JP5401526B2 - Novel lactam ring-opening enzyme and its use - Google Patents

Description

The present invention relates to a novel lactam ring-opening enzyme and use thereof. More specifically, a novel lactam ring-opening enzyme having an activity to open a lactam of a streptosricin derivative, a method for producing a streptosricin derivative in which a lactam is opened using the enzyme, and a streptosyl ring in which a lactam is opened The present invention relates to an antibacterial agent containing a thin derivative.

Streptothricin (sometimes abbreviated as “ST”) (see FIG. 1) is a broad antibiotic isolated from Streptomyces lavendulae for the first time in 1943 (Non-Patent Document 1: Waksman, SA). (1943) J. Bacteriol. 46, 299-310). All STs are β-
Consists of a carbamoylated D-gulosamine group and an amide type abnormal amino acid streptolidine (streptolidine lactam) to which a lysine homopolymer (1-7 residues) is attached. Since ST is a potent protein biosynthesis inhibitor in prokaryotic cells and also strongly inhibits the growth of eukaryotic cells such as yeast, fungi, protozoa, insects, plants, etc., in some of these organisms, Recombinant
Used as an effective selection agent for DNA technology. However, ST is not used for treatment because it shows nephrotoxicity.

To date, many ST resistance genes have been identified in transposable elements such as Tn1825 and Tn1826 isolated from ST-resistant bacteria (Non-patent Document 2), and this type of transposable element is Shiga toxin-producing. It has also been isolated from clinically problematic pathogens such as E. coli (Non-patent Document 3) and Shigella strain (Non-patent Document 4). Bacterial resistance to antibiotics that inhibit protein biosynthesis (such as aminoglycosides) is attributed to three causes: reduced uptake and accumulation of antibiotics, modification of 16S RNA or ribosomal proteins, and enzymatic modification of antibiotics. Caused by (
Non-patent document 5). However, with regard to bacterial resistance to ST, to date, β-lysine β-
The only common mechanism of resistance brought about by modification of the ST molecule by monoacetylation of the amino group (position 16) (see FIG. 1) has been elucidated. In fact, Streptomyces lavendula
Even in ST-producing strains such as e (Non-patent document 6), Streptomyces rochei (Non-patent document 7), Streptomyces noursei (Non-patent document 8 and Non-patent document 9), N-acetyltransferase (NA)
An ST resistance gene encoding T) has been identified and its role in self-resistance to self-produced ST has been studied. Based on this resistance mechanism and the fact that ST-D is more antibacterial than ST-F against many bacteria, the β-lysine group has been shown to play an important role in antibiotic activity. It was. On the other hand, the group of Inamori (Non-patent document 10) and Taniyama (Non-patent document 11) is ST-F-acid chemically synthesized from ST-F (see Fig. 1; in their paper, racenomycin-A-acid). Independently reported no antibiotic activity against bacteria, fungi or plants. This result confirmed that streptolidine lactam is also essential for antibiotic activity. However, the antibiotic activity of ST-D-acid has not been tested.

Waksman, S. A. (1943) J. Bacteriol. 46, 299-310 Partridge, S. R., & Hall, R. M. (2005) J. Clin. Microbiol. 43, 4298-4300 Singh, R., Schroeder, CM, Meng, J., White, DG, McDermott, PF, Wagner, DD, Yang, H., Simjee, S., Debroy, C., Walker, RD, & Zhao, S. (2005) J. Antimicrob. Chemother. 56, 216-219 Peirano, G., Agerso, Y., Aarestrup, F. M., & dos Prazeres Rodrigues, D. (2005) J. Antimicrob. Chemother. 55, 301-305 Vakulenko, S. B., & Mobashery, S. (2003) Clin. Microbiol. Rev. 16, 430-450 Horinouchi, S., Furuya, K., Nishiyama, M., Suzuki, H., & Beppu, T. (1987) J. Bacteriol. 169, 1929-1937 Fernandez-Moreno, M. A., Vallin, C., & Malpartida, F. (1997) J. Bacteriol. 179, 6929-6936 Krugel, H., Fiedler, G., Haupt, I., Sarfert, E., & Simon, H. (1988) Gene. 62, 209-217 Grammel, N., Pankevych, K., Demydchuk, J., Lambrecht, K., Saluz, H. P., & Krugel, H. (2002) Eur. J. Biochem. 269, 347-357 Inamori, Y., Tominaga, H., Okuno, M., Sato, H., & Tsujibo, H. (1988) Chem. Pharm. Bull. (Tokyo) 36, 1577-80 Taniyama, H., Sawada, Y., & Kitagawa, T. (1971) J. Antibiot. (Tokyo) 24, 662-666

In the above situation, an enzyme having an activity capable of reducing the antibiotic activity (ie, toxicity) against eukaryotic cells without losing the antibiotic activity against prokaryotic cells against streptococrine, an antibiotic against prokaryotic cells There has been a demand for a streptosricin derivative that retains its activity and has reduced toxicity to eukaryotic cells and a method for producing the same.

As a result of intensive studies to solve the above problems, the present inventors have found that SEQ ID NOs: 2, 4, 6
, 8, 10, 12, 14 or 16 has been found to have an activity to open a lactam of streptococrine. Furthermore, it was found that a derivative of streptolysin D (ST-D-acid) whose lactam was ring-opened by the enzyme has reduced toxicity to eukaryotic cells while retaining antibiotic activity against prokaryotic cells. . As a result of further studies based on these findings, the present invention has been completed.

（式中、ｎは１〜７の整数を表す。）
で表される化合物である、上記（１２）に記載の製造方法、
（１４） 式（Ｉ）で表される化合物が、式（ＩＩ）

で表される化合物である、上記（１３）に記載の製造方法、
（１５） 式（ＩＩ）

で表される化合物またはその塩を含有する抗菌剤
などを提供する。 That is, the present invention
(1) The polynucleotide according to any one of the following (a) to (f):
(a) a polynucleotide containing a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15;
(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(c) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 And a polynucleotide containing a polynucleotide encoding a protein having lactam ring-opening activity;
(d) having an amino acid sequence having 60% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 and having a lactam ring-opening activity A polynucleotide containing a polynucleotide encoding the protein;
(e) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15;
And a polynucleotide comprising a polynucleotide encoding a protein having lactam ring-opening activity; and
(f) a polynucleotide consisting of a base sequence complementary to the base sequence of a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 and a stringent A polynucleotide containing a polynucleotide that hybridizes under mild conditions and encodes a protein having lactam ring-opening activity,
(2) The polynucleotide according to (1) above, which is any of the following (g) to (i):
(g) in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 or in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16; A polynucleotide comprising a polynucleotide consisting of an amino acid sequence having 1 to 10 amino acids deleted, substituted, inserted and / or added and having a lactam ring-opening activity;
(h) having an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 and having a lactam ring-opening activity A polynucleotide containing a polynucleotide encoding the protein; and
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, or SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 A polynucleotide comprising a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence described in 1. under a highly stringent condition and encodes a protein having a lactam ring-opening activity;
(3) The polynucleotide according to (1) above, which comprises a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
(4) The polynucleotide according to (1) above, which contains a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.
(5) The polynucleotide according to any one of (1) to (4) above, which is DNA,
(6) a protein encoded by the polynucleotide according to any one of (1) to (5) above,
(7) The protein according to (6) above, comprising the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16.
(8) The protein according to (7) above, comprising the amino acid sequence set forth in SEQ ID NO: 2.
(9) A recombinant vector containing the polynucleotide according to any one of (1) to (5) above,
(10) A transformant introduced with the recombinant vector according to (9) above,
(11) The method for producing the protein according to (6), including a step of culturing the transformant according to (10) and generating the protein according to (6).
(12) A method for producing a streptosricin derivative having a ring opened by a lactam or a salt thereof, comprising a step of opening a lactam using the protein according to (6) above,
(13) A streptosricin derivative in which a lactam has been opened is represented by the formula (I)

(In the formula, n represents an integer of 1 to 7.)
The production method according to (12) above, which is a compound represented by:
(14) The compound represented by the formula (I) is represented by the formula (II)

The production method according to (13), which is a compound represented by:
(15) Formula (II)

The antibacterial agent containing the compound represented by these, or its salt is provided.

Since the protein of the present invention can reduce the antibiotic activity (ie, toxicity) against eukaryotic cells without losing the antibiotic activity against prokaryotic cells by opening the lactam of streptococrine, It can be used for the production of streptoslicin derivatives that can be developed or made into lead compounds in drug discovery.
Moreover, since the polynucleotide of the present invention can confer resistance to streptococrine to eukaryotic cells (for example, yeast), it can be suitably used for recombinant DNA technology as an antibiotic resistance marker gene. .
Furthermore, ST-D-acid retains antibiotic activity against prokaryotic cells while reducing antibiotic activity (ie, toxicity) against eukaryotic cells.
Alternatively, it can be used as a lead compound in such antibacterial drug discovery.

It is a figure which shows the chemical structure of streptothricin (ST). FIG. 2 is a schematic diagram of a clone 2.9 kb fragment involved in ST resistance and an ORF estimated by sequencing. The hatched square indicates the DNA fragment cloned into the pWHM3 plasmid. The start codon candidates (1st to 8th positions) of the sttH gene are surrounded by a gray square. The PCR primers used in this study are schematically indicated by arrows. It is a figure which shows the result of the HPLC analysis of the substance produced | generated by rSttH. ST-F (C) reacted with rSttH, ST-F (B) when rSttH was not added, and the reaction solution and ST-F standard (A) were analyzed by reverse phase HPLC. HPLC conditions are as described in “Materials and Methods”. It is a figure which shows the result of the ESI-MS / MS analysis of the substance (ST-F-acid) produced | generated from ST-F by the effect | action of ST-F and rSttH. The ESI-MS and MS / MS spectra of ST-F (A) and ST-F-acid (B) dissolved in 0.2% formic acid + 50% acetonitrile are shown.

Hereinafter, the present invention will be described in more detail.
The inventors have identified Streptomyces a which is considered as a non-producing strain of streptococci (ST).
We succeeded in isolating ST resistance conferring gene (sttH) from lbulus NBRC14147, which shows a new mechanism. In vivo and in vitro analysis of SttH demonstrated that this enzyme confers ST resistance by catalyzing the hydrolysis of the streptolidine lactam amide bond. Interestingly, ST-F (one β-lysine residue) loses toxicity to both prokaryotic and eukaryotic cells (yeast) due to the action of SttH, but has three β-lysine residues The selective toxicity of ST-D is st
Conversion from broad to bacterial specific by hydrolysis of reptolidine lactam. ST has never been clinically developed because it is toxic to mammals. However, in this study, ST-D (which may be referred to as ST-D-acid) hydrolyzed by SttH and ring-opened with lactam still has strong antibacterial activity even if its toxicity to eukaryotic cells is reduced. ST-Da
The results suggest that cid can be clinically developed or a new lead compound in drug discovery. Furthermore, the use of ST-D in combination with the sttH gene is considered to be a very powerful technique in recombinant DNA technology using eukaryotic cells such as yeast.

Based on these findings, the present invention provides a protein (novel lactam) that can reduce antibiotic activity (ie, toxicity) against eukaryotic cells without losing antibiotic activity against prokaryotic cells of streptolysin (ST). A ring-opening enzyme), a polynucleotide encoding the protein, a vector containing the polynucleotide, a transformant introduced with the vector or polynucleotide, a method for producing the protein using the transformant, and using the protein A method for producing a streptoslicin derivative having reduced antibiotic activity (ie, toxicity) against eukaryotic cells while retaining antibiotic activity against prokaryotic cells, ST
An antibacterial agent containing -D-acid is provided.

1. Polynucleotide of the Present Invention In the present specification, a polynucleotide containing a polynucleotide having the base sequence set forth in SEQ ID NO: 1 may be referred to as “sttH gene”.
First, the present invention relates to (a) a polynucleotide containing a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 (specifically, DNA, hereinafter these (Also simply referred to as “DNA”); and (b) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14
Alternatively, a polynucleotide containing a polynucleotide encoding a protein consisting of 16 amino acid sequences is provided. The DNA targeted by the present invention is the above Streptomyces albulus.
It is not limited to DNA encoding a novel lactam ring opening enzyme derived from NBRC14147, but includes other DNA encoding a protein functionally equivalent to this protein. Examples of functionally equivalent proteins include (c) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 1
In the amino acid sequence described in 6, a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added and having lactam ring-opening activity can be mentioned. Examples of such proteins include SEQ ID NOs: 2, 4, 6, 8, 10, 12,
In the amino acid sequence of 14 or 16, for example, 1 to 100, 1 to 70, 1 to 50, 1
-30, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5
A protein having an amino acid sequence in which one, one to four, one to three, one to two, or one amino acid residue is deleted, substituted, inserted and / or added, and having a lactam ring-opening activity Can be mentioned. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. Moreover, as such a protein, (d) SEQ ID NO: 2, 4, 6, 8,
10, 12, 14 or 16 amino acid sequences and about 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3% More than 99.5%, 99
A protein having an amino acid sequence having an identity of .7% or more, 99.8% or more, or 99.9% or more and having a lactam ring-opening activity. In general, the larger the homology value, the better. In addition, the identity of amino acid sequences and base sequences is determined by BLAST (for example, Altzshul SF
et al., J. Mol. Biol. 215, 403 (1990), etc.) and FASTA (Pearson WR, M
ethods in Enzymology 183, 63 (1990), etc.). When using BLAST or FASTA, the default parameters of each program are used. Here, the lactam ring-opening activity can be measured by a usual method or a method according thereto, for example, by a method described in Examples described later. The lactam ring-opening activity in the present invention is more specifically an activity for opening a lactam of streptosricin, and more specifically an activity for opening a streptolidine lactam of streptosricin. The lactam ring opening is specifically performed by hydrolysis.

The present invention also relates to (e) a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 under stringent conditions. A polynucleotide comprising a polynucleotide encoding a protein that is soy and has lactam ring-opening activity; and (f) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14
Alternatively, it encodes a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence encoding a protein comprising the amino acid sequence of 16 and has a lactam ring-opening activity. Also included are polynucleotides containing the polynucleotide.

Here, “a polynucleotide (DNA) that hybridizes under stringent conditions”
"Is a DNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 or SEQ ID NO: 2, 4, 6, 8, 10, DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of the DNA encoding the amino acid sequence described in 12, 14 or 16 as a probe. Specifically, using a filter on which colony or plaque-derived DNA is immobilized, 0.7 to 1.0 mol /
After hybridization at 65 ° C. in the presence of L NaCl, a 0.1- to 2-fold SSC (Saline-sodium citrate) solution (the composition of the 1-fold SSC solution is 150 mmol)
1 / L sodium chloride, 15 mmol / L sodium citrate)
Polynucleotides that can be identified by washing the filter under conditions can be mentioned.

Hybridization was performed using Sambrook J. et al., Molecular Cloning: A Laboratory.
Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Cloning 3rd Edition), Ausbel FM et al., Current Protocols in Mo
lecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997), Glover DM
and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second E
It can be carried out according to the method described in experiments such as dition, Oxford University Press (1995).

As used herein, the “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 32
It is a condition of ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The tighter the conditions, the higher the complementarity required for duplex formation. Specifically, for example, under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, factors affecting the stringency of hybridization include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

In addition, when using a commercially available kit for hybridization, for example, Alkphos Direct
Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol supplied with the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. , Hybridized DNA can be detected.

As other hybridizable DNAs, SEQ ID NOs: 2, 4, 6, 8, 10 when calculated using default parameters by an analysis program such as FASTA or BLAST.
About 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99 and DNA encoding the amino acid sequence described in % Or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more of DNA.

One or more amino acids are deleted, substituted, inserted and / or with respect to a certain amino acid sequence
Alternatively, a polynucleotide encoding a protein having an added amino acid sequence can be obtained by site-directed mutagenesis (for example, Gotoh, T. et al., Gene 152, 271-275 (1995), Zoller, M.
J., and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al., Nucl
eic Acids Res. 12, 9441-9456 (1984), Kramer W, and Fritz HJ, Methods. Enzymol.
154, 350-367 (1987), Kunkel, TA, Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985)
), Kunkel, Methods Enzymol. 85, 2763-2766 (1988), etc.), methods using amber mutation (eg, Gapped duplex method, Nucleic Acids Res. 12, 9441-9456 (1984), etc.), etc. Can be obtained.

PCR (for example, Ho SN et al., Gene 77, 51) using a pair of primers each having a sequence introduced with the target mutation (deletion, addition, substitution and / or insertion) at the 5 ′ end thereof.
(See (1989), etc.) can also introduce mutations into polynucleotides.
A polynucleotide encoding a partial fragment of a protein that is a kind of deletion mutant is:
Primers of oligonucleotides having a sequence matching the 5 'end base sequence of the region encoding the partial fragment to be prepared in the polynucleotide encoding the protein and oligonucleotides having a sequence complementary to the 3' end base sequence And can be obtained by performing PCR using a polynucleotide encoding the protein as a template.

Specific examples of the polynucleotide of the present invention include the polynucleotides described in any of (a) to (f) above. The polynucleotide of the present invention includes (g) SEQ ID NO:
Amino acid sequence according to 2, 4, 6, 8, 10, 12, 14 or 16 or SEQ ID NO:
In the amino acid sequence of 2, 4, 6, 8, 10, 12, 14 or 16, it consists of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and has lactam ring opening activity A polynucleotide containing a polynucleotide encoding the protein; (h) an amino acid having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 A polynucleotide comprising a polynucleotide having a sequence and encoding a protein having lactam ring-opening activity; and (i) a base according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 A polynucleotide comprising a sequence or a polynucleotide comprising a base sequence complementary to the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. A polynucleotide containing a polynucleotide that hybridizes with tide under highly stringent conditions and encodes a protein having lactam ring-opening activity is preferred, and SEQ ID NOs: 2, 4, 6, 8, 10, 12 ,
A polynucleotide comprising a polynucleotide encoding a protein having the amino acid sequence described in 14 or 16, and a polynucleotide comprising the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15; The containing polynucleotide is preferred. Among these, in terms of the lactam ring-opening activity of the encoded protein, the polynucleotide comprises a polynucleotide encoding a protein having the amino acid sequence described in SEQ ID NO: 2 and the base sequence described in SEQ ID NO: 1. Polynucleotides containing polynucleotides are preferred.

2. Protein of the present invention The present invention also provides a protein encoded by the polynucleotide of the present invention. More specifically, it is a protein encoded by any one of the polynucleotides (a) to (i), and preferably described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. Or a protein consisting of the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14
Or a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence described in 16, and having lactam ring-opening activity, and more preferably SEQ ID NO: A protein comprising the amino acid sequence described in 2, 4, 6, 8, 10, 12, 14, or 16. Especially, the protein which consists of an amino acid sequence of sequence number: 2 is preferable at the point of lactam ring-opening activity.

The amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and a lactam Examples of the protein having ring-opening activity include SEQ ID NOs: 2, 4,
The amino acid sequence according to 6, 8, 10, 12, 14 or 16, comprising the amino acid sequence in which the number of amino acid residues as described above is deleted, substituted, inserted and / or added;
And proteins having lactam ring-opening activity. In addition, such a protein has an amino acid sequence having homology as described above with the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, and a lactam Examples include proteins having ring activity. Such proteins are described in "Molecular Cloning 3rd Edition", "Current Protocols in Molecular Biology", "Nuc. Acids
Res., 10, 6487 (1982) ”,“ Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Ge
ne, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad.
Sci. USA, 82, 488 (1985) "etc., and can be obtained using the site-directed mutagenesis method.

The deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues in one and a plurality of amino acid sequences in the same sequence It means that there are amino acid residue deletion, substitution, insertion and / or addition, and two or more of deletion, substitution, insertion and addition may occur simultaneously.
Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine,
t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; C
Group: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-
Hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine.

In addition, the protein of the present invention comprises Fmoc method (fluorenylmethyloxycarbonyl method), tB
It can also be produced by a chemical synthesis method such as the oc method (t-butyloxycarbonyl method).
Chemical synthesis using peptide synthesizers such as Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instruments, Syntheselvega, Perceptive, Shimadzu, etc. You can also.

3. Furthermore, the present invention provides a recombinant vector and a transformant containing the above-described polynucleotide (DNA) of the present invention. The recombinant vector of the present invention contains the polynucleotide (DNA) described in any of the above (a) to (i). The recombinant vector of the present invention is introduced into the transformant of the present invention so that the polynucleotide (DNA) of the present invention can be expressed.

(1) Preparation of recombinant vector The recombinant vector of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the present invention into an appropriate vector. More specifically, it can be obtained by cleaving the purified DNA with an appropriate restriction enzyme, inserting it into a restriction enzyme site or a multiple cloning site of an appropriate vector, and ligating it to the vector. The vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, bacteriophages, animal viruses and the like. Examples of the plasmid include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YE).
p24, YCp50, etc.). Examples of the bacteriophage include λ phage. Examples of animal viruses include retroviruses, vaccinia viruses, insect viruses (eg, baculoviruses) and the like.

The polynucleotide of the present invention is usually operably linked downstream of a promoter in an appropriate vector. As the promoter to be used, when the host to be transformed is an animal cell, an SV40-derived promoter, a retroviral promoter,
Metallothionein promoter, heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferable. When the host is Escherichia, a Trp promoter, T7 promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP
A promoter, ADH1 promoter, GAL promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.
In addition to the above, the recombinant vector of the present invention may include those containing an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a selection marker, and the like as desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

(2) Preparation of transformant By introducing the recombinant vector containing the polynucleotide of the present invention (that is, the DNA encoding the protein of the present invention) thus obtained into a suitable host. A transformant can be prepared. The host is not particularly limited as long as it can express the DNA of the present invention, and examples thereof include Escherichia, Bacillus, Pseudomonas, Rhizobium, yeast, animal cells and insect cells. can give. Examples of the genus Escherichia include Escherichia coli. Examples of the genus Bacillus include Bacillus subtilis. Examples of the genus Pseudomonas include Pseudmonas putida (Pseud
omonas putida). Examples of the genus Rhizobium include Rhizobium meliloti. Examples of the yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe (Schizosacchar
omyces pombe). Examples of animal cells include COS cells and CHO cells. Examples of insect cells include Sf9 and Sf21.

The introduction method of the recombinant vector into the host and the transformation method thereby can be performed by various general methods. Examples of methods for introducing a recombinant vector into a host cell include, for example, the calcium phosphate method (Virology, 52, 456-457 (1973)), the lipofection method (
Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), electroporation method (EMBO J.
, 1, 841-845 (1982)). As a transformation method of the genus Escherichia,
Examples thereof include the methods described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and the like. As a method for transforming Bacillus, for example, Molecula
r & General Genetics, 168, 111 (1979). Examples of yeast transformation methods include the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). Animal cell transformation methods include, for example, Virology, 52, 456 (1973
) And the like. Insect cell transformation methods include, for example, Bio / Techn
ology, 6, 47-55 (1988). In this way, a transformant transformed with a recombinant vector containing a DNA encoding the protein of the present invention can be obtained.

4). Production of protein of the present invention The present invention also provides a method for producing the protein of the present invention, comprising the steps of culturing the transformant to produce the protein of the present invention. The protein of the present invention can be produced by culturing the transformant under conditions capable of expressing the DNA encoding the protein of the present invention, generating and accumulating the protein of the present invention, and separating and purifying the protein. .

(Culture of transformants)
The transformant of the present invention can be cultured according to a usual method used for host culture. By the culture, the protein of the present invention is produced by the transformant, and the protein of the present invention is accumulated in the transformant or in the culture solution.

As a medium for cultivating transformants whose hosts are Escherichia or Bacillus genus, it contains a carbon source, nitrogen source, inorganic salts, etc. necessary for the growth of the transformant, so that transformants can be cultured efficiently. Any natural or synthetic medium may be used as long as it can be performed automatically. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When cultivating a transformant 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 cultivating a transformant transformed with an expression vector using the Lac promoter, transformation by transforming isopropyl-β-D-thiogalactopyranoside (IPTG) with an expression vector using the trp promoter When culturing the body, indoleacrylic acid (IAA) or the like may be added to the medium.

When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation is added. When the host is Bacillus, the culture is usually about 30 to 40 ° C.
For about 6 to 24 hours, and if necessary, add aeration or stirring.

As a medium for culturing a transformant whose host is yeast, for example, Burkholder (Bu
rkholder) minimal medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) and SD medium (Proc. Natl. Acad. Sci. USA, 81, 5330 (1984) containing 0.5% casamino acid. )). The pH of the medium is preferably adjusted to about 5-8. Culture is usually about 2 at about 20 to 35 ° C.
Perform for 4 to 72 hours, adding aeration and stirring as necessary.

As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium (Science, 122, 501 (1952)) containing about 5 to 20% fetal bovine serum, a DMEM medium (Virology, 8,
396 (1959)). The pH is preferably about 6-8. Culture is usually about 3
It is carried out at 0 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and stirring are added as necessary.

As a medium for cultivating transformants whose host is insect cells, Grace's Insect Medium (N
ature, 195, 788 (1962)) to which an additive such as 10% bovine serum that has been inactivated is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually about 27
Carry out at 3 ° C for about 3 to 5 days, and add aeration and stirring as necessary.

(Separation and purification of the protein of the present invention)
From the culture, the protein of the present invention can be obtained by separating and purifying the protein of the present invention. Here, the culture means any of a culture solution, cultured cells or cultured cells, or disrupted cultured cells or cultured cells. Separation and purification of the protein of the present invention can be carried out according to a usual method.

Specifically, when the protein of the present invention accumulates in cultured cells or cells, the cells or cells are disrupted after culturing by ordinary methods (for example, ultrasound, lysozyme, freeze-thawing, etc.). After that, a crude extract of the protein of the present invention can be obtained by a usual method (for example, centrifugation, filtration, etc.). When the protein of the present invention is accumulated in the culture solution, the cells or cells and the culture supernatant are separated from each other by the usual method (for example, centrifugation, filtration, etc.) after completion of the culture. A culture supernatant containing the protein can be obtained.

Purification of the protein of the present invention contained in the extract or culture supernatant thus obtained can be performed according to a usual separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in appropriate combination. be able to.

5. Production of Streptosricin Derivative with Ring Opened by Lactam Furthermore, the present invention includes a step of ring opening of lactam using the protein of the present invention. There is provided a manufacturing method. Since the protein of the present invention has the activity of ring-opening the lactam of streptococrine, by using the protein of the present invention,
A streptosricin derivative in which a lactam is opened can be produced.

（式中、ｎは１〜７の整数を表す。） Hereinafter, according to the following reaction formula 1, a method for producing a streptosricin derivative in which the lactam of the present invention is opened will be described.
[Reaction Formula 1]

(In the formula, n represents an integer of 1 to 7.)

Compound (III) is streptosricin used as a raw material, and streptosricin X (n = 7), A (n = 6), B (n = 5), C having different β-lysine side chain lengths (N = 4),
D (n = 3), E (n = 2), and F (n = 1) are known. Streptoslicin (ie, compound (III)) is commercially available, or is a method described in literature (Reference 1, Ikuo Umezawa,
Tomio Takeuchi, Eiji Kurosu, (1949) J. Antibiot, 3, 232-235; Reference 2, Taniyam H., Sawads Y
., Kitagawa T. (1971) J. Antibiot, 24, 390-392; Reference 3, Miyashiro S, Ando T, Hi
rayama K, Kida T, Shibai H, Murai A, Shiio T, Udaka S. (1983) J. Antibiot, 36, 16
38-1643; Reference 4, Ando T, Miyashiro S, Hirayama K, Kida T, Shibai H, Murai A, Ud
aka S. (1987) J. Antibiot, 40, 1140-1145).

Examples of the solvent used in the reaction include buffer solutions such as sodium phosphate buffer and Tris-HCl buffer. The pH of the reaction solution is usually about 4.5 to 8.0, preferably about 6.0 to 8.
0, more preferably about 6.5. The reaction temperature is usually about 25 to 65 ° C., preferably about 3
5 to 65 ° C, more preferably about 45 ° C. The reaction time is usually about 30 minutes to 2 hours, but can be appropriately set according to the reaction rate and the like.

Compound (I) produced by the above reaction can be isolated and purified by a commonly used method such as reverse phase high performance liquid chromatography, gel filtration chromatography, or extraction.

When the compound (I) thus obtained is obtained in a free form, it can be converted into a salt by a usual method. Conversely, when compound (I) is obtained in the form of a salt, it can be converted to a free form or other salt by a conventional method. As such a salt, a salt with a physiologically acceptable acid (for example, inorganic acid, organic acid, etc.) or a base (for example, alkali metal, etc.) is preferable. Examples of the salt with an inorganic acid include salts with hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid and the like. Examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, citric acid, gluconic acid, tartaric acid, lactic acid, phthalic acid, fumaric acid, oxalic acid, maleic acid, succinic acid, malic acid, methanesulfonic acid, Examples thereof include salts with p-toluenesulfonic acid, adipic acid, propionic acid, sorbic acid, benzoic acid, ascorbic acid and the like. Examples of the salt with a base include a metal salt, an ammonium salt, and a salt with an organic base. Examples of the metal salt include salts with alkali metal salts such as sodium and potassium; salts with alkaline earth metals such as calcium, magnesium and barium; and aluminum salts. Examples of the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine,
Examples thereof include salts with cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine and the like.

6). Antibacterial Agent Containing Streptolysine Derivative of the Present Invention The present invention also relates to a derivative of streptosricin D (ST-D-acid) in which a lactam is ring-opened
Or the antibacterial agent containing the streptosylcin derivative of this invention, or its salt, such as its salt, is provided. The lactam-opened streptosricin D derivative (ST-D-acid) or a salt thereof that can be produced by the above production method has reduced antibiotic activity (ie, toxicity) against eukaryotic cells. However, it retains antibiotic activity against prokaryotic cells. Accordingly, mammals including humans (eg, dogs, cats, guinea pigs, rats, mice,
Pigs, sheep, cattle, etc.), birds, and reptiles can be used as antibacterial agents that can be used safely.

The lactam ring-opened streptosricin derivative used in the antibacterial agent of the present invention is streptosricin D with lactam ring-opened in terms of antibiotic activity against prokaryotic cells.
A derivative (ST-D-acid), that is, a compound represented by the above formula (II) is preferred.

Prokaryotic organisms in which the streptosricin derivative with the lactam ring-opened according to the present invention exhibits antibacterial activity (
Examples include bacteria such as E. coli and Gram-negative bacteria; pathogenic bacteria such as Mycobacterium tuberculosis, Bacillus subtilis, and Staphylococcus aureus.

When the streptosricin derivative or the salt thereof in which the lactam of the present invention is opened is used as the above-mentioned antibacterial agent, it can be carried out by a usual method. Specifically, for example,
It can be carried out as described below. When the streptosricin derivative or the salt thereof in which the lactam of the present invention is opened is used as an antibacterial agent, for example, as oral or parenteral, intravenous, oral, rectal, vaginal, transdermal, as a pharmaceutical composition. It can be administered via the skin, the nasal route or via inhalation, but is preferably administered orally. Pharmaceutical compositions for oral administration include tablets (including sugar-coated tablets, coated tablets, dry-coated tablets, sublingual tablets, buccal patches, orally disintegrating tablets), pills, capsules (hard capsules, soft capsules, microcapsules) Capsules), powders, granules, fine granules, troches, liquids (including syrups, emulsions, suspensions) and the like. Examples of the pharmaceutical composition for parenteral administration include injections, creams, ointments, suppositories and the like. Such a pharmaceutical composition can be produced, for example, by mixing with physiologically acceptable excipients, carriers and the like according to a conventional method. Examples of physiologically acceptable excipients and carriers include excipients, binders, disintegrants, lubricants in solid preparations; solvents, solubilizers, suspending agents, and buffering agents in liquid preparations. , Thickeners and emulsifiers. In addition, formulation additives such as coloring agents, sweetening agents, and antioxidants can be used as necessary.

Examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose (for example, microcrystalline cellulose),
Examples include low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate, and the like. Examples of the binder include pregelatinized starch, sucrose, gelatin, macrogol, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose (
HPC), hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (
PVP). Examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, crosslinked polyvinylpyrrolidone, carmellose sodium, croscarmellose sodium, sodium carboxymethyl starch, light anhydrous silicic acid, low-substituted hydroxypropylcellulose, Examples include cation exchange resin, partially pregelatinized starch, and corn starch. Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, talc, waxes, colloidal silica, DL-leucine, sodium lauryl sulfate, magnesium lauryl sulfate, macrogol, and aerosil.

Examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, medium chain fatty acid triglyceride (MCT), vegetable oil (for example, safflower oil, sesame oil, corn oil, olive oil, cottonseed oil, soybean Lecithin, etc.). Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol,
Examples include trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.
Examples of the thickener include natural gums and cellulose derivatives. Examples of emulsifiers include fatty acid esters (eg, sucrose fatty acid ester, glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester), wax (eg, beeswax, rapeseed hydrogenated oil, safflower hydrogenated oil, palm Hydrogenated oil,
Sitosterol, stigmasterol, campesterol, brush casterol, cacao butter powder, carnauba wax, rice wax, molasses, paraffin, etc.), lecithin (eg, egg yolk lecithin, soybean lecithin, etc.).

Examples of the colorant include water-soluble edible tar dyes (eg, edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2 and the like, water-insoluble lake dyes (eg, Examples of the water-soluble edible tar pigment include aluminum salts, natural pigments (eg, β-carotene, chlorophyll, bengara, etc.) Examples of the sweetener include sucrose, lactose, sodium saccharin, dipotassium glycyrrhizinate, Examples include aspartame, stevia, etc. Examples of the antioxidant include sulfite, ascorbic acid, and alkali metal salts and alkaline earth metal salts thereof.

Tablets, granules, fine granules and the like may be coated by a usual method using a coating substrate for the purpose of masking taste, improving light stability, improving appearance or enteric properties. Examples of the coating base include sugar coating base, water-soluble film coating substrate, enteric film coating substrate and the like. Examples of the sugar coating base include sucrose, and one or more kinds selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination. Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer Synthetic polymers such as E (Eudragit E (registered trademark)) polyvinylpyrrolidone; polysaccharides such as pullulan. Examples of the enteric film coating base include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L (Eudragit L (registered trademark)) ), Acrylic acid-based polymers such as methacrylic acid copolymer LD (Eudragit L-30D55 (registered trademark)) methacrylic acid copolymer S (Eudragit S (registered trademark)); natural products such as shellac. These coating bases may be coated singly or in combination of two or more at an appropriate ratio, or two or more may be coated sequentially.

In the antibacterial agent of the present invention, the content of the streptosricin derivative or the salt thereof in which the lactam of the present invention is opened is usually 0.01% to 100% by weight, preferably 1 to 99% by weight.

The dose of the streptosricin derivative or the salt thereof in which the lactam of the present invention is opened may be within the range of the effective amount of the antibacterial action, and varies depending on the target disease, administration subject, administration method, symptoms, etc. Usually from about 0.001 to about 1000 mg per kg body weight per day. More specifically, for example, when orally administered to a patient infected with a pathogenic bacterium as described above, the streptococrine derivative in which the lactam of the present invention is opened per day per kg of body weight. About 0.01-100 mg, preferably 0.05-50 mg, more preferably 0.1-10 mg is administered. When administered parenterally, about 0.001-50 of a streptoslicin derivative with the lactam of the present invention opened per day per kg of body weight.
mg, preferably 0.005 to 20 mg, more preferably 0.01 to 10 mg.

EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to these.

[Materials and methods]
First, materials used in the present invention and experimental methods will be described.
(1) Chemicals Streptosricin (ST) (clone NAT, ST-F and ST-D mixture; ST-F and ST-D ratio is about 5: 1) WERNER BioAgents (Meisenweg, Jena, Germany) Obtained from All other reagents were designated as analytical grades.

(2) General methods for manipulation of bacterial strains, plasmids, and DNA
S. albulus NBRC14147 DNA was used for cloning of the sttH gene. S. albulus NBRC
The medium and growth conditions of 14147 were as previously reported (Takagi, H., Hoshino, Y., Nakamo
ri, S., & Inouye, S. (2000) J. Biosci. Bioeng. 89, 94-96). E. coli-Streptomyce
sShuttle vector pWHM3 (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, K
F., & Hopwood, DA (2000) Practical Streptomyces Genetics (John Innes Founda
tion, Norwich, UK)) and S. lividans TK23 (Kieser, T., Bibb, MJ, Buttner, MJ)
, Chater, KF, & Hopwood, DA (2000) Practical Streptomyces Genetics (John I
nnes Foundation, Norwich, UK)) was used to clone the sttH gene. S. lavendulae NBRC12789 was used as the ST producing strain. The nat gene encoding NAT for ST is p
It was excised from HN15 plasmid (WERNER BioAgents). pQE30 plasmid, E. coli M15 (
pREP4) (Qiagen) and E. coli XL1-Blue MRF (Toyobo, Osaka, Japan) were used for overexpression of the recombinant protein DNA recombination of E. coli and Streptomyces strains is a standard technique. (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, &
Hopwood, DA (2000) Practical Streptomyces Genetics (John Innes Foundation, No
rwich, UK), Sambrook, J., & Russell, DW (2001) Molecular Cloning: A Laborator
y Manual (Cold Spring Harbor Lab. Press, Plainview, New York)). Southern blot analysis was performed using an ECL direct nucleic acid labeling and detection system (Amersham Bioscience, Piscataway, NJ). The expression of sttH and nat genes in S. cerevisiae
The cerevisiae CKY8 strain (MAT (ura3-52 leu2-3, 112) and the yeast episome-like pAD4 plasmid were used. This plasmid contains an ampicillin resistance gene (for E. coli) and an LEU2 gene (for yeast) selection marker. coli-Saccharomyces shuttle vector CKY8
The transformation of the strain is BD Yeastmaker Yeast Transformation System 2 (BD Biosciences Clon
tech, Palo Alto, Calif.). S. cerev with pAD4 derivative
The isiae CKY8 strain is a synthetic complete medium that does not contain L-leucine (SC-Leu) (Sherman, F. (1991)
Methods Enzymol. 194, 3-21) or YPD medium (Sherman, F. (1991) Methods Enzymol. 1
94, 3-21). In the minimum inhibitory concentration (MIC) test of ST and ST-acid, S. cerevisiae S288C (with the same genetic background as CKY8 strain), S. pombe L972, E. c as microorganisms
oli W3110, B. subtilis NBRC13169, S. aureus AB, and S. aureus FIR1169 (Igarash
i, H., Fujikawa, H., Usami, H., Kawabata, S., & Morita, T. (1984) Infect. Immun.
44, 175-181).

(3) PCR amplification of the gene encoding N-acetyltransferase (NAT) of Streptomyces strain
S. lavendulae (Horinouchi, S., Furuya, K., Nishiyama, M., Suzuki, H., & Beppu,
T. (1987) J. Bacteriol. 169, 1929-1937), S. rochei (Fernandez-Moreno, MA,
Vallin, C., & Malpartida, F. (1997) J. Bacteriol. 179, 6929-6936), S. noursei (
Krugel, H., Fiedler, G., Haupt, I., Sarfert, E., & Simon, H. (1988) Gene. 62, 20
9-217, Grammel, N., Pankevych, K., Demydchuk, J., Lambrecht, K., Saluz, HP, &
Based on the highly conserved NAT amino acid sequence of Krugel, H. (2002) Eur. J. Biochem. 269, 347-357), 5 (-GACGC (G / C) GA (A / G) GC ( G / C) ATCGA (A / G) G (G / C) (G / C) CT (G / C) GA-3 (
(SEQ ID NO: 17) and 5 (-GTTST (C / T) GTT (G / C) GT (G / C) AC (C / T) TC (G / C) AGCCA-3 ((SEQ ID NO: 18) Two primers were designed: PCR amplification was denatured at 94 ° C for 1 minute and 1 at 60 ° C.
Annealing for 1 minute and extension at 72 ° C. for 1 minute were performed under 30 cycles.

(4) Cloning of the sttH gene of S. albulus NBRC14147
The genomic DNA of S. albulus NBRC14147 was partially digested with Sau3AI. A Sau3AI fragment exceeding 2.0 kb was incorporated into the BamHI site of the pWHM3 plasmid carrying the thiostrepton resistance gene. S. lividans TK23 was transformed with the integrated DNA thus prepared, and ST (1
00 μg / mL) and thiostrepton (20 μg / mL)
Agar (Kieser, T., Bibb, MJ, Buttner, MJ, Chater, KF, & Hopwood, D.
A. (2000) Practical Streptomyces Genetics (John Innes Foundation, Norwich, UK)
Isolated from. From the 13 transformants, one of the transformants carrying a plasmid with a 2.9 kb insert (pWHM3-st11) was selected for subsequent experiments. After determining the complete nucleotide sequence of this 2.9 kb fragment, using pWHM3, ORF2-ORF3 (pWHM3-orf2-3), ORF1 (pWHM3-or
Two plasmids with each of f1) were made.

(5) Estimating the start codon of the sttH (ORF2) gene
In order to estimate the start codon of the sttH gene, seven forward primers and one reverse primer were designed as schematically shown in FIG. 2 and used for amplification of the sttH gene. BamH
I site (5 (-GGGGGATCC-3 () is added to all forward primers and HindIII site (5 (-
ACCAAGCTT-3 () was added to the reverse primer. PCR was performed under standard conditions. After sequence confirmation, each of the seven amplified fragments was inserted into the same site of the pQE30 plasmid, and pQE30-SHF1
R (with PCR fragment amplified with SH-F1 and SH-R primers), pQE30-SHF2R (SH-F2 and SH-R), p
QE30-SHF3R (SH-F3 and SH-R), pQE30-SHF4R (SH-F4 and SH-R), pQE30-SHF5R (SH-F5 and SH-R)
), PQE30-SHF6R (SH-F6 and SH-R), and pQE30-SHF7R (SH-F7 and SH-R) plasmids. Each plasmid was introduced into E. coli XL1-Blue MRF (the ST MIC in these transformants was expressed as ampicillin (100 (g / mL), isopropyl-(-D-thiogalactoside (IPTG) 0.1).
Luria-Bertani (LB) agar plate (Sambrook, J., & Russel) containing mM, ST (0-100 g / mL)
l, DW (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab.
Press, Plainview, New York)).

(6) Identification of the ST-F and ST-D-derived compounds produced by SttH recombination enzyme (rSttH) and r
SttH rate reaction test (kinetic analysis)
According to the manufacturer's (Qiagen) protocol, the reaction solution (500 μL) is E. coli containing 100 mM sodium phosphate buffer (pH 6.5), ST-F or ST-D 1 mg / mL, pQE30-SHF6R. M15
It was prepared using rSttH 100 μg / mL purified from (pREP4). The reaction solution was reacted with rSttH or without adding rSttH for 1 hour at 30 ° C., followed by chloroform extraction for protein removal. The aqueous layer was analyzed by reverse phase HPLC using an ion pair reagent. The analysis conditions are
Column, _C18 reverse phase column [COSMOSIL 5C18-AR-II (250 × 4.6 mm) (Nacalai Tesque, Kyoto, Japan)]; column temperature, 30 ° C .; detection, 210 nm; flow rate, 1 mL / min . Two different mobile phases were used: 0.1% heptafluorobutyric acid + 18% acetonitrile (for ST-F reaction) and 0.1% heptafluorobutyric acid + 23% acetonitrile (for ST-D reaction). The reaction rate test is
The test was carried out under the same conditions as described above except that the enzyme concentration (2 μg / mL) and the reaction time (5 minutes) were reduced in order to adapt to the measurement of the reaction rate parameter in the stable state. All tests were performed within the linearity range. After the reaction was completed by adding 15 μl of 2N HCl, analysis was performed using HPLC.
The reaction rate constant was estimated using the Lineweaver-Burk plot. Sodium sulfate 100
COSMOSIL 5Diol-300 buffered with 20 mM sodium phosphate buffer (pH 7.0) containing mM (7.
The native molecular weight of rSttH was estimated by gel filtration using an 8 mm × 600 mm) column (Nacalai Tesque).

(7) Purification of ST-compound (ST and ST-derived compound) Basically the same as described above, except for column size (250 × 10 mm, flow rate (4.72 mL / min), acetonitrile concentration (25%) by reverse-phase HPLC. ST-F and ST-D were purified from commercially available ST under the conditions described below: After removing the organic solvent from the HPLC fraction, the aqueous phase was lyophilized to produce a white powder of the compound.
Enzymatically synthesized ST-F-acid and ST-D-acid were purified by procedures similar to those described for purification of ST-F and ST-D.

(8) Structure determination of ST-F-acid and ST-D-acid
ESI-MS / MS of ST compounds using Finnigan MAT TSQ 7000 (quadrupole tandem mass spectrometer)
The spectrum was calculated. The ¹ H-NMR spectrum data of ST-F-acid and ST-D-acid are JEOL L
Recordings were made at 500 MHz using an NM-LA500 spectrometer. (i) ST-F-acid, ¹ H-NMR (500 MHz, D ₂ O)
δ: 1.60 (4H, m, H-17, 18), 2.51 (1H, dd, J = 8 and 17 Hz, H-15), 2.62 (1H, dd,
J = 5 and 17 Hz, H-15), 2.86 (2H, br s, H-19), 2.94 (1H, dd, J = 10 and 13 Hz, H
-4), 3.08 (1H, dd, J = 3 and 13 Hz, H-3), 3.49 (1H, m, H-16), 3.55 (2H, d, J = 6
Hz, H-12), 3.98 (2H, m, H-5), 3.99 (1H, t, J = 3 Hz, H-9), 4.05 (1H, dd, J = 3
and 10 Hz, H-8), 4.14 (1H, t, J = 6 Hz, H-11), 4.31 (1H, d, J = 5 Hz, H-2), 4.59
(1H, d, J = 4 Hz, H-10), 4.95 (1H, d, J = 9 Hz, H-7). (Ii) ST-D-acid, ¹ H-NMR (5
00 MHz, D ₂ O) δ: 1.43 (4H, m, H-18, 24), 1.51 (4H, m, H-17, 23), 1.60 (4H, m, H-
29, 30), 2.40 (1H, dd, J = 8 and 16 Hz, H-27), 2.43 (1H, dd, J = 8 and 16 Hz, H-
21), 2.48 (1H, dd, J = 8 and 16 Hz, H-15), 2.50 (1H, dd, J = 5 and 17 Hz, H-27),
2.53 (1H, dd, J = 5 and 17 Hz, H-21), 2.57 (1H, dd, J = 5 and 17 Hz, H-15), 2.8
5 (2H, m, H-31), 2.86 (2H, br s, H-19), 2.94 (1H, dd, J = 10 and 13 Hz, H-4), 3.0
5 (3H, m, Acetyl), 3.08 (1H, dd, J = 3 and 13 Hz, H-3), 3.45 (4H, m, H-19, 25), 3
.48 (3H, m, H-16, 22, 28), 3.54 (2H, d, J = 6 Hz, H-12), 3.98 (1H, t, J = 3 Hz, H
-9), 3.99 (2H m, H-5), 4.06 (1H, dd, J = 3 and 10 Hz, H-8), 4.14 (1H, t, J = 6 Hz,
H-11), 4.31 (1H, d, J = 5 Hz, H-2), 4.58 (1H, d, J = 4 Hz, H-10), 4.94 (1H, d, J =
9 Hz, H-7).

(9) ST of E. coli and S. cerevisiae strains overexpressing sttH gene or nat gene
-D resistance profile study
In order to produce E. coli strains that overexpress the nat gene, the following primers were designed and PC
Used for R. 5 (-GGG GGATCC ACCACTCTTGACGACACGGCT-3 ((forward) (SEQ ID NO: 19
), 5 (-ACC AAGCTT TCA GGGGCAGGGCATGCTCAT-3 ((reverse) (SEQ ID NO: 20). Restriction enzyme site (GGATCC or AAGCTT, underlined) and stop codon (TCA, underlined) were inserted into these primers. PQE30 was inserted. An expression vector (pQE30-nat) incorporating the amplified fragment with these primers was prepared, and E. coli carrying pQE30-nat and pQE30-SHF6R, respectively.
XL1-Blue MRF (ST-F and ST-D MICs in strains were determined using ampicillin (100 (g / mL), IPTG
Determined on LB agar plates containing 0.1 mM ST (0-4 mM). Further, S. cerevisiae CKY8 strain overexpressing each of the nat gene and the sttH gene was prepared by the following procedure. The following two sets of primers with restriction sites (AAGCTT or CTGCAG, underlined) and stop codons (TCA, underlined) were designed and used for PCR:
5 (-ACC AAGCTT AATATGACCACTCTTGACGACACG-3 ((forward primer of nat gene) (SEQ ID NO: 21),
5 (-AAA CTGCAG TCA GGGGCAGGGCATGCTCAT-3 ((reverse primer of nat gene) (SEQ ID NO: 22),
5 (-ACC AAGCTT ACCATGCCCCCCGAGACCGCCGCG-3 ((sttH gene forward primer) (SEQ ID NO: 23),
5 (-AAA CTGCAG TCA GCGCGCTGGAGCGGGCGG-3 ((reverse primer for sttH gene) (SEQ ID NO: 24).
After confirming the sequence, the amplified fragment was inserted into the same site of pAD4 to prepare pAD4-nat and pAD4-sttH in which these genes are expressed under the control of a constant promoter of yeast alcohol dehydrogenase. S. cerevisiae CKY8 strains harboring each of pAD4-nat and pAD4-sttH were transformed into ST (ST-
F- or ST-D (0-4 mM) containing SC-Leu medium and ST-F and ST-D MICs were determined.

Example 1 : Cloning and nucleotide sequencing of a streptosricin (ST) resistant gene Interestingly, a minimum inhibitory concentration (MIC) of ST using a Streptomyces strain considered to be an ST non-producing strain ) By testing, Streptomyce
s albulus NBRC14147 was found to be more resistant to ST than the ST-producing strain S. lavendulae NBRC12789 (Table 1). Furthermore, N-acetyltransferase (N-acetyltran) acting on ST
When a primer designed for a gene such as the nat gene encoding sferese (NAT) was used and PCR was performed using the genomic DNA of the NBRC14147 strain as a template, no amplified fragment was observed. On the other hand, when the genomic DNA of S. lavendulae NBRC12789 was used as a template, a specific amplified fragment was detected. The NBRC14147 strain was considered to have no homologous gene encoding NAT and was selected for isolation of the ST resistance gene. By using Streptomyces lividans TK23 strain as a heterologous host exhibiting ST and thiostrepton sensitivity, and using thiostrepton (20 μg / mL) and ST (400 μg) A large number of transformants of TK23 strains were isolated that were resistant to both> / mL, a mixture of ST-F and ST-D). Southern blot analysis performed with the 2.9 kb fragment as a probe showed that this 2.9 kb fragment was found in all plasmids isolated from these transformants, so that from these transformants, 2 .
One transformant carrying the pWHM3 plasmid (pWHM3-st11, FIG. 2 and Table 1) with a 9 kb fragment was selected.
Determination of nucleotide sequence of 2.9 kb DNA fragment and codon frame analysis of Streptomyces strain (Bibb, M. J
., Findlay, PR, & Johnson, MW (1984) Gene. 30, 157-166)
ORF1 and 2) and one partial ORF (ORF3) were shown (FIG. 2). To elucidate the function of individual ORFs, BLAST (Altschul, SF, Gish, W., Miller, W., Myers, EW, & Lipman, D. J
(1990) J. Mol. Biol. 215, 403-410) and 3D-PSSM (Kelley, LA, MacCallum, R.
M., & Sternberg, MJ (2000) J. Mol. Biol. 299, 499-520) was used to perform a database (UniPro) search for these translation products. The results are summarized in FIG. In summary, each ORF was similar to esterase and β-lactamase (ORF1), hydrolytic enzymes such as isochorismatase (ORF2), lipase (ORF3). Therefore, no homology to the nat gene was found on this fragment for any gene. Which gene is ST
PWHM3-orf1 with each of ORF1 and ORF2-3 to see if it is involved in resistance
And pWHM3-orf2-3 plasmid was prepared (FIG. 2) and introduced into S. lividans TK23. MIC test confirmed that transformants harboring pWHM3-orf2-3 show ST resistance (Table 1).
. In view of the fact that the pWHM3-orf2-3 plasmid has part of ORF3, ORF2 was shown to confer ST resistance. In this study, OR was found to be similar to the hydrolase gene.
F2 was named sttH.

Example 2 : Estimation of start codon of sttH gene Based on the results of nucleotide sequence determination and frame analysis, eight start codon candidates (ATG and GTG at the 1st to 8th positions shown in Fig. 2) are confirmed in the sttH gene. It was done. Furthermore, since there is no clear and characteristic base sequence of the set of promoter region and ribosome binding site of generally known Streptomyces strains, it is difficult to predict the start codon based on the base sequence information.
Therefore, we developed six types of SttH recombination enzymes (“rSttH” each with a different N-terminal region.
May be abbreviated. ) Was prepared as an N-terminal 6 × His Tag fusion protein, and the start codon was estimated based on the respective enzyme activities judged by the MIC value. PQE30-SHF1R (having the sttH gene from the first position), pQE30-SHF2R (second position), pQE30-SHF3R (third and fourth positions), pQE30-SHF4R (fifth position) ), PQE30-SHF5R (sixth and seventh positions) and pQE30-SHF6R (eighth position) were prepared. The 9th position GTG codon was not considered a start codon candidate due to the very short peptide chain of the translation product, but pQE30-SHF7R was also produced. After introducing these seven plasmids and pQE30 (no insertion) into E. coli, ST MIC was 12.5 μg / mL (E. coli strain carrying pQE30), 50 μg / mL (pQE30-SHF1R), 50 μg. / ML (pQE30-SHF2
R), 50 μg / mL (pQE30-SHF3R), 100 μg / mL (pQE30-SHF4R), 100 μg
/ ML (pQE30-SHF5R),> 100 μg / mL (pQE30-SHF6R), and 12.5 μg /
mL (pQE30-SHF7R), suggesting that the 8th position codon is the start codon of S. albulus NBRC14147.

Example 3: Identification and structure determination of ST-F and ST-D derived compounds converted by rSttH
Highly purified rSttH using Ni affinity chromatography was reacted with ST-F. The eluted rSttH-dependent product was detected in a reaction solution having a retention time on reverse phase HPLC longer than that of ST-F and not containing any additive such as a cofactor or metal ion (FIG. 3C). Similarly, rSttH-dependent products were detected when ST-D was used as the substrate. RSttH obtained in this way
These compounds were purified and analyzed by electrospray ionization mass spectrometry (ESI-MS) and NMR in positive ion mode to determine the structure of the dependent products. As a result of ESI-MS analysis, the molecular weight of the ST-F-derived compound was 521, and it was confirmed that the molecular weight was increased by 18 from the molecular weight of ST-F (503 Da) (FIG. 4). Tandem mass spectrometry (ESI-MS / MS) showed that this molecular weight change occurred at the streptolidine lactam group. A similar event is ST-
It was also found in D-derived compounds. These results strongly suggested that rSttH catalyzes the hydrolysis of streptolidine lactam amide bond. When NMR analysis was performed to confirm these predicted structures, the characteristics of the ¹ H NMR spectrum of the resulting streptolidine group (see “Materials and Methods”) were found in Zabriskie et al. (Jackson, MD, Gould, SJ, & Zabrisk
ie, TM (2002) J. Org. Chem. 67, 2934-2941)
It was completely consistent with the characteristics of ine. Therefore, ST-F generated by the action of rSttH and
ST-D-derived compounds are ST-F-acid (a compound where n = 1 in the formula (I)) and ST-D-acid (a compound where n = 3 in the formula (I)), respectively. It was confirmed that.

Example 4: Enzyme properties of rSttH
The optimum pH was measured using several buffer solutions of 100 mM with different pH (sodium phosphate (NaPB), pH 4.5-7; Tris-HCl, pH 7-10). Maximum activity is observed at pH 6.5, and the pH decreases (approximately
4) Then activity decreased rapidly. In addition, the effect of temperature on enzyme activity can be determined using NaPB buffer 1
It examined in 25-75 degreeC using 00 mM (pH 6.5). Enzyme activity was maximal at 45 ° C, but about 90% of the maximum activity was detected at 65 ° C. The reaction rate parameters are summarized in Table 2. rStt
The K _m value of H was calculated to be 0.96 ± 0.19 mM for ST-F and 5.74 ± 0.99 mM for ST-D, indicating that this enzyme has high affinity for ST compounds with short-chain β-lysine polymers. . But rSttH V _{ma
The x} value was slightly higher for ST-D compared to ST-F. When V _max / K _m value in the reaction with ST-F was calculated, it was 4 times higher than ST-D. The native molecular weight of rSttH ORF2 was estimated to be 50 kDa by gel filtration, suggesting that rSttH exists as a homodimer.

Example 5: Examination of ST-D resistance profile in E. coli and yeast cells and other microorganisms overexpressing SttH gene or nat gene The biological activity of chemically synthesized ST-F-acid is ignored in microorganisms and plants Although it has been reported previously (20,21), ST-D-acid remains unclear.
So we have E. coli and Saccharomy overexpressing the sttH or nat gene
ST-F and ST-D MICs in ces cerevisiae were examined (Table 3). As previously reported, E. coli (pQE-nat) and S. cerevisiae (pAD4-nat) strains with the nat gene were resistant to both ST-F and ST-D. Interestingly, however, the MIC value of ST-D is higher in E. coli (pQE30-SHF6R) overexpressing rSttH, in contrast to the results in S. cerevisiae (pAD4-sttH) overexpressing rSttH. It was found to be very low, suggesting that ST-D-acid still exhibits antibiotic activity against prokaryotic cells. To confirm this, we examined the selective toxicity of ST-acid and ST against various microorganisms such as Gram-positive and Gram-negative bacteria, clinically isolated pathogenic bacteria, and yeast. In contrast to the MIC test, where ST-F-acid antibiotic activity was almost completely lost in both prokaryotic and eukaryotic cells, ST-D-acid was successfully treated with S. cerevisiae and Schizosaccharomyces. Although it did not show activity against eukaryotic cells such as pombe, it actually showed high activity against bacteria such as E. coli, Bacillus subtilis, and Staphylococcus aureus (Table 3). C / a in parentheses in the table is ST-F -ac
The ratio of id and the minimum growth inhibitory concentration of ST-F is represented, and d / b represents the ratio of the minimum growth inhibitory concentration of ST-D-acid and ST-D.

Since the protein of the present invention can reduce the antibiotic activity (ie, toxicity) against eukaryotic cells without losing the antibiotic activity against prokaryotic cells by opening the lactam of streptococrine, It is useful for the production of streptosricin derivatives that can be developed or used as lead compounds in drug discovery.
Moreover, since the polynucleotide of the present invention can confer resistance to streptococrine to eukaryotic cells (eg, yeast), it is useful as an antibiotic resistance marker gene that can be used in recombinant DNA technology. ST and nat genes are now prokaryotic and eukaryotic recombinant D
Used in NA technology, it will replace the combination of ST-D (an antibiotic with higher activity than ST-F) and the sttH gene in eukaryotic cells, especially yeast.
Furthermore, ST-D-acid retains antibiotic activity against prokaryotic cells while reducing antibiotic activity (ie, toxicity) against eukaryotic cells.
Alternatively, it is useful as a lead compound in such antibacterial drug discovery. That is, as shown in Table 3, in yeast, the rate at which the bioactivity of ST-D-acid was inactivated was about 4 to 17 times higher than that of bacteria. In particular, S. aureus AB, a clinically isolated pathogenic bacterium (
Unreported enterotoxin AB producing strain) and S. aureus FIR 1169 (toxic shock syndrome exotoxin producing strain) (Igarashi, H., Fujikawa, H., Usami, H., Kawabata, S
, & Morita, T. (1984) Infect. Immun. 44, 175-181.) ST-D-acid showed strong antibacterial activity. ST is a powerful antibiotic against a variety of bacteria but has never been clinically developed because it is toxic to mammals. However, in the present invention, ST-D-acid
Has been shown to still have strong antibacterial activity even though its toxicity to eukaryotic cells has been reduced, revealing the possibility that ST-D-acid can be clinically developed or a new lead compound in drug discovery It was.

[SEQ ID NO: 1] This shows the base sequence of the sttH gene (starting from the 8th position in FIG. 2).
[SEQ ID NO: 2] SttH encoded by the sttH gene comprising the nucleotide sequence set forth in SEQ ID NO: 1
The amino acid sequence of the protein is shown.
[SEQ ID NO: 3] This shows the base sequence of DNA encoding rSttH started from position 7 in FIG.
[SEQ ID NO: 4] This shows the amino acid sequence of rSttH encoded by DNA consisting of the base sequence shown in SEQ ID NO: 3.
[SEQ ID NO: 5] This shows the base sequence of DNA encoding rSttH started from position 6 in FIG.
[SEQ ID NO: 6] This shows the amino acid sequence of rSttH encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 5.
[SEQ ID NO: 7] This shows the base sequence of DNA encoding rSttH started from the 5th position in FIG.
[SEQ ID NO: 8] This shows the amino acid sequence of rSttH encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 7.
[SEQ ID NO: 9] This shows the base sequence of DNA encoding rSttH started from the 4th position in FIG.
[SEQ ID NO: 10] This shows the amino acid sequence of rSttH encoded by DNA consisting of the base sequence shown in SEQ ID NO: 9.
[SEQ ID NO: 11] This shows the base sequence of DNA encoding rSttH started from the third position in FIG.
[SEQ ID NO: 12] This shows the amino acid sequence of rSttH encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 11.
[SEQ ID NO: 13] This shows the base sequence of DNA encoding rSttH started from the second position in FIG.
[SEQ ID NO: 14] This shows the amino acid sequence of rSttH encoded by DNA consisting of the base sequence shown in SEQ ID NO: 13.
[SEQ ID NO: 15] This shows the base sequence of DNA encoding rSttH started from the first position in FIG.
[SEQ ID NO: 16] This shows the amino acid sequence of rSttH encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 15.
[SEQ ID NO: 17] This shows the base sequence of the primer used in [Materials and methods] (3) of the above Examples.
[SEQ ID NO: 18] This shows the base sequence of the primer used in [Materials and Methods] (3) of the above Examples.
[SEQ ID NO: 19] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.
[SEQ ID NO: 20] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.
[SEQ ID NO: 21] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.
[SEQ ID NO: 22] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.
[SEQ ID NO: 23] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.
[SEQ ID NO: 24] This shows the base sequence of the primer used in [Materials and Methods] (9) of the above Examples.

Claims (2)

Formula (II)

An antibacterial agent against at least one pathogenic bacterium selected from the group consisting of a gram-negative bacterium and a gram-positive bacterium, containing the compound represented by : Formula (II)

Or a salt thereof.

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