FI93860C - Method for producing antibiotics, useful DNA sequences therein, hybrid DNA constructs and those containing microbe strains - Google Patents

Description

5,93860

Method for producing antibiotics, useful DNA sequences, recombinant DNA constructs and microbial strains containing them

The present invention relates to a method in which known microorganisms are biotechnologically produced anthracycline group antibiotics which are not naturally produced by these microorganisms by transferring certain genes to them from certain microbial strains which produce antibiotics of closely related structure. The invention also relates to microorganisms formed by recombinant DNA technology, recombinant DNA constructs and the DNA sequences required therein for such a method. The Kek-15 invention belongs to the field of biotechnological production of antibiotics, and concerns the application of hybrid antibiotic technology to anthracycline group antibiotics.

Hybrid antibiotics are molecules in which the same molecule has the structural features of two antibiotics that are not naturally produced by a single microorganism. Such molecules can, in principle and in some cases in practice, be produced by biotransformation, i.e. by administering an antibiotic produced by one microorganism for 25 min to another, molecule-modifying microbe. However, the use of the term is well established to mean that the biosynthetic genes of one antibiotic are transferred by recombinant DNA technology to a microbe that produces another antibiotic, and the latter microbe is thus made to produce antibiotics that neither it nor the gene donor strain naturally produces. The hybrid antibiotic technique is described, e.g., in H.G. In Floss, "Hybrid Antibiotics - the contribution of the new gene com-35 binations" (Trends in Biotechnology vol. 5, 1987, pp. 111-115) and references cited therein.

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An antibiotic molecule is formed in the microorganism that produces it by an enzymatic pathway, which typically involves 10 to 20 enzymes. The first enzymes in the chain use the normal intermediates of cellular metabolism-5 nan as substrates, but as the molecule progresses in the reaction chain, several so-called on the basis of primary metabolism considering quite exotic structural features. An important property for the production of hybrid antibiotics is that these enzymes have a relatively low substrate specificity, i.e. that they are also able to use as their substrate compounds which differ in structure from those present in the original microbes.

15 Another important feature of the study of the genetics of antibiotic biosynthesis in the Streptomyces microbial genus is that the antibiotic biosynthetic genes are clustered, i.e. they are located close together in the microbial DNA. In several cases, this has made it possible to isolate other genes involved in antibiotic biosynthesis when one gene involved in biosynthesis has been identified by some procedure.

25 Anthracyclines are a broad class of compounds with a common backbone structure of 7,8,9,10-tetrahydro-5,12-naphthasene quinone of general formula I

R1 o R1 1 R1 o

30 r JL JL jL

vCyC ^ · R4 o r6 r7

In typical anthracyclines, this backbone is associated with several substituents, the most important group of which is 35 3 93860, some sugar derivatives. In particular, compounds of formula I may be prepared in accordance with this invention wherein R 1 and R 2 represent hydrogen, R 1 and R 6 represent hydroxyl, R 1 is -CH 2 CH 3, R 1 'is hydroxyl, R 10 and R 4 represent hydrogen or hydroxyl, and a sugar moiety R7 is rhodosamine or rhodosamine-2-deoxy-L-fucose-L-cinerulose A or rhodosamine-2-deoxy-L-fucose-L-cinerulose B.

10 Several substances in the anthracycline group are used as cytostatic drugs in the treatment of cancer, such as daunorubicin, doxorubicin and aclarubicin. Antibiotics of the anthracycline group are described, for example, in the article "Anthracycline 15 Antibiotics" by A. Fujiwara and T. Hoshino (CRC Critical Reviews in Biotechnology, Vol. 3, 1986, pp. 133-157) and references cited therein. Anthracyclines belong to the so-called polyketide-structured antibiotics.

20 The relatively complex structure of anthracyclines has hampered the development of new and improved compounds. A large number of anthracyclines have been able to be produced synthetically, but an important source of new anthracyclines has also been the screening of soil-producing microorganisms, mainly belonging to the genus Streptomyces. This procedure is unsatisfactory because it does not allow a systematic change in the anthracycline structure, but the discovery of new anthracyclines is random.

30

The principle of hybrid antibiotics seems to allow for a systematic modification of the anthracycline structure. Although the first hybrid antibiotics were described in 1985 (Hopwood et al., 1985b), only a few successful attempts to obtain hybrid antibiotics have been described to date. Hutchinson et al. (1989) list four publications describing a successful 4,93860 method for producing hybrid antibiotics. They point to several factors that may preclude the production of hybrid antibiotics: Some hosts may not be able to transform foreign DNA due to restriction or expression barriers, foreign genes may inhibit the production of host antibiotics by the regulatory sequences they contain, may require control factors that the host may not have. To this can be added the common problem of identifying and distinguishing from other genes those genes that are involved in antibiotic biosynthesis in the donor. Finally, the substrate specificity of the enzymes to which the genes encoding the gene are transferred may, however, be so stringent that the substrates available in the host are not converted to new compounds.

Thus, while it can be predicted that transferring genes involved in biosynthesis from one Streptomyce-20 to another could yield new compounds, it is not possible to predict what will be accomplished and whether the experiment will succeed at all. Thus, the identification of a gene sequence and the construction of a recombinant DNA construct that successfully yields hybrid antibiotics using an appropriate host are significant and. an industrially applicable invention.

We have now discovered a method for causing certain anthracycline-producing microorganisms of the genus Streptomyces to produce new anthracycline antibiotics for them. We have inserted into them DNA sequences derived from rhodomycin-producing microorganisms belonging to the species Streptomyces purpurascens, as described below, using a suitable recombinant DNA construct and recombinant DNA technology.

4 5 93860 The starting point for our effort to find DNA sequences that could provide anthracycline-group hybrid antibiotics was the finding by Malpartida et al. isolation when a specific probe containing a similar DNA sequence 10 between different species is used in a DNA hybridization technique when the biosynthetic genes of a single polyketide antibiotic are available. However, this procedure does not reliably detect biosynthetic genes, but Stutzman-Engwall and Hutchinson (1989), for example, found five different gene regions in S. peucetius that contained regions homologous to the actl probe: only one of them has been shown to contain doxorubicin. biosynthetic genes. It should be noted that the acm probe described below identifies S. peucetius from just five of these possible gene regions.

20

Based on published data, the biosynthesis of various anthracyclines is characterized by the first formation of an anthracycline aglycone called aclavinone, from which other anthracyclines are formed as a result of the activity of several modifying enzymes. Thus, we concluded that a suitable host microbe into which the expression of these modifying enzymes could be detected would be the glycopride-producing Strepto-royces strain of aclavinone. Streptomyces galilaeus ATCC 31615 and ATCC 31133 proved to be suitable microbial strains.

In selecting the microbial strain from which to attempt to transfer the genes of the modifying enzymes, we ended up with the microbial species Streptomyces purpurascens because it is assumed to have 35 enzyme activities modifying several aglycone moieties, namely the modifying activities of positions 1, 10 and 11. Thus, S. purpurascens ATCC 25489, a type strain of S. purpurascens, was selected as the gene donor strain.

The strategy that led to the discovery of the DNA sequences described in this application was thus as follows: Isolate a region homologous to the actl region described by S. purpurascens from Malpartida et al., Transfer DNA sequences from the surrounding region to S. galilaeus, grow the resulting recombinant DNA strains under conditions in which the host strain naturally produces the antibiotic, and analyzing the antibiotics produced to determine whether any DNA sequence produces new compounds. A portion of the above actl region was used as a probe, with 15 notations actIO.8.

When the actI0.8 probe was hybridized to S. galilaeus and S. purpurascens DNAs transferred to the membrane by Southern technique, S. galilaeus DNA was found to give 20 clear signals corresponding to an approximately 3 kb BamHI fragment. acetate. S. purpurascens, on the other hand, gave a moderately weak signal corresponding to an approximately 8 kb BamHI fragment.

Because of the difficulty of cloning genes by weak cross-hybridization, biosynthetic genes were isolated from S. purpurascens by first isolating the DNA sequence corresponding to act1 from S. galilaeus. and then using this as a probe, assuming that it would give a better hybridization signal as a biosynthetic fragment of a structurally closer antibiotic (Figure 1).

A region homologous to act10.8 isolated from S. galilaeus, designated acm, proved to be a very useful probe for isolating the biosynthetic genes of at least some anthracyclines. In addition to the 35 hybrid antibiotic-producing genes described in this application, this probe specifically identified the doxorubicin biosynthetic genes from S. peucetius, 739360, described by Stutzman-Engwall and Hutchinson (1989).

When Southern hybridization was repeated using the acm probe-5, S. purpurascens DNA was found to give a clear hybridization signal corresponding to an approximately 8 kb BamHI fragment. Around the DNA sequence giving this signal, several DNA sequences were isolated and transferred by a procedure similar to that described in Example 10 below to S. galilaeus. However, of the DNA sequences tested, only the sequence of the invention produced new compounds.

The strains of the invention may be reproduced as described below; in addition, the reproducibility of the invention has been ensured by depositing key microbial strains and plasmids in a deposit facility under the Budapest Treaty. It will be apparent to one skilled in the art that the steps used in recombinant DNA technology are known per se, but the inventive step lies in the fact that these steps are carried out according to a defined strategy in order to obtain a new result. It will also be apparent to those skilled in the art that alternative methods for carrying out the individual steps have often been described which, in accordance with good professional skill, can replace the steps set forth in this description.

The present invention therefore relates to a method for producing hybrid antibiotics of the anthracycline group, which comprises isolating a DNA sequence from an anthracycline-producing strain of Streptomyces purpascens to effect the expression of anthracycline group hybridian antibiotics, constructing this DNA sequence. DNA construct, transform the resulting recombinant DNA construct 5 into an aclavinone glycoside-producing

Streptomyces galilaeus host, the resulting transformed strain is cultured under conditions in which the host strain naturally produces an antibiotic, and the resulting hybrid antibiotic is recovered.

The present invention also relates to said DNA sequence isolated from Streptomyces 15 purpurascens, which produces the described hybrid antibiotics.

In particular, the invention relates to a DNA-20 sequence useful in generating anthracycline family hybrid antibiotics, which is approximately 12,000 base pairs of functional sequences between the two EcoRI restriction endonuclease recognition sites of plasmid pH2008 deposited in Streptomyces galilaeus (DSM 6403) shown in Figure 4. The invention also relates to a recombinant DNA construct comprising this sequence.

Said recombinant DNA construct can be constructed by inserting such a DNA sequence according to the invention into a suitable vector, which is preferably a vector amplifiable in microorganisms of the genus Streptomyces-30, for example plasmid pIJ486 (Ward et al., 1986). When such a recombinant DNA construct is transformed into a S. galilaeus father, preferably one that produces aclavinone glycosides, especially the aforementioned S. Galila-35 eus strains, an anthracycline group antibiotic-producing Streptomyces strain is obtained.

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The compounds produced are novel anthracycline antibiotics that could be shown cytostatic activity (Example 12). Thus, these compounds of the invention are interesting compounds for further development for possible clinical use. The production of these compounds by a method other than the hybrid strain Ferment o ima 11a is remarkably difficult.

As an example of 10 preferred embodiments of the invention, the isolation of a DNA sequence of the invention from S. purpurascens strain ATCC 25489, the production of hybrid antibiotics by the obtained strain, and the purification and structural analysis of aglycone portions of the produced hybrid antibiotics are described in detail. Except for the properties described in Figures-15, this can be ATCC

The DNA sequence that produces the hybrids of 25489 is identified by the fact that it contains the nucleotide sequence from the left BamHI recognition site on the left in the restriction map (Figure 3):

1 GATCCCTATG CCAGAGCACC GTCAGCAACG GGCNCTCCGC ATGGGCGTGA 51 TCGGCACGGC GAACATCGCG ATTCGCCGGA TCATGCCCGT GCNCNCCGCG 101 CATGACCACG TCGACCTTGT CGCGGTGGCC AGCCGGGACA AGGCCCGGGC 151 CGAGCGGGTG GGGGCCGCTT TCGGCTGCGG TGGCGTGGGG GATTACGCGG 201 CGCTCGTCGA GCGGACGACC TTGACNCGTC TATATTCCGC TGCCGCCCGG 251 CATGCATCAC GAGTGGGCGC TGCGGGCTTT GCGTTCGGGA AAGCACGTGC 301 TGGTCGAGAA ACCGATGTCG GACACGTACG AGAAAACTCT CGAGCTGATG 351 TCGACCGCGT CGGAACTCGG ACTCGTGCTC GCCGAGAACT TCATGTTCCT 401 GCACCATTCC CAGCACGCNG CGGTACNNCG ATGCTCGACG AGTCCGTGGG 451 AGAACTGCGG CTCTTCTCCN GNNCGTTCNC CGTNNGCCGC TGGCACCCGA 501 GTGTTCCGGT ACCAG

wherein N represents one or more nucleotides that have not been identified.

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Lvhvt description of the drawings

Figure 1 Schematic representation of the procedure by which a region homologous to the conserved region of the biosynthetic genes of polyketide antibiotics 5 was identified from S. purpurascens DNA.

Figure 2 Restriction map of plasmid pIJ2345.

10 Figure 3 Restriction map of rdm clones cloned from S. purpurascens and the part of the chromosome covered by them, the region recognized by the acm probe marked in shaded.

15 Figure 4 Restriction map of plasmid pH2008.

Figure 5 A thin plate when fractionating an aglycone mixture is obtained from du fractions.

20 Figure 6 Proton NMR spectrum and structure of Aglycon IV

Figure 7 Mass spectrum of Aglycon IV (chemical ionization, negative ions) 25 Figure 8 Proton NMR spectrum of hybrid glycoside IVT ψ

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Figure 9 Mass spectrum of hybrid glycoside IVT Solutions and media used 30 SGYEME per liter

Yeast extract (Difco) 3 g

Bacto-peptone (Difco) 5 g

Malt extract (Oxoid) 3 g 35 Glucose 10 g

Sucrose 100 g 11 93860

Sterilize by autoclaving for 20 min at 121 ° C. After autoclaving, sterile 2M MgCl2 solution is added per ml of 2 ml and sterile 10% glycine solution is added per 50 ml.

5

lysozyme

Sucrose 0.3 M

Tris (tris-hydroxymethylaminomethane) pH 8, 25 mM EDTA (ethylenediaminotetraacetic acid) pH 8, 25 mM

Lysozyme (Sigma) 2 mg / ml

FENOLISEOS

Phenol (Ultrapure, Gibco BRL) 500 g 8-hydroxyquinoline 0.5 g

Saturate with 50 mM Tris-HCl buffer, pH 8 20 "2 * KIRBY MIXTURE"

Sodium triisopropylnaphthalenesulfonate (Fluka) 2 g Sodium 4-aminosalicylate 12 g 2 M Tris-HCl buffer pH 8, 5 ml 25 Phenol mixture 6 ml H2O ad 100 ml

Of phenol-chloroform

30 Phenol mixture 50 ml

Chloroform 50 ml * 'Isoamyl alcohol 1 ml

YOU

Tris-HCl buffer, pH 8.0 10 mM

EDTA, pH 8.0 1 mM

35 5 12 93860

SSC

Different dilutions are used, such as 2 * SSC, 6 * SSC, etc., prepared from stock solution 20 * SSC: 20 * SSC per liter

NaCl 175.3 g

Na citrate 88.2 g The pH is adjusted to 7 with NaOH; sterilized by autoclaving.

DENHARDT SOLUTION (Maniatis et al., P. 448) 15

Stock solution 50 *:

Ficoll® (Pharmacia) 5 g

Polyvinylpyrrolidone 5 g

Bovine serum albumin 5 g 20 Dist. water ad 500 ml

Filter through a 0,45 μιη sterile filter, divide into 5 ml portions and store in a freezer at -20 ° C.

25 EL SUBSTANCE

Per liter:

Glucose 20 g 30 Soluble starch 20 g

Pharmamedia ^ 5 g

Yeast extract 2.5 g K2HPO4. 3H 2 O 1.3 g

MgSO 4. 7H 2 O 1 g 35 NaCl 3 g

CaCO 3 3 g 13 93860

Stir in tap water and adjust to pH 7.5 with NaOH. Sterilize by autoclaving.

5 EXAMPLE l. Probe fabrication

The actl probe described in Malpartida et al., I.e. a 2.2 kb (= kilobase, thousand base pairs, unit of measurement of DNA molecular length) fragment from the actl region in the vector pBR329 = pIJ2345, was obtained from Professor DA Hopwood (John Innes Institute, Norwich, UK). A 0.8 kb BglII fragment was used as a probe. A restriction map of plasmid pIJ2345 is shown in Figure 2.

15

E. coli strain W 5445 containing plasmid pIJ2345 was grown, the plasmid was isolated therefrom by neutral SDS digestion (Maniatis et al., P. 92), and the plasmid was purified by cesium chloride-ethidium bromide gradient centrifugation (Hopwood et al. 1985a, p. 83, steps 17-21). The plasmid fraction taken from the gradient was extracted with isopropanol and precipitated with ethanol (Hopwood et al. 1985a, p. 127).

The 0.8 kb BglII fragment was isolated by digesting the above-prepared plasmid pIJ2345 with BglII endonuclease (Boehringer Mannheim or New England Biolabs) according to the manufacturer's instructions, and separating the fragment from the rest of the plasmid by preparative agarose gel electrophoresis (1985). , p. 137). The gel excised fragment was purified from agarose using the GeneClean reagent kit (Bio 101) according to the manufacturer's instructions.

For hybridization, approximately 100 ng of isolated probe fragment was labeled with α32β-deoxyadenosine phosphate (New England Nuclear NEG-021H, 3000 Ci / mmol) using 14,93860

Boehringer Mannheim random prime labeling reagent kit according to the manufacturer's instructions. Labeled DNA was separated from the radioactive nucleotide on a Nick column (Pharmacia) according to the manufacturer's instructions.

5 EXAMPLE 2. Isolation of total DNA from Streptomyces strains

Streptomyces strains ATCC 31615 and ATCC 25489 were obtained from the American Type Culture Collection. For isolation of total DNA, they were grown in 50 ml of SGYEME medium in 250 ml Erlenmeyer flasks shaken at 250 rpm at 28.5 ° C for about 50 hours.

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Total DNA was isolated as follows, modified from that described by Hopwood et al. (Hopwood et al. 1985a, p. 77).

The culture was separated by centrifugation for 10 min at about 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 gently mixed by inverting the tube for about 1 min. I drive. 25

8 ml of phenol-chloroform were added, mixed as above and centrifuged for 10 min. time at about 3000 g. The upper aqueous phase was transferred from the tube with an open-tip pipette to another test tube containing 3 ml of phenol-30 chloroform and mixed and centrifuged as above. The aqueous phase was transferred with an open-ended pipette to another test tube, 1/10 of the volume of the aqueous phase was added to 3 M sodium acetate, the pH of which had been adjusted to 6 with acetic acid, and mixed. An equal volume of isopropanol was then carefully pipetted onto the aqueous phase 35 and the high molecular weight DNA precipitating at the interface between the aqueous and isopropanol phases was gently "spun" sterile.

Iin around the glass rod. The glass rod with DNA precipitation was transferred to another test tube containing 7 ml of 70% ethanol, the precipitate was removed from the glass rod into an empty test tube, and dried in a vacuum desiccator for 5-10 5 min. DNA was dissolved in 1-2 ml of TE. Its concentration and molecular size were measured by running the sample on an agarose gel electrophoresis run (Hopwood et al. 1985a, p. 136, TAE buffer, 0.3-0.6% agarose 4 V / cm).

EXAMPLE 3. Southern hybridization Transfer of DNA to a hybridization membrane

The Streptomyces DNA preparations isolated above were digested with BamHI endonuclease (Boehringer Mannheim or

New England Biolabs) according to the manufacturer's instructions, and Diges-tit was fractionated by agarose gel electrophoresis (Hopwood et al. 1985a, p. 137, TAE buffer, 0.8% agarose, 0.5 V / cm, run time 16 hours).

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The fractionated DNA was transferred from the gel to a Hybond N-membrane (Amersham) using a VacuGene (LKB 2016, Pharmacia LKB Biotechnology) according to the manufacturer's instructions (Preliminary Instruction Manual, LKB 2016 VacuGene Vacuum Blot-25 Ting System, No. 90 02 5378, Pharmacia LKB Biotechnology AB, Bromma, Sweden) with the modifications that depurination was performed for 10 min. time (instructions 4 min.), denaturation 15 min. (3 min.), Neutralization 15 min. (3 min.) And transfer for 1 h (20-60 min.). The membrane to which the DNA was transferred was washed with 2 * SSC, dried at room temperature and exposed to UV to attach the DNA for 2 min. during LKB 2011 with Macro Vue Transilluminator (Pharmacia LKB Biotechnology).

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Hybridization

The membranes to be hybridized were sealed in a plastic bag (Hyb-aid) by heat sealing. Approximately 50 ml of precursor hybrid-5 dissolution solution was prepared: 1% Na dodecyl sulfate 1 M NaCl 5 * Denhardt's solution 10

The hybridization bag was filled with an amount of pre-hybridization solution that could easily remove air bubbles, and the pre-hybridization solution was poured from the bag into a graduated container. Carrier-15 DNA (DNA from calf thymus, Boehringer Mannheim 104 167) was denatured to a concentration of 100 hg / ml in the prehybridization mixture by heating in a boiling water bath for 10 min. time and cooling in an ice bath for 5 min. I drive. Denatured carrier DNA was added to the prehybridization solution, which was returned to the hybridization bag, air bubbles were removed as closely as possible, and prehybridized at 65 ° C in a shaking water bath for at least 6 h.

The labeled probe was added to an equal volume of carrier DNA as above and denatured in the same manner. The prehybridization mixture was removed from the bag and the denatured probe and carrier were added. The mixture was returned to the bag, air bubbles removed, and hybridized overnight under the same conditions as prehybridization.

Laundry and autoradiography

The hybridization mixture was removed from the bag and replaced with 35,100 ml of wash solution:

• I

17,93860 1% Na dodecyl sulfate

2 * SSC

Stir, wash off the wash solution and repeat the wash. Then 300 ml of washing solution was taken into the bag and shaken for 30 min. At 65 ° C in a water bath. The wash solution was poured off and repeated. Membranes were applied to glass plates and covered with plastic film. Autoradiography was performed by superimposing a plastic film-protected membrane, autoradiographic film (Hyperfilm-MP, Amersham) and a reinforcement plate (Cronex Quanta Fast Detail, Dupont) and exposing for 1-2 days at -80 ° C.

EXAMPLE 4. Preparation of a gene bank from S. aalila-eus DNA

A gene bank for the λ vector EMBL3 was prepared from S. galilaeus DNA prepared as described above. DNA was partially digested with 5au3A endonuclease (Maniatis et al., Pp. 282-285) and fractionated by sucrose gradient centrifugation according to the same protocol. A DNA fraction of approximately 20 kb was ligated with the vector (EMBL3 BamHI Arms Cloning System, Promega Biotech.) According to the manufacturer's instructions and packaged into λ particles using the same manufacturer's Packagene reagent kit according to the manufacturer's instructions. Escherichia coli strain GM2163 (E. coli Genetic Stock Center, Department of Biology 255 OML, Yale University, New Haven, USA) was used as a host. Host cells were prepared for infection according to the instructions of Maniatis et al. (P. 63). The host cells were infected with the resulting kit mixture and plated on Prom-gan instructions.

EXAMPLE E Isolation of the 5th acm strand from a gene bank of 150 mm plates with infected host cells with approximately 10,000 35 18 93860 plaques formed by λ phage / plate was immobilized on phage DNA Colony / Plaque Screen (New England Nuclear) membranes according to the manufacturer's instructions. by making two membranes from each plate. The membranes thus obtained were hybridized and autoradiographed using the actIO.8 probe as described above. Plaques that gave a positive signal from both membranes on autoradiography were picked apart and phages were eluted from them (Maniatis et al., P. 64).

10

To obtain pure positive phage populations, a dilution was prepared from each candidate to give approximately 300 plaques / plate upon infection of E. coli host strain LE392 (Promega), immobilized on phage DNA membranes as above, and hybridized as above. For each candidate, one distinct plaque was picked from which the phages were eluted and the host strain LE392 was infected using a dilution such that the so-called confluent lysis. Phage stock solutions were prepared from these plates and the titer, i.e. the concentration of phages, was determined (typically 1010 / ®1).

From each clone thus obtained, λ phage were prepared on a% liter scale by infecting the NM538 host (Promega) as described in Maniatis et al., Pp. 77-78. X-phage DNA was purified from the lysate by the method of Kaslow (1986) with the modification that the phage DNA was mixed with isopropanol instead of polyethylene glycol (Hop-wood et al. 1985a, p. 124).

30 EXAMPLE 6. Acm Probe Manufacturing

The X-DNA of the resulting clones was digested with BamHI endonuclease and fractionated by agarose gel electrophoresis-35 Ia, transferred to a membrane and hybridized using the actI0.8 probe as described above for chromosomal DNA. One of the clones, designated X-acm5, gave an approximately 3 kb BamHI fragment that gave a clear hybridization signal; the fragment hybridizing to the other positive clones was not cleaved from the vector by BamHI endonuclease.

X-acm5 DNA was digested with BamHI endonuclease and digested by preparative agarose gel electrophoresis. The 3 kb BamHI fragment was isolated and purified as described above and ligated into the BamHI endonuclease-opened plasmid vector pBR322 (Bolivar et al., 1977 and Sutcliffe, 1978) (Maniatis et al., P. 391). The plasmid is generally available, e.g. In the E. coli strain ATCC 15 37017 of the American Type Culture Collection.

Competent E. coli HB101 cells were transformed with the ligation mixture (Hopwood et al. 1985a, p. 120), and a clone containing a 3 kb BamHI fragment was searched for the resulting transformants by preparing plasmid DNA from the clones on a small scale (Hopwood et al. 1985a, p. 120). 85), by digesting the samples with BamHI endonuclease and identifying the clones with the insert by agarose gel electrophoresis. The plasmid containing the insert was named pacm5: k-25 si and was prepared on a large scale as described above for plasmid pIJ2345. The probe, abbreviated acm, is a 3 kb BamHI fragment contained in plasmid pacm5, which was isolated in a similar manner to the actIO.8 probe from pIJ2345.

EXAMPLE 7. Preparation of a gene bank from S. purpurascens DNA A gene bank was prepared from S. purpurascens DNA as described above for S. galilaeus, except that the vector used was X-EMBL4 and a vector preparation was used. "X-EMBL4 BamHI Arms", RPN.1254X, Amersham International pic.

EXAMPLE 8. S ♦ Screening of the Purpurascens Gene Bank 5 and Mapping of Clones Clones giving a positive hybridization signal from the S. purpurascens gene bank were screened by the same procedure as described above for the S. galilaeus gene bank 10 using an acm probe, the clones were purified and cloned. Λ-DNA was prepared from the clones as described above, and the clones were mapped for the recognition sites of some restriction endonucleases (Maniatis et al., Pp. 374-378). The map thus obtained of the clones designated by the abbreviation rdm and the sequence number is shown in Figure 3.

EXAMPLE 9. Transfer of S. purpurascens DNA Axes to S. aalilae and Finding a Producer of the Hybrid Antibiotic-20 Pathway

Inserts from the resulting clones were transferred to Strep-tomyces galilaeus to detect possible new products. Because S. galilaeus apparently has a fairly strong restriction system, it was not possible to transform ligation mixtures directly into S. galilaeus, but the clones had to be transferred to an easily transformable Streptomyces host using S. lividans TK24 (Hopwood et al. 1985a, p. 266 and Kieser et al.

30 1982) obtained by Professor D.A. John from Hopwood

Innes Institutesta (Norwich, UK).

Recombinant phage X-rdm6 was digested with JFcoRI (Boehringer Mannheim) according to the manufacturer's instructions. The approx. 12 35 kb insert was run apart from the arms on a 0.5% SeaPlaque LGT agarose gel (FMC Corporation) at 8 V overnight. The insert DNA was recovered using the GeneClean gel kit (Bioiol) according to the manufacturer's instructions.

Streptomyces plasmid pIJ486 (Ward et al., 1986; plasmid 5 was obtained from Prof. Hopwood, John Innes Institute) was linearized with EcoRI as recombinant phage and treated with alkaline phosphatase (Calf Intestinal Alkaline Phosphatase, CIAP, Boehringer Mannheim 713023) as follows: vector DNA and 0.5 U CIAP 10 were incubated in a +37 eC water bath in a volume of 100 μl (50 mM Tris-HCl, pH 8 and 0.1 mM EDTA) for 0.5 h. was possible by heating at 65 ° C for about 0.5 h and then extracting twice with neutral phenol-chloroform (1: 1). The plasmid was precipitated with 2-propanol, washed with about 70% ethanol and dissolved in TE buffer (about 1 pq / μl).

Plasmid pIJ486 and insert DNA were ligated with T4 DNA ligase (Boehringer Mannheim) according to the manufacturer's instructions in a volume of 20 μl.

The ligation mixture was transformed into S. lividans TK24 protoplasts stored at -20 ° C. Protoplast preparation and transformation was performed according to the method of Hopwood et al. (1985a) ku-25 dowel (pages 12-14 and 108-109).

After 1 day, thiostrepton (tsr) as an aqueous suspension of 0.5 mg / plate was added to the plates. Regenerated protoplasts were collected with toothpicks after about 6 days on ISP4 plates (Difco) supplemented with thiostrepton 50 30 μg / ml. Culture was transferred from ISP4 plates by loop

Falcon tubes with 3 ml TSB medium (Oxoid Tryptone Soya Broth 30 g / l) and thiostrepton 5 Mg / ml to maintain selection pressure. After 3 days, plasmids were isolated from 42 cultures by the method described by Kieser (1984). Of the isolated plasmids, 8 were inserted. Of these, 1 (pH2008) was grown in 500 ml of TSB medium, and the plasmid was isolated by the Kieser method. A map of plasmid pH2008 is shown in Figure 4.

The 5 changes required for the transformation of Streptomyces galilaeus to the above-mentioned transformation method used for S. lividans have been described previously (Ylihon-ko, K. Master's thesis, University of Turku, 1986). For the preparation of protoplasts, SGYEME medium (see above) supplemented with 0.8% glycine was used instead of YEME medium. 1-2 μq of the plasmid preparation was transformed into S. galilaeus strain ATCC 31615. Transformants were obtained 1.

Strain ATCC 31615 / pH2008 was grown in E1 medium supplemented with 5 μg / ml thiostrepton, and after 7 days the products were extracted from the culture with toluene-methanol (1: 1). As a control, the products of strain ATCC 31615 were isolated accordingly. Approximately 1 ml of culture was used to isolate the products, from which the cells were separated from the medium by centrifugation. Only cells were used for extraction because only a small portion of the products remained in the supernatant. The products were separated from the toluene phase of the extract by thin layer chromatography, using a toluene-ethyl acetate-methanol-formic acid (50: 50: 15: 10) -25 mixture as eluent. Strain ATCC 31615 / pH2008 was found to form anthracyclines that are not naturally produced by host strain ATCC 31615.

The properties were confirmed to be derived from plasmid 30 by re-transforming the recombinant plasmid pH2008 into ATCC 31615, whereby new compounds were also found to be formed with this strain. Growth of the recombinant strain in the production medium E1 without thiostrepton-induced selection pressure caused the loss of production of new anthracycline-35 lines.

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The hybrid products were hydrolyzed by heating for 0.5-1 hours in 0.1 M HCl solution. The aglycones were separated on an oxalic acid-treated TLC plate and compared to the aglycones produced by strain ATCC 31615. The eluent was chloroform-acetone (10: 1). The Rf values shown in Table I were obtained for the products:

Rf value ATCC 31615 ATCC 31615 / pH2008 0.11 - + 0.15 - + 0.19 - + 0.21 + + 0.30 - + 0.36 - + 0.42 - + 0.46 + + 0 , 50 - + 0.53 - + 0.56 + + 0.61 - + - = Product is not in stock + = Stock produces product corresponding to Rf

Table I.

• EXAMPLE 10, Production of Hybrid Antibiotics and Purification of Aqlv Machines

Fermentation 5 S. galilaeus ATCC 31615 / pH2008 was inoculated from an ISP4 + tsr plate into a shake flask containing 60 ml of E1 medium supplemented with 5 Mg / ml thiostrepton and grown for 4 days at 300 rpm on a shaker at 10-30 ° C. A 7 L fermentor was prepared with 5.5 L of E1 medium, 5 mL of antifoam (Polypropylene glycol P 2000, Fluka) and 5 Mg / mL thiostrepton.

* 24 93860

The fermentor was inoculated. pre-culture and fermented for 5 days. At a temperature of 30 ° C, stirring at 350 rpm and an air supply of 6 1 / min.

5 Extraction

A 10 L reaction flask was prepared for extraction by adding:

Celatom00 400 g 10 Na 2 HPO 4. 2H 2 O 47 g citric acid 24.4 g water 500 g methyl ethyl ketone (MEK) 3 L The medium was transferred from the fermentor under reduced pressure to an extraction vessel and stirred for 45 min. The mixture was filtered through a Bvichner filter, and the filter cake was washed with 300 mL of MEK. 500 g of NaCl was dissolved in the filtrate and allowed to separate in a separatory funnel overnight.

20

The lower layer (aqueous phase) was removed. The upper layer was placed in a vessel weighed with 400 g of Na 2 SO 4. Stirred for 10 min. and filtered through a Biichner filter. The filter cake was washed with 200 ml of MEK.

25

Separation of glycosides The crude MEK extract thus obtained was evaporated to near dryness on a rotary evaporator. Toluene (200 ml) was added and evaporated to dryness. Make up to 150 ml with toluene. The following was added: “isopropanol 150 ml 0.1 M HCl 150 ml hexane 75 ml 35

Transfer to a separatory funnel, mix and allow the layers to separate. The lower layer (aqueous phase) was taken up in a second separatory funnel and 40 ml of dichloromethane were added. Stirred and allow the phases to separate. The lower phase (dichloromethane) was poured into a vessel containing 30 ml of 1 M phosphate buffer, pH 7.0. To the aqueous phase remaining in the separatory funnel was added 10 ml of dichloromethane, extracted, and the MeCl 2 phase was combined with the MeCl 2 phase under the above phosphate buffer. To the toluene phase of the first extract was added: 10 isopropanol 75 ml 0.1 M HCl 150 ml

The extraction into methylene chloride was repeated as above, and the methylene chloride phases were combined. The methylene chloride solution was evaporated to dryness on a rotary evaporator. About 10 ml of methanol were added and evaporated to dryness again. The evaporation residue was dissolved in 200 ml of toluene.

Hydrolysis of glycosides to aglycones 20

To the toluene solution obtained above was added 200 ml of 0.1 M HCl, stirred and the aqueous phase was separated. The aqueous phase was incubated for 2 hours in a water bath at 85 ° C. The solution was cooled and extracted three times with 100 ml of toluene. The combined toluene phase was then extracted with 150 ml of 0.1 M Na phosphate buffer, pH 7.0. The toluene phase was evaporated to dryness on a rotary evaporator, 20 ml of methanol were added, re-evaporated to dryness and dissolved in 10 ml of toluene.

30

Chromatographic separation of aglycones

The aglycones were chromatographically separated in two steps. The first chromatography run was performed on an oxal-35 acid-silica gel column as follows: 26,93860

Oxalic acid-silica gel was prepared by mixing 100 g of Kieselgel 60 (Merck) in 200 ml of 0.25 M oxalic acid, removing most of the oxalic acid solution on a Biichner filter and drying the silica gel overnight in an oven at 110 5 ° C. The oxalic acid-silica gel thus obtained was slurried in toluene and packed on a 4 cm diameter chromatography column.

The aglycone mixture obtained above in toluene was applied to column 10 and eluted with a solution of 10% acetone in toluene. Approximately 15 ml fractions were collected from the eluate, and the fractions were analyzed by thin layer chromatography as described above. Figure 5 is a photograph of the sheet thus obtained, on the basis of which the eluate was divided into four fractions: I, II, III and IV.

Fractions I and II appeared to contain two main components, which is why they were rechromatographed using oxalic acid-silica gel, but the column used was 1.5 cm in diameter and about 50 cm in length. 5% acetone in methylene chloride was used for elution. In this way, the main components of fractions I and II, designated IA, IB, IIA and IIB, were purified.

The resulting aglycones were crystallized from methanol.

Structural studies of aglycones

The resulting aglycone fractions were characterized by thin layer chromatography, mass spectrometry and proton NMR. IA was found to be aclavinone, an aglycone produced by the host strain ATCC “31615. IB is e-rhodomycinone, an aglycone produced by the gene donor strain S. purpurascens, as is IIB, which is O-rhodomycinone. IIA is 10-35 decarbomethoxyaclavinone, which has been described as a product of chemical demethylation and decarboxylation of aclavinone (Tanaka et al., 1980), but the authors have not found information in the 27,93860 literature that no microbes produce it. III was found to be the 7-epimer of IIA and is likely to be formed by hydrolysis.

For Aglycon IV, the structure shown in Figure 6, in connection with the proton NMR spectrum, was obtained, which the authors have not found in the literature.

Thus, aglycones IIA and IV are products that are not naturally produced by its recipient gene as well as the donor strain, i.e. hybrid antibiotics. Agly-noise IV is also absolutely new.

EXAMPLE 11. Production of hybrid antibiotics and purification of qlv-15 cosides

Fermentation, extraction and separation of glycosides were performed as in Example 10.

Purification of Glycosides IVA and IVB 2 ml of the glycoside extract obtained above was applied as a narrow band to a thin layer chromatography plate (20 cm x 20 cm x 0.5 mm, Kieselgel 60, Merck). The plate was eluted with eluent 25 chloroform: methanol: acetic acid (20: 5: 1). Compounds IVA (yellow product, Rf 0.40) and IVB (yellow product, Rf 0.55) were scraped from the plate and extracted into methanol.

30 Partial hydrolysis of glycosides

About 200 ml of glycoside extract was extracted by adding 200 ml of 0.05 M HCl, stirring and separating the aqueous phase. The aqueous phase was incubated for 30 min. In a water bath at 55 ° C.

After incubation, the solution was neutralized by adding 20 ml of 1 M phosphate buffer, pH 7.

28 93860

The solution was extracted three times with 100 ml of chloroform. The chloroform form was evaporated to dryness and dissolved in 20 ml of dichloromethane.

Purification of IVT IVT was purified from the partially hydrolyzed glycoside fraction obtained above by chromatography in two steps. Both steps were performed on a silica gel column.

In the first step, Kieselgel 60 was slurried in dichloromethane and packed into a 4 cm diameter column. The glycoside mixture obtained above was applied to the column and eluted with eluent dichloromethane: methanol: acetic acid (100: 20: 1). Approximately 15 ml fractions were collected from the eluate and the fractions were analyzed by thin layer chromatography using eluent chloroform: methanol: acetic acid (20: 5: 1). Fractions containing predominantly IVT (product K-20, Rf 0.21) were pooled. Acetic acid was extracted from the combined fractions into water by neutralizing the solution with 1 M NaOH solution. The yellow dichloromethane phase was evaporated to dryness and dissolved in 2 ml of methanol, which was stirred in 20 ml of toluene.

25

In the second step, the anthracycline in the toluene-methanol solution was applied to a chromatography column prepared from Kieselgel 60 slurried in toluene. The column was eluted with eluent toluene: methanol (1: 1) and fractions of about 15 ml were collected. Based on thin layer chromatography, IVT-containing fractions were selected as described above. The fractions were combined and evaporated to dryness.

Structural studies of glycosides IVA, IVB and IVT 35 IVA, IVB and IVT were found to give aglycone IV upon hydrolysis with 1 M HCl in a boiling water bath.

t - 29 93860 IVA and IVB were found to give IVT upon hydrolysis with 0.05 M HCl for 30 min. In a water bath at 55 ° C. With this treatment, IVT was slightly converted to aglycone. Based on the mass and HNMR spectra (Figures 8 and 9), IVT 5 proved to be a monosugar glycoside with rhodosamine as the sugar moiety.

Biotransformation of Glycoside IVB to IVA About 1 mg of IVB was dissolved in 1 ml of methanol.

60 ml of ATCC 31615 culture grown for 2 days in E1 medium was washed by centrifugation and suspension of the cells in 60 ml of physiological saline and transferred to a shake flask. IVB dissolved in methanol was added to the flask and the flask was shaken for 6 h at 300 rpm at 30 ° C. After incubation, the products were extracted from the culture as in Example 9. Based on thin layer chromatography, it was found that IVB was converted to IVA.

20

Strain ATCC 31615 has been found to convert aclasinomycin B to A under these conditions (Hoshino et al., 1983). The reaction has also been found to occur with compounds whose aglycone moiety differs from aclavinone, but the sugar moiety is the same as that of aclasinomycin B. Based on this reaction, it can be concluded that the structure of IVA corresponds to aclasinomycin A and the structure of IVB to aclasinomycin B. This structural determination is also supported by the results obtained from hydrolysis.

30 EXAMPLE 12. IVA of glycosides. IVB and IVT biological activity

The activity of the compounds was determined by a cytotoxicity assay which measured the ability of the compounds to inhibit the growth of mouse leukemia-35 myocyte strain L1210 (ATCC CCL 219) in vitro (Matsuzawa et al., 1981). Aclasinomycin A was used as a reference. The following ED 2 values were obtained for the compounds: 30,93860

Compound EDjo / ninol / l

Aclasinomycin A 10 5 IVA 75 IVB 24 IVT 22

10 STORED MICRO-ORGANISMS

The following microorganism strains have been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), 15 Mascheroder Weg 1 B, D-3300 Braunschweig, Germany, in accordance with the Budapest Treaty.

Micro-organism Deposit number Deposit date

Streptomyces galilaeus 20 ATCC 31615 / pH2008 DSM 6403 4.3.1991

Escherichia coli HB101 / pacm5 DSM 6404 4.3.1991 93860 31

Viiteiulkaisut

Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heynecker, H.L., Boyer, H.W., Crosa, J.H. and Fal-5 Kow, S .: Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2.

(1977) 95

Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., 10 Bruton, C.J., Kieser, H.M., Lyndiate, D.J., Smith, C.P., Ward, J.M. and Schremp, H .: Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich, UK, (1985a) 15 Hopwood, DA, Malpartida, F., Kieser, HM, Ikeda, H., Duncan, J., Fujii, I., Rudd, BAM, Floss, HG and Omura, S .: Production of 'hybrid' Antibiotics by Genetic engineering, Nature 314 (1985b) 642-644 20 Hoshino, Y., Sekine, Y. and Fujiwara, A .: Microbial conversion ov aclacinomycin B to aclacinomycin A, J .Anti-Biot. 36 (1983) 1458-1462

Hutchinson, C.R., Borell, C.W., Otten, S.L., Stutzman-25 Engwall, K.J. and Wang, Y .: Drug Discovery and Development through the Genetic Engineering of Antibiotic-Producing Microorganisms, J. Med. Chem. 32_ (1989) 929-937

Kaslow, D.C .: A rapid biochemical method for Purifying X-30 DNA from phage lysates, Nucl. Acids Res. 14 (1986) 6767

Kieser, T .: Plasmid 12 (1984) 19-36

Kieser, T., Hopwood, D.A., Wright, H.M. and Thompson, 35 C.J .: Mol. Gen. Genet. 185 (1982) 223-228 32 93860

Malpartida, F., Hallam, S.E., Kieser, H.M., Motamedi, H., Hutchinson, C.R., Butler, M.J., Sugden, D.A., Warren, M., McKillop, C., Bailey, C.R., Humphreys, G.O. and Hopwood, D.A .: Homology between Streptomyces genes coding for 5 Synthesis of different polyketides used to Clone antibiotic biosynthetic genes, Nature 325 (1987) 818-821

Maniatis, Fritsch, and Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory, New York, (1982) 10

Matsuzawa, Y., Oki, T., Takeuchi, T., and Umezawa, H .: Structure - activity relationships of anthracyclines relative to cytotoxicity and effects on macromolecular Synthesis in L1210 leukemia cells, J. Antibiot. 34 (1981) 15 1596-1606

Stutzman-Engvall, K.J. and Hutchinson, C.R .: Multigene families for anthracycline antibiotic production in Streptomyces peucetius, Proc. Natl Acad. Sci. USA, 86 20 (1989) 3135-3139

Sutcliffe, J.G .: Complete nucleotide sequence of the Escherichia coli Plasmid pBR322, Cold Spring Harbor Symp. Quant. Biol. 43 (1978) 77.

25

Tanaka, H., Yoshioka, T., Shimauchi, Y., Matsuzawa, Y., Oki, T., and Inui, T .: Chemical modification of anthracycline Antibiotics. I. Demethoxycarbonylation, 10-epimerization and 4-O-methylation of aclacinomycin A, J. Anti-30 Biot. 33 (1980) 1323-1330 * 'Ward, J.M., Janssen, G.R., Kieser, T., Bibb, M.J., Buttner, M.J. and Bibb, M.J .: Construction and characterization of a series of multi-copy promoter-probe Plasmid 35 vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as an indicator. Molec. Gen. Genet. 203 (1986) 468-478.

Claims (10)

1. DNA Fragments Useful in Providing Hybrid Antibiotic Group Antibiotics, Characterized in That It is the rdm-6 fragment with about 12000 base pairs between the two Eco RI restriction endonuclease recognition sites of plasmid pH2008 in Fig. 4 and deposited in the strain Streptomyces galilaeus (DSM 6403), or a functional portion thereof. 10 2. DNA fragment according to claim 1, characterized in that it was isolated from Streptomyces purpurascens strain ATCC 25489. 3. A hybrid DNA construct useful in obtaining hybrid antibiotics from the anthracycline group, characterized in that a DNA fragment according to claim 1 is inserted into a vector propagated in microorganisms of the genus Streptomyces. 20 4. A hybrid DNA construct according to claim 3, characterized in that it is the plasmid pH2008, the restriction map of which is indicated in Fig. 4, and which can be isolated from Streptomyces galilaeus strain DSM 6403. 5. A Streptomyces strain producing hybrid antibiotics of the anthracycline group, characterized in that a hybrid DNA construct of claim 3 or 4 is inserted into a Streptomyces galilaeus strain which naturally produces glycosides of aclavinone. The Streptomyces strain according to claim 5, characterized in that the Streptomyces galilaeus strain, which is used as host and which produces glycosides of aclavinone, is ATCC 31615 or ATCC 31133. 37 93860 Streptomyces strain according to claim 5 or 6, characterized in that the hybrid DNA construct is the plasmid pH2008, the restriction map of which is indicated in Fig. 4 and deposited in Streptomyces galilaeus 5 strain ATCC 31615 / pH2008 with deposit number DSM 6,403th Streptomyces strain according to claim 5, characterized in that it is Streptomyces galilaeus 10 DSM 6403. 9. A method of producing hybrid antibiotics of the anthracycline group, characterized in that a Streptomyces strain according to any one of claims 5-8 is cultivated in conditions where the host strain naturally produces antibiotics, the antibiotics produced are separated from the plant medium and the obtained hybrid antibiotics are purified. 20 10. A process according to claim 9, characterized in that the hybrid antibiotic produced is IVA, IVB or IVT, whose structural formulas are 3893860 O OH · O OH (ϋΐΛϋΐϋΐΛϋΐ Ο κ ^ ι · OH O OH J OH O OH J „p» p N (CH) _ N (CHJ _ O o J 1 pp oOH OA ° \ 1 0 -jpo O OH OH O OH IO ιντ Λ— ° x HO 1 N ( CH 3)