Process for Preparing Daunorubicin and Doxorubicin

The present invention concerns a process for improving daunorubicin and doxorubicin production by means of a recombinant strain bearing a mutation inactivating a gene of daunorubicin metabolism involved in the glycosylation of daunorubicin to acid-sensitive, baumycin-like compounds.

Anthracyclines of the daunorubicin group such as doxorubicin, carminomycin and aclacinomycin and their synthetic analogs are among the most widely employed agents in antitumoral therapy (F. Arcamone, Doxorubicin, Academic Press N.Y., 1981, pp. 12-25; A. Grein, Process Biochem., 16:34, 1981; T. Kaneko, Chimicaoggi May 11, 1988; C. E. Myers et al., "Biochemical mechanism of tumor cell kill" in Anthracycline and Anthracenedione-Based Anti-cancer Agents (Lown, J. W., ed.) Elsevier Amsterdam, pp. 527-569, 1988; J. W. Lown, Pharmac. Ther. 60:185-214, 1993). Anthracyclines of the daunorubicin group are naturally occurring compounds produced by various strains of Streptomyces and by Actinomyces carminata. Doxorubicin is mainly produced by strains of S. peucetius while daunorubicin is produced by many other Actinomycetes. In particular daunorubicin and doxorubicin are synthesized in Streptomyces peucetius ATCC 29050 and 27952 from malonic acid, propionic acid and glucose by the pathway summarized in Grein, Advan. Applied Microbiol. 32;203, 1987 and in Eckart and Wagner, J. Basic Microbiol. 28:137, 1988. Aklavinone (11-deoxy-.epsilon.-rhodomycinone), .epsilon.-rhodomycinone and carminomycin are established intermediates in this process. The final step in this pathway involves the hydroxylation of daunorubicin to doxorubicin, which is reported to occur only in S. peucetius species.

Daunorubicin is known to be converted to 4'-O-glycosides called baumycins in Streptomyces species (Y. Takahashi, H. Naganawa. T. Takeuchi, H. Umezawa, T. Komiyama, T. Oki and T. Inui. J. Antibiot. 30:622, 1977) thus decreasing also the amount of doxorubicin potentially obtainable through oxidation of daunorubicin. For recovering daunorubicin at the end of fermentation, baumycins are converted to daunorubicin by acid hydrolysis. However this process presents certain drawbacks in that the amount of doxorubicin thus produced is low and the process is complicated by the acid hydrolysis step. The present invention solves this problem by providing a mutated Streptomyces strain in which one of the genes responsible for the conversion of daunorubicin to baumycins has been insertionally inactivated.

The present invention provides a process for preparing daunorubicin and doxorubicin by means of a bacterial recombinant strain bearing a mutation inactivating the function of a gene of daunorubicin metabolism involved in the glycosylation of daunorubicin to acid-sensitive baumycin-like compounds. The inactivating mutation increases daunorubicin and doxorubicin production levels and causes the disappearance of baumycin-like products resulting in daunorubicin and doxorubicin secretion directly into the culture medium. Consequently, there is no need to acidify the cultures at the end of fermentation. Preferably the bacterial strain is a strain of Streptomyces sp. producing daunorubicin and also doxorubicin, having a mutation inactivating the function of a gene of daunorubicin metabolism. Said inactivated gene is preferably comprised in the DNA fragment having the configuration of restriction sites shown in FIG. 1 or in a fragment derived therefrom containing a gene, dnrX, encoding for a protein involved in the metabolism of daunorubicin to acid-sensitive, baumycin-like compounds. Thus, the present invention provides a mutant strain of S. peucetius, obtained from S. peucetius ATCC 29050, having a mutation inactivating the function of the dnrX gene. A cluster of genes for daunorubicin and doxorubicin biosynthesis and resistance have been obtained from S. peucetius 29050 and S. peucetius 27952 by cloning experiments as described in Stutzman-Engwall and Hutchinson (Proc. Natl. Acad. Sci. U.S.A. 86: 3135 (1988)) and Otten et al., (J. Bacteriol. 172: 3427 (1990)). As explained in more details in the following description, the dnrX mutant was obtained disrupting the dnrX gene of the anthracycline biosynthetic gene cluster by insertion of the neomycin/kanamycin resistance gene (aphII) in the NcoI restriction site of dnrX.

FIG. 1 is a restriction map analysis of the DNA of the invention. Said DNA is a BamHI fragment of 3.3 kb, containing the dnrX gene, obtained from the cosmid clone pWHM335 described in Stutzman-Engwall and Hutchinson (Proc. Natl. Acad. Sci. U.S.A. 86: 3135 (1988)) and Otten et al., (J. Bacteriol. 172: 3427 (1990)). The fragment was inserted into the unique BamHI restriction site of the polylinker region of plasmid pUC19 (Yanish-Perron C. et al., Gene 33:103-119 (1985)). The map shown in FIG. 1 does not necessarily provide an exhaustive listing of all restriction sites present in the DNA fragment. However, the reported sites are sufficient for an unambiguous recognition of the DNA segment. [Restriction site abbreviations: Ba, BamHI; Nc, NcoI; Sc, ScaI; Xm, XmaI; Aw, AlwNI; Bg, BglII; Pv, PvuI.]

FIG. 2 is a representation of plasmid pWHM225 in which the 4.3 kb EcoRI-NindIII fragment from plasmid pWHM223 is inserted in the EcoRI-HindIII sites of plasmid pKC1139. The construction of plasmid pWHM225 is described in Example 1. The map shown in FIG. 2 does not necessarily provide an exhaustive listing of all restriction sites present in the DNA molecule.

SEQ ID NO:1 is a schematic illustration of the dnrX DNA nucleotide sequence. Said DNA corresponds to that encoding a protein required for daunorubicin metabolism. The sequence covers the region between the ScaI and the BamHI restriction sites and shows the coding strand in the 5' to 3' direction. The derived amino acid sequence of the translated open reading frame encoding a protein required for daunorubicin glycosylation is shown under SEQ ID NO:2.

The present invention provides a bacterial recombinant strain bearing a mutation inactivating the function of the daunorubicin metabolism gene dnrX involved in the glycosylation of daunorubicin to acid-sensitive baumycin-like compounds.

The bacterial strain may be one that is daunorubicin- or doxorubicin-sensitive, i.e. cannot grow in the presence of a certain amount of daunorubicin or doxorubicin, or that is daunorubicin- or doxorubicin-resistant. Strains belonging to the Streptomyces genus constitute a preferred embodiment of the invention; a Streptomyces peucetius strain constitutes a particularly preferred embodiment of the invention. Most preferred is the S. peucetius strain WMH1654. The strain WMH1654 has been deposited at the American Type Culture Collection, Rockville, Md., USA, under the accession number ATCC 55936.

Strain WMH1654 has been obtained from the S. peucetius ATCC 29050 strain by replacing the dnrX gene with a mutated dnrX gene insertionally inactivated by introduction of the neomycin/kanamycin resistance gene (aphII) from pFDNEO-S plasmid described by F. Danis and R. Brzezinski, (FEMS Microbiology Letters 81:261-264 (1991)). The aphII gene has been inserted at the NcoI site of dnrX to disrupt its function, as better explained in Example 1.

The expert in the art will recognize that any other technique to inactivate the gene can be suitably employed in the present invention.

The bacterial recombinant strain may be any other microorganism transformed with plasmids or transfected with phage DNA containing an anthracycline gene cluster able to produce daunorubicin and/or doxorubicin and/or baumycins.

In another aspect, the present invention provides a process for preparing daunorubicin and doxorubicin, which process comprises:

(i) culturing a bacterial recombinant strain of the invention, and

(ii) isolating daunorubicin and doxorubicin from the culture.

In this process the bacterial recombinant strain may be cultured at from 20 to 40.degree. C., for example from 26 to 37.degree. C. The culture is preferably carried out with agitation. In order to obtain the bacterial recombinant strain of the invention, the dnrX gene was isolated from clones described in Stutzman-Engwall and Hutchinson, (Proc. Acad. Sci. USA 86:3135 (1989) and Otten et al., (J. Bacteriol. 172:3427 (1990). The gene dnrX is contained in a 3.3 kb DNA fragment obtained by digestion of the clone pWHM335 with the restriction endonuclease BamHI.

The dnrX gene consists essentially of the sequence of SEQ. ID NO. 1, which sequence will be referred to as the "dnrX" sequence. The deduced amino acid sequence of the daunorubicin and doxorubicin metabolism protein encoded by SEQ. ID NO. 1 is shown in SEQ. ID NO. 2. The isolated dnrX gene was subsequently subcloned into an appropriate DNA cloning vector. Any autonomously replicating and/or integrating agent comprising a DNA molecule to which one or more additional DNA segments can be added may be used. Typically, however, the vector is a plasmid. Preferred plasmids are pUC19 (Yanish-Perron et al., Gene 33: 103 (1985)) and pWHM3 (Vara et al., J. Bacteriol. 171:5872 (1989)). Any suitable technique may be used to insert the insert DNA into the vector. Insertion can be achieved by ligating the DNA into a linearized vector at an appropriate restriction site. For this, direct combination of sticky or blunt ends, homopolymer tailing, or the use of a linker or adapter molecule may be employed. The recombinant plasmid is then digested with a suitable restriction enzyme and ligated with the aphII gene. This construction is transferred into a suitable vector for homologous integration. Among the possible vectors that can be used, pKC1139 (M. Bierman et al., Gene 116:43-49 (1992)) is preferred. This is an E. coli-Streptomyces shuttle vector that contains a temperature-sensitive replicon that functions well at temperature below 34.degree. C. and bears the apramycin resistance gene. The recombinant vector thus obtained is used to transform, typically by protoplast transformation, an appropriate Streptomyces strain; the final step in this inactivation protocol is the isolation of kanamycin resistant transformant in which the recombinant plasmid has recombined with the dnrX gene and inactivated it (see also Example 1).

On the basis of the information provided herein, the expert in the art can easily obtain the 3.3 kb BamHI DNA fragment by:

a) preparing a library of the genomic DNA of S. peucetius 29050 or a strain derived therefrom;

b) screening the library for clones positive to a labelled probe, of at least 24 nucleotides, synthesized according to the sequence of SEQ ID NO: 1;

c) obtaining an insert DNA, from a recombinant vector, that forms part of the library and that has been screened as positive for the ability to convert daunorubicin into the acid-sensitive metabolite form in the S. peucetius dnrX mutant.

To obtain the DNA fragment, the library may be prepared in step a) by partially digesting the genomic DNA of S. peucetius 29050 or a strain derived therefrom; or by screening a library of Streptomyces genomic DNA that has been enriched for the cluster of daunorubicin/doxorubicin biosynthesis genes. Generally the restriction enzyme MboI is preferably used for genomic DNA, but for the libraries containing the cluster of daunorubicin biosynthesis genes, the restriction enzyme BamHI is preferred. The DNA fragments thus obtained can be size fractionated; fragments from 1 to 7 kb in size are preferred for libraries containing the cluster of daunorubicin and doxorubicin biosynthesis genes. These fragments are ligated into a linearized vector such as pWHM3 or pKC505 ((M. A. Richardson et al, Gene 61:231 (1987)). E.coli DH5.alpha. and DH1 are respectively transformed or transfected with the ligation mixtures.

In step b) the colonies obtained by the transformantion are transferred to nylon membranes and screened by colony hybridization for plasmids or cosmids which hybridized to the labelled probe, of at least 24 nucleotides, synthesized according to the sequence of SEQ ID No. 1

In step c) plasmid DNA from the clones which hybridized to the probe is isolated and used to transform protoplasts of host cells. The hosts may be microorganisms that do not produce acid-sensitive, baumycin-like products. The S. peucetius dnrX mutant strain (ATCC 55936) that does not produce acid-sensitive, baumycin-like compounds represents a particularly suitable host. Clones containing DNA fragments which include the 3.3 kb BamHI DNA fragment of the invention are recognized by the appearance in non-acidified fermentation cultures of acid-sensitive, baumycin-like compounds.

Bacterial strains and plasmids:

E. coli strains DH5.alpha. or JM109, which are sensitive to ampicillin and apramycin, are used for subcloning DNA fragments. S. peucetius dnrX mutant that does not produce acid-sensitive daunorubicin metabolites is used for expression of the dnrX gene. The plasmid cloning vectors are pUC18/19 ((Yanish-Perron et al., Gene 33:103 (1985)) and pWHM3 (Vara et al., J. Bacteriol. 171:5872 (1989)). The vector for homologous integration is pKC1139 (M. Bierman et al., Gene 116:43-49 (1992)), a shuttle E. coli-Streptomyces vector. The pFDNEO-S plasmid described by F. Danis and R. Brzezinski (FEMS Microbiology Letters 81:261-264 (1991)) is used to get the neomycin/kanamycin resistance gene (aphII).

Media and buffer:

E. coli strains DH5.alpha. and JM109 are maintained on LB agar (Sambrook et al., Molecular cloning. A laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). When selecting for transformants, ampicillin or apramycin are added at concentrations of 100 micrograms/ml and 50 micrograms/ml, respectively. S. peucetius dnrX is maintained on ISP4 agar (Difco Laboratories, Detroit, Mich.) for the preparation of spores and on R2YE agar (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) for regeneration of protoplasts. When selecting for transformants, the plates are overlayed with soft agar containing 350 micrograms/ml apramycin corresponding to a final concentration in the plates of 40 micrograms/ml apramycin.

Subcloning DNA fragments:

DNA samples are digested with appropriate restriction enzymes and separated on an agarose gel by standard methods (Sambrook et al., Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Agarose slices containing DNA fragments of interest are excised from a gel and the DNA is isolated from these slices using the GENECLEAN device (Bio101, La Jolla, Calif.) or an equivalent. The isolated DNA fragments are subcloned using standard techniques (Sambrook et al., Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989) into E. coli for routine manipulations, including DNA sequencing, and E. coli-Streptomyces shuttle vectors or Streptomyces vectors for expression experiments and fermentations.

DNA sequencing of the dnrX gene.

In order to sequence the fragment containing the gene of interest, the following synthetic oligonucleotide primers for pUC19 have been synthesized:

pUC Sequencing Primer (-47) 24-mer:

PUC Reverse Sequencing (-48) 24-mer:

The sequence determination has been performed simultaneously on both strands.

Transformation of Streptomyces species and E. coli:

Competent cells of E. coli are prepared by the calcium chloride method (Sambrook et al., Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989) and transformed by standard techniques (Sambrook et al., Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). S. peucetius 29050 or S. peucetius dnrX mycelium is grown in R2YE medium (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) and harvested after 48 hr. The mycelial pellet is washed twice with 10.3% (wt/vol) sucrose solution and used to prepare protoplasts according to the method outlined in the Hopwood manual (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985). The protoplast pellet is suspended in about 300 microlitres of P buffer (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) and a 50 microlitre aliquot of this suspension is used for each transformation. Protoplasts are transformed with plasmid DNA according to the small scale transformation method of Hopwood et al.. (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall and Hutchinson, (Proc. Natl. Acad. Sci. U.S.A. 86: 3135 (1988) or Otten et al. (Otten et al., J. Bacteriol. 172:3427 (1990)). After 17 hr of regeneration on R2YE medium at 30.degree. C., the plates are overlayed with 40 micrograms/ml of apramycin and allowed to grow at 30.degree. C. until sporulated.

Doxorubicin and daunorubicin production:

S. peucetius dnrX mutant strain is inoculated into 25 ml of liquid R2YE medium with 40 micrograms/ml of kanamycin sulfate in a 300 ml flask and incubated at 30.degree. C. and 300 rpm on a rotary shaker. After 2 days of growth 2.5 ml of this culture are transferred to 25 ml of APM production medium: ((g/l) glucose (60), yeast extract (8), malt extract (20), NaCl (2), 3-(morpholino)propanesulfonic acid (MOPS) sodium salt (15), MgSO.sub.4 0.7H.sub.2 O (0.2), FeSO.sub.4 0.7H.sub.2 O (0.01), ZnSO.sub.4 0.7H.sub.2 O (0.01), and incubated in a 300 ml flask at 30.degree. C. and 300 rpm on a rotary shaker for 120 hr. Each culture is divided in two aliquots of 10 g each: one is acidified with 250 mg oxalic acid and incubated at 30.degree. C. overnight and then extracted. The other is immediately extracted. Cultures are extracted with an equal volume of acetonitrile:methanol (1:1) at 30.degree. C. and 300 rpm for 2 hr. The extract is filtered and the filtrate is analyzed by reversed-phase high pressure liquid chromatography (RP-HPLC). RP-HPLC is performed by using a Vydac C.sub.18 column (4.6.times.250 mm; 5 micrometers particle size) at a flow rate of 0.385 ml/min. Mobile phase A is 0.2% trifluoroacetic acid (TFA, from Pierce Chemical Co.) in H.sub.2 O and mobile phase B is 0.078% TFA in acetonitrile (from J: T: Baker Chemical Co.). Elution is performed with a linear gradient from 20 to 60% phase B in phase A in 33 minutes and monitored with a diode array detector set at 488 nm (bandwith 12 micrometers). Daunorubicin and doxorubicin (10 micrograms/ml in methanol) are used as external standards to quantitate the amount of these metabolites isolated from the cultures.

Disruption of dnrX:

pWHM223 is constructed by subcloning the 3.3 kb BamHI fragment obtained from the pWHM335 cosmid clone, described by Stutzman-Engwall and Hutchinson ((Proc. Acad. Sci. USA 86:3135 (1989)) and Otten et al. (J. Bacteriol. 172:3427 (1990)), in the BamHI site of pUC19. This recombinant plasmid is NcoI digested, filled in with the Klenow fragment and ligated with the blunt-ended SalI aphII gene from the pFDNEO-S plasmid described by F. Danis and R. Brzezinski, (FEMS Microbiology Letters 81:261-264 (1991)). This construction is transferred as an EcoRI-HindIII fragment into plasmid pKC1139 (M. Bierman et al., Gene 116:43-49 (1992)) to obtain pWHM225 (FIG. 2). The pKC1139 vector contains a temperature-sensitive replicon that functions well at temperature below 34.degree. C. and bears the apramycin resistance gene. Protoplasts of 29050 strain are transformed with pWHM225, and transformants are selected with apramycin sulfate (40 micrograms/ml) at 30.degree. C. Colonies from this transformation are transferred to ISP4 agar supplemented with kanamycin sulfate (40 micrograms/ml) and incubated at 39.degree. C. for 7 days to eliminate the autonomous replicating vector and to select transformants in which homologous recombination between the cloned DNA and the chromosome has occurred. Three of these cultures are isolated on ISP4 agar with kanamycin sulfate (40 micrograms/ml) and incubated at 39.degree. C. for 7 days. Plates with about 100 colonies are replicated on ISP4 with apramycin sulfate (40 micrograms/ml) and incubated at 39.degree. C. for 7 days. Colonies that don't grow on apramycin are selected from kanamycin plates and checked in fermentation in presence of kanamycin. Two colonies with the kanamycin-resistance, apramycin-sensitive phenotype are examined by Southern analysis to verify the disruption of the dnrX gene. Chromosomal DNA from the 29050 strain and the two dnrX mutants is digested with BamHI and probed with a 500 bp internal fragment of the aphII gene from the pFDNEO-S. The probe hybridizes to 4.3 kb fragment for the two dnrX mutants, which is consistent with the insertion of the aphII resistance gene in the dnrX gene.

Enhanced doxorubicin and daunorubicin production and baumycin-like compounds disappearance in fermentation broth of dnrX mutant.

The dnrX mutant is grown for 10 days at 30.degree. C. on slants of ISP4 agar medium added with 40 micrograms of kanamycin sulfate. The spores of this culture are collected and suspended in 300 ml Erlenmeyer flasks containing 25 ml of R2YE liquid medium containing 40 micrograms of kanamycin sulfate and the flasks are shaken for 2 days at 30.degree. C. on a rotary shaker running at 300 rpm in a 5 cm diameter circle. 2.5 ml of this culture are used to inoculate 25 ml of APM medium containing 40 micrograms of kanamycin sulfate in 300 ml Erlenmeyer flasks. The flasks are incubated at 30.degree. C. for 120 hr under the same conditions described for the seed cultures. The metabolites are extracted from the cultures according to the methods described in the Materials and Methods section. The production values are indicated in Tables 1, A and B wherein R.T. stands for Retention Time.

Complementation of the dnrX mutation with the wild-type dnrX gene

To confirm that the disappearance of the baumycin-like products in the fermentation broth is due to dnrX disruption, the dnrX mutant is transformed with the pWHM226 containing, in the BamHI site of the polylinker of pWHM3, the 3.3 kb fragment of the invention including the dnrX. pWHM226 is introduced in the S. peucetius dnrX mutant by the protoplast-mediated transformation method described above, using thiostrepton (50 .mu.g/ml) for selection of the recombinant strains. Transformants are checked for doxorubicin and daunorubicin production, in presence of thiostrepton (10 .mu.g/ml), according to the fermentation conditions described in Materials and Methods. Extracts of the non-acidified fermentation broths of two transformants show in RP-HPLC the appearance of the two peaks with retention times of 35.9 and 39.3 min, respectively, corresponding to the two acid-sensitive, baumycin-like compounds, while extracts of the dnrX mutant fermentation broth obtained under the same conditions do not show these peaks.