WO1992016629A1 - Procede de production d'antibiotiques, sequences d'adn, structures d'adn recombine et souches microbiennes utilisees - Google Patents

Procede de production d'antibiotiques, sequences d'adn, structures d'adn recombine et souches microbiennes utilisees Download PDF

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WO1992016629A1
WO1992016629A1 PCT/FI1992/000084 FI9200084W WO9216629A1 WO 1992016629 A1 WO1992016629 A1 WO 1992016629A1 FI 9200084 W FI9200084 W FI 9200084W WO 9216629 A1 WO9216629 A1 WO 9216629A1
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dna
streptomyceε
strain
antibiotics
galilaeuε
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PCT/FI1992/000084
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English (en)
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Jarmo Niemi
Juha Hakala
Anja Kopio
Raimo PÄRSSINEN
Kristiina Ylihonko
Klaus Lampi
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Leiras Oy
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention concerns a process by which, by means of known microorganisms, by transferring into them specific genes from certain microbial strains which produce structurally closely related antibiotics, such antibiotics of the anthracycline group are biotechnically produced, which these microorganisms do not produce naturally.
  • the invention concerns also microorganisms needed in such a process and formed by recombinant-DNA- technology, recombinant-DNA-constructions and DNA-sequen- ces needed therein.
  • the invention belongs to the field of biotechnical production of antibiotics and relates to the application of hybrid antibiotic technology to anti ⁇ biotics of the anthracycline group.
  • hybrid anti ⁇ biotics Molecules, which in the same molecule have structural features from two such antibiotics which are not produced naturally by one microorganism, are called hybrid anti ⁇ biotics.
  • Such molecules can in principle, and in some cases also in practice be produced by biotransformation, i.e. by giving an antibiotic, produced by one microorga ⁇ nism, to another microbe which converts the molecule.
  • biotransformation i.e. by giving an antibiotic, produced by one microorga ⁇ nism
  • the use of the term is, however, established to mean the fact that biosynthetic genes of one antibiotic are transferred by recombinant-DNA-technology into a microbe producing another antibiotic, and thus the latter microbe is made to produce antibiotics, which it does not itself, nor the gene donor strain produce naturally.
  • the hybrid anti ⁇ biotics technique is described e.g. in H. G.
  • Hybrid antibiotics the contribution of the new gene combinations
  • An antibiotic molecule is formed in a microorganism, which produces it by an enzymatic reaction cascade in ⁇ cluding typically from 10 to 20 enzymes.
  • the first en ⁇ zymes in the chain use as their substrates the normal intermediates of the cell metabolism, but as the molecule proceeds in the reaction chain, usually various rather exotic structural features are formed therein, when considering the matter on the basis of the so called primary metabolism.
  • An important characteristic from the point of view of obtaining hybrid antibiotics is the fact that these enzymes are believed to have relatively minor substrate specificity, i.e. that they are able to use as their substrate also compounds which differ as to their structure from those which are found in the original microbe.
  • biosynthetic genes of an antibiotic are clustered which means that they exist in the microbial DNA close to each other. This has in many cases made it possible to isolate other genes, which participate in the biosyn ⁇ thesis of the antibiotic, one gene participating in the biosynthesis having been identified by some procedure.
  • the anthracyclines are a broad group of compounds having the common skeletal structure of 7,8,9,10-tetrahydro- 5,12-naphthacene quinone of the general formula I
  • anthracyclines various substituents are linked to this skeletal structure, the most important group of which are formed by some sugar derivatives.
  • Several of the substances of the anthracycline group are in use as cytostatic drugs in the treatment of cancer, such as e.g. daunorubicin, doxorubicin and aclarubicin.
  • Antibiotics of the anthracycline group are presented e.g. in the article of A. Fujiwara and T. Hoshino: "Anthra ⁇ cycline antibiotics" (CRC Critical Reviews in Biotechno- logy, vol. 3, 1986, pages 133-157) and in the references cited therein.
  • the anthracyclines belong to the anti ⁇ biotics with a so-called polyketide structure.
  • the relatively complex structure of the anthracyclines has retarded the development of novel compounds with better characteristics. It has been possible to prepare synthetically a large group of anthracyclines, but screening of microorganisms belonging mainly to the genus Streptomyces from the soil has also formed an important source of new anthracyclines. This procedure is not satisfactory, because it does not make it possible to convert the anthracycline structure systematically, but discovery of new anthracyclines is random.
  • peuce- tius five different gene regions containing domains homological to actl probe from these only one has been shown to include the biosynthetic genes of doxorubicin. It is to be noted that the acm probe described hereafter identifies from S. peucetius exactly this gene region of five possible regions.
  • the gene donor strain was selected to be S. purpura ⁇ cen ⁇ ATCC 25489, which is a type strain of the species S. purpura ⁇ cens .
  • the strains according to the invention can be reproduced according to the following description; in addition, the reproducibility of the invention has been secured by depositing the crucial microbial strains and plasmids in a depository according to the Budapest treaty. It is obvious to a man skilled in the art that the process steps used in the recombinant DNA techniques are known as such, but the inventive step lies in the fact that these steps are carried out according to a specific strategy to give a new result. It is also obvious to a man skilled in the art that for carrying out individual steps, alterna- tive processes have often been described, by which the steps given in this description can be replaced by using good professional skill.
  • the present invention thus relates to a process for producing hybrid antibiotics of the anthracycline group which process comprises - isolating from a Streptomyces purpurascens strain producing anthracyclines a DNA sequence leading to the expression of hybrid anti ⁇ biotics of the anthracycline group,
  • the present invention also relates to the said DNA se- quences isolated from the bacterium Streptomyce ⁇ purpu ⁇ ra ⁇ cen ⁇ , which lead to the production of the hybrid antibiotics as described.
  • the said recombinant-DNA-construction can be constructed by ligating such a DNA sequence according to the inven ⁇ tion to a suitable vector, which is preferably a vector amplifying in microorganisms of genus Streptomyce ⁇ , e.g. the plasmid pIJ486 (Ward et al . , 1986).
  • a suitable vector which is preferably a vector amplifying in microorganisms of genus Streptomyce ⁇ , e.g. the plasmid pIJ486 (Ward et al . , 1986).
  • a recombinant-DNA-construction is transformed to a S. gali- laeus host, preferably to a host, which produces akla ⁇ vinone glycosides, especially to the above mentioned S. galilaeu ⁇ strains, a Streptomyce ⁇ strain producing anti ⁇ biotics of the anthracycline group is obtained.
  • the compounds produced are new anthracycline antibiotics, for which cytostatic activity has been shown (Example 12) .
  • the compounds of the present invention are interesting compounds to be further developed for possi ⁇ ble clinical use.
  • the production of the said compounds by some other process than the fermentation of a hybrid strain is very difficult.
  • N refers to one or more nucleotides which have not been identified.
  • Fig. 1 A schematic presentation of the procedure, by which the region homological to the conserved region actl of the biosynthetic genes of the polyketide antibiotics was recognized in S. purpurascens-DNA.
  • Fig. 2 The restriction map of the plasmid pIJ2345.
  • Fig. 3 The restriction map of the rdm-clones cloned from S. purpurascens and the part of the chro ⁇ mosome covered by them. The region recognized by the acm-probe is marked as shaded.
  • Fig. 4 The restriction map of the plasmid pH2008.
  • Fig. 5 The thin layer of the fractions obtained when fractioning the aglycon mixture.
  • Tris tris-hydroxymethylaminomethane pH 8 25 mM
  • EDTA ethylenediaminotetraacetic acid pH 8 25 mM
  • Phenol (Ultrapure, Gibco BRL) 500 g 8-hydroxyquinoline 0.5 g
  • Tris-HCl buffer pH 8.0 10 mM EDTA, pH 8.0 1 mM
  • E. coli strain W 5445 carrying the plasmid pIJ2345 was grown, the plasmid was isolated therefrom by neutral SDS degradation (Maniatis et al . , p. 92) and the plasmid was purified by centrifuging in a cesium chloride-ethidium bromide gradient (Hopwood et al. 1985a, p. 83, steps 17- 21). The plasmid fraction from the gradient was ex- tracted with isopropanol and precipitated with ethanol (Hopwood et al . 1985a, p. 127).
  • the 0.8 kb Bglll fragment was isolated by digesting the pIJ2345-plasmid prepared above, by Bglll endonuclease (Boehringer Mannheim or New England Biolabs) according to the instructions of the manufacturer, and by separating the fragment from the rest of the plasmid by preparative agarose gel electrophoresis (Hopwood et al. 1985a, p. 137). The fragment cut away from the gel was purified from the agarose using the GeneClean reagent kit (Bio 101) according to the instructions of the manufacturer.
  • the isolated probe fragment was labelled with ⁇ 32 P-deoxyadenosine phosphate (New England Nuclear NEG-021H, 3000 Ci/mmol) for the hybridization, using the random prime labelling reagent kit of Boehringer Mannheim according to the instructions of the manufacturer.
  • the labelled DNA was separated from the radioactive nucleo ⁇ tide in a Nick column (Pharmacia) according to the inst ⁇ ructions of the manufacturer.
  • the Streptomyce ⁇ strains ATCC 31615 and ATCC 25489 were obtained from the American Type Culture Collection. -In order to isolate the total DNA they were grown in 50 ml of SGYEME medium in 250 ml erlen eyer flasks, which were shaken at 250 rpm at 28.5 °C for ca 50 hours.
  • the total DNA was isolated in the following manner, which is a modification of that described by Hopwood et al. (Hopwood et al. 1985a, p. 77).
  • the culture was separated by centrifuging for 10 min at ca 3000 g, resuspended in 3 ml of lysozyme solution and incubated for 10 min in a 37 ⁇ C water bath. 4 ml of "2 * Kirby mixture” reagent was added and stirred carefully by turning around the tube for ca 1 min.
  • Streptomyce ⁇ DNA preparations isolated above were digested with BamHI endonuclease (Boehringer Mannheim or New England Biolabs) according to the instructions of the manufacturer, and the digests were fractionated by agaro ⁇ se gel electrophoresis (Hopwood et al. 1985a, p. 137, TAE buffer, 0.8% agarose, 0,5 V/cm, running time 16 hours).
  • the fractionated DNA was transferred from the gel to Hybond N membrane (Amersham) using the VacuGen apparatus (LKB 2016, Pharmacia LKB Biotechnology) according to the instructions of the manufacturer (Preliminary Instruction Manual, LKB 2016 VacuGene Vacuum Blotting System, n:o 90 02 5378, Pharmacia LKB Biotechnology AB, Bromma, Sweden) with the modifications that depurination was performed for 10 min (in the instructions 4 min), denaturation for 15 min (3 min), neutralization for 15 min (3 min) and transferring for 1 h (20-60 min).
  • the membrane, onto which the DNA had been transferred, was washed with 2 * SSC, dried in room temperature and exposed to UV for 2 minutes by LKB 2011 Macro Vue Transilluminator (Pharmacia LKB Biotechnology) in order to fix the DNA.
  • the membranes to be hybridized were enclosed in a plastic bag (Hybaid) by heat-sealing. About 50 ml of a prehybri- dization solution was prepared:
  • the hybridization bag was filled with such an amount of the prehybridization solution that air bubbles could be easily removed therefrom, and the prehybridization solu ⁇ tion was poured from the bag into a container with a scale.
  • a carrier DNA DNA from calf thymus, Boehringer Mannheim 104 167
  • the denatured carrier DNA was added into the prehybridization solution, which was returned to the hybridization bag, the air bubbles were removed as carefully as possible and pre- hybridization was performed at 65 ⁇ C in a shaking water bath at least for 6 h.
  • the labelled probe was added into an equal amount of the carrier DNA as above, and denatured similarly.
  • the pre ⁇ hybridization mixture was removed from the bag and the denatured probe as well as the carrier were added there ⁇ to. The mixture was returned to the bag, the air bubbles were removed and hybridization was performed overnight in similar conditions as the prehybridization.
  • the hybridization mixture was removed from the bag and in its place 100 ml of washing solution was introduced:
  • the mixture was stirred, the washing solution was poured away, and the washing was repeated. Subsequently 300 ml of the washing solution was taken into the bag and it was shaken for 30 min in a 65 ⁇ C water bath. The washing solution was poured away and the washing was repeated.
  • the membranes were spread on glass plates and covered by plastic film.
  • the autoradiography was performed by placing one on top of the other the membrane protected by the plastic film, the autoradiography film (Hyperfilm-MP, Amersham) and the amplifying plate (Cronex Quanta Fast Detail, Dupont) and by exposing 1-2 days at -80 ⁇ C.
  • a gene bank was prepared into the ⁇ -vector EMBL3 from the S. galilaeus DNA prepared as described above.
  • the DNA was partly digested with Sau3A-endonuclease (Maniatis et al . , p. 282-285) and fractionated by sucrose gradient centri- fuging according to the same instructions.
  • the ca 20 kb DNA fraction was ligated with the vector (EMBL3 BaroHI . Arms Cloning System, Promega Biotech. ) according to the instructions of the manufacturer, and was packed to ⁇ - particles by using the Packagene-reagent kit of the same manufacturer according to the instructions of the manu ⁇ facturer.
  • the E ⁇ cher ⁇ chia coll strain GM2163 E.
  • the host cells were prepared for the infection according to the instructions of Maniatis et al. (p. 63). The host cells were infected with the obtained packing mixture and spread onto the plates according to the instructions of Promega.
  • Phage, base solutions were prepared from these plates, the titers of which, i.e. the concentration of the phages were determined (typically 10 10 /ml).
  • ⁇ -phage DNA was prepared from the lysate according to the method of Kaslow (1986) with the modifi- cation that the phage DNA was precipitated with isopro ⁇ panol rather than with polyethylene glycol (Hopwood et al. 1985a, p. 124).
  • the ⁇ -DNA of the clones obtained was digested with BamHI endonuclease and it was fractionated by agarose ' gel electrophoresis, transferred onto a membrane, and hybri ⁇ dized using the actI0.8 probe as described above for the chromosomal DNA.
  • One of the clones, designated ⁇ -acm5 gave a ca 3 kb BamHI fragment, which gave a distinct hybridization signal; the fragment hybridizing with the other positive clones did not separate from the vector with BamHI endonuclease.
  • ⁇ -acm5-DNA was digested with BamHI endonuclease and the digest was fractionated by preparative agarose gel elec ⁇ trophoresis.
  • the 3 kb BaznHI fragment was isolated and purified as described above, and it was ligated into the plasmid vector pBR322 (Bolivar et al., 1977 and Sutclif- fe, 1978) opened with BamHI endonuclease (Maniatis et al., p. 391).
  • the plasmid is commonly available, a.o. in the E. coll strain ATCC 37017 of American Type Culture Collection.
  • Competent E. coll HB101 cells were transformed with the ligation mixture (Hopwood et al. 1985a, p. 120) and among the obtained transformants a clone containing the 3 kb BamHI fragment was searched by preparing plasmid DNA from the clones in a small scale (Hopwood et al. 1985a, p.
  • the plasmid carrying the insert was designated pacmS, and it was prepared in a large scale, as described above for the plasmid pIJ2345.
  • the probe, abbreviated acm is the 3 kb BamHI fragment contained in the plasmid pacm5, which fragment was isolated as de ⁇ scribed for the actl ⁇ .8 probe from pIJ2345.
  • the gene bank was prepared from S. purpurascens DNA as described above for S. galilaeus, however so that the vector used was ⁇ -EMBL4, and as the vector preparation
  • EXAMPLE 8 Screening of the gene bank of S. pur ⁇ purascens and mapping of the clones
  • the clones giving a positive hybridization signal were screened from the S. purpura ⁇ cen ⁇ gene bank by the same procedure as described above for the S. galilaeu ⁇ gene bank by using the ac probe, the clones were purified, and phage base solutions were prepared thereof. From the clones ⁇ -DNA was prepared as described above, and the clones were mapped for some restriction endonuclease recognition sites (Maniatis et al . , p. 374-378). The so obtained map from the clones, which were designated by the abbreviation rdm and a serial number, is given in the Figure 3.
  • EXAMPLE 9 Transfer of the S. purpurascens DNA seguences to S. cralilaeu ⁇ and disco ⁇ very of the producer of hybrid anti ⁇ biotics
  • the recombinant phage ⁇ -rdm6 was digested with EcoRI
  • Streptomyce ⁇ plasmid pIJ486 (Ward et al . , 1986; the plasmid was obtained from prof. Hopwood, John Innes
  • alkaline phosphatase (Calf Intes ⁇ tinal Alkaline Phosphatase, CIAP, Boehringer Mannheim 713023) as follows: 10 ⁇ g of linear vector DNA and 0.5 U of CIAP were incubated in a +37 °C water bath in a volume of 100 ⁇ l (50 mM Tris-HCl, pH 8 and 0.1 mM EDTA) 0.5 h. CIAP was inactivated by heating at 65 C C for ca 0.5 h and by extracting subsequently two times with a neutral phenol-chloroform (1:1) mixture. The plasmid was precipi ⁇ tated with 2-propanol, washed with ca 70% ethanol and dissolved in TE buffer (about 1 ⁇ g/ ⁇ l).
  • the plasmid pIJ486 and the insert-DNA were ligated with T4-DNA-ligase (Boehringer Mannheim) according to the instructions of the manufacturer in a volume of 20 ⁇ l.
  • the ligation mixture was transformed into S. lividan ⁇ TK24 protoplasts stored in -20 °C.
  • the preparation and the transformation of the protoplasts was performed according to the method described by Hopwood et a . (1985a) (pages 12-14 and 108-109).
  • thio- streptone (tsr) was added onto the plates as a water suspension, 0.5 mg/plate.
  • the regenerated protoplasts were collected with tooth picks after ca 6 days to ISP4 plates (Difco), on which 50 ⁇ g/ml of thiostreptone had been added.
  • plasmids were isolated from 42 cultures according to the method de ⁇ scribed by Kieser (1984). 8 of the isolated plasmids carried the insert.
  • pH2008 was grown in 500 ml of TSB medium, and the plasmid was isolated there ⁇ from according to the method of Kieser.
  • the map of the plasmid pH2008 is given in Figure 4. The modifications to the above mentioned transformation method used for S.
  • lividan ⁇ which are needed for the transformation of Streptomyce ⁇ galilaeu ⁇ , have been described earlier (Ylihonko, K. Thesis for M.Sc, Univer ⁇ sity of Turku, 1986).
  • SGYEME medium was used instead of YEME medium (see above), wherein 0.8 % of glycine had been added.
  • 1-2 ⁇ g of the plasmid preparation was transformed into the S. galilaeus strain ATCC 31615. One transformant was obtained.
  • the strain ATCC 31615/pH2008 was grown in El growth medium, whereto 5 ⁇ g/ml of thiostreptone had been added and after 7 days the products were extracted from the culture with a toluene-methanol (1:1) mixture.
  • the products of the strain ATCC 31615 were isolated correspondingly.
  • About 1 ml of the culture was used for the isolation of the products, wherefrom the cells were separated from the medium by centrifuging. Only the cells were used for the extraction, because only a small part of the products were left in the super ⁇ natant.
  • the products were separated from the toluene phase of the extract by thin layer chromatography, using as the eluant the mixture toluene - ethyl acetate - methanol - formic acid (50:50:15:10). It was found that the strain ATCC 31615/pH2008 had formed anthracyclines, which the host strain ATCC 31615 does not produce natu ⁇ rally.
  • S. galilaeus ATCC 31615/pH2008 was inoculated from the ISP4 + tsr plate to a shaking flask, which contained 60 ml of the medium El, whereto 5 ⁇ g/ml of thiostreptone had been added, and it was grown for 4 days in a shaker at 300 rpm at a temperature of 30 ⁇ C.
  • a 7 1 fermentor was prepared, which contained 5,5 1 of the medium El, 5 ml of anti-foam substance (Polypropylenglycol P 2000, Fluka) and 5 ⁇ g/ml of thiostreptone.
  • the fermentor was inocu ⁇ lated with the above mentioned preculture and fermented for 5 days at a temperature of 30 "C by stirring at 350 rpm and using an air supply of 6 1/min.
  • Celatom 400 g Na 2 HP0 4 . 2H 2 0 47 g citric acid 24.4 g water 500 g methylethylketone (MEK) 3 1
  • the culture broth was transferred from the fermentor by vacuum to the extraction container and stirred for 45 min.
  • the mixture was filtered in a Biichner funnel and the filter cake was washed with 300 ml of MEK.
  • 500 g of NaCl was dissolved and was allowed to separate overnight in a separatory funnel.
  • the lower phase (water phase) was removed.
  • the upper phase was run into a container, whereto 400 g of Na 2 S0 4 had been weighed. It was stirred for 10 min and filtered in a Biichner funnel. The filter cake was washed with 200 ml of MEK.
  • the raw MEK extract so obtained was evaporated by rotary evaporator to nearly dryness. 200 ml of toluene was added and evaporated to dryness. The mixture was filled with toluene to 150 ml. 150 ml of isopropanol, 150 ml of 0.1 M HC1 and 75 ml of hexane were added.
  • the mixture was transferred into a separatory funnel, stirred and the layers were allowed to separate.
  • the lower layer (water phase) was taken into another separa ⁇ tory funnel, and 40 ml of dichloromethane was added thereto. It was stirred and the phases were allowed to separate.
  • the lower phase (dichloromethane) was run into a container, which contained 30 ml of 1M phosphate buff ⁇ er, pH 7.0.
  • To the toluene phase of the first extraction 75 ml of isopropanol and 150 ml of 0,1 M HC1 were added.
  • the aglycones were separated chromatographically in two steps.
  • the first chromatographic run was performed in an oxalic acid - silica gel column as follows:
  • the oxalic acid - silica gel was prepared by mixing 100 g of Kieselgel 60 (Merck) with 200 ml of 0.25M oxalic acid, by removing most of the oxalic acid solution in a Biichner funnel and by drying the silica gel overnight in a 110 °C hot cupboard.
  • the oxalic acid - silica gel so obtained was slurried in toluene and packed into a 4 cm diameter chromatographic column.
  • aglycon mixture in toluene obtained above was applied to the column and eluted with a solution, which contained 10 % acetone in toluene. About 15 ml fractions were collected from the eluate and ⁇ e fractions were analyzed by thin layer chromatography as described above.
  • Fig. 5 shows a photograph of the thin layer so obtained, on the basis of which the eluate was divided into four frac ⁇ tions: I, II, III and IV.
  • fractions I and II seemed to contain two main com- ponents, wherefore they were rechromatographed using oxalic acid - silica gel, but the column used had a diameter of 1.5 cm and length of ca 50 cm. For the elu- tion, 5 % acetone in methylene chloride was used. Thus the main components of the fractions I and II could be purified, which were designated IA, IB, IIA and IIB.
  • the aglycones obtained were chrystallized from methanol.
  • IA was found to be aklavinone, i.e. the aglycone produced by the host strain ATCC 31615.
  • IB is 6-rhodo- mycinone, which is the aglycone produced by the gene donor strain S. purpura ⁇ cen ⁇ , as well as IIB, which is ⁇ - rhodomycinone.
  • IIA is 10-decarbomethoxy aklavinone, which has been described as the product of chemical demeth- ylation and decarboxylation of aklavinone (Tanaka et al., 1980), but the inventors have not found any information in the literature, that any microbe would produce it. Ill was found to be the 7-epimer of IIA, and it has evidently been formed in the connection with the hydrolysis.
  • aglycones IIA and IV are thus products, which are neither produced naturally by the gene recipient strain nor the donor strain, i.e. they are hybrid antibiotics. Aglycone IV is also absolutely new.
  • glycoside extract About 200 ml of the glycoside extract was extracted by adding 200 ml of 0.05M HC1, by stirring and separating the water phase. The water phase was incubated for 30 min on a 55 C C water bath. After the incubation the solution was neutralized by adding 20 ml of IM phosphate buffer, pH 7.
  • the solution was extracted three times with 100 ml of chloroform.
  • the chloroform phase was evaporated to dry ⁇ ness and dissolved in 20 ml of dichloromethane.
  • IVT was purified chromatographically in two steps from the partly hydrolyzed glycoside fraction obtained above. Both of the steps were performed in a silica gel column.
  • Kieselgel 60 was slurried into dichlo ⁇ romethane and packed into a column of 4 cm diameter.
  • the glycoside mixture obtained above was applied to the column and eluted with the eluant dichloromethane:meth ⁇ anol:acetic acid (100:20:1).
  • About 15 ml fractions were collected from the eluate and the fractions were analyzed by thin layer chromatography using the eluant chloro ⁇ form:methanol:acetic acid (20:5:1).
  • the fractions con ⁇ taining mainly IVT (yellow product, Rf 0.21) were pooled.
  • Acetic acid was extracted from the pooled fractions into water by neutralizing the solution with IM NaOH solution.
  • the yellow dichloromethane phase was evaporated to dry ⁇ ness and dissolved in 2 ml of methanol, which was mixed with 20 ml of toluene.
  • the anthracycline in the toluene-methanol solution was applied in the second step onto a chromatography column prepared from Kieselgel 60 slurried in toluene.
  • the column was eluted with the eluant toluene:methanol (1:1) and ca 15 ml fractions were collected.
  • the fractions containing IVT were selected as above. The fractions were combined and evaporated to dryness.
  • IVA, IVB and IVT were found to give aglycon IV when hydrolyzed with IM HCl on a boiling water bath.
  • IVA and IVB were found to give IVT when hydrolyzed with 0.05M HCl for 30 min on a 55 °C water bath.
  • IVT converted in small amounts to aglycone.
  • IVT proved to be a glycoside with one sugar, the sugar moiety of which is rhodinose.
  • the activities of the compounds were determined by a cytotoxicity test, in which the ability of the compounds to inhibit growth of the mouse leukemia cell line L1210 (ATCC CCL 219) in vitro (Matsuzawa et al . , 1981) was measured.
  • As the comparative substance aclacinomycin A was used as the comparative substance aclacinomycin A.
  • the following ED 50 values were obtained for the compounds:
  • microorganism strains have been deposited according to the Budapest treaty to Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM). Mascheroder Weg 1 B, D-3300 Braunschweig, Germany.

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Abstract

L'invention se rapporte à un procédé au moyen duquel on prépare des antibiotiques du groupe anthracycline, à partir de microorganismes biotechnologiquement connus du genre Streptomyces dans lesquels on introduit des gènes spécifiques provenant de certaines souches microbiennes produisant des antibiotiques à structures étroitement apparentées, que lesdits microorganismes ne produisent pas naturellement. L'invention se rapporte également aux microorganismes nécessaires à ce procédé et formés selon la technique de recombinaison de l'ADN, ainsi qu'aux structures d'ADN recombiné et aux séquences d'ADN nécessaires audit procédé. L'invention appartient au domaine de la production biotechnique d'antibiotiques et se rapporte à l'application d'une technologie relative aux antibiotiques hybrides à des antibiotiques du groupe anthracycline.
PCT/FI1992/000084 1991-03-25 1992-03-23 Procede de production d'antibiotiques, sequences d'adn, structures d'adn recombine et souches microbiennes utilisees WO1992016629A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI911441 1991-03-25
FI911441A FI93860C (fi) 1991-03-25 1991-03-25 Menetelmä antibioottien tuottamiseksi, siinä käyttökelpoisia DNA-jaksoja, yhdistelmä-DNA-konstruktioita ja näitä sisältäviä mikrobikantoja

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WO1992016629A1 true WO1992016629A1 (fr) 1992-10-01

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010581A1 (fr) * 1994-09-30 1996-04-11 Galilaeus Oy Procedes de production des anthracyclines et de leurs intermediaires
US5843735A (en) * 1995-02-03 1998-12-01 Korea Institute Of Science And Technology Aklavinone C-11 hydroxylase, gene coding for same, expression vector therefor, and process for preparing hybrid antibiotics by using said vector
WO1999058544A1 (fr) * 1998-05-13 1999-11-18 Galilaeus Oy Anthracyclines hybrides derivees de souches streptomyces galilaeus obtenues par genie genetique
WO2000000620A2 (fr) * 1998-06-26 2000-01-06 Regents Of The University Of Minnesota Adn codant pour la methymycine et la pikromycine
US6600029B1 (en) 1995-12-19 2003-07-29 Regents Of The University Of Minnesota Metabolic engineering of polyhydroxyalkanoate monomer synthases
US7862552B2 (en) 2005-05-09 2011-01-04 Boston Scientific Scimed, Inc. Medical devices for treating urological and uterine conditions

Citations (4)

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Publication number Priority date Publication date Assignee Title
EP0050724A1 (fr) * 1980-10-27 1982-05-05 F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft Préparation de glycosides d'anthracyclines
EP0131181A2 (fr) * 1983-06-25 1985-01-16 Hoechst Aktiengesellschaft Dérivés d'anthracyclines, une méthode microbiologique pour leur préparation et leur emploi comme agents cytostatiques
EP0298385A2 (fr) * 1987-07-09 1989-01-11 BEHRINGWERKE Aktiengesellschaft Dérivés d'anthracycline à activité cytostatique
EP0299350A2 (fr) * 1987-07-09 1989-01-18 BEHRINGWERKE Aktiengesellschaft Dérivés d'anthracycline à activité cytostatique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0050724A1 (fr) * 1980-10-27 1982-05-05 F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft Préparation de glycosides d'anthracyclines
EP0131181A2 (fr) * 1983-06-25 1985-01-16 Hoechst Aktiengesellschaft Dérivés d'anthracyclines, une méthode microbiologique pour leur préparation et leur emploi comme agents cytostatiques
EP0298385A2 (fr) * 1987-07-09 1989-01-11 BEHRINGWERKE Aktiengesellschaft Dérivés d'anthracycline à activité cytostatique
EP0299350A2 (fr) * 1987-07-09 1989-01-18 BEHRINGWERKE Aktiengesellschaft Dérivés d'anthracycline à activité cytostatique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOTECHNOLOGY, September 1984, CHRISTOPHER R. BAILEY et al., "Cloning a streptomyces clavuligerus genetic locus involved in clavulanic acid biosynthesis ", pp. 808-811. *
NATURE, Vol. 314, April 1985, D.A. HOPWOOD et al., "Production of hybrid antibiotics by genetic engineering", pp. 642-644. *
NATURE, VoL. 325, february 1987, F. MALPARTIDA et al., "Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes", pp. 818-821. *
PROC. NATL. ACAD. SCI., Vol. 86, May 1989, KIM J. STUTZMAN-ENGWALL et al., "Multigene families for anthracycline antibiotic production in Streptomyces peucetius", pp. 3135-3139. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010581A1 (fr) * 1994-09-30 1996-04-11 Galilaeus Oy Procedes de production des anthracyclines et de leurs intermediaires
US5986077A (en) * 1994-09-30 1999-11-16 Galilaeus Oy Process for producing anthracyclines and intermediates thereof
US5843735A (en) * 1995-02-03 1998-12-01 Korea Institute Of Science And Technology Aklavinone C-11 hydroxylase, gene coding for same, expression vector therefor, and process for preparing hybrid antibiotics by using said vector
US6600029B1 (en) 1995-12-19 2003-07-29 Regents Of The University Of Minnesota Metabolic engineering of polyhydroxyalkanoate monomer synthases
WO1999058544A1 (fr) * 1998-05-13 1999-11-18 Galilaeus Oy Anthracyclines hybrides derivees de souches streptomyces galilaeus obtenues par genie genetique
US6399583B1 (en) 1998-05-13 2002-06-04 Galilaeus Oy Hybrid anthracyclines from genetically engineered streptomyces galilaeus strains, process for production and uses thereof
WO2000000620A2 (fr) * 1998-06-26 2000-01-06 Regents Of The University Of Minnesota Adn codant pour la methymycine et la pikromycine
WO2000000620A3 (fr) * 1998-06-26 2000-04-13 Univ Minnesota Adn codant pour la methymycine et la pikromycine
US6265202B1 (en) 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin
US7862552B2 (en) 2005-05-09 2011-01-04 Boston Scientific Scimed, Inc. Medical devices for treating urological and uterine conditions

Also Published As

Publication number Publication date
FI911441A0 (fi) 1991-03-25
FI93860B (fi) 1995-02-28
FI93860C (fi) 1995-06-12
FI911441A (fi) 1992-09-26
AU1446192A (en) 1992-10-21

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