JP2002533067A - Micromonospora echinospora gene encoding calicheamicin biosynthesis and self-resistance to calicheamicin - Google Patents

Abstract

  (57) [Summary] A gene cluster isolated from Micromonospora echinospora that encodes calicheamicin biosynthesis. This biosynthetic gene cluster contains genes encoding proteins and enzymes used in the biosynthesis of calicheamicin, including aryl tetrasaccharides and aglycones. This gene cluster also contains genes that confer calicheamicin resistance. The present invention also provides a group of genes isolated from the biosynthetic cluster and proteins corresponding to those genes. Furthermore, the present invention also relates to a DNA that hybridizes to the calicheamicin gene cluster and genes isolated from this cluster. An expression vector comprising a gene of the biosynthetic gene cluster and a functional variant thereof is also provided. The invention also relates to host cells which mate with DNA isolated from the genome of Micromonospora echinospora sp.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to the field of Micromonospora echinospora.
It relates to a biosynthetic gene cluster of the species Calichensis. Among them, the gene cluster that biosynthesizes calicheamicin consists of genes encoding proteins and enzymes used to form aryl tetrasaccharides and aglycones of calicheamicin in the biosynthetic pathway, and genes that confer calicheamicin resistance. Contains. The invention also relates to genes isolated from the biosynthetic gene cluster and their corresponding proteins. Furthermore, the present invention provides a calicheamicin gene cluster and DNs that hybridize with genes isolated from the cluster.
Regarding A. The present invention also relates to an expression vector comprising a biosynthetic gene cluster, individual genes, or functional variants thereof.

BACKGROUND OF THE INVENTION [0002] Energy antibiotics discovered in the 1980s have long been appreciated for their novel molecular structure, surprising biological activity, and attractive modes of action. Enewin antibiotics were originally obtained by fermenting microorganisms such as Micromonospora, Actinomadura, and Streptomyces. Rothstein, DM, "Enegine Antibiotics as Anticancer Agents", page 2, 1995. The enediyne antibiotics are considered to be the strongest and most active group of anticancer drugs discovered so far. Rothstein, DM, "Enegine Antibiotics as Anticancer Agents", Preface, 1995.

[0003] To date, at least twelve members of this family of antibiotics have been discovered, which fall into two broad categories. The first category of enediynes are classified as chromoprotein enediynes because of their novel 9-membered chromophore core structure. This core structure further requires special satellite proteins to stabilize the chromophore. The second category of enediynes is classified as non-chromoprotein enediynes. This engin contains a 10-membered ring, but no additional stabilizing factors are required.

This cyclic structure of enediyne is often referred to as a “warhead”. This warhead causes DNA damage. The damage is often a double-strand break and appears irreparable. This type of DNA damage is usually not repairable by the cell and is often fatal. Because of these amazing chemical and biological properties, both the pharmaceutical and academia have been eager to study these substances with the aim of developing new anticancer drugs that could be used in therapy.

A subfamily of the nine-membered chromoprotein enediynes is the neocarzinostatin from Streptomyces carzinostanicus (Myers, AG et al., J. Am. Chem. Soc., Vol. 110, Pages 7212-7214,
1988); Kedarcidin from actinomycetes L585-6 (REIT, JE et al., J. Am. Chem. S.
oc., 114, 7946-7948, 1992), N1999A2 from Streptomyces globisporus (Yoshida, K. et al., Tetrahedron L.
ett., 34, pp. 2637-2640, 1993), Actino Madura Madurea (
Acturomadura madurea) from Maduropeptin (Schroeder, DR et al., J.A.
m. Chem. Soc., 116, 9351-9352, 1994);

[0006] N1999A2 from Streptomyces sp. AJ9493 (Schroeder, DR et al., J. Am. Ch.
em. Soc., 116, 9351-9352, 1994); Actinoxanthin from Actinomyces globisporus (Koklov, AS
Et al., J. Antibiot., XXII, pp. 541-544, 1969); largomycin from Streptomyces pluricolorescens (Yamaguchi, T. et al., J. Antibiot., XXIII). Vol. 369-372, 1970);
Streptomyces macromomyceticus
Auromomycin from Yamashita, T. et al., J. Antibiot., XXXII, 330-33
9 pages, 1979),

[0007] Streptosporangium pseudovulg
sporamycin (Komiyama, K. et al., J. Antibiot., XXX, 202-2).
08 pages, 1977). All are believed to have a novel bicyclo [7.3.0] dodecadininene chromophore core structure that is essential for biological activity. Furthermore, except for N1999A2, the required apoproteins serve as stabilizers for unstable chromophores and as specific carriers for transporting unstable chromophores to interact with target DNA. Works.

[0008] The subfamily of non-chromophore enediynes is calicheamicin from Micromonospora echinospora sp. Calikensis; polycincraton lysostratum (
Polysincraton lithostrotum); esperamycin from Actinomadura verrucosospora; dynemycin from Micromonospora chersina.

[0009] The enediyne antibiotics can be anticancer drugs because of their ability to cut DNA, but many are too toxic to use at present in clinical trials.
To date, only calicheamicin has been used in clinical trials, with promising results as an anticancer drug. Enegine may be used as an antiinfective if it can control toxicity.

[0010] Because of the toxicity of enediyne compounds such as calicheamicin, it is important that these compounds cleave only the DNA of interest, for example, the DNA of cancer cells, but not the DNA of the host. The biggest problem. Because calicheamicin has a strong ability to cut DNA, researchers suggest that calicheamicin-producing organisms may
AWe continue to study the mechanisms that protect ourselves from cutting forces. Rothstein,
DM, "Enegine Antibiotics as Anticancer Agents", p. 77, 1995. Until the present invention, nothing was known about the gene encoding the non-chromophore enediyne self-resistance.

[0011] Understanding how micromonospora self-resistance genes and gene products regulate the toxic effects of calicheamicin opens new avenues for clinical research.
For example, understanding the mechanisms behind calicheamicin resistance provides the means needed to use calicheamicin at higher doses while reducing the toxic effects of calicheamicin on non-cancer cells Will be able to do so. Furthermore, understanding the mechanisms behind calicheamicin self-resistance can help to understand the self-resistance of other enediyne antibiotics, and consequently may be too toxic for therapeutic use. These previously conceived enediyne antibiotics could be useful.

According to the calicheamicin self-resistance mechanism revealed by the present invention,
For example, gene therapy can be achieved in which an enediyne resistance gene is introduced into bone marrow cells to increase the tolerance to calicheamicin dose in chemotherapy and to make the dose tolerant to the dose. Banaghery, D. et al., Stem Cells,
Volume 12, pages 378-385, 1994. Thus, understanding calicheamicin self-resistance would be of great use in continuing clinical research on calicheamicin and enegine. The present invention has been made in consideration of this requirement. According to the present invention, it is possible to isolate and characterize a resistance gene of any non-pigment protein, enedyne, and a protein corresponding thereto.

Calicheamicin has two distinct structural domains. That is, aryl tetrasaccharides and aglycones (also known as warheads). Aryl tetrasaccharides are substances in which glycosides, thioesters, and hydroxylamines are extremely unusually linked. Drugs are used to bind drugs to specific regions (5'-TCCT-3 'and 5'-TTTT-3') in the small groove of DNA. ) To help deliver. The calicheamicin aglycone consists of a highly activated bicyclo [7.3.1] tridecadininene core structure with allyl trisulfide acting as a trigger. McGullen, WJ, et al., "Enegine Antibiotics as Anticancer Agents"
75-86, 1995.

When the aryltetrasaccharide is firmly fixed, the site-specific oxidized form of the DNA of interest is aromatized by the 1,4-dehydrobenzene-diradical of the bicyclo [7.3.1] tricadienene core structure. Double-strand breaks occur. Zain, N. et al., Science,
Volume 240, pp. 1198-1201, 1988. The aglycone reacts to generate a carbon-centered diradical. This diradical cuts DNA. The great interest of the pharmaceutical industry in this activity has led to the recent success of calicheamicin-antibody conjugate (CMA-676) in phase III clinical trials in the treatment of acute myeloid leukemia (AML). In addition, a similar strategy has been used in Phase 1 clinical trials for breast cancer treatment.

[0015] A large-scale project to study calicheamicin combined with another delivery system has just begun. Haman, PR et al., The 87th Annual Meeting of the American Cancer Research Society, Washington DC, p. 471, 1996; Hinman, LM et al., Cancer Re.
53, 3336, 1993; Hinman, LM, et al., "Enegine Antibiotics as Anticancer Agents", pp. 87-105, 1995; Seavers, EL, et al., Blood, ed.
93, 3678-3684, 1999; Siegel, MM et al., Anal. Chem., 69, 2
716-2726, 1997; Erestad, G., personal communication.

[0016] The elucidation of the biological activity and molecular structure of calicheamicin has led to active attempts to search for potentially useful analogs. One of many laboratories synthesizing analogs suggests a novel calicheamicin θ ^I 1 that effectively suppresses the growth and systemic dissemination of liver metastases in the same genotype model of murine neuroblastoma Was synthesized. Loud, HN et al., Cancer Res., 58, 295-22928, 1998; Urashiduro, W. et al., Acta Oncologica, 34, 157-164, 1995. In addition to synthesizing calicheamicin analogs, studies have been conducted to screen for mutant strains that induce random mutations in Micromonospora echinospora and have improved biosynthetic ability. Rothstein, DM, "Enegine Antibiotics as Anticancer Agents", pp. 107-126, 1995.

The complete synthesis of calicheamicin was first reported in 1992 by Nicolau et al. However, the synthesis of this complex antibiotic has many problems. For example, the Nacelle method also requires 47 steps to achieve a yield of only about 0.007%.
Halcom, RL, "Enegine Antibiotics as Anticancer Agents", pp. 383-439,
1995. Therefore, isolation of calicheamicin from a large-scale fermentation product of Micromonospora echinospora is still mainstream at present, rather than complete synthesis of calicheamicin. Therefore, there is still a need for methods of producing calicheamicin and potentially useful calicheamicin variants in large quantities. Fantini, A. et al., "Enegine Antibiotics as Anticancer Agents", pp. 29-48, 1995.

[0018] Calicheamicin-producing bacterial strains, such as calicheamicin in E. coli
Transformation and introduction of DNA may be able to meet this need. At present, the genes of Micromonospora echinospora have not been cloned, and only a limited series of studies on putative promoters of Micromonospora echinospora are known. Lin, LS et al., J. G
En. Microbiol., 138: 1881-1885, 1992; Lynn, LS et al., J. Bact.
eriol., Vol. 174, pp. 3111-3117, 1992; Baum, EZ et al., J. Bacteriol.
171: 6503-6510, 1989; Baum, EZ et al., J. Bacteriol., 17
Volume 0, pages 71-77, 1988.

[0019] Since calicheamicin DNA was obtained, the calicheamicin biosynthesis could be analyzed from the gene aspect. Because such analysis requires the ability to obtain high quality calicheamicin DNA. For example, the biosynthesis of calicheamicin can be studied by mutagenesis of Micromonospora echinospora. Specific examples include isolation and characterization of mutants that block calicheamicin biosynthesis, followed by analysis of defective or partially calicheamicin products. Research.

Furthermore, after subcloning the gene and placing it in a host such as Escherichia coli, a specific enzyme is over- or under-expressed, and the function of the enzyme is revealed by studying the result of the over-expression. I can do it. In addition, if the biosynthetic gene can be cloned, the biosynthetic gene encoding the rate-limiting enzyme is cloned, and this gene product is returned to the organism that produces the corresponding gene product and expressed. Can ultimately be increased. By cloning the biosynthetic gene into a strain that produces the relevant compound, a new product could be produced. Such a gene could provide the host organism with the ability to cause a new reaction in the enediyne nucleus, thus allowing the host organism to produce new drugs.

Calicheamicin has attracted attention as a target for research on the biosynthesis of natural products because of its useful biological activity due to its special molecular structure and its potential as a therapeutic agent. Radical-based mechanism of oxidative DNA cleavage by calicheamicin (ie, site-specific oxidative cleavage as a result of aromatization of bicyclo [7.3.1] tridecadininene core structure by 1,4-dehydrobenzene-diradical Although double-stranded DNA breaks occur), it was not known before the present invention how micromonospora produced calicheamicin. Therefore, it is necessary to clarify and understand the biosynthesis of calicheamicin.

Prior to this discovery, nothing was known about the genes encoding the biosynthesis of the non-pigment protein enegine. Thus, the present invention relates to the world's first work on the identification, isolation, and cloning of a non-chromoprotein enediyne biosynthesis gene cluster, and mapping of genes in this cluster and analysis of nucleic acid sequences. The present invention provides the entire calicheamicin biosynthesis cluster and the results of biochemical studies on the biosynthesis of aryl tetrasaccharides.

In addition, the calicheamicin self-resistance gene and its corresponding protein have been isolated in the same manner as the genes required for the steps in the calicheamicin cascade and the corresponding enzymes. According to the present invention, a method for constructing an enediyne overproducing strain,
Also provided are methods for rationally modifying bioactive secondary metabolites by biosynthesis, clues to new drugs, and combinatorial biosynthesis programs for enediyne.

Accordingly, the present invention also relates to a method of modifying a biologically active secondary metabolite through combinatorial biosynthesis of enediyne. Drug clues are often based on inspiration from natural compounds, and the challenge of synthesizing this metabolite raises the possibility of creating new drugs by genetically manipulating sugar adducts on this metabolite . As such, the emerging field of combinatorial biosynthesis represents a rich new source of modified sugar scaffolds that do not exist in nature. Marsden, A. et al., Scienc
e, Volume 279, pp. 199-201, 1998.

A problem inherent in the engineering of sugar adducts is that natural biologically active secondary metabolites have unusual carbohydrate ligands, which serve as essential molecular recognition elements for biological activity. Has to do with the fact that "The Chemistry, Biology, and Practical Use of Macrolide Antibiotics", 1984. In the absence of these crucial sugar adducts, most clinically important secondary metabolites will have a complete loss of biological activity or a sharp decline. At present, genetic engineering techniques for sugar adducts of a given metabolite focus on altering and / or deleting the small number of genes necessary to make up and add each desired sugar moiety. It has become.

[0026] Therefore, there is a need to develop alternative strategies to construct and add sugars that do not exist in nature. The present invention addresses this need. The present invention takes advantage of the fact that glycosyltransferases that ultimately glycosylate certain secondary metabolites are more prone to indiscriminately mixing with nucleotide sugar donors. Cao, L. et al., J. Am. Chem. Soc., 120, 12159-12160, 1988.

[0027] The lack of selectivity of glycosyltransferases can alter the pattern of glycosylation, which is crucial in glycosylating natural or unnatural secondary metabolite scaffolds, in a combinatorial manner. . The present invention discloses a method for constructing a complex gene cluster for synthesizing and adding sugars that do not exist in nature by utilizing the reinforcing and cooperative functions of sugar genes from various biochemical pathways.

SUMMARY OF THE INVENTION According to the present invention, a micromonospora gene encoding a gene derived from the non-pigment protein enegine biosynthesis gene cluster, or a protein coding region of this gene, or a biologically active fragment of this gene. Provided is a nucleic acid molecule isolated from Echinospora. In particular, the present invention provides an isolated nucleic acid molecule, gene or gene cluster involved in the biosynthesis of calicheamicin, derived from Micromonospora echinospora sp. Calikensis. According to another embodiment, the present invention relates to a nucleic acid molecule capable of hybridizing with a nucleic acid from Micromonospora echinospora sp. Calikensis encoding one or more genes from the non-chromoprotein enegine biosynthesis gene cluster. Related to

In yet another embodiment, the present invention provides an expression vector comprising a nucleic acid molecule isolated from the non-pigment protein enegine biosynthetic gene cluster from Micromonospora echinospora sp. Calikensis. In yet another embodiment, the present invention provides a cosmid comprising an isolated nucleic acid molecule comprising a nucleic acid sequence from Micromonospora echinospora sp. Calikensis that encodes a non-pigmented protein enediyne biosynthesis gene cluster. . In a preferred embodiment, the present invention provides an isolated nucleic acid molecule of SEQ ID NO: 1, 3, 5.

In yet another embodiment, the invention provides a host cell transformed with a nucleic acid molecule isolated from the non-pigment protein enegine biosynthesis gene cluster from Micromonospora echinospora sp. . Host cells can be cells from bacteria, yeast, insects, plants, fungi, or mammals, and the cells can be transformed according to standard methods. In a preferred embodiment, the host cell is a bacterium, such as E. coli or Streptomyces.
In yet another embodiment, the invention provides that an expression vector encoding the gene calC is used, or is operably linked to a regulatory sequence that is a functional derivative of the gene that allows expression of the gene. A host cell transformed with an expression vector encoding the derivative is provided.

[0031] In yet another embodiment, the invention provides that an expression vector encoding the gene calH is used, or operably linked to a regulatory sequence that is a functional derivative of the gene and allows the expression of the gene. Provided is a host cell transformed with an expression vector encoding the derivative. Similarly, according to the present invention, the gene calG
Or a host transformed with an expression vector encoding a derivative which is a functional derivative of this gene and is operably linked to a regulatory sequence enabling expression of this gene A cell is provided.

Further according to the present invention, a method for expressing a protein includes using an expression vector comprising a nucleic acid molecule isolated from Micromonospora echinospora, encoding a gene from the non-pigment protein enediyne biosynthesis gene cluster. The present invention provides a method of culturing a host cell transformed by using the method, and culturing the host cell under a time and under conditions capable of expressing a protein. According to another embodiment, the present invention provides a method for purifying calicheamicin using affinity chromatography. For the affinity matrix containing the protein with CalC attached,
The calicheamicin-containing sample is contacted with the calicheamicin for a time and under conditions that allow the calicheamicin to bind to the matrix, and the calicheamicin is eluted from the matrix and the calicheamicin is recovered.

According to yet another embodiment, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NOs: 2, 4, and 6. According to yet another embodiment, the present invention produces the following two novel macrolides:

[0034]

Embedded image

[0035]

Embedded image Furthermore, according to the present invention, there is provided a method for imparting calicheamicin resistance to a test subject, wherein cells are collected from the test subject, and the cells are transformed with a calicheamicin self-resistance gene. A method is provided that includes returning to a subject. In addition, attention can be paid to the calicheamicin self-resistance gene, and the calicheamicin self-resistance gene can be delivered to a desired host cell through a known gene therapy delivery system.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the isolation and characterization of a calicheamicin biosynthesis cluster. This cluster encodes deoxysugar synthesis (aryl tetrasaccharide), polyketide biosynthesis to synthesize calicheamicin (aglycone), and genes encoding proteins and enzymes involved in calicheamicin resistance. ing. Twenty-one structural genes (about 20 kb) encoding sugar ligands for aryl tetrasaccharides have been identified, and about eight modules (about 40 kb) are required for the 15-carbon aglycone.
4 proteins involved in transport and uptake, 1 protein that confers resistance, 1
Two regulatory proteins have been identified.

The calicheamicin biosynthesis gene cluster contains the following genes. That is, calA, calB, calC, calD, calE, calF, calG, calH, calI, calJ, calK
, CalL, calM, calN, calO, calP, calQ, calR, calS, calT, orf1, orf2, orf3
, Orf4, orf5, orf6, orf7, IS-factor gene. These genes encode the following polypeptides.

That is, CalA (328 amino acids), CalB (561 amino acids), CalC (181 amino acids), CalD (263 amino acids), CalE (420 amino acids), CalF (245 amino acids), CalG
(990 amino acids), CalH (338 amino acids), CalI (568 amino acids), CalJ (332 amino acids), CalK (440 amino acids), CalL (562 amino acids), CalM (416 amino acids), C
alN (398 amino acids), CalO (331 amino acids), CalP (about 179 amino acids), CalQ (45 amino acids)
3 amino acids), CalR (265 amino acids), CalS (1113 amino acids), CalT (280 amino acids), Orf1 (322 amino acids), Orf2 (654 amino acids), Orf3 (209 amino acids), Orf
4 (521 amino acids), Orf5 (175 amino acids), Orf6 (139 amino acids), Orf7 (187 amino acids) and IS-factor (402 amino acids).

To elucidate the calicheamicin biosynthesis gene cluster, the inventors
We started with a genomic library containing the genome of Micromonospora echinospora sp. According to methods well known in the art, chromosomal DNA of Micromonospora echinospora species calikensis was isolated, the chromosomal DNA was fragmented, and the DNA was inserted into a cosmid vector to create a cosmid library. This method can be applied to any kind of micromonospora.

Based on studies of the metabolic labeling of enediyne, it was postulated that the aglycone of calicheamicin would be derived from polyketide. Polyketide metabolites can take on a variety of structures, but still have a common biosynthetic mechanism.
Hutchinson, CR et al., Chem. Rev., Vol. 97, pp. 2525-2536, 1997; Strol, WR et al., "Biotechnology of Antibiotics", pp. 577-657;
Chem. Rev., 97, pp. 2511-2523, 1997; Hopwood, DA.
Chem. Rev., Vol. 97, pp. 2465-2497, 1997; Hopwood, DA et al.,
Ann. Rev. Genet., 24, pp. 37-66, 1990; Staunton, J. et al., Chem.
Rev., Vol. 97, pages 261-2629, 1997.

Of great importance, the polyketide synthase ("PKS") gene (interpathway,
Often, the sequences are highly homologous (between organisms) and form clusters with genes encoding self-resistance and genes encoding biosynthesis of deoxysugar ligands. Hopwood, DA et al., Chem. Rev., Vol. 97, pp. 2465-2497, 1997; Hopwood, DA et al., Ann. Rev. Genet., Vol. 24, pp. 37-66, 1990; Staunton, J. et al., Chem. Rev., 97, 261-2629, 1997.
Year.

Using a degenerate primer based on a conserved region within the PKS gene in Southern hybridization, clones containing genes thought to be the PKS gene were identified from the genomic library of Micromonospora echinospora. Southern hybridization was performed using methods known in the art. By performing Southern hybridization on a genomic library of Micromonospora echinospora cosmid using a DNA probe designed to target the type I PKS gene (KS ^I ) (Kakavas, SJ et al., J. Am.
Bacteriol., Vol. 179, pp. 7515-7525, 1997), showing a positive response 5
Two clones were revealed.

They were named 4b, 10a, 13a, 56, 60. See FIG. Type II DNA
Re-screening of the genomic library with the probe (actI) identified the same five clones. See FIG. Although this preliminary analysis clarified the presence of a homologue in the PKS gene of Micromonospora, PKS hybridization analysis could incorrectly hybridize with the gene cluster encoding spore pigment biosynthesis. Because of this, it was often a problem, so a second screening was performed.

The second round of screening was based on the assumption that the calicheamicin biosynthetic cluster would also contain a gene encoding the synthesis of a deoxysugar ligand. In addition, the synthesis of the macromolecule sugar begins in many organisms with similar common intermediates, so that all hexopyranosyl and
The ligand was assumed to have diverged from the common 4-keto-6-deoxy TDP-D-glucose (30) (FIG. 5). Therefore, the cluster encoding calicheamicin biosynthesis contains both the common glucose-1-phosphate nucleotidyltransferase gene and the NDP-α-D-glucose 4,6-dehydratase gene in addition to the PKS coding region. I thought it must have been included. The former is an enzyme E _p, it believed latter encodes the enzyme E _od. See FIG.

These enzymes convert sugar (12) (FIG. 5) into a common virtual intermediate, 4-keto-6-deoxy.
Required to convert to TDP-D-glucose (30). Characterization of 4,6-dehydratase analogs has previously been performed on Escherichia coli, Salmonella, and Streptomyces. In addition, nucleotides from Salmonella
As a result of the characterization, the transferase was found to be α-D-glucose-1-thymidylyl phosphate transferase.

Assuming that the calicheamicin synthesis of Micromonospora echinospora starts from a precursor similar to that found in E. coli, Streptomyces, Salmonella,
In addition, a second screening was performed using a probe that assumed that dehydratase was required to convert this precursor to the common intermediate, 4-keto-6-deoxy TDP-D-glucose (30). Done. In particular, a DNA probe ( _{designated Eod} ^I ) designed based on the conserved NAD ^- binding site of bacterial NDP-α-D-glucose 4,6-dehydratase was used. He, X. et al., Biochem. 35: 4727-14731, 1996.
Year.

The genome of Micromonospora, Ekinosupora cosmid by using the E _od ^I probe
Performing Southern hybridization on the library revealed cross-hybridization with clones 4b, 10a, 13a, 56, 60. Two additional clones (named 58, 66) were also identified by this screen. See FIG. With this second screening,
The genes encoding both polyketide and deoxysugar biosynthesis were found to be clustered.

Since the biosynthetic genes of the secondary metabolites generally form clusters with the resistance gene of actinomycetes, various clones that showed a positive reaction in hybridization were subjected to various tests for final confirmation. The ability to grow in the presence of concentrations of calicheamicin was tested. In this final screen, six of the seven hybridized clones showed different levels of calicheamicin resistance (4b ≒ 10a ≒ 13a ≧ 56 ≧ 66> 60) (see FIG. 1). In contrast, clone 58 had no ability to grow in the presence of calicheamicin.

In addition, this resistance screen revealed that clones 4b, 10a, 13a confer much higher calicheamicin resistance than the other clones. By rescreening the genomic library for calicheamicin resistant clones, three additional clones (3a, 4a, 16a) were found to confer similar levels of resistance. Considering these results together, clones 4b, 10a, 13a, 56, and 60 contain PKS I homologs, PKS II homologs, deoxyglycosylation biosynthesis genes, and genes that cause calicheamicin self-resistance. I knew I was coding.

Biosynthetic clusters were mapped using clones that showed a positive response to PKS I, PKS II, homologues of the deoxysaccharide biosynthesis gene and the calicheamicin resistance gene. By Southern hybridization, clones 3a, 4a, 4b, 10
It turned out that there was similarity between a, 13a, 16a and 56. In addition, clones 4b, 13a
, 56 were found to have overlapping nucleotide sequences. See FIG.

The construction of restriction maps and Southern hybridization of these clones suggest that the cosmid clones that showed a positive response corresponded to a continuous region extending beyond 100 kb of the Micromonospora echinospora chromosome. Was done.
Thus, the present invention provides a cosmid having a nucleic acid molecule derived from Micromonospora echinospora, which encodes a non-pigment protein enediyne biosynthesis cluster.

After isolating and sequencing the biosynthetic gene cluster, the open reading frame ("orf") was identified. Translated amino acid sequence of orf
By directly searching the database using BLAST (Basic Local Alignment Search Tool), you can search the amino acid sequences of gene products whose functions are known for those similar to the translated orf amino acid sequence. We searched and determined a tentative gene. Karlin et al., Proc. Natl. Acad. Sci. USA, 87, pp. 2264-2268, 1990.
Natl. Acad. Sci. USA, 90, 5873-5877, 19
93; Altsur, Nature Genet., Vol. 6, pp. 119-129, 1994. The structure of the gene cluster is shown in FIG.

A temporary gene was determined based on the analysis result using BLAST. The genes involved in aryl tetrasaccharide composition include: a) genes encoding sugar nucleotide biosynthesis (calG, H, K, O, Q, S); b) genes encoding aryl tetrasaccharides. Gene (calE, N); c) gene (c) that encodes a “tailoring” reaction
alD, F, J).

One aspect of the present invention relates to transforming host cells with DNA from Micromonospora echinospora. By this method, it is possible repeatedly to achieve a transformation efficiency of DNA per about ¹⁰³ kanamycin-resistant transformants 1μg using a vector based pKC1139. Further, according to the present invention, the host cells can be, but are not limited to, bacterial, yeast, fungal, insect, plant, or mammalian cells. Transformation of bacterial, yeast, fungal, insect, plant, or mammalian cells employs methods known in the art.

According to the present invention, isolation and characterization of a gene encoding calicheamicin resistance are realized. One aspect of the invention relates to an isolated DNA strand comprising the gene calC and having the DNA sequence of SEQ ID NO: 1. The present invention also relates to an isolated protein CalC having the amino acid sequence of SEQ ID NO: 2. The present invention also provides a CalC gene fragment encoding a biologically active CalC. CalC polypeptide is 181
And confers calicheamicin resistance. The invention also provides a CalC fragment that confers calicheamicin resistance.

The calC locus was isolated by identifying a calicheamicin genomic cosmid clone that was able to grow on Luria Bertani (“LB”) agar plates containing ampicillin and calicheamicin. Isolation of DNA from clones that showed a positive response (clones grown on plates containing calicheamicin) followed by restriction mapping yielded the desired phenotype (calicheamicin resistance).
The position was determined. Next, this DNA was sequenced and the open reading frame was analyzed to confirm the orf encoding the desired phenotype.
In vitro studies have confirmed that CalC has the ability to suppress DNA cleavage.

When DNA containing calC was cloned and introduced into an induction vector by a known method, overexpression of calC occurred. The polypeptide product (CalC) was then isolated and purified to homogeneity. Analysis of the purified CalC revealed that it was a non-heme iron metalloprotein. This CalC has a function of suppressing calicheamicin-induced DNA cleavage in vitro. Another aspect of the invention is a cal
An expression vector containing C or an expression vector containing a fragment of calC encoding a bioactive molecule. Also provided is a transformed host cell comprising calC or a fragment of calC encoding a bioactive molecule. Preferably, the host cell is a bacterium, more preferably E. coli.

The present invention provides a method for transforming human cells using the calC gene. This method allows, for example, harvesting bone marrow cells from a patient being treated with calicheamicin, transforming the cells with calC, and returning the transformed cells to the patient. Because human cells transformed with calC and returned to the patient are calicheamicin-resistant, this method allows patients to tolerate treatment with calicheamicin and allows patients to use larger doses of calicheamicin. Be able to accept keamycin. Transformation is performed by methods known in the art. This embodiment of the invention may be applicable to many diseases that are treated with caremycin.

Another aspect of the present invention relates to an isolated DNA strand comprising the calH gene having the DNA sequence of SEQ ID NO: 3. The present invention also relates to a CalH polypeptide having the amino acid sequence of SEQ ID NO: 4. Further, according to the present invention, there is also provided a calH gene fragment encoding a biologically active CalH. CalH is involved in the formation of the aryltetrasaccharide 4,6-dideoxy-4-hydroxylamino-D-glucose moiety. CalH converts intermediate (30) to intermediate (3
It functions as a catalyst for conversion to 9) (Fig. 5).

CalH is TDP-6-deoxy-D-glycerol-L-threo-4-hexulose 4-transaminase, which converts glutamate to amino-6-deoxy by transpyridoxal phosphate (“PLP”)-dependent transamination. It functions as a catalyst to make TDP-D glucose (intermediate 39) (Figure 5). The present invention also provides a CalH fragment that retains biological activity. An expression vector comprising the calH gene or an expression vector comprising a calH gene fragment encoding a biologically active polypeptide is also provided. CalH was overexpressed as a (histidine) 10 fusion protein and was purified by nickel affinity chromatography.

According to an analysis using BLAST, calH was very similar to perosamine synthase. Perosamine synthase is used in Escherichia coli to express TDP-perosamine (TDP-
During the biosynthesis of 4,6-dideoxy-4-amino-D-mannose), compound 30 was converted to compound 39.
It is an enzyme that converts to One, L. et al., Infect. Immunol. 66: 3455-3551, 1998. Therefore, we were convinced that CalH was 4-ketohexose aminotransferase. Combinatorial biosynthesis was performed to confirm the functions tentatively determined by BLAST.

Specifically, the calH gene from calicheamicin was integrated into a mutant strain of Streptomyces venezuelan. The 4-dehydrase gene (des1) in the methymycin / picromycin pathway was removed from this mutant. Streptomyces
A promoter sequence from the Methymycin / Picromycin cluster of Venezuela was incorporated into the expression vector to facilitate the expression of a foreign gene (calH of calicheamicin) in Streptomyces venezuela. It is known that wild-type Streptomyces venezuela produces methymycin, neomethymycin, picromycin, and narbomycin from the methymycin / picromycin pathway. See FIG.

Since the mutant des1 was removed, TDP-4-keto-6-deoxyglucose (Compound 30, FIG. 6), a substrate for CalH, accumulated. An expression vector constructed using a Streptomyces venezuelan promoter expressed the calH gene and produced CalH protein. CalH acted on substrate 30 to produce compound 39 (FIG. 6). This time compound 39 was subjected to the action of Streptomyces venezuelan DesVII (glycosyltransferase) to produce two methimicin / picromycin-calicheamicin hybrid compounds 40 and 41. See FIG.

These hybrid compounds include the calicheamicin 4-aminohexose ligand. This result indicates that the hypothesized calH gene
There is no doubt that it encodes TDP-6-deoxy-D-glycero-L-threo-4-hexulose 4-aminotransferase. CalH is TDP-4-keto
Acted on -deoxyglucose substrate (Compound 30) to produce Compound 39 (Figure 5)
.

Furthermore, these results indicate that the corresponding glycosyltransferase (DesVII)
Has non-specific properties. This is because the glycosyltransferase (DesVII) of the Streptomyces venezuelan pathway has been shown to be able to recognize another sugar substrate whose structure is significantly different from the original amino sugar substrate, TDP-D-desosamine. is there. These results also clearly show that by rational selection of gene combinations, glycosylation of secondary metabolites can be achieved by genetic engineering. The successful expression of the CalH protein in Streptomyces venezuela with the newly constructed expression vector strongly suggests that this system can be used to express other foreign genes in this strain. .

Thus, one aspect of the invention also relates to constructing a complex gene cluster having the ability to produce and add non-natural sugars. The present invention further provides an expression vector having a calicheamicin gene operably linked to a regulatory sequence for controlling the expression of a calicheamicin protein. Preferably, the regulatory sequence is a Streptomyces promoter. The present invention
It also relates to the two newly synthesized sugars 11 and 12 (Figure 7). Compound 11 has the following chemical formula:

[0067]

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The spectral data for compound 11 were as follows: ¹ H NMR (500 MHz, CDCl ₃ , J is expressed in Hertz) δ6.75 (1H, dd, J = 16.0, 5.5, 9
-H), 6.44 (1H, dd, J = 16.0, 1.2, 8-H), 5.34 (1H, d, J = 8.0, NH), 4.96
(1H, m, 11-H), 4.27 (1H, d, J = 7.5, 1-H), 3.66 (1H, dd, J = 9.5, 8.0, 4 '
-H), 3.60 (1H, d , J = 10.5,3-H), 3.50 (1H, l, J = 9.5,3'-H), 3. D (1H, m
, 5'-H), 3.4 (1H, m, 2'-H), 2.84 (1H, dq, J = 10.5, 7.5, 2-H), 2.64 (1H
, M, 10-H), 2.53 (1H, m, 6-H), 2.06 (3H, s, Me-C = 0), 1.7 (1H, m, 12-H)
), 1.66 (1H, m, 5-H), 1.56 (1H, m, 12-H), 1.4 (1H, M, 5-H), 1.36 (3H
, D, J = 7.5, 2-Me), 1.25 (311, D, J = 6.5, 5'-Me), 1.24 (1H, m, 4-H), 1.
21 (3H, d, J = 7.5, 6-Me), 1.10 (3H, d, J = 6.5, 10-Me), 0.99 (3H, d, J = 6.
0,4-Me), 0.91 (3H, t, J = 7.2, 12-Me); ¹³ C NMR (125 MHz, CDCl ₃ , J is expressed in Hertz) δ205.3 (C-7), 175.1 (C- 1), 171.9 (Me-CO), 147.1 (C-9), 1
26.1 (C-8), 103.0 (C-1 '), 85.8 (C-3), 75.8 (C-5'), 75.8 (C-3 '), 74.
1 (C-11), 70.8 (C-2 '), 57.6 (C-4'), 45.3 (C-6), 44.0 (C-2), 38.1 (C
-10), 34.2 (C-5), 33.6 (C-4), 25.4 (C-12), 23.7 (Me-CO), 18.1 (C-6)
'), 17.9 (6-Me), 17.6 (4-Me), 16.4 (2-Me), 10.5 (12-Me), 9.8 (10-Me
). The calculated high-resolution FAB-MS value for C ₂₅ H ₄₂ NO ₈ (M + H ⁺ ), 484.2910, is 484.2
It turned out to be 303.

Compound 12 has the following chemical formula:

Embedded image

The spectral data for compound 12 were as follows: ¹ H NMR (500 MHz, CDCl ₃ , J is expressed in Hertz) δ 6.69 (1 H, dd, J = 16.0, 6.0, 1
1-H), 6.09 (1H, dd, J = 16.0, 1.5, 10-H), 5.35 (1H, d, J = 8.5, NH), 4.9
6 (1H, m, 13-H), 4.36 (1H, d, J = 7.5, 1'-H), 4.19 (1H, m, 5-H), 3.83 (
1H-q, J = 6.5, 2-H), 3.68 (1H, dt, J = 10.0, 8.5, 4'-H), 3.52 (1H, t, J = 8.
5, 3'-H), 3.50 (1H, m, 5-H), 3.42 (1H, t, J = 7.5, 2'-H), 2.92 (1H, dq
, J = 7.0, 5.0, 4-H), 2.81 (1H, m, 8-H), 2.73 (1H, t, J = 7.5, 2'-H), 2.0
6 (3H, a, Me-CO), 1.8 (1H, m, 6-H), 1.6 (1H, m, 14-H), 1.55 (1H, m,
7-H), 1.37 (3H, d, J = 6.5, 2-Me), 1.32 (3H, d, J = 7.0, 4-Me), 1.3 (1H,
m, H-14), 1.27 (3H, d, J = 6.5, 5'-Me), 1.25 (1H, m, 7-H), 1.12 (3H, d
, J = 6.0, 8-Me), 1.11 (3H, d, J = 6.5, 12-Me), 1.07 (3H, d, J = 6.0, 6-Me)
, 0.91 (3H, l, J = 7.2,1-Me); C 28 H 46 NO 2 (M + H +) High Resolution F calculated for
AB-MS value 540.3172 was found to be 540.3203.

One aspect of the present invention relates to an isolated DNA strand comprising the calG gene and having the DNA sequence of SEQ ID NO: 5. Another aspect of the present invention is a protein CalG having the amino acid sequence of SEQ ID NO: 6. Based on analysis using BLAST, calG was estimated to encode 4,6-dehydratase. Dehydratase from E. coli, Salmonella, Streptomyces has been characterized (Thompson, M. et al., J. Gen. Microbiol., 138, 779-786, 1992; Vala, JA et al., J. Am. Biol. Chem., 263, 14992-14995, 1988).

The analog, NDP-D-glucose 4,6-dehydratase, has been characterized from various organisms. Liu, H.-w. et al., Ann
Rev. Microbiol. 48: 223-256, 1994; Harris, TM et al., Acc.
Chem. Res., Printing, 1999. Based on these findings to date, the overall transformation catalyzed by 4,6-dehydratase is that NAD- bound to the enzyme receives 4-H as a hybrid in an oxidative half-reaction, It was found to be an intramolecular oxidation-reduction in which an equivalent reducing substance was transferred to the dehydrated product C-6 in a reductive half-reaction.

Thus, it appears that CalG is required for the formation of the 4,6-dideoxy-4-hydroxylamino-D-glucose moiety of the aryl tetrasaccharide. CalG appears to be a TDP-D-glucose 4,6-dehydratase that functions as a catalyst to convert intermediate 13 to intermediate 30 (FIG. 5). Another aspect of the invention is an expression vector comprising calG, or an expression vector comprising a fragment of calG encoding a bioactive molecule. Also provided is a transformed host cell comprising calG or a fragment of calG that encodes a bioactive molecule. The host cell is preferably a bacterium, and more preferably Escherichia coli.

[0074] An isolated DNA strand containing the calS gene is also disclosed herein. Based on the fact that the sequence is homologous to other P450-oxidases, CalS is considered to be a homologue of P450-oxidase and oxidize intermediate 39 to intermediate 42 (
Figure 5). Oxidation can occur at the sugar nucleotide level or during hydroxylamine formation after the sugar has been transferred to the aglycone. An expression vector comprising calS or an expression vector comprising a fragment of calS encoding a biologically active molecule is also provided. Also provided is a transformed host cell comprising calS or a fragment of calS that encodes a bioactive molecule. The host cell is preferably a bacterium, and more preferably Escherichia coli.

According to the present invention, biosynthetic gene clusters can be genetically engineered to produce calicheamicin analogs. The present invention provides a method for producing an analog of calicheamicin by deleting or substituting a gene involved in the biosynthesis of an aryltetrasaccharide. The invention further provides an in vitro glycosylation method that produces additional analogs by altering the pattern of glycosylation of calicheamicin (using a glycosyltransferase). The present invention further provides a method of altering the calicheamicin aglycone by genetically modifying the gene encoding warhead biosynthesis. For example, a method known in the prior art is used to generate a deletion or substitution by genetic manipulation.

According to the present invention, there is provided a method for purifying calicheamicin by affinity chromatography. CalC functions as a calicheamicin sequestration / binding protein because it is homologous to calicheamicin. Affinity chromatography is performed using methods known in the art.

The present invention relates to a method for expressing a gene located in a biosynthetic gene cluster. This method uses a method known in the art to insert a gene into an appropriate expression vector, functionally associate the gene with a regulatory sequence to control the expression of the gene, and the inserted gene encodes To produce the protein you are doing. The present invention also provides a method for expressing a biologically active protein. In this method, a fragment of a gene group encoding a bioactive protein selected from a biosynthetic gene cluster is inserted into an appropriate expression vector using a method known in the art. . By functionally relating these genes to regulatory sequences, expression will be controlled.

Examples Example 1 For rapid nucleotide sequence determination, thermal cycle sequencing from pUC or pBluescript based subclones (using M13 primers and primer walking) and isolation (through primer walking) Cycle sequencing was also performed directly from the cosmids.

Nucleotide sequence data was acquired using two Applied Biosystems automated 310 gene analyzers and applied to Applied Biosystems Auto
Sequences were integrated using Assembler® DNA sequence integration software. Deer, S. et al., Nucl. Acids Res., 14, pp. 3907-3911, 1991; Juan,
X., Genomics, Vol. 14, pages 18-25, 1992. Computer program MacVector (
Orf assignment was performed using a combination of Brujene® and 6.0. MacVe
ctor is a commercially available software package.

Using MacVector, a codon usage table for micromonospora is constructed (from the known sequence of micromonospora) and then used to determine the optimal or
You can search for f. Ficket, JW, Nucl. Acids Res., Volume 10, 530
3-5318, 1982. The shareware program Brujene is designed specifically for Streptomyces bacteria and gives priority to orfs that show consistently high G / C% at the fluctuation position.

Embodiment 2 FIG. Isolation and Characterization of calC To isolate the gene responsible for calicheamicin resistance in micromonospora on LB plates containing ampicillin (50 μg / ml) and calicheamicin (0.25 μg / ml) A clone conferring calicheamicin resistance was selected by growing a bivalent cosmid library of the micromonospora genome in. In this selection, six clones (3a, 4a, 4b, 10a, 13a, 16a) showed calicheamicin resistance. When restriction maps of these clones were created, the desired phenotype was DNA
Was found to be located in a PstI-SacI fragment of about 2 kb (FIG. 2). The maximum allowable concentration of calicheamicin on the LB plate was confirmed. The results are as follows.

[0082]

[Table 1]

Analysis of the nucleotide sequence of the PstI-SacI fragment showed that the four possible
It was suggested to contain orf. The proximal 1 kb of this fragment contained one orf (calD). In the distal 1 kb, three candidates for orf overlapped (calC, calC ', calC
"). These three orfs (calC, calC ', calC") were translated by a computer, and the corresponding proteins CalC, CalC', and CalC "were analyzed using BLAST.
No homologous known proteins were found. However, the translation product of calC gene is
A weak alignment with the apoprotein of the chromoprotein enegine was shown.

The calD-translated protein CalD was found to contain three amino acid motifs that are typically conserved in S-adenosylmethionine-utilizing O-methyltransferase. Therefore, it was hypothesized that calD was not responsible for calicheamicin resistance. To deny that calD is responsible for calicheamicin resistance, a genetically engineered subclone containing the complete calD but excluding the calC, calC ', and calC "genes was created (pjT1224).

This subclone failed to confer calicheamicin resistance. Next, a subclone containing the calC region was constructed (pjT1232). This subclone conferred calicheamicin resistance. See table above. calC '(pAP6) and calC "(pR
Subclones containing E1) were constructed and tested for calicheamicin resistance. These clones failed to confer calicheamicin resistance. See table above.

In order to confirm the amino acid sequence of CalC and to know its characteristics, cloning calC
Placed in the pMAL-C2 vector (pMAL-C2 itself failed to confer calicheamicin resistance; see table above). The resulting plasmid, pRE7, contained calC and conferred calicheamicin resistance. See table above. Next, plasmid pRE7 was induced with isopropyl β-D-thiogalactoside (“IPTG”) to overexpress CalC. The induced pRE7 conferred calicheamicin resistance and produced a maltose binding protein-CalC fusion protein (mbp-CalC). Such overexpression of CALC, calicheamicin resistance rose to 10 ^twice in vivo. See table above.

Embodiment 3 FIG. Expression of the protein CalC For further analysis, the mbp-CalC protein was overexpressed and purified. mb
p-CalC was purified from pRE7 / E. coli to homogeneity. Homogeneity was determined by SDS-PAGE. A culture (containing 50 mg / ml ampicillin and 50 ng / ml calicheamicin) from a fresh pRE7 / E. Coli colony grown overnight on LB is grown at 37 ° C., 250 rpm to A ₆₀₀ = 0.5, Induction was performed with 0.5 mM IPTG and growth was continued overnight. The cells were collected (4,000 × g, 4 ° C., 20 minutes) and buffer solution A (50 mM Tris HCl, pH 7.5, 200 mM
M NaCl, 1 mM EDTA) and disrupted by sonication.

The cell residue was removed by centrifugation (5,000 × g, 4 ° C., 20 minutes). The supernatant was applied to an amylose affinity column (1.5 × 7.0 cm, 1 mL / min).
The desired mbp-CalC protein was eluted with buffer solution A containing 10 mM maltose. The eluate was concentrated and subjected to an S-300 column (50 mM Tris HCl, pH 7.5, 200 mM NaCl
). Active fragments were used immediately or stored frozen at -80 ° C.

Embodiment 4 FIG. Analysis of protein CalC Next, the metal content in the purified mbp-CalC was analyzed. The purified mbp-CalC was yellow when concentrated. Subsequently, an inductively coupled plasma atomic mass spectrometer (“IC
Analysis of the metals using P-MS ") revealed the presence of iron (Fe). When combined with quantitative amino acid hydrolysis and determined for iron stoichiometry, (mbp 2.23 iron per mole of mbp-CalC based on the molecular weight of the monomer calculated from the known nucleotide sequence of the -calC gene fusion, 63.576 daltons (this value is consistent with the molecular weight of the subunit as determined by SDS-PAGE). It was shown to be ± 0.3 mol.

The exact concentration of mbp-CalC was determined by quantitative amino acid hydrolysis at the Rockefeller University Protein / DNA Technology Center. Then, four separate mbp-CalC
For the preparations, trace metal content in aliquots of the hydrolyzate was determined using ICP-MS. At the same time, only the buffer solution and / or the maltose binding protein alone were analyzed as controls.

The results were independently confirmed using the methodology used in spectrophotometric determination of iron. Fish, WW, Meth. Enzymol., 158, pp. 357-364, 1988. FIG. 4 shows the electronic absorption spectrum of mbp-CalC. In addition to the absorption of A280 protein (ε280 = 99,300 / M · cm), a clear maximum of the absorption is observed at the position of 411 nm (ε411 = 6,000 / M · cm). Electron paramagnetic resonance ("EPR") confirmed the metal content in CalC.

When the oxidized CalC protein was subjected to EPR measurement in the X band,
A standard orthorhombic EPR signal appeared at g = 4.3 (E / D = 0.33) (Fig. 4 (a
) Inset). Iron was contained at 90 ± 10 μM as a metal (about 72 ± 10% of the total amount of iron found by ICP-MS; FIG. 4 (a)). Spectroscopy results suggest that Fe ⁺³ is solely central in CalC, consistent with the absence of cysteine in the primary sequence of CalC. Palmer, G., Biochem. Soc. Tran
s., vol. 13, pages 548-560, 1985.

Embodiment 5 FIG. Validation of CalC calicheamicin resistanceCalicheamicin cleaves double-stranded DNA, and CalC confers calicheamicin resistance in vivo, so that calicheamicin-induced DNA cleavage assays can be performed in vitro.
It was expected that DNA cleavage could be suppressed by adding alC. To test this idea, a preliminary assay was performed using supercoiled pBluescript plasmid DNA ("pBS") as the template and dithiothreitol ("DTT") as the reducing initiator. In a typical assay, purified mbp-CalC (
15.0 nM) and 30.0 nM calicheamicin at 40 mM in a total volume of 25 μl
In Tris HCl, pH 7.5 at 37 ° C. for 15 minutes.

Next, 2.5 μl of a 10 mM DTT stock solution was added to the assay solution, and the assay solution was further cultured at 37 ° C. for 1 hour. The fragmented DNA was evaluated by electrophoresis on a 1% agarose gel and staining with ethidium bromide. This assay, mbp-CALC, in concentrations calicheamicin excessive ten ^3-fold, was found to be completely inhibited cleavage of DNA induced by calicheamicin. Preculture of mbp-CalC and DTT, removal of protein by forced dialysis, and subsequent use of DTT solution as reducing agent did not significantly affect the amount of DNA cut.

As shown in FIG. 4 (b), in the absence of DTT or calicheamicin, no DNA cleavage was observed (lanes a and b). In contrast, cleavage occurred effectively when DTT and calicheamicin were present (lane c). As expected, the addition of mbp-CalC resulted in DN induced by calicheamicin.
The cleavage of A was completely suppressed (lane f). In contrast, when only mbp was added as a control, calicheamicin-induced DNA cleavage could not be suppressed (lane d).

Furthermore, when mpp-CalC was pre-cultured together with DTT (not shown),
Preliminary culture of mbp-CalC (lack of iron cofactor) (lane e) also
It was not possible to suppress DNA cleavage induced by isin. But Fe^{+ Two} Or Fe⁺³Was added to Apo-mbp-CalC, the activity of CalC could be reproduced.
(Lane g). Apo-mbp-CalC reconstitution was performed at 1 mM prior to performing the activity assay described above.
FeSO4 (Fe⁺²) Or FeCl3 (Fe⁺³) Was achieved by pre-culture.

Embodiment 6 FIG. Metimycin / picromycin-calicheamicin hybridisation
It was amplified calH gene 1.2kb from _pJST1192 kpn7 in production polymerase chain reaction compound (PCR) method. pJST1192kpn7 is a subclone containing a 7.0 kb KpnI fragment of cosmid 13a. The amplified gene was cloned, and the expression vector pDHS617 was Eco-
It was introduced at the RI / XbaI site. This expression vector contains an apramycin resistance marker. Plasmid pDHS617 is derived from pOJ1446 (Bierman, M., Gene
116, pp. 43-49, 1992).

[0098] A promoter sequence from the Streptomyces venezuelan methymycin / picromycin cluster was inserted into the plasmid to promote expression of the foreign gene in Streptomyces venezuela. The resulting plasmid pLZ-C242 (calH
The gene insert and the promoter sequence were introduced into the previously constructed Streptomyces venezuelan mutant desI by conjugation using E. coli S17-1 (Borisova, S. et al., Org. Lett., No. 1). Vol. 133-136, 1999). In this DesI mutant, desI was replaced with a neomycin resistance gene that confers kanamycin resistance.

[0099] Streptomyces venezuelan-DesI colonies containing PLS-C242 were identified based on resistance to apramycin antibiotics. One of the positively responding colonies, DesI / calH-1, was grown in 100 ml of seed medium at 29 ° C. for 48 hours, and then cultured in 5 liters of plant medium to grow. I let it. Kane, DE et al., J. Am. Chem. Soc., 115, 522-526, 1993. The culture was centrifuged to remove cell residues and mycelium. The pH of the supernatant was adjusted to 9.5 using concentrated KOH, and then extracted using chloroform.

The crude product (700 mg) was flash chromatographed on silica gel using chloroform containing a 1-20% gradient of methanol. The main product, 10-deoxymethinolide (about 400 mg), and small amounts of a mixture of the two macrolide compounds were obtained. The two macrolides were further purified by high performance liquid chromatography (HPLC) on a _C18 column using an acetonitrile / water (1: 1) isocratic mobile phase. The two macrolides were later identified as compound (11) and compound (12) by spectral analysis (FIG. 7).

[0101]

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[0102]

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[0103]

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[0104]

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[0105]

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[0106]

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[Sequence list]

[Brief description of the drawings]

FIG. 1 is a table summarizing cosmid clones isolated from a genomic library of Micromonospora echinospora. This table shows the results of screening a genomic library of clones having a calicheamicin biosynthesis cluster.

FIG. 2 is a restriction map showing a part of cosmid clones 4b, 13a, and 56 and a corresponding position of a cal gene derived from Micromonospora echinospora.

FIG. 3 is a table of open reading frames (“orf”) within the calicheamicin biosynthesis cluster. This table lists the polypeptide encoded by the gene and the proposed or actually determined function of the polypeptide in the biosynthetic pathway.

FIG. 4 (a) is a graph of an ultraviolet-visible light spectrum of purified mbp-CalC. Purified mbp-CalC was analyzed in a solution consisting of 52 μM mbp-CalC; 10 mM Tris HCl, pH 7.5. The upper right figure inserted into the graph shows the result of analyzing CalC by X-band EPR at low temperature (4.3K). 25 moles of iron per mole of CalC 25
0 μM mbp-CalC was analyzed in 10 mM Tris HCl, pH 7.5. The settings of the spectrometer are as follows. Magnetic field = 2050 G; scan width = 4,000 G; time constant = 82 seconds; modulation amplitude = 16 G; microwave power = 31 μW; frequency = 9.71 GHz; gain = 1000; determined spin value = 90 ± 10 μM Fe.

FIG. 4 (b) shows the results of an in vitro assay for mbp-CalC.

FIG. 5 shows pathways envisioned as required sugar nucleotide biosynthesis pathways. Enzymes are described as follows. _E deox = de oxygenase; E _am = aminotransferase; E _ep = epimerase; E _{the met} = methyltransferase; E _od = 4,6-dehydratase; E _ox = oxidase; E _p = nucleotidyl transferase; E _red = reductase; E _sh = sulfhydryl transferase.

FIG. 6 shows in vivo picromycin / methimicin-calicheamicin.
FIG. 2 is a schematic diagram when a hybrid metabolite is produced.

FIG. 7 shows the Streptomyces venezuela methimycin / picromycin gene cluster. 8 in this cluster
One open reading frame (desI-desVIII) has been found to be a gene involved in desosamine biosynthesis. This figure also shows the hybrid pathway when new methimicin / picromycin derivatives (11 and 12) are generated after heterologous expression of the calicheamicin calH gene in a mutant of Streptomyces venezuelan.

FIG. 8 shows calicheamicin (6) 4 which is crucial for the tight binding of DNA.
Shows two special sugars. The sugar (9) is 4-amino-4,6-dideoxyglucose (
8) and is part of a NO bond restricted to the position between sugar A and sugar B. Compound 8 is derived from the corresponding 4-keto sugar (7) through a transamination reaction. The calH gene encodes a desired C-4 aminotransferase and can convert compound (7) to compound (8).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/21 1/19 9/00 1/21 15/00 ZNAA 5/10 5/00 A 9/00 A61K 37/02 F term (reference) 4B024 AA01 AA20 BA07 BA67 BA80 CA03 DA06 EA06 GA11 HA17 4B050 CC03 DD02 LL01 LL10 4B065 AA26X AA35Y AB01 BA02 CA24 CA27 CA44 4C057 CC03 DD01 KK11 4C08A AA02A

Claims (43)

[Claims] 1. The method of claim 1, wherein the nonchromoprotein is enediy.
ne) a nucleic acid sequence encoding a gene derived from a biosynthetic gene cluster, or a protein coding region of the gene, or a biologically active fragment in the gene,
A nucleic acid molecule isolated from Micromonospora echinospra. 2. The gene is calA, calB, calC, calD, calE, calF, calG
, CalH, calI, calJ, calK, calL, calM, calN, calO, calP, calQ, calR, calS
2. The isolated nucleic acid molecule of claim 1, which is a gene, calT, orf1, orf2, orf3, orf4, orf5, orf6, orf7, or an IS-factor gene. 3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes more than one of said genes. 4. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a full-length biosynthetic gene cluster. 5. The non-chromoprotein enediyne (enchromin)
2. The isolated nucleic acid molecule of claim 1, wherein the ediyne is calicheamicin. 6. The method of claim 1, wherein the non-pigment protein enediyne biosynthesis gene cluster comprises
An isolated nucleic acid molecule capable of hybridizing with a nucleic acid from Micromonospora echinospora sp. Calichensis encoding one or more genes. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule encodes a protein having the same activity as at least one gene from the biosynthetic gene cluster.
4. The isolated nucleic acid molecule of claim 1. 8. The gene is calA, calB, calC, calD, calE, calF, calG
, CalH, calI, calJ, calK, calL, calM, calN, calO, calP, calQ, calR, calS
7. The isolated nucleic acid molecule of claim 6, which is a calT, orf1, orf2, orf3, orf4, orf5, orf6, orf7, or IS-factor gene. 9. The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO: 1. 10. The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO: 3. 11. The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO: 5. 12. P4 from Micromonospora echinospora species calikensis
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a polypeptide encoding 50 oxidase. 13. A membrane transporter derived from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis.
2.) The isolated nucleic acid molecule of claim 1 which encodes a polypeptide that encodes). 14. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a polypeptide encoding an O-methyltransferase from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. . 15. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule encodes a polypeptide encoding a glycosyltransferase from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. 16. The isolated bacterium of claim 1, which encodes a polypeptide encoding an N, N-dimethyltransferase from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. Nucleic acid molecule. 17. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule encodes a polypeptide encoding a dipeptide transporter from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. 18. The isolation of claim 1, which encodes a polypeptide encoding an L-cysteine / cysteine CS-lyase derived from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora sp. Calikensis. Nucleic acid molecule. 19. The isolated nucleic acid of claim 1, wherein the nucleic acid encodes a polypeptide encoding an oligopeptide transporter protein derived from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. molecule. 20. The isolated nucleic acid molecule of claim 1, which encodes a polypeptide that encodes a regulatory protein derived from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. 21. The isolated nucleic acid molecule of claim 1, which encodes a polypeptide encoding hexopyranosyl-2--3-reductase from the gene cluster of Micromonospora echinospora sp. Calikensis. 22. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule encodes a polypeptide encoding a desaturase from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. 23. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule encodes a polypeptide encoding UDP-D-glucose 6-dehydrogenase from the gene cluster of Micromonospora echinospora sp. Calikensis. 24. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a polypeptide that encodes a transcriptional regulator derived from the calicheamicin biosynthesis gene cluster of Micromonospora echinospora species calikensis. 25. An expression vector comprising a nucleic acid molecule encoding a protein coding sequence, wherein the nucleic acid molecule is selected from the nucleic acid molecules according to any one of claims 1 to 24. An expression vector, characterized in that: 26. The expression vector according to claim 25, wherein the nucleic acid molecule is operably linked to a control sequence for controlling the expression of the protein. 27. The expression vector according to claim 26, wherein the control sequence is a Streptomyces promoter. 28. A host cell transformed with the nucleic acid molecule according to any one of claims 1 to 24. 29. A host cell transformed with the nucleic acid molecule of claim 25. 30. A host cell transformed with the nucleic acid molecule of claim 26. 31. The host cell according to claim 28, wherein said host cell is a bacterial, yeast, insect, plant, fungal, or mammalian cell. 32. The host cell according to claim 28, wherein the host bacterium is Escherichia coli or Streptomyces. 33. An isolated cosmid comprising a nucleic acid molecule derived from Micromonospora echinospora sp. Calikensis, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a non-chromoprotein enediyne biosynthesis gene cluster. 34. The nucleic acid sequence according to claim 31, wherein the nucleic acid sequence is calA, calB, calC, calD, calE, calF,
calG, calH, calI, calJ, calK, calL, calM, calN, calO, calP, calQ, calR,
34. The cosmid according to claim 33, which encodes calS, calT, orf1, orf2, orf3, orf4, orf5, orf6, orf7, or an insertion factor gene. 35. A method for expressing a protein, comprising culturing a host cell transformed with the expression vector according to claim 25 for a time and under conditions allowing expression of the protein. 36. The method of claim 35, wherein said host cell is a bacterial, yeast, insect, plant, fungal, or mammalian cell. 37. A method for purifying calicheamicin using affinity chromatography, comprising supplying a solution containing calicheamicin to an affinity column to which CalC is bound, and recovering calicheamicin. . 38. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 39. A polypeptide comprising the amino acid sequence of SEQ ID NO: 4. 40. A polypeptide comprising the amino acid sequence of SEQ ID NO: 6. 41. A method for conferring calicheamicin resistance on a subject, comprising collecting cells from the subject, transforming the cells with a calicheamicin self-resistance gene, and subjecting the cells to the subject. A method that includes the operation of returning to. 42. The following structure: A compound having the formula: 43. The following structure: A compound having the formula: