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United States Patent Application 20160367585
Kind Code A1
SUMIDA; Naomi ;   et al. December 22, 2016

NOVEL AMINOGLYCOSIDE ANTIBIOTICS, PROCESS FOR PRODUCING THE SAME, AND PHARMACEUTICAL USE THEREOF

Abstract

The present invention relates to novel aminoglycoside antibiotics, a process for producing the same, and pharmaceutical use thereof. More specifically, the present invention relates to compounds represented by formula (I), a process for producing the same, and use of the same as antimicrobial agents. ##STR00001## wherein R represents amino or hydroxyl.


Inventors: SUMIDA; Naomi; (Odawara-shi, JP) ; YANAI; Koji; (Minamiashigara-shi, JP) ; TANI; Masato; (Kawasaki-shi, JP) ; FUKUSHIMA; Takayoshi; (Odawara-shi, JP) ; OTA; Yasumasa; (Odawara-shi, JP) ; GOMI; Shuichi; (Tokyo-to, JP) ; NAKANE; Akitaka; (Kawasaki-shi, JP)
Applicant:
Name City State Country Type

MEIJI SEIKA KAISHA CO., LTD.

Tokyo-to

JP
Family ID: 1000002165249
Appl. No.: 15/257158
Filed: September 6, 2016


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
12734848Jun 14, 20109469863
PCT/JP2008/071795Dec 1, 2008
15257158

Current U.S. Class: 1/1
Current CPC Class: A61K 31/7036 20130101; C12R 1/465 20130101; C12P 19/485 20130101
International Class: A61K 31/7036 20060101 A61K031/7036; C12R 1/465 20060101 C12R001/465; C12P 19/48 20060101 C12P019/48

Foreign Application Data

DateCodeApplication Number
Nov 30, 2007JP2007-310618

Claims



1-23. (canceled)

24. A kanamycin producing strain of the genus Streptomyces capable of producing a compound represented by formula (I), ##STR00005## wherein 2-deoxy-scyllo-inosose synthase has been inactivated.

25. The kanamycin producing strain according to claim 24 which is 2-deoxystreptamine-dependent.

26. The kanamycin producing strain according to claim 24, wherein a gene that codes for a polypeptide selected from the following polypeptides (a) to (d) has been integrated: (a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO:1 having a mutation in which aspartic acid at position 136 has been changed to asparagine, (b) a polypeptide consisting of the amino acid sequence defined in (a) in which one or more amino acids have been substituted, deleted, added, or inserted, the polypeptide having an activity functionally equivalent to the polypeptide defined in (a), and (c) a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence defined in (a), the polypeptide having an activity functionally equivalent to the polypeptide defined in (a).

27. The strain according to claim 26, wherein the polypeptide defined in (b) or (c) holds the mutation defined in (a).

28. The strain according to claim 26, wherein the one or more amino acids in (b) is 1 to 40 amino acids.

29. The strain according to claim 26, wherein the homology in (c) is not less than 90%.

30. The strain according to claim 24, wherein the strain is S. Kanamyceticus-DOS.

31. The strain according to claim 24, capable of producing the compound represented by formula (I) in combination with a component selected from streptamine and myo-inositol.

32. A composition comprising the kanamycin producing strain according to claim 24 and a component selected from streptamine and myo-inositol.

33. A composition comprising the kanamycin producing strain according to claim 25 and a component selected from streptamine and myo-inositol.

34. A composition comprising the kanamycin producing strain according to claim 26 and a component selected from streptamine and myo-inositol.

35. A composition comprising the kanamycin producing strain according to claim 27 and a component selected from streptamine and myo-inositol.

36. A composition comprising the kanamycin producing strain according to claim 28 and a component selected from streptamine and myo-inositol.

37. A composition comprising the kanamycin producing strain according to claim 29 and a component selected from streptamine and myo-inositol.

38. A composition comprising the kanamycin producing strain according to claim 30 and a component selected from streptamine and myo-inositol.

39. A composition comprising the kanamycin producing strain according to claim 31 and a component selected from streptamine and myo-inositol.

40. A method for treating or preventing an infectious disease, comprising administering an effective amount of a compound represented by formula (I) or its pharmacologically acceptable salt or their solvates to an animal including human: ##STR00006## wherein R represents amino or hydroxyl.

41. The process according to claim 40, wherein the infectious disease is derived from Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 310618/2007, filed on Nov. 30, 2007, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to novel aminoglycoside antibiotics, a process for producing the same, and pharmaceutical use thereof.

BACKGROUND ART

[0003] Aminoglycoside antibiotics are a generic term for glycoside antibiotics containing amino sugar or aminocyclitol and exclude a group of antibiotics such as macrolides, nucleosides, and anthracyclines. Up to now, a number of aminoglycoside antibiotics have been discovered from culture of actinomyces or bacteria. Among them, streptomycin, neomycin, kanamycin, gentamicin, ribostamycin, tobramycin and the like have been used as useful chemotherapeutic agents. On the other hand, the widespread use of these aminoglycoside antibiotics in clinical practice has led to a problem of the appearance of bacteria resistant to aminoglycoside antibiotics.

[0004] Kanamycins (kanamycin A, kanamycin B, and kanamycin C) are aminoglycoside antibiotics produced by Streptomyces kanamyceticus. Kanamycins have a wide spectrum of antimicrobial activity. Since, however, many infecting bacteria become rapidly resistant to kenamycins, in recent years, the clinical adaptation of kanamycins is limited to diseases, mainly tuberculosis.

[0005] In kanamycins, kanamycin derivatives such as dibekacins, amicacins, and arbekacins effective also against resistant bacteria have been developed based on studies on a resistant mechanism. However, bacteria resistant to these agents are appearing. Under such circumstances, the development of novel aminoglycoside antibiotics that are effective against resistant bacteria and can reduce nephrotoxicity that is a problem common to aminoglycoside antibiotics has been expected.

[0006] Regarding aminoglycoside antibiotics comprising 2-deoxystreptamine as one constituent sugar, studies have been made on the production of novel aminoglycoside antibiotics by acquiring a mutant strain which produces an aminoglycoside antibiotic 2-deoxystreptamine-dependently, adding a 2-deoxystreptamine analog to the mutant strain, and cultivating the mixture. Also in kanamycins, there is a report that antibiotics different from kanamycins are produced by acquiring a mutant strain having a phenotype of 2-deoxystreptamine-dependent kanamycin production, adding 2-epistreptamine to the mutant strain, and cultivating the mixture (U.S. Pat. No. 3,669,838). Further, there is a report that 4-O-(.alpha.-D-glucopyranosyl)6-O-(3-amino-3-deoxy-.alpha.-D-glucopyranos- yl)1-N-methyl-2-deoxystreptamine or 4-O-(.alpha.-D-glucopyranosyl)6-O-(3-amino-3-deoxy-.alpha.-D-glucopyranos- yl)2-epi-streptamine is produced by adding 1-N-methyl-deoxystreptamine or myo-inosadiamine-1,3(2-epistreptamine) to a mutant strain having a phenotype of 2-deoxystreptamine-dependent kanamycin production and cultivating the mixture (Kojima, M. and Satoh, A., "Journal of Antibiotics", (Japan), 1973, Vol. 26, p. 784-786). Furthermore, there is a report that 4-O-(6-amino-6-deoxy-.alpha.-D-glucopyranosyl)6-O-(3-amino-3-deoxy-.alpha- .-D-glucopyranosyl)streptamine (LL-BM27.alpha.) and 4-O-(6-amino-6-deoxy-.alpha.-D-glucopyranosyl)6-O-(.alpha.-D-glucopyranos- yl)streptamine (LL-BM2713) are produced by adding streptamine to a mutant strain having a phenotype of 2-deoxystreptamine-dependent kanamycin production and cultivating the mixture (Borders, D. B. et al., "Journal of Antibiotics", (Japan), 1982, Vol. 35, p. 1107-1110). Here LL-BM27.alpha. is synonymous with 2-hydroxykanamycin A.

[0007] The amounts of aminoglycoside antibiotics produced by the addition of the substances and cultivation of the mixture are so small that the industrial applicability of the aminoglycoside antibiotics is low. Accordingly, it can be said that novel aminoglycoside antibiotics which are clinically useful and have potent antimicrobial activity are still demanded.

SUMMARY OF THE INVENTION

[0008] The present inventors have now found that novel aminoglycoside antibiotics having potent antimicrobial activity can be produced by cultivating a kanamycin producing strain derived from the genus Streptomycin together with a specific 2-deoxystreptamine analog. The present invention has been made based on such finding.

[0009] Accordingly, an object of the present invention is to provide novel aminoglycoside antibiotic agents possessing potent antimicrobial activity and a process for producing the same.

[0010] According to the present invention, there are provided aminoglycoside antibiotics that are compounds represented by formula (I) or their pharmacologically acceptable salts or their solvates.

##STR00002##

[0011] wherein R represents amino or hydroxyl.

[0012] According to another aspect of the present invention, there is provided a process for producing compounds represented by formula (I), the process comprising cultivating a kanamycin producing strain of the genus Streptomyces in a medium which comprises streptamine and/or myo-inositol to produce the compound.

[0013] The compounds according to the present invention have potent antimicrobial activity against bacteria causative of various infectious diseases and can be advantageously utilized in the treatment of infectious diseases. Further, the production process according to the present invention can simply and stably supply the above compounds.

DETAILED DESCRIPTION OF THE INVENTION

Deposit

[0014] The strain S. Kanamyceticus-DOS according to the present invention has been deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (address: Tsukuba Central 6 Tsukuba-shi, Higashi 1-1-1, Ibaraki, 305-8566 Japan) (original deposited date: Nov. 1, 2007) under accession number FERM BP-11052.

DEFINITION

[0015] The term "2-hydroxykanamycin A" as used herein refers to a compound having hydroxyl introduced into the 2-position of kanamycin A. The term "2-hydroxykanamycin" refers to a group of compounds having hydroxyl introduced into the 2-position of kanamycins (kanamycin A, kanamycin B, and kanamycin C). Specifically, the group of compounds includes 2-hydroxykanamycin A, 2-hydroxykanamycin B, and 2-hydroxykanamycin C.

[0016] Further, the kanamycin producing strain, as used herein, which is "deoxystreptamine dependent" refers to a mutant strain, among kanamycin producing bacteria, that can restore the capability to produce kanamycins by adding deoxystreptamine.

[0017] The polypeptide "having activity functionally equivalent to" or "having functionally equivalent activity" as used herein refers to the following polypeptide.

[0018] In the polypeptide, in addition to polymorphisms or mutants of genes coding for the polypeptide, structural mutants may occur in an amino acid sequence, for example, by a modification reaction. However, it is known that some polypeptides, despite the presence of these mutants, have substantially the same physiological and biological activity as polypeptides not having a mutant. The polypeptide which, despite the structural difference, does not have a large difference in function refers to a polypeptide "having functionally equivalent activity."

[0019] Compounds Represented by Formula (I) or their Pharmacologically Acceptable Salts or their Solvates

[0020] One characteristic feature of the compounds represented by formula (I) according to the present invention is that hydroxyl has been introduced into the 2-position of kanamycin B or C. The compounds having the structure have a broad antimicrobial spectrum ranging from gram-positive bacteria to gram-negative bacteria including Pseudomonas aeruginosa and have potent antimicrobial activity.

[0021] According to one embodiment of the present invention, in the compounds represented by formula (I), R represents amino. This compound (hereinafter referred to as "hydroxykanamycin B") has a structure represented by formula (1).

##STR00003##

[0022] According to another embodiment of the present invention, in the compounds represented by formula (I), R represents hydroxyl. This compound (hereinafter referred to as "2-hydroxykanamycin C") has a structure represented by formula (2).

##STR00004##

[0023] The compounds represented by formula (I) may be present as salts. Such salts include, for example, pharmaceutically acceptable salts. Specific examples thereof include hydrohalides such as hydrofluorides, hydrochlorides, hydrobromides, or hydroiodides, inorganic acid salts such as sulfates, phosphates, perchlorates, or carbonates, salts of carboxylic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, hydroxyacetic acid, lactic acid, citric acid, tartaric acid, oxalic acid, benzoic acid, mandelic acid, butyric acid, maleic acid, propionic acid, formic acid, or malic acid, salts of amino acids such as alginic acid, aspartic acid, or glutamic acid, or salts of sulfonic acid such as methanesulfonic acid or p-toluenesulfonic acid. Preferred are hydrohalides such as hydrochlorides and inorganic acid salts such as sulfates.

[0024] The compounds represented by formula (I) or its pharmaceutically acceptable salts may exist as their solvates. Preferred solvates include hydrates, methanolates, or ethanolates.

[0025] Producing Strains

[0026] The compounds represented by formula (I) can be produced by various methods. For example, as described above, the compounds represented by formula (I) can be produced by cultivating a kanamycin producing strain of the genus Streptomyces in a medium comprising streptamine and/or myo-inositol.

[0027] Examples of such suitable producing strains include deoxystreptamine-dependent kanamycin producing strains. More preferred are strains of the genus Streptomyces wherein 2-deoxy-scyllo-inosose synthase catalyzing a first reaction in 2-deoxystreptamine biosynthesis from glucose-6-phosphoric acid has been inactivated. It is surprising that, when such strains are cultivated together with a deoxystreptamine analog, the compounds according to the present invention can be selectively produced without producing kanamycins.

[0028] Accordingly, according to still another aspect of the present invention, there is provided a kanamycin producing strain of the genus Streptomyces capable of producing compounds represented by formula (I) wherein 2-deoxy-scyllo-inosose synthase has been inactivated.

[0029] The mutant producing strain can be acquired, for example, by treating kanamycin producing bacteria which are derived from the genus Streptomyces including Streptomyces kanamyceticus, for example, by artificial mutation methods including ultraviolet (UV) irradiation or nitrosoguanidine (NTG). The acquisition of the desired mutant strain can be confirmed, for example, by acquiring a deoxystreptamine-dependent kanamycin producing strain, then measuring the activity of intracellular 2-deoxy-scyllo-inosose synthase in each mutant strain by a conventional method (Kudo, F. et al., "Journal of Antibiotics", (Japan), 1999, Vol. 52, p. 81-88), and selecting a mutant strain that does not have enzyme activity.

[0030] Regarding the kanamycin producing strain in which 2-deoxy-scyllo-inosose synthase has been inactivated, genes coding for 2-deoxy-scyllo-inosose synthase have already been clarified (Japanese Patent Application Laid-Open No. 173537/2004). Accordingly, a desired mutant strain can also be acquired by gene recombination technology. For example, a desired mutant strain may be acquired by destroying a gene coding for 2-deoxy-scyllo-inosose synthase. Further, the mutant strain can also be acquired by preparing a mutant gene having an amino acid substitution that provides inert 2-deoxy-scyllo-inosose synthase, and subjecting the mutant gene to gene substitution with a wild-type gene on a chromosome. The gene can be destroyed or substituted by methods commonly used in Actinomyces ("Practical Streptomyces Genetics", "The John Innes Foundation", (England), Norwick, 2000, p. 311-338). The disclosures of the document are incorporated herein by reference.

[0031] For example, a mutation in which aspartic acid at position 136 of the amino acid sequence of the synthase represented by SEQ ID No. 1 is substituted by asparagines may be mentioned as one example of mutation by which 2-deoxy-scyllo-inosose synthase is inactivated. Accordingly, the strain that can produce the compound represented by formula (I) can be acquired by integrating a gene coding a polypeptide having the mutation or a gene coding for an analog functionally equivalent to the polypeptide into a strain.

[0032] According to a preferred embodiment of the present invention, the kanamycin producing strain capable of producing the compound represented by formula (I) is the strain into which a gene coding for a polypeptide selected from the following polypeptides (a) to (c) has been integrated:

[0033] (a) a polypeptide consisting of an amino acid sequence represented by SEQ ID No. 1 having a mutation in which aspartic acid at position 136 has been changed to asparagine,

[0034] (b) a polypeptide consisting of the amino acid sequence defined in (a) wherein one or more amino acids have been substituted, deleted, added, or inserted, the polypeptide having an activity functionally equivalent to the polypeptide defined in (a), and

[0035] (c) a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence defined in (a), the polypeptide having an activity functionally equivalent to the polypeptide defined in (a)

[0036] S. Kanamyceticus-DOS may be mentioned as an example of suitable kanamycin producing strains corresponding to (a).

[0037] In (b), the "one or more amino acids" is preferably 1 to 40 amino acids, more preferably 1 to 8 amino acids, still more preferably 1 to 4 amino acids.

[0038] In (d), the homology is preferably not less than 90%, more preferably not less than 95%.

[0039] Further, when stable production of the compound represented by formula (I) is taken into consideration, preferably, the polypeptide described in (b) or (c) holds a mutation in which aspartic acid at position 136 in the amino acid sequence described in (a) or at a position corresponding to the position 136 has been changed to asparagine. The presence or absence of the mutation in the amino acid sequence described in (a) to (c) and the determination of sequence homology can be properly determined by a person having ordinary skill in the art by comparing the amino acid sequence represented by SEQ ID No. 1 with the amino acid sequence in (a) to (c) by a conventional method.

[0040] The functional equivalency between the polypeptide described in (b) or (c) and the polypeptide described in (a) can be confirmed by measuring 2-deoxy-scyllo-inosose synthase activity for both the polypeptides according to the above-described method described in Kudo, F. et al., "Journal of Antibiotics", (Japan), 1999, Vol. 52, p. 81-88 and comparing the measured results. Further, the functional equivalency can be indirectly confirmed by measuring the deoxystreptamine dependency or antimicrobial activity of kanamycin producing strain with genes coding for the polypeptides integrated thereinto by a method described in Example 2 or Test Example 1 and statistically comparing the measured results.

[0041] Production Process

[0042] As described above, the compound represented by formula (I) according to the present invention can be produced by cultivating a kanamycin producing strain derived from the genus Streptomycin in a medium which comprises a 2-deoxystreptamine analog selected from streptamine and myo-inositol.

[0043] When selective production of the compound represented by formula (I) is taken into consideration, as described above, the kanamycin producing strain derived from the genus Streptomycin is preferably a kanamycin producing strain in which 2-deoxy-scyllo-inosose synthase has been inactivated. However, a kanamycin producing strain in which 2-deoxy-scyllo-inosose synthase has not been inactivated may also be used. Examples of suitable strains in which 2-deoxy-scyllo-inosose synthase has not been inactived include Streptomyces kanamyceticus.

[0044] Further, in the production process according to the present invention, a combination of the kanamycin producing strain with the 2-deoxystreptamine analog added to the medium may be properly determined by taking the type of the desired contemplated compound into consideration.

[0045] According to one embodiment of the present invention, the kanamycin producing strain is one in which 2-deoxy-scyllo-inosose synthase has been inactivated, and the medium comprises streptamine. According to this embodiment, the compound represented by formula (I) can be produced together with 2-hydroxykanamycin A. This is advantageous in that 2-hydroxykanamycins (2-hydroxykanamycins A to C) can be simultaneously produced. Further, according to another embodiment of the present invention, the kanamycin producing strain is one in which 2-deoxy-scyllo-inosose synthase has been inactivated, and the medium comprises myo-inositol. According to this embodiment, 2-hydroxykanamycin C, which is a compound represented by formula (I) wherein R represents hydroxyl, can be selectively produced.

[0046] The addition amount of streptamine or myo-inositol may be properly varied depending upon cultivation conditions, but is preferably 100 to 50,000 .mu.g/ml, more preferably 1,000 to 10,000 .mu.g/ml.

[0047] Conventional substances may be properly added as ingredients of the medium in the production process according to the present invention.

[0048] Medium ingredients other than streptamine and myo-inositol usable herein include, for example, carbon sources such as glucose, sucrose, starch syrups, dextrins, starches, glycerol, syrups, animal and vegetable oils; and nitrogen sources such as soybean meals, wheat germ oils, corn steep liquors, cottonseed cakes, meat extracts, polypeptone, malt extracts, yeast extracts, ammonium sulfate, sodium nitrate, and urea. Further, if necessary, the addition of sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid (for example, dipotassium hydrogenphosphate), sulfuric acid (for example, magnesium sulfate) and inorganic salts which can produce other ions is also effective. Furthermore, if necessary, various vitamins such as thiamines (for example, thiamine hydrochloride), glutamic acid (for example, sodium glutamate), amino acids such as asparagine (for example, DL-asparagine), micronutrients such as nucleotides, and selected drugs such as antibiotics may also be added. Furthermore, organic and inorganic substances that aid the growth of bacteria and promote the production of 2-hydroxykanamycin B and 2-hydroxykanamycin C can be properly added.

[0049] The medium is preferably approximately pH 5.5 to pH 9.

[0050] The cultivation may be carried out by solid cultivation, shaking cultivation, aeration agitation cultivation, or deep aerobic cultivation under aerobic conditions. Among them, the deep aerobic cultivation is preferred.

[0051] For example, 15.degree. C. to 40.degree. C. is suitable as the cultivation temperature. In many cases, however, bacteria are grown at a temperature around 25.degree. C. to 35.degree. C.

[0052] The amount of the compound represented by formula (I) produced varies depending upon the medium, cultivation conditions, and cultivation method used, but reaches the largest, for example, 2 days to 15 days.

[0053] Preferably, when the amount of the compound represented by formula (I) in the medium has reached the largest, the cultivation is stopped. The compound is then harvested from the culture and is purified.

[0054] In order to harvest the compound of the present invention from the culture, a conventional separation means utilizing the properties of the compound may be used. The separation means may be, for example, solvent extraction, ion exchange resin methods, adsorption or partition column chromatography, gel filtration, dialysis, precipitation, and crystallization, either alone or in an appropriate combination. For example, the culture is filtered to give a filtrate. The filtrate is then adsorbed on a cation exchange resin such as Amberlite IRC-50 or Amberlite FPC3500, and elution is carried out with aqueous ammonia. The eluate is further purified with a cation exchange resin such as Dowex 50W or Amberlite CG-50 and is if necessary purified by ion-exclusion chromatography with Dowex 1 or by adsorption chromatography with HP20ss, whereby each 2-hydroxykanamycin represented by formula (I) can be isolated.

[0055] Use

[0056] The compounds according to the present invention have potent antimicrobial activity and are useful for administration as a medicines to animals including human. Thus, according to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a compound represented by formula (I) or its pharmacologically acceptable salt or their solvates. The composition is preferably used as an antimicrobial agent.

[0057] According to another aspect of the present invention, there is provided use of a compound represented by formula (I) or its pharmacologically acceptable salt or their solvates, in the manufacture of a pharmaceutical composition. According to still another aspect of the present invention, there is provided use of a compound represented by formula (I) or its pharmacologically acceptable salt or their solvates, in the manufacture of an antimicrobial agent.

[0058] When the compound according to the present invention is used as a pharmaceutical composition, the pharmaceutical composition may be formulated according to various dosage forms or usage forms by conventional methods. Pharmaceutical preparations for oral administration include tablets, pills, granules, capsules, powders, liquid formulations, suspensions, syrups, and sublingual agents. Pharmaceutical preparations for parenteral administration include injections, transdermal agents, inhalants, and suppositories. Pharmaceutical additives such as surfactants, excipients, stabilizers, wetting agents, disintegrants, dissolution aids, tonicity adjusting agents, buffers, colorants, and flavoring agents are properly used in the formulation.

[0059] Pharmaceutically acceptable carriers may be used as the carrier for the pharmaceutical composition. The type and composition of the carrier may be properly determined according to administration routes and administration methods. For example, liquid carriers usable herein include water, alcohols, soybean oils, and sesame oils. Example of solid carriers include sugars such as maltose and sucrose, amino acid salts such as lysine, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, and cellulose derivates such as hydroxylpropyl cellulose.

[0060] The compounds according to the present invention having antimicrobial activity are preferably used in the treatment or prevention of infectious diseases. Accordingly, according to a further aspect of the present invention, there is provided a method for treating or preventing an infectious disease, comprising administering an effective amount of a compound represented by formula (I) or its pharmacologically acceptable salt or their solvates to an animal including human. The term "treatment" as used herein means ameliorating an established disease state, and the term "prevention" as used herein means preventing the establishment of a disease state in the future.

[0061] The compounds according to the present invention can be applied to bacteria causative of various infectious diseases. Bacteria causative of infectious diseases include, for example, Staphylococcus aureus, Staphylococcus epidermidis, Entercoccus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella, or Acinetobacter. Preferred is Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

[0062] When the compound represented by formula (I) is 2-hydroxykanamycin B, the bacteria causative of infectious diseases are preferably Staphylococcus aureus, Staphylococcus epidermidis, Entercoccus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella, or Acinetobacter, more preferably Staphylococcus aureus, Staphylococcus epidermidis, Entercoccus, Escherichia coli, Bacillus subtilis, Salmonella, or Acinetobacter, still more preferably Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, Salmonella, or Acinetobacter.

[0063] When the compound represented by formula (I) is 2-hydroxykanamycin C, the bacteria causative of infectious diseases are preferably Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, Salmonella, or Acinetobacter, more preferably Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, Salmonella, or Acinetobacter, still more preferably Staphylococcus epidermidis, Bacillus subtilis, Salmonella, or Acinetobacter.

[0064] The effective dose of the compound according to the present invention may be properly determined by physicians in consideration of particular conditions, for example, the age, weight, type and severity of patients, and administration route. When the compound is orally administered to human, for example, the compound can be administered, for example, at a dose of 0.01 to 1000 mg/kg per adult per day. On the other hand, when the compound is intravenously administered, the compound can be administered at a dose of 0.001 to 100 mg/kg per adult per day.

EXAMPLES

[0065] The present invention is further illustrated by the following Examples that are not intended as a limitation of the invention.

[0066] In the following Examples, LC/MS analyses were carried out under the following conditions.

[0067] Conditions for LC/MS Analyses

[0068] (HPLC part: Waters 2690)

[0069] Column: Capcell Pak C18 MG, 4.6.times.150 mm, 5 .mu.m (manufactured by Shiseido Company, Limited)

[0070] Mobile phase: A: 0.2% Aqueous pentafluoropropionic acid solution

[0071] B: Acetonitrile

[0072] C: H.sub.2O

[0073] Liner gradient: 0 min (A/B/C=10/10/80).fwdarw.15 min (A/B/C=10/30/60)

[0074] Flow rate: 0.4 ml/min, temp.: 30.degree. C.

[0075] (MS part: Waters ZQ) ESI method

[0076] Ion source temp.: 100.degree. C.

[0077] Desolvation temp.: 380.degree. C.

[0078] Desolvation gas flow: 350 L/hr

[0079] Cone gas flow: 50 L/hr

[0080] Capillary voltage: 3.5 kV

[0081] Cone voltage: positive: 30 V

Example 1

Construction of Plasmid pDDOI for Introduction of 2-Deoxy-Scyllo-Inosose Synthase Gene (Orf11) Mutation of Streptomyces kanamyceticus

[0082] Regarding 2-deoxy-scyllo-inosose synthase (SEQ ID No. 1), aspartic acid at position 136 conserved as an amino acid residue important to binding to a substrate, glucose-6-phosphoric acid, was changed to asparagine to inactivate enzyme protein. Preparation was carried out by a PCR reaction using pKM9 (see Japanese Patent Application Laid-Open No. 173537/2004, Example 2, FERM P-19117) as a template.

[0083] A kanamycin biosynthetic cluster region utilized is shown in SEQ ID No. 2.

[0084] The following primers, i.e., a primer including Hind III or Xba I digestion site and a primer which had been designed so that aspartic acid (GAT) at position 136 from an initiation codon of a DOI synthase gene is mutated to asparagine (AAC), were used.

TABLE-US-00001 Km-Mu-Hind III (SEQ ID No. 3) 5'-GGGAAGCTTGACCTTGGAGGTATGTGT-3' Km-Mu-L (SEQ ID No. 4) 5'-GTTCAGCATGGCCACCACGGTGGT-3' (The underlined part represents mutation introduced part) Km-Mu-R (SEQ ID No. 5) 5'-TCGGTGCTCTCGCTCAAGCAG-3' Km-Mu-Xba I (SEQ ID No. 6) 5'-GGGTCTAGATGCCGTCCTGGTGGTAGT-3'

[0085] A PCR reaction was carried out using a primer combination of Km-Mu-Hind III (SEQ ID No. 3) with Km-Mu-L (SEQ ID No. 4) and a primer combination of Km-Mu-R (SEQ ID No. 5) with Km-Mu-Xba I (SEQ ID No. 6). The reaction was carried out using about 1 .mu.g of genomic DNA, 0.3 .mu.M of each primer, and KOD plus DNA polymerase (manufactured by TOYOBO CO., LTD.) under conditions of 94.degree. C./2 min (94.degree. C./15 sec, 50.degree. C./30 sec, and 68.degree. C./1.5 min).times.25 cycles. As a result, about 1.5 kbp DNA fragments were specifically amplified. The DNA fragments were purified by a QIAquick PCR purification kit (manufactured by QIAGEN K.K.). The blunt end was phosphorylated (manufactured by NIPPON GENE CO., LTD.), and the phosphorylated DNA fragments were digested with Hind III and Xba I, followed by cloning into Hind III and Xba I sites of pUC119. The base sequence of the cloned DNA fragments were analyzed. As a result, it could be confirmed that the DNA fragments contained a 2-deoxy-scyllo-inosose synthase (orf11) gene (SEQ ID No. 7) with contemplated substitution by asparagine inserted thereinto.

[0086] Plasmid pSET152 (Bierman, M. et al., "Gene", (Netherlands), 1992, Vol. 116, p. 43-49) for conjugation transfer of Actinomyces was digested with Sph I and was blunted with T4 DNA polymerase. A Hind III linker (manufactured by TAKARA SHUZO CO., LTD.) was then linked thereto to construct pSET153. An about 2.8 kbp Hind III-Xba I fragment derived from pSET153 was linked to an about 3 kbp Hind III-Xba I fragments containing the gene subjected to amino acid substitution to obtain plasmid pDDOI for orf11 gene mutation introduction that had a conjugation transfer ability.

Example 2

Creation of Deoxystreptamine-Non-Producing Strain by Plasmid pDDOI for Orf11 Gene Mutation Introduction

[0087] Streptomyces kanamyceticus which is a kanamycin producing bacterium was coated onto an MS agar medium (2% S soybean meal, 2% mannitol, 2% agar) and was cultivated at 28.degree. C. for 3 days. After the cultivation, hyphae were scraped with 3 ml of 20% glycerol and were collected to prepare a hypha liquid of a host.

[0088] On the other hand, the E. coli (Escherichia coli) ET12567/pUZ8002 strain carrying plasmid pDDOI was inoculated into 100 ml of an LB liquid medium (1% Difco Bacto tryptone, 0.5% Difco yeast extract, 0.5% NaCl, and 0.1% glucose) containing 25 .mu.g/ml of chloramphenicol, 25 .mu.g/ml of kanamycin, and 50 .mu.g/ml of apramycin and was cultivated at 37.degree. C. overnight to prepare a preculture. The culture was inoculated into the same liquid medium as the precultivation so that the final concentration of the preculture was 1%, followed by cultivation at 37.degree. C. for about 4 hr. The culture was washed twice with an LB liquid medium and was finally suspended in 10 ml of an LB liquid medium to prepare an E. coli liquid.

[0089] The hypha liquid of the host (500 .mu.l) prepared above and 500 .mu.l of the E. coli liquid were mixed together for harvesting. The harvested bacteria were then coated on an MgCl.sub.2-added MS agar medium so that the final concentration of the bacteria was 10 mM. After cultivation at 28.degree. C. for 20 hr, 1 ml of sterilized water containing 1 mg of apramycin and 1.5 mg of nalidixic acid was overlayered, and the cultivation was continued at 28.degree. C. for 5 days to obtain an apramycin-resistant strain.

[0090] A genomic DNA was prepared from the apramycin-resistance strain with a MagExtractor genomic DNA extractor (manufactured by TOYOBO CO., LTD.) according to a protocol, and it was confirmed by PCR and an southern blot analysis that pDDOI was inserted into the chromosome by homologous recombination.

[0091] The homologous recombinant was inoculated into a modified YEME medium (50 ml), followed by shake cultivation at 28.degree. C. for 2 days, and 1 ml of the culture was further inoculated into a fresh modified YEME medium (50 ml) to perform successive cultivation. This procedure was repeated five times. Thereafter, the culture diluted to a suitable vial cell count was coated onto an MS agar medium, followed by cultivation at 28.degree. C. for 4 days. The grown colony was replicated onto an MS agar medium containing 20 .mu.g/ml of apramycin and onto an apramycin-free MS agar medium, and 18 apramycin-sensitive strains that cannot grow in the apramycin-containing medium were selected.

[0092] Genomic DNAs of the apramycin-sensitive strains were prepared, and a PCR reaction was carried out using a primer combination of Km33 (5'-CTTCGTGAATCCCCCTT-3': SEQ ID No. 8) with Km35 (5'-GCCCACCGCCTCGATCA-3': SEQ ID No. 9) to obtain about 3.5 kbp amplified DNA fragments. The base sequence of these amplified DNA fragments was analyzed. As a result, one strain was a mutant strain in which substitution by asparagines as designed was observed, and, for 17 strains, the base sequences remained unchanged.

[0093] In order to examine the productivity of kanamycin, these strains were inoculated into 30 ml of a liquid growing medium (Umezawa, H. et al., "The Journal of Antibiotics", (Japan), 1977, Vol. 30, p. 181-188) prepared in a 250 ml-volume conical flask, followed by cultivation at 28.degree. C. for two days. Thereafter, 1 ml of the culture was inoculated in 30 ml of a liquid producing medium (in which the amount of starch was increased from 1.2% to 6%), and shake cultivation was carried out at 26.degree. C. for 7 days. In order to analyze the product, the culture was adjusted to pH 2.5 with 50% H.sub.2SO.sub.4, was placed in a 1.5-ml Eppendorf tube, and was centrifuged under conditions of 17,400.times.g and 10 min, and the supernatant was subjected to an LC/MS analysis. As a result, for one orf11 mutation introduced strain, the production of kanamycins was not observed, and 17 strains returned to the same base sequence as the parent strain produced kanamycin A (retention time 8.2 min, m/z 485) and kanamycin B (retention time 10.4 min, m/z 484).

[0094] Next, the kanamycin non-producing strain was coated onto an agar medium prepared by adding deoxystreptamine to the liquid medium, diluted to a half concentration, so as to give a concentration of 200 .mu.g/ml, followed by cultivation at 28.degree. C. for 7 days. Thereafter, the agar was frozen and thawed to extract the product which was then bioassayed. The assay bacterium was Bacillus subtilis ATCC6633. As a result, the product obtained by deoxystreptamine-free cultivation did not have antimicrobial activity, whereas, for the product obtained by deoxystreptamine-added cultivation, an inhibition circle indicating antimicrobial activity was detected. Accordingly, the product obtained by deoxystreptamine-added cultivation was subjected to an LC/MS analysis. As a result, kanamycin A and kanamycin B could be detected from the product. Thus, it was confirmed that the substitution of amino acid at position 136 of orf11 created a deoxystreptamine-dependent kanamycin producing strain, S. Kanamyceticus-DOS.

Example 3

Streptamine-Added Cultivation Utilizing Deoxystreptamine-Dependent Kanamycin Producing Strain

[0095] The deoxystreptamine-dependent kanamycin producing strain obtained in Example 2 was cultivated in the liquid producing medium at 26.degree. C. for 7 days. On the second and third days, streptamine adjusted so as to have a final concentration of 2,000 .mu.g/ml was added.

[0096] Radio Light #800 was added to 2,000 L of the culture, and the mixture was filtered. The filtrate was adsorbed on 50 ml-volume Amberlite FPC3500 (NH.sub.4.sup.+ type, Rohm and Haas Japan K.K.). The resin was washed with water, and elution was then carried out with 0.5 N aqueous ammonia. The eluate was adjusted to pH 6 and was adsorbed on 50 ml-volume Dowex 50W (NH.sub.4.sup.+ type, Muromachi Technos CO., LTD.), and elution was carried out with 0.04 N to 0.2 N aqueous ammonia to obtain 57.6 mg of 2-hydroxykanamycin A, 32.8 mg of 2-hydroxykanamycin B, and 513.2 mg of 2-hydroxykanamycin C. The structures of these hydrokykanamycins were determined by an HR-FAB/MS (JEOL JMS-700, JEOL Ltd.) and NMR (JEOL JNM-LA400, JEOL Ltd.) spectral analysis.

Example 4

Myo-Inositol-Added Cultivation Utilizing Deoxystreptamine-Dependent Kanamycin Producing Strain

[0097] The deoxystreptamine-dependent kanamycin producing strain obtained in Example 2 was coated onto the agar medium to which myo-inositol had been added to a concentration of 500 .mu.g/ml, followed by cultivation at 28.degree. C. for 7 days. Thereafter, the agar was frozen and thawed to extract the product which was then subjected to an LC/MS analysis. As a result, a peak attributable to 2-hydroxykanamycin C (retention time 8.3 min, m/z 501) was detected.

Example 5

Streptamine or Myo-Inositol-Added Cultivation in Kanamycin Producing Bacteria

[0098] In kanamycin producing bacteria (Streptomycin kanamyceticus), agar cultivation with the addition of 500 .mu.g/ml of streptamine or myo-inositol was carried out in the same manner as in Example 4. The product was analyzed by LC/MS. As a result, in both the addition of streptamine and the addition of myo-inositol, it was detected that, in addition to kanamycin A (retention time 8.2 min, m/z 485) and kanamycin B (retention time 10.4 min, m/z 484), 2-hydroxykanamycin B (retention time 10.7 min, m/z 500) and 2-hydroxykanamycin C (retention time 8.3 min, m/z 501) were produced.

Example 6

Confirmation of Physicochemical Properties of Hydroxykanamycins B and C

[0099] The physicochemical properties of hydroxykanamycins B and C acquired in Examples 3 to 5 were examined and were found to be as follows.

[0100] Physicochemical Properties of 2-Hydroxykanamycin B

[0101] (1) Color and properties: Colorless powder

[0102] (2) Molecular formula: C.sub.18H.sub.37N.sub.5O.sub.11

[0103] (3) Mass spectrum (HR-FAB/MS): measured value 500.2563 (M+H).sup.+, calculated value 500.2568

[0104] (4) Specific rotation: [.alpha.]D.sup.25=+127.1.degree. (c=1, H.sub.2O)

[0105] (5) Ultraviolet absorption spectrum .lamda.max nm: terminal adsorption (H.sub.2O)

[0106] (6) Infrared absorption spectrum vmax cm.sup.-1 (KBr): 3351, 2910, 1585, 1477, 1368, 1032

[0107] (7) .sup.1H-NMR spectrum (400 MHz, D.sub.2O) .delta. (ppm): 2.87 (1H, dd, H-1), 3.14 (1H, dd, H-2), 2.84 (1H, dd, H-3), 3.40 (1H, dd, H-4), 3.77 (1H, dd, H-5), 3.32 (1H, dd, H-6), 5.37 (1H, d, H-1'), 2.79 (1H, dd, H-2'), 3.58 (1H, dd, H-3'), 3.32 (1H, dd, H-4'), 3.81 (1H, m, H-5'), 2.84 (1H, dd, H-6'a), 3.06 (1H, m, H-6'b), 5.05 (1H, d, H-1''), 3.52 (1H, dd, H-2''), 3.02 (1H, dd, H-3''), 3.35 (1H, dd, H-4''), 3.93 (1H, dt, H-5''), 3.78 (2H, br d, H-6'')

[0108] [TSP=0 ppm]

[0109] (8).sup.13C-NMR spectrum (100 MHz, D.sub.2O) .delta. (ppm): 57.0 (d, C-1), 73.5 (d, C-2), 55.7 (d, C-3), 82.9 (d, C-4), 74.7 (d, C-5), 84.7 (d, C-6), 100.7 (d, C-1'), 55.9 (d, C-2'), 74.1 (d, C-3'), 72.0 (d, C-4'), 73.3 (d, C-5'), 42.1 (t, C-6'), 100.7 (d, C-1''), 72.4 (d, C-2''), 54.9 (d, C-3''), 69.8 (d, C-4''), 72.7 (d, C-5''), 60.9 (t, C-6'')

[0110] [Dioxane=67.4 ppm]

[0111] (9) Solubility: soluble in water, and insoluble in ethyl acetate and chloroform

[0112] Physicochemical Properties of 2-Hydroxykanamycin C

[0113] (1) Color and properties: Colorless powder

[0114] (2) Molecular formula: C.sub.18H.sub.36N.sub.4O.sub.12

[0115] (3) Mass spectrum (HR-FAB/MS): measured value 501.2398 (M+H).sup.+, calculated value 501.2408

[0116] (4) Specific rotation: [.alpha.]D.sup.25=+114.3.degree. (c=1, H.sub.2O)

[0117] (5) Ultraviolet absorption spectrum .lamda.max nm: terminal absorption (H.sub.2O)

[0118] (6) Infrared absorption spectrum vmax cm.sup.-1 (KBr): 3358, 2920, 1591, 1457, 1369, 1032

[0119] (7) .sup.1H-NMR spectrum (400 MHz, D.sub.2O) .delta. (ppm): 2.88 (1H, dd, H-1), 3.14 (1H, dd, H-2), 2.83 (1H, dd, H-3), 3.39 (1H, dd, H-4), 3.76 (1H, dd, H-5), 3.33 (1H, dd, H-6), 5.32 (1H, d, H-1'), 2.81 (1H, dd, H-2'), 3.60 (1H, dd, H-3'), 3.41 (1H, dd, H-4'), 3.86 (1H, m, H-5'), 3.76 (1H, dd, H-6'a), 3.88 (1H, m, H-6'b), 5.06 (1H, d, H-1''), 3.54 (1H, dd, H-2''), 3.04 (1H, dd, H-3''), 3.37 (1H, dd, H-4''), 3.94 (1H, dt, H-5''), 3.79 (2H, br d, H-6'')

[0120] [TSP=0 ppm]

[0121] (8).sup.13C-NMR spectrum (100 MHz, D.sub.2O) .delta. (ppm): 57.1 (d, C-1), 73.5 (d, C-2), 56.0 (d, C-3), 83.6 (d, C-4), 74.8 (d, C-5), 84.8 (d, C-6), 101.1 (d, C-1'), 56.1 (d, C-2'), 74.3 (d, C-3'), 70.6 (d, C-4'), 73.7 (d, C-5'), 61.4 (t, C-6'), 100.9 (d, C-1''), 72.4 (d, C-2''), 55.1 (d, C-3''), 69.7 (d, C-4''), 72.8 (d, C-5''), 60.9 (t, C-6'')

[0122] [Dioxane=67.4 ppm]

[0123] (9) Solubility: soluble in water, and insoluble in ethyl acetate and chloroform

Test Example 1

Antimicrobial Activity of 2-Hydroxykanamycin B and 2-Hydroxykanamycin C

[0124] The antimicrobial activity of 2-hydroxykanamycin B and 2-hydroxykanamycin C was measured as the minimum inhibitory concentration (MIC) by an agar dilution method. Test bacteria which had been cultivated overnight in a growth medium were adjusted to 10.sup.6 cells/ml, and one platinum loop thereof was inoculated in a Mueller-Hinton agar medium containing 2-hydroxykanamycin B or 2-hydroxykanamycin C (manufactured by Difco Laboratories Inc.), followed by cultivation at 37.degree. C. for 18 to 20 hr.

[0125] The results are shown in Table 1.

TABLE-US-00002 TABLE 1 Antimicrobial activity of 2-hydroxykanamycins B and C MIC (.mu.g/mL) 2-Hydroxy- 2-Hydroxy- Strain kanamycin B kanamycin C Staphylococcus aureus 2 128 ATCC29213 Staphylococcus aureus 2 128 209P JC-1 Staphylococcus epidermidis 1 32 ATCC14990 Bacillus subtilis 0.5 16 ATCC6633 Enterococcus faecalis 64 >128 ATCC29212 Enterococcus faecium 64 >128 ATCC19434 Salmonella typhimurium 1 32 ATCC13311 Acinetobacter calcoaceticus 1 32 ATCC23055 Escherichia coli 4 128 ATCC25922 Pseudomonas aeruginosa 128 >128 PA01

Sequence CWU 1

1

91390PRTStreptomyces kanamyceticus 1Met Gln Val Thr Thr Ile Thr Met Asp Asp Val Gln Tyr Pro Tyr Arg 1 5 10 15 Leu Gly Thr Asp Cys Leu Asp Gly Ile Val Thr Arg Leu Gly Glu Leu 20 25 30 Gly Ala Ser Arg Tyr Leu Ile Val Ser Asp Pro Arg Val Ala Glu Leu 35 40 45 Tyr Gly Gln Gly Leu Arg Glu Arg Leu Ala Glu Gln Ala Gly Pro Ala 50 55 60 Glu Leu Ile Thr His Ala Ser Gly Glu Gln Asn Lys Gly Leu Pro Ala 65 70 75 80 Leu His Asp Leu Ala Glu Glu Ala Leu Arg Arg Gly Ala Asp Arg Gln 85 90 95 Ser Ile Val Val Ala Leu Gly Gly Gly Val Thr Gly Asn Ile Ala Gly 100 105 110 Leu Leu Ala Ala Leu Leu Phe Arg Gly Ile Arg Leu Val His Val Pro 115 120 125 Thr Thr Val Val Ala Met Leu Asp Ser Val Leu Ser Leu Lys Gln Ala 130 135 140 Val Asn Ala Gly Val Gly Lys Asn Leu Val Gly Thr Phe Tyr Gln Pro 145 150 155 160 Val Glu Val Leu Ala Asp Thr Ala Met Leu Arg Thr Leu Pro Val Arg 165 170 175 Glu Val Arg Ser Gly Met Cys Glu Val Val Lys Asn Ser Leu Ala Ile 180 185 190 Arg Pro Ser Met Ile Asp Gln Leu Ser Ala Gly Leu Arg Pro Asp Gly 195 200 205 Arg Tyr Pro Asp Asp Thr Met His Trp Ile Ile Tyr Glu Ser Leu Ala 210 215 220 Ala Lys Ala Gln Val Thr Ala Tyr Asp Lys Tyr Glu Arg Gly Glu Gly 225 230 235 240 Leu Ile Leu Glu Tyr Gly His Thr Val Gly His Ala Val Glu His Ser 245 250 255 Ser Gln Gly Ala Val Pro His Gly Ala Ala Val Ala Leu Gly Met Ile 260 265 270 Ala Ala Ala Gln Val Ser His Arg Ala Gly Trp Ala Ser Ala Glu Leu 275 280 285 Val Asp Leu His Arg Glu Leu Val Ala Lys Thr Gly Val Ala Arg Arg 290 295 300 Ile Pro Ser Asp Ile Pro Leu Ser Ala Val Arg His Arg Leu Ser Phe 305 310 315 320 Asp Asn Lys Arg Gly Tyr Leu Pro Ala Ser Ala Asp Thr Tyr Pro Met 325 330 335 Val Leu Leu Glu Ser Pro Gly Lys Val Leu Arg Ser Glu Gly Thr Val 340 345 350 Leu Thr Ala Ala Pro Arg Asp Leu Val Asp Ala Val Val Asp Glu Leu 355 360 365 Ala Glu Pro Pro Arg Pro Ala Ala Ala Arg Thr Asp Asp Ala Ala Thr 370 375 380 Val Leu Gly Gly Ala Gly 385 390 25261DNAStreptomyces kanamyceticus 2gtgcgcatca tctccgtaca ggaggcggcc acccgcacct tcgacgtctg cgtggtgggc 60agcggcgcct ccggtgccat caccgccgcg gtgctcgccg aacggggcct gtccgtgctc 120atcctcgaac agggcaccgc gatcccgccg ggcaccgacc acgacgacgt cgaggacccc 180gacacctggg cgtacgcacg cgacggggaa ggctggagca aggagggcta tccctggagc 240gccatggcgt tcggcggcgg cacggtgttc tacggcggca tctccttccg ctacgaacag 300cgcgacctcg acccgccgcc cgcgctcctc ggcgacgccg actacgcgca ctggcggctg 360cggctcgacg aactggagcc gcactacgac tgggtggagg accggctcgg cgtgagcggc 420ccgtcccacg gccgggtggg cgactacgcc ttcccccact acgcgcgggg ctcgctgccg 480cacaccccga tgggcggcgc gctggcccgg ggcgccgacg ccctggggct gaccccgctg 540tccaccccga tggcgatcag cggggccagg gaccggcacg gccccggctg cgccgagctg 600acgccgtgca ccggcttcac ctgcccggtc aacgccaagg ccgatgtgat cagccgcatc 660ctggcgcgcg ccgaggggga cgtgtccgtc gccctggaca ccagagcggt gcggttcgtg 720gcctcggcac cggggcgggc gaaacgtctc gaagtgctcg gcggcagtcc ccgcagccgc 780cgctcggtcc atgccgaccg cttcgtcctc gcggccaacg ccatccagtc cgccgcgctc 840ctgctgcgtt cggcggaccg gcgcgagccg gacggcatgg ggaactccag cggtcaggtg 900ggccgccacc tggccatgaa gaacagcgtc tacgtccgcg gcaggaccca ggagcggatc 960gtcgcccacc agccgctgcg ccatcgctac tccagcgtct gcgtcctgga ccacctgcgc 1020ggtgcggagt tccccgggca gctgggcggg atcatctacg aggccaaccc gtgggaggac 1080cccgaggccg accgtcctgg cgccggttcg ctgctccagc tggagtgcct cctcggcgac 1140cgcccgcagg cccgcaacat ggtgcggctc gccaggagcc gggaccggga cgggctccag 1200cgcatcgtca tggactaccg ccagcacccc ttggacggcg aacgcctgga cgtgctccag 1260gggaaggcga aggatgtact gcacgcggcc ggggccgagc gcaccgagtc cgtcgacagc 1320gatttcgcga ccggcagcac gcatctgcac ggcacgctgc gcgccggtga cgaccccgcg 1380acctcggtga ccgaccggac gggccggctg cacgactacg acaacgtgtg gtcggcggac 1440ggcgcgacgt tccccttcgc cgggaacttc aatcccaccc tcaccatcca ggccaatgcc 1500cggcggatcg cggtcggcat ctcctgacgg ccgccccctc tcccctcccc cgtacgccct 1560gccgcccttc cccgtggcga gccaccccct tcgtgaatcc cccttcgtga cgcgcgaact 1620gtcttcgacc ttggaggtat gtgtcatgcc cctgcaaagt tcacggcttg cggtcgacaa 1680cggaaccccc gtccgcggca agccctggcc ggtgtggccg cagcccaccg acggcaccct 1740cgacgccctc tcccgcgtcc tgcgttccgg ccgctgggcc atcagcggcc cctaccgggg 1800cgtggagtcg gccgaacgcc gcttcgcccg ccggttcgcc gactaccacc gcatcgccca 1860ctgcgtgccc gcctccagcg gtacggcgag cctgatgctg gccctggagg cctgtggcgt 1920cggcgcggga gacgaggtca tcctgcccgg cgtcacctgg gtcgcctccg cctccacggt 1980ggtgggcgtc aacgcggtgc cggtgttcgc cgacatcgac ccggacaccc tctgcctcga 2040cccggacgcc gtcgaggcgg ccatcacccc ggccaccaag gcgatcgtcg tcgtccacct 2100ctacgcggcc gtcgccgacc tcacccgcct caaggaggtg gccgaccggc acggcatcgt 2160gctcatcgag gactgcgcgc aggcccacgg cgccgagttc gaaggccaca aggtcggcac 2220cttcggcgcg gtcggcacct tcagcatgca gcagagcaag gtcctgacca gcggcgaggg 2280cggcgccgcc atcaccgccg acccggtgct cgcccgccgg atggaacacc tgcgcgcgga 2340cggccgctgc taccgcgatc aggcgccgcc ctccggccac atggagctcg tcgagacggg 2400cgagctgatg ggcagcaacc gctgcatctc cgagttccag gcagcggtcc tgaccgagca 2460gctgggcgaa ctcgaccggt tcaacgccct gcgacggcac aacgcggaac tcctcgacgc 2520gctgctgacc gacgtcggat accgcccgca gcgcagcacg cccggcacca ccgcccgcac 2580gtactacacc tacgtcgccg agctgcccga cgcggaactg cccggcgcgg acatcaccaa 2640ggtcaccgag gcgctgaccg ccgaactcgg cttcccggtg gcaccggcct actcgccgct 2700caacgccaac cccctgtacg acccggccag tcgcagccgg ttcgccctcg gaccgcagca 2760cgagaagctc atcgaccccg cccgattcgt gctcccggtg agcggccgcc tgacgcgtcg 2820gctcgtcacc ttccaccacg ccgccctgct cggcgacgag tcggacatga gggacatcgc 2880ggaagcgttc accaaggtgc tccagcaccg ggccgtcctg gccgcttgag ccgaagccgt 2940cacacacgcc ttcaggattg gggacagacc atgcaggtca ccaccatcac gatggatgac 3000gtccagtatc cctaccgatt aggcacggac tgcctcgacg gcatcgtcac gcgcctcggc 3060gaactcggcg ccagccgcta cctgatcgtc agcgacccca gggtcgccga gctgtacggg 3120caggggctgc gcgaacggct cgcggagcag gcgggacccg ccgagctgat cacccatgcc 3180tcgggagaac agaacaaggg cctgcccgca ctgcacgacc tggccgagga ggcgctgcgg 3240cgcggcgccg accggcagag catcgtcgta gcactcggcg gcggtgtcac cgggaacatc 3300gcggggctgc tggccgcgct gctcttccgc ggcatccgtc tggtgcacgt gcccaccacc 3360gtggtggcca tgctggattc ggtgctctcg ctcaagcagg ccgtgaacgc gggagtcggc 3420aagaacctgg tcggcacctt ctaccagccc gtcgaagtgc tcgccgacac cgcgatgctg 3480cgcaccctgc cggtccgcga ggtcaggtcg gggatgtgcg aggtggtgaa gaactcgctc 3540gccatccgcc ccagcatgat cgaccagctg tcggccgggc tgcgccccga cggccgctat 3600cccgacgaca cgatgcactg gatcatctac gagagcctgg ccgccaaggc ccaggtcacg 3660gcgtacgaca agtacgagcg cggcgaggga ctcatcctgg agtacgggca caccgtcggg 3720cacgccgtgg agcactcctc gcagggagcc gtgccgcacg gcgccgccgt cgcgctcggc 3780atgatcgccg ccgcccaggt ctcccaccgg gcgggctggg cctcggccga actcgtcgac 3840ctgcaccggg agctcgtcgc caagaccggg gtcgcgcggc gcatcccgtc cgacataccg 3900ctctccgccg tcaggcaccg cctctccttc gacaacaagc ggggctacct cccggcctcc 3960gccgacacct atccgatggt gctgctcgaa tcccccggca aggtgctgcg cagcgagggc 4020accgtcctga cggcggcgcc acgggacctg gtcgacgcgg tggtcgacga actcgcggaa 4080cccccacggc ccgcggccgc gaggaccgac gacgccgcca ccgtcctcgg cggtgccggg 4140tgagcgcccc cgtgcgcgtc ggcgtcgtcg gtgcggggtt catgggcggg gtgcacgccg 4200aggtggtggc ggctcatccc ggcgcccggc tcgaagcggt gcacgacctc gaccccgccg 4260ccgccaggga cctggccgag cggttccgcg ccgagcgggc cgagccctcc tgggcggacc 4320tgctcgccga ccccgcgatc gacctgctca tcatcaccac gcccaacggg ctgcaccacc 4380ggcaggcggc cgaggcgctg cgggcgggca agcacgtact ggtggagaag ccgctcggtg 4440tcacgccgga gcaggtggcc gagctcgtcg aactcgccgg acggcacgac cgggtccttg 4500cccacggaag caacttcgtg cacagcccga agttcgtccg ggcccgtcaa ctggtcgcgg 4560acaccgaggc gttcggacgg ccgcacctgg tccgggtcgt cttccgcaac tcgggccccg 4620aggccgcctg ggccgcgtcc aaggacctcg cgggcggcgg agccctcctg gacctgggct 4680gtcacgcggt ggagctgtgc cggtggctgc tcgacggcgc cgacgtcgag tcggtcagcg 4740cccgactgca gcgggtgcgg ccgccccacg acgccgaagc ggaccgcgcg tccggcaccg 4800cgggaaccgc gcgggtcgcg ctggaggacc aggcgctgct ggtcatggag ttcgccgacg 4860gcgcggtcgg gcagtgcgac gtctcctggg tcacccaggg cggtgagcag gtcaccgcgg 4920agatcatcgg caccaagggc agggtcgagg tcgacctgtg gaccggcatg gggctgcgcg 4980cctactcgga caagggctat caggacgtct gggatcccga gcagggctgg gtgcatccgg 5040aatgggagtg gatccgggcg agcggctact accaccagga cggcaccgtg atcgaggcgg 5100tgggccaggg catccccctc acccacggcc ccgcggaagc gctcgcctcg gcccgtgtcc 5160tggccaccgg ttaccgcagt cacgcggagg ggcgggtact gcggctgtcc ggcgcgccgg 5220tcggccctgg cgcgtcgacg acggcggcgg gctcggaatg a 5261327DNAArtificial SequenceSynthetic construct; primer 3gggaagcttg accttggagg tatgtgt 27424DNAArtificial SequenceSynthetic construct; primer 4gttcagcatg gccaccacgg tggt 24521DNAArtificial SequenceSynthetic construct; primer 5tcggtgctct cgctcaagca g 21627DNAArtificial SequenceSynthetic construct; primer 6gggtctagat gccgtcctgg tggtagt 2771176DNAStreptomyces kanamyceticus 7atgcaggtca ccaccatcac gatggatgac gtccagtatc cctaccgatt aggcacggac 60tgcctcgacg gcatcgtcac gcgcctcggc gaactcggcg ccagccgcta cctgatcgtc 120agcgacccca gggtcgccga gctgtacggg caggggctgc gcgaacggct cgcggagcag 180gcgggacccg ccgagctgat cacccatgcc tcgggagaac agaacaaggg cctgcccgca 240ctgcacgacc tggccgagga ggcgctgcgg cgcggcgccg accggcagag catcgtcgta 300gcactcggcg gcggtgtcac cgggaacatc gcggggctgc tggccgcgct gctcttccgc 360ggcatccgtc tggtgcacgt gcccaccacc gtggtggcca tgctggattc ggtgctctcg 420ctcaagcagg ccgtgaacgc gggagtcggc aagaacctgg tcggcacctt ctaccagccc 480gtcgaagtgc tcgccgacac cgcgatgctg cgcaccctgc cggtccgcga ggtcaggtcg 540gggatgtgcg aggtggtgaa gaactcgctc gccatccgcc ccagcatgat cgaccagctg 600tcggccgggc tgcgccccga cggccgctat cccgacgaca cgatgcactg gatcatctac 660gagagcctgg ccgccaaggc ccaggtcacg gcgtacgaca agtacgagcg cggcgaggga 720ctcatcctgg agtacgggca caccgtcggg cacgccgtgg agcactcctc gcagggagcc 780gtgccgcacg gcgccgccgt cgcgctcggc atgatcgccg ccgcccaggt ctcccaccgg 840gcgggctggg cctcggccga actcgtcgac ctgcaccggg agctcgtcgc caagaccggg 900gtcgcgcggc gcatcccgtc cgacataccg ctctccgccg tcaggcaccg cctctccttc 960gacaacaagc ggggctacct cccggcctcc gccgacacct atccgatggt gctgctcgaa 1020tcccccggca aggtgctgcg cagcgagggc accgtcctga cggcggcgcc acgggacctg 1080gtcgacgcgg tggtcgacga actcgcggaa cccccacggc ccgcggccgc gaggaccgac 1140gacgccgcca ccgtcctcgg cggtgccggg tgatga 1176817DNAArtificial sequenceSynthetic construct; primer 8cttcgtgaat ccccctt 17917DNAArtificial SequenceSynthetic construct; primer 9gcccaccgcc tcgatca 17

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