JP2003510081A - Gene cluster involved in achracinomycin biosynthesis and its use for genetic engineering - Google Patents

Abstract

(57) Abstract: The present invention relates to a gene cluster for aracinomycin biosynthesis contained in a 7 kb XhoI-NotI fragment derived from Streptomyces galilaeus and an adjacent 8.5 kb BglII fragment, and a hybrid antibiotic. To the use of said genes in said gene cluster to obtain or to increase the yield of achracinomycin or related antibiotics.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a gene cluster for aclacinomycin biosynthesis from Streptomyces galilaeus, and to obtain hybrid antibiotics or of aclacinomycin or related antibiotics. It relates to the use of said genes comprised in said gene cluster for increasing the yield.

BACKGROUND OF THE INVENTION Anthracyclines are widely used anticancer agents. Seven anthracyclines are in clinical use worldwide: Daunorubicin, Doxorubicin, Idarubicin, Epirubicin, Pirarubicin, Zorubicin and Aclarubicin. A representative compound is doxorubicin, which is the most effective,
Acts against a wide range of malignant diseases. Various toxic effects, such as the cumulative cardiotoxin seen with doxorubicin, can often interrupt treatment. Furthermore, there are also types of malignancy that do not respond to available anthracyclines. Although the mechanism of action of anthracyclines that reflects the clinical efficacy of anthracyclines is not clear, most researchers consider inhibition of topoisomerase II as a desirable effect. It has been suggested that the generation of free radicals derived from a quinone structure is involved in side effects such as cardiotoxin. Anthracyclines were recently reviewed by Professor Strohl and his group (1
997).

Aclacinomycin A first disclosed by Oki et al. (1975)
(Aclarubicin) is Streptomyces grillaeus ATCC31133
And the anthracycline antibiotic produced by Streptomyces galileaeus ATCC 31615. It acts on tumor cells and exhibits reduced toxic properties compared to doxorubicin. However, its activity is
It falls short of solid tumors and its use in treating leukemia is limited. Aclarubicin differs from its other counterparts in its structure. Rhodosamine which is a trisaccharide moiety
2-Deoxyfucose-Cinellulose A is linked at C-7 by a glycosidic bond, whereas at the corresponding position in daunomycin, only one sugar residue, daunosamine, is bound.

[0004] Despite the long history of anthracyclines of around 30 years, research into their biosynthesis is still ongoing and it has been sought to obtain new molecules for the development of cancer chemotherapeutic agents. Has an aim. The current method for finding new molecules for drug screening is genetic engineering. Cloning of genes for anthracycline biosynthesis facilitates the production of hybrid anthracyclines, which can be used in combinatorial biosynthesis to obtain novel molecules. With respect to the chemistry of anthracyclines currently in clinical use, aclarubicin has the unique characteristic that its biosynthetic genes are involved in the creation of new products.

Regarding the gene for the deoxyhexose pathway, Madduri et al., (1998) generated a hybrid anthracycline that changes the sugar moiety when a gene derived from the avermectin biosynthetic cluster is introduced into a Streptomyces piucetius strain. Reported that caused. The product obtained was epirubicin, a commercially important anthracycline. In this case, the hydroxy group in the daunosamine moiety had the opposite stereochemistry due to the action of the avermectin biosynthesis gene.

[0006] Using Streptomyces galileaeus as a host, Streptomyces
Using genes from the rhodomycin pathway from S. purpurascens (Niemi et al., 1994) and from the nogaramycin biosynthetic cluster from Streptomyces nogarater (Ylihonko et al. , 199
6a), a hybrid anthracycline was produced. Genes for the nogaramycin pathway have been used to generate hybrid anthracycline production in S. steffisburgensis, which typically produces stepfimycin (Kunnari et al., 1997). Previously, a biosynthetic gene for actinorhodin was expressed in Streptomyces galileaeus to form aloesaponarin (Strohl et al.,
1991). These hybrid compounds were modified in the aglycone part. Recently, a hybrid compound was obtained using a biosynthetic gene involved in the deoxyhexose pathway of nogaramycin and a Streptomyces galileaeus mutant strain as a host (Finnish Patent Application No. 982295).

As indicated above, a novel molecule has been obtained using Streptomyces galileaeus as a cloning host, while its use for providing genes has not been disclosed. As the identified genes involved in aclacinomycin biosynthesis, polyketide reductase gene (Tsukamoto et al., 1994), akranonate methyl ester cyclase (GeneBank, ACCESSION AF043550) and polyketide synthase gene (Hutchinson and Fujii, 1995; Sequence not available)
Is mentioned.

SUMMARY OF THE INVENTION The present invention relates to gene clusters where most of the genes are from the deoxyhexose pathway for rhodosamine, 2-deoxyfucose and / or rosinose. The gene cluster is Streptomyces galileaeus AT
It has been cloned from CC 31615 and is involved in the biosynthesis of aclacinomycin.

DETAILED DESCRIPTION OF THE INVENTION The experimental methods of the present invention include biochemical and chemical methods conventional in the art. A detailed disclosure of the technique, not described herein, is provided by Hopwood et.
al., “Genetic manipulation of Streptomyces: a laboratory manual” The Jo
hn Innes Foundation, Norwich (1985) and Sambrook et al., (1989) “Mole
cular cloning: a laboratory manual ”. The publications, patents and patent applications cited in this specification are fully listed in the literature list.

The invention relates in particular to the discovery of gene clusters for aclacinomycin biosynthesis. When the cluster is introduced into the Streptomyces piucetius strain,
Causes the production of hybrid antibiotics modified in their sugar moieties.

Several strategies are available for cloning genes for antibiotics. When E. coli is used as the host for the gene library,
Hybridization is the most advantageous screening strategy. The hybridization probe may be any known fragment that shows sufficient homology to a biosynthetic cluster for aclarubicin sugar so that it can hybridize with the cluster. DNA fragments that are identical to the desired region are preferred. Such a fragment, termed Sg-dht, was obtained by PCR amplification of Streptomyces galileaeus chromosomal DNA using degenerate oligonucleotides that anneal to the conserved region of the 4,6-dehydratase gene. 4,6-Dehydratase is the first enzyme involved in the reaction series that converts nucleotide-bound glucose molecules to 6-deoxy sugars commonly found in antibiotics. With this probe it was possible to clone clusters of the deoxyhexose pathway from a restriction gene library. To simplify the cloning strategy, the library is cloned into an E. coli pUC-based plasmid (eg, pBlue).
prepared in script or pWHM1109).

The strategy for cloning the genes involved in aclacinomycin biosynthesis of the present invention was simply as follows: total DNA was Streptomyces
It was isolated from Galileaeus (ATCC 31615) and digested with several restriction enzymes resulting in an average 10 kb fragment. The restriction fragments were analyzed by Southern hybridization using the homologous DNA fragment Sg-dht as a probe. BglII gave an 8.5 kb hybridized fragment and double digestion with XhoI and NotI gave a 7 kb hybridized fragment. (I) BglII and (ii) Xh
DNA digestion was carried out with oI-NotI and the fragments were digested with BamH, respectively.
E. coli-Streptomyces shuttle vector pWHM11 digested with I
09, and ligated with pBluescript digested with XhoI-NotI. The ligation mixture was transformed with E. coli X showing a relaxed restriction-modification system.
It was introduced into L1Blue MRF '. 200-60 colonies per plate
Diluted to 0 cfu (colony forming units) and plated on agar plates. The sufficiently grown colonies were transferred to a nylon membrane for hybridization, and hybridization was performed using Sg-dht probe. B
Hybridization signals were obtained from 6 out of 786 glII digested clones, and these clones 152 carrying the XhoI-NotI fragment.
Hybridization signals were obtained from 7 out of 3. Hybridization and washing were performed under low salt concentration and stringent conditions at 65 ° C. Several techniques for probe labeling and hybridization are possible, but are described by Boehringer Mannheim in “The D”.
IG System User's Guide for Filter Hybridization ”is preferred.
Colonies for which a hybridization signal was obtained were cultivated for plasmid isolation. The plasmid was analyzed by Southern hybridization to confirm the reliability of colony hybridization. The desired DNA fragment (
The plasmids containing Sg4 and Sg5) were named pSgc4 (BglII-fragment) and pSgc5 (XhoI-NotI fragment) (
See FIG. 2).

E. coli vector p for sequencing the Sg4 and Sg5 fragments
Subcloned into UC19 and pBluescript. A total of 30 subclones were used to obtain the nucleotide sequences of Sg4 and Sg5. The sequenced cluster revealed 13 genes involved in aclacinomycin biosynthesis. By comparison with the sequences found in the sequence library, sga2 for activator, sga3 for dehydratase, sga4 for oxidoreductase, sga5 for dTDP-glucose-4,6-dehydratase, sga6 for glycosyl transferase (GTF), Sga7 for putative isomerase, sga8 for aclabiketone reductase
, Sga9 for putative polyketide assembler, sg for putative cyclase
The functions of a10, sga11 for aminomethylase, sga12 for glucose-1-phosphate thymidylyltransferase, and sga13 for aminotransferase were shown. The function of sga1 is not shown based on similarity searches. Nine genes are involved in the glycosylation pathway based on their putative function. Genes involved in the formation of aglycones are sga8, s
ga9 and sga10. The activator Sga2 can regulate both the glycosylation system and the formation of aclabinone via the polyketide pathway.

Sg4 from pSgc4 was cloned in Streptomyces expression vector pIJ486 in Streptomyces lividans TK24 (Ylihonko et al.
., 1996b), pSgs4 was obtained. This vector is a high copy number plasmid that replicates in several Streptomyces species (Ward et al., 1986), which contains the promoter ermE, which is constitutively expressed upstream of the multiple cloning site. (Bibb et al., 1985). Plasmid pSgs isolated from TK24
4 was introduced into a Streptomyces galileaeus strain that is blocked in the deoxyhexose pathway of aclacinomycin biosynthesis, and introduced into a Streptomyces piucetius mutant strain that produces ε-rhodomycinone based on damage in the glycosylation gene. did. The ability to produce aclacinomycin was restored by three Streptomyces galileaeus mutant strains H063, H054 and H065. Mutant H063 accumulates aclabinone, which is the plasmid pSgs
4 completely complemented. Instead, H054 and H065, which produce an aclabinone glycoside with a neutral sugar but not rhodosamine, are
It was only partially complemented by s4. Surprisingly, H063 carrying pSgs4 (H063 / pSgs4) was able to produce twice as much aclacinomycin as wild type Streptomyces galileaeus. Streptomyces piucetius M18 and M90, which produce ε-rhodomycinone, have pSg
Selected as host for s4. L-Rhamnosyl-ε-rhodomycinone (El Kha
med et al., 1977) was obtained when pSgs4 was expressed in mutant strains M18 and M90, and further, M18 / pSgs4 produced L-daunosaminyl-ε-rhodomycinone (Essery and Doyle, 1980). . The structure, although not new, demonstrates the ability of the gene cluster of the invention to give rise to hybrid products in heterologous hosts. To produce a hybrid compound, we supplemented E with a suitable antibiotic, in this case thiostrepton.
One medium was chosen to maintain the selective pressure for the plasmid containing strain.
The product was extracted with an organic solvent and purified by chromatography to obtain a highly pure compound for structure elucidation.

[0015]   Examples are provided below to further illustrate the present invention.

Experimental Materials The restriction enzymes used were purchased from Promega (Madison, WI, USA), Fermentas (Lithuania) or Boehringer Mannheim (Germany), and alkaline phosphatase was Boehringer
Purchased from Mannheim and used according to manufacturer's instructions. Proteinase K was purchased from Promega and lysozyme was purchased from Sigma. The Hybond ^™ -N nylon membrane used in the hybridization was purchased from Amersham (Buckinghamshire, UK) and purchased from DIG DNA La.
belling Kit and DIG Luminescent Detecti
on Kit was purchased from Boehringer Mannheim. Qiagen
Qiaquick Gel Extracti obtained from Hilden, Germany
on Kit was used to isolate DNA from agarose.

Bacterial strains and their use Escherichia coli XL1 Blue MRF '(Stratagene, La Jolla, Calif.) Was used for cloning. Streptomyces lividans TK24 was the primary cloning host for gene expression. The strain is sold by John Innes Center of England.
Center Prof. Sir David Hopwood). Wild-type Streptomyces galileus ATCC 31615 produces aclacinomycin. It was used to obtain the gene of the invention. Streptomyces galileaeus H039 (Ylihonko et al., 1994)
It produces Akv- (Rho) _0-3 . It was used as an expression host for pSgs4 and was transformed more easily than other mutants or wild type. Streptomyces galileaeus H054 (Ylihonko et al., 1994)
Akv-Rho-dF- (CinA) _0-1 , Akv-dF-dF- (CinA) _0-1 and Ak
Produces v-dF-Rho-Rho. It was used as an expression host for pSgs4. Streptomyces galileaeus H063 produces aclabinone. It is a mutant strain derived from wild-type Streptomyces galileaeus. H063
Was used as the expression host for pSgs4. Streptomyces galileaeus H065 produces aclavinone with a neutral glycoside. It is a mutant strain derived from wild-type Streptomyces galileaeus. H065 was used as an expression host for pSgs4. The ε-rhodomycinone producing Streptomyces piucetius M18 and M90 are Streptomyces piucetius var.
ar. caesius) (ATCC 27952). They are pSg
Used as an expression host for s4.

Plasmids E. coli cloning vectors pBluescript SK (Stratagene) and pUC19 (Pharmacia, Sweden) were used to prepare subclones for sequencing, and pBluescript was used as a vector for gene libraries. Was also used. pWHM1109 (provided by Professor prof CR Hutchinson, Wisconsin, USA) is a shuttle vector that replicates in E. coli and Streptomyces. It was used as a vector for a gene library. pIJ486 is a high copy plasmid vector provided by Professor David Hopwood of John Innes Centre, England (Ward et al., 1986).
). pIJE486 (Ylihonko et al., 1996b) is an expression vector containing ermE to promote the expression of cloned genes (Bibb et al., 1985).

Nutrient medium and solution TSB medium was used for culturing Streptomyces galileaeus for total DNA isolation. Lysozyme solution (0.3 M sucrose, 25 mM Tris (pH 8)
And total DNA was isolated using 25 mM EDTA (pH 8). D with TE buffer (10 mM Tris (pH 8.0) and 1 mM EDTA)
NA was dissolved.

Tryptone-soy broth (TSB) Per liter: Oxoid tryptone-soy broth powder 30 g. ISP4 Bacto ISP medium 4, Difco; 37 g / l. E1 Per liter of tap water: Glucose 20 g Soluble starch 20 g Farmamedia 5 g Yeast extract 2.5 g K ₂ HPO ₄ / 3H ₂ O 1.3 g MgSO ₄ / 7H ₂ O 1 g NaCl 3 g CaCO _{3 3} g Before autoclaving The pH was adjusted to 7.4.

General Method NMR data were taken using a JEOL JNM-GX 400 spectrometer. ¹ H and ¹³ C NMR samples had TMS as internal reference. Anthracycline metabolites were measured by (i) LiChroCART RP-18 column by HPLC (LaChrom, Merck Hitachi, pump L-7100, detector L-7400 and integrator D-7500). Acetonitrile as mobile phase: potassium hydrogen phosphate buffer (6
0 mM, adjusted to pH 3.0 with citric acid) was used. Compounds were fractionated using a gradient system starting from 65% to 30% potassium dihydrogen phosphate buffer. Flow rate was 1 ml / min, detection at 480 nm, (ii) Toluene: Ethyl acetate:
Performed by TLC using pre-coated Kieselgel 60 F ₂₅₄ glass plates (Merck, Darmstadt, Germany) with an eluent consisting of methanol: formic acid (50: 50: 15: 3). Plasmid-bearing cultures were maintained using ISP4 plates supplemented with thiostrepton (50 μg / ml).

Example 1 Cloning of the gene cluster for aclacinomycin biosynthesis 1.1 Selection of clones by hybridization Streptomyces galileaeus supplemented with 0.5% glycine TSB for isolation of total DNA. It was grown in 50 ml of medium for 4 days. 12 ml Falc
Harvest the cells by centrifugation in an on tube for 15 minutes (3900 xg),
Stored at -20 ° C. DNA was isolated using cells from 50 ml of culture. 5
5 ml of lysozyme solution containing mg / ml lysozyme was added to the cells of each Falcon tube and incubated at 37 ° C for 20 minutes. Proteinase K 0.7
500 μl of 10% SDS containing mg was added to the cells, incubated at 62 ° C. for 80 minutes, and 10% SD containing proteinase K 0.7 mg was added.
500 μl S was added and the incubation was continued for 60 minutes. The sample was cooled on ice, 600 μl of 3M NaAc (pH 5.8) was added and the mixture was extracted with equilibrated phenol (Sigma). The phases were separated by centrifugation (1400 xg) for 10 minutes. DNA was precipitated from the aqueous phase using the same amount of isopropanol, collected by winding with a glass rod, washed by immersion in 70% ethanol, air dried and dissolved in 500 μl TE buffer.

Southern hybridizations to determine suitable restriction enzymes for the preparation of restriction plasmid libraries were performed with BglII, XhoI, NotI and combinations thereof. A fragment of approximately 9 kb that hybridized with the Sg-dht probe was preferred. For hybridization, 600 ng of digested Streptomyces galileaeus DNA was loaded on an agarose gel, and after electrophoresis, the DNA was transferred from the gel to a nylon membrane by vacuum brotting. Boehringer Mannheim Manual “The DIG System User's Guide fo
Hybridization was performed according to "r Filter Hybridization".
-634, which is internal to the dehydratase gene and is shown in SEQ ID NO: 14
Streptomyces galileaeus DN corresponding to the fragment of 5 to 6861
By amplifying the gene fragment derived from A, a hybridization probe Sg-dht, which was also used for colony hybridization, was obtained. PCR was used for amplification and the sequence of the degenerate oligonucleotide primer was
5'-CSGGSGSSGCGSGSTTCATSGG-3 '(forward, SEQ ID NO: 15) and 5'-GGGGWRCTGGYRSGGSCCCGTTAGTG-
It was 3 '(reverse, SEQ ID NO: 16). A suitable fragment is 9 kb B
It was the glII fragment and the 7 kb XhoI-NotI fragment.

10 μg of chromosomal DNA was digested with BglII. DNA fragments were separated by agarose gel electrophoresis and the 8-9 kb band was cleaved from 0.6% low gel temperature SeaPlaque ^R agarose. Qiagen Gel Extr
DNA bands were isolated from the gel using the action Kit. The isolated fragment was ligated to BamHI digested and dephosphorylated pWHM1109 plasmid vector at a ratio of 3 moles of insert DNA to 1 mole of vector DNA. The ligated DNA was introduced into E. coli XL1Blue MRF 'by electroporation. Using the entire ligation mixture, 786 colonies were obtained. The colonies were grown on agar plates for at least 12 hours and transferred to nylon membranes. Hybridization of the colony membrane was performed in the same manner as Southern using Sg-dht as a probe. Signals for hybridization were obtained from 6 clones and the corresponding colonies were plated on agar and inoculated into 3 ml LB medium for isolation of plasmid DNA. Southern hybridization was used to study whether the plasmid from the clone carried the desired insert. Four of these plasmids contained the 4,6-dehydratase gene fragment and provided the same restriction map, thus
It carried the same fragment presenting both orientations. Sg the fragment
4 and the plasmid containing the fragment was named pSgc4.

Similarly, a 7 kb XhoI-Not from Streptomyces galileaeus
A plasmid library displaying the I DNA fragment was constructed. pBl
uescript was digested with XhoI-NotI to construct a library containing a gene fragment of about 7 kb. A total of 1523 colonies hybridized, 7 of which were the desired clones. The clone was cloned into Xho as described above.
Experiments were performed on the I-NotI fragment. The insert fragment was named Sg5 and the plasmid was named pSgc5. Obtained strain E. coli XL1Blur
MRF '/ pSgc5 was released on August 12, 1999 by Deutsche Samlunk von Microorganism und Zerkulturen Gesellschaft.
Mitt Beschlenktel Haftung (Deutsche Sammlung von Mikroorganis
It has been deposited with men und Zellkulturen GmbH (DSMZ) in accordance with the rules of the Budapest Treaty and has been given the deposit number DSM 12999. Fragments Sg4 and S
g5 is 836 bp corresponding to bases 6181 to 7016 in SEQ ID NO: 14
Duplicated within.

1.2 Subcloning of Fragments for Sequencing Subclones suitable for determining the nucleotide sequence of the entire cluster of Sg4 and Sg5 were constructed. A convenient restriction site was used to subclon the 14806 bp region in plasmids pUC19 and pBluescript. 19 subclones are required to sequence Sg4 and Sg5
Required 11 subclones.

E. coli XL1Blue MRF ′ cells containing subcloned plasmids were cultured overnight at 37 ° C. in 5 ml of LB medium supplemented with 50 μg / ml ampicillin. To isolate the plasmid for the sequencing reaction, Promega Wi
zard Plus Minipreps DNA Purification S
system kit, or Biomedizinische Analytik GmbH, Biomedizinische Analytik GmbH
Biometa Silica Spin Plasmid DNA Mini
The prep kit was used according to the manufacturer's instructions. DNA sequencing was performed using an automated ABI DNA sequencer (Perkin-Elmer) according to the manufacturer's instructions.

1.3 Sequence Analysis and Deduced Function of the Gene Sequence analysis was performed using the GCG sequence analysis software package (version 8; Genetics Computer Group, Madison, WI, USA).
tics Computer Group)). The translation table was changed so that GTG was also regarded as a start codon. Codon usage was analyzed using published data (Wright and Bibb 1992).

According to the CODEN PREFERENCE program, the sequenced DNA fragments consisted of 11 complete open reading frames (ORFs),
And the 5'ends of two other ORFs (sga1 and sga13). The function of the gene was determined by comparing the amino acid sequences translated from their base sequences with the known sequences in the databank. The results are shown in Table 1 which cites the sequence data provided herein.

The function demonstrated for the gene fits well with the proposed biosynthetic pathway for the sugar of aclacinomycin (FIG. 3). The last residue in the trisaccharide portion of aclacinomycin is rosinose, which is enzymatically converted to sinerulose. Aclacinomycin N, the precursor of aclarubicin, contains rosinose as the third sugar residue.

[0031]

[Table 1]

1.4 Expression Cloning in Streptomyces Strains The 8 kb BamHI-HindIII fragment from pSgc4 was inserted into pIJ.
Ligation at E486 gave pSgs4. The plasmid pSgs4 was introduced into Streptomyces lividans TK24 by protoplast transformation. The obtained Streptomyces lividans TK24 / pSgs4 strain was
August 12, 1999 Deutsche Samlunk von Microorganismen
It has been deposited with Und Zerkulturen Geselschaft Mitt Beschlenktel Haftung (DSMZ) in accordance with the rules of the Budapest Treaty and has been given accession number DSM 12998. The plasmid pSgs4 was isolated from the strain and further
It was introduced into the Streptomyces galileaeus mutant strain H039. Then H03
The plasmid preparation isolated from 9 was introduced into the H063, H054 and H065 mutant strains deficient in the glycosylation system of aclacinomycin. The use of H039 as the primary Streptomyces galileaeus host was due to its good efficiency in uptake of foreign DNA.

PSgs4 in E1 medium supplemented with thiostrepton (10 μg / ml)
Streptomyces galileaeus mutants were studied for complementation by culturing clones containing The product from a 500 μl sample of culture broth was extracted with toluene: methanol (1: 1) at pH 7. Metabolites from both transformed clones and mutants were analyzed by TLC and HPLC to determine pSgs4
We found the differences caused by. H06 that endogenously produces aclabinone
3 reverted to an aclacinomycin producer using pSgs4. No aclabinone was found in the culture broth of H063 / pSgs4. However, when pSgs4 was expressed in H054 and H065, the complementarity was:
It wasn't perfect. Both mutants produce aclabinone with a neutral glycoside. Possibly incomplete complementation was due to plasmid deletion in some bacterial cells during culture, or low expression of the gene required as an activator was absent in pSgs4.

Similarly, pSgs4 isolated from TK24 was introduced into Streptomyces piucetius mutant strains M18 and M90. The characteristic product for these mutants is ε-rhodomycinone. The plasmid-containing M18 / pSgs4 and M90 / pSgs4 strains were cultured in E1 medium supplemented with thiostrepton (10 μg / ml), and the metabolites therein were analyzed by TLC and HPLC. Both clones showed an altered production profile compared to the product obtained from the mutant strain. M90 / pSgs4 accumulated glycosylation products, yielding ε-rhodomycinone as an aglycone. The compound is described by El Khamed et al. (19
77) previously synthesized by L-rhamnosyl-ε-rhodomycinone (CAS = 6
3252-11-9).

M18 / pSgs4 produced two compounds that differed from the parent strain. HPLC
And according to TLC data, one compound is L-rhamnosyl-ε-rhodomycinone identical to that produced by M90 / pSgs4 and another compound was previously characterized by Essery and Doyle (1980). It was L-daunosaminyl-ε-rhodomycinone.

[0036]

[Table 2]

1.5 Application of pSgs4 for strain improvement Since H063 was not completely complemented by pSgs4, the production level of aminoglycosides was studied. For this purpose, H063 / pSgs4, H063 and wild-type Streptomyces galileaeus were transformed into E.
The cells were cultured in 1 medium for 4 days. Two 2 ml samples from each culture were first extracted with toluene: methanol (1: 1) under acidic conditions to remove neutral glycosides and aglycones. This extraction process was repeated until neutral glycosides and aglycones disappeared from the aqueous phase. The amount of anthracycline metabolites in the toluene phase was measured and is shown in Table 3. Aclacinomycin containing rhodosamine was extracted from the aqueous phase with chloroform. Both toluene and chloroform extracts were
Analyzed by LC, the toluene phase mainly contained aclabinone and decomposition products. The chloroform phase contained mainly aminoglycosides,
A small amount of aglycone was also detected. The extract was evaporated to dryness, then methanol 1
Dissolved in ml. The amount of anthracycline metabolites was detected spectrophotometrically at 430 nm. The extinction coefficient of 13000 was used to calculate the relative amount to the absorbance.
The results obtained as mg per liter of culture broth are shown in Table 3. H063
The production of aclacinomycin by / pSgs4 was at least 2-fold better than that obtained with wild type.

[0038]

[Table 3]

The ability of pSgs4 to increase aclacinomycin yield in the mutant strain H063 indicates that the gene of the present invention is useful for strain improvement.

Example 2 Compound Obtained by pSgs4 Plasmid-containing strain M18 / pSgs4 or M90 / pSgs that is a seed culture
180 ml of E1 culture solution of 4 was supplemented with thiostrepton (5 μg / ml) E
It was obtained by culturing each strain in three 250 ml Erlenmeyer flasks containing 50 ml of one medium for 4 days at 30 ° C. and 330 rpm. The combined culture broth (180 ml) was used to inoculate 13 liters of E1 medium in a fermentor (Biostat E). Fermentation was carried out at 28 ° C. for 5 days (330 rpm, aeration: 45
0 liter / minute).

The cells were harvested by centrifugation. Bacterial cells were disrupted with 2.6 liters of methanol. Anthracycline metabolites were extracted from a methanol solution at pH 8 with 2 liters of ethyl acetate and the extracts were evaporated to dryness. The viscous residue was loaded onto a 4 × 10 cm silica column and toluene: ethyl acetate: formic acid (50: 50: 3) was used as eluent with increasing amounts of methanol. Pure fractions were pooled and extracted with 1M phosphate buffer (pH 8.0) and water.
The organic phase was dried over anhydrous Na ₂ SO ₄ , then treated with hexane and precipitated. The pure compound, which appeared as a red powder, was dried under vacuum.

A complete structure determination of the compound was made by NMR. Proton and carbon assignments were based on conventional NOE differences, pHSQC and HMBC measurements. The binding, in particular, relies heavily on HMBC experiments. Inferred from the data shown in Table 4, the structure revealed was L- shown in FIG.
Rhamnosyl-ε-rhodomycinone (1) and L-daunosaminyl-ε-rhodomycinone (2). Although these structures are not new, the availability of a hybrid product with genes involved in the glycosylation portion of aclacinomycin biosynthesis has been shown to be pSgs4.
The gene has been shown to be functional and easy to use to find new molecules in drug discovery.

Deposited Microorganisms Germany-De-38124 Braunschweig, Mascherodawek 1
Bee's Deutsche Samrunk von Microorganism und Zerkulturen Gesellschaft Mitt Beschlenktel Haftung (D
The following microorganisms have been deposited with SMZ) according to the Budapest Treaty.

[0044] Microorganism deposit number Deposit date Streptomyces lividans TK24 / pSgs4 DSM 12998 August 12, 1999 E. coli XL1 BlueMRF '/ pSgc5 DSM 12999 August 12, 1999

[0045]

[Table 4]

[0046]

[Table 5]

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tsaelae, P. 1994. Hybrid anthracycline antibiotics: production of new an
thracyclines by cloned genes from Streptomyces purpurascens in Streptomy
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[Sequence list]

[Brief description of drawings]

FIG. 1 shows the structures of aclacinomycin, daunomycin and ε-rhodomycinone.

FIG. 2 is a diagram showing a gene cluster relating to aclacinomycin biosynthesis.

FIG. 3 shows the proposed biosynthetic pathway for sugars found in aclacinomycin.

FIG. 4. M18 / pSgs4 (1 and 2) and M90 / pSgs4 (
The figure which shows the structure of the hybrid compound produced by 2).

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yuha Hakara             Finland, FIEN-20540 Tu             Luk, Hemaintier 34 F-term (reference) 4B024 AA01 BA67 CA01 DA06 EA04                       GA11                 4B064 AH05 CA04 CA19 CC24 DA02                       DA05                 4B065 AA50X AA50Y AB01 AC14                       BA02 CA34 CA44

Claims (13)

[Claims] 1. Streptomyces galilae
us) 7 kb XhoI-NotI fragment of the genome and 8.5 kb flanking B
isolated and purified D, a gene cluster for the anthracycline biosynthetic pathway of the bacterium Streptomyces galileaeus, contained in a glII fragment.
NA fragment. 2. The DNA fragment according to claim 1, comprising the nucleotide sequence shown in SEQ ID NO: 14, or a part thereof having similar characteristics, or a sequence showing at least 84% homology to the sequence. . 3. A DNA fragment according to claim 1 or 2 which is cloned in a plasmid which replicates in Streptomyces or E. coli or a part thereof having similar characteristics. Recombinant DNA. 4. Streptomyces with accession number DSM 12998.
The recombinant DNA according to claim 3, which is the plasmid pSgs4 deposited in the S. lividans TK24 / pSgs4 strain. 5. E. coli XL1Bl with accession number DSM 12999
The recombinant DNA according to claim 3, which is the plasmid pSgc5 deposited in the ueMRF '/ pSgc5 strain. 6. The method according to claim 1 or 2 in the production of anthracycline metabolites.
Use of genes derived from the described DNA fragments. 7. Use of a gene derived from the DNA fragment according to claim 1 or 2 for increasing aclacinomycin production. 8. The gene has an activator, dehydratase, oxidoreductase, dTDP-glucose 4,6-dehydratase, glycosyltransferase, isomerase, aclaviketone reductase, polyketide assembler, cyclase, aminomethylase, glucose-1-phosphate thymidylyl. Rutransferase and aminotransferase are encoded.
Or the use according to 7. 9. A step of introducing the DNA fragment according to claim 1 or 2 into a Streptomyces host, a step of culturing the obtained recombinant strain, and a step of isolating the produced aclacinomycin. A method of increasing aclacinomycin production in a bacterial host. 10. The method according to claim 9, wherein the Streptomyces host is a Streptomyces galileaeus host. 11. The method according to claim 10, wherein the Streptomyces galileus host is a mutant strain derived from Streptomyces galileus ATCC 31615. 12. A metabolite comprising the steps of introducing the DNA fragment according to claim 1 or 2 into a Streptomyces host, culturing the obtained recombinant strain, and isolating the produced compound. Manufacturing method. 13. A step of introducing the DNA fragment according to claim 1 or 2 into a Streptomyces peucetius host, culturing the obtained recombinant strain, and isolating the produced compound. Including the process,
A method for producing anthracycline metabolites.