EP1137796A2 - Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto - Google Patents

Abstract

An isolated gene cluster of Micromonospora echinospora which codes for calicheamicin biosynthesis. The biosynthetic gene cluster contains genes encoding for proteins and enzymes used in the biosynthetic production of calicheamicin, including the aryltetrasaccharide and aglycone. The gene cluster also includes the gene conferring calicheamicin resistance. The invention also provides isolated genes of the biosynthetic cluster and their corresponding proteins. In addition, the invention relates to DNA hybridizing with the calicheamicin gene cluster and the isolated genes of that cluster. Expression vectors containing genes of the biosynthetic gene and their functional variants are also provided. The invention also relates to host cells conjugated with DNA isolated from the Micromonospora echinospora spp. calichensis genome.

Description

Micromonospora ec inospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto

This application claims benefit from provisional application 60/1 1 1,325 filed on

December 7, 1998, which application is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to a biosynthetic gene cluster of Micromonospora

echinospora spp. calichensis. In particular, the calicheamicin biosynthetic gene cluster

contains genes encoding for proteins and enzymes used in the biosynthetic pathway and

construction of calicheamicin^" s aryltetrasaccharide and aglycone, and the gene conferring

calicheamicin resistance. The present invention also relates to isolated genes of the

biosynthetic cluster and their corresponding proteins. In addition, the invention relates to

DNA hybridizing with the calicheamicin gene cluster and the isolated genes of that

cluster. The invention also relates to expression vectors containing the biosynthetic gene

cluster, the individual genes, or functional variants thereof.

Background of the Invention

The enediyne antibiotics, which were discovered in the 1980's, have long been

appreciated for their novel molecular architecture, their remarkable biological activity,

and their fascinating mode of action. Enediyne antibiotics were originally derived by

fermentation of microorganisms, including Micromonospora, Actinomedura, and

Streptomyces. Rothstein, D. M.. Enediyne Antibiotics as Antitumor Agents, p. 2 (1995). As a class, the enediyne antibiotics have been referred to as the most potent and highly

active antitumor reagents yet discovered. Rothstein, D. M.. Enediyne Antibiotics as

Antitumor Agents, preface (1995).

To date, at least twelve members of this family of antibiotics have been

discovered, all of which fall roughly into two categories. The first category of enediynes

is classified as chromoprotein enediynes because they possess a novel 9-membered ring

chromophore core structure, which also requires a specific associated protein for

chromophore stabilization. The second category of enediyne is classified as non-

chromoprotein enediynes. These enediynes contain a 10-membered ring, which requires

no additional stabilization factors. This enediyne ring structure is often referred to as the

"warhead." The warhead induces DNA damage, which is frequently a double-stranded

cleavage and appears to be irreparable. This type of DNA damage is usually

nonrepairable for the cell and is most often lethal. Because of these remarkable chemical

and biological properties, there has been an intense effort by both the pharmaceutical

industry and academia to study these substances with the goal of developing new and

clinically useful therapeutic anti-tumor agents.

The 9-membered ring chromoprotein enediyne subfamily is comprised of:

neocarzinostatin from Streptomyces carzinostaticus, (Myers, A.G., et al., J. Am. Chem.

Soc, 110, 7212-7214 (1988)); kedarcidin from Actinomycete L585-6, (Leet, J.E., et al., J.

Am. Chem. Soc, 114, 7946-7948 (1992)), N1999A2 from Streptomyces globisporus,

(Yoshida, K., et al. Tetrahedron Lett., 34, 2637-2640 (1993)), maduropeptin from

Actinomadura madurea, (Schroeder, D.R., et al., J. Am. Chem. Soc, 116, 9351-9352

(1994)); N1999A2 from Streptomyces sp. AJ9493, (Schroeder, D.R., et al., J. Am. Chem. Soc, 116, 9351 -9352 (1994)): actinoxanthin from Actinomyces globisporus. (Khokhlov,

A.S., et al., J. Antibiot., XXII. 541-544 (1969)); largomycin from Streptomyces

pluricolorescens, (Yamaguchi. T.. et al., J Antibiot.. XXIII 369-372 (1970));

auromomycin from Streptomyces macromomyceticus, (Yamashita, T., et al., J. Antibiot.,

XXXII, 330-339 (1979)), and sporamycin from Strepto porangium pseudovulgare.

(Komiyama, K, et al., J. Antibiot., XXX, 202-208 (1977)) all of which are believed to

possess a novel bicylo[7.3.0.]dodecadiynene chromophore core structure essential for

biological activity. In addition, with the exception of N1999A2. a required apoprotein

acts as a stabilizer and specific carrier for the unstable chromophore, and for its transport

and interaction with target DNA.

The non-chromophore enediyne subfamily is comprised of calicheamicin from

Micromonospora echinospora spp. calichensis; namenamicin from Polysyncraton

lithostrotum; esperamicin from Actinomadura verrucosospora; and dynemicin from

Micromonospora chersina.

Enediyne antibiotics have potential as anticancer agents because of their ability to

cleave DNA, however, many of these compounds are too toxic to be used currently in

clinical studies. Today, only calicheamicin is known to be currently used in clinical trials

and it has provided promising results as an anticancer agent. The enediynes potentially

have utility as anti-infective agents, provided that toxicity can be managed.

The toxicity of the enediyne compounds, including calicheamicin, centers on the

problem of directing the compound to the cleave only the DNA of interest, such as tumor

cell DNA, and not the DNA of the host. Due to calicheamicin' s powerful ability to cleave

DNA, scientists have investigated the mechanism by which calicheamicin-producing organism protects itself against the DNA-cleaving activity of the molecule. Rothstein, D.

M., Enediyne Antibiotics as Antitumor Agents, p. 77 (1995). Prior to this invention,

knowledge of genes encoding for non-chromoprotein enediyne self resistance was

completely lacking. Insight into how Micromonospora self resistance gene and gene

products act to control the toxic effects of calicheamicin offers new avenues of clinical

research. For example, knowledge of the mechanisms underlying calicheamicin

resistance could provide the means necessary to use higher doses of calicheamicin while

simultaneously inhibiting the toxic effects of the drug on non-cancer cells. Additionally,

understanding the mechanism behind calicheamicin^" s self-resistance may aid in the

understanding of self-resistance in other enediyne antibiotics, thereby potentially making

useful those enediynes once thought to be too toxic to be viably used as therapeutic

agents. The calicheamicin self-resistance mechanisms elucidated utilizing the present

invention provide gene therapy approaches, for example, via introduction of enediynes

resistance genes into bone marrow cells, thereby increasing resistance and allowing

tolerance to chemotherapeutic doses of calicheamicin. Banerjee, D., et al., Stem Cells, 12,

378-385 (1994). Thus, understanding calicheamicin self-resistance will significantly aid

continuing clinical studies involving calicheamicin and the enediynes. The present

invention addresses this need as it provides for the isolation and characterization of a

resistance gene and its associated protein for any nonchromoprotein enediynes.

Calicheamicin has two distinct structural regions: the aryltetrasaccharide and the

aglycone (also known as the warhead). The aryltetrasaccharide displays a highly unusual

series of glycosidic, thioester, and hydroxylamine linkages and serves to deliver the drug

to specific tracts (5'-TCCT-3' and 5'-TTTT-3') within the minor groove of DNA. The aglycone of calicheamicin consists of a highly functionalized

bicyclo[7.3.1]tridecadiynene core structure with an allylic trisulfide serving as the

triggering mechanism. McGahren. W.J.,et al.. Enediyne Antibiotics as Antitumor Agents,

pp. 75-86 (1995). Once the aryltetrasaccharide is firmly docked, aromatization of the

bicyclo[7.3.1]tridecadiynene core structure, via a 1 ,4-dehydrobenzene-diradical, results in

the site specific oxidative double strand scission of the targeted DNA. Zein, N.,et al.,

Science, 240, 1 198-1201 (1988). The aglycone undergoes a reaction that yields carbon-

centered diradicals, which are responsible for DNA cleavage. This activity has sparked

considerable interest in the pharmaceutical industry culminating in the recent success of

calicheamicin-antibody conjugates (CMA-676) to treat acute myelogenous leukemia

(AML) in phase III trials. Additionally, similar strategies have been used in phase I trials

to treat breast cancer. A massive program to examine calicheamicin conjugated to

alternative delivery systems has also recently been undertaken. Hamann, P.R., et al., 87th

Annual Meeting of the American Association of Cancer Research, Washington, D.C., pp.

471 (1996); Hinman, L.M., et al. Cancer Res., 53, 3336 (1993); Hinman, L. M., et al.,

Enediyne Antibiotics as Antitumor Agents, pp. 87- 105 (1995); Sievers, E.L., et al., Blood,

93, 3678-3684 (1999); Siegel. M.M.. ei a\., Anal. Chem., 69, 2716-2726 (1997); Ellestad,

G. personal communication.

The biological activity and molecular architecture of calicheamicin has also

prompted a search for the potentially useful analogs. Of the numerous laboratories

producing synthetic analogs, one group has produced a novel calicheamicin θ¹, shown to

effectively suppress growth and dissemination of liver metastases in a syngeneic model of

murine neuroblastoma. Lode, H. N., et al, Cancer Res., 58, 2925-2928 (1998); Wrasidlo, W., et al., Ada Oncologica, 34, 157-164 (1995). In addition to synthesizing

calicheamicin analogs, random mutagenesis of M. echinospora and screening for mutant

strains with improved biosynthetic potential has also been pursued. Rothstein, D. M,

Enediyne Antibiotics as Antitumor Agents, pp. 107-126 (1995).

The first total synthesis of calicheamicin was reported by Nicolaou and coworkers

in 1992. Synthesizing this complex antibiotic, though, presents many disadvantages. For

example, Nacelle's procedure only provides approximately a 0.007% yield and requires

47 steps. Halcomb, R.L., Enediyne Antibiotics as Antitumor Agents, pp. 383-439 (1995).

Thus, the total synthesis of calicheamicin remains secondary to the isolation of

calicheamicin from large fermentations of M. echinospora. Therefore, methods to

produce mass amounts of calicheamicin and potentially useful variants are still needed.

Fantini, A., et al., Enediyne Antibiotics as Antitumor Agents, pp. 29-48 (1995).

Transforming calicheamicin DNA into producing strains of bacteria, E. coli for example,

would address this need. Currently there are no cloned M. echinospora genes and only a

set of limited studies upon putative M. echinospora promoters are available. Lin, L.S., et

al., J Gen. Microbiol, 138, 1881-1 ^5 (1992); Lin, L.S., et al., J Bacteriol., 174, 3111-

3117 (1992); Baum, E.Z., et al., J. Bacteriol, 171, 6503-6510 (1989); Baum. E.Z., et al.,

J Bacteriol, 170, 71-77 (1988).

Having calicheamicin DNA opens the door for genetic analysis of calicheamicin

biosynthesis, as such analysis requires the ability to obtain large qualities of calicheamicin

DNA. For example, one can study calicheamicin biosynthesis by mutagenesis of M.

echinospora, including the isolation and characterization of mutants blocked in

calicheamicin biosynthesis and the subsequent analysis of their defective or partial calicheamicin products. Additionally, particular enzyme could be overexpressed or

underexpressed after subcloning its gene into a host such as E. coli. and the results of

such overexpression studied to reveal the enzyme's function. Furthermore, the cloning of

biosynthetic genes can ultimately result in increased yields of the gene product by cloning

and expressing the biosynthetic gene encoding the rate-limiting enzyme back into the

producing organism. It may also be possible to generate novel products by cloning

biosynthetic genes into strains that make related compounds. Such genes could endow

the host organism with the ability to carry out new reactions on the enediyne nucleus, and

thus produce novel drugs.

Calicheamicin^'s molecular architecture in conjunction with its useful biological

activity and potential therapeutic value brand calicheamicin an target for the study of

natural product biosynthesis. While the radical-based mechanism of oxidative DNA

cleavage by calicheamicin (i.e. aromatization of the bicyclo[7.3.1]tridecadiynene core

structure, via a 1 ,4-dehydrobenzene-diradical, resulting in the site specific oxidative

double strand DNA cleavage) is well understood, it was unknown, prior to this invention,

how Micromonospora constructs calicheamicin. As a result, there is a need to discover

and understand calicheamicin biosynthesis. Prior to this discovery, knowledge of genes

encoding for nonchromoprotein enediyne biosynthesis was completely lacking. Thus,

this invention relates to the first identification, isolation, and cloning of a

nonchromoprotein enediyne biosynthetic gene cluster and mapping and nucleotide

sequence analysis of the genes within the cluster. The invention provides the entire

calicheamicin-biosynthetic cluster and biochemical studies of aryltetrasaccharide

biosynthesis. Furthermore, the calicheamicin self-resistance gene and protein have been isolated as have the genes and resulting enzymes for steps within the calicheamicin

cascade. The invention also provides for construction of enediyne overproducing strains,

for rational biosynthetic modification of bioactive secondary metabolites, for new drug

leads, and for an enediyne combinatorial biosynthesis program.

The present invention thus, also relates to a biosynthetic modification of bioactive

secondary metabolites through enediyne combinatorial biosynthesis. As most

pharmaceutical drug leads are inspired by naturally occurring compounds, and given the

challenge posed in synthesizing these metabolites, genetic manipulation of the sugar

appendage on the metabolites offers avenues for creating potential new drugs. Thus the

emerging field of combinatorial biosynthesis has become a rich new source for modified

non-natural sugar scaffolds. Marsden, A., et al., Science 1998, 279, 199-201. Problems

inherent with the genetic manipulation of the sugar appendage relate to the fact that

naturally occurring bioactive secondary metabolites possess unusual carbohydrate

ligands, which serve as molecular recognition elements critical for biological activity.

Macrolide Antibiotics, Chemistry. Biology and Practice, 1984. Without these essential

sugar attachments, the biological activities of most clinically important secondary

metabolites are either completely abolished or dramatically decreased. Currently,

techniques for the genetic manipulation of the sugar appendage for a given metabolite

rely mainly on the alteration and/or deletion of a small subset of genes required to

construct and attach each desired sugar moiety. Thus there is a need to develop alternate

strategies to construct and attach non-naturally occurring sugars. The present invention

addresses this need. The present invention utilizes the fact that glycosyltransferases,

which are responsible for the final glycosylation of certain secondary metabolites, show a high degree of promiscuity toward the nucleotide sugar donor. Zhao. L.. et al.. J. Am.

Chem. Soc. 1988, 120, 12159-12160. This unselectivity of the glycosyltransferases has

the potential for allowing modification of the crucial glycosylation pattern of natural, or

non-natural, secondary metabolite scaffolds in a combinatorial fashion. The present

invention discloses a method using the recruitment and collaborative action of sugar

genes from a variety of biosynthetic pathways to construct composite gene clusters, which

make and attach non-natural sugars.

Summary of the Invention

The present invention provides an isolated nucleic acid molecule from

Micromonospora echinospora encoding for a gene from a nonchromoprotein enediyne

biosynthetic gene cluster, the protein coding region of the gene or a biologically active

fragment of the gene. In particular, the present invention provides an isolated nucleic acid

molecule, gene, or gene cluster from Micromonospora echinospora spp. calichensis that

is involved in the biosynthesis of calicheamicin. In another embodiment, the present

invention also relates to nucleic acids capable of hybridizing with a nucleic acid molecule

from Micromonospora echinospora spp. calichensis coding for one or more genes from a

nonchromoprotein enediyne biosynthetic gene cluster. In a further embodiment the

invention provides an expression vector comprising an isolated nucleic acid molecule

from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora

echinospora. In yet a further embodiment the invention provides a cosmid comprising an

isolated nucleic acid molecule from Micromonospora echinospora comprising a nucleic

acid sequence encoding for a nonchromoprotein enediyne biosynthetic gene cluster. In preferred embodiments, the invention provides the isolated nucleic acid

molecules of SEQ ID Nos. 1. 3. and 5.

In an additional embodiment, the present invention provides a host cell

transformed with an isolated nucleic acid molecule from a nonchromoprotein enediyne

biosynthetic gene cluster from Micromonospora echinospora. Host cells can optionally

be of bacterial, yeast, fungal, insect, plant or mammalian origin and can be transformed

according to standard methods. In a preferred embodiment, the host cell is the bacterium

E. coli or Streptomyces. In a further embodiment, the invention is directed to a

transformed host cell with an expression vector encoding gene calC. or a functional

derivative thereof, operably linked to regulatory sequences that enable expression ofcalC.

In a yet further embodiment, the invention provides a transformed host cell with an

expression vector encoding the gene calH, or a functional derivative thereof, operably

linked to regulatory sequences that enable expression of calH. Likewise, the invention

provides a transformed host cell with an expression vector encoding the gene calG, or a

functional derivative thereof, operably linked to regulatory sequences that enable

expression of calG.

The invention further provides a method of expressing a protein by culturing a

host cell transformed with an expression vector comprising an isolated nucleic acid

molecule from Micromonospora echinospora encoding for a gene from a

nonchromoprotein enediyne biosynthetic gene cluster, and incubating the host cell for a

time and under conditions allowing for protein expression. In another embodiment the

invention provides a method of purifying calicheamicin using affinity chromatography. A

sample containing calicheamicin is contacted with an affinity matrix having the protein CalC bound thereto, for a time and under conditions allowing calicheamicin to bind to the

matrix, eluting calicheamicin from the matrix, and recovering calicheamicin.

In a further embodiment the present invention provides polypeptides having the

amino acid sequences of SEQ ID Nos. 2, 4, and 6.

In yet a further the invention provides the production of the following two new

macrolides:

The invention further provides a method of conferring calicheamicin resistance to

a subject comprising obtaining cells from the subject, transforming the cells with the

calicheamicin self-resistance gene, and returning the cells to the subject. Alternatively,

the calicheamicin self-resistance gene can be targeted and delivered to the desired host

cells through known gene therapy delivery systems.

Brief Description of the Figures

Figure 1 depicts the summary of the cosmid clones isolated from M. echinospora

genomic library. This figure illustrates the results of the screening of the genomic library

for clones carrying the calicheamicin biosynthetic cluster.

Figure 2 shows a restriction map of a portion of cosmid clones 4b, 13a, and 56 and

the corresponding location of cal genes from M. echinospora. Figure 3 is a table of the open reading frames ("orfs^") in the calicheamicin

biosynthetic cluster. This table lists the polypeptides that the genes encode for as well as

their proposed or actual determined function in the biosynthetic pathway.

Figure 4 is a graph of the UV-visible absorption spectra of purified mbp-CalC.

The purified mpb-CalC was analyzed in the following solution: 52 μM mpb-CalC; 10

mM Tris-HCl. pH 7.5). The inset shows the results of low temperature (4.3 K) the X-

band EPR analysis of CalC. 250 μM mpb-CalC containing 0.5 mol Fe per mol CalC was

analyzed in 10 mM Tris-HCl, pH 7.5. The spectrometer settings were as follows: field

set = 2050 G; scan range = 4.000G; time constant = 82 s; modulation amplitude =16 G;

microwave power = 31 μW; frequency = 9.71 Ghz; gain = 1000; determined spin

quantitation = 90 ± 10 μM Fe.

Figure 4(b) provides the results of the mbp-CalC in vitro assay.

Figure 5 depicts the postulated routes for the biosynthesis of required nucleotide

sugars. The enzymes are depicted as follows: E_deox = deoxygenase; E_am =

aminotransferase; E_ep = epimerase; E_met = methyltransferase; E_od = 4,6-dehydratase; E_ox =

oxidase; E_p = nucleotidyltransferase; E_red = reductase; E_sh = sulfhydrytransferase.

Figure 6 illustrates a schematic representation of the in vivo production of

pikromycin/methymycin-calicheamicin hybrid metabolites.

Figure 7 depicts the Streptomyces Venezuela methymycin/pikromycin gene cluster.

Eight open reading frames (desl-desWlll) in this cluster have been assigned as genes

involved in desoamine biosynthesis. This figure also depicts the hybrid pathway toward

new methymycin/pikromycin derivatives ( 11 and 12) produced after heterologous

expression of the calH gene of calicheamicin in a S. Venezuela mutant. Figure 8 illustrates calicheamicin's (6) four unique sugars which are crucial to

tight DNA binding. Sugar (9) is derived from 4-amino-4,6-dideoxyglucose (8) and is part

of the restricted N-O connection between sugars A and B. Compound 8 is derived from

the corresponding 4-ketosugar (7) via a transamination reaction. The gene calH encodes

the desired C-4 aminotransferase allowing conversion of compound (7) to compound (8).

Detailed Description of the Invention

The present invention is directed to the isolation and characterization of the

calicheamicin biosynthetic cluster. This cluster encodes the genes that encode the

proteins and enzymes that are involved in the deoxysugar synthesis (the

aryltetrasaccharide), polyketide biosynthesis (the aglycone) of calicheamicin synthesis,

and calicheamicin resistance. Twenty-one structural genes have been identified that

encode for the aryltetrasaccharide sugar ligands (-20 kb); approximately eight modules

(-40 kb) are required for the 15-carbon aglycone. Four proteins involved in transport and

uptake, one protein conferring resistance, and one regulatory protein have been identified.

The calicheamicin biosynthetic gene cluster comprises the following genes: calA.

calB, calC, calD, calE, calF, calG. calH, call, call, calK, calL, ca M, calN, calO, cal?,

calQ, calR, calS, call, orfλ, orfl, orβ, or 4, or/5, orf , orfl , and an IS-element gene.

The above listed genes encode for the following polypeptides: 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),

Call (568 amino acids), CalJ (332 amino acids), CalK (440 amino acids), Cal L (562

amino acids), Cal M (416 amino acids), CalN (398 amino acids), CalO (331 amino acids), Cal P (approximately 179 amino acids). CalQ (453 amino acids). CalR (265 amino acids),

CalS (11 13 amino acids), CalT (280 amino acids). Orfl (322 amino acids). Orf2 (654

amino acids). Orf3 (209 amino acids). Orf4 (521 amino acids), Orf5 (175 amino acids),

Orf6 (139 amino acids), Orf7 (187 amino acids), and IS-element (402 amino acids).

In elucidating the calicheamicin biosynthetic gene cluster, the inventors began

with a genomic library containing the genome of Micromonospora echinospora spp.

calichensis. The cosmid library was generated by isolating chromosomal DNA of

Micromonospora echinospora spp. calichensis. fragmenting that chromosomal DNA,

inserting the DNA into a cosmid vector and generating a cosmid library according to

methods well known in the art. This procedure can be performed in any species of

Micromonospora.

Based upon prior enediyne metabolic labeling studies it was postulated that the

calicheamicin aglycone would be polyketide derived. Polyketide metabolites encompass

a vast variety of structural diversities yet share a common mechanism of biosynthesis.

Hutchinson, C.R., et al., Chem. Rev., 97, 2525-2535 (1997); Strohl, W.R., et al,

Biotechnology of Antibiotics pp. 577-657; Fujii, I., et al., Chem. Rev. 97, 2511-2523

(1997); Hopwood, D.A., et al.. Chem. Rev, 97, 2465-2497 (1997); Hopwood, D.A., et al.,

Ann. Rev. Genet., 24, 37-66 (1990); Staunton, J., et al., Chemical Reviews, 97, 261 1-2629

(1997). Most important, polyketide synthase ("PKS") genes display a high degree of

sequence homology (from pathway to pathway and organism to organism) and are often

clustered with genes encoding self resistance and deoxysugar ligand biosynthesis.

Hopwood, D.A., et al., Chem. Rev., 97, 2465-2497 (1997); Hopwood, D.A., et al., Ann.

Rev. Genet., 24, 37-66 (1990); Staunton, J., et al., Chem. Rev., 97, 261 1-2629 (1997). Degenerate primers based upon conserved regions within PKS genes were used in

Southern hybridizations to identify clones from the M. echinospora genomic library that

carried putative PKS genes. The Southern hybridizations were performed by methods

known in the art. Southern hybridization of the genomic M. echinospora cosmid library

with a DNA probe designed to target type I PKS genes (KS' ), (Kakavas, S.J., et al., J.

Bacteriol, 179, 7515-7522 (1997)). unveiled five positive clones, which were designated

clones 4b, 10a, 13a, 56, and 60. See Figure 1. The same five clones were also identified

upon rescreening the genomic library with type II DNA probe (actl). See Figure 1.

Although this preliminary analysis clearly demonstrated the presence of Micromonospora

PKS gene homologues. a secondary screen was performed as PKS hybridization analyses

are often plagued by false hybridization to gene clusters that encode spore pigment

biosynthesis.

The second screening was based on the assumption that calicheamicin's

biosynthetic cluster would also contain genes encoding for deoxysugar ligand synthesis.

Further, it was postulated that all hexopyranosyl ligands of calicheamicin diverged from

the common intermediate 4-keto-6-deoxy TDP-D-glucose (30), Figure 5, as

macromolecule-sugar synthesis in many organisms began with a similar common

intermediate. Thus, it was believed that the cluster encoding for calicheamicin

biosynthesis should, in addition to carrying a PKS-encoding region, would carry both a

common glucose-1-phosphate nucleotidyltransferase and a NDP- -D-glucose 4,6-

dehydratase gene, encoding the putative enzymes E_p and E_od. respectively. See figure 5.

These enzymes are necessary to convert a sugar (12)(figure 5) to the hypothesized

common intermediate, 4-keto-6-deoxy TDP-D-glucose (30). Analogs to 4,6-dehydratases have been previously characterized from E. coli. Salmonella, and Streptomyces.

Additionally, a nucleotide transferase from Salmonella has been characterized as an

alpha-D-glucose-1 -phosphate thymidylyltransferase. The secondary screen was

performed using a probe based upon the postulation that the M. echinospora's

calicheamicin synthesis would begin from a similar precursor found in E. coli,

Streptomyces and Salmonella, and that this precursor required a dehydratase to convert it

into the common intermediate, 4-keto-6-deoxy TDP-D-glucose (30). In particular, a

DNA probe (designated Ε_od') was designed from the conserved NAD -binding site of

bacterial NDP- -D-glucose 4.6-dehydratases. He. X.. et al., Biochem.. 35, 4721-4731

(1996). Southern hybridization of the genomic M echinospora cosmid library with the

E_od' probe revealed cross-hybridization with clones 4b, 10a, 13a. 56. and 60. Two

additional clones, designated 58 and 66, were also identified in this screen. See Figure 1.

This secondary hybridization indicated the clustering of genes encoding both polyketide

and deoxysugar biosynthesis.

For final corroboration. since secondary metabolite biosynthesis is typically

clustered with resistance genes in actinomycetes, all hybridization-positive clones were

tested for their ability to grow in the presence of varying concentrations of calicheamicin.

In this final screen, six of the seven hybridizing clones displayed differing levels of

resistance to calicheamicin (4b= 10a= 13a>56>66>60)(See Figure 1) while clone 58

lacked the ability to grow in the presence of calicheamicin. In addition, these resistance

screens revealed that clones 4b, 10a, 13a conferred much higher levels of resistance to

calicheamicin than the other clones. Upon rescreening the genomic library for

calicheamicin-resistant clones, three additional clones (3a, 4a, and 16a) were found to confer similar levels of resistance. Cumulatively, the results demonstrated that clones 4b,

10a. 13a, 56, and 60 carried PKS I and II homologues and deoxy sugar biosynthetic

genes, as well as encoded the gene responsible for conferring calicheamicin-self

resistance.

The clones positive for PKS I and II and deoxy sugar biosynthesis homology and

calicheamicin resistance were used to map the biosynthetic cluster. Southern

hybridization established similarity between clones 3a, 4a. 4b, 10a, 13a. 16a and 56. In

addition, nucleotide sequence overlaps were found between clones 4b. 13a. and 56. See

Figure 1. Restriction mapping and Southern hybridization of these clones indicated that

the positive cosmid clones corresponded to a continuous region of the M. echinospora

chromosome spanning > 100 kb. The present invention thus provides for cosmids having

a nucleic acid molecule from Micromonospora echinospora encoding for a

nonchromoprotein enediyne biosynthetic cluster.

After isolating the biosynthetic gene cluster and elucidating the sequence, open

reading frames ("Orfs") were assigned. Tentative gene assignments were derived from

amino acid sequence similarity of translated orfs to gene products of known function via

direct BLAST (Basic Local Alignment Search Tool) database searches on the amino acid

level. Karlin, et al., Proceed Natl. Acad. Sci, U.S.A., 87, 2264-2268 (1990); Karlin, et al.,

Proceed Natl Acad. Sci., U.S.A., 90, 5873-5877 (1993); Altchul. Nature Genet, 6, 1 19-

129 (1994). The gene cluster organization is provided in figure 1.

Based on BLAST analysis tentative gene assignments were made. It was

deducted that genes participating in the construction of the aryltetrasaccharide include: a)

genes encoding nucleotide sugar biosynthesis (calG H, K, O, Q, and S); b) genes encoding for aryltetrasaccharide assembly (calE and N); and c) genes encoding for

'"tailoring" reactions (calD. F. and J).

One aspect of the invention relates to transformation of a host cell with M.

echinospora DNA. This method provides a reproducible transformation efficiency of

~10³ kanamycin resistant transformants/μg DNA using a pKCl 139-based vector. The

invention further provides that the host cell can be but is not limited to bacteria, yeast,

fungus, insect, plant or mammalian. Transformations of bacteria, yeast, fungus, insect,

plant or mammalian cells are performed by methods known in the art.

The present invention also provides the isolation and characterization of the gene

encoding for calicheamicin resistance. One aspect of the invention relates to an isolated

DNA strand having the gene calC and having the DNA sequence SEQ. ID No.: 1. The

present invention also relates to an isolated protein CalC, having the amino acid sequence,

SEQ ID. NO. 2. The invention further provides for calC gene fragments coding for a

bioactive CalC. The polypeptide, CalC, confers calicheamicin resistance and has 181

amino acids. The invention also provides for CalC fragments conferring calicheamicin

resistance.

The calC locus was isolated by identifying calicheamicin genomic cosmid clones

that were able to grow on luria bertani ("LB") agar plates containing ampicillin and

calicheamicin. The DNA of the positive clones (clones that grew on the plates containing

calicheamicin) was isolated and subsequent restriction mapping localized the desired

phenotype (calicheamicin resistance). The DNA was then sequenced and the open

reading frames analyzed to ascertain the orf encoding for the desired phenotype. In vitro

studies were also performed and confirmed the ability of CalC to inhibit DNA cleavage. DNA containing calC was cloned into an inducible vector, using known methods,

resulting in overexpression of calC. The polypeptide product (CalC) was then isolated

and purified to homogeneity. Analysis of the purified CalC revealed that it is a non-heme

iron metalloprotein that functions via inhibition of calicheamicin-induced DNA cleavage

in vitro. Another aspect of the invention is an expression vector containing calC or a

fragment of calC encoding for a bioactive molecule. There is also provided a transformed

host cell, preferably bacteria, more preferably, E. coli containing calC or a fragment of

calC encoding for a bioactive molecule.

The present invention provides for the transformation of human cells with the

calC gene. This allows bone marrow cells, for example, to be removed from a patient

being treated with calicheamicin. and to transform these cells with calC, and return the

transformed cells to the patient. This allows the patient to tolerate treatment with

calicheamicin or allows the patient to receive higher doses of calicheamicin as the

returned human-cα/C-transformed cells have calicheamicin resistance. The

transformation is performed by methods known in the art. The embodiment of the

invention would be applicable to many diseases being treated with calicheamicin.

Another aspect of the invention relates to an isolated DNA strand containing the

caM. gene having the DNA sequence SΕQ ID. No: 3. The invention also relates to the

polypeptide CalH, having amino acid sequence SΕQ ID. No. 4. The invention further

provides for calti gene fragments coding for a bioactive CalH. CalH is involved in the

formation of the aryltetrasaccharide 4,6-dideoxy-4-hydroxylamino-D-glucose moiety.

CalH catalyzes the conversion of intermediate (30) to intermediate (39) (figure 5). CalH is

a TDP-6-deoxy-D-glycerol-L-threo-4-hexulose 4-transaminase, which catalyzes a pyridoxal phosphate ("PLP^")-dependent transamination from glutamate to provide 4-

amino-6-deoxy TDP-D glucose (intermediate 39)(figure 5). The invention also provides

for CalH fragments that retain bioactivity. There is also provided an expression vector

containing the calH gene or fragments of the calH gene that encode for a bioactive

polypeptide. CalH were overexpressed as a (histidine),₀-fusion protein and subsequently

purified by nickel affinity chromatography.

According to BLAST analysis, calH closely resembled perosamine synthase, an

enzyme which converts compound 30 to compound 39 (See figure 5) en route to the

biosynthesis of TDP-perosamine (TDP-4.6-dideoxy-4-amino-D-mannose) in E. coli.

Wang, L.. et al., Infect. Immunol. 66. 3545-3551 (1998). Thus CalH was believed to be a

4-ketohexose aminotransferase. To confirm the tentative BLAST assigned function, a

combinatorial biosynthesis was performed. Specifically the caM gene from calicheamicin

was incorporated into a mutant strain of Streptomyces Venezuela. The 4-dehydrase gene

(des\) in the methymycin/pikromycin pathway was deleted in this mutant strain. A

promoter sequence from the S. Venezuela methymycin/pikromycin cluster was

incorporated in the expression vector to drive the expression of foreign genes (the calH of

calicheamicin) in S. Venezuela. In wild type S. Venezuela methymycin/pikromycin

pathway is known to produce methymycin, neomethymycin, pikromycin, and narbomycin.

See figure 6. Deletion of the des\ gene in the mutant strain led to the accumulation of the

CalH substrate, TDP-4-keto-6-deoxyglucose (compound 30, figure 6). The constructed

expression vector with the S. Venezuela promoter expressed the calW gene to make the

CalH protein. CalH acted on the substrate, 30, to produce compound 39 (figure 6).

Compound 39 in turn, with the action of S. Venezuela's DesVII (a glycosyltransferase) produced two methymycin pikromycin-calicheamicin hybrid compounds. See Figure 6.

compounds 40 and 41. These hybrid compounds carry the 4-aminohexose ligand of

calicheamicin. This work provides indisputable support for the calH gene assignment as

encoding the TDP-6-deoxy -D-glycero-L-threo-4-hexulose 4-aminotransferase of the

calicheamicin pathway. The CalH acted on the TDP-4-keto-deoxyglucose substrate

(compound 30) to produce compound 39. (Figure 5).

In addition, these results reinforce the indiscriminate nature of the corresponding

glycosyltransferase (DesVII) as it reveals that the glycosyltransferase (DesVII) of the S.

Venezuela pathway can recognize alternative sugar substrates whose structures are

considerably different from the original amino sugar substrate, TDP-D-desoamine. The

results also clearly demonstrate the ability to engineer secondary metabolite glycosylation

through a rational selection of gene combinations. The successful expression of the CalH

protein in S. Venezuela by the newly constructed expression vector highlights the potential

of using this system to express other foreign genes in this strain.

Thus, one aspect of the present invention further relates to the construction of a

composite gene cluster having the ability to make and attach non-natural sugars. The

invention further provides an expression vector having a calicheamicin gene operably

linked to regulatory sequences to control expression of the calicheamicin protein and

preferably the regulatory sequence is a Streptomyces promoter. The present invention also

relates to two newly synthesized sugars, compound (1 1) and compound (12)(figure 7).

Compound 11 has the formula:

11 The spectral data of compound 1 1 was as follows:

'H NMR (500 MHz CDCI,. J in hertz) δ 6.75 (III, dd, J = 16.0. 5.5, 9-H) 6.44 (I H,

dd, J = 16.0, 1.2. 8-H), 5.34 (IH, d, j = 8.0, N-H). 4.96 (IH. m, 1 1-H). 4.27 (IH, d. J=7.5,

1-H), 3.66 (IH, dd, J = 9.5, 8.0. 4'-H). 3.60 (IH, d, J = 10.5. 3-H). 3.50 (IH, 1 , J - 9.5.

3'H), 3.^d (IH, m. 5'-H), 3.4 (IH. m. 2'-H), 2.84 (I H, dq, J = 10.5. 7.5. 2-H). 2.64 (IH. m.

10-H), 2.53 (IH, m, 6-H), 2.06 (3H. s. Me-C=0), 1.7 (IH, m, 12-H). 1.66 (IH, m, 5-H).

1.56 (lH. m. 12-H), 1.4 (I H, M. 5-H). 1.36 (3H, d.. J=7.5. 2-Me). 1.25 (31 1. D, J = 6.5. 5'-

Me). 1.24 (I H, 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); ^l3C NMR (125 MHz. CDC1₃) δ 205.3 (C-7),

175.1 (C-l), 171.9 (Me-C-O), 147.1 (C-9), 126.1 (C-8), 103.0 (C-l'). 85.8 (C-3), 75.8 (C-

5'), 75.8 (C-3¹), 74.1 (C-l 1) 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-C-O), 18.1 (C-6'), 17.9 (6 Me), 17.6 (4-Me),

16.4 (2-Me), 10.5 (12-Me), 9.8 (10-Me). High-resolution FAB-MS calculated for C₂₅H₄₂-

NO_g (M + H⁺) 484.2910. found 484.2303.

Compound 12 has the formula:

/: The spectral data of compound 12 was as follows:

Η NMR (500 MHz. CDC1, J in hertz) δ 6.69 ( I H. dd. J = 16.0. 6.0. 1 1-H). 6.09

(IH, dd, J = 16.0. 1.5, 10-H), 5.35 (I H. d. J = 8.5. N-H). 4.96 ( IH. m. 13-H). 4.36 (IH. d,

J = 7.5. l 'H). 4.19 (I H, m. 5-H). 3.83 (lFI-q. J=6.5, 2-H). 3.68 (IH, dt, J=10.0, 8.5. 4'H),

3.52 (IH, t, J = 8.5, 3-'H), 3.50 (IH. m. 5-H), 3.42 (IH. t, J = 7.5, 2'-H). 2.92 (IH, dq, J =

7.0, 5.0, 4-H). 2.81 (IH, m, 8-H). 2.73 (IH. t, J=7.5, 2'-H). 2.06 (3H. a, Me-C-O), 1.8 (IH,

m. 6-H). 1.6 (I H. m, 14-H), 1.55 (IH. m. 7-H). 1.37 (3H, d, J = 6.5. 2-Me). 1.32 (3H. d.

J = 7.0. 4-Me), 1.3 (I H, m. H-14). 1.27 (3H. d. J = 6.5, 5'-Me), 1.25 (I H, m. 7-H). 1.12

(3H. d. J =6.0. 8-Me). 1.1 1 (3H. d. J = 6.5. 12-Me). 1.07 (3H, d. J = 6.0. 6-Me), 0.91 (3H.

1 , J -7.2. 1 + Me); high resolution FAB MS calculated for C₂₈ H ₆ N0₂ (M+H^{^})

540.3172.found 540.3203.

One aspect of the invention relates to an isolated DNA strand containing the calG

gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is

the protein, CalG, having amino acid sequence SEQ ID. No.: 6. Based on BLAST analysis

it was presumed that calG encoded a 4,6-dehydratase. Dehydratases had been

characterized from E. coli, Salmonella and Streptomyces, (Thompson, M. et al., J. Gen.

Microbiol. 138, 779-786 (1992); Vara, J.A., et al., J Biol. Chem., 263, 14992-14995

(1988)), and analogous NDP-D-glucose 4,6-dehydratases had been characterized from a

variety of organisms. Liu, H.-w.. et a\.. Ann. Rev. Microbiol, 48, 223-256 (1994): Hallis.

T.M., et al., Ace Chem. Res., in press (1999). Based upon these prior studies, it was

known that the overall transformation catalyzed by 4,6-dehydratases is an intramolecular

oxidation-reduction where an enzyme-bound NAD^' receives the 4-H as a hydride in the

oxidative half-reaction and passes the reducing equivalents to C-6 of the dehydration product in the reductive half-reaction. Thus, it appears that Cal G is necessary for the

formation of the aryltetrasaccharide 4.6-dideoxy-4-hydroxylamino-D-glucose moiety.

CalG appears to be a TDP-D-glucose 4,6-dehydratase which catalyzes the conversion of

intermediate 13 into intermediate 30. (See figure 5). Another aspect of the invention is an

expression vector containing calG or a fragment of calG encoding for a bioactive

molecule. There is also provided a transformed host cell, preferably bacteria, more

preferably, E. coli. containing calG or a fragment of calG encoding for a bioactive

molecule.

There is also disclosed an isolated DNA strand containing the calS gene. Based on

sequence homology with other P450-oxidases, CalS appears to be a P450-oxidase

homolog which performs the oxidation of intermediate 39 to intermediate 42 (figure 5).

The oxidation may occur at the nucleotide sugar level or hydroxylamine formation after

the sugar has been transferred to the aglycone. There is also provided an expression vector

containing the calS gene or a fragment of calS encoding for a bioactive molecule. There is

also provided a transformed host cell, preferably bacteria, more preferably, E. coli,

containing calG or a fragment of calG encoding for a bioactive molecule.

The present invention allows genetic manipulation of the biosynthetic gene cluster

to produce calicheamicin analogs. The present invention provides for producing

calicheamicin analogs by constructing deletions or substitutions of the genes involved in

biosynthesis of the aryltetrasaccharide. The invention further provides for in vitro

glycosylation by altering the glycosylation pattern of calicheamicin (via a

glycosyltransferase) to produce additional analogs. The invention also provides for

alteration of the calicheamicin aglycone by genetic manipulation of the genes encoding the biosynthesis of the warhead. Genetic manipulation, such as producing deletions or

substitutions are performed using methods known in the art.

The invention provides for a method of purifying calicheamicin through affinity

chromatography. CalC. because of its homology with calicheamicin functions as a

calicheamicin-sequestering/binding protein. Affinity chromatography is performed using

methods known in the art.

The invention relates to the expression of the genes located in the biosynthetic gene

cluster by using methods known in the art to insert the genes into a suitable expression

vector and operably linking the gene to regulatory sequences to control expression of the

gene to produce the protein encoded by the inserted gene. The present invention also

provides for expression of biologically active proteins by inserting fragments of genes

selected from the biosynthetic gene cluster, which encode for biologically active proteins,

into a suitable expression vector, using methods known in the art. The genes would be

operably linked to regulatory sequences to control their expression.

EXAMPLES

Example 1

To rapidly elucidate the nucleotide sequence, thermocycle sequencing was

accomplished from pUC- or pBluescript-based subclones (using Ml 3 primers and primer

walking) as well as directly from isolated cosmids (via primer walking). Nucleotide

sequence data was acquired using two Applied Biosystems automated 310 genetic

analyzers and sequences were subsequently assembled using the Applied Biosystems

AutoAssembler™ DNA sequence assembly software. Dear, S., et al., Nucl Acids Res., 14, 3907-391 1 (1991 ): Huang. X.. Genomics, 14. 18-25 ( 1992). Orf assignments were

accomplished using a combination of the computational programs Mac Vector™ 6.0 and

Brujene. MacVector is a commercially available software package which provides the

ability to construct a Micromonospora codon bias table (from known Micromonospora

sequences) and subsequently use this codon bias table to search for optimal orfs. Fickett,

J.W., Nucleic Acids Research, 10, 5303-5318 (1982). Alternatively, the shareware

program Brujene was specifically designed for streptomycetes and assigns priority to orfs

that illustrate a consistency high G/C% in the wobble position.

Example 2: Isolating and Characterizing calC

To isolate the gene(s) responsible for calicheamicin resistance in Micromonospora.

clones conferring calicheamicin resistance were selected by growth of a Micromonospora

genomic bifunctional cosmid library on LB plates containing ampicillin (50 μg ml^"1) and

calicheamicin (0.25 μg ml^"1). In this selection, six clones (3a, 4a, 4b, 10a. 13a and 16a)

displayed resistance to calicheamicin. Restriction mapping of these clones localized the

desired phenotype to a ~2kb Pstl-Sacl fragment of DNA. (Figure 2). Maximum tolerated

concentrations of calicheamicin on the LB plates was ascertained. The results are as

follows:

Nucleotide sequence analysis of the Pstl-Sacl fragment suggested that it contained

four possible orfs. The proximal 1 kb of this fragment carried a single orf (calD). The

distal 1 kb presented three overlapping orf candidates (calC. calC, and calC"). Computer

translation of these three orfs (calC. calC. and calC") was performed and subsequent

BLAST analysis of their corresponding proteins. CalC. CalC. and CalC". respectively,

revealed no homology with known proteins, while the translation of gene calC displayed a

weak alignment with apoproteins of the chromoprotein enediynes. Translation of calD to

its respective protein, CalD, revealed the presence of three amino acid motifs typically

conserved in S-adenosylmethionein-utilizing O-methyltransferases. Therefore it was

hypothesized that calD was not responsible for calicheamicin resistance. To rule out calD

as being responsible for calicheamicin resistance, a subclone was engineered (pjT1224) to

contain an intact calD, but truncated calC, calC, and calC" genes. This subclone was not

able to confer resistance to calicheamicin. Next, a subclone containing the calC region

was constructed (pjT1232). This clone conferred calicheamicin resistance. See above

chart. Subclones containing calC (pAP6) and calC" (pREl) were constructed and tested

for calicheamicin resistance. These clones could not confer resistance to calicheamicin.

See above chart. To ascertain the amino acid sequence of CalC and learn its properties. calC was

cloned into a pMAL-C2 vector. (pMAL-C2 by itself could not confer calicheamicin

resistance. See above chart.) The resulting plasmid. pRE7, which contained calc.

conferred resistance to calicheamicin. See above chart. Plasmid pRE7 was then induced

with Isopropyl Beta-D-thiogalactoside ("IPTG") to overexpress CalC. Induced pRE7

conferred resistance to calicheamicin and produced a maltose-binding protein CalC fusion

protein (mbp-CalC). This resulting overexpression of CalC increased calicheamicin

resistance 10²-fold in vivo. See above chart.

Example 3: Expression of protein CalC

The protein mbp-CalC was overexpressed and purified for further analysis. The

mbp-CalC was purified from pRE7/E. coli to homogeneity as judged by SDS-PAGΕ. An

overnight LB culture (containing 50 mg ml^"1 ampicillin and 50 ng ml^"' calicheamicin from

a fresh pRΕ7/E. coli colony was grown at 37 °C, 250 rpm to an A₆₀₀=0.5, induced with 0.5

mM IPTG and growth continued overnight. Cells were harvested (4.000 x g, 4 °C, 20

minutes), resuspended in buffer A (50mM Tris-Cl, pH 7.5, 200 mM NaCl, ImM ΕDTA)

and disrupted by sonication. The cell debris was removed by centrifugation (5,000xg,

4°C, 20 minutes). The supernatant was applied to an amylose affinity column (1.5 x 7.0

cm, 1 mL min^"1). The desired mbp-CalC protein was eluted with buffer A containing 10

mM maltose. The eluate was concentrated and chromatographed on an S-300 column

(50mM Tris-Cl, pH 7.5, 200 mM NaCl). Active fractions were used immediately or

frozen at -80° C for storage. Example 4: Analysis of Protein CalC

The purified mbp-CalC was then analyzed for metal content. Purified mbp-CalC

displayed a yellow color in concentrated form and subsequent metal analysis, using

inductively coupled plasma atomic mass spectrometry ("ICP-MS^"). revealed the presence

of iron (Fe). Determination of the Fe stoichiometry, accomplished in conjunction with

quantitative amino acid hydrolysis, indicated 2.23 ± 0.3 mol Fe per mol mbp-CalC (based

upon the monomeric molecular weight of 63.576 dalton calculated from the known

nucleotide sequence of the mbp-calC gene fusion, which is consistent with the determined

subunit molecular weight determined by SDS-PAGE). The precise mbp-CalC

concentration was determined by quantitative amino acid hydrolysis by the Rockefeller

University Protein/DNA Technology Center. Trace metal content of an aliquot of the

hydrolysate was subsequently determined by ICP-MS on four distinct mbp-CalC

preparations with buffer alone and/or maltose-binding protein alone analyzed in parallel as

controls. These results were independently confirmed by methodologies used for

spectrophotometric iron determination. Fish, W.W., Meth. Enzymol 1988, 158, 357-364.

The electronic absorption spectrum of mbp-CalC is shown in Figure 4. In addition, to the

A_2g0 protein absorbance (<Ξ₂₈₀ = 99.300 M^"1 cm^"1), a clear absorbance maxima at 41 1 nm

(e_4U = 6.000 M^"1 cm^"1) can be observed. Electron para magnetic resonance ("EPR") was

performed to ascertain the metal content of CalC. The X-band EPR measurements on the

oxidized CalC proteins exposed a standard rhombic EPR signal at g = 4.3 (E/D =

0.33)(Figure 4, inset). The metal content was 90 ± 10 μM Fe (approximately 72 ± 10% of

total iron as seen by ICP-MS. Figure 4. The spectroscopic evidence indicates the presence of a mononuclear Fe^'3 center in CalC is consistent with the lack of cysteins in the primary

sequence of CalC. See Palmer. G.. Biochem. Soc. Trans. 1985, 13. 548-560.

Example 5: Verification of CalC's calicheamicin resistance

Given that calicheamicin leads to double strand DNA cleavage and CalC provides

calicheamicin-resistance in vivo, it was expected that the addition of CalC to an in vitro

calicheamicin-induced DNA cleavage assay would inhibit DNA cleavage. To test this

theory, preliminary assays were performed with supercoiled pBlusecript plasmid DNA

("pBS") as the template, and dithiothreitol ("DTT") as the reductive initiator. In a typical

assay, purified mbp-CalC (15.0 nM) and 30.0 nM calicheamicin were preincubated for 15

min. in a total volume of 25 μl 40 mM Tris-Cl, pH 7.5, at 37 °C. Then 2.5 μl lOmM DTT

stock solution was added to the assay solution, and the assay was incubated an additional 1

hour at 37°C. DNA fragmentation was assessed by electrophoresis on a 1% agarose gel

stained with ethidium bromide. Using this assay, it was found that mbp-CalC could

completely inhibit calicheamicin-induced DNA cleavage at concentrations nearing 10³-

fold excess of calicheamicin. Preincubation of mbp-CalC and DTT, protein removal via

forced dialysis, and the subsequent use of the DTT solution as reductant did not noticeably

affect the amount of DNA cleavage.

As indicated in Figure 4(b), no DNA cleavage was observed in the absence of DTT

or calicheamicin (lanes a and b), while efficient cleavage was demonstrated in the presence

of DTT and calicheamicin (lane c). As expected, the addition of mbp-CalC completely

inhibited calicheamicin-induced DNA cleavage (lane f) while the addition of mbp alone

(lane d) as a control, failed to inhibit calicheamicin-induced DNA cleavage. Furthermore, preincubation of mbp-CalC with DTT (not shown), or α/ -mbp-CalC (lacking the Fe

cofactor)(lane e). also failed to inhibit calicheamicin-induced DNA cleavage. However,

the addition of Fe ^'2 or Fe^"3 to the -vpo-mbp-CalC assay could reconstitute CalC activity

(lane g). Reconstitution of -vpo-mbp-CalC was accomplished by preincubation with 1 mM

FeSO₄ (Fe^{^2}) or FeCl₃ (Fe^*3) prior to the activity assay as previously described.

Example 6: Production of methymycin/pikromycin-calicheamicin hybrid compounds

The 1.2 kb calH gene was amplified by polymerase chain reaction (PCR) from

pJSTl 192 _pn7, which is a subclone containing a 7.0 kb Kpnl fragment of cosmid 13a. The

amplified gene was cloned into the EcoR /Xbal site of the expression vector pDHS617.

This expression vector contains an apramycin resistance marker. The plasmid pDHS617

was derived from pOJ1446 (Bierman. M. et al.. Gene 1992, 1 16, 43-49). A promoter

sequence from the S. Venezuela methymycin/pikromycin cluster was incorporated in the

plasmid to drive the expression of foreign genes in S. Venezuela. The resulting plasmid,

pLZ-C242 (containing the calΗ gene insert and the promoter sequence) was introduced by

conjugal transfer using E.coli S 17-1 into a previously constructed S. Venezuela mutant,

desl. (Borisova, S. et al., Org. Lett. 1999. 1. 133-136). In the Desl mutant, the desl was

replaced by the ncomycin resistance gene, which confers resistance to kanamycin The

PLS-C242-containing S. venezuela-Desl colonies were identified on the basis of their

resistance to apramycin antibiotic. One of these positive colonies, DesI/calH-1 was grown

in 100 ml of seed medium at 29 °C for 48 hours and then inoculated and grown in five

Liters of vegetative medium. Cane, D.E., et al., J. Am. Chem. Soc, 1993, 115, 522-526.

The culture was centrifuged to remove cellular debris and mycella. The supernatant was adjusted to pH 9.5 with concentrated KOH. followed by chloroform extraction. The crude

products (700 mg) were subjected to flash chromatography on silica gel using a gradient of

1-20% methanol in chloroform. A major product. 10-deoxymethynolide (ca. 400 mg). and

a mixture of two minor macrolide compounds were obtained. The two macrolides were

further purified by HPLC on a C,₈ column using an isocratic mobile phase of

acetonitrile/H₂O (1 : 1). They were later identified as compound (1 1) and compound

(12)(figure 7) by spectral anaylses.

Sequence listing 1 — calC gene:

ATGACTCAGGAGAAGACCGCACCGGCCGCGAAGAGCACGACCACCAAGAGCA

CCGCCGCGAAGAAGCCGAAGCCCCCGAACTACGACCCGTTCGTCCGGCACAG

CGTCACTGTCAAGGCCGACCGCAAGACCGCCTTCAAGACGTTCCTCGAAGGCT

TTCCGGAGTGGTGGCCGAACAACTTCCGCACCACCAAGGTCGGGGCCCCGCTG

GGCGTCGACAAGAAGGGCGGCCGCTGGTACGAGATCGACGAGCAGGGCGAGG

AGCACACCTTCGGCCTGATCCGGAAGGTGGACGAGCCGGACACGCTGGTCATC

GGCTGGCGGCTCAACGGCTTCGGCCGGATCGACCCGGACAACTCGAGCGAGTT

CACCGTGACCTTCGTGGCCGACGGCCAGAAGAAGACCCGGGTGGACGTCGAG

CACACCCACTTCGACCGGATGGGCACCAAGCACGCCAAGCGGGTCCGCAACG

GCATGGACAAGGGCTGGCCGACGATCCTCCAGTCGTTCCAGGACAAGATCGAC

GAGGAAGGGGCGAAGAAGTGA

Sequence Listing 2 —CalC protein:

(Note that in protein sequences amino acids are designated in one-letter code)

MTQEKTAPAAKSTTTKSTAAKKPKPPNYDPFVRHSVTVKADRKTAFKTFLEGFPE

WWPNNFRTTKVGAPLGVDKKGGRWYEIDEQGEEHTFGLIRKVDEPDTLVIGWRL

NGFGRIDPDNSSEFTVTFVADGQKKTRVDVEHTHFDRMGTKHAKRVRNGMDKG

WPTILQSFQDKIDEEGAKK

Sequence Listing 3 — calH gene:

GTGGCAACTAGCGAGAGGGGTGTCATGATCCCGCTGTCCAAGGTCGCCATGTC

TCCGGACGTCAGCACCCGCGTCTCCGCCGTCCTGAGCAGTGGCCGGCTGGAGC

ACGGGCCGACCGTCGCCGAGTACGAGGCGGCCGTGGGCAGTCGTATCGGCAA

CCCCCGGGTGGTCTCGGTCAACTGCGGCACGGCCGGGCTCCACCTGGCGCTGA

GCCTCGCCGCGCGGCCGGGGGCCGGCGAGTCGGAGCACGACGGCCCGGGCGA

GGTGCTCACCACGCCGCTGACCTTCGAGGGCACGAACTGGCCGATCCTCGCCA

ACGGGCTGCGCATCCGGTGGGTGGACGTCGACCCGGCCACCCTCAACATGGAC

CTCGACGACCTGGCCGCGAAGATCTCGCCCGCCACCCGGGCCATCGTGGTGGT

CCACTGGCTCGGCTACCCGGTGGACCTCAACCGGCTGCGCGCCGTCGTGGACC

GGGCCACGGCGGGATACGACCGCCGCCCGCTGGTCGTGGAGGACTGCGCGCA

GGCGTGGGGCGCCACCTACCGGGGCGCGCCGCTGGGCACGCACGGCAACGTC

TGCGTGTACAGCACCGGCGCGATCAAGATCCTGACGACCGGCAGCGGCGGCTT

CGTCGTGCTGCCCGACGACGACCTGTACGACCGGCTCCGGCTGCGCCGCTGGC

TCGGCATCGAGCGGGCGTCGGACCGGATC ACCGGCGACTACGACGTCGCCGA

GTGGGGCTACCGGTTCATCCTCAACGAGATCGGCGGGGCGATCGGCCTGTCCA

ACCTGGAACGCGTCGACGAGCTGCTGCGCCGGCACCGGGAGAACGCCGCGTT

CTACGACAAGGAACTGGCCGGCATCGACGGCGTCGAGCAGACCGAGCGGGCC

GACGACCGGGAGCCCGCGTTCTGGATGTACCCGCTGAAGGTCCGCGACCGTCC

CGCCTTCATGCGCCGGCTGCTCGACGCCGGCATCGCCACCAGCGTCGTGTCGC

GCCGCAACGACGCGCACAGCTGCGTCGCGTCGGCCCGCACCACCCTGCCCGGG

CTGGACCGGGTGGCGGACCGCGTGGTCCACATCCCGGTGGGCTGGTGGCTCAC

CGAGGACGACCGCTCCCACGTCGTCGAAACGATCAAGTCCGGCTGGTGA Sequence Listing 4 —CalH protein:

MATSERGVMIPLSKVAMSPDVSTRVSAVLSSGRLEHGPTVAEYEAAVGSRIGNPR

VVSVNCGTAGLHLALSLAARPGAGESEHDGPGEVLTTPLTFEGTNWPILANGLRIR

WVDVDPATLNMDLDDLAAKISPATRAIVVVHWLGYPVDLNRLRAVVDRATAGY

DRRPLVVEDCAQAWGATYRGAPLGTHGNVCVYSTGAIKILTTGSGGFVVLPDDD

LYDRLRLRRWLGIERASDRITGDYDVAEWGYRFILNEIGGAIGLSNLERVDELLRR

HRENAAFYDKELAGIDGVEQTERADDREPAFWMYPLKVRDRPAFMRRLLDAGIA

TSVVSRRNDAHSCVASARTTLPGLDRVADRVVHIPVGWWLTEDDRSHVVETIKS GW

Sequence Listing 5 — calG gene:

GTGCCCAGATCCCTGGTCACCGGCGGCTTCGGCTTCGTCGGCAGTCACGTCGT

CGAACGGCTGGTCCGCCGGGGTGACGAGGTCGTCGTCTACGACCTCGCCGACC

CGCCGCCCGACCTGGAGCACCCGCCGGGCGCGATCCGGCACGTCCGCGGCGA

CGTCCGGGACGCCGACGGGCTGGCGGCCGCCGCCACCGGCGTGGACGAGGTC

TACCACCTCGCGGCGGTCGTCGGCGTCGACCGGTACCTCAGCCGGCCGCTGGA

CGTGGTCGAGATCAACGTGGACGGCACCCGGAACGCGTTGCGCGCCGCACTG

CGCGCCGGTGCCCGGGTCGTGGTGTCCAGCACCAGCGAGGTGTACGGGCGCA

ATCCGCGGGTGCCGTGGCGGGAGGACGACGACCGGGTGCTCGGCAGCACGGC

GACGGACCGGTGGTCGTACTCGACGAGCAAGGCGGCGGCCGAGCACCTGGCC

TTCGCCTTCCACCGGCAGGAGGGCCTGCCGGTGACGGTGCTGCGGTACTTCAA

CGTCTACGGCCCACGCCAGCGCCCGGCGTACGTCCTCAGCCGCACCGTCGCCC

GCCTGCTGCGGGGCGTTCCGCCCGTGGTGTACGACGACGGCCGCCAGACGCGG

TGCTTCACCTGGATCGACGAGGCGGCCGAGGCGACCCTGCTGGCCGCCGCCCA

CCCGCGGGCCGTCGGCGAGTGTTTCAACATCGGCAGCAGCGTGGAGACCACC

GTCGCCGAGGCGGTCCGGCTGGCCGGCACGGTGGCCGGGGTGCCGGTGGCGG

CCCAGACCGCGGACACCGGAGCCGGGCTCGGCGCCCGCTACCAGGACATTCC

CCGCCGCGTACCGGACTGCGGCAAGGCCGCCGCGCTGCTGGACTGGCGGGCC

CGGGTGCCGCTGGTGACCGGCCTGCGCCGGACCGTCGAGTGGGCCCGCCGCA

ACCCGTGGTGGACCGCCCAGGCCGACGACGGACTGGTCGTCAGGTAG Sequence Listing 6 — CalG protein:

MPRSLVTGGFGFVGSHVVERLVRRGDEVVVYDLADPPPDLEHPPGAIRHVRGDV

RDADGLAAAATGVDEVYHLAAVVGVDRYLSRPLDVVEINVDGTRNALRAALRA

GARVVVSSTSEVYGRNPRVPWREDDDRVLGSTATDRWSYSTSKAAAEHLAFAFH

RQEGLPVTVLRYFNVYGPRQRPAYVLSRTVARLLRGVPPVVYDDGRQTRCFTWI

DEAAEATLLAAAHPRAVGECFNIGSSVETTVAEAVRLAGTVAGVPVAAQTADTG

AGLGARYQDIPRRVPDCGKAAALLDWRARVPLVTGLRRTVEWARRNPWWTAQ

ADDGLVVR

Claims

1. An isolated nucleic acid molecule from Micromonospora echinospora comprising

a nucleic acid sequence encoding for a gene from a nonchromoprotein enediyne

biosynthetic gene cluster, the protein coding region of said gene or a biologically

active fragment of said gene.

2. The isolated nucleic acid molecule of Claim 1 , wherein said gene is cal A. calB,

calC. calD, calE, calF, calG. calH. call. cali. calK. calL, calM. calN, calO. cal?.

calQ. calR, calS. caϊϊ, orfl. orfl. or 3, or 4, or 5, or/6, or 7, or an IS-element

gene.

3. The isolated nucleic acid molecule of Claim 1. wherein said molecule encodes two

or more of said genes.

4. The isolated nucleic acid molecule of Claim 1, wherein said molecule encodes the

full biosynthetic gene cluster.

5. The isolated nucleic acid molecule of Claim 1 , wherein said nonchromoprotein

enediyne is calicheamicin.

6. An isolated nucleic acid molecule capable of hybridizing with a nucleic acid from

Micromonospora echinospora spp. calichensis encoding for one or more genes

from a nonchromoprotein enediyne biosynthetic gene cluster.

7. The isolated nucleic acid molecule of Claim 6, wherein said molecule encodes a

protein having the activity of at least one gene from the biosynthetic gene cluster.

8. The isolated nucleic acid molecule of Claim 6, wherein said gene is cal A, calB,

calC, calD, calE, calF, calG, caM, call, cali, calK, calL, calM, caM, calO, cal?, calQ. calR. calS. call. orfl . orfl. or/3, or/4, or/5, or/6, orfl. or an IS-element

gene.

9. The isolated nucleic acid molecule of Claim 1. comprising SEQ ID No.l .

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. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for a P₄₅₀ oxidase from Micromonospora echinospora spp. calichensis.

13. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for a membrane transporter from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

14. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for an 0-methyltransferase from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

15. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for a glycosyltransferase from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

16. The isolated nucleic acid molecule of Claim 1, which encodes a polypeptide

encoding for a NN-dimethyltransferase from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

17. The isolated nucleic acid molecule of Claim 1, which encodes a polypeptide

encoding for a dipeptide transporter from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

18. The isolated nucleic acid molecule of Claim 1. which encodes a polypeptide

encoding for a L-cysteine/cystine C-S-lyase from a gene cluster of

Micromonospora echinospora spp. calichensis coding for calicheamicin

biosynthesis.

19. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for an oligopeptide transporter protein from a gene cluster of

Micromonospora echinospora spp. calichensis coding for calicheamicin

biosynthesis.

20. The isolated nucleic acid molecule of Claim 1. which encodes a polypeptide

encoding for a regulatory protein from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

21. The isolated nucleic acid molecule of Claim 1, which encodes a polypeptide

encoding for a hexopyranosyl-2-3-reductase from Micromonospora echinospora

spp. calichensis.

22. The isolated nucleic acid molecule of Claim 1, which encodes a polypeptide

encoding for a desaturase from a gene cluster of Micromonospora echinospora spp.

calichensis coding for calicheamicin biosynthesis.

23. The isolated nucleic acid molecule of Claim 1 , which encodes a polypeptide

encoding for an UDP-D-glucose 6-dehydrogenase from Micromonospora

echinospora spp. calichensis.

24. The isolated nucleic acid molecule of Claim 1, which encodes a polypeptide

encoding for a transcriptional regulator from a gene cluster of Micromonospora

echinospora spp. calichensis coding for calicheamicin biosynthesis.

25. An expression vector comprising a nucleic acid molecule encoding a protein

coding sequence, wherein the nucleic acid molecule is selected from any of Claims

1 through 24.

26. The expression vector of Claim 25, wherein said nucleic acid molecule is operably

linked to regulatory sequences to control expression of said protein.

27. The expression vector of Claim 26. wherein the regulatory sequence is a

Streptomyces promoter.

28. A host cell transformed with a nucleic acid molecule from any one of Claims 1

through 24.

29. A host cell transformed with a nucleic acid molecule from Claim 25.

30. A host cell transformed with a nucleic acid molecule from Claim 26.

31. The host cell of Claim 28. wherein said host cell is a bacterium, yeast, insect, plant,

fungi, or mammalian cell.

32. The host cell of Claim 28, wherein the host bacteria is E coli or Streptomyces.

33. A cosmid comprising an isolated nucleic acid molecule from Micromonospora

echinospora spp. calichensis, comprising a nucleic acid sequence encoding for a

nonchromoprotein enediyne biosynthetic gene cluster.

34. The cosmid of Claim 33, wherein said sequence encodes cal A, calB. calC, calD,

calE, calF, calG, calR, call, cal], calK, calL, calM, calN, calO, cal?, calQ, calR,

calS, call, orf , orfl, orβ, or 4, or 5, or/6, orfl, and an IS-element gene.

35. A method of expressing a protein comprising culturing a host cell with an

expression vector from Claim 25 for and incubating under time and conditions that

allow for protein expression.

36. The method of Claim 35. wherein said host cell is a bacterium, yeast, insect, plant,

fungi, or mammalian cell.

37. A method of purifying calicheamicin using affinity chromatography, comprising

providing a solution containing calicheamicin to an affinity column having CalC

bound thereto, and recovering calicheamicin.

38. A polypeptide comprising an amino acid sequence SEQ ID. No.: 2.

39. A polypeptide comprising an amino acid sequence SEQ ID. No.: 4.

40. A polypeptide comprising an amino acid sequence SEQ ID. No.: 6.

41. A method of conferring calicheamicin resistance to a subject comprising obtaining

cells from the subject, transforming the cells with the calicheamicin self resistance

gene and returning the cells to the subject.

42. A compound having the structure:

43. A compound having the structure:

;