CN1198930C - Process for preparing doxorubicin - Google Patents
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- CN1198930C CN1198930C CNB998054003A CN99805400A CN1198930C CN 1198930 C CN1198930 C CN 1198930C CN B998054003 A CNB998054003 A CN B998054003A CN 99805400 A CN99805400 A CN 99805400A CN 1198930 C CN1198930 C CN 1198930C
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Abstract
The ability to convert daunorubicin into doxorubicin can be improved by transforming a host cell with a recombinant vector comprising a DNA molecule comprising: a DNA region or fragment containing the gene doxA encoding daunorubicin 14-hydroxylase and a DNA region or fragment containing one or more gene conferring daunorubicin and doxorubicin resistance.
Description
The present invention relates to a kind ofly by improving the method that daunorubicin to Zorubicin transforms with the recombinant vectors transformed host cells, described recombinant vectors comprises the DNA of coding daunorubicin C-14 hydroxylase and gives gene to the antibiotic resistance of anthracyclines.
Daunorubicin-type anthracene nucleus element (for example Zorubicin, carminomycin and aclacinomycin and synthetic analogue thereof) is the medicament (F.Arcamone of widespread use in the antitumor therapy, Zorubicin (Doxorubicin), Academic Press New York, 1981, pp.12; A.Grein, biological process (Process Biochem.), 16:34,1981; T.Kaneko, Chimicaoggi, on May 11st, 1988; C.E.Myers etc., " biochemical mechanism of tumor cytotoxicity " (" Biochemical mechanism of tumour cellkill " in Anthracycline and Anthracenedione-Based Anti-cancerAgents) (Lown of the carcinostatic agent of and amerantrone plain based on anthracene nucleus, J.W. edit), Elsevier Amsterdam, pp.527～569,1988; J.W.Lown, pharmacotherapy (Parmac.Ther.), 60:185,1993).
Daunorubicin-type anthracene nucleus element is normally by various streptomyces (Streptomyces) [ripple match streptomycete (S.peucetius), sky blue little red streptomyces (S.coeruleorubidus), Galilee streptomycete (S.galilaeus), streptomyces griseus (S.griseus), ash red streptomyces (S.griseoruber), mark streptomycete (S.insignis), green color-producing streptomycete (S.viridochromogenes), S.bifurcus and streptomycete bacterial strain C5 (S.sp.strainC5)] bacterial strain and the natural compounds produced by Actinomyces carminata.Zorubicin is mainly produced by ripple match streptomycete bacterial strain.Specifically, daunorubicin and Zorubicin are produced in ripple match streptomycete ATCC 29050 and the steel gray subspecies of Bo Sai streptomycete (S.peucetiussubsp.caesius) ATCC 27952.The plain Zorubicin of anthracene nucleus be by ripple match streptomycete 27952 by the method summarized in the following document from propanedioic acid, propionic acid and glucose production: Grein, applied microbiology progress (Advan.Applied Microbiol.), 32:203,1987 and Eckart and Wagner, basis JOURNAL OF MICROBIOLOGY (J.BasicMicrobiol.) 28:137,1988.Acker quinomycin (11-deoxidation-ε-rhodomycinon), ε-rhodomycinon, rhodomycin D, carminomycin and daunorubicin all are the intermediates that produces in this process.Final step in this approach relates to the C-14 hydroxylation of daunorubicin to Zorubicin.
Be used for the biosynthetic gene of daunorubicin and obtain (Stutzman-Engwall and Hutchinson from ripple match streptomycete 29050 and Bo Sai streptomycete 27952 by clonogenic assay, newspaper (Proc.Natl.Acad.Sci.USA) 86:3135 of institute of NAS, 1988; Otten etc., bacteriology magazine (J.Bacteriol.) 172:3427,1990).As described in WO96/27014 (date of publication is on September 6th, 1996), the gene of coding daunorubicin C-14 hydroxylase (it makes daunorubicin change into Zorubicin) obtains from ripple match streptomycete 29050 and mutant thereof by clonogenic assay, and, it in the host cell of streptomycete and colon bacillus (Escherichia coli) by overexpression.
Two kinds of genes (drrA and drrB that the daunorubicin biosynthesizing clusters, they give the resistance of shallow Streptomyces glaucoviolaceus (Streptomyces lividans) anti-Zorubicin and daunorubicin) be from ripple match streptomycete ATCC 29050 bacterial strains (Guilfoile and Hutchinson, institute of NAS reports 88:8553,1991) (registration number of going into to hide of Genbank is M73758) and from ripple match streptomycete 7600 mutant (EP-0371,112-A and Colombo etc., the bacteriology magazine, 174:1641,1992) clone.Link coupled protein in two kinds of translations of these genes encodings, they the two all be that daunorubicin and Zorubicin resistance are required among this host.The sequence of the expectation product of one of these two kinds of genes is similar to the product (it should be noted that the P-glycoprotein from mammalian tumor cell most) of other transporter gene and resistant gene.(it gives the resistance of anti-daunorubicin and Zorubicin to another kind of gene drrC, have the sequence similarity of height with colon bacillus that relates in the excision reparation of DNA and micrococcus luteus (Micrococcus luteus) UvrA protein) from ripple match streptomycete ATCC 29050 clone (Lomovskaya etc., bacteriology magazine 178:3238,1996).
The invention provides and a kind ofly in host cell, improve the method that daunorubicin to Zorubicin transforms by recombinant vectors, described recombinant vectors comprises: contain coding daunorubicin c-14 hydroxylase gene dxrA DNA district or fragment and contain a kind ofly, two or three is selected from the DNA district or the fragment of the gene of drrA, the drrB of the resistance of giving anti-daunorubicin and Zorubicin and drrC.Three kinds of last genes give anti-Zorubicin (product in the described conversion process) high-caliber resistance in host cell, make this process more effective than former, process is in the past used with recombinant vectors (only carrying the dna fragmentation that contains the dxrA gene) transformed host cells (being described among the WO96/27014, even used strong promoter).
DNA of the present invention preferably comprises drrA, drrB and drrC gene whole three kinds or only contain drrA and two kinds of genes of drrB.
Described DNA can be cloned in the carrier by near the restriction site place that correct form is connected on the allos transcriptional control sequence or the transcriptional control sequence that suitably is in the carrier.Usually, this carrier is a kind of plasmid.Described recombinant vectors can be used to transform proper host cell.This host can be actinomycetes (Actinomycetes) bacterial strain (it does not produce or produce anthracyclines), preferably streptomyces bacterial strain.
(a～c) has set forth the structure of the plasmid pIS156 that describes among the embodiment 1 to Fig. 1.This plasmid is by at strong promoter ermE
*(Bibb etc., molecular microbiology (Molec.Microbiol.) 14:533,1994) control is inserted plasmid pWHM3 (Vara etc., bacteriology magazine 171:5872 with the fragment of 2.9kb down, 1989) make up, described fragment comprises the C-terminal portions of doxA (being dxrA in the past), dnrV (being dnrORF10 in the past) and dnrU (Δ dnrU was dnrORF9 in the past) gene, derives from recombinant plasmid pIS70 (WO 96/27014 and A.InventiSolari etc., GMBIM ' 96, P58).
In order to describe the present invention better, we provide the sequence 1 of 2.867nt, and it comprises the C-terminal portions (complementary strand of coding strand) of doxA, dnrV and dnrU (Δ dnrU) gene.
(a～d) has set forth the structure of the plasmid pIS284 that describes among the embodiment 1 to Fig. 2.This plasmid comprises the 2.9kb fragment, and it has the C-terminal portions of doxA, dnrV and dnrU gene, derives from recombinant plasmid pIS70, is subjected to strong promoter ermE
*Control, and 2.3Kb comprises the dna fragmentation of drrA and drrB resistant gene, described drrA and drrB resistant gene derive from the plasmid pWHM603 (P.Guilfoile and C.R.Hutchinson, institute of NAS reports 88:8553,1991) that is gone into plasmid pWHM3 by subclone.
(a～c) has set forth the structure of the plasmid pIS287 that describes among the embodiment 2 to Fig. 3.Described plasmid is by at strong promoter ermE
*Control under, (it comprises doxA (being dxrA in the past), dnrV (being dnr-ORF10 in the past) and dnrU (Δ dnrU with the BamHI-HindIII fragment of 2.9kb, be dnr-ORF9 in the past) the C-terminal portions of gene, derive from recombinant plasmid pIS70 (WO 96/727014)) contain the Xbal-HindIII dna fragmentation of drrA and drrB resistant gene and EcoRI-HindIII fragment that 3.9kb contains the drrC resistant gene with 2.3kb and insert plasmid pWHM3 and make up.
Figure shown in Fig. 1,2 and 3 does not need detailed exist in the described dna fragmentation whole that list Restriction site. Yet the site of report is enough to the described DNA sections of clear identification.
Restriction site abbreviation: Ap, apramycin; Tsr, bryamycin; Amp, the ampicillin; B, BamHI; G, BgIII; N, NotI; K, KpnI; E, EcoRI; H, HindIII; P, PstI; S, SphI; X, XbaI; L, BglI; T, SstI.
The invention provides a kind of dna molecular, wherein, will comprise coding daunorubicin C-14 hydroxyl Change enzyme gene the DNA district or segment ligation is a kind of to comprising, two or three be selected from drrA, (their codings are given the anti-of the anti-daunorubicin of host cell and adriamycin for drrB and drrC gene The protein of property) in the heterogeneic DNA district or fragment.
The DNA district of gene that comprises coding daunorubicin C-14 hydroxylase is preferably by deriving from the 2.9kb DNA district of recombinant plasmid pIS70 (being described among the patent WO96/27014) with the BamHI-HindIII enzymic digestion.This fragment comprises the doxA gene of coding C-14 hydroxylase.Daunorubicin C-14 hydroxylase changes into Zorubicin with daunorubicin.This 2.9kb dna fragmentation also comprises the dnrV gene that is between the NotI-KpnI site and comprises the NotI-SphI fragment of the C-terminal portions of dnrU (Δ dnrU) gene.
Preferably, the 2.9kb dna fragmentation of coding daunorubicin C-14 hydroxylase is connected to the 2.3kbXbal-HindIII dna fragmentation that comprises the drrA that derives from plasmid pWHM603 and drrB resistant gene and contains on the 3.9kbEcoRI-HindIII fragment of the drrC gene that derives from plasmid pWHM264; In another preferred embodiment, this 2.9kb dna fragmentation only is connected on the 2.3kb Xbal-HindIII dna fragmentation.
Being described in all DNA molecule among the WO 96/27014, coding daunorubicin C-14 hydroxylase all can be applicable among the present invention.
Particularly, dna molecular of the present invention can comprise the whole of 2.9kb dna fragmentation or be this segmental part, at least 1.2kb length is corresponding to the KpnI-BamHI fragment of the dna molecular that contains doxA, the doxA daunorubicin C-14 hydroxylase (it changes into Zorubicin with daunorubicin) of encoding.This dna molecular comprises the sequence of report among the patent application WO 96/27014 basically, and this sequence is called as " dxrA " sequence.In addition, the deduction aminoacid sequence of daunorubicin C-14 hydroxylase also is shown in this patent application.
Dna molecular of the present invention can comprise the 2.3kbXbal-HindIII dna fragmentation of 2247nt at least, and this fragment comprises drrA and drrB gene, and these genes encodings are given the protein of the resistance of anti-daunorubicin of host cell and Zorubicin.
It is segmental all or part of that dna molecular of the present invention can comprise the 3.9kb EcoRI-HindIII that contains the drrC resistant gene, at least 2.5kb length is corresponding to the SstI-SphI fragment of the dna molecular that contains drrC, and the drrC coding is given the protein of the resistance of anti-daunorubicin of host cell and Zorubicin.
The present invention also comprises such DNA: it comprises the gene of the resistance of giving anti-daunorubicin and Zorubicin, sequence at least 80% and drrA and drrB gene (Guilfoile and the Hutchinson of this gene, institute of NAS reports 88:8553,1991) sequence and/or drrC gene (Lomovskaya etc., bacteriology magazine 178:3238,1996) sequence is identical.
Near the DNA of the present invention correctly restriction site place that is connected on the allos transcriptional control sequence or the transcriptional control sequence that suitably is in the carrier of form is cloned in the carrier.Preferably, heterogeneic transcribing can be passed through common strong promoter such as ermE
*Coordinate (Bibb etc., molecular microbiology 14:533,1994).
Dna molecular of the present invention can be connected into any self-replicating agent and/or integrated agent (it comprises a kind of dna molecular, can be added one or more other DNA sections in this dna molecular).But, usually, described carrier is a kind of plasmid.Plasmid pWHM 3 that a kind of preferred plasmid is a high copy number or pIJ702 (Katz etc., general microbiology magazine (J.Gen.Microbiol.) 129:2703,1983).Other suitable plasmid is pIJ680 (Hopwood etc. the, " genetic manipulation of the mould Pseudomonas of Key.Experiment guide " (GeneticManipulation of Streptomyces.A laboratory Manual), John InnesFoundation, Norwich; UK; 1985) and pWHM601 (Guilfoile and Hutchinson, institute of NAS reports 88:8553, and 1991).
Can use any suitable technique described DNA is inserted carrier.Can realize inserting by this DNA is connected into linearized vector in suitable site.For this reason, can use the direct combination of sticky end or flush end, the homopolymer tailing is perhaps used linkers or adaptor molecule.
Can use recombinant vectors and transform the proper host cell of not producing or producing ring anthra class.
Host cell can be daunorubicin or Zorubicin sensitivity (that is, can not grow in the presence of a certain amount of daunorubicin or Zorubicin) or daunorubicin or Zorubicin resistance.Under any circumstance, the recombinant clone of generation (by what obtain with new recombinant vectors conversion of the present invention) shows the resistance of anti-daunorubicin of higher level and Zorubicin than parental generation host.The Zorubicin resistance level of recombinating in the shallow Streptomyces glaucoviolaceus is higher than observed level in ripple match streptomycete ATCC29050 that produces the anthracene nucleus element and ATCC 27952 bacterial strains far away.
Described host can be microorganism (a for example bacterium).The actinomyces bacterial strain (especially shallow Streptomyces glaucoviolaceus strain and other streptomyces strain) of not producing anthracyclines can be transformed.Compare with ripple match streptomycete dnrN mutant, shallow Streptomyces glaucoviolaceus TK 23 is hosts more suitably, and described mutant is with recombinant plasmid pIS70 conversion, and this pIS70 comprises and is used for the dxrA gene (WO 96/27014) of daunorubicin to the Zorubicin bio-transformation.
Recombinant vectors of the present invention also can be used to transform the proper host cell of producing daunorubicin, so that strengthen the conversion of daunorubicin to Zorubicin.
Therefore, the ripple match streptomycete ATCC29050 and ATCC 27952 bacterial strains of mutant that comprise their production anthracyclines can be transformed.Particularly, can use ripple match streptomyces strain WMH1654, it is the mutant strain that derives from ripple match streptomycete ATCC 29050, and be deposited in American type culture collection, 10801 UniversityBoulevard, Manassas, Virginia 20110-2209, USA, going into to hide registration number is ATCC55936.
The transformant of streptomyces bacterial strain generally can obtain by protoplast transformation.
The present invention includes the method that Zorubicin that improvement transforms by daunorubicin is produced, this method is included among the host who does not produce anthracyclines the fermenting process that the daunorubicin that adds is become the biotransformation of Zorubicin and produce Zorubicin in the host of direct production daunorubicin.
Daunorubicin is to the biotransformation of Zorubicin
This method comprises:
1) cultivate the recombinant host cell of not producing daunorubicin that transforms with carrier of the present invention, the past daunorubicin that wherein adds, and
2) separate Zorubicin from culture.
In the method, can under the temperature of 20 ℃～40 ℃ (for example 24 ℃～37 ℃), cultivate recombinant bacterial strain.In 24～96 hours vegetative period, in substratum, add daunorubicin.Preferably under vibration, cultivate.Incubation time in the presence of daunorubicin can be 12～72 hours.The concentration of daunorubicin can be 20～1000mcg/ml (for example 100～400mcg/ml) in the nutrient solution.
By the fermentative production Zorubicin
This method comprises:
1) cultivate the reorganization daunorubicin that transforms with carrier of the present invention and produce host cell, and
2) separate Zorubicin from culture.
In the method, can under the temperature of 20 ℃～40 ℃ (for example 26 ℃～34 ℃), cultivate recombinant bacterial strain.Under vibration, cultivate.Incubation time can be 72～168 hours.
Raw material and method
Bacterial isolates and plasmid:Colon bacillus strain DH5 α (it is Ampicillin Trihydrate and apramycin sensitivity) is used to the subclone dna fragmentation.The shallow Streptomyces glaucoviolaceus TK23 of host derives from D.A.Hopwood (John Innes Institute, Norwich, United Kindom), host Bo Sai streptomycete WMH1654 is the mutant strain that derives from ripple match streptomycete ATCC 29050, has been deposited in American type culture collection, 10801 UniversityBoulevard, Manassas, Virginia 20110-2209, USA, going into to hide registration number is ATCC55936.Plasmid cloning vector be pGem-7Zf (+) with relevant plasmid (Promega, Madison, WI), pIJ4070 (D.A.Hopwood) and colon bacillus-streptomyces shuttle vectors pWHM3 (Vara etc., bacteriology magazine 171:5872,1989).
Substratum and damping fluid:Colon bacillus strain DH5 α is maintained at (Sambrook etc., " molecular cloning on the LB agar.Experiment guide " (Molecular Cloning.A Laboratory manual), the 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989).When selecting transformant, add Ampicillin Trihydrate or apramycin with the concentration of 100 μ g/ml.Shallow Streptomyces glaucoviolaceus TK23 and Bo Sai streptomycete WMH1654 are maintained at R2YE (Hopwood etc. the, " genetic manipulation of the mould Pseudomonas of key respectively.Experiment guide ", John InnesFoundation, Norwich, UK, 1985) and ISP4 (Difco, Detroit is MI) on the nutrient agar.When selecting transformant, contain the soft agar that concentration is 50 μ g/ml Thiactins at dull and stereotyped upper berth one deck.
The subclone dna fragmentation: on sepharose, separate (Sambrook etc., " molecular cloning with suitable restriction enzyme dna digestion sample and by standard method.Experiment guide ", the 2nd edition, ColdSpring Harbor Press, Cold Spring Harbor, NY, 1989).Comprise the agarose section of interested dna fragmentation from gel excision, use the GENECLEAN device (Bio101, La Jolla, CA) or equivalent from these slice separation DNA.Application standard technology (Sambrook etc., " molecular cloning.Experiment guide "; the 2nd edition, Cold Spring HarborPress, Cold Spring Harbor; NY, 1989) streptomyces carrier that separated DNA fragment subclone is gone into to be used for the colon bacillus of routine operation and colon bacillus-streptomyces shuttle vectors or is used to express experiment.
The conversion of streptomycete and colon bacillus:By Calcium Chloride Method (Sambrook etc., " molecular cloning.Experiment guide "; the 2nd edition, Cold Spring Harbor Press, Cold SpringHarbor; NY; 1989) the preparation colon bacillus competent cell and by standard technique (Sambrook etc., " and molecular cloning. experiment guide ", the 2nd edition; Cold Spring HarborPress; Cold Spring Harbor, NY, 1989) transform.Shallow Streptomyces glaucoviolaceus TK23 is grown in (Hopwood etc. the, " genetic manipulation of the mould Pseudomonas of Key in the liquid R2YE substratum.Experiment guide ", John Innes Foundation, Norwich, UK, 1985), behind 48hr, gather in the crops.With 10.3% (wt/vol) sucrose solution with the hyphostoma particle washed twice and be used for method (Hopwood etc. the, " genetic manipulation of streptomyces summarized by the Hopwood guide.Experiment guide ", John Innes Foundation, Norwich, UK, 1985) the preparation protoplastis.With the protoplastis particle suspension in about 300 microlitre P damping fluid (Hopwood etc., " genetic manipulations of streptomyces.Experiment guide ", John Innes Foundation, Norwich, UK, 1985), the five equilibrium sample of 50 these suspension of microlitre is used for each conversion.Protoplastis is by (" genetic manipulations of streptomyces such as Hopwood with plasmid DNA.Experiment guide ", John InnesFoundation, Norwich; UK; 1985), the small-scale conversion method of Stutzman-Engwall and (bacteriology magazine 172:3427,1990) such as Hutchinson (institute of NAS reports 86:3135,1988) or Otten transforms.Behind regeneration 17hr on 30 ℃ the R2YE substratum,, allow it grow until sporulation down at 30 ℃ at the Thiactin of dull and stereotyped upper berth one deck 200 μ g/ml.
The assessment of daunorubicin and Zorubicin resistance level: this resistance level is measured with minimum inhibition concentration (MIC) expression and by the standard double dilution method of using the R2YE substratum.Bacterial strain placed on the R2YE medium slant cultivate and cultivated 8～10 days down at 28 ℃.Recombinant bacterial strain is to grow in the same medium of having added 20 μ g/ml Thiactins.Comprise about 10
6～10
7The bacterial cultures of viable cell/ml be under 28 ℃, 280rpm, trypticase soy broth (Tryptic SoyBroth) the culture preparation of having grown 48 hours (Difo).By granulated glass sphere homogenizing culture.Get on the agar plate that culture after the loopful homogenizing is seeded in the daunorubicin that contains 0.39～800 μ g/ml different concns and Zorubicin.Under 30 ℃, this agar plate was cultivated 7 days, measured MICs with the minimum concentration that prevents visible growth.
Daunorubicin is to the bio-transformation of Zorubicin: the shallow Streptomyces glaucoviolaceus TK23 transformant that will have a plasmid of the present invention is inoculated in the liquid R2YE substratum that 25ml contains 40 μ g/ml Thiactins.Culture is grown in the 300ml Erlenmeyer flask, on the gyrate shaker of 30 ℃ of following 280rpm, cultivate.Grow after 2 days, this culture of 2.5ml is transferred to 25ml APM produce substratum: ((g/l) glucose (60), yeast extract (8), malt extract (20), NaCl (2), 3-(morpholino) propanesulfonic acid (MOPS sodium salt) (15), MgSO
47H
2O (0.2), FeSO
47H
2O (0.01), ZnSO
47H
2O (0.01) has added 20 μ g/ml Thiactins.48hr adds 400 μ g/ml daunorubicins in vegetative period.Culture is grown in the 300ml Erlenmeyer flask, on the gyrate shaker of 30 ℃ of following 280rpm, cultivate 72hr.With each culture of 25mg/ml oxalic acid acidifying, after cultivating 30min on the gyrate shaker of 30 ℃ of following 280rpm, use the equal-volume acetonitrile: methyl alcohol (1: 1) 30 ℃ with 300rpm under extract 2hr.With extracting liquid filtering, analyze filtrate by reversed-phase high pressure liquid chromatography (RP-HPLC).RP-HPLC uses Vydac C
18Post (4.6 * 250mm; 5 μ m particle diameters) with the operated in flow rate of 0.385ml/min.Mobile phase A is that 0.2% trifluoroacetic acid (TFA derives from Pierce Chemical Co.) is (in H
2Among the O), Mobile phase B is that 0.078%TFA[is in acetonitrile (deriving from J.T.Baker Chemical Co.)].In 33 minutes, carry out wash-out and use diode-array detector fixing on 488nm (12 microns of bandwidth) monitoring with 20～60% linear gradients that are dissolved in the B phase of A in mutually.Daunorubicin and Zorubicin (10 μ g/ml are in methyl alcohol) be used as external standard and quantitative analysis from the amount of isolating these metabolites of culture.
Zorubicin is produced: transform ripple match streptomycete WMH1654 mutant with plasmid of the present invention.Transformant is inoculated the R2YE substratum of having added 20 μ g/ml Thiactins into 25ml.Culture is grown in the 300ml Erlenmeyer flask on 30 ℃ and the 280rpm gyrate shaker.Grow after 2 days, this culture of 2.5ml is transferred to the APM substratum that 25ml has added 20 μ g/ml Thiactins.Culture was grown 96～120 hours in the 300ml Erlenmeyer flask on 28 ℃ and the 280rpm gyrate shaker.With each culture of 25mg/ml oxalic acid acidifying, after cultivating 45min on the gyrate shaker of 30 ℃ of following 280rpm, use the equal-volume acetonitrile: methyl alcohol (1: 1) 30 ℃ with 300rpm under extract 2hr.With extracting liquid filtering, by RP-HPLC by the same methods analyst filtrate that is used to analyze described bioconversion product.
Embodiment 1
Embodiment 1(Fig. 1 (a～c) and Fig. 2 (a～d)).
In order to remove nonessential region, plasmid pIS70 (WO96/27014) digests with EcoRI-HindIII in advance, 3.5kb fragment subclone is gone into plasmid pGEM-7Zf (+) (Promega, Madison-WIUSA) same loci of multiple clone site sequence and obtain another kind of BamHI restriction site.Novel plasmid pGendoxAUV is BamHI digestion, at strong promoter ermE
*Control down, with described fragment (now being reduced to 2.9kb) be transferred to plasmid pIJ4070 (derive from John InnesInstitute, Norwich, UK).This novel plasmid (being called as p7doxAUV) is the BgIII of digestion, and described fragment is inserted plasmid pWHM3 (J.Vara etc., bacteriology magazine 171:5872～5881,1989) and obtained plasmid pIS156 (Fig. 1 c).
The 2.3kb BgII fragment that comprises drrA and drrB resistant gene is transferred to the SmaI site of plasmid pBluescript II SK+ (Stratagene) from plasmid pWHM603 and is obtained plasmid pdrrAB after doing into flush end, again the XbaI-HindIII fragment is transferred to carrier pIJ4070 and obtains pIS278 from pdrrAB.Then, pIS278 is inserted into after the EcoRI-XbaI digestion among the EcoRI-XbaI plasmid pWHM3 and obtains plasmid pIS281. and digest this plasmid with XbaI, inserts the XbaI fragment of plasmid pIS156 and obtains plasmid pIS284.
Embodiment 2
The structure of plasmid pIS287 (Fig. 3 (a～c)): excise the drrC resistant gene that is contained among the plasmid pWHM264 by EcoRI-HindIII digestion, again it is inserted plasmid pIJ4070 and obtains plasmid pIS282.From this plasmid the drrC resistant gene is transferred to pIS252 (this plasmid is the modified forms that comprises near the pWHM3 in the extra BglII site in EcoRI site) and obtained plasmid pIS285 as the BglII fragment.Plasmid pIS285 is EcoRI digestion, it is connected with the 5.5kb dna fragmentation that excises from plasmid pIS284 and obtains plasmid pIS287.
Embodiment 3
Above-mentioned recombinant plasmid is to the resistance of Zorubicin: by operation of describing in " raw material and method ", on the R2YE substratum, measured the resistance level (shallow Streptomyces glaucoviolaceus TK23 that transforms with shallow Streptomyces glaucoviolaceus TK23, with carrier pWHM3 and anthracene nucleus be plain produces ripple match streptomycete ATCC 29050 and ATCC 27952 bacterial strains relatively) of the anti-daunorubicin of shallow Streptomyces glaucoviolaceus TK23 that transforms with recombinant plasmid pIS70, pIS284 or pIS287 and Zorubicin as MICs.The plasmid pIS287 that comprises drrA, drrB and drrC resistant gene has obtained the daunorubicin and the Zorubicin resistance of maximum horizontal.The Zorubicin resistance level that only comprises the plasmid of drrA and drrB resistant gene has also increased 64 times (table 1).
Table 1. recombinant bacterial strain is to the resistance of Zorubicin
The MIC of the relevant Zorubicin of bacterial strain (μ g/ml)
Ripple match streptomycete ATCC 29,050 12.5
Ripple match streptomycete ATCC 27,952 12.5
Shallow Streptomyces glaucoviolaceus TK23 12.5
Shallow Streptomyces glaucoviolaceus TK23 (pWHM3) 12.5
Shallow Streptomyces glaucoviolaceus TK23 (pIS284) 800
Shallow Streptomyces glaucoviolaceus TK23 (pIS287)＞800
Embodiment 4
Transforming with the plasmid that comprises doxA daunorubicin C-14 '-hydroxylase gene and different resistant genes Shallow Streptomyces glaucoviolaceus TK23 in, the daunorubicin bio-transformation of interpolation becomes Zorubicin: by the operation of describing in " raw material and method " part, pIS70, pIS284 or pIS287 plasmid are introduced shallow Streptomyces glaucoviolaceus TK23 by the conversion of selecting the Thiactin resistance.Use the APM substratum and test shallow Streptomyces glaucoviolaceus TK23 (pIS70), the shallow Streptomyces glaucoviolaceus TK23 (pIS284) of generation and shallow Streptomyces glaucoviolaceus TK23 (pIS287) transformant bio-transformation high-content (400 μ g/ml) daunorubicin ability as described above to Zorubicin.Shallow Streptomyces glaucoviolaceus TK23 (pIS70) transforms physical efficiency and is converted into many 11.5% daunorubicin that adds to Zorubicin (table 2).Shallow Streptomyces glaucoviolaceus TK23 (pIS284) and shallow Streptomyces glaucoviolaceus TK23 (pIS287) transform physical efficiency and transform the daunorubicin of nearly 73.5% interpolation to Zorubicin (table 2).
Table 2. by shallow Streptomyces glaucoviolaceus bacterial strain bio-transformation daunorubicin to Zorubicin
Bacterial strain anthracene nucleus element (μ g/ml)
DOX DNR 13-dihydro DNR
Shallow Streptomyces glaucoviolaceus TK23 (pIS70) (contrast) 46 250 70
Shallow Streptomyces glaucoviolaceus TK23 (pIS284) 294 33 21
Shallow Streptomyces glaucoviolaceus TK23 (pIS287) 288 24 35
Embodiment 5
Transforming with the plasmid that comprises doxA daunorubicin C-14 '-hydroxylase gene and different resistant genes Ripple match streptomycete WMH1654dnrX mutant in Zorubicin production: by the operation of describing in " raw material and method " part, pIS284 and pIS287 plasmid are introduced ripple match streptomycete WMH1654 dnrX mutant strain by the protoplast transformation of selecting the Thiactin resistance.With the ripple match streptomycete transformant fermentation that generates, press the methods analyst fermented liquid that preamble is described.After the 120hr fermentation, ripple match streptomycete WMH11654 (pIS284) produces nearly 81 μ g/ml Zorubicins and nearly 18 μ g/ml daunorubicins (table 3).Ripple is matched streptomycete WMH1654 (pIS287) but is produced the daunorubicin (table 3) that reaches 92 μ g/ml Zorubicins and do not have detection limit.
Table 3. the Zorubicin production of ripple match streptomycete WMH1654 dnrX bacterial strain
Bacterial strain anthracene nucleus element (μ g/ml)
DOX DNR 13-dihydro DNR
Ripple match streptomycete WMH1654 41 35 18
Ripple match streptomycete WMH1654 (pIS284) 81 18 6
Ripple match streptomycete WMH1654 (pIS287) 92 00
Sequence 1
1 GGATCCGCAC?CGGGTACACG?GCACGGGACC?GCCCACCGCG?CGGTGCGCGG
51 TGGGCGGTCC?CGTGCCGGTC?GCGGCCGGCG?GATCAGCGCA?GCCAGACGGG
101?CAGTTCGGTG?AGCCGCGCCG?TCTGGGCCCC?CTTCCGGCAC?CACCGCAACT
151?CGTCGTACGG?CACGGCCAGT?CGGGCCTCGG?GGAACCTGCT?GCGCAGTACG
201?CCGATCATCG?TGCGCGACTC?CAGCTGGGCG?AGCTGCTCCC?CGATGCAGTA
251?GTGCGGCCCG?TCGCCGAAGG?TGAGCCGCCG?CCACGAGGGA?CGGTCCGGGT
301?GGAAGGCGTG?CGGGGCGTCG?TGATGGCGGC?CGTCGGTGTT?GGTGCCCTCG
351?ATGTCCACCA?GCACCGGCGC?TCCGCGGGGC?AGCCGGACGC?CGCCGATGGT
401?CACCTCCGTG?GCAGCGAACC?TCCACAACGT?GTAGGGCACC?GGCGGGTGGT
451?AGCGCAGCGC?CTCCTCCACG?AACCGGGAGA?CGGCGTCCTC?GTCGGCATCC
501?GCCGCGAGGC?GGCCCGCCAG?GACCTCCGCG?AGCAGGAAGC?CCAGGAAGGA
551?GCCGGTGGTG?TCGTGGCCGG?CGAAGATGAG?CCCGGTGATC?ATGTAGACGA
601?GCTGGTCGTC?GGAGACCGAG?CCGAACTCGG?CCTGCGCGCG?CTCGTACAGC
651?ACGCGGGTCA?TGGTCGGGGT?GTCGTTCCGC?CGGGCTGAGT?GCACGGCTTC
701?GAGGAGCAGG?CTCTCCAGGG?CCGAGGTGTC?CGGCACGCCC?CCGGCAGGGT
751?CCGTGCCGTC?ACCCCCGCCG?CTCTGCGGGC?CGCCGAGGCC?GAGTGCCTTG
801?AGAACGCTGA?CGGCCTCGCG?GGCCATCGCC?GGATCGGTGA?CCGGCACACC
851 GAGCAGCTCG?CAGATGACCA?ACAGCGGGAA?GTGGTACGCG?AAGCCGCCGA
901 TCAGCTCGGC?CGGTTTGCCC?GACCGGCCGG?AGGCGTCGGC?GAGTTCGGTG
951 AGCAGCCGGC?CGGCGATCGC?GGCGATGCGA?TCCGTCCGCT?CGGCCAGCCG
1001?GCGCGGGTTG?AACGCAGGTG?CGTGGATGCG?GCGCAGGCGC?CGGTGGGCCT
1051?CGCCGTCCAC?GGCGATGAGC?GTGAACGGAC?GCAGCTCCGG?AACGGGGATG
1101?TCGAGACCGT?CGTCCACCCC?CCGCCAGGCG?GCGGGGGCGA?GGTCGGGGTC
1151?CTTCACGAAC?CGGGGATCGG?CCAGCACCTC?GCGGGCGAGG?GCGTCATCGG
1201?TGATGACCCA?GGCGGGTCCG?CCCGCGGGGG?CGTTCACCTC?GACGACCGGG
1251?CCCGCCTCCC?GGAAGGCGTC?GTGCACCTCG?GGCTTGCGCT?GCATGGTCAT
1301?CATGGGACAC?GCGAACGGGT?CGACGGCCAC?CCGGGGCGCC?TCGCCGCTCA
1351?CGAGGCACCG?CCCGCCGCCG?CGGGGTACCC?CTCCCGCAGT?TCGACCACCG
1401?AGAAGCCGGC?CCCGTGCGGG?TCGAGCAGGT?CCGCCCGCCG?CCCCCTGGGC
1451?GTGTCGGCGG?GCTCGTTCTC?GACGGAGCCG?CCGAGTTCAA?CGGCGCGCCG
1501?GACCGTCGCG?TCGCAGTCGT?GCACGGCGAA?CAGCACGGCC?CAGTGCGGCC
1551?GTACCGCGCC?GGTGACGCCC?AGCTCCTGGG?TGCCGGCGAC?CGGTGTGTCA
1601?CCGATGTGCC?AGACCGGGTC?GGTGACGCCC?TTCAGTCCGG?TGTCGGCCGG
1651?AGCCAGGCCG?AGGGTCGCCG?GGTAGAAGTC?CCGGGCGGCC?CCGATGCCGT
1701?CGGTCACCAG?CTCGACCCAG?CCGACCGAGC?CGGGCACGCC?CGTCACCTCC
1751?GCGCCCTCCA?TGACTCCCTT?GCGCCAGACC?GCGAACGCGG?CCCCGGCGGG
1801?GTCGGCGAAG?ACCGCCATCC?GGCCGAGGCC?GAGGACGTCC?ATCGGAGTCA
1851?TGATGACCTC?GCCGCCCGCC?GTCTCGACCC?GCTTGGTCAG?TGCGTCGGCG
1901?TCGTCGGTGG?CGAAGTACAC?GGTCCAGATG?GCCGGCATGC?CGTGCTGGTC
1951?GTTCCCGGGC?CCGTACGGCC?GGTGGTAGGG?GGTGTCGATC?TGGTGGCGGG
2001?CGACCGCGGC?GACCAGCTTC?CCGTCGGAGC?TGAACGTCGT?GTATCCCCCG
2051?GCGCCCGGGT?CGCTGACCAC?GGTGGCGGTC?CAGCCGAACA?GGCCGGTGTA
2101?GAAGTCGGCC?GAGGCGGCGA?CATCGGGCGA?ACCGAGGTCG?AACCATGCGG
2151?GGGCGCCGGG?CGCGAACCTG?GTCACGAATC?GTTCCTTTCG?ATGGATCGGC
2201?ACACGAGCGT?CTGCGCTCGC?GGATGAGACG?GACATCTCGC?GGATGAGACG
2251?GACATGCGGG?CGGGGCGGGC?CGCCGCCGTC?AGTGCGCGGT?GTCGCCGACG
2301?GCGGCCGCGC?CGGCCTCCCA?GAGCTTCGCC?GCGAGGCCGG?CGTCGGCGGT
2351?CGGGCCGCTC?ACCGGGGACA?GCCGCCGGTC?GCTGTAGTAG?CCGCCCGTGG
2401?TCAACTCCTC?GGCCGGCGCG?GACGCCAGCC?ACACGAGGGT?GTCGGCGCCC
2451?TTCGCCGCGG?AGCGCAGGAA?GGGGTTGAAC?CGGAAGTAGG?ACGAGGCGAC
2501?CGTGCCCCGT?CCGATGCGGG?TGCGGACCTC?ACCGGGGTGA?TAGCTGACCG
2551?CCAGCACGTC?CGGCCAGCGC?CTGGCGGCCT?CCGCCGCGGT?CATGATGTTG
2601?GCCTGTTTGG?ACGTGCCGTA?CGCCTGGCCG?GCGCTGTAGC?GGTGACGGTC
2651?GCCGTTGAGG?TCGTCCGGGT?CGATCCGGCC?CTGGGTGTAC?GCGTCGGACG
2701?AGGTGAGGAT?CAGCCGCCCG?CCCGCGAGCC?GCTCCCGCAG?CAGCCGTGCC
2751?AGCAGGAAGC?CTGCGAGGTG?ATTGACCTGG?ATGGTGGCCT?CGAACCCGTC
2801?CTGGGTCGTG?GTGCGCGACC?AGAACATGCC?GCCGGCGTTG?CTGGCCATGA
2851?CATCGATGCG?CGGGTACCGG
Sequence table
<110>PHARMACIA?&?UPJOHN?S.P.A.
＜120〉prepare the method for Zorubicin
<130>1615-9003
<140>PCT/UNKNOWN
<141>1999-04-22
<150>09/065,606
<151>1998-04-24
<160>1
<170>PatentIn?Ver.2.0
<210>1
<211>2870
<212>DNA
＜213〉ripple match streptomycete
<220>
<221>misc_feature
<222>Comolement((1)..(2870))
＜223〉complementary strand of coding strand
<400>1
ggatccgcac?cgggtacacg?gcacgggacc?gcccaccgcg?cggcgcgcgg?cgggcggtcc?60
cgtgccggtc?gcggccggcg?gatcagcgca?gccagacggg?cagttcggtg?agccgcgccg?120
tctgggcccc?cttccggcac?caccgcaact?cgtcgtacgg?cacggccagc?cgggcctcgg?180
ggaacctgct?gcgcagtacg?ccgatcatcg?tgcgcgactc?cagctgggcg?agctgctccc?240
cgatgcagta?gtgcggcccg?tcgccgaagg?tgagccgccg?ccacgaggga?cggtccgggt?300
ggaaggcgtg?cggggcgtcg?tgatggcggc?cgtcggtgtt?ggtgccctcg?atgtccacca?360
gcaccggcgc?tccgcggggc?agccggacgc?cgccgatggt?cacctccgcg?gcagcgaacc?420
tccacaacgt?gtagggcacc?ggcgggtggt?agcgcagcgc?ctcctccacg?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?1440
ccccctgggc?gtgtcggcgg?gctcgttctc?gacggagccg?ccgagttcaa?cggcgcgccg?1500
gaccgtcgcg?tcgcagtcgt?gcacggcgaa?cagcacggcc?cagtgcggcc?gtaccgcgcc?1560
ggtgacgccc?agctcctggg?tgccggcgac?cggtgtgtca?ccgatgtgcc?agaccgggtc?1620
ggtgacgccc?ttcagtccgg?tgtcggccgg?agccaggccg?agggtcgccg?ggtagaagtc?1680
ccgggcggcc?ccgatgccgt?cggtcaccag?ctcgacccag?ccgaccgagc?cgggcacgcc?1740
cgtcacctcc?gcgccctcca?tgactccctt?gcgccagacc?gcgaacgcgg?ccccggcggg?1800
gtcggcgaag?accgccatcc?ggccgaggcc?gaggacgtcc?atcggagtca?tgatgacctc?1860
gccgcccgcc?gtctcgaccc?gcttggtcag?tgcgtcggcg?tcgtcggtgg?cgaagtacac?1920
ggtccagatg?gccggcatgc?cgtgctggtc?gttcccgggc?ccgtacggcc?ggtggtaggg?1980
ggtgtcgatc?tggtggcggg?cgaccgcggc?gaccagcttc?ccgtcggagc?tgaacgtcgt?2040
gtatcccccg?gcgcccgggt?cgctgaccac?ggtggcggtc?cagccgaaca?ggccggtgta?2100
gaagtcggcc?gaggcggcga?catcgggcga?accgaggtcg?aaccatgcgg?gggcgccggg?2160
cgcgaacctg?gtcacgaatc?gttcctttcg?atggatcggc?acacgagcgt?ctgcgctcgc?2220
ggatgagacg?gacatctcgc?ggatgagacg?gacatgcggg?cggggcgggc?cgccgccgtc?2280
agtgcgcggt?gtcgccgacg?gcggccgcgc?cggcctccca?gagcttcgcc?gcgaggccgg?2340
cgtcggcggt?cgggccgctc?accggggaca?gccgccggtc?gctgtagtag?ccgcccgtgg?2400
tcaactcctc?ggccggcgcg?gacgccagcc?acacgagggt?gtcggcgccc?ttcgccgcgg?2460
agcgcaggaa?ggggttgaac?cggaagtagg?acgaggcgac?cgtgccccgt?ccgatgcggg?2520
tgcggacctc?accggggtga?tagctgaccg?ccagcacgtc?cggccagcgc?ctggcggcct?2580
ccgccgcggt?catgatgttg?gcctgtttgg?acgtgccgta?cgcctggccg?gcgctgtagc?2640
ggtgacggtc?gccgttgagg?tcgtccgggt?cgatccggcc?ctgggtgtac?gcgtcggacg?2700
aggtgaggat?cagccgcccg?cccgcgagcc?gctcccgcag?cagccgtgcc?agcaggaagc?2760
ctgcgaggtg?attgacctgg?atggtggcct?cgaacccgtc?ctgggtcgtg?gtgcgcgacc?2820
agaacatgcc?gccggcgttg?ctggccatga?catcgatgcg?cgggtaccgg 2870
Claims (17)
1. dna molecular, it comprises: the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one are contained at least a DNA district that gives the gene of daunorubicin and Zorubicin resistance, and the wherein said gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC.
2. the dna molecular of claim 1, it further comprises a kind of strong promoter.
3. the dna molecular of claim 2, wherein, described strong promoter is ermE
*
4. the dna molecular of claim 1, wherein, the described gene of giving daunorubicin and Zorubicin resistance is drrA and drrB gene.
5. the dna molecular of claim 1, wherein, the described gene of giving daunorubicin and Zorubicin resistance is drrA, drrB and drrC gene.
6. the dna molecular of claim 1, wherein, the length in district that comprises the gene doxA of coding daunorubicin 14-hydroxylase is 2.9kb.
7. the dna molecular of claim 6, wherein, the district that comprises gene doxA is corresponding to the KpnI-BamHI fragment that comprises the doxA nucleotide sequence.
8. the dna molecular of claim 4, wherein, the described district that comprises described drrA and drrB gene is a 2.3kb XbaI-HindIII dna fragmentation.
9. carrier that comprises the dna molecular of claim 1.
10. the carrier of claim 9, wherein, described carrier is a plasmid.
11. the plasmid of claim 10, wherein, described plasmid is selected from pIS284 and pIS287.
12. host cell that transform or transfection of the carrier with claim 9.
13. the host cell of claim 12, wherein, described host cell is not produced daunorubicin.
14. the host cell of claim 12, wherein, described host cell is a bacterial cell of producing daunorubicin.
15. the host cell of claim 12, wherein, described host cell is the streptomyces cell.
16. a method that is used for the daunorubicin bio-transformation is become Zorubicin, it comprises the steps:
In containing the substratum of daunorubicin, cultivate a kind of recombinant host cell, wherein, described host cell comprises a kind of dna molecular, this dna molecular comprises the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one and contains at least a DNA district that gives the gene of daunorubicin and Zorubicin resistance, wherein, the described gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC, and described host cell is not produced daunorubicin, and
The Zorubicin that separates any generation from described substratum.
17. the method by the fermentative production Zorubicin, it comprises the steps:
In substratum, cultivate a kind of recombinant host cell, wherein, described host cell comprises a kind of dna molecular, this dna molecular comprises the DNA district of a gene doxA who contains coding daunorubicin 14-hydroxylase and one and contains the DNA district that one or more give the gene of daunorubicin and Zorubicin resistance, wherein, the described gene of giving daunorubicin and Zorubicin resistance is selected from drrA, drrB and drrC, and described host cell is a bacterial cell of producing daunorubicin, and
The Zorubicin that separates any generation from described substratum.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6560698A | 1998-04-24 | 1998-04-24 | |
| US09/065,606 | 1998-04-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1298453A CN1298453A (en) | 2001-06-06 |
| CN1198930C true CN1198930C (en) | 2005-04-27 |
Family
ID=22063870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB998054003A Expired - Fee Related CN1198930C (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 |
| US8383392B2 (en) | 2007-09-14 | 2013-02-26 | Meiji Seika Pharma Co., Ltd. | Transformant and 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 JP JP2000545975A patent/JP2002512784A/en active Pending
- 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 CN CNB998054003A patent/CN1198930C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2326500A1 (en) | 1999-11-04 |
| JP2002512784A (en) | 2002-05-08 |
| WO1999055829A3 (en) | 1999-12-23 |
| WO1999055829A2 (en) | 1999-11-04 |
| EP1071746A2 (en) | 2001-01-31 |
| EP1071746A4 (en) | 2003-04-23 |
| CN1298453A (en) | 2001-06-06 |
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