 Ala Gly Ala Gly Met Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Leu 385 390 395 400 caa cgg ggc tac gac cag atc gcc cat gac gtg gcg ctg cag aac c Gly Tyr Asp Gln Ile Ala His Asp Val Ala Leu Gln Asn Leu 405 410 415 ccc gtc cgc ttc gtg atc gac cgg gcg ggg ctc gtg ggg gcc gac ggt 1296 Pro Val Arg Phe Val Ile Asp Arg Ala Gly Leu Val Gly Ala As p Gly 420 425 430 gcg acc cat gcg ggg gcc ttc gat gtg ggc ttc ctc acg tcg ctg ccc 1344 Ala Thr His Ala Gly Ala Phe Asp Val Gly Phe Leu Thr Ser Leu Pro 435 440 445 aat atg acc gtg atg gcc ggg gcc gag gcc gag ctc atc cac atg 1392 Asn Met Thr Val Met Ala Ala Ala Asp Glu Ala Glu Leu Ile His Met 450 455 460 atc gcc acc gcc gtg gcc ttc gac gag ggc ccc att gcc ttc cgc ttc 1440 Ile Ala Thr Ala Val Phe Asp Glu Gly Pro Ile Ala Phe Arg Phe 465 470 475 480 ccg cgg ggc gag ggg gtg ggc gtc gag atg ccc gag cgc ggg acc gtg 1488 Pro Arg Gly Glu Gly Val Gly Val Glu Met Pro Glu Arg Gly Thr Val 485 490 ctg gaa ccc ggc cgg ggc cgc gtg gtg cgc gag ggg acg gat gtg gcg 1536 Leu Glu Pro Gly Arg Gly Arg Val Val Arg Glu Gly Thr Asp Val Ala 500 505 510 atc ctt tcc ttc ggc gc g cat gg gc cat gg gc cat gcg g cat g gc g g cat g gc g g cat g g gcg aaa 1584 Ile Leu Ser Phe Gly Ala His Leu His Glu Ala Leu Gln Ala Ala Lys 515 520 525 ctc ctc gag gcc gag ggg gtg agc gtg acc gtg gcc gac gcc cgc ttc 1632 Leu Leu Glu Ala Glu Gly Val Ser Val Ala Asp Ala Arg Phe 530 535 540 tcg cgc ccg ctc gac acg ggg ctc att gac cag ctc gtg cgc cat cac 1680 Ser Arg Pro Leu Asp Thr Gly Leu Ile Asp Gln Leu Val Arg His His 545 550 550 555 ct gcc gg acg gtg gag cag ggg gcc atg ggc ggc ttc ggc gct 1728 Ala Ala Leu Val Thr Val Glu Gln Gly Ala Met Gly Gly Phe Gly Ala 565 570 575 cat gtc atg cac tat ctc gcc aat tcc ggc ggc ggc ggc ggc ggc gg Val Met His Tyr Leu Ala Asn Ser Gly Gly Phe Asp Gly Gly Leu 580 585 590 gcg ctc cgg gtc atg acg ctg ccc gac cgc ttc atc gag cag gcg agc 1824 Ala Leu Arg Val Met Thr Leu Pro Asp Arg Phe Ile Glu Ser 595 600 605 ccc gag gac atg tat gcc gat gcg ggg ctg cgg gcc gag gat atc gcg 1872 Pro Glu Asp Met Tyr Ala Asp Ala Gly Leu Arg Ala Glu Asp Ile Ala 610 615 620 gcc acc gcg cgg ggc ggg gc gc cgc gtg atg ccg ctc cgg 1920 Ala Thr Ala Arg Gly Ala Leu Ala Arg Gly Arg Val Met Pro Leu Arg 625 630 635 640 cag acg gca aag ccg cgg gcg gtc 1944 Gln Thr Ala Lys Pro Arg Ala Val 645 <210> 30 < twenty one 1> 394 <212> PRT <213> Rhodobacter sphaeroides <400> 30 Met Arg Ser Leu Ser Ile Phe Gly Ala Thr Gly Ser Ile Gly Glu Ser 1 5 10 15 Thr Phe Asp Leu Val Met Arg Lys Gly Gly Pro Glu Ala Phe Arg Thr 20 25 30 Val Ala Leu Thr Gly Gly Arg Asn Ile Arg Arg Leu Ala Glu Met Ala 35 40 45 Arg Ala Leu Lys Ala Glu Leu Ala Val Thr Ala His Glu Asp Cys Leu 50 55 60 Pro Ala Leu Arg Glu Ala Leu Ala Gly Thr Gly Thr Glu Val Ala Gly 65 70 75 80 Gly Ala Gln Ala Ile Ala Glu Ala Ala Asp Arg Pro Ala Asp Trp Thr 85 90 95 Met Ser Ala Ile Val Gly Ala Ala Gly Leu Val Pro Gly Met Arg Ala 100 105 110 Leu Lys His Gly Arg Thr Leu Ala Leu Ala Asn Lys Glu Ser Leu Val 115 120 125 Thr Ala Gly Gln Leu Leu Met Arg Thr Ala Gln Glu Asn Gly Ala Thr 130 135 140 Ile Leu Pro Val Asp Ser Glu His Ser Ala Val Phe Gln Ala Leu Ala 145 150 155 160 Gly Glu Asp Thr Ala Cys Val Glu Arg Val Ile Ile Thr Ala Ser Gly 165 170 175 Gly Pro Phe Arg Asp Trp Ser Leu Glu Arg Ile Arg Ala Cys Thr Val 180 185 190 Ala Glu Ala Gln Ala His Pro Asn Trp Ser Me t Gly Gln Arg Ile Ser 195 200 205 Ile Asp Ser Ala Ser Met Phe Asn Lys Ala Leu Glu Leu Ile Glu Thr 210 215 220 Arg Glu Phe Phe Gly Phe Glu Pro Asp Arg Ile Glu Ala Val Val His 225 230 235 240 Pro Gln Ser Ile Val His Ala Met Val Gly Phe Cys Asp Gly Gly Leu 245 250 255 Met Ala His Leu Gly Pro Ala Asp Met Arg His Ala Ile Gly Phe Ala 260 265 270 Leu Asn Trp Pro Gly Arg Gly Glu Val Pro Val Ala Arg Ile Asp Leu 275 280 285 Ala Gln Ile Ala Ser Leu Thr Phe Gln Lys Pro Asp Glu Glu Arg Phe 290 295 300 Pro Ala Leu Arg Leu Ala Arg Asp Val Met Ala Ala Arg Gly Leu Ser 305 310 315 320 Gly Ala Ala Phe Asn Ala Ala Lys Glu Ile Ala Leu Asp His Phe Ile 325 330 335 Ala Gly Arg Ile Gly Phe Leu Asp Met Ala Ala Val Val Glu Glu Thr 340 345 350 Leu Ala Gly Val Ser Thr Asp Pro Leu Phe Gly Lys Val Pro Asp Ala 355 360 365 Leu Glu Glu Val Leu Ala Met Asp His Leu Ala Arg Arg Ala Ala Glu 370 375 380 Glu Ala Ala Gly Leu Arg Gln Gln Lys Arg 385 390 <210> 31 <211> 1182 <212> DNA <213> Rhodobacter sphaeroides < 220> <221> C DS <222> (1) .. (1182) <400> 31 atg cgc agc ctg tcg atc ttt ggg gcc acc ggc tcc atc ggc gaa tcc 48 Met Arg Ser Leu Ser Ile Phe Gly Ala Thr Gly Ser Ile Gly Glu Ser 1 5 10 15 acc ttc gac ctc gtc atg cgg aag ggc ggg ccc gag gcg ttc cgc acc 96 Thr Phe Asp Leu Val Met Arg Lys Gly Gly Pro Glu Ala Phe Arg Thr 20 25 30 gtc gct ctg acc ggc ggg cgc aac atc cgg cga ctg gcc gaa atg gcg 144 Val Ala Leu Thr Gly Gly Arg Asn Ile Arg Arg Leu Ala Glu Met Ala 35 40 45 cgt gcg ctg aag gcg gag ctt gcc gtc acc gcg cat gag gac tgc ctg 192 Arg Ala Leu Lyu Ala Glu Val Thr Ala His Glu Asp Cys Leu 50 55 60 ccc gcg ctg cgc gag gcg ctg gcc ggg acg ggc acc gag gtc gcg ggc 240 Pro Ala Leu Arg Glu Ala Leu Ala Gly Thr Gly Thr Glu Val Ala Gly 65 70 75 80 ggg gcg cag gcc atc gcc gag gcc gcc gac cgg ccg gcc gac tgg acc 288 Gly Ala Gln Ala Ile Ala Glu Ala Ala Asp Arg Pro Ala Asp Trp Thr 85 90 95 atg tcg gcc atc gtg ggc gcc gcg ggc ctc gtg ccc gg atg 336 Met Ser Ala Ile Val Gly Ala Ala Gly Leu Val Pro Gly Me t Arg Ala 100 105 110 ctg aag cac ggc cgc acg ctg gcg ctc gcc aac aag gaa agc ctc gtg 384 Leu Lys His Gly Arg Thr Leu Ala Leu Ala Asn Lys Glu Ser Leu Val 115 120 125 acg gca ggg caa ctc ctg acg gcc cag gag aac ggc gcc acg 432 Thr Ala Gly Gln Leu Leu Met Arg Thr Ala Gln Glu Asn Gly Ala Thr 130 135 140 atc ctg ccg gtg gac agc gag cac tcc gcg gtc ttt cag gcg ctg gcg Asp Leu Ser Glu His Ser Ala Val Phe Gln Ala Leu Ala 145 150 155 160 ggc gag gac acg gcc tgc gtc gag cgc gtc atc atc atg acg gcg tcc ggc 528 Gly Glu Asp Thr Ala Cys Val Glu Arg Val Ile Ile Thr Ala Ser Gly 165 170 175 ggg ccg ttc cgc gac tgg agc ctc gag cgc atc cgc gcc tgc acc gtg 576 Gly Pro Phe Arg Asp Trp Ser Leu Glu Arg Ile Arg Ala Cys Thr Val 180 185 190 gcc gag gcg cag gcc cat ccc aac tgg tcc atg cgg atc tcc 624 Ala Glu Ala Gln Ala His Pro Asn Trp Ser Met Gly Gln Arg Ile Ser 195 200 205 atc gac agc gcc tcg atg ttc aac aag gcg ctc gag ctg atc gag acg 672 Ile Asp Ser Ala Ser Met Phe Asn Lys A Le u Glu Leu Ile Glu Thr 210 215 220 cgc gaa ttc ttc ggc ttc gag ccg gac cgg atc gag gcg gtc gtc cat 720 Arg Glu Phe Phe Gly Phe Glu Pro Asp Arg Ile Glu Ala Val Val His 225 230 235 240 ccg caacc gtc cat gcg atg gtg ggc ttc tgc gac ggg ggc ctg 768 Pro Gln Ser Ile Val His Ala Met Val Gly Phe Cys Asp Gly Gly Leu 245 250 255 atg gcc cat ctc ggc ccc gcc gac atg cgc cac gcc atc gga ttc Ala His Leu Gly Pro Ala Asp Met Arg His Ala Ile Gly Phe Ala 260 265 270 ctg aac tgg ccg ggt cgc ggc gag gtg ccc gtc gcc cgg atc gac ctc 864 Leu Asn Trp Pro Gly Arg Gly Glu Val Pro Val Ala Arg Ile Leu 275 280 285 gca cag att gcg agc ctc acc ttc cag aag cct gac gag gaa cgc ttt 912 Ala Gln Ile Ala Ser Leu Thr Phe Gln Lys Pro Asp Glu Glu Arg Phe 290 295 300 ccg gcc ctg agg ctt gcg cg cg cg gcg gcg cgc ggc ctg tcg 960 Pro Ala Leu Arg Leu Ala Arg Asp Val Met Ala Ala Arg Gly Leu Ser 305 310 315 320 ggc gcc gcc ttc aac gcg gcc aag gag atc gcg ctc gat cat ttc atc Ala Ala Aly A la Lys Glu Ile Ala Leu Asp His Phe Ile 325 330 335 gcc gga cgc atc ggg ttt ctg gac atg gcg gcg gtg gtc gag gag acg 1056 Ala Gly Arg Ile Gly Phe Leu Asp Met Ala Ala Val Val Glu Glu Thrc 340 350 gcg ggc gtt tcg acc gac ccc ctg ttc gga aaa gtg ccc gac gcc 1104 Leu Ala Gly Val Ser Thr Asp Pro Leu Phe Gly Lys Val Pro Asp Ala 355 360 365 ctt gag gaa gtg ctg gcc atg gac cat ctc gct cgg gaga gc gag 1152 Leu Glu Glu Val Leu Ala Met Asp His Leu Ala Arg Arg Ala Ala Glu 370 375 380 gaa gcc gcc ggt ctc cgc cag cag aaa agg 1182 Glu Ala Ala Gly Leu Arg Gln Gln Lys Arg 385 390 <210> 32 <211 > 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 32 aagctgatct gggacgtggg gca 23 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 33 tgctatccgc acaagatcct gac 23 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 34 gcatgctgtt ccgcgatgcc gac 23

[0225]

[Sequence List Free Text] SEQ ID NO: 12: Synthetic DNA SEQ ID NO: 13: Synthetic DNA SEQ ID NO: 14: Synthetic DNA SEQ ID NO: 16: Synthetic DNA SEQ ID NO: 17: Synthetic DNA SEQ ID NO: 18: Synthetic DNA SEQ ID NO: 19: synthetic DNA SEQ ID NO: 20: synthetic DNA SEQ ID NO: 21: synthetic DNA SEQ ID NO: 23: synthetic DNA SEQ ID NO: 24: synthetic DNA SEQ ID NO: 25: synthetic DNA SEQ ID NO: 32: synthetic DNA SEQ ID NO: 33 Synthetic DNA SEQ ID NO: 34: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of reaction temperature on the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

FIG. 2 is a graph showing the influence of the reaction solution pH on the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Each p in 100 mM Tris-HCl buffer
H showed activity. 100 activity at pH 8.0
The activity at each pH was shown as a relative activity as%.

FIG. 3 is a diagram showing a method for disrupting a yaeM gene on a chromosome using homologous recombination.

FIG. 4 shows the effect of fosmidomycin on 1-deoxy-D-xylulose 5-phosphate reductoisomerase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12P 7/04 C12R 1:19) (C12P 7/04 C12R 1:18) (C12P 7/04 (C12R 1:01) (C12P 7/26 C12R 1:19) (C12P 7/26 C12R 1:18) (C12P 7/26 C12R 1:01) (C12P 13/00 C12R 1:19) (C12P 13/00 (C12R 1:18) (C12P 13/00 C12R 1:01) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:18) (C12P 21/02 C12R 1:01) (72) Inventor Akio Ozaki 3-9-13 Nakamachi, Machida City, Tokyo (72) Inventor Haruo Seto 100-5 Uenocho, Hachioji City, Tokyo (72) Inventor Tomohisa Kuzuyama 2-11-5, Daizawa, Setagaya-ku, Tokyo (72) Invention Shunji Takahashi, Chiba City, Chiba Prefecture Subdivision Yahagi-cho 641 Green House Building B No. 102 F-term (reference) 4B024 AA01 AA03 BA07 BA80 CA04 DA05 DA06 EA04 GA11 GA14 GA19 HA01 HA12 4B064 AB04 AD92 AD94 CA02 CA19 CC24 CE02 CE08 DA01

Claims (13)

[Claims] 1. The following (a), (b), (c),
A DNA containing at least one DNA selected from (d), (e) and (f) is incorporated into a vector, the resulting recombinant DNA is introduced into a prokaryotic host cell, and the resulting transformant is obtained. Is cultured in a medium, the isoprenoid compound is produced and accumulated in the culture, and the isoprenoid compound is collected from the culture. (A) a DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvate and glyceraldehyde 3-phosphate (b) encoding a farnesyl pyrophosphate synthase D
NA (c) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and an isoprenoid compound (D) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4, or one or several amino acids in the amino acid sequence of the protein (E) a DNA encoding a protein comprising an amino acid sequence having a deleted, substituted or added amino acid sequence and having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound; 2-C-methyl-D-erythritol 4-phosphate is DN encoding a protein having an activity of catalyzing the Jill reaction
A (f) Hybridizes with a DNA selected from (a), (b), (c), (d) and (e) under stringent conditions, and has a protein encoded by the selected DNA DNA encoding a protein having substantially the same activity 2. A DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate is represented by SEQ ID NOs: 1, 26 and DNA encoding a protein having the amino acid sequence of any one of 28.
Or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and which is composed of pyruvic acid and glyceraldehyde 3-phosphate and 1-deoxy-D-xylulose 5-phosphate.
The production method according to claim 1, wherein the DNA is a DNA encoding a protein having an activity of catalyzing a reaction for producing phosphate. 3. The method according to claim 1, wherein the DNA comprises SEQ ID NOs: 6, 27 and 29.
The production method according to claim 1 or 2, which is a DNA having any one of the nucleotide sequences described above. 4. The DNA encoding farnesyl pyrophosphate synthase is a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, or one or several amino acids are deleted or substituted in the amino acid sequence of the protein. Or a DNA comprising an added amino acid sequence and encoding farnesyl pyrophosphate synthase
The production method according to claim 1, wherein 5. The method according to claim 1, wherein the DNA is a DNA having the nucleotide sequence of SEQ ID NO: 7. 6. A DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, wherein the DNA is SEQ ID NO: 5 or 30. A DNA encoding a protein having the amino acid sequence of 30 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and comprising 1-deoxy-D-xylulose. The production method according to claim 1, which is a DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 5-phosphate. 7. The DNA according to claim 1, wherein the DNA has the nucleotide sequence of SEQ ID NO: 10 or 31.
The manufacturing method as described. 8. The production method according to claim 1, wherein the DNA has a base sequence selected from the base sequences set forth in SEQ ID NOs: 8 and 9. 9. The isoprenoid compound is ubiquinone,
A isoprenoid compound selected from vitamin K ₂ and carotenoids, The process of claim 1 wherein. 10. A protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound selected from the following (a), (b) and (c): (A) a protein having the amino acid sequence of SEQ ID NO: 3,
Or a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein; (b) a protein having the amino acid sequence of SEQ ID NO: 4,
Or a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein; (c) a protein having the amino acid sequence of SEQ ID NO: 5,
Or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein 11. A recombinant DNA obtained by incorporating a DNA encoding the protein according to claim 10 into a vector.
Is introduced into a host cell, the resulting transformant is cultured in a medium, the protein is produced and accumulated in a culture, and the protein is collected from the culture, thereby producing an isoprenoid compound. A method for producing a protein having an activity capable of improving efficiency. 12. The transformant may be a microorganism belonging to the genus Escherichia , a microorganism belonging to the genus Rhodobacter or Erwinia.
The production method according to claim 1 or 11, which is a microorganism belonging to the genus. 13. A DNA encoding a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound according to any one of the following (a) to (g): (A) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 3 (b) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4 (c) Encoding a protein having the amino acid sequence of SEQ ID NO: 5 DNA (d) DNA having the base sequence of SEQ ID NO: 8 (e) DNA having the base sequence of SEQ ID NO: 9 (f) DNA having the base sequence of SEQ ID NO: 10 (g) (a) to (f) DNA that hybridizes with any of the DNAs under stringent conditions