WO2008093912A1

WO2008093912A1 - Novel ansamycin derivatives and the method for mutational biosynthesis thereof

Info

Publication number: WO2008093912A1
Application number: PCT/KR2007/002378
Authority: WO
Inventors: Young-Soo Hong; Jung Joon Lee; Kyeong Lee; Woncheol Kim; Xingfu Cai; Dongho Lee
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-01-30
Filing date: 2007-05-15
Publication date: 2008-08-07
Also published as: KR100861697B1; KR20080071338A

Abstract

The present invention relates to a method for producing ansamycin derivatives by using a mutant strain with the mutation of 3-amino-5-hydroxylbenzoic acid (AHBA) biosynthesis gene, more precisely a method for producing novel ansamycin derivatives by adding various types of AHBA analogs to the growth medium of the transformant with the mutation of an AHBA biosynthesis gene. Ansamycin derivatives produced by the method of the present invention have ansamycin-like Hsp90 inhibitory effect, so that it can be effectively used as antibiotics, antifungal agents, antiviral agents, immunosuppressants, anti-inflammatory agents or anticancer agents.

Description

[DESCRIPTION]

[invention Title]

NOVEL ANSAMYCIN DERIVATIVES AND THE METHOD FOR MUTATIONAL BIOSYNTHESIS THEREOF

[Technical Field]

The present invention relates to a novel preparation method for ansamycin derivatives using AHBA biosynthesis gene knock-out strain.

[Background Art]

Ansamycin antibiotics are exemplified by geldanamycin, herbimycin, macbecin, reblastatin, rifamycin and ansamitocin, etc. These ansamycin antibiotics are the compounds having a handle-shaped chemical structure based on polyketide backbone synthesized by using 3-amino-5-hydroxybenzoic acid

(AHBA) as a starter unit. After priming the start unit, this is followed by the sequential addition of extender units, such as acetate, propionate, and glycolate, to form a polyketide backbone, which then undergoes further downstream processing. The functions of these compounds as antibiotics (Takahashi A. et al. (2003) PNAS 100(20) 11777-11782; Agbessi S. et al. (2003) Appl. Microbiol. Biotechnol 62, 233-238), antifungal agents (Cardenas M. E. et al. (1999) Clinical Microbiology

Review 12(4) 583-611), antiviral agents (Li Y. et al.

(2004) Antimicrobial Agents and Chemotherapy 48(3) 867-

872), immunosuppressants (Owens-Grillo J. et al. (1995) J. Biological Chemistry 270(35) 20479-20484), degenerative nervous disease treatment agents (Sittler

A. et al. (2001) Human Molecular Genetics 10(12) 1307-

1315; Waza M. et al. (2005) Nature medicine 11(10)

1088-1095), anti-inflammatory agents (Pittet J. et al. (2005) The Journal of Immunology 174, 384-394; Hsu H. et al. (2006) Molecular Pharmacology, Web pub. JuI 25) and anticancer agents have been confirmed between 1970 and 2000 (DeBoer C. et al. J. Antibiotic. , 23(9) 442- 447, 1970; Omura, S. et al., J. Antibiotic. 32, 255-261, 1979; Muroi, M. et. al., J. Antibiotic. 33, 205-212, 1980; Neckers L. et al . , Invest. New Drugs 17, 361-373, 1999; Piper P. W., Curr. Opin. Investing Drugs 2(11) 1606-1610, 2001) .

Among those ansamycin compounds, herbimycin, macbecin, reblastatin and geldanamycin which have benzoquinone structure have been known to inhibit heat shock protein 90 (Hsp90) having protein chaperone activity. In particular, geldanamycin binds to the ATP (adenosine triphosphate) -binding site of heat shock protein (Hsp) 90 and inhibits the chaperone activity of the protein, which leads to the destabilization of the Hsp90 client proteins which are critical in signal transduction pathways. Because human Hsp90 client proteins are importantly involved in signal transduction and transcription on the cancer cells, geldanamycin and its derivatives are potential anticancer chemotherapeutic agents (Whitesell L. et. al. Proc. Natl. Acad. Sci. USA, 91, 8324-8328, 1994; Prodromou C. et. al. Cell, 90, 65-75, 1997; Walter S. and Buchner J., Angew. Chem. Int. Ed. 41, 1098-1113, 2002; Piper P. W., Current opinion in Investigational Drugs 2, 1606-1610, 2001) .

Considering the physiological significance of Hsp90, several Hsp90 inhibitors, including 17- allylamino-17-demethoxygeldanamycin (17-AAG) and 17- DMAG, are in various stages of clinical trials as novel antitumor agents.

Based on predictions from sequence homology and the results of feeding experiment with 3H and 14C- labeled precursor, it was proposed that the final product, geldanamycin needs several modification steps including O-carbamoylation, hydroxylation, O- methylation and oxidation to the initial polyketide synthase product. However, beyond sequences and putative functions deduced from sequence homologies, a few have so far been learned about the post-PKS modification genes and the tailoring process leading from initial polyketide to geldanamycin. Among them, PCT/KR2005/002601 describes the development of geldanamycin derivatives based on O-carbamoylation that converts the primary polyketide to geldanamycin. The nucleotide sequences of genes involved in the biosynthesis of AHBA, a geldanamycin precursor, exhibit high homology with enzymes involved in the biosynthesis of AHBA of rifamycin (Hong Y. S et. al., GenBank DQ249342; Rascher, A. et al., FEMS. Microbiol. Lett. 218 223-230, 2003), and also genes involved in the biosynthesis of herbimycin have been confirmed to have high homology with genes involved in the biosynthesis of geldanamycin (Rascher, A. et al. Appl . Environ. Microbiol. 71(8) 4862-4871, 2005). In the case of rifamycin, AHBA synthase gene (rifK) knock-out strain did not produce rifamycin, and the production of rifamycin was resumed by the addition of exogenous AHBA (Yu. T. W. et. al., J. Biol. Chem. 276(16) 12546-12555, 2001) . However, the experiments using AHBA analogs showed that polyketide formation was not fully processed with the AHBA analogs and only the short polyketide chains, which came apart from polyketide synthase, were discovered (Admiraal, S. J. et. al., Biochemistry 41(16), 5313-5324. 2002 & Biochemistry 40(20) 6116-6123, 2001; Hunziker, D. et al., J. Am. Chem. Soc, 120(5) 1092-1093 1998).

The biosynthesis of ansamycin compounds is as follows; Biosynthesis of ansamycins is initiated by priming the AHBA as a common starter unit. This is followed by the sequential addition of extender units, such as acetate, propionate and glycolate, to form a polyketide backbone. The polyketide chain is fallen apart from PKS by the action of amide synthase, which is located in the last domain of PKS, and at the same time forms a ring structure by amide bond. Then, the tailoring process leading from initial polyketide to final ansamycin. Most PKS enzymes have thioesterase (TE) in their last domains, so that they are able to separate polyketide chains from PKS proteins and then form a ring structure by ester bond. For example, the last TE domain of DEBS ( 6-deoxyerythronolide B synthase), an erythromycin PKS, catalyze bounded polyketide chain from the adjacent ACP (acyl carrier protein) domain to itself active serine residue. And then a lactonized ring, called a 6-deoxyerythroronolide B, is formed by intramolecular nucleophilic attack (S. Donadio. et al. (1992) Gene 115:97-103; R. Aggarwal. et al. (1995) J. Chem. Soc. Chem. Commun. 15:1519-1520; S. E. O'Connor (2006) Nature Chem. Biol. 2 (10) : 511-512 ). There several reports are presented that the TE is involved in a ring formation of various polyketides (R. S. Gokhale et al. (1999) Chemistry & Biology 6: 117-125; W. He. et al. (2006) Bioorg. Medi . Chem. Lett. 16:391-394; H. Lu et al. (2002) Biochemistry 41:12590-12697).

In this invention, a gene cluster involved in the biosynthesis of AHBA of Streptomyces hygroscopicus subsp. duamyceticus producing geldanamycin has been confirmed. And then napK (that exhibits high homology with rifK of the above rifamycin gene cluster) gene- knock out strain (AC2) has been generated. This AC2 mutant is unable to produce any geldanamycin related compounds, but the production of geldanamycin can be restored by supplementing synthetic AHBA to culture. Also, when different concentrations of AHBA were fed to culture medium, extracts exhibited precursor concentrate-dependent geldanamycin productivity. Based on those results, producing of expected novel derivatives by the addition of AHBA analogs were examined by LC/MS analysis. Thus, the inventors showed that the novel derivatives can be purified by depending on the LC/MS data analysis. The present inventors made AHBA gene knock-out mutant strain (AC2) and further completed this invention by providing a production method for ansamycin comprising the step of adding AHBA analogs to the culture medium of AHBA gene knock-out mutant and also providing novel ansamycin derivatives produced by the same.

[Disclosure]

[Technical Problem]

It is an object of the present invention to provide a production method for ansamycin derivatives including the step of addition of AHBA analogs to the medium where mutant strain (AC2), which is prepared by eliminating AHBA biosynthetic gene, is growing and novel ansamycin derivatives produced therefrom.

[Technical Solution] To achieve the above object, the present invention provides a method for producing ansamycin derivatives comprising the following steps:

(1) Preparing a construct wherein AHBA biosynthesis gene is inactivated; (2) Constructing a recombinant vector containing the construct of step (1) ;

(3) Generating a recombinant mutant by transforming host cells with the recombinant vector of step (2) ; and

(4) Adding 3-amino-5-hydroxylbenzoic acid (AHBA) or its derivatives to the culture medium of the mutant of step (3) and obtaining ansamycin derivatives from the culture medium. The present invention also provides ansamycin derivatives produced by the above method for biosynthesis of ansamycin derivatives.

The present invention further provides novel ansamycin derivatives. The present invention also provides a napK gene knock-out construct in which napK gene represented by

SEQ. ID. NO: 7 is inactivated.

The present invention also provides a recombinant vector comprising the napK gene knock-out construct. The present invention also provides a recombinant

Streptomyces hygroscopicus mutant strain generated by transforming Streptomyces hygroscopicus subsp. duamyceticus with the above vector.

The present invention also provides an ansamycin derivative producing strain having ester bond and characterized by being produced by the transformation of the above mutant strain with an expression vector harboring thioesterase gene.

In addition, the present invention provides antibiotics, antifungal agents, antiviral agents, immunosuppressants, degenerative nervous disease treatment agents, anti-inflammatory agents or anticancer agents containing any of the ansamycin derivatives, the recombinant vector above, the mutant strain or its culture solution, the ansamycin derivatives produced by the mutant strain, the ansamycin derivative producing strain having ester ring or its culture solution or the ansamycin derivatives produced by the ansamycin derivative producing strain having ester ring as an effective ingredient.

Hereinafter, the present invention is described in detail.

The gene cluster (Hong Y. S et. al., GenBank DQ249342; Rascher, A. et al., Appl . Environ. Microbiol.

71(8) 4862-4871, 2005) involved in AHBA biosynthesis especially in the biosynthesis of geldanamycin among notified ansamycins (see Fig. 1) contains 6 AHBA biosynthesis genes and polyketide synthase gene whose function has not been explained, though (see Fig. 2 and Table 1). The present inventors identified the geldanamycin polyketide synthase gene presumed by the sequencing of the ansamycin gene cluster involved in Streptomyces hygroscopicus subsp. duamyceticus and the gene cluster involved in the biosynthesis of AHBA acting as a starter unit (see Fig. 3) .

The amino acid sequence of the gene involved in AHBA biosynthesis of geldanamycin exhibited significant homology with the amino acid sequence encoding the gene involved in AHBA biosynthesis of ansamitosin. So, each gene above is presumed to be directly associated with biosynthesis of AHBA, as previously confirmed in the case of rifamycin (see Fig. 4 and Table 2) . AHBA biosynthesis genes of rifamycin have already been confirmed to play a key role in AHBA synthesis. So, the present inventors picked napK gene that exhibits significant homology with rifK gene, an AHBA synthase gene of rifamycin, and inserted kanamycin gene to the napK to inhibit its functions, resulting in the construction of AC2 strain with restricted functions.

TA cloning vector (see Fig. 5) was used for the cloning of napK gene presumed to encode AHBA synthase of AHBA biosynthesis gene cluster and an antibiotic (such as kanamycin) resistant gene was inserted in the cloned napK gene to generate an inactive construct. The prepared napK gene knock-out construct was inserted in pKC1139 vector (see Fig. 6) to construct the recombinant pKC-AHBA vector. The recombinant vector containing the napK gene knock-out construct was inserted in Streptomyces hygriscopicus to generate a mutant strain, which was named as Streptomyces hygriscopicus AC2 (Accession No: KCTC 10676BP) (see Fig. 7) . The mutant strain (AC2 strain) was prepared by transforming a host strain with recombinant pKC-AHBA vector, precisely by the homologous recombination using the sequence homology to substitute wild-type napK gene with the inactivated mutant form. PCR was performed with the mutant strain AC2 to confirm the successful insertion of the napK gene knock-out construct (see Fig. 8) . When the mutant strain AC2 was cultured under the same conditions as for the culture of wild-type, the mutant strain could not produce geldanamycin. But, when synthetic AHBA was added to the culture medium, the production of geldanamycin was recovered AHBA dose- dependently (see Fig. 9 and Fig. 10) . Therefore, the mutant strain AC2 cannot produce AHBA and ansamycin by itself but is able to produce ansamycin derivatives by the addition of exogenous AHBA and its derivatives (see Fig. 11) . The present inventors constructed an expression vector containing thioesterase gene (see Fig. 12). The expression vector containing thioesterase was inserted in Streptomyces hygriscopicus AC2, which was further cultured. 3-hydroxy benzoic acid or 3, 5-dihydroxy benzoic acid was co-treated to the culture (see Fig. 14) . As a result, ansamycin derivatives forming ester ring were produced.

[Table l]

The present invention provides a method for producing ansamycin derivatives comprising the following steps:

(1) Preparing a construct wherein the AHBA biosynthesis gene is inactivated;

(2) Constructing a recombinant vector containing the construct of step (1); (3) Generating a recombinant mutant by transforming host cells with the recombinant vector of step (2) ; and

(4) Adding 3-amino-5-hydroxylbenzoic acid (AHBA) or its derivatives to the culture medium of the mutant of step (3) and obtaining ansamycin derivatives from the culture medium.

The AHBA biosynthesis gene of step (1) is napK gene represented by SEQ. ID. NO: 7 but not always limited thereto, and any other gene that has at least 70% homology with the gene and has equal biological functions can be used. The inactivating construct can be prepared by inserting an antibiotic resistant gene into napK gene and kanamycin is preferably used but not always limited thereto and any antibiotic resistant gene that can be effectively manipulated by those in the art can be accepted.

The recombinant vector of step 2) is preferably the recombinant vector pKC-AHBA which is prepared by conjugating the inactivating construct to the vector pKC1139 (Bierman, M. et . al . , (1992) Gene 116, 43-49, see Fig. 6) pre-digested with EcoRI/Hindlll, but not always limited thereto. The backbone structure vector is preferably pKC1139 but not always limited thereto and any other vector that is accepted by those in the art can be used.

The host cell of step (3) can be one of well- known ansamycin producing strains harboring the gene cluster that plays a key role in AHBA biosynthesis and napK gene or genes having at least 70% homology with napK and AHBA synthase activity. In this invention, napK was eliminated and thus a mutant strain with restricted production of AHBA was generated. But, a strain with restricted production of AHBA can also be produced by eliminating any other AHBA biosynthesis related gene (see Fig. 4 ) ., To produce geldanamycin derivatives, Streptomyces hygroscopicus or Streptomyces melanosporofaciens can be preferably used and precisely the above Streptomyces hygroscopicus is preferably selected from the group consisting of Streptomyces hygrqscopicus var. geldanus NRRL 3602, Streptomyces hygroscopicus subsp. duamyceticus and Streptomyces hygroscopicus 17997. And, Streptomyces hygroscopicus var. geldanus NRRL 3602 or Streptomyces melanosporofaciens EF-76 is more preferred. Geldanamycin producing strain is exemplified by Streptomyces violaceusniger and herbimycin producing strain is exemplified by Streptomyces hygroscopicus AM- 3672. Reblastatin producing strain is exemplified by Streptomyces sp. S6699 and macbecin producing strain is exemplified by Nocardia sp. C-14919. Ansamitocin producing strain is exemplified by Actinosynnema pretiosum ATCC 31565 and rifamycin producing strain is exemplified by Amycolatopsis mediterranei S699. In addition, any strain that is informed to those in the art can be used. A mutant strain was generated by transforming such host cells mentioned above with the recombinant vector above, which was then named "Streptomyces hygroscopicus AC2" and deposited at KCTC on August 3, 2004 (Accession NO: 10676BP) .

AHBA or its derivatives of step (4) can be any AHBA or its derivatives that can act as an ansamycin precursor and can be represented by the following formula 1. The preferable treatment concentration of AHBA is mg/m£ ~ 20 mg/m£ but not always limited thereto.

Wherein, Ri is preferably H, OH, NH₂, halogen or ally group. R₂ is preferably H, OH, NH₂, halogen or ally group. R₃ is preferably H, OH, NH₂, halogen or ally group. R₄ is preferably H, NH₂, halogen or ally group. Halogen is preferably F, Cl, Br or I.

Ansamycin derivatives were extracted from a microorganism culture solution with an organic solvent and then proceed to chromatography for separation and purification. A method for separation and purification can be selected from a variety of methods that have been well-known to those in the art.

The above method can additionally include the following steps after step (3): (4) Constructing a recombinant expression vector containing thioesterase gene; (5) Introducing the recombinant expression vector prepared in step (4) to the mutant strain generated in step (3); and (6) Adding an AHBA analog to the transformed mutant strain in order to form ester bond and culturing thereof, and then obtaining ansamycin derivatives from the culture solution.

In the above step (4), the thioesterase is preferably DEBS ( β-deoxyerythronolide B synthase) thioesterase or pikromycin thioesterase but not always limited thereto. In step (6), the AHBA analog is preferably 3- hydroxy benzoic acid or 3, 5-dihydroxy benzoic acid but not always limited thereto. The preferable treatment concentration of the AHBA analog is 0.1 rag/ml ~ 20 mg/ml but not always limited thereto.

In step (6), the ansamycin derivative is characterized by ester ring.

The present invention provides ansamycin derivatives prepared by the above method for biosynthesis of ansamycin derivatives. Ansamycin derivatives of the present invention can be prepared by the above described method and herein the polyketide compound that has a handle shaped ring having benzene structure as a basic structure is defined as ansamycin derivatives .

The present inventors confirmed that not only geldanamycin but also linear polyketide compound represented by formula 2 and ansamycin derivatives with an authentic macrolactam ring represented by formula 3 can be produced by the addition of AHBA or its analogs to the mutant strain AC2 growth medium (see Fig. 10) . So, different kinds of ansamycin derivatives can be generated according to the kind of AHBA analogs added. In the previous study with rifamycin, polyketide formation was not completed with an AHBA-like precursor and short polyketide chains was alone fallen apart from the PKS protein during the polyketide condensing reaction. However, according to the method of the invention, polyketide formation was completed and thus a ring structure was formed by the following amide synthase reaction. It is judged that when naphthobenzoquinone compound such as rifamycin is used, naphthobenzoquinone ring formation takes place during polyketide formation reaction and thus polyketide reaction is interrupted (Yu. T. W. et. al._r Proc. Natl. Acad. Sci. 96, 9051-9056, 1999) . In the meantime, when benzoquinone compound such as geldanamycin is used, benzoquinone ring is not interrupted during polyketide reaction, suggesting that polyketide formation reaction can be continued successfully. Therefore, it is expected that polyketide formation using an AHBA-like precursor can be generally achieved by using benzoquinone ansamycin.

The present invention also provides novel ansamycin derivatives represented by the following formulas 2 - 4.

R = H or OH; single bond or double bond between C₅.

Ri = H or OH ; R₂ = H or OH; single bond or double bond between C₄ and C₅ .

The present invention also provides a napK gene knock-out construct, in which napK gene represented by SEQ. ID. NO: 7 is inactivated.

The napK gene knock-out construct is characterized by the insertion of an antibiotic resistant gene in a target gene. In the present invention, kanamycin resistant gene was used as the antibiotic resistant gene, but not always limited thereto.

The present invention also provides a recombinant vector containing the napK knock-out construct. Herein, pKC1139 vector was used as a backbone structure vector for the recombinant vector but not always limited thereto.

The present invention also provides a recombinant Streptomyces hygriscopicus mutant strain that is generated by transformation of Streptomyces hygroscopicus subsp. duamyceticus by the recombinant vector of the invention. The transformation above is preferably performed by one of the well-known methods accepted by those in the art. The present inventors named the generated mutant strain "Streptomyces hygroscopicus AC2" and deposited at KCTC on August 3, 2004 (Accession No: 10676BP).

In addition, the present invention provides an ansamycin derivative producing strain having an ester ring, which is prepared by the transformation of the mutant strain prepared above with the expression vector harboring thioesterase .

The thioesterase is preferably DEBS-thioesterase or pikromycin thioesterase but not always limited thereto. The present invention also provides antibiotics, antifungal agents, antiviral agents, immunosuppressants, degenerative nervous disease treatment agents, antiinflammatory agents or anticancer agents containing any of the ansamycin derivatives, the recombinant vector above, the mutant strain or its culture solution, the ansamycin derivatives produced by the mutant strain, the ansamycin derivative producing strain having ester ring or its culture solution or the ansamycin derivatives produced by the ansamycin derivative producing strain having ester ring as an effective ingredient .

Geldanamycin, an ansamycin anticancer agent, has been confirmed to be bound to ATP (adenosine triphosphate) binding site of Hsp90 (heat shock protein

90) that has the activity of protein chaperone

(Whitesell L. et. al., Proc. Natl. Acad. Sci . 91, 8324-

8328, 1994; Prodromou C. et. al., Cell 90, 65-75, 1997,

USA) . By this founding, it is confirmed that the anticancer activity of the conventional geldanamycin was attributed not to the inhibition of tyrosin kinase having oncogenic protein function but to the inhibition of Hsp90 playing a key role in structural stability of Hsp90 client proteins including tyrosin kinase. Again, the anticancer activity of geldanamycin is generated by inhibiting the stability of oncogenic proteins. Therefore, ansamycin including geldanamycin can be effectively used as antibiotics, antifungal agents, antiviral agents, immunosuppressants, degenerative nervous disease treatment agents, anti-inflammatory agents and anticancer agents.

The treatment agent of the present invention can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration. Pharmaceutically acceptable carrier can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol and ethanol. Other general additives such as anti-oxidative agent, buffer solution, bacteriostatic agent, etc, can be added. In order to prepare injectable solutions, pills, capsules, granules or tablets, diluents, dispersing agents, surfactants, binders and lubricants can be additionally added. The composition of the present invention can further be prepared in suitable forms for each disease or according to ingredients by following a method represented in Remington' s Pharmaceutical Science (the newest edition) , Mack Publishing Company, Easton PA .

The treatment agent of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection) . The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The preferable dosage is 0.1 ~ 500 mg/kg. Administration frequency is once a day or preferably a few times a day.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig. 1 is a diagram illustrating structures of ansamycins synthesized in various strains using AHBA as a precursor.

Fig. 2 is a diagram showing the gene map of a

55.6 kb fraction of the AHBA biosynthesis gene cluster involved in geldanamycin biosynthesis, obtained by the sequencing of genomic cosmid DNA of Streptomyces hygroscopicus subsp. duamyceticus. Fig. 3 is a diagram illustrating the biosynthesis process of geldanamycin using AHBA as a precursor.

Fig. 4 is a diagram illustrating the biosynthesis pathway of AHBA confirmed to be a common precursor for ansamycin antibiotics by the investigation of biosynthesis pathway of rifamycin, the most representative ansamycin antibiotics, and enzymes involved in each step.

Fig. 5 is the vector map of pCR2.1-TOPO. Fig. 6 is the vector map of pKC1139.

Fig. 7 is a diagram illustrating the elimination of napK_r AHBA synthase gene, among AHBA precursor biosynthesis genes.

Fig. 8 is a diagram illustrating the insertion of kanamycin gene in napK gene of AC2 chromosome where

AHBA synthase gene was eliminated and a photograph illustrating the result of PCR confirming the generation of the mutant strain AC2.

Fig. 9 is a HPLC graph confirming the recovery of geldanamycin production by the addition of synthetic AHBA to the culture of AC2.

Fig. 10 is a diagram illustrating the structures of geldanamycin and ansamycin derivatives and the preparation method thereof. Fig. 11 is a diagram illustrating the method for production of ansamycin derivatives by the addition of AHBA derivatives to the AHBA synthase gene knock-out strain.

Fig. 12 is a diagram illustrating the construction of a thioesterase expression vector.

Fig. 13 is a diagram illustrating the production of ansamycin derivatives forming ester ring after transformation of the mutant with the thioesterase expression vector. Fig. 14 is a diagram illustrating the biosynthesis of ansamycin derivatives having ester ring.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Generation of the mutant strain AC2

To generate the mutant strain AC2, PCR was performed with primers represented by SEQ. ID. NO: 1, NO: 2, NO: 3 and NO: 4 under the following conditions (Table 3). Chromosomal DNA of Streptomyces hygroscopicus subsp. duamyceticus was used as a template. Primers (25 pmol ) , template DNA, Ex Taq polymerase (Takara) , dNTP mixture and distilled water were mixed to make the final volume 50 μi . PCR was performed as follows; predenaturation at 97 ^°C for 5 minutes, denaturation at 95 ^°C for 1 minute, annealing at 55 ^°C for 1 minute, polymerization at 72 ^°C for 1 minute, 30 cycles from denaturation to polymerization, and final extension at 72 °C for 10 minutes. The PCR product obtained by PCR using primers represented by SEQ. ID. NO: 1 and NO: 2 was cloned into TA cloning vector (pCR2.1-TOPO, InvitrogenTM life technologies, USA, Fig. 5) to construct the recombinant vector pTA- AHBA-N (front part containing AHBA biosynthesis gene) and another PCR product obtained by PCR using primers represented by SEQ. ID. NO: 3 and NO: 4 was cloned into TA cloning vector to construct the recombinant vector pTA-AHBA-C (end part containing AHBA biosynthesis gene) . pFD-neoS (Denis, F. & Brzezinski, R. (1991) FEMS Microbiol. Lett. 81, 261-264) containing kanamycin resistant gene aphll was digested with Kpnl and Pstl. The obtained 1.1-kb DNA fragment was used to construct a selection marker and a gene disrupted construct. For gene inactivation experiments, EcoRI/Kpnl fraction of pTA-AHBA-N, Pstl/Hindlll fraction of pTA- AHBA-C and 1.1-kb Pstl/Kpnl fraction {aphll gene) of pFD-neoS were conjugated to pKC1139 (Bierman, M. et. al., (1992) Gene 116, 43-49, Fig. 6) which was pre- digested with EcoRI/Hindlll to construct the recombinant vector pKC-AHBA.

The recombinant vector pKC-AHBA was inserted in E. coli ET12567/pUZ8002 (Allen, I. W. & D. A. Ritchie. (1994) MoI. Gen. Genet. 243:593-599) and 5. hygroscopicus JCM4427 was transformed with the resultant strain according to the conjugation method of Flett, F. et al (Flett, F. et. al., (1997). FEMS Microbiol. Lett. 155, 223-229) (Fig. 7).

For the chromosomal integration of pKC-AHBA gene into S. hygroscopicus JCM4427 chromosome, the transformant was cultured in YEME (yeast extract-malt extract agar) liquid medium supplemented with kanamycin at 37 ^°C for 4 days. Owing to the genetic characteristics of pKC1139 vector, the duplication of the vector was terminated at 37 ^°C and only the strain containing kanamycin resistant gene in its chromosome survived (Bierman, M. et . al., (1992) Gene 116, 43-49). The survived strain was cultured in a solid medium supplemented with kanamycin or apramycin to select the kanamycin resistant but apramycin sensitive recombinant mutant strain. The recombinant mutant strain was confirmed by PCR with the total genomic DNA thereof (Fig. 8) . The PCR was performed by the following conditions with the primers represented by SEQ. ID. NO: 1, NO: 4, NO: 5 and NO: 6 (Table 3). The strain was named as "Streptomyces hygriscopicus AC2" and deposited at KCTC on August 3, 2004 (Accession No: KCTC 10676BP⁾.

[Table 3]

Example 2: Construction of thioesterase (referred js 'TE' hereinafter) expressing transformant

The actinomyces high-expressing promoter ermE* is the well-known promoter by previous reports. The present inventors performed PCR to amplify nucleotide sequence of the ermE* promoter region by using pIJ471 vector as a template (M. J. Bibb, et al. (1994) MoI.

Microbiol. 14 (3) : 533-545) . PCR was performed with primers represented by SEQ. ID. NO: 8 and NO: 9 (Table

3) . Primers (25 pmol), template DNA, Ex Taq polymerase

(Takara) , dNTP mixture and distilled water were mixed to make the final volume 50 μJt. PCR was performed as follows; predenaturation at 97^°C for 5 minutes, denaturation at 95^°C for 1 minute, annealing at 55^°C for 1 minute, polymerization at 72^°C for 1 minute, 30 cycles from denaturation to polymerization, and final extension at 72 ^°C for 10 minutes. The PCR product was cloned into a TA cloning vector (pCR2.1-TOPO, InvitrogenTM life technologies, USA, Fig. 5) to construct the recombinant vector pCR-erm53 (ermE* promoter region) .

To amplify DEBS-TE, the chromosomal DNA (GenBank AY6615ββ) of erythromycin producing strain Saccharopolyspora erythraea was used as a template and to amplify PikAV-TE, the chromosome (GenBank AF079138) of pikromycin producing strain Streptomyces venezuelae was used as a template. For the PCR with DEBS-TE, primers represented by SEQ. ID. NO: 10 and NO: 11 were used, while primers represented by SEQ. ID. NO: 12 and NO: 13 were used for the PCR with pikAV-TE. The PCR products were cloned into a TA cloning vector (pCR2.1- TOPO, InvitrogenTM life technologies, USA, Fig. 5) respectively to construct pCR-dTE and pCR-pTE (Fig. 12) . As shown in Table 3, the ermE* promoter region (pCR-erm53) was digested with EcoRI and BgIII and each TE site was digested with BgIII and HindIII . The resultant fragments were obtained from agarose gel and conjugated to pKC1139 digested with EcoRI and HindIII to construct the recombinant vectors pKC-dTE and pKC- pTE.

The recombinant vectors pKC-dTE and pKC-pTE were introduced into E. coli ET12567/pUZ8002 (Allen, I. W. & D. A. Ritchie. (1994) MoI. Gen. Genet. 243:593-599). The prepared strain was inserted into S. hygroscopicus AC2 for transformation according to the conjugation method of Flett, F. et al (Flett, F. et. al., (1997). FEMS Microbiol. Lett. 155, 223-229), to generate AC2/dTE and AC2/pTE strains (Fig. 13) .

Example 3: Preparation method of ansamycin derivatives using the mutant strain AC2

The mutant strain AC2 prepared in Example 1 and wild type strain were cultured in 25 m£ of solid YEME at 28 ^°C for 7 days. On the 3^rd day of culture, synthetic AHBA was dissolved in 1 ml of distilled water at the concentration of 1 mg/25 ml and 10 mg/25 mi respectively and then added to each growing medium, followed by further culture for 5 days at 28 ^°C . Extraction was performed twice for each culture medium using EtOAc and each extract was filtered to eliminate insoluble impurities, concentrated, and then fractionated by using EtOAc and H₂O to give an organic extract. For the fractionation of each culture extract, silica gel chromatography was preformed using CHCI₃- MeOH as a moving phase. The obtained fractions were analyzed by HPLC [YMC J' sphere ODS-H80, 150 x 4.6 mm i.d., MeOH-H₂O (0.05% acetic acid) gradient, 1 m£/min] using Waters Delta Prep 3000 system (Waters, USA) . As a result, the wild type strain cultured in AHBA-free medium produced geldanamycin, the ansamycin anticancer agent, whereas the mutant strain AC2 did not produce geldanamycin in AHBA-free medium. But, the mutant strain AC2 recovered its capability of producing geldanamycin when AHBA was added to the medium (Fig. 9) . The compounds represented by formula 1 were also added in the culture medium of mutant strain AC2 at the concentration of 10 mg/nι£ by the same manner as described above. Then, each medium was extracted and analyzed. The analysis was performed by LC/MS using Finnigan LCQ Advantage Max mass spectrophotometer

(Thermo, USA) . In this LC/MS analysis, it was investigated by Xcalibur software (version 1.3 SP2,

Thermo Electron) whether there was the mass peak of an expected compound added with AHBA-like compound, for which the basic background peak was eliminated in advance by using Metabolite ID 2.0 software (Thermo

Electron Co., USA). As a result, as shown in Table 4, the mass of the expected ansamycin compound in the culture solution added with AHBA analogue was observed.

The expected compounds exhibited ansamycin specific mass fragmentation, that is mass value with reduced carbamoyl residue (CONH₂= M. W. 43) in MS² was observed.

[Table 4]

Mass peaks of compounds by LC/MS in AC2 medium using substrates respectively

Example 4: Preparation method of ansamycin derivatives using thioesterase transformant

AC2/dTE strain and AC2/pTE strain prepared in Example 2 were cultured in 25 mi of solid YEME for 7 days at 28 ^°C . In the course of the culture of the mutant strain AC2, 3-hydroxyl benzoic acid or 3,5- dihydroxy benzoic acid dissoLved in 1 mi of distilled water at the concentration of 1 rag/25 mi and 10 rag/25 mi were added to each growing medium cultured for 3 days, followed by further culture for 5 days at 28 "C. Extraction of each medium was performed twice using EtOAc. The extracts were filtered to eliminate insoluble impurities, concentrated, and then fractionated by using EtOAc and H₂O to give organic extract .

The extract includes the compounds represented by the following formula.

compound 6 R1-H, R2-H, carbon 4,5 = single bond compound 7 R1-H, R2-0H, carbon 4,5 = single bond compound 8 R1-H, R2-H, carbon 4,5 = double bond compound 9 R1-H, R2-0H, carbon 4,5 = double bond compound 10 R1-0H, R2-H, carbon 4,5 = single bond compound 11 R1-0H, R2-0H, carbon 4,5 = single bond compound 12 R1-0H, R2-H, carbon 4,5 = double bond compound 13 R1-0H, R2-0H, carbon 4,5 = double bond

Example 5: Identification of the structure of the novel ansamycin compound using the mutant strain AC2 To identify the structure of the novel compound prepared in Example 2, the mutant strain AC2 was cultured in 100 ml of liquid YEME for 3 days at 28^°C. 75 mi of the culture medium was distributed on a Petri dish containing 2 L of solid YEME, followed by culture for one more day at 28^°C. After confirming the normal growth of AC2, 700 mg of 3-hydroxylbenzoic acid and 3- aminobenzoic acid were respectively added to each culture medium, followed by culture for 7 days at 28 ^°C. Extraction was performed by the same manner as described above and fractionation was performed to give an organic extract. For the fractionation of each culture extract, silica gel chromatography was preformed using CHCl₃-MeOH as a moving phase. The obtained fractions were analyzed by ESIMS.

To identify the compounds produced and deposited below, melting point was measured with Electrothermal 9100 (Electrothermal, England) without modification. Specific rotation ([a] D²⁵) and UV were measured by JASCO DIP-370 polarimeter (JASCO, Japan) and Shimadzu UV-1601 spectrophotometer (Shimadzu, Japan) . Every NMR test was carried out by Bruker DMX 600 NMR spectrophotometer (Bruker, USA) at CDCl₃. ESIMS and HRFABMS were performed with Finnigan LCQ Advantage Max mass spectrophotometer (Thermo, USA) and JEOL JMS- HX110A/HX110A Tandem mass spectrophotometer (Jeol, Japan) . HPLC was performed using Waters Delta Prep 3000 system (Waters, USA) . The identified compounds are as follows (Fig. 10) .

Geldanamycin; ¹H and ¹³C NMR data, Table 5 and Table 6; ESIMS m/z 561 [M + H]⁺, 559 [M - H]^"

Compound 1; ¹H and ¹³C NMR data, Table 5 and Table 6. Compound 2; ¹H and ¹³C NMR data, Table 5 and Table 6; ESIMS m/z 501 [M - H]^" 503 [M+H]⁺ HRFABMS m/z 503.31156, C₂₈H₄₃O₆N₂H calculation value, 503.31156.

Compound 3; ¹H and ¹³C NMR data, Table 5 and Table 6; ESIMS m/z 517 [M- H]^" 519 [M+H]⁺. HRFABMS m/z 519.30664, C₂₈H₄₃O₇N₂H calculation value, 519.30648.

[Table 5]

¹H NMR spectrums of compounds 1, 2, and 3 were compared with that of geldanamycin.

[Table 6]

C -13 NMR spectrums of compounds 1, 2, and 3 were compared with that of geldanamycin.

Hereinafter, manufacturing examples for the present invention is described, but these examples cannot limit the spirit and scope of the present invention.

Manufacturing Example 1: Preparation of pharmaceutical formulations

<!-!> Preparation of powders Ansamycin derivative 2 g

Lactose i g Powders were prepared by mixing all the above components and filled airtight bags with them.

<l-2> Preparation of tablets Ansamycin derivative 100 mg

Corn starch 100 rag

Lactose 100 mg

Magnesium stearate 2 nig

Tablets were prepared by mixing all the above components by the conventional method for preparing tablets.

<l-3> Preparation of capsules

Ansamycin derivative 100 rag Corn starch 100 rag

Lactose 100 mg

Magnesium stearate 2 rag

Capsules were prepared by mixing all the above components and filled gelatin capsules with them according to the conventional method for capsules.

<l-4> Preparation of pills

Ansamycin derivative 1 g

Lactose 1.5 g Glycerin 1 g Xylitol 0.5 g

Pills were prepared by mixing all the above components by the conventional method for preparing pills (4 g/pill) .

<l-5> Preparation of granules

Ansamycin derivative 150 mg

Soybean extract 50 nig

Glucose 200 rag Starch 600 mg The above components were mixed and 30% ethanol was added thereto. The mixture was dried to make granules and filled bags with them.

<l-6> Preparation of injectable solutions

Ansamycin derivative 10 βg/\ut

Weak HCl BP added until pH reached 3.5

Injectable NaCl BP upto 1 ml

Ansamycin derivatives were dissolved in injectable NaCl BP and pH of the solution was adjusted to 3.5 by using weak HCl BP. The volume was regulated by using injectable NaCl BP and the solution was mixed well enough. Transparent type I ampoules (5 m#) were filled with the solution. The ampoules were sealed by melting the necks of the glass ampoules. The ampoules were sterilized by autoclave for at least 15 minutes at 120^°C.

[industrial Applicability] As explained hereinbefore, the present invention provides a preparation method for geldanamycin derivatives from the mutant strain of Streptomyces hygroscopicus subsp. duamyceticus with transformation of geldanamycin biosynthesis gene therein. This preparation method enables the production of novel ansamycin derivatives by the addition of AHBA and its derivatives. Ansamycin derivatives prepared by the method of the invention has Hsp90 inhibitory activity which is as effective as that of geldanamycin, so that the derivatives can be effectively applied to antibiotics, antifungal agents, antiviral agents, immunosuppressants, degenerative nervous disease treatment agents, anti-inflammatory agents and anticancer agents

[Sequence List Text]

SEQ. ID. NO: 1 is the NK-I primer sequence.

SEQ. ID. NO: 2 is the NK-2 primer sequence.

SEQ. ID. NO: 3 is the NK-3 primer sequence. SEQ. ID. NO: 4 is the NK-4 primer sequence. SEQ. ID. NO: 5 is the NH-I primer sequence.

SEQ. ID. NO: 6 is the Neo-L primer sequence.

SEQ. ID. NO: 7 is the napK sequence.

SEQ. ID. NO: 8 is the ermE5 primer sequence. SEQ. ID. NO: 9 is the ermE3 primer sequence.

SEQ. ID. NO: 10 is the dTEl primer sequence.

SEQ. ID. NO: 11 is the dTE2 primer sequence.

SEQ. ID. NO: 12 is the pTEl primer sequence.

SEQ. ID. NO: 13 is the pTE2 primer sequence.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]

[Claim l]

A method for biosynthesis of ansamycin derivatives comprising the following steps: (1) Preparing a construct wherein 3-amino-5- hydroxylbenzoic acid (AHBA) biosynthesis gene is inactivated;

(2) Constructing a recombinant vector containing the construct of step (1); (3) Generating a recombinant mutant strain by transforming host cells with the recombinant vector of step (2) ; and

[Claim 2]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the following steps are additionally included after step

(3) :

(4) Constructing a recombinant expression vector containing thioesterase gene;

(5) Introducing the recombinant expression vector prepared in step (4) to the mutant strain generated in step (3) ; and

(6) Adding an AHBA analog to the transformed mutant strain in order to form ester bond and culturing thereof, and then obtaining ansamycin derivatives from the culture solution.

[Claim 3] The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the AHBA biosynthesis gene of step 1) is napK gene represented by SEQ. ID. NO: 7.

[Claim 4]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the AHBA biosynthesis gene of step 1) is the gene having at least 70% homology with the gene represented by SEQ. ID. NO: 7.

[Claim 5]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the napK gene knock-out construct of step 1) includes an antibiotic resistant gene.

[Claim β] The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the host cell of step 3) is Streptomyces hygroscopicus or Streptomyces melanosporofaciens.

[Claim 7]

The method for biosynthesis of ansamycin derivatives according to claim 6, wherein the Streptomyces hygroscopicus is selected from the group consisting of Streptomyces hygroscopicus var. geldanus, Streptomyces hygroscopicus subsp. duamyceticus and Streptomyces hygroscopicus 17997.

[Claim 8]

The method for biosynthesis of ansamycin derivatives according to claim 6, wherein the Streptomyces hygroscopicus geldanus is geldanus NRRL 3602.

[Claim 9] The method for biosynthesis of ansamycin derivatives according to claim 6, wherein the Streptomyces melanosporofaciens is Streptomyces melanosporofaciens EF-76.

[Claim 10]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the mutant strain of step 3) is Streptomyces hygroscopicus AC2 KCTC 10676BP.

[Claim 11]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the AHBA or its analog added to the culture of the mutant strain of step 4) has the structure represented by the following formula .

Ri= H, OH, NH₂, halogen or ally group, R2= H, OH, NH₂, halogen or ally group, R3= H, OH, NH₂, halogen or ally group, R₄= H, NH₂, halogen or ally group, Halogen = F, Cl, Br or I.

[Claim 12]

The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the concentration of the AHBA or its analog of step 4) is 0.1 mg/m£ ~ 20 mg/m£.

[Claim 13] The method for biosynthesis of ansamycin derivatives according to claim 1, wherein the ansamycin of step 4) is selected from the group consisting of geldanamycin, herbimycin, macbecin, reblastatin and ansamitocin.

[Claim 14] The method for biosynthesis of ansamycin derivatives according to claim 2, wherein the thioesterase is DEBS ( β-deoxyerythronolide B synthase) thioesterase or pikromycin thioesterase.

[Claim 15]

The method for biosynthesis of ansamycin derivatives according to claim 2, wherein the AHBA analog of step 6) is 3-hydroxy benzoic acid or 3,5- dihydroxy benzoic acid.

[Claim 16]

The method for biosynthesis of ansamycin derivatives according to claim 2, wherein the concentration of the AHBA analog of step 6) is 0.1

[Claim 17]

Ansamycin derivatives prepared by the method of claim 1.

[Claim 18 ]

Ansamycin derivatives represented by the following formula.

[Claim 19]

Ansamycin derivatives represented by the following formula.

R = H or OH; single bond or double bond between C₄ and C₅.

[Claim 20]

Ansamycin derivatives represented by the following formula.

[Claim 21]

A napK gene knock-out construct in which the napK gene represented by SEQ. ID. NO: 7 is inactivated by mutation.

[Claim 22]

The napK gene knock-out construct according to claim 21, wherein an antibiotic resistant gene is introduced.

[Claim 23]

A recombinant vector containing the napK gene knock-out construct of claim 21.

[Claim 24 ]

A recombinant Streptomyces hygroscopicus mutant strain prepared by transforming Streptomyces hygroscopicus subsp. duamyceticus with the vector of claim 23.

[Claim 25]

The Streptomyces hygriscopicus mutant strain according to claim 24, wherein the mutant strain is Streptomyces hygroscopicus AC2 KCTC 10676 BP.

[Claim 26]

A strain producing ansamycin derivatives having ester ring, prepared by transforming the mutant strain of claim 24 with the expression vector harboring thioesterase gene.

[Claim 27] The strain producing ansamycin derivatives according to claim 26, wherein the thioesterase is DEBS-thioesterase or picromycin thioesterase.

[Claim 28] An antibiotics, antifungal or anti-viral agent containing:

(a) ansamycin derivatives of claim 17;

(b) ansamycin derivatives of claim 18 or claim 19;

(c) ansamycin derivatives of claim 20;

(d) recombinant vector of claim 23;

(e) mutant strain or its culture medium of claim 24; or (f) ansamycin derivatives produced by the Streptomyces hygriscopicus mutant strain of claim 24;

(g) ansamycin derivative producing strain of claim 26 or its culture medium; or

(h) ansamycin derivatives produced by the ansamycin derivative producing strain of claim 26, as an effective ingredient.

[Claim 29]

An immunosuppressant or anti-inflammatory agent containing one of a) , b) , c) , d) , e) , f) , g) and h) of claim 28 as an effective ingredient.

[Claim 30] A degenerative nervous disease treatment agent containing one of a) , b) , c) , d) , e) , f) , g) and h) of claim 28 as an effective ingredient.

[Claim 3l]

An anticancer agent containing one of a) , b) , c) , d) , e) , f) , g) and h) of claim 28 as an effective ingredient .