WO1999055829A2

WO1999055829A2 - Process for preparing doxorubicin

Info

Publication number: WO1999055829A2
Application number: PCT/US1999/007016
Authority: WO
Inventors: Augusto Inventi Solari; Giovanna Zanuso; Silvia Filippini; Francesca Torti; Sharee Otten; Anna Luisa Colombo; Charles R. Hutchinson
Original assignee: Pharmacia & Upjohn S.P.A.
Priority date: 1998-04-24
Filing date: 1999-04-22
Publication date: 1999-11-04
Also published as: EP1071746A4; CA2326500A1; WO1999055829A3; CN1198930C; EP1071746A2; JP2002512784A; CN1298453A

Abstract

The ability to convert daunorubicin into doxorubicin can be improved by transforming a host cell with a recombinant vector comprising a DNA molecule comprising: a DNA region or fragment containing the gene doxA encoding daunorubicin 14-hydroxylase and a DNA region or fragment containing one or more gene conferring daunorubicin and doxorubicin resistance.

Description

Process for Preparing Doxorubicin.

Field of the Invention

The present invention concerns a process for improving daunorubicin to doxorubicin conversion by means of host cells transformed with recombinant vectors comprising DNA encoding a daunorubicin C-14 hydroxylase together with genes conferring resistance to anthracycline antibiotics. Background of the Invention

Anthracyclines of 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 New York, 1981 , pp. 12; 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, 1993).

Anthracyclines of the daunorubicin group are naturally occurring compounds produced by various strains of Streptomyces (S. peucetius, S.coeruieorubidus, S.galilaeus, S.griseus, S.g seoruber, S.insignis, S.viridochromogenes, S.bifurcus and S.sp. strain C5) and by Actinomyces carminata. Doxorubicin is mainly produced by strains of S. peucetius. In particular daunorubicin and doxorubicin are synthesized in Streptomyces peucetius ATCC 29050 and in S. peucetius subsp. caesius ATCC 27952. The anthracycline doxorubicin is made by S.peucetius 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-e-rhodomycinone), e-rhodomycinone, rhodomycin D, carminomycin and daunorubicin are established intermediates in this process. The final step in this pathway involves the C-14 hydroxylation of daunorubicin to doxorubicin.

Genes for daunorubicin biosynthesis have been obtained from S.peucetius 29050 and S.peucetius 27952 by cloning experiments (Stutzman-Engwall and Hutchinson, Proc.Natl.Acad.Sci.USA 86:3135,1988; Otten et al., J.Bacteriol. 172:3427, 1990).The gene encoding the daunorubicin 14-hydroxylase, which converts daunorubicin to doxorubicin has been obtained from S.peucetius 29050 and its mutants by cloning experiments and it was overexpressed in the host cells of Streptomyces species and Escherichia coli as described in WO 96/27014, publication date Sept.6,1996.

Two genes of the daunorubicin biosynthetic cluster, drrA and drrB, which confer doxorubicin and daunorubicin resistance to Streptomyces lividans have been cloned from S. peucetius ATCC 29050 strain (Guilfoile and Hutchinson, Proc.Natl.Acad.Sci.USA 88:8553, 1991 ) (Accession Number M73758 of Genbank) and from the S.peucetius 7600 mutant (EP-0371.112-A and Colombo et al., J.Bacteriol.174:1641 , 1992). These genes encode two translationally coupled proteins, both of which are required for daunorubicin and doxorubicin resistance in this host. The sequence of the predicted product of one of the two genes is similar to the products of other transport and resistance genes, most notably the P-glycoproteins from mammalian tumor cells. Another gene, drrC, which confers resistance to daunorubicin and doxorubicin with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA has been cloned from S.peucetius ATCC 29050 (Lomovskaya et al., J.Bacteriol.178:3238, 1996). Summary of the invention

The present invention provides a process for improving daunorubicin to doxorubicin conversion in host cells by means of recombinant vectors comprising a DNA region or fragment containing the gene dxrA encoding daunorubicin 14- hydroxylase together with a DNA region or fragment containing one, two or three genes, selected from the group consisting of drrA, drrB and drrC, conferring resistance to daunorubicin and doxorubicin. The last three genes confer a high level of resistance in the host cells to doxorubicin, the product of the conversion process, making the process more efficient than the previous one obtained using host cells transformed with the recombinant vectors carrying only the DNA fragment containing the dxrA gene, described in WO 96/27014, even when a strong promoter is used.

The DNA of the invention comprises preferably all three of the drrA, drrB and drrC genes or only the two drrA and drrB genes.

The DNA may be ligated to a heterologous transcriptional control sequence in the correct fashion or cloned into a vector at the restriction site appropriately located near a transcriptional control sequence in a vector. Typically, the vector is a plasmid. The recombinant vectors may be used to transform a suitable host cell. The host may be strains of Actinomycetes that do not or do produce anthracyclines, preferably strains of Streptomyces . Brief description of the drawings

Fig. 1 (a-c) illustrate the construction of the plasmid plS156 described in Example 1. This plasmid was constructed by insertion of the 2.9 kb fragment containing the doxA (formerly dxrA), the dnrV (formerly dnrORFIO) and the C-terminal part of the dnrU (Δdnril, formerly dnrORF9) genes, obtained from the recombinant plasmid plS70 (WO 96/27014 and A. Inventi Solari et al., GMBIM '96, P58), under the control of the strong promoter ermE^* (Bibb et al., Moiec. Microbiol. 14:533, 1994) into the plasmid pWHM3 (Vara et al., J. Bacteriol. 171 :5872, 1989). in order to better describe the invention, we provide the SEQ.ID. No:1 of 2.867 nt consisting of the doxA, dnrV and the C-terminal part of the dnrU (AdnrU) genes (complementary strand to the coding strand).

Fig. 2 (a-d) illustrate the construction of the plasmid plS284 described in Example 1. This plasmid contains the 2.9 kb fragment encompassing the doxA, the dπrVand the C-terminal part of the dnrU genes, obtained from the recombinant plasmid plS70, under the control of the strong promoter ermE* together with a DNA fragment of 2.3 Kb including the drrA and drrB resistance genes obtained from the plasmid pWHM603 (P. Guilfoile and CR. Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553, 1991 ) subcloned into the plasmid pWHM3.

Fig. 3 (a-c) illustrate the construction of the plasmid plS287 described in Example 2. Said plasmid was constructed by insertion of the 2.9 kb BamHI-Hindlll fragment containing the doxA formerly, dxrA), dnrV (formerly dnr-ORF10) and the C- terminal part of the dnrU (ΔdnrU, formerly, dnr-ORF9) genes, obtained from the recombinant plasmid plS70 (WO 96/727014), under the control of the strong promoter ermE^* together with the 2.3 kb Xbal-Hindlll DNA fragment containing the drrA and drrB resistance genes and the 3.9 kb EcoRI-Hindlll fragment containing the drrC resistance gene into the plasmid pWHM3.

The maps shown in Figs. 1 ,2 and 3 do not necessarily provide an exhaustive listing of all restriction sites present in the DNA fragments. However, the reported sites are sufficient for an unambiguous recognition of the DNA segments.

Restriction sites abbreviations: Ap, apramycin;ter, thiostrepton, amp, ampicillin; B, Bam , G, eg/I I; N, Λ/ofl; K, Kpn\; E, EcoRI; H, HindWV, P, Pst\; S, SphY, X, Xba\, L, Bgl\; T, Sst\.

Detailed description of the invention. The present invention provides a DNA molecule in which a DNA region or fragment containing the gene encoding a daunorubicin C-14 hydroxylase is joined to a DNA region or fragment containing one, two or three different genes selected from the group consisting of drrA, drrB, drrC genes encoding proteins conferring to the host cells resistance to daunorubicin and doxorubicin. The DNA region containing the gene encoding a daunorubicin C-14 hydroxylase is preferably the 2.9 kb DNA region obtained from the recombinant plasmid plS70 described in the patent WO 96/27014 by digestion with BamH\-Hind\\\ enzymes. This fragment contains the doxA gene, encoding the C-14 hydroxylase. Daunorubicin C-14 hydroxylase converts daunorubicin to doxorubicin. The 2.9 kb DNA fragment also comprises the dnrV gene between the Not\-Kpn\ sites and a Not\-Sph\ fragment containing the C-terminal part of the dnrU (ΔdnrU ) gene.

Preferably, this 2.9 kb DNA fragment encoding a daunorubicin C-14 hydroxylase was ligated to both the 2.3 kb Xba\-Hind\\\ DNA fragment containing the drrA and drrB resistance genes obtained from the plasmid pWHM603 and the 3.9 kb EcoRI-H/ndlll fragment containing the drrC gene obtained from the plasmid pWHM264; in another preferred embodiment, the 2.9 kb DNA fragment is ligated to the 2.3 kb Xba\ - Hind\\\ DNA fragment only.

All the DNA molecules encoding a daunorubicin C-14 hydroxylase described in WO 96/27014 may be employed in the present invention. In particular the DNA molecule of the present invention may comprise all of the 2.9 kb DNA fragment or only a part of the fragment, at least 1.2 kb in length corresponding to the Kpn\-BamH\ fragment containing the DNA molecule of doxA, encoding a daunorubicin C-14 hydroxylase, which converts daunorubicin to doxorubicin. This DNA molecule consists essentially of the sequence reported in the patent application WO 96/27014, which sequence is referred to as the "dxrA" sequence. Also, the deduced amino acid sequence of the daunorubicin C-14 hydroxylase is shown in that patent application.

The DNA molecule of the present invention may comprise at least 2247 nt of the 2.3 kb / al-/-//t7dlll DNA fragment containing the drrA and drrB genes encoding proteins conferring to host cells resistance to daunorubicin and doxorubicin.

The DNA molecule of the invention may comprise all or part of the 3.9 kb EcoRI- Hind\\\ fragment containing the drrC resistance gene, at least 2.5 kb in length corresponding to the Sst\-Sph\ fragment containing the DNA molecule of drrC, encoding a protein conferring to host cells resistance to daunorubicin and doxorubicin.

The present invention also includes DNA comprising genes conferring resistance to doxorubicin and daunorubicin having a sequence at least 80% identical to the sequences of the drrA and drrB genes (Guilfoile and Hutchinson,

Proc.Natl.Acad.Sci.USA 88:8553, 1991 ) and or drrC gene (Lomovskaya et al., J.Bacteriol.178:3238, 1996).

The DNA molecule of the invention may be ligated to a heterologous transcriptional control sequence in the correct fashion or cloned into a vector at a restriction site appropriately located near a transcriptional control sequence in the vector. Preferably the transcription of the different genes may be coordinated by a common strong promoter such as ermE*(Bibb et al., Molec. Microbiol. 14:533, 1994).

The DNA molecule of the invention may be ligated into any autonomously replicating and/or integrating agent comprising a DNA molecule to which one or more additional DNA segments can be added. Typically, however, the vector is a plasmid. A preferred plasmid is the high-copy number plasmid pWHM3 or plJ702 (Katz et al., J. Gen. Microbiol. 129:2703, 1983). Other suitable plasmids are plJ680 (Hopwood et al., Genetic Manipulation of Streptomyces. A laboratory Manual, John Innes Foundation, Norwich, UK.1985) and pWHM601 (Guilfoile and Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553, 1991 ).

Any suitable technique may be used to insert the 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 vector may be used to transform a suitable host cells that do not or do produce anthracyclines.

The host cells may be ones that are daunorubicin or doxorubicin sensitive, i.e., cannot grow in the presence of a certain amount of daunorubicin or doxorubicin, or that are daunorubicin or doxorubicin resistant. In any case the resulting recombinant clones obtained by transformation with the new recombinant vectors of the invention show higher level of resistance to daunorubicin and doxorubicin than the parental host. The level of doxorubicin resistance in recombinant S. lividans is much higher than the level observed in anthracycline producing strains S. peucetius ATCC 29050 and ATCC 27952. The host may be a microorganism such as a bacterium. Strains of

Actinomycetes, in particular strains of S. lividans and other strains of Streptomyces species that do not produce anthracyclines may be transformed. S. lividans TK 23 is a more suitable host in comparison to the S. peucetius dnrN mutant transformed with the recombinant plasmid plS70 containing the dxrA gene used for daunorubicin to doxorubicin bioconversion (WO 96/27014).

The recombinant vectors of the invention may also be used to transform a suitable host cell which produces daunorubicin, in order to enhance the conversion of daunorubicin to doxorubicin. S. peucetius ATCC 29050 and ATCC27952 strains including their mutants that produce anthracyclines may therefore be transformed. In particular S. peucetius strain WMH1654, a mutant strain obtained from S.peucetius ATCC 29050 and deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, under the accession number ATCC55936 may be used. Transformants of Streptomyces strains are typically obtained by protoplast transformation.

The invention includes processes for improving doxorubicin production by conversion of daunorubicin, which processes comprise a bioconversion process of added daunorubicin into doxorubicin in hosts which do not produce anthracyclines and a fermentation process for producing doxorubicin in hosts which directly produce daunorubicin.

Bioconversion process of daunorubicin to doxorubicin. This process comprises:

1 ) culturing the recombinant host cells not producing daunorubicin transformed with the vectors of the invention to which daunorubicin is added and

2) isolating doxorubicin from the culture.

In this process the recombinant strain may be cultured at temperatures from

20°C to 40°C, for example from 24°C to 37°C. The daunorubicin is added to the culture medium from 24 to 96 hours of the growth phase. The culture is preferably carried out with shaking. The duration of the culture in the presence of daunorubicin may be from

12 to 72 hours. The concentration of daunorubicin in the culture may be from 20 to

1000 mcg/ml; for example from 100 to 400 mcg/ml.

Doxorubicin production by fermentation.

This process comprises: 1 ) culturing recombinant daunorubicin-producing host cells transformed with the vectors of the invention and

2) isolating doxorubicin from the culture.

In this process the recombinant strain may be cultured at temperature from 20°C to 40°C; for example from 26°C to 34°C. The culture is carried out with shaking. The duration of the culture may be from 72 to 168 hours. Materials and Methods

Bacterial strains and plasmids: F. coli strain DH5α, which is sensitive to ampicillin and apramycin is used for subcloning DNA fragments. The host S. lividans TK23 was obtained from D. A. Hopwood (John Innes Institute, Norwich, United Kingdom) and the host S. peucetius WMH1654 is a mutant strain obtained from S.peucetius ATCC 29050 and has been deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, under the accession number ATCC55936.

The plasmid cloning vectors are pGem-7Zf(+) and related plasmids (Promega, Madison, WI), plJ4070 (D. A. Hopwood) and the E.coli-Streptomyces shuttle vector pWHM3 (Vara et al., J. Bacteriol. 171 :5872, 1989).

Media and buffer: E. coli strain DH5 is maintained on LB agar (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). When selecting for transformants, ampicillin or apramycin are added at concentrations of 100 micrograms/ml. S. lividans TK23 and S. peucetius WMH1654 are maintained on R2YE (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) and ISP4 (Difco, Detroit, Ml) agar media, respectively. When selecting for transformants, the plates are overlayed with soft agar containing thiostrepton at a concentration of 50 micrograms/ml.

Subcloning DNA fragments: DNA samples are digested with appropriate restriction enzymes and separated on agarose gels by standard methods (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 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, CA) 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, NY, 1989) into E. coli for routine manipulations, and E. coli-Streptomyces shuttle vectors or Streptomyces vectors for expression experiments.

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, NY, 1989) and transformed by standard techniques (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). S. lividans TK23 is grown in liquid 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 50 microlitres 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. (Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall and Hutchinson (Proc. Natl. Acad. Sci. USA. 86:3135, 1988) or Often et al. (J. Bacteriol. 172: 3427, 1990). After 17 hr of regeneration on R2YE medium at 30°C, the plates are overlayed with 200 micrograms/ml of thiostrepton and allowed to grow at 30°C until sporulated. Fvaluation of daunorubicin and doxorubicin resistance level: The level of resistance is expressed as Minimal Inhibitory Concentration (MIC) and is determined by the standard two-fold dilution method using R2YE medium. The strains are cultured in slants of R2YE medium and incubated at 28°C for 8-10 days. Recombinant strains are grown in the same medium added with 20 micrograms/ml of thiostrepton. Bacterial cultures containing approximately 10^6"10⁷ viable cells/ml are prepared from cultures grown at 28 °C at 280 rpm for 48 hours in Tryptic Soy Broth (Difco). The cultures are homogenized by glass beads. One loopful of the homogenized cultures is inoculated on the agar plates containing different concentrations of daunorubicin and doxorubicin from 0.39 to 800 micrograms/ml. The agar plates are incubated at 30°C for 7 days and the MICs are determined as the lowestconcentrations that prevent visible growth.

Daunorubicin to Doxorubicin bioconversion: S. lividans TK23 transformants harboring a plasmid of the invention are inoculated into 25 ml of liquid R2YE medium with 40 micrograms/ml of thiostrepton. Cultures are grown in 300 ml Erlenmeyer flasks and incubated on a rotary shaker at 280 rpm at 30 C°. 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), NaCI (2), 3-(morpholino)propanesulfonic acid (MOPS sodium salt) (15), MgS0₄ .7H₂0 (0.2), FeS0₄ .7H₂0 (0.01 ), ZnS0₄.7H₂0 (0.01), supplemented with 20 micrograms/ml of thiostrepton. 400 micrograms/ml of daunorubicin are added at 48 hr.of the growth phase. Cultures are grown in 300 ml Erlenmeyer flasks and incubated on a rotary shaker at 280 rpm at 30 C° for 72 hr. Each culture is acidified with 25 milligrams/ml of oxalic acid and after incubation at 30°C on a rotary shaker at 280 rpm for 30 min. is extracted with an equal volume of acetonitrile:methanol (1 :1) at 30°C and 300 rpm for 2 hr. The extract is filtered and the filtrate is analyzed by reversed-phasehigh pressure liquid chromatography (RP-HPLC). RP-HPLC is performed by using a Vydac C₁₈ column (4.6 x 250 millimeters; 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 H₂0 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 (bandwidth 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.

Doxorubicin production: The S. peucetius WMH1654 mutant is transformed with a plasmid of the invention. Transformants are inoculated into 25 ml of R2YE medium supplemented with 20 micrograms/ml thiostrepton. Cultures are grown in 300 ml Erlenmeyer flasks on a rotary shaker at 280 rpm at 30°C. After 2 days of growth, 2.5 ml of this culture are transferred to 25 ml of APM medium supplemented with 20 micrograms/ml thiostrepton. Cultures are grown in 300 ml Erlenmeyer flasks on a rotary shaker at 280 rpm at 28°C for 96 - 120 hours. Each culture is acidified with 25 milligrams/ml of oxalic acid and, after 45 min. incubation at 30°C on a rotary shaker at 280 rpm, is extracted with an equal volume of acetonitrile:methanol (1 :1 ) at 30°C and 300 rpm for 2 hr. The extract is filtered and the filtrate is analyzed by RP-HPLC following the same method used to analyze the bioconversion products.

Example 1

Example 1 (Fig. 1 (a-c) and Fig. 2 (a-d).

In order to remove a non-essential region, the plasmid plS70 (WO96/27014) is before digested EcoRI-Hindlll and the 3.5 kb fragment is subcloned into the same sites of the multiple cloning site sequence of the plasmid pGEM-7Zf (+) (Promega, Madison-WI USA) to obtain another BamHI restriction site. The new plasmid pGendoxAUV was BamHI digested and the fragment, now reduced to 2.9 kb, was transferred into the plasmid plJ4070 (from the John Innes Institute, Norwich, UK) under the control of strong promoter ermE*. This new plasmid, named p7doxAUV, was digested Bglll and the fragment inserted into the plasmid pWHM3 (J.Vara et al., J. Bacteriol. 171 :5872-5881 , 1989) to obtain the plasmid plS156 (fig. 1c). The 2.3 kb Bgll fragment containing the drrA and drrB resistance genes is transferred after blunt ending from the plasmid pWHM603 into the Smal site of the plasmid pBluescript II SK + (Stratagene) to obtain the plasmid pdrrAB and an Xbal-Hindlll fragment is transferred from pdrrAB into the vector plJ4070 to obtain plS278. Afterwards, plS278 is digested with EcoRI-Xbal and inserted into the EcoRI-Xbal plasmid pWHM3 to obtain the plasmid plS281. This plasmid is digested with Xbal and the Xbal fragment of plasmid plS156 is inserted to obtain the plasmid plS284.

Example 2

Construction of the plasmid ρlS287 (Fig.3 (a-c)): The drrC resistance gene contained in the plasmid pWHM264 is excised by EcoRI-H/πdlll digestion and inserted into the plasmid plJ4070 to obtain the plasmid plS282. From this plasmid, the drrC resistance gene is transferred as a BglW fragment to plS252 (this plasmid is a modified form of pWHM3 containing an extra BglW site close to the EcoRI site) to obtain the plasmid plS285. plS285 is EcoRI digested and ligated with the 5.5 kb DNA fragment excised from plasmid plS284 to obtain the plasmid plS287. Example 3

Resistance of the above recombinant plasmids to doxorubicin: The level of resistance to daunorubicin and doxorubicin of S. lividans TK23 transformed with the recombinant plasmids plS70, plS284 or plS287 in comparison with S. lividans TK23, S. lividans TK23 transformed with the vector pWHM3 and the anthracycline producing S. peucetius ATCC 29050 and ATCC 27952 strains is determined as MICs on R2YE medium following the procedure described in Materials and Methods. The maximum level of daunorubicin and doxorubicin resistance is obtained with the plasmid plS287 containing the drrA, drrB and drrC resistance genes. The level of doxorubicin resistance was increased 64 times also with the plasmid containing only the drrA and drrB. resistance genes (Table 1).

Table 1. Resistance of recombinant strains to doxorubicin.

Strain MIC for doxorubicin (micrograms/ml)

S. peucetius ATCC 29050 12.5 S. peucetius ATCC 27952 12.5

S. lividans TK23 12.5

S. lividans TK23(pWHM3) 12.5

S. lividans TK23(plS284) 800

S. lividans TK23(plS287) >800

Example 4

Bioconversion of added daunorubicin to doxorubicin in S. lividans TK23 transformed with plasmids containing the doxA daunorubicin C-14 hydroxylase gene together with different resistance genes: The plS70, plS284 or plS287 plasmids are introduced into S. / ividans TK23 by transformation with selection for thiostrepton resistance, according to the procedures described in the Materials and Methods section. The resulting S. lividans TK23(plS70), S. lividans TK23(plS284) and S. lividans TK23(plS287) transformants are tested for the ability to bioconvert a high level (400 micrograms/ml) of daunorubicin to doxorubicin using the APM medium as described above. S. lividans TK23(plS70) transformants can convert up to 11.5% of added daunorubicin to doxorubicin (Table 2). S. lividans TK23(plS284) and S. lividans TK23(plS287) transformants can convert up to 73.5% of added daunorubicin to doxorubicin (Table 2). Table 2. Bioconversion of daunorubicin to doxorubicin by S. lividans strains. Strain Anthracycline (micrograms/ml)

DOX DNR 13-dihydroDNR S. lividans TK23(plS70) (control) 46 250 70

S. lividans TK23(plS284) 294 33 21

S. lividans TK23(plS287) 288 24 35

Example 5

Doxorubicin production in the S. peucetius WMH1654 dnrX mutant transformed with plasmids containing the doxA daunorubicin C-14 hydroxylase gene together with different resistance genes: The plS284 and plS287 plasmids are introduced into S. peucetius WMH1654 dnrX mutant strain by protoplasts transformation with selection for thiostrepton resistance, according to the procedures described in the Materials and Methods section. The resulting S. peucetius transformants are fermented and the fermentation broths analyzed according to the method previously described. S. peucetius WMH1654(plS284) produced up to 81 micrograms/ml of doxorubicin and up to 18 micrograms/ml of daunorubicin after a 120 hr fermentation (Table 3). S.peucetius WMH1654(plS287) produced up to 92 micrograms/ml of doxorubicin and no detectable amount of daunorubicin (Table 3).

Table 3. Doxorubicin production by S. peucetius WMH1654 dnrX strains. Strain Anthracycline (micrograms/ml)

DOX DNR 13-dihydroDNR

S. peι/cetøγs WMH1654 41 35 18 S. peucetius WMH1654(plS284) 81 18 6

S. peucetius WMH1654(plS287) 92 0 0

SEQ ID.1

1 GGATCCGCAC CGGGTACACG GCACGGGACC GCCCACCGCG CGGTGCGCGG

51 TGGGCGGTCC CGTGCCGGTC GCGGCCGGCG GATCAGCGCA GCCAGACGGG

101 CAGTTCGGTG AGCCGCGCCG TCTGGGCCCC CTTCCGGCAC CACCGCAACT

151 CGTCGTACGG CACGGCCAGT CGGGCCTCGG GGAACCTGCT GCGCAGTACG

201 CCGATCATCG TGCGCGACTC CAGCTGGGCG AGCTGCTCCC CGATGCAGTA

251 GTGCGGCCCG TCGCCGAAGG TGAGCCGCCG CCACGAGGGA CGGTCCGGGT

301 GGAAGGCGTG CGGGGCGTCG TGATGGCGGC CGTCGGTGTT GGTGCCCTCG

351 ATGTCCACCA GCACCGGCGC TCCGCGGGGC AGCCGGACGC CGCCGATGGT

401 CACCTCCGTG GCAGCGAACC TCCACAACGT GTAGGGCACC GGCGGGTGGT

451 AGCGCAGCGC CTCCTCCACG AACCGGGAGA CGGCGTCCTC GTCGGCATCC

501 GCCGCGAGGC GGCCCGCCAG GACCTCCGCG AGCAGGAAGC CCAGGAAGGA

551 GCCGGTGGTG TCGTGGCCGG CGAAGATGAG CCCGGTGATC ATGTAGACGA

601 GCTGGTCGTC GGAGACCGAG CCGAACTCGG CCTGCGCGCG CTCGTACAGC

651 ACGCGGGTCA TGGTCGGGGT GTCGTTCCGC CGGGCTGAGT GCACGGCTTC

701 GAGGAGCAGG CTCTCCAGGG CCGAGGTGTC CGGCACGCCC CCGGCAGGGT

751 CCGTGCCGTC ACCCCCGCCG CTCTGCGGGC CGCCGAGGCC GAGTGCCTTG

801 AGAACGCTGA CGGCCTCGCG GGCCATCGCC GGATCGGTGA CCGGCACACC 851 GAGCAGCTCG CAGATGACCA ACAGCGGGAA GTGGTACGCG AAGCCGCCGA

901 TCAGCTCGGC CGGTTTGCCC GACCGGCCGG AGGCGTCGGC GAGTTCGGTG

951 AGCAGCCGGC CGGCGATCGC GGCGATGCGA TCCGTCCGCT CGGCCAGCCG

1001 GCGCGGGTTG AACGCAGGTG CGTGGATGCG GCGCAGGCGC CGGTGGGCCT

1051 CGCCGTCCAC GGCGATGAGC GTGAACGGAC GCAGCTCCGG AACGGGGATG

1101 TCGAGACCGT CGTCCACCCC CCGCCAGGCG GCGGGGGCGA GGTCGGGGTC

1151 CTTCACGAAC CGGGGATCGG CCAGCACCTC GCGGGCGAGG GCGTCATCGG

1201 TGATGACCCA GGCGGGTCCG CCCGCGGGGG CGTTCACCTC GACGACCGGG

1251 CCCGCCTCCC GGAAGGCGTC GTGCACCTCG GGCTTGCGCT GCATGGTCAT

1301 CATGGGACAC GCGAACGGGT CGACGGCCAC CCGGGGCGCC TCGCCGCTCA

1351 CGAGGCACCG CCCGCCGCCG CGGGGTACCC CTCCCGCAGT TCGACCACCG

1401 AGAAGCCGGC CCCGTGCGGG TCGAGCAGGT CCGCCCGCCG CCCCCTGGGC

1451 GTGTCGGCGG GCTCGTTCTC GACGGAGCCG CCGAGTTCAA CGGCGCGCCG

1501 GACCGTCGCG TCGCAGTCGT GCACGGCGAA CAGCACGGCC CAGTGCGGCC

1551 GTACCGCGCC GGTGACGCCC AGCTCCTGGG TGCCGGCGAC CGGTGTGTCA

1601 CCGATGTGCC AGACCGGGTC GGTGACGCCC TTCAGTCCGG TGTCGGCCGG

1651 AGCCAGGCCG AGGGTCGCCG GGTAGAAGTC CCGGGCGGCC CCGATGCCGT 1701 CGGTCACCAG CTCGACCCAG CCGACCGAGC CGGGCACGCC CGTCACCTCC

1751 GCGCCCTCCA TGACTCCCTT GCGCCAGACC GCGAACGCGG CCCCGGCGGG

1801 GTCGGCGAAG ACCGCCATCC GGCCGAGGCC GAGGACGTCC ATCGGAGTCA

1851 TGATGACCTC GCCGCCCGCC GTCTCGACCC GCTTGGTCAG TGCGTCGGCG

1901 TCGTCGGTGG CGAAGTACAC GGTCCAGATG GCCGGCATGC CGTGCTGGTC

1951 GTTCCCGGGC CCGTACGGCC GGTGGTAGGG GGTGTCGATC TGGTGGCGGG

2001 CGACCGCGGC GACCAGCTTC CCGTCGGAGC TGAACGTCGT GTATCCCCCG

2051 GCGCCCGGGT CGCTGACCAC GGTGGCGGTC CAGCCGAACA GGCCGGTGTA

2101 GAAGTCGGCC GAGGCGGCGA CATCGGGCGA ACCGAGGTCG AACCATGCGG

2151 GGGCGCCGGG CGCGAACCTG GTCACGAATC GTTCCTTTCG ATGGATCGGC

2201 ACACGAGCGT CTGCGCTCGC GGATGAGACG GACATCTCGC GGATGAGACG

2251 GACATGCGGG CGGGGCGGGC CGCCGCCGTC AGTGCGCGGT GTCGCCGACG

2301 GCGGCCGCGC CGGCCTCCCA GAGCTTCGCC GCGAGGCCGG CGTCGGCGGT

2351 CGGGCCGCTC ACCGGGGACA GCCGCCGGTC GCTGTAGTAG CCGCCCGTGG

2401 TCAACTCCTC GGCCGGCGCG GACGCCAGCC ACACGAGGGT GTCGGCGCCC

2451 TTCGCCGCGG AGCGCAGGAA GGGGTTGAAC CGGAAGTAGG ACGAGGCGAC

2501 CGTGCCCCGT CCGATGCGGG TGCGGACCTC ACCGGGGTGA TAGCTGACCG 2551 CCAGCACGTC CGGCCAGCGC CTGGCGGCCT CCGCCGCGGT CATGATGTTG

2601 GCCTGTTTGG ACGTGCCGTA CGCCTGGCCG GCGCTGTAGC GGTGACGGTC

2651 GCCGTTGAGG TCGTCCGGGT CGATCCGGCC CTGGGTGTAC GCGTCGGACG

2701 AGGTGAGGAT CAGCCGCCCG CCCGCGAGCC GCTCCCGCAG CAGCCGTGCC

2751 AGCAGGAAGC CTGCGAGGTG ATTGACCTGG ATGGTGGCCT CGAACCCGTC

2801 CTGGGTCGTG GTGCGCGACC AGAACATGCC GCCGGCGTTG CTGGCCATGA

2851 CATCGATGCG CGGGTACCGG

Claims

1. A DNA molecule comprising a DNA region containing a gene doxk encoding daunorubicin 14-hydroxylase and a DNA region containing at least one gene conferring daunorubicin and doxorubicin resistance.

2. A DNA molecule according to claim 1 , further comprising a strong promoter.

3. A DNA molecule according to claim 2, wherein said strong promoter is ermE*.

4. A DNA molecule according to claim 1 , wherein said gene conferring daunorubicin and doxorubicin resistance is selected from the group consisting of drrk, drrB and drrC genes and any mixtures thereof.

5. A DNA molecule according to claim 4, wherein said genes conferring daunorubicin and doxorubicin resistance are drrk and drrB genes.

6. The DNA molecule according to claim 4, wherein said genes conferring daunorubicin and doxorubicin resistance are drrk, drrB and drrC genes.

7. The DNA molecule according to claim 1 , wherein the region containing the gene doxA encoding daunorubicin 14-hydroxylase is 2.9 kb in length.

8. The DNA molecule according to claim 7, wherein the fragment containing the gene doxA corresponds to the Kpn\-BamH\ fragment containing the doxk nucleotide sequence.

9. The DNA molecule according to claim 5, wherein said region containing said drrk and ofrt╬▓ genes is a 2.3 kb Xba\-HindW\ DNA fragment.

10. The DNA molecule according to claim 1 , wherein said genes conferring daunorubicin and doxorubicin resistance are at least 80% identical to genes selected from the group consisting of drrA, drrB and drrC genes.

11. A vector containing a DNA molecule according to claim 1.

12. A vector according to claim 11 wherein said vector is a plasmid.

13. A plasmid according to claim 12, wherein said plasmid is selected from the group consisting of plS284 and plS287.

14. A host cell transformed or transfected with a vector according to claim 11.

15. The host cell according to claim 14, wherein said host cell does not produce daunorubicin.

16. The host cell according to claim 14, wherein said host cell is a bacterial cell which produces daunorubicin.

17. The recombinant host cell according to claim 14, wherein said host cell is a Streptomyces cell .

18. A process for bioconverting daunorubicin into doxorubicin, comprising the steps of: culturing a recombinant host cell in a culture medium containing daunorubicin, wherein said host cell contains a DNA molecule comprising a DNA region containing a gene doxA encoding daunorubicin 14-hydroxylase and a DNA region containing at least one gene conferring daunorubicin and doxorubicin resistance, wherein said host cell does not produce daunorubicin, and isolating any resulting doxorubicin from the culture medium.

19. A process for producing doxorubicin by fermentation, comprising the steps of: culturing a recombinant host cell in a culture medium, wherein said host cell contains a DNA molecule comprising a DNA region containing a gene doxA encoding daunorubicin 14-hydroxylase and a DNA region containing one or more genes conferring daunorubicin and doxorubicin resistance, wherein said host cell is a bacterial cell which produces daunorubicin, and isolating any resulting doxorubicin from the culture medium.