THE GENE CLUSTER INVOLVED IN ACLACINOMYCIN BIOSYNTHESIS, AND ITS USE FOR GENETIC ENGINEERING

Abstract: This invention relates to the gene cluster for aclacinomycin biosynthesis being included in a 7kb <i>Xho</i>I-<i>Not</i>I fragment and a flanked 8.5kb <i>Bgl</i>II fragment derived from <i>Streptomyces galilaeus</i>, and the use of the genes included therein to obtain hybrid antibiotics, or to increase yields of aclacinomycins or related antibiotics.

Description: The gene cluster involved in aclacinomycin biosynthesis, and its use for genetic engineering Field of the invention This invention relates to the gene cluster for aclacinomycin biosynthesis derived from Streptomyces galilaeus, and the use of the genes included therein to obtain hybrid antibiotics, or to increase yields of aclacinomycins or related antibiotics Background of the invention Anthracyclines are widely used anticancer agents Seven different anthracyclines are in worldwide clinical use daunorubicin, doxorubicin, ldarubicin, epirubicin, pirarubicin, zorubicin and aclarubicin A representative compound is doxorubicin, being the most efficient and acting on a wide array of malignancies A variety of toxic effects, like cumulative cardiotoxicity found with doxorubicin has sometimes led to discontinuation of the treatment Furthermore, there are some type of malignancies which do not respond to available anthracyclines The mechanism of action of anthracyclines, reflecting to their clinical efficiencies, is not clear, although most researchers consider inhibition of topo- isomerase II as a desired effect Generation of free radicals derived from quinonic structures is suggested to be related to side effects such as cardiotoxicity Anthracyclines have recently been reviewed by Professor Strohl and his group (1997) Aclacinomycin A (aclarubicin) first described by Oki et al (1975) is an anthracycline anti- biotic produced by Streptomyces galilaeus ATCC 31133 and S galilaeus ATCC 31615 It is active against tumor cells and exhibits alleviated toxic properties as compared with doxorubicin However, its activity does not reach solid tumors, limiting its use in leukemia treatment Aclarubicin differs from the other counterparts in its structure A trisacchaπde moiety, rhodosamιne-2-deoxyfucose-cιnerulose A is attached at C-7 by a glycosidic bond, whereas at the corresponding position of daunomycins only one sugar residue, daunos- amme, is attached Despite the long history of anthracyclines, three decades or so, the studies on their biosynthesis are still going on, and there is further interest to obtain novel molecules for the development of cancer chemotherapeutics A method currently used for finding novel molecules for drug screening is genetic engineering Cloning the genes for anthracyc ne biosynthesis facilitates the production of hybrid anthracyclines, as well as their use in combinatorial biosynthesis to generate novel molecules As regards the chemical nature of anthracyclines currently in clinical use, aclarubicin has unique features which make its biosynthetic genes interesting in creating novel products Regarding the genes for deoxyhexose pathway, Madduπ et al (1998) have reported that a gene derived from avermectin biosynthesis cluster caused the production of hybrid anthracyclines altering a sugar moiety when transferred into a S peucetius strain The product obtained was epirubicin, a commercially important anthracychne In this case a hydroxy group in the daunosamine moiety was in the opposite stereochemistry due to the action of an avermectin biosynthesis gene S galilaeus has been used as the host to prepare hybrid anthracyclines using the genes derived from rhodomycin pathway from S purpurascens (Niemi et al , 1994) and from nogalamycin biosynthesis cluster from S nogalater (Yhhonko et al , 1996a) The genes for nogalamycin pathway were used to generate the hybrid anthracychne production in S steffisburgensis producing typically steffimycin (Kunnaπ et al , 1997) Previously, biosynthesis genes for actinorhodin have been expressed in S galilaeus, resulting in the formation of aloesaponarin (Strohl et al , 1991) These hybrid compounds were modified in the aglycone moiety Recently, the biosynthesis genes involved in deoxyhexose pathway of nogalamycin were used to generate hybrid compounds using the S galilaeus mutants as hosts (FI pat appln No 982295) As shown above, S galilaeus has been used as a cloning host to generate novel molecules, whereas its use to donate the genes has not been described The identified genes involved in aclacinomycin biosynthesis include polyketide reductase gene (Tsukamoto et al , 1994), aklanomc acid methyl ester cyclase (GeneBank, ACCESSION AF043550) and genes for polyketide synthase (Hutchinson and Fujn, 1995, the sequence not available) Summary of the invention The present invention concerns a gene cluster, most of the genes of which are derived from deoxyhexose pathway for rhodosamine, 2-deoxyfucose and/or rhodmose The gene cluster was cloned from S galilaeus ATCC 31615 and it is involved in biosynthesis of aclacino- mycins Detailed description of the invention The experimental procedures of the present invention include biochemical and chemical methods conventional in the art Detailed description of the techniques not explained here are given in the manuals by Hopwood et al 'Genetic manipulation of Streptomyces a laboratory manual' The John Innes Foundation, Norwich (1985) and by Sambrook et al (1989) 'Molecular cloning a laboratory manual' The publications, patents and patent applications cited herein are given in the reference list in their entirety The present invention concerns particularly the discovery of the gene cluster for aclacino- mycin biosynthesis The cluster, when introduced into S peucetius strains caused the production of hybrid antibiotics modified in their sugar moiety Several strategies may be adopted to clone genes for an antibiotic Using E coh as a host for a gene library, hybridization is the most advantageous screening strategy The probe for hybridization may be any known fragment that shows sufficient homology to the bio- synthetic cluster for aclarubicin sugars, to be able to hybridize with said cluster A DNA fragment which is identical to the desired region is preferred Such a fragment, called Sg- dht, was obtained by PCR amplification of S galilaeus chromosomal DNA, using degenerated oligonucleotides annealing to the conserved region of 4,6-dehydratase gene 4,6- dehydratase is the first enzyme participating to a reaction series that converts a glucose molecule bound to a nucleotide into 6-deoxy sugars generally found in antibiotics Using this probe it was possible to clone the cluster of deoxyhexose pathway from a restricted gene library To simplify the cloning strategy the library was prepared in a pUC-based plasmid (e g pBluescript or pWHMl 109) replicating in E. coh The strategy to clone the genes involved in aclacinomycin biosynthesis according to the invention was in brief Total DNA was isolated from S. galilaeus (ATCC 31615) and digested with several restriction enzymes that yield fragments of 10 kb in average Restriction fragments were analyzed by Southern hybridization using a homologous DNA fragment, Sg-dht, as a probe. Bglϊl gave a hybridized fragment of 8 5 kb, and a double digestion with Xhol and Notl gave a hybridized fragment of 7 kb DΝ A digestion using (i) Bglll and (ii) Xhol-Notl was carried out and the fragments were ligated to the E. coli- Streptomyces shuttle vector, pWHMl 109, digested with fømHI and to the pBluescript digested with Xhol-Notl, respectively The ligation mixtures were introduced into E. coh XLlBlueMRF' that exhibits alleviated restriction-modification systems Colonies were plated on the agar plates in the dilution to give 200 to 600 cfu (colony forming units) per plate Well grown colonies were transferred in nylon membranes for hybridization, which was carried out using the Sg-dht probe Six out of the 786 .βg/II-digested clones gave hybridization signal and 7 out of 1523 of those clones carrying Xhol-Notl fragments Hybridization and washes were carried out in the stringent conditions of 65 ° C in a low salt concentration Several techniques for the labeling of the probe and for hybridization are possible, but the procedure according to Boehringer Mannheim's "The DIG System User's Guide for Filter Hybridization" is preferred The colonies giving hybridization signals were cultivated for plasmid isolation The plasmids were analyzed by Southern hybridization to confirm the reliability of the colony hybridization Plasmids containing the desired DΝA fragments (Sg4 and Sg5) were designated as pSgc4 (i?g/II-fragment) and pSgc5 (Xhol-Notl fragment)(see Fig. 2). The fragments, Sg4 and Sg5, were subcloned for sequencing in E. coh vectors pUC19 and pBluescript In total 30 subclones were used to obtain the nucleotide sequence of Sg4 and Sg5 The sequenced cluster revealed thirteen genes involved in biosynthesis of aclacino- mycins Comparison with the sequences found in the sequence library suggested the functions as sga.2 for an activator, sga3 for a dehydratase, sga4 for oxidoreductase, sga5 for dTDP-glucose 4,6-dehydratase, sga6 for glycosyl transferase (GTF), sgal for a putative isomerase, sgaS for aklaviketone reductase, sga9 for a putative polyketide assembler, sgalO for a putative cyclase, sgal 1 for aminomethylase, sga\2 for glucose- 1 -phosphate thymidylyl transferase, sga\3 for aminotransferase The function of sgal is not suggested based on similarity searches Based on the deduced functions, nine genes are involved in glycosyla- tion pathway The genes involved in the formation of aglycone are sga8, sga9, and sgalO The activator, Sga2, may control both the glycosylation system and the formation of akla- vinone via polyketide pathway Sg4 derived from pSgc4 was cloned in the Streptomyces expression vector pIJE486 (Ylihonko et al , 1996b) in S hvidans TK24 to give pSgs4 This vector is a high copy number plasmid that replicates in several Streptomyces spp (Ward et al , 1986) and it contains a constitutively expressed promoter, ermE (Bibb et al , 1985) upstream from the multiple cloning site The plasmid pSgs4 isolated from TK24 was introduced into the S galilaeus strains that are blocked in deoxyhexose pathway of aclacinomycin biosynthesis and into the S peucetius mutants producing ε-rhodomycinone based on a lesion in glycosylation genes The ability of aclacinomycin production was restored by three S galilaeus mutants, H063, H054 and H065 The mutant strain H063 accumulates aklavinone and it was completely complemented by the plasmid pSgs4 Instead, H054 and H065 producing aklavinone glycosides sharing neutral sugars, but not rhodosamine, were only partially complemented by ρSgs4 Surprisingly, H063 carrying pSgs4 (H063/pSgs4) was able to produce aclacinomycins two-fold to that of the wild type S galilaeus S peucetius Ml 8 and M90 which produce ε-rhodomycinone were selected to hosts for pSgs4 L- rhamnosyl-ε-rhodomycinone (El Khamed et al , 1977) was obtained when pSgs4 was expressed in the mutants Ml 8 and M90 and, in addition, M18/pSgs4 produced L-daunos- aminyl-ε-rhodomycinone (Essery and Doyle, 1980) The structures were not new ones but this demonstrates the ability of the gene cluster according to the present invention to generate hybrid products in a heterologous host To produce hybrid compounds we prefer to use El medium supplemented with a suitable antibiotic, in this case, thiostrepton, to maintain the selection pressure for the plasmid containing strains The products were extracted by organic solvents and purified by chromatography to obtain the compounds in high purity for structural elucidation Examples to further illustrate the invention are given hereafter Brief description of the drawings FIG. 1 shows the structures of aclacinomycin, daunomycin and ε-rhodomycinone FIG. 2 is a diagram of the gene cluster for aclacinomycin biosynthesis FIG. 3 describes the proposed biosynthesis pathway for sugars found in aclacinomycins FIG. 4 shows the structures of the hybrid compounds produced by M18/pSgs4 (1 and 2) and M90/pSgs4 (2) EXPERIMENTAL Materials used Restriction enzymes used were purchased from Promega (Madιson,Wιsconsιn, USA), Fermentas (Lithuania) or Boehringer Mannheim (Germany), alkaline phosphatase from Boehringer Mannheim, and used according to manufacturers' instructions Proteinase K was purchased from Promega and lysozyme from Sigma Hybond™-N nylon membranes used in hybridization were purchased from Amersham (Buckinghamshire, England), DIG DNA Labelling Kit and DIG Luminescent Detection Kit from Boehringer Mannheim Qiaquick Gel Extraction Kit from Qiagen (Hilden, Germany) was used for isolating DNA from agarose Bacterial strains and their use Escherichia coh XLlBlueMRF' (Stratagene, La Jolla, California) was used for cloning Streptomyces lividans TK24 was the first cloning host for gene expression The strain was provided by prof Sir David Hopwood, John Innes Centre, UK The wild type, Streptomyces galilaeus ATCC 31615, produces aclacinomycins It was used here to donate the genes of the invention Streptomyces galilaeus H039 (Ylihonko et al, 1994) produces Akv-(Rho) 0 _ 3 It was used as an expression host for pSgs4 being more easily transformed than the other mutants or the wild type Streptomyces galilaeus H054 (Ylihonko et al, 1994) produces Akv-Rho-dF-(CinA) 0 _ι, Akv-dF-dF-(CinA) 0 . 1 and Akv-dF-Rho-Rho It was used as an expression host for pSgs4 Streptomyces galilaeus H063 produces aklavinone It is a mutant strain derived from the wild type S. galilaeus H063 was used as an expression host for pSgs4 Streptomyces galilaeus H065 produces aklavinone with neutral glycosides It is a mutant strain derived from the wild type S. galilaeus H065 was used as an expression host for pSgs4 Streptomyces peucetius Ml 8 and M90 producing ε-rhodomycinone are the mutants derived from S. peucetius var caesius (ATCC 27952) They were used as expression hosts for pSgs4 Plasmids E. coh cloning vectors pBluescript SK (Stratagene) and pUC19 (Pharmacia, Sweden) were used for making the subclones for sequencing and pBluescript was used also as a vector of a gene library pWHMl 109 (provided by prof CR Hutchinson, Wisconsin, USA) is a shuttle vector replicating in E. coh and in streptomycetes. It was used as a vector of a gene library pIJ486 is a high copy plasmid vector provided by prof. Sir David Hopwood, John Innes Centre, UK (Ward et al, 1986) pIJE486 (Ylihonko et al , 1996b) is an expression vector containing ermE (Bibb et al , 1985) to promote expression of the cloned genes Nutrient media and solutions For cultivation of S galilaeus for total DNA isolation TSB medium was used Lysozyme solution (0 3 M sucrose, 25 mM Tris, pH 8 and 25mM EDTA, pH 8) was used to isolate total DNA TE buffer (10 mM Tris, pH 8 0 and ImM EDTA) was used to dissolve DNA TRYPTONE-SOYA BROTH (TSB) Per litre Oxoid Tryptone Soya Broth powder 30 g ISP4 Bacto ISP-medium 4, Difco, 37 g/1 El Per litre in 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 0 i g NaCl 3 g CaCO 3 3 g pH adjusted to 7 4 before autoclaving General methods: NMR data was collected with a JEOL JNM-GX 400 spectrometer Η and 13 C NMR samples were internally referenced to TMS The anthracychne metabolites were determined by (l) HPLC (LaChrom, Merck Hitachi, pump L-7100, detector L-7400 and integrator D-7500) using a LiChroCART RP-18 column Acetomtrile potassium hydrogen phosphate buffer (60 mM, pH 3 0 adjusted with citric acid) was used as a mobile phase Gradient system starting from 65 % to 30 % of potassium dihydrogen phosphate buffer was used to separate the compounds The flow rate was 1 ml/min and the detection was carried out at 480 nm, and (ii) by TLC using precoated Kieselgel 60 F 254 glass plates (Merck, Darmstadt, Germany) with an elution solution of toluene ethyl acetate methanol formic acid (50 50 15 3) ISP4 plates supplemented with thiostrepton (50 μg/ml) were used to maintain the plasmid carrying cultures Example 1. Cloning the gene cluster for aclacinomycin biosynthesis 1.1 Selection of clones by hybridization For isolation of total DNA, Streptomyces galilaeus was grown for four days in 50 ml of TSB medium supplemented with 0 5% glycine The cells were harvested by centrifuging for 15 min (3900 x g) in 12 ml Falcon tubes, and stored at -20 °C Cells from a 50 ml culture were used to isolate DNA 5 ml of lysozyme solution containing 5 mg/ml of lysozyme was added on the cells of each Falcon tube, and incubated for 20 min at 37°C 500 μl of 10% SDS contaimng 0 7 mg of proteinase K was added on the cells, and incubated for 80 min at 62 °C, another 500 μl of 10% SDS containing 0 7 mg of proteinase K was added, and incubation was continued for 60 min The sample was chilled on ice and 600 μl of 3M NaAc, pH 5 8 was added, and the mixture was extracted with equilibrated phenol (Sigma) The phases were separated by centrifuging (1400 x g) for 10 min The DNA was precipitated from the water phase with an equal volume of isopropanol and collected by spooling with a glass rod and washed by dipping into 70%) ethanol, air dried and dissolved in 500 μl of TE-buffer Southern hybridization to determine suitable restriction enzymes for preparing the restricted plasmid libraries was carried out using BgHl, Xhol, Noil and their combinations A fragment of about 9 kb hybridizing with the Sg-dht probe was preferred For hybridization 600 ng of digested S. galilaeus DNA was loaded onto the agarose gel and after electrophoresis, the DNA was transferred from the gel to a nylon membrane by vacuum blotting Hybridization was carried out according to Boehringer Mannheim's manual 'The DIG System User's Guide for Filter Hybridization' The probe for hybridization, Sg-dht, which was used for colony hybridization as well, was obtained by amplifying a gene fragment from the S galilaeus DNA which is internal to the 4,6-dehydratase gene and corresponds to the fragment of 6345 to 6861 shown in SEQ ID NO 14 PCR was used for amplification, and the sequences for the degenerated o gonucleotide primers were 5 -CSGGSGSSGCS- GGSTTCATSGG-3' (forward, SEQ ID NO 15) and 5'-GGGWRCTGGYRSGGSCCG- TAGTTG-3' (reverse, SEQ ID NO 16) Suitable fragments were a 9 kb BgHl fragment and a 7 kb Xhol-Notl fragment Ten micrograms of the chromosomal DNA was digested with BgHl The DNA fragments were separated by agarose gel electrophoresis and the band of 8 to 9 kb were cut from the 0 6% low gelling temperature SeaPlaque® agarose The DNA band was isolated from the gel using Qiagen Gel Extraction Kit The isolated fragment was ligated to pWHMl 109 plasmid vector digested with BamYQ. and dephosphorylated, in the ratio of 3 moles of the insert DNA to 1 mole of the vector DNA The ligated DNA was introduced into E coh XLlBlueMRF' by electroporation Using the whole gation mixture 786 colonies were obtained The colonies were grown on agar plates for at least 12 h and transferred to nylon membranes Hybridization of colony membranes was carried out as Southern using Sg-dht as a probe Six clones gave signal in hybridization and the corresponding colonies were plated on agar and inoculated in 3 ml of LB medium for isolation of the plasmid DNA Southern hybridization was used to study whether the plasmids derived from the clones carried the desired insert Four of these plasmids contained the 4,6-dehydratase gene fragment and gave the identical restriction map thus carrying the same fragment representing both oπentations The fragment was designated as Sg4 and the plasmid containing the fragment as pSgc4 In the same manner the plasmid library representing a 7 kb Xhol-Notl DNA fragment derived from S galilaeus was constructed pBluescript was digested with Xhol-Notl and the library containing the gene fragments of around 7 kb was constructed In total 1523 colonies were hybridized and seven turned to be the desired clone As described above, the clones were studied for the Xhol-Notl fragment The insert fragment was designated as Sg5 and the plasmid as pSgc5 The strain E coh XLlBlue MRF'/pSgc5 obtained was deposited according to the rules of the Budapest Treaty at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on August 12, 1999 with the accession number DSM 12999 The fragments Sg4 and Sg5 overlap within 836 bp corresponding bases from 6181 to 7016 in SEQ ID NO 14 1.2. Subcloning the fragments for sequencing To determine the nucleotide sequence of the whole cluster of the Sg4 and Sg5 suitable subclones were constructed The convenient restriction sites were used for subcloning the 14806 bp region in the plasmids pUC19 and pBluescript Nineteen subclones were needed to sequence Sg4, and 1 1 subclones for Sg5 E coh XLlBlueMRF' cells containing the subcloned plasmids were cultivated overnight at 37° C in 5 ml of LB-medium supplemented with 50 μg/ml of ampicil n To isolate plasmids for sequencing reactions Wizard Plus Mimpreps DNA Purification System kit of Promega or Biometra Silica Spin Disc Plasmid DNA Mimprep kit of Biomedizimsche Analytik Gmbh were used according to the manufacturers' instructions DNA sequencing was performed using the automatic ABI DNA sequencer (Perkin-Elmer) according to the manufacturer's instructions 1.3 Sequence analysis and the deduced functions of the genes Sequence analyses were made using the GCG sequence analysis software package (Version 8, Genetics Computer Group, Madison, Wis , USA) The translation table was modified to accept also GTG as a start codon Codon usage was analyzed using published data (Wright According to the CODONPREFERENCE program the sequenced DNA fragment revealed 11 complete open reading frames (ORFs), and two 5' ends of the other ORFs (sga and sgal3) The functions of the genes were concluded by comparing the amino acid sequences translated from their base sequences to the known sequences in the data banks The results are shown in Table 1 referring to the sequence data given in the application The suggested functions for the genes match well with a proposed biosynthetic pathway of sugars of aclacinomycins (Fig. 3) The last residue in a trisaccharide moiety of aclacinomycins is rhodinose that is enzymatically converted to cinerulose Aclacinomycin N, a precursor of aclarubicin, contains rhodinose as the third sugar residue Table 1. 1.4 Expression cloning in Streptomyces strains The 8 kb BamYH-Hindlll fragment from pSgc4 was ligated in pIJE486 to give pSgs4 Plasmid pSgs4 was introduced into S. lividans TK24 by protoplast transformation The strain S. lividans TK24/pSgs4 obtained was deposited according to the rules of the Budapest Treaty at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on August 12, 1999 with the accession number DSM 12998 The plasmid pSgs4 was isolated from the strain, and further transferred into S. galilaeus mutant H039 The plasmid preparate isolated from H039 was subsequently introduced into H063, H054, and H065 mutants deficient of glycosylation system of aclacinomycins The usage of H039 as a primary S. galilaeus host was due to the better efficiency for the intake of foreign DNA S. galilaeus mutants were studied for complementation by cultivating the clones containing pSgs4 in El medium supplemented with thiostrepton (10 μg/ml) The products from a 500 μl sample of the culture broth were extracted with toluene methanol (1 1) at pH 7 The metabolites from both the transformed clones and the mutants were analyzed by TLC and HPLC to find the differencies caused by pSgs4 H063 producing endogenously aklavinone was restored to aclacinomycin producer with pSgs4 No aklavinone was found in the culture broth of H063/pSgs4 However, complementation was not completed when pSgs4 was expressed in H054 and H065 Both of the mutants produce aklavinone with neutral glycosides Incomplete complementation was presumably due to the loss of the plasmids of some bacterial cells during cultivation, or a low expression of the genes needed as an activator is not present in pSgs4 In the same manner, pSgs4 isolated from TK24 was introduced into the S. peucetius mutants Ml 8 and M90 The characteristic product for these mutants is ε-rhodomycinone The strains M18/pSgs4 and M90/pSgs4 containing the plasmid were cultivated in El medium supplemented with thiostrepton (10 μg/ml), and the metabolites therein were analyzed by TLC and HPLC Both of the clones revealed an altered production profile as compared with the products obtained from the mutants M90/pSgs4 accumulated a glycosylated product, yielding ε-rhodomycinone as the aglycone The compound was identified as L-rhamnosyl-ε-rhodomycinone which has been previously synthesized (CAS=63252-11-9) by El Khamed et al. (1977) M18/pSgs4 produced two compounds differing from the parental strain According to the HPLC and TLC data one compound was the same as was produced by M90/pSgs4, L- rhamnosyl-ε-rhodomycinone, and the other one was L-daunosaminyl-ε-rhodomycinone, which was previously characterized by Essery and Doyle (1980) Table 2: TLC and HPLC data of the hybrid products 1.5 Applicability of pSgs4 for strain improvement Since H063 was completely complemented by pSgs4, the production level of aminogly- cosides was studied For this purpose, H063/pSgs4, H063 and the wild type S. galilaeus were cultivated in El medium in the Erlenmeyer bottles for four days Two samples of 2 ml from each culture were extracted first with toluene methanol (1 1) in acidic conditions to remove the neutral glycosides and the aglycones The extraction procedure was repeated until neutral glycosides and the aglycones had disappeared from the water phase The amount of anthracychne metabolites in toluene phase was determined and is shown in Table 3 Aclacinomycins containing rhodosamine were extracted from the water phase by chloroform Both toluene and chloroform extracts were analyzed by TLC and toluene phases contained mostly aklavinone and the degradative products Chloroform phases contained mainly aminoglycosides, although minor amounts of the aglycones were also detected Extracts were evaporated to dryness and subsequently dissolved into 1 ml of methanol The amounts of anthracychne metabolites were detected by spectrophotometer at 430 nm The amounts related to absorbance were calculated using an extinction coefficient of 13000 The results given as mg/1 of cultivation broth are shown in Table 3. The production of aclacinomycins by H063/pSgs4 was at least twofold better than obtained by the wild type Table 3. The ability to increase the yield of aclacinomycins by pSgs4 in the mutant H063 suggests that the genes according to the present invention are useful in strain improvement. Example 2. Compounds generated by pSgs4 The seed culture, 180 ml of El culture of the plasmid containing strains, M18/pSgs4 or M90/pSgs4, was obtained by cultivating each of the strains in three 250 ml Erlenmeyer flasks containing 50 ml of El-medium supplemented with thiostrepton (5 μg/ml) for four days at 30°C, 330 rpm. The combined culture broths (180 ml) were used to inoculate 13 1 of El -medium in a fermentor (Biostat E) Fermentation was carried out for five days at 28°C (330 rpm, aeration: 450 1/min). The cells were harvested by centrifuging. 2.6 1 of methanol was used to brake the bacterial cells. The anthracychne metabolites were extracted from methanol solution at pH 8 using 2 1 of ethyl acetate and the extract was evaporated to dryness. The viscous residue was loaded onto a silica column of 4 * 10 cm and toluene:ethyl acetate: formic acid (50:50:3) with increasing amount of methanol was used as an eluent. Pure fractions were pooled and extracted with 1M phosphate buffer (pH 8.0) and water. Organic phase was dried with anhydrous Na 2 SO 4 and then treated with hexane to effect precipitation. Pure compounds appeared as red powders dried under vacuum. Complete structural determination of the compounds were accomplished by NMR Proton and carbon assignments were based on a conventional NOE difference, pHSQC and HMBC measurements. Connectivities in particular relied heavily on HMBC experiment As deduced from the data given in Table 4, the structures revealed were L-rhamnosyl-ε- rhodomycinone (1) and L-daunosaminyl-ε-rhodomycinone (2) shown in Figure 4. Although these structures were not novel, the generation of the hybrid products by the genes involved in glycosylation portion of aclacinomycin biosynthesis well demonstrates that the genes of pSgs4 are functional and ready to use in drug discovery for finding novel molecules Deposited microorganisms The following microorganisms were deposited according to the Budapest Treaty at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, Germany. Microorganism Accession number Date of deposit S. lividans TK24/pSgs4 DSM 12998 12 August 1999 E. coh XLlBlueMRF7pSgc5 DSM 12999 12 August 1999 Table 4. Η and 13 C chemical shifts of 1 (DMSO d6 ) and 2 (trace of TFA in DMSO d6 ) in 400 and 100 MHz, respectively References Bibb, M. J., Janssen, G. R., and Ward, J. M. 1985 Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus Gene 38: 215-226 El Khamed, H.S., Swartz, D.L., and Cermak, R.C. 1977 Synthesis of ε-rhodomycinone glycosides J Med Chem 20: 957-960 Essery, J.M., and Doyle, T.W. 1980 The synthesis of daunosaminyl-ε-rhodomycinone, daunosamιnyl-10-epi-ε-rhodomycinone, daunosaminyl-ε-pyrromycinone and 10-descarbo- methoxy-ε-pyrromycin Can J Chem 58: 1869-1874 Hutchinson, C.R., and Fujii, I. 1995 Polyketide synthase gene manipulation A structure- function approach in engineering novel antibiotics Annu Rev Microbiol 49: 201-238 Kunnari, T., Tuikkanen, J., Hautala, A., Hakala, J., Ylihonko, K., and Mantsala, P. 1997 Isolation and characterization of 8-demethoxy steffimycins and generation of 2,8- demethoxy steffimycins in Streptomyces steffisburgensis by the nogalamycin biosynthesis genes J Antibiot 50: 496-501 Madduri, K., Kennedy, J., Rivola,G., Inventi-Solari, A., Filippini, S., Zanuso, G., Colombo, A.L., Gewain, K.M., Occi, J.L., MacNeil, D.J., and Hutchinson, C.R. 1998 Production of the antitumor drug epirubicin (4'-epidoxorubicin) and its precursor by a genetically engineered strain oϊ Streptomyces peucetius Nature Biotech 16: 69-74 Niemi, J., Ylihonko, K., Hakala, J., Kopio, A., Parssinen, R., and Mantsala, P. 1994 Hybrid anthracychne antibiotics production of new anthracyclines by cloned genes from Streptomyces purpurascens in Streptomyces galilaeus Microbiol 140: 1351-1358 Oki, T., Matsuza a, Y., Yoshimoto, A., Numata, K., Kitamura, I., Hori, S., Takamatsu, A., Umezawa, H., Ishizuka, M., Naganawa, H., Suda, H., Hamada, M, and Takeuchi, T. 1975 New antitumor antibiotics, Aclacinomycins A and B. J Antibiot 28: 830-834 Strohl, W. R., Dickens, M. L., Rajgarhia, V. B., Woo, A. J., and Priestley, N. D. 1997 Anthracyclines in Biotechnology of Antibiotics, ed Strohl, W R Marcel Dekker Inc , New York, pp 577-657 Strohl, W.R., Bartel, P.L. Li, Y., Connors, N.C., and Woodman, R.H 1991 Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes J Ind Microbiol 7: 3 163-174 Tsukamoto, N., Fujii, I., Ebizuka, Y., and Sanka a, U. 1994 Nucleotide sequence of the aknA. region of the aklavinone biosynthetic gene cluster oϊ Streptomyces galilaeus J Bacteriol 176: 2473-2475. Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J., and Bibb, M. J. 1986 Construction and characterization of a series of multicopy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase from Tn5 as indicator Mol Gen Genet 203: 468-478 Wright, F., and Bibb, M. J. 1992 Codon usage in the G+C-rich Streptomyces genome Gene 113: 55-65 Ylihonko K., Hakala J., Kunnari T., and Mantsala P. 1996a Production of hybrid anthracychne antibiotics by heterologous expression oϊ Streptomyces nogalater nogalamycin biosynthesis genes Microbiol 142: 1965-1972 Ylihonko, K., Tuikkanen, J., Jussila, S., Cong, L., and Mantsala, P. 1 96b A gene cluster involved in nogalamycin biosynthesis from Streptomyces nogalater sequence analysis and complementation of early-block mutations in the anthracychne pathway Mol Gen Genet 251: 113-120 Ylihonko, K., Hakala, J., Niemi, J., Lundell, J., and Mantsala, P. 1994 Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants Microbiol 140: 1359-1365

Claims: Claims 1. An isolated and purified DNA fragment, which is the gene cluster for the anthracychne biosynthetic pathway of the bacterium Streptomyces galilaeus, being included in a 7 kb Xhol-Notl fragment and a flanked 8.5 kb Bglϊl fragment of S. galilaeus genome. 2. The DNA fragment according to claim 1, which comprises the nucleotide sequence given in SEQ ID NO: 14, or a part thereof having similar characteristics, or a sequence showing at least 84 %> homology to said sequence. 3. A recombinant DNA, which comprises the DNA fragment of claim 1 or 2, or a part thereof having similar characteristics, cloned in the plasmid replicating in Streptomyces or in E. coli. 4. The recombinant DNA according to claim 3, which is the plasmid pSgs4 deposited in S. lividans strain TK24/pSgs4 with the accession number DSM 12998. 5. The recombinant DNA according to claim 3, which is the plasmid pSgc5 deposited in E. coli strain XLlBlueMRF'/pSgc5 with the accession number DSM 12999. 6. Use of the genes derived from the DNA fragment of claim 1 or 2 in the production of anthracychne metabolites. 7. Use of the genes derived from the DNA fragment of claim 1 or 2 to increase aclacino- mycin production. 8. Use according to claim 6 or 7, wherein the genes are encoding an activator, a dehydratase, an oxidoreductase, a dTDP-glucose 4,6-dehydratase, a glycosyl transferase, an isomerase, an aklaviketone reductase, a polyketide assembler, a cyclase, an aminomethylase, a glucose- 1 -phosphate thymidylyl transferase, and an aminotransferase. 9. A process for increasing aclacinomycin production in a bacterial host, comprising transferring the DNA fragment of claim 1 or 2 into a Streptomyces host, cultivating the recombinant strain obtained, and isolating the aclacinomycins produced. 10. The process according to claim 9, wherein the Streptomyces host is a Streptomyces galilaeus host. 11. The process according to claim 10, wherein the Streptomyces galilaeus host is a mutant strain derived from S. galilaeus ATCC 31615. 12. A process for producing metabolites, comprising transferring the DNA fragment of claim 1 or 2 into a Streptomyces host, cultivating the recombinant strain obtained, and isolating the compounds produced. 13. A process for producing anthracychne metabolites, comprising transferring the DNA fragment according to claim 1 or 2 into a Streptomyces peucetius host, cultivating the recombinant strain obtained, and isolating the compounds produced.

Inventors: HAKALA JUHA, RAETY KAJ, YLIHONKO KRISTIINA, HAKALA, JUHA, RAETY, KAJ, YLIHONKO, KRISTIINA
Applicants: GALILAEUS OY, HAKALA JUHA, RAETY KAJ, YLIHONKO KRISTIINA, HAKALA, JUHA, RAETY, KAJ, YLIHONKO, KRISTIINA
Application year: 2000
Main classification: C12N15
IPC codes: C12N1, C12N15, C12P1, C12P19