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EP1071746A2 - Procede de preparation de doxorubicine - Google Patents

Procede de preparation de doxorubicine

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Publication number
EP1071746A2
EP1071746A2 EP99919767A EP99919767A EP1071746A2 EP 1071746 A2 EP1071746 A2 EP 1071746A2 EP 99919767 A EP99919767 A EP 99919767A EP 99919767 A EP99919767 A EP 99919767A EP 1071746 A2 EP1071746 A2 EP 1071746A2
Authority
EP
European Patent Office
Prior art keywords
daunorubicin
doxorubicin
host cell
dna molecule
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99919767A
Other languages
German (de)
English (en)
Other versions
EP1071746A4 (fr
Inventor
Augusto Inventi Solari
Giovanna Zanuso
Silvia Filippini
Francesca Torti
Sharee Otten
Anna Luisa Colombo
Charles R. Hutchinson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Italia SRL
Original Assignee
Pharmacia and Upjohn SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacia and Upjohn SpA filed Critical Pharmacia and Upjohn SpA
Publication of EP1071746A2 publication Critical patent/EP1071746A2/fr
Publication of EP1071746A4 publication Critical patent/EP1071746A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin

Definitions

  • 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.
  • 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.
  • 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 e-rhodomycinone
  • rhodomycin D 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 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.
  • 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 .
  • 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 ( ⁇ dnril, 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.
  • 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 d ⁇ rVand 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 ( ⁇ dnrU, 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.
  • 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, ⁇ /ofl; K, Kpn ⁇ ; E, EcoRI; H, HindWV, P, Pst ⁇ ; S, SphY, X, Xba ⁇ , L, Bgl ⁇ ; T, Sst ⁇ .
  • 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 ( ⁇ dnrU ) gene.
  • 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.
  • DNA molecules encoding a daunorubicin C-14 hydroxylase described in WO 96/27014 may be employed in the present invention.
  • 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,
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • 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 comprises:
  • the recombinant strain may be cultured at temperatures from
  • 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
  • the concentration of daunorubicin in the culture may be from 20 to 22 hours.
  • the concentration of daunorubicin in the culture may be from 20 to 22 hours.
  • This process comprises: 1 ) culturing recombinant daunorubicin-producing host cells transformed with the vectors of the invention and
  • 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.
  • F. coli strain DH5 ⁇ 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).
  • 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).
  • 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.
  • the plates are overlayed with soft agar containing thiostrepton at a concentration of 50 micrograms/ml.
  • 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.
  • 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.
  • 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" 10 7 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°.
  • APM production medium ((g/l) glucose (60), yeast extract (8), malt extract (20), NaCI (2), 3-(morpholino)propanesulfonic acid (MOPS sodium salt) (15), MgS0 4 .7H 2 0 (0.2), FeS0 4 .7H 2 0 (0.01 ), ZnS0 4 .7H 2 0 (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 reversed-phasehigh pressure liquid chromatography
  • RP-HPLC is performed by using a Vydac C 18 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 H 2 0 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.
  • 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 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.
  • 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.
  • the drrC resistance gene contained in the plasmid pWHM264 is excised by EcoRI-H/ ⁇ dlll 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.
  • 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).
  • 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)
  • 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.
  • 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).

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Abstract

On peut améliorer la capacité de conversion de la daunorubicine en doxorubicine en transformant une cellule hôte avec un vecteur recombinant comprenant une molécule d'ADN présentant une région ou un fragment d'ADN ou contenant le gène doxA codant pour la daunorubicine 14-hydroxylase et une région ou un fragment d'ADN contenant un ou plusieurs gènes conférant une résistance à la daunorubicine et à la doxorubicine.
EP99919767A 1998-04-24 1999-04-22 Procede de preparation de doxorubicine Withdrawn EP1071746A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US6560698A 1998-04-24 1998-04-24
US65606 1998-04-24
PCT/US1999/007016 WO1999055829A2 (fr) 1998-04-24 1999-04-22 Procede de preparation de doxorubicine

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EP1071746A2 true EP1071746A2 (fr) 2001-01-31
EP1071746A4 EP1071746A4 (fr) 2003-04-23

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EP (1) EP1071746A4 (fr)
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CN (1) CN1198930C (fr)
CA (1) CA2326500A1 (fr)
WO (1) WO1999055829A2 (fr)

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WO2006111561A1 (fr) * 2005-04-21 2006-10-26 Dsm Ip Assets B.V. Production amelioree d'anthracyclines d'origine microbienne
CN101016533B (zh) * 2005-11-09 2010-05-19 上海医药工业研究院 一种蒽环类抗生素产生工程菌及其应用
CN1962869B (zh) * 2005-11-09 2010-08-11 上海医药工业研究院 一种调控蛋白SnpR及其基因和应用
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WO1999055829A3 (fr) 1999-12-23
CN1198930C (zh) 2005-04-27
EP1071746A4 (fr) 2003-04-23

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