CN1198930C - Process for preparing doxorubicin - Google Patents

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

The method for preparing doxorubicin

The present invention relates to a method for improving daunorubicin to doxorubicin transformation by host cells transformed with a recombinant vector comprising DNA encoding daunorubicin C-14 hydroxylase and endowing anthracycline Antibiotic resistance genes.

Daunorubicin anthracyclines (such as doxorubicin, carmine and aclarmycin and their synthetic analogues) are the most widely used agents in antineoplastic therapy (F. Arcamone, Doxorubicin (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., based on anthracycline "Biochemical mechanism of tumor cellkill" in Anthracycline and Anthracenedione-Based Anti-cancer Agents (Lown, J.W. ed.), Elsevier Amsterdam, pp.527～ 569, 1988; J.W. Lown, Pharmac Therapy (Parmac. Ther., 60:185, 1993).

Daunorubicin anthracyclines are usually produced by various Streptomyces (S.peucetius), S.coeruleorubidus, S.galilaeus ), S. griseus, S. griseoruber, S. insignis, S. viridochromogenes, S. bifurcus and Streptomyces strain C5 ( S.sp.strainC5)] strains and natural compounds produced by Actinomyces carminata. Doxorubicin is mainly produced by strains of Streptomyces persay. Specifically, daunorubicin and doxorubicin are produced in S. peucetius subsp. caesius ATCC 27952 and S. peucetius subsp. caesius. The anthracycline doxorubicin is produced by Streptomyces persei 27952 from malonate, propionate and glucose by methods outlined in: Grein, Advan. Applied Microbiol., 32:203, 1987 and Eckart and Wagner, J. Basic Microbiol. 28:137,1988. Acroquinone (11-deoxy-ε-rhodomycin), ε-rhodomycin, rhodomycin D, carmine and daunorubicin are all intermediates produced in this process. The final step in this pathway involves the C-14 hydroxylation of daunorubicin to doxorubicin.

The gene for daunorubicin biosynthesis was obtained by cloning experiments from S. persei 29050 and S. persais 27952 (Stutzman-Engwall and Hutchinson, Proc. Natl. Acad. Sci. USA ) 86:3135, 1988; Otten et al., J. Bacteriol. 172:3427, 1990). As described in WO96/27014 (published September 6, 1996), the gene encoding daunorubicin C-14 hydroxylase (which converts daunorubicin to doxorubicin) was obtained by cloning experiments It is obtained from Streptomyces persei 29050 and its mutants, and it is overexpressed in Streptomyces and Escherichia coli host cells.

Two genes in the daunorubicin biosynthetic cluster (drrA and drrB, which confer resistance to doxorubicin and daunorubicin in Streptomyces lividans) were obtained from S. persei ATCC 29050 strain ( Guilfoile and Hutchinson, Proceedings of the National Academy of Sciences USA 88:8553, 1991) (Genbank Accession No. M73758) and mutants from Streptomyces persay 7600 (EP-0371,112-A and Colombo et al., J. Bacteriology, 174:1641, 1992) cloned. 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 these two genes is similar to that of other transporter and resistance genes (most notably P-glycoprotein from mammalian tumor cells). Another gene, drrC (which confers resistance to daunorubicin and doxorubicin), has a high degree of sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in DNA excision repair sex) was cloned from S. persay ATCC 29050 (Lomovskaya et al., J. Bacteriol. 178:3238, 1996).

The invention provides a method for improving daunorubicin to doxorubicin transformation in host cells through a recombinant vector, the recombinant vector comprising: a DNA region containing a gene dxrA encoding daunorubicin c-14 hydroxylase or fragments and DNA regions or fragments 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 high levels of resistance in the host cell against doxorubicin (the product of the transformation process), making the process more efficient than previous ones, which used recombinant vectors (carrying only dxrA containing DNA fragment of the gene) transformed host cells (described in WO96/27014, even if a strong promoter is used).

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

The DNA can be ligated in the correct form to a heterologous transcriptional control sequence or cloned into the vector at restriction sites suitably located near the transcriptional control sequence in the vector. Typically, the vector is a plasmid. The recombinant vector can be used to transform suitable host cells. The host may be a strain of Actinomycetes (which does not or does not produce anthracyclines), preferably a strain of Streptomyces.

Figures 1 (a-c) illustrate the construction of the plasmid pIS156 described in Example 1. This plasmid was obtained by inserting a 2.9 kb fragment into plasmid pWHM3 (Vara et al., Journal of Bacteriology 171:5872) under the control of the strong promoter ermE ^* (Bibb et al., Molec. Microbiol.) 14:533,1994). , 1989), said fragment contains the C-terminal part of doxA (formerly dxrA), dnrV (formerly dnrORF10) and dnrU (ΔdnrU, formerly dnrORF9) genes, obtained from recombinant plasmid pIS70 (WO 96/27014 and A. Inventi Solari et al., GMBIM '96, P58).

To better describe the present invention, we provide Sequence 1 of 2.867 nt, which contains the C-terminal part of the doxA, dnrV and dnrU (ΔdnrU) genes (complementary strand of the coding strand).

Figure 2(a-d) illustrates the construction of plasmid pIS284 described in Example 1. This plasmid consists of a 2.9 kb fragment, which possesses the C-terminal part of the doxA, dnrV and dnrU genes, obtained from the recombinant plasmid pIS70, under the control of the strong promoter ermE ^* , and a 2.3 Kb DNA fragment containing the drrA and drrB resistance genes, The drrA and drrB resistance genes were obtained from plasmid pWHM603 subcloned into plasmid pWHM3 (P. Guilfoile and CR Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553, 1991).

Figure 3(a-c) illustrates the construction of plasmid pIS287 described in Example 2. The plasmid was obtained by inserting the 2.9 kb BamHI-HindIII fragment (which contained doxA (formerly dxrA), dnrV (formerly dnr-ORF10) and dnrU (ΔdnrU, formerly dnr-ORF10) under the control of the strong promoter ermE ^* C-terminal portion of ORF9) gene obtained from recombinant plasmid pIS70 (WO 96/727014)) with a 2.3 kb Xbal-HindIII DNA fragment containing the drrA and drrB resistance genes and a 3.9 kb EcoRI-HindIII fragment containing the drrC resistance gene It was constructed by inserting the plasmid pWHM3 together.

The diagrams shown in Figures 1, 2 and 3 are not necessarily exhaustive listings of all restriction sites present in the DNA fragments. However, the reported positions are sufficient to unambiguously identify the DNA segment.

Restriction site abbreviations: Ap, apramycin; tsr, mossycin; amp, ampicillin; B, BamHI; G, BgIII; N, NotI; K, KpnI; E, EcoRI; H, HindIII; P, PstI ; S, SphI; X, XbaI; L, BglI; T, SstI.

The present invention provides a DNA molecule wherein a DNA region or fragment comprising a gene encoding daunorubicin C-14 hydroxylase is linked to a gene comprising one, two or three genes selected from drrA, drrB and drrC On DNA regions or fragments of different genes that encode proteins that confer resistance to daunorubicin and doxorubicin in host cells.

The DNA region comprising the gene encoding daunorubicin C-14 hydroxylase is preferably the 2.9 kb DNA region obtained from recombinant plasmid pIS70 (described in patent WO96/27014) by digestion with BamHI-HindIII enzyme. This fragment contains the doxA gene encoding C-14 hydroxylase. Daunorubicin C-14 hydroxylase converts daunorubicin to doxorubicin. This 2.9 kb DNA fragment also contained the dnrV gene between the NotI-KpnI sites and the NotI-SphI fragment comprising the C-terminal portion of the dnrU (ΔdnrU) gene.

Preferably, the 2.9 kb DNA fragment encoding daunorubicin C-14 hydroxylase is ligated to a 2.3 kb Xbal-HindIII DNA fragment containing the drrA and drrB resistance genes from plasmid pWHM603 and containing the drrC gene from plasmid pWHM264 On the 3.9kb EcoRI-HindIII fragment; In another preferred embodiment, this 2.9kb DNA fragment is only connected to the 2.3kb Xbal-HindIII DNA fragment.

All DNA molecules encoding daunorubicin C-14 hydroxylase described in WO 96/27014 are applicable in the present invention.

In particular, the DNA molecule of the present invention may comprise all or only a part of a 2.9 kb DNA fragment, at least 1.2 kb in length corresponding to the KpnI-BamHI fragment of a DNA molecule containing doxA encoding daunorubicin C-14 hydroxyl enzyme (which converts daunorubicin to doxorubicin). This DNA molecule essentially comprises the sequence reported in patent application WO 96/27014, which is referred to as the "dxrA" sequence. In addition, the deduced amino acid sequence of daunorubicin C-14 hydroxylase is also shown in this patent application.

The DNA molecules of the present invention may comprise a 2.3 kb Xbal-HindIII DNA fragment of at least 2247 nt comprising the drrA and drrB genes encoding proteins that confer resistance to daunorubicin and doxorubicin in host cells.

The DNA molecule of the present invention may comprise all or part of the 3.9kb EcoRI-HindIII fragment containing the drrC resistance gene, at least 2.5kb in length corresponding to the SstI-SphI fragment of the DNA molecule containing drrC, the drrC encoding endows the host cell with resistance to Daunorubicum Proteins that are resistant to adriamycin and doxorubicin.

The present invention also includes DNA comprising a gene conferring resistance to daunorubicin and doxorubicin that is at least 80% identical in sequence to the drrA and drrB genes (Guilfoile and Hutchinson, Proc. 8553, 1991) and/or the sequence of the drrC gene (Lomovskaya et al., J. Bacteriol. 178:3238, 1996) is identical.

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

The DNA molecules of the present invention can be linked into any autonomous replicating and/or integrating agent (which comprises a DNA molecule to which one or more additional DNA segments have been added). Typically, however, the vector is a plasmid. A preferred plasmid is the high copy number plasmid pWHM3 or pIJ702 (Katz et al., J. Gen. Microbiol. 129:2703, 1983). Other suitable plasmids are pIJ680 (Hopwood et al., "Genetic Manipulation of Streptomyces. A laboratory Manual", John Innes Foundation, Norwich, UK, 1985) and pWHM601 (Guilfoile and Hutchinson, National Academy of Sciences, USA Proceedings of the Academy 88:8553, 1991).

The DNA can be inserted into the vector using any suitable technique. Insertion can be achieved by ligating the DNA into a linearized vector at the appropriate site. For this, a direct combination of cohesive or blunt ends, homopolymer tailing, or the use of linker molecules or adapter molecules can be used.

Suitable host cells that do not produce or produce cyclothracene can be transformed with the recombinant vector.

The host cell may be daunorubicin or doxorubicin sensitive (ie, unable to grow in the presence of an amount of daunorubicin or doxorubicin) or daunorubicin or doxorubicin resistant. In any case, the resulting recombinant clones (obtained by transformation with the novel recombinant vectors of the present invention) showed higher levels of resistance to daunorubicin and doxorubicin than the parental host. The level of doxorubicin resistance in recombinant S. lividans was much higher than that observed in the anthracycline-producing S. persei ATCC 29050 and ATCC 27952 strains.

The host may be a microorganism (eg, a bacterium). Actinomyces strains that do not produce anthracyclines (especially S. lividans and other Streptomyces strains) can be transformed. S. lividans TK 23 is a more suitable host than the S. persei dnrN mutant transformed with the recombinant plasmid pIS70 containing dxrA gene (WO 96/27014).

The recombinant vector of the present invention can also be used to transform suitable host cells producing daunorubicin, so as to enhance the transformation of daunorubicin into doxorubicin.

Therefore, the strains of S. persei ATCC 29050 and ATCC 27952 containing their anthracycline-producing mutants can be transformed. In particular, Streptomyces persay strain WMH1654, which is a mutant strain obtained from Streptomyces persay ATCC 29050 and deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, may be used. The Tibetan registration number is ATCC55936.

Transformants of Streptomyces strains can generally be obtained by protoplast transformation.

The present invention includes a method for improving the production of doxorubicin by daunorubicin transformation comprising the biotransformation process of adding daunorubicin to doxorubicin in a host that does not produce anthracyclines and the direct Fermentation process for the production of doxorubicin in a daunorubicin-producing host.

Biotransformation Process of Daunorubicin to Adriamycin

The method includes:

1) cultivating a recombinant host cell transformed with the vector of the present invention that does not produce daunorubicin, adding daunorubicin thereto, and

2) Isolation of doxorubicin from the culture.

In this method, the recombinant strain can be cultured at a temperature of 20°C to 40°C (eg, 24°C to 37°C). Daunorubicin was added to the medium during the 24-96 hour growth phase. Incubation is preferably performed with shaking. The incubation time in the presence of daunorubicin can be 12-72 hours. The concentration of daunorubicin in the culture solution can be 20-1000mcg/ml (for example, 100-400mcg/ml).

doxorubicin by fermentation

The method includes:

1) cultivating recombinant daunorubicin production host cells transformed with the vector of the present invention, and

2) Isolation of doxorubicin from the culture.

In this method, the recombinant strain can be cultured at a temperature of 20°C to 40°C (eg, 26°C to 34°C). Incubate with shaking. The incubation time may be 72 to 168 hours.

Raw Materials and Methods

Bacterial Strains and Plasmids: Escherichia coli strain DH5[alpha] (which is ampicillin and apramycin sensitive) was used for subcloning of DNA fragments. The host Streptomyces lividans TK23 is obtained from DAHopwood (John Innes Institute, Norwich, United Kindom), and the host Streptomyces persay WMH1654 is a mutant strain obtained from Streptomyces persay ATCC 29050, which has been deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, accession number ATCC55936. Plasmid cloning vectors were pGem-7Zf(+) and related plasmids (Promega, Madison, WI), pIJ4070 (DAHopwood) and the Escherichia coli-Streptomyces shuttle vector pWHM3 (Vara et al., J. Bacteriology 171:5872, 1989).

Media and buffers: Escherichia coli strain DH5α was maintained on LB agar (Sambrook et al., "Molecular Cloning. A Laboratory manual", 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor , NY, 1989). When transformants were selected, ampicillin or apramycin was added at a concentration of 100 µg/ml. S. lividans TK23 and S. persei WMH1654 were maintained at R2YE (Hopwood et al., "Genetic Manipulation of Keyomyces. A Laboratory Guide", John Innes Foundation, Norwich, UK, 1985) and ISP4 (Difco, Detroit, MI) respectively. on agar medium. When transformants were selected, a layer of soft agar containing 50 μg/ml syringomycin was spread on the plate.

Subcloning DNA fragments : DNA samples were digested with appropriate restriction enzymes and separated on agarose gels by standard methods (Sambrook et al., "Molecular Cloning. A Laboratory Guide", 2nd Edition, ColdSpring Harbor Press, Cold Spring Harbor, NY, 1989). Agarose slices containing the DNA fragment of interest are excised from the gel and DNA is isolated from these slices using a GENECLEAN apparatus (Bio101, La Jolla, CA) or equivalent. The isolated DNA fragments were subcloned into Escherichia coli and E. coli-Streptomyces shuttle vector or Streptomyces vector for expression experiments.

Transformation of Streptomyces and Escherichia coli: Preparation of Escherichia coli by the Calcium Chloride Method (Sambrook et al., "Molecular Cloning. A Laboratory Guide", 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) Competent cells were obtained and transformed by standard techniques (Sambrook et al., "Molecular Cloning. A Laboratory Guide", 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). Streptomyces lividans TK23 was grown in liquid R2YE medium (Hopwood et al., "Genetic Manipulation of Keyomyces. A Laboratory Guide", John Innes Foundation, Norwich, UK, 1985) and harvested after 48 hrs. Mycelium pellets were washed twice with 10.3% (wt/vol) sucrose solution and used as outlined in the Hopwood guide (Hopwood et al., "Genetic manipulation of Streptomyces. A guide to experiments", John Innes Foundation, Norwich, UK , 1985) to prepare protoplasts. The protoplast pellet was suspended in about 300 microliters of P buffer (Hopwood et al., "Genetic Manipulation of Streptomyces. Experimental Guide", John Innes Foundation, Norwich, UK, 1985), and an aliquot of 50 microliters of this suspension was sample for each conversion. Protoplasts were prepared using plasmid DNA according to Hopwood et al. ("Genetic Manipulation of Streptomyces. A Laboratory Guide", John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall and Hutchinson (Proceedings of the National Academy of Sciences USA 86:3135, 1988) or Otten et al (Journal of Bacteriology 172:3427, 1990) transformed by the small-scale transformation method. After regeneration on R2YE medium at 30°C for 17hr, spread a layer of 200 μg/ml physomycin on the plate and let it grow at 30°C until sporulation.

Evaluation of daunorubicin and doxorubicin resistance levels : The resistance levels are expressed as minimal inhibitory concentrations (MIC) and determined by standard two-fold dilutions using R2YE medium. The strains were cultured on R2YE medium slant and cultured at 28°C for 8-10 days. Recombinant strains were grown in the same medium supplemented with 20 μg/ml Physicine. Bacterial cultures containing approximately 10 ⁶ -10 ⁷ viable cells/ml were prepared from cultures grown in Tryptic Soy Broth (Difo) for 48 hours at 28° C., 280 rpm. Homogenize the culture by glass beads. Take a loop of homogenized culture and inoculate it on agar plates containing different concentrations of daunorubicin and doxorubicin in the range of 0.39-800 μg/ml. The agar plates were incubated for 7 days at 30°C and MICs were determined at the lowest concentration that prevented visible growth.

Biotransformation of daunorubicin to doxorubicin : the Streptomyces lividans TK23 transformant carrying the plasmid of the present invention was inoculated into 25 ml of liquid R2YE medium containing 40 μg/ml moscomycin. Cultures were grown in 300 ml Erlenmeyer flasks at 30°C on a rotary shaker at 280 rpm. After 2 days of growth, 2.5 ml of this culture was 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 ₄ 7H ₂ O (0.2), FeSO ₄ 7H ₂ O (0.01), ZnSO ₄ 7H ₂ O (0.01), added 20 μg/ml Physicine. Add 400 μg/ml Daunorubicin at 48 hr of the growth phase. Grow the culture in a 300 ml Erlenmeyer flask on a rotary shaker at 280 rpm at 30 °C for 72 hr. Acidify with 25 mg/ml oxalic acid Each culture was incubated on a rotary shaker at 280rpm at 30°C for 30min, then extracted with an equal volume of acetonitrile:methanol (1:1) at 30°C and 300rpm for 2hr. The extract was filtered and passed through a reverse-phase high-pressure liquid Phase chromatography (RP-HPLC) analyzes filtrate.RP-HPLC is to apply Vydac C ₁₈ post (4.6 * 250mm; 5 μm particle diameter) with the flow rate operation of 0.385ml/min.Mobile phase A is 0.2% trifluoroacetic acid (TFA , from Pierce Chemical Co.) (in H ₂ O), mobile phase B is 0.078% TFA [in acetonitrile (from JTBaker Chemical Co.)]. Use 20-60% of phase B dissolved in phase A The linear gradient of elution is carried out in 33 minutes and application diode array detector is fixed at 488nm (bandwidth 12 microns) monitoring.Daunorubicin and doxorubicin (10 μ g/ml in methanol) are used as external standard and quantitative analysis Amounts of these metabolites isolated from cultures.

Doxorubicin production : Transformation of Streptomyces persaicin WMH1654 mutant with the plasmid of the present invention. The transformants were inoculated into 25 ml of R2YE medium supplemented with 20 μg/ml Physicine. Cultures were grown in 300ml Erlenmeyer flasks at 30°C and 280rpm rotary shaker. After 2 days of growth, 2.5 ml of this culture was transferred to 25 ml of APM medium supplemented with 20 μg/ml Physicine. Cultures were grown in 300 ml Erlenmeyer flasks at 28°C and 280 rpm rotary shaker for 96-120 hours. Each culture was acidified with 25 mg/ml oxalic acid, incubated at 30°C on a rotary shaker at 280 rpm for 45 min, and extracted with an equal volume of acetonitrile:methanol (1:1) at 30°C and 300 rpm for 2 hr. The extract was filtered and the filtrate was analyzed by RP-HPLC in the same way as for the biotransformation product.

Example 1

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

In order to remove non-essential regions, plasmid pIS70 (WO96/27014) was digested with EcoRI-HindIII in advance, and the 3.5kb fragment was subcloned into the same position of the multiple cloning site sequence of plasmid pGEM-7Zf (+) (Promega, Madison-WIUSA) point 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 plasmid pIJ4070 (obtained from the John Innes Institute, Norwich, UK) under the control of the strong promoter ermE ^* . The new plasmid (termed p7doxAUV) was digested BgIII and the fragment was inserted into plasmid pWHM3 (J. Vara et al., J. Bacteriol. 171:5872-5881, 1989) to obtain plasmid pIS156 (Fig. 1c).

The 2.3kb BgII fragment containing the drrA and drrB resistance genes was blunted and transferred from the plasmid pWHM603 into the SmaI site of the plasmid pBluescript II SK+ (Stratagene) to obtain the plasmid pdrrAB, and then the XbaI-HindIII fragment was transferred from pdrrAB into the vector pIJ4070 to obtain pIS278. Then, pIS278 was digested with EcoRI-XbaI and inserted into EcoRI-XbaI plasmid pWHM3 to obtain plasmid pIS281. This plasmid was digested with XbaI, and the XbaI fragment of plasmid pIS156 was inserted to obtain plasmid pIS284.

Example 2

Construction of plasmid pIS287 (Figure 3 (a-c)) : The drrC resistance gene contained in plasmid pWHM264 was excised by EcoRI-HindIII digestion, and then inserted into plasmid pIJ4070 to obtain plasmid pIS282. From this plasmid the drrC resistance gene was transferred as a BglII fragment into pIS252 (this plasmid is a modified version of pWHM3 containing an additional BglII site close to the EcoRI site) to obtain plasmid pIS285. Plasmid pIS285 was EcoRI digested and ligated with the 5.5 kb DNA fragment excised from plasmid pIS284 to obtain plasmid pIS287.

Example 3

Resistance of the above recombinant plasmids to doxorubicin : The resistance of Streptomyces lividans TK23 transformed with recombinant plasmids pIS70, pIS284 or pIS287 to Daunorubicum was measured on R2YE medium as MICs according to the operation described in "Materials and Methods". Levels of resistance to doxorubicin and doxorubicin (compared to S. lividans TK23, S. lividans TK23 transformed with the vector pWHM3, and anthracycline-producing S. persaicin ATCC 29050 and ATCC 27952 strains). Plasmid pIS287 containing the drrA, drrB and drrC resistance genes acquired the greatest level of daunorubicin and doxorubicin resistance. The level of doxorubicin resistance was also increased 64-fold for plasmids containing only the drrA and drrB resistance genes (Table 1).

Table 1. Resistance of recombinant strains to doxorubicin

Strain MIC of doxorubicin (μg/ml)

Streptomyces persay ATCC 29050 12.5

Streptomyces persay ATCC 27952 12.5

Streptomyces lividans TK23 12.5

Streptomyces lividans TK23(pWHM3) 12.5

Streptomyces lividans TK23(pIS284) 800

Streptomyces lividans TK23(pIS287) ＞800

Example 4

Bioconversion of added daunorubicin to doxorubicin in Streptomyces lividans TK23 transformed with a plasmid containing the doxA daunorubicin C-14 hydroxylase gene and a different resistance gene : as per "Materials and methods" The pIS70, pIS284 or pIS287 plasmids were introduced into Streptomyces lividans TK23 by selecting transformations for mossomycin resistance following the procedure described in the section. The resulting S. lividans TK23 (pIS70), S. lividans TK23 (pIS284) and S. lividans TK23 (pIS287) transformants were tested as described above for biotransformation of high-level (400 μg/ml) daunorubicum using APM medium. Ability to prime to doxorubicin. S. lividans TK23(pIS70) transformants were able to convert up to 11.5% of added daunorubicin to doxorubicin (Table 2). S. lividans TK23 (pIS284) and S. lividans TK23 (pIS287) transformants were able to convert up to 73.5% of added daunorubicin to doxorubicin (Table 2).

Table 2. Bioconversion of daunorubicin to doxorubicin by Streptomyces lividans strains

Strains Anthracycline (μg/ml)

DOX DNR 13-Dihydro DNR

Streptomyces lividans TK23 (pIS70) (comparison) 46 250 70

Streptomyces lividans TK23(pIS284) 294 33 21

Streptomyces lividans TK23(pIS287) 288 24 35

Example 5

Doxorubicin production in S. persaici WMH1654dnrX mutant transformed with a plasmid containing the doxA daunorubicin C-14 hydroxylase gene and a different resistance gene : as described in the "Materials and methods" section, The pIS284 and pIS287 plasmids were introduced into the S. persaicum WMH1654 dnrX mutant by protoplast transformation with selection for moscomycin resistance. The resulting S. persaii transformants were fermented and the fermentation broth analyzed as previously described. After 120 hr fermentation, S. persei WMH11654 (pIS284) produced up to 81 μg/ml doxorubicin and up to 18 μg/ml daunorubicin (Table 3). S. persei WMH1654 (pIS287) produced up to 92 μg/ml doxorubicin and no detectable amounts of daunorubicin (Table 3).

Table 3. Doxorubicin Production by Streptomyces persay WMH1654 dnrX Strain

Strains Anthracycline (μg/ml)

DOX DNR 13-Dihydro DNR

Streptomyces persay WMH1654 41 35 18

Streptomyces persay WMH1654(pIS284) 81 18 6

Streptomyces persay WMH1654(pIS287) 92 0 0

sequence 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

Sequence Listing

<110>PHARMACIA & UPJOHN S.P.A.

<120> method for preparing doxorubicin

<130>1615-9003

<140>PCT/UNKNOWN

<141>1999-04-22

<150>09/065,606

<151>1998-04-24

<160>1

<170>PatentIn Ver.2.0

<210>1

<211>2870

<212>DNA

<213> Streptomyces persay

<220>

<221>misc_feature

<222>Comolement((1)..(2870))

<223> Complementary strand of coding strand

<400>1

ggatccgcac cgggtacacg gcacgggacc gcccaccgcg cggcgcgcgg cgggcggtcc 60

cgtgccggtc gcggccggcg gatcagcgca gccagacggt cagttcggtg agccgcgccg 120

tctgggcccc cttccggcac caccgcaact cgtcgtacgg cacggccagc cgggcctcgg 180

ggaacctgct gcgcagtacg ccgatcatcg tgcgcgactc cagctgggcg agctgctccc 240

cgatgcagta gtgcggcccg tcgccgaagg tgagccgccg ccacgaggga cggtccgggt 300

ggaaggcgtg cggggcgtcg tgatggcggc cgtcggtgtt ggtgccctcg atgtccacca 360

gcaccggcgc tccgcggggc agccggacgc cgccgatggt cacctccgcg gcagcgaacc 420

tccacaacgt gtagggcacc ggcgggtggt agcgcagcgc ctcctccacg aaccggggaga 480

cggcgtcctc gtcggcatcc gccgcgaggc ggcccgccag gacctccgcg agcaggaagc 540

ccaggaagga gccggtggtg tcgtggccgg cgaagatgag cccggtgatc atgtagacga 600

gctggtcgtc ggagaccgag ccgaactcgg cctgcgcgcg ctcgtacagc acgcgggtca 660

tggtcggggt gtcgttccgc cgggctgagt gcacggcttc gaggagcagg ctctccaggg 720

ccgaggtgtc cggcacgccc ccggcagggt ccgtgccgtc accccccgccg ctctgcgggc 780

cgccgaggcc gagtgccttg agaacgctga cggcctcgcg ggccatcgcc ggatcggtga 840

ccggcacacc gagcagctcg cagatgacca acagcgggaa gtggtacgcg aagccgccga 900

tcagctcggc cggtttgccc gaccggccgg aggcgtcggc gagttcggtg agcagccggc 960

cggcgatcgc ggcgatgcga tccgtccgct cggccagccg gcgcgggttg aacgcaggtg 1020

cgtggatgcg gcgcaggcgc cggtggggcct cgccgtccac ggcgatgagc gtgaacggac 1080

gcagctccgg aacggggatg tcgagaccgt cgtccacccc ccgccaggcg gcgggggcga 1140

ggtcggggtc cttcacgaac cggggatcgg ccagcacctc gcgggcgagg gcgtcatcgg 1200

tgatgaccca ggcgggtccg cccgcggggg cgttcacctc gacgaccggg cccgcctccc 1260

ggaaggcgtc gtgcacctcg ggcttgcgct gcatggtcat catgggacac gcgaacgggt 1320

cgacggccac ccggggcgcc tcgccgctca cgaggcaccg cccgccgccg cggggtaccc 1380

ctcccgcagt tcgaccaccg agaagccggc cccgtgcggg tcgagcaggt ccgcccgccg 1440

ccccctgggc gtgtcggcgg gctcgttctc gacggagccg ccgagttcaa cggcgcgccg 1500

gaccgtcgcg tcgcagtcgt gcacggcgaa cagcacggcc cagtgcggcc gtaccgcgcc 1560

ggtgacgccc agctcctggg tgccggcgac cggtgtgtca ccgatgtgcc agaccgggtc 1620

ggtgacgccc ttcagtccgg tgtcggccgg agccaggccg agggtcgccg ggtagaagtc 1680

ccgggcggcc ccgatgccgt cggtcaccag ctcgacccag ccgaccgagc cgggcacgcc 1740

cgtcacctcc gcgccctcca tgactccctt gcgccagacc gcgaacgcgg ccccggcggg 1800

gtcggcgaag accgccatcc ggccgaggcc gaggacgtcc atcggagtca tgatgacctc 1860

gccgcccgcc gtctcgaccc gcttggtcag tgcgtcggcg tcgtcggtgg cgaagtacac 1920

ggtccagatg gccggcatgc cgtgctggtc gttcccgggc ccgtacggcc ggtggtaggg 1980

ggtgtcgatc tggtggcggg cgaccgcggc gaccagcttc ccgtcggagc tgaacgtcgt 2040

gtatcccccg gcgcccgggt cgctgaccac ggtggcggtc cagccgaaca ggccggtgta 2100

gaagtcggcc gaggcggcga catcgggcga accgaggtcg aaccatgcgg gggcgccggg 2160

cgcgaacctg gtcacgaatc gttcctttcg atggatcggc acacgagcgt ctgcgctcgc 2220

ggatgagacg gacatctcgc ggatgagacg gacatgcggg cggggcgggc cgccgccgtc 2280

agtgcgcggt gtcgccgacg gcggccgcgc cggcctccca gagcttcgcc gcgaggccgg 2340

cgtcggcggt cgggccgctc accggggaca gccgccggtc gctgtagtag ccgcccgtgg 2400

tcaactcctc ggccggcgcg gacgccagcc acacgagggt gtcggcgccc ttcgccgcgg 2460

agcgcaggaa ggggttgaac cggaagtagg acgaggcgac cgtgccccgt ccgatgcggg 2520

tgcggacctc accggggtga tagctgaccg ccagcacgtc cggccagcgc ctggcggcct 2580

ccgccgcggt catgatgttg gcctgtttgg acgtgccgta cgcctggccg gcgctgtagc 2640

ggtgacggtc gccgttgagg tcgtccgggt cgatccggcc ctgggtgtac gcgtcggacg 2700

aggtgaggat cagccgcccg cccgcgagcc gctcccgcag cagccgtgcc agcaggaagc 2760

ctgcgaggtg attgacctgg atggtggcct cgaacccgtc ctgggtcgtg gtgcgcgacc 2820

agaacatgcc gccggcgttg ctggccatga catcgatgcg cgggtaccgg 2870

Claims (17)

1. dna molecular, it comprises: the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one are contained at least a DNA district that gives the gene of daunorubicin and Zorubicin resistance, and the wherein said gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC.

2. the dna molecular of claim 1, it further comprises a kind of strong promoter.

3. the dna molecular of claim 2, wherein, described strong promoter is ermE ^*

4. the dna molecular of claim 1, wherein, the described gene of giving daunorubicin and Zorubicin resistance is drrA and drrB gene.

5. the dna molecular of claim 1, wherein, the described gene of giving daunorubicin and Zorubicin resistance is drrA, drrB and drrC gene.

6. the dna molecular of claim 1, wherein, the length in district that comprises the gene doxA of coding daunorubicin 14-hydroxylase is 2.9kb.

7. the dna molecular of claim 6, wherein, the district that comprises gene doxA is corresponding to the KpnI-BamHI fragment that comprises the doxA nucleotide sequence.

8. the dna molecular of claim 4, wherein, the described district that comprises described drrA and drrB gene is a 2.3kb XbaI-HindIII dna fragmentation.

9. carrier that comprises the dna molecular of claim 1.

10. the carrier of claim 9, wherein, described carrier is a plasmid.

11. the plasmid of claim 10, wherein, described plasmid is selected from pIS284 and pIS287.

12. host cell that transform or transfection of the carrier with claim 9.

13. the host cell of claim 12, wherein, described host cell is not produced daunorubicin.

14. the host cell of claim 12, wherein, described host cell is a bacterial cell of producing daunorubicin.

15. the host cell of claim 12, wherein, described host cell is the streptomyces cell.

16. a method that is used for the daunorubicin bio-transformation is become Zorubicin, it comprises the steps:

In containing the substratum of daunorubicin, cultivate a kind of recombinant host cell, wherein, described host cell comprises a kind of dna molecular, this dna molecular comprises the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one and contains at least a DNA district that gives the gene of daunorubicin and Zorubicin resistance, wherein, the described gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC, and described host cell is not produced daunorubicin, and

The Zorubicin that separates any generation from described substratum.

17. the method by the fermentative production Zorubicin, it comprises the steps:

In substratum, cultivate a kind of recombinant host cell, wherein, described host cell comprises a kind of dna molecular, this dna molecular comprises the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one and contains the DNA district that one or more give the gene of daunorubicin and Zorubicin resistance, wherein, the described gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC, and described host cell is a bacterial cell of producing daunorubicin, and

The Zorubicin that separates any generation from described substratum.

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