CA2326500A1 - Process for preparing doxorubicin - Google Patents
Process for preparing doxorubicin Download PDFInfo
- Publication number
- CA2326500A1 CA2326500A1 CA002326500A CA2326500A CA2326500A1 CA 2326500 A1 CA2326500 A1 CA 2326500A1 CA 002326500 A CA002326500 A CA 002326500A CA 2326500 A CA2326500 A CA 2326500A CA 2326500 A1 CA2326500 A1 CA 2326500A1
- Authority
- CA
- Canada
- Prior art keywords
- daunorubicin
- doxorubicin
- host cell
- dna
- dna molecule
- 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.)
- Abandoned
Links
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
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 s 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 io 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, is 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.coeruleorubidus, S.galilaeus, S.griseus, S.griseoruber, S.insignis, S.viridochromogenes, S.bifurcus and S.sp. strain C5) and by Acfinomyces carminata. Doxorubicin is mainly produced by 2o strains of S. peucefius. 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.
2s 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. peucefius 27952 by cloning experiments (Stutzman-Engwall and 3o Hutchinson, Proc.NatLAcad.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.peucefius 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, dn-A and drr8, which confer doxorubicin and daunorubicin resistance to Streptomyces lividans have been cloned from S. peucetius ATCC 29050 strain (Guilfoile and Hutchinson, s Proc.NatLAcad.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 io 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.Bacterio1.178:3238, 1996).
~5 Summani 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 2o genes, selected from the group consisting of drrA, drr8 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, 2s described in WO 96/27014, even when a strong promoter is used.
The DNA of the invention comprises preferably all three of the drrA, drr8 and drrC genes or only the two drrA and drr8 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 .
s Brief descrir~tion of the drav~ina~
Fig. 1 (a-c) illustrate the construction of the plasmid pIS156 described in Example 1. This plasmid was constructed by insertion of the 2.9 kb fragment containing the doxA (formerly dxrA), the dnrV (formerly dnrORF10) and the C-terminal part of the dnrU (ddnrU, formerly dnrORF9) genes, obtained from the recombinant plasmid pIS70 io (WO 96/27014 and A. Inventi Solari et al., GMBIM '96, P58), under the control of the strong promoter ermE* (Bibb et al., Molec. Microbiol. 14:533, 1994) into the plasmid pWHM3 (Vara et ai., 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 (ddnrU) genes is (complementary strand to the coding strand).
Fig. 2 (a-d) illustrate the construction of the plasmid pIS284 described in Example 1. This plasmid contains the 2.9 kb fragment encompassing the doxA, the dnrV and the C-terminal part of the dnrU genes, obtained from the recombinant plasmid pIS70, under the control of the strong promoter ermE* together with a DNA
fragment 20 of 2.3 Kb including the drrA and drr8 resistance genes obtained from the plasmid pWHM603 (P. Guilfoile and C.R. Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553, 1991 ) subcloned into the plasmid pWHM3.
Fig. 3 (a-c) illustrate the construction of the plasmid pIS287 described in Example 2. Said plasmid was constructed by insertion of the 2.9 kb BamHl-Hindlll 2s fragment containing the doxA formerly, dxrA), dnrV (formerly dnr-ORF10) and the C
terminal part of the dnrU (ddnrU, formerly, dnr-ORF9) genes, obtained from the recombinant plasmid pIS70 (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 drr8 resistance genes and the 3.9 kb EcoRl-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 s are sufficient for an unambiguous recognition of the DNA segments.
Restriction sites abbreviations: Ap, apramycin;tsr, thiostrepton, amp, ampicillin;
B, BamHl; G, Bglll; N, Notl; K, Kpnl; E, EcoRl; H, Hindlll; P, Psfl; S, Sphl;
X, Xbal, L, Bgll ; T, Ssfl .
Detailed d rir~tion of the in~Pntion.
to 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.
is 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 pIS70 described in the patent WO 96/27014 by digestion with BamHl-Hindlll enzymes.
This fragment contains the doxA gene, encoding the C-14 hydroxylase. Daunorubicin C-hydroxylase converts daunorubicin to doxorubicin. The 2.9 kb DNA fragment also 2o comprises the dnrV gene between the Notl-Kpnl sites and a Notl-Sphl fragment containing the C-terminal part of the dnr(J (~dnrU ) gene.
Preferably, this 2.9 kb DNA fragment encoding a daunorubicin C-14 hydroxylase was ligated to both the 2.3 kb Xbal-Hindlll DNA fragment containing the drrA and drrB
resistance genes obtained from the plasmid pWHM603 and the 3.9 kb EcoRl-HindlIl 2s 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 Xbal -Hindlll 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.
s 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 Kpnl-BamHl fragment containing the DNA molecule of doxA, encoding a daunorubicin C-14 hydroxylase, which converts daunorubicin to doxorubicin.
s 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 io 2.3 kb Xbal-Hindlll 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 EcoRl-Hindlll fragment containing the drrC resistance gene, at least 2.5 kb in length corresponding to the Sstl-Sphl fragment containing the DNA molecule of drrC, encoding is 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.NatLAcad.Sci.USA 88:8553, 1991 ) and or drrC gene (Lomovskaya et al., 2o 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 2s 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 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, Proc. Natl. Acad.
Sci.
s 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 Pinker or adapter molecule may be employed.
io 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 is 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.
2o The host may be a microorganism such as a bacterium. Strains of Actinomycetes, in particular strains of S. lividans and other strains of Strepfomyces 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 pIS70 containing the dxrA gene used for daunorubicin to 2s 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. )n 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.
s 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 io a fermentation process for producing doxorubicin in hosts which directly produce daunorubicin.
Bioconversion procPS~ of da ~nnmbi in to doxo »ir9n This process comprises:
1 ) culturing the recombinant host cells not producing daunorubicin transformed with the is 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 2o 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 ferment~~: ;;, This process comprises:
2s 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 s Bacterial strains and r~lasmidw E, coli strain DHSa, 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 lnnes Institute, Norwich,United Kingdom) and the host S. peucetius WMH1654 is a mutant strain obtained from S.peucetius ATCC
and has been deposited at the American Type Culture Collection, 10801 University io Boulevard, Manassas, Virginia 20110-2209, USA, under the accession number ATCC55936.
The plasmid cloning vectors are pGem-7Zf(+) and related plasmids (Promega, Madison, WI), pIJ4070 (D. A. Hopwood) and the E.coli-Streptomyces shuttle vector pWHM3 (Vara et al., J. Bacteriol. 171:5872, 1989).
is Media and bcff .r~ E. coli strain DHSa 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.
2o 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, MI} agar media, respectively.
When selecting for transformants, the plates are overlayed with soft agar containing thiostrepton at a concentration of 50 microgramslml.
2s Subcloning D A fragm n : 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) s into E. coli for routine manipulations, and E, coli-Streptomyces shuttle vectors or Streptomyces vectors for expression experiments.
Transformation of tr ,otom Pt Sp~GI~S and E coli: Competent cells of E. coli are prepared by the calcium chloride method (Sambrook et al., Molecular Cloning. A
i~ 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) is 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 20 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
LaboraforyManual, John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall and 2s Hutchinson (Proc. Natl. Acad. Sci. USA. 86:3135, 1988) or Otten 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.
WO 99/55829 PC'T/US99/07016 F_val~ation of daunor~bicin and doxorubicin resistan hPVel: 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 s 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 106-10'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 to 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 lowest_concentrations that prevent visible growth.
I5 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/I) glucose (60), yeast 2o 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.
zs 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-phase high pressure liquid chromatography (RP-HPLC).
RP-HPLC is performed by using a Vydac C,e column (4.6 x 250 millimeters; 5 micrometers particle size) at a flow rate of 0.385 ml/min. Mobiie 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 s 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.
I~xer~bis'ttion: The S. peucetius WMH1654 mutant is transformed with a plasmid of the invention. Transformant~ arP i~~~~~m+o.~ ir,f~, ~~ .",i ..r onvr .r_~:____ 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 is 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 Ex~mpl~1 (Fig. 1 (a-c) and Fig. 2 (a-d).
In order to remove a non-essential region, the plasmid pIS70 (W096/27014) is before 2s digested EcoRl-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 BamHl restriction site. The new plasmid pGendoxAUV was BamHl digested and the fragment, now reduced to 2.9 kb, was transferred into the plasmid pIJ4070 (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 pIS156 (fig. 1c).
s 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 pIJ4070 to obtain pIS278.
Afterwards, pIS278 is digested with EcoRl Xbal and inserted into the EcoRl Xbal to plasmid pWHM3 to obtain the plasmid pIS281. This plasmid is digested with Xbal and the Xbal fragment of plasmid pIS156 is inserted to obtain the plasmid pIS284.
Example 2 is Con tr ~ ion of th _ la mid I 87 tFig~~~; The drrC resistance gene contained in the plasmid pWHM264 is excised by EcoRl-Hindlll digestion and inserted into the plasmid pIJ4070 to obtain the plasmid pIS282. From this plasmid, the drrC
resistance gene is transferred as a Bglll fragment to pIS252 (this plasmid is a modified form of 2o pWHM3 containing an extra Bglll site close to the EcoRl site) to obtain the plasmid pIS285. pIS285 is EcoRl digested and ligated with the 5.5 kb DNA fragment excised from plasmid pIS284 to obtain the plasmid pIS287.
Example 3 2s R istanc~ of th abov r ombinant olas~~~ids t~ 'IOXOtLhlr'~n~ The level of resistance to daunorubicin and doxorubicin of S. lividans TK23 transformed with the recombinant plasmids pIS70, pIS284 or pIS287 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 pIS287 containing the drrA, drr8 and drrC resistance genes. The level of doxorubicin resistance was increased 64 times also with the plasmid containing only the drrA and s 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 to S. peucetius ATCC 27952 12.5 S. lividans TK23 12.5 S. lividans TK23(pWHM3) 12.5 S. lividans TK23(pIS284) 800 S. lividans TK23(pIS287) >800 is Example 4 2o different r i tan P n~PrnPs: The pIS70, pIS284 or pIS287 plasmids are introduced into S. l 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(pIS70), S. lividans TK23(pIS284) and S, lividans TK23(pIS287) transformants are tested for the ability to bioconvert a high level (400 micrograms/ml) 2s of daunorubicin to doxorubicin using the APM medium as described above. S.
lividans TK23(pIS70) transformants can convert up to 11.5% of added daunorubicin to doxorubicin (Table 2). S. lividans TK23(pIS284) and S. lividans TK23(pIS287) 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 S. lividans TK23(pIS70) (control) 46 250 70 S. lividans TK23(pIS284) 294 33 21 S. iividans TK23(pIS287) 288 24 35 to Example 5 different resistan~r~P a ne : The pIS284 and pIS287 plasmids are introduced into S.
is 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.
peucefius WMH1654{pIS284) produced up to 81 micrograms/ml of doxorubicin and up 2o to 18 micrograms/ml of daunorubicin after a 120 hr fermentation (Table 3).
S.peucefius WMH1654(pIS287) produced up to 92 micrograms/ml of doxorubicin and no detectable amount of daunorubicin (Table 3). .
TabIP ~. Doxorubicin production by S. peucefius WMH1654 dnrX strains.
Strain Anthracycline (micrograms/ml) DOX DNR 13-dihydroDNR
S. peucetius WMH1654 41 35 18 s S. peucetius WMH1654(pIS284) 81 18 g S. peucetius WMH1654(pIS287) g2 p WO 99/55829 PC"T/US99/07016 SEQ ID.1 SEQUENCE LISTING
<110> PHARMACIA & UPJOHN S.P.A.
<120> .PROCESS FOR PREPARING DOXORUBICIN
<130> 1615-9003 <140> PCT/UNKNOWN
<141> 1999-04-22 <150> 09/065,606 <151> 1998-09-24 <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 2870 <212> DNA
<213> Streptomyces peucetius <220>
<221> misc feature <222> Complement((1)..(2870)) <223> Complementary strand to the coding strand <400> 1 ggatccgcac cgggtacacg gcacgggacc gcccaccgcg cggtgcgcgg tgggcggtcc 60 cgtgccggtc gcggccggcg gatcagcgca gccagacggg cagttcggtg agccgcgccg 120 tctgggcccc cttccggcac caccgcaact cgtcgtacgg cacggccagt 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 cacctccgtg gcagcgaacc 420 tccacaacgt gtagggcacc ggcgggtggt agcgcagcgc ctcctccacg aaccgggaga 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 acccccgccg 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 cggtgggcct 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 1490 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 gcgaggcc 2340 gg cgtcggcggt cgggccgctc accggggaca gccgccggtc gctgtagtag ccgcccgtgg 2900 tcaactcctc ggccggcgcg gacgccagcc acacgagggt gtcggcgccc ttcgccgcgg 2460 agcgcaggaa ggggttgaac cggaagtagg acgaggcgac cgtgccccgt ccgatgcggg 2520 tgcggacctc accggggtga tagctgaccg ccagcacgtc cggccagcgc ctggc ccgccgcggt catgatgttg gcctgtttgg acgtgccgta cgcctggcc g gcgctgtagc 2690 ggtgacggtc cc tt a tc tcc t c atcc 9gcct 2580 g g g gg g ggg g ggcc ctgggtgtac gcgtcggacg 2700 aggtgaggat cagccgcccg cccgcgagcc gctcccgcag cagccgtgcc agcaggaagc 2760 ctgcgaggtg attgacctgg atggtggcct cgaacccgtc ctgggtcgtg gtgcgcgacc 2820 agaacatgcc gccggcgttg ctggccatga catcgatgcg cgggtaccgg 2870
Field of the Invention The present invention concerns a process for improving daunorubicin to s 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 io 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, is 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.coeruleorubidus, S.galilaeus, S.griseus, S.griseoruber, S.insignis, S.viridochromogenes, S.bifurcus and S.sp. strain C5) and by Acfinomyces carminata. Doxorubicin is mainly produced by 2o strains of S. peucefius. 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.
2s 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. peucefius 27952 by cloning experiments (Stutzman-Engwall and 3o Hutchinson, Proc.NatLAcad.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.peucefius 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, dn-A and drr8, which confer doxorubicin and daunorubicin resistance to Streptomyces lividans have been cloned from S. peucetius ATCC 29050 strain (Guilfoile and Hutchinson, s Proc.NatLAcad.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 io 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.Bacterio1.178:3238, 1996).
~5 Summani 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 2o genes, selected from the group consisting of drrA, drr8 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, 2s described in WO 96/27014, even when a strong promoter is used.
The DNA of the invention comprises preferably all three of the drrA, drr8 and drrC genes or only the two drrA and drr8 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 .
s Brief descrir~tion of the drav~ina~
Fig. 1 (a-c) illustrate the construction of the plasmid pIS156 described in Example 1. This plasmid was constructed by insertion of the 2.9 kb fragment containing the doxA (formerly dxrA), the dnrV (formerly dnrORF10) and the C-terminal part of the dnrU (ddnrU, formerly dnrORF9) genes, obtained from the recombinant plasmid pIS70 io (WO 96/27014 and A. Inventi Solari et al., GMBIM '96, P58), under the control of the strong promoter ermE* (Bibb et al., Molec. Microbiol. 14:533, 1994) into the plasmid pWHM3 (Vara et ai., 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 (ddnrU) genes is (complementary strand to the coding strand).
Fig. 2 (a-d) illustrate the construction of the plasmid pIS284 described in Example 1. This plasmid contains the 2.9 kb fragment encompassing the doxA, the dnrV and the C-terminal part of the dnrU genes, obtained from the recombinant plasmid pIS70, under the control of the strong promoter ermE* together with a DNA
fragment 20 of 2.3 Kb including the drrA and drr8 resistance genes obtained from the plasmid pWHM603 (P. Guilfoile and C.R. Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553, 1991 ) subcloned into the plasmid pWHM3.
Fig. 3 (a-c) illustrate the construction of the plasmid pIS287 described in Example 2. Said plasmid was constructed by insertion of the 2.9 kb BamHl-Hindlll 2s fragment containing the doxA formerly, dxrA), dnrV (formerly dnr-ORF10) and the C
terminal part of the dnrU (ddnrU, formerly, dnr-ORF9) genes, obtained from the recombinant plasmid pIS70 (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 drr8 resistance genes and the 3.9 kb EcoRl-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 s are sufficient for an unambiguous recognition of the DNA segments.
Restriction sites abbreviations: Ap, apramycin;tsr, thiostrepton, amp, ampicillin;
B, BamHl; G, Bglll; N, Notl; K, Kpnl; E, EcoRl; H, Hindlll; P, Psfl; S, Sphl;
X, Xbal, L, Bgll ; T, Ssfl .
Detailed d rir~tion of the in~Pntion.
to 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.
is 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 pIS70 described in the patent WO 96/27014 by digestion with BamHl-Hindlll enzymes.
This fragment contains the doxA gene, encoding the C-14 hydroxylase. Daunorubicin C-hydroxylase converts daunorubicin to doxorubicin. The 2.9 kb DNA fragment also 2o comprises the dnrV gene between the Notl-Kpnl sites and a Notl-Sphl fragment containing the C-terminal part of the dnr(J (~dnrU ) gene.
Preferably, this 2.9 kb DNA fragment encoding a daunorubicin C-14 hydroxylase was ligated to both the 2.3 kb Xbal-Hindlll DNA fragment containing the drrA and drrB
resistance genes obtained from the plasmid pWHM603 and the 3.9 kb EcoRl-HindlIl 2s 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 Xbal -Hindlll 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.
s 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 Kpnl-BamHl fragment containing the DNA molecule of doxA, encoding a daunorubicin C-14 hydroxylase, which converts daunorubicin to doxorubicin.
s 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 io 2.3 kb Xbal-Hindlll 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 EcoRl-Hindlll fragment containing the drrC resistance gene, at least 2.5 kb in length corresponding to the Sstl-Sphl fragment containing the DNA molecule of drrC, encoding is 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.NatLAcad.Sci.USA 88:8553, 1991 ) and or drrC gene (Lomovskaya et al., 2o 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 2s 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 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, Proc. Natl. Acad.
Sci.
s 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 Pinker or adapter molecule may be employed.
io 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 is 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.
2o The host may be a microorganism such as a bacterium. Strains of Actinomycetes, in particular strains of S. lividans and other strains of Strepfomyces 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 pIS70 containing the dxrA gene used for daunorubicin to 2s 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. )n 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.
s 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 io a fermentation process for producing doxorubicin in hosts which directly produce daunorubicin.
Bioconversion procPS~ of da ~nnmbi in to doxo »ir9n This process comprises:
1 ) culturing the recombinant host cells not producing daunorubicin transformed with the is 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 2o 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 ferment~~: ;;, This process comprises:
2s 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 s Bacterial strains and r~lasmidw E, coli strain DHSa, 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 lnnes Institute, Norwich,United Kingdom) and the host S. peucetius WMH1654 is a mutant strain obtained from S.peucetius ATCC
and has been deposited at the American Type Culture Collection, 10801 University io Boulevard, Manassas, Virginia 20110-2209, USA, under the accession number ATCC55936.
The plasmid cloning vectors are pGem-7Zf(+) and related plasmids (Promega, Madison, WI), pIJ4070 (D. A. Hopwood) and the E.coli-Streptomyces shuttle vector pWHM3 (Vara et al., J. Bacteriol. 171:5872, 1989).
is Media and bcff .r~ E. coli strain DHSa 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.
2o 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, MI} agar media, respectively.
When selecting for transformants, the plates are overlayed with soft agar containing thiostrepton at a concentration of 50 microgramslml.
2s Subcloning D A fragm n : 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) s into E. coli for routine manipulations, and E, coli-Streptomyces shuttle vectors or Streptomyces vectors for expression experiments.
Transformation of tr ,otom Pt Sp~GI~S and E coli: Competent cells of E. coli are prepared by the calcium chloride method (Sambrook et al., Molecular Cloning. A
i~ 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) is 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 20 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
LaboraforyManual, John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall and 2s Hutchinson (Proc. Natl. Acad. Sci. USA. 86:3135, 1988) or Otten 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.
WO 99/55829 PC'T/US99/07016 F_val~ation of daunor~bicin and doxorubicin resistan hPVel: 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 s 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 106-10'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 to 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 lowest_concentrations that prevent visible growth.
I5 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/I) glucose (60), yeast 2o 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.
zs 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-phase high pressure liquid chromatography (RP-HPLC).
RP-HPLC is performed by using a Vydac C,e column (4.6 x 250 millimeters; 5 micrometers particle size) at a flow rate of 0.385 ml/min. Mobiie 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 s 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.
I~xer~bis'ttion: The S. peucetius WMH1654 mutant is transformed with a plasmid of the invention. Transformant~ arP i~~~~~m+o.~ ir,f~, ~~ .",i ..r onvr .r_~:____ 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 is 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 Ex~mpl~1 (Fig. 1 (a-c) and Fig. 2 (a-d).
In order to remove a non-essential region, the plasmid pIS70 (W096/27014) is before 2s digested EcoRl-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 BamHl restriction site. The new plasmid pGendoxAUV was BamHl digested and the fragment, now reduced to 2.9 kb, was transferred into the plasmid pIJ4070 (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 pIS156 (fig. 1c).
s 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 pIJ4070 to obtain pIS278.
Afterwards, pIS278 is digested with EcoRl Xbal and inserted into the EcoRl Xbal to plasmid pWHM3 to obtain the plasmid pIS281. This plasmid is digested with Xbal and the Xbal fragment of plasmid pIS156 is inserted to obtain the plasmid pIS284.
Example 2 is Con tr ~ ion of th _ la mid I 87 tFig~~~; The drrC resistance gene contained in the plasmid pWHM264 is excised by EcoRl-Hindlll digestion and inserted into the plasmid pIJ4070 to obtain the plasmid pIS282. From this plasmid, the drrC
resistance gene is transferred as a Bglll fragment to pIS252 (this plasmid is a modified form of 2o pWHM3 containing an extra Bglll site close to the EcoRl site) to obtain the plasmid pIS285. pIS285 is EcoRl digested and ligated with the 5.5 kb DNA fragment excised from plasmid pIS284 to obtain the plasmid pIS287.
Example 3 2s R istanc~ of th abov r ombinant olas~~~ids t~ 'IOXOtLhlr'~n~ The level of resistance to daunorubicin and doxorubicin of S. lividans TK23 transformed with the recombinant plasmids pIS70, pIS284 or pIS287 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 pIS287 containing the drrA, drr8 and drrC resistance genes. The level of doxorubicin resistance was increased 64 times also with the plasmid containing only the drrA and s 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 to S. peucetius ATCC 27952 12.5 S. lividans TK23 12.5 S. lividans TK23(pWHM3) 12.5 S. lividans TK23(pIS284) 800 S. lividans TK23(pIS287) >800 is Example 4 2o different r i tan P n~PrnPs: The pIS70, pIS284 or pIS287 plasmids are introduced into S. l 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(pIS70), S. lividans TK23(pIS284) and S, lividans TK23(pIS287) transformants are tested for the ability to bioconvert a high level (400 micrograms/ml) 2s of daunorubicin to doxorubicin using the APM medium as described above. S.
lividans TK23(pIS70) transformants can convert up to 11.5% of added daunorubicin to doxorubicin (Table 2). S. lividans TK23(pIS284) and S. lividans TK23(pIS287) 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 S. lividans TK23(pIS70) (control) 46 250 70 S. lividans TK23(pIS284) 294 33 21 S. iividans TK23(pIS287) 288 24 35 to Example 5 different resistan~r~P a ne : The pIS284 and pIS287 plasmids are introduced into S.
is 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.
peucefius WMH1654{pIS284) produced up to 81 micrograms/ml of doxorubicin and up 2o to 18 micrograms/ml of daunorubicin after a 120 hr fermentation (Table 3).
S.peucefius WMH1654(pIS287) produced up to 92 micrograms/ml of doxorubicin and no detectable amount of daunorubicin (Table 3). .
TabIP ~. Doxorubicin production by S. peucefius WMH1654 dnrX strains.
Strain Anthracycline (micrograms/ml) DOX DNR 13-dihydroDNR
S. peucetius WMH1654 41 35 18 s S. peucetius WMH1654(pIS284) 81 18 g S. peucetius WMH1654(pIS287) g2 p WO 99/55829 PC"T/US99/07016 SEQ ID.1 SEQUENCE LISTING
<110> PHARMACIA & UPJOHN S.P.A.
<120> .PROCESS FOR PREPARING DOXORUBICIN
<130> 1615-9003 <140> PCT/UNKNOWN
<141> 1999-04-22 <150> 09/065,606 <151> 1998-09-24 <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 2870 <212> DNA
<213> Streptomyces peucetius <220>
<221> misc feature <222> Complement((1)..(2870)) <223> Complementary strand to the coding strand <400> 1 ggatccgcac cgggtacacg gcacgggacc gcccaccgcg cggtgcgcgg tgggcggtcc 60 cgtgccggtc gcggccggcg gatcagcgca gccagacggg cagttcggtg agccgcgccg 120 tctgggcccc cttccggcac caccgcaact cgtcgtacgg cacggccagt 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 cacctccgtg gcagcgaacc 420 tccacaacgt gtagggcacc ggcgggtggt agcgcagcgc ctcctccacg aaccgggaga 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 acccccgccg 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 cggtgggcct 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 1490 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 gcgaggcc 2340 gg cgtcggcggt cgggccgctc accggggaca gccgccggtc gctgtagtag ccgcccgtgg 2900 tcaactcctc ggccggcgcg gacgccagcc acacgagggt gtcggcgccc ttcgccgcgg 2460 agcgcaggaa ggggttgaac cggaagtagg acgaggcgac cgtgccccgt ccgatgcggg 2520 tgcggacctc accggggtga tagctgaccg ccagcacgtc cggccagcgc ctggc ccgccgcggt catgatgttg gcctgtttgg acgtgccgta cgcctggcc g gcgctgtagc 2690 ggtgacggtc cc tt a tc tcc t c atcc 9gcct 2580 g g g gg g ggg g ggcc ctgggtgtac gcgtcggacg 2700 aggtgaggat cagccgcccg cccgcgagcc gctcccgcag cagccgtgcc agcaggaagc 2760 ctgcgaggtg attgacctgg atggtggcct cgaacccgtc ctgggtcgtg gtgcgcgacc 2820 agaacatgcc gccggcgttg ctggccatga catcgatgcg cgggtaccgg 2870
Claims (19)
1. 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.
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 drrA, 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 drrA and drrB genes.
6. The DNA molecule according to claim 4, wherein said genes conferring daunorubicin and doxorubicin resistance are drrA, 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 Kpnl-BamHl fragment containing the doxA
nucleotide sequence.
nucleotide sequence.
9. The DNA molecule according to claim 5, wherein said region containing said drrA and drrB genes is a 2.3 kb XbaI-HindIII 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 pIS284 and pIS287.
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.
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.
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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6560698A | 1998-04-24 | 1998-04-24 | |
| US09/065,606 | 1998-04-24 | ||
| PCT/US1999/007016 WO1999055829A2 (en) | 1998-04-24 | 1999-04-22 | Process for preparing doxorubicin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2326500A1 true CA2326500A1 (en) | 1999-11-04 |
Family
ID=22063870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002326500A Abandoned CA2326500A1 (en) | 1998-04-24 | 1999-04-22 | Process for preparing doxorubicin |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1071746A4 (en) |
| JP (1) | JP2002512784A (en) |
| CN (1) | CN1198930C (en) |
| CA (1) | CA2326500A1 (en) |
| WO (1) | WO1999055829A2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006111561A1 (en) * | 2005-04-21 | 2006-10-26 | Dsm Ip Assets B.V. | Improved microbial production of anthracyclins |
| CN1962869B (en) * | 2005-11-09 | 2010-08-11 | 上海医药工业研究院 | Regulation protein SnpR and its gene and application |
| CN101016533B (en) * | 2005-11-09 | 2010-05-19 | 上海医药工业研究院 | Engineering bacterium capable of producing anthracene ring antibiotics and application of the same |
| CN101802168B (en) * | 2007-09-14 | 2013-06-12 | 明治制果株式会社 | Method for production of non-natural antibiotic |
| CN102229906B (en) * | 2011-03-09 | 2013-04-10 | 北京赛诺百奥生物技术有限公司 | Daunorubicin and adriamycin producing engineered pseudomonas |
| CN104059892A (en) * | 2013-03-18 | 2014-09-24 | 江苏禾昌生物科技有限公司 | Daunorubicin C-14 hydroxylase mutant and production method of genetically engineered bacteria thereof |
| CN111187746B (en) * | 2018-11-15 | 2022-11-22 | 上海医药工业研究院 | Genetically engineered bacterium and preparation method and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5665564A (en) * | 1988-05-27 | 1997-09-09 | Pharmacia & Upjohn S.P.A. | Isolation and characterisation of genes resistant to anthracycline antibiotics |
| US5364781A (en) * | 1991-11-18 | 1994-11-15 | Farmitalia Carlo Erba S.R.L | Process for preparing daunorubicin |
| US5695966A (en) * | 1995-02-27 | 1997-12-09 | Pharmacia & Upjohn S.P.A. | DNA encoding daunorubicin 14-hyroxylase and method for preparing doxorubicin |
| US5801032A (en) * | 1995-08-03 | 1998-09-01 | Abbott Laboratories | Vectors and process for producing high purity 6,12-dideoxyerythromycin A by fermentation |
| US5976830A (en) * | 1996-05-24 | 1999-11-02 | The Ohio State University Research Foundation | Methods of producing doxorubicin |
| US5652125A (en) * | 1996-06-10 | 1997-07-29 | Pharmacia S.P.A. | Process for preparing daunorubicin |
-
1999
- 1999-04-22 EP EP99919767A patent/EP1071746A4/en not_active Withdrawn
- 1999-04-22 CA CA002326500A patent/CA2326500A1/en not_active Abandoned
- 1999-04-22 WO PCT/US1999/007016 patent/WO1999055829A2/en active Application Filing
- 1999-04-22 JP JP2000545975A patent/JP2002512784A/en active Pending
- 1999-04-22 CN CNB998054003A patent/CN1198930C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999055829A3 (en) | 1999-12-23 |
| WO1999055829A2 (en) | 1999-11-04 |
| CN1198930C (en) | 2005-04-27 |
| JP2002512784A (en) | 2002-05-08 |
| CN1298453A (en) | 2001-06-06 |
| EP1071746A2 (en) | 2001-01-31 |
| EP1071746A4 (en) | 2003-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5786190A (en) | Daunorubin 14-hydroxylase | |
| US5801032A (en) | Vectors and process for producing high purity 6,12-dideoxyerythromycin A by fermentation | |
| CA2326500A1 (en) | Process for preparing doxorubicin | |
| US6210930B1 (en) | Process for preparing doxorubicin | |
| EP0567630B1 (en) | Dna-sequence coding for carminomycin 4-o-methyltransferase | |
| EP2042608B1 (en) | Genetically modified Streptomyces strains for biotransformations in anthracycline production | |
| RU2115731C1 (en) | Polypeptide showing carminomycin-4-o-methyltransferase activity and a method of its preparing | |
| US5652125A (en) | Process for preparing daunorubicin | |
| US5955319A (en) | Process for preparing doxorubicin | |
| Tornus et al. | Identification of four genes from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22 involved in the biosynthesis of L-rhodinose | |
| US5989869A (en) | Process for preparing daunorubicin and doxorubicin | |
| WO2010028667A1 (en) | Genetically modified strains for biotransformations in anthracycline production | |
| EP0792285B1 (en) | Process for producing anthracyclines and intermediates thereof | |
| IL167854A (en) | Nucleotide sequences encoding polypeptides involved in spiramycin biosynthesis, expression vector comprising same and uses thereof | |
| Nastro et al. | Streptomyces peucetius dpsY The | |
| Inventi-Solari et al. | Doxorubicin Overproduction in | |
| MXPA96005018A (en) | Process for the preparation of doxorrubic | |
| EP1133562A1 (en) | The gene cluster involved in aclacinomycin biosynthesis, and its use for genetic engineering |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |