DNA-SEQUENCE CODING FOR CARMINOMYCIN 4-O-METHYLTRANSFERASE

Abstract: The ability to convert carminomycin to daunorubicin can be conferred on a host by transforming the host with a recombinant vector comprising a gene coding for carminomycin 4-O-methyltransferase.

Description: DNA-sequence coding for carminomycin 4-0-methyltransferase Field of the Invention The present invention concerns a way to produce anthracyclines useful in the treatment of cancer by modifying the biosynthesis of daunorubicin so as to improve the production of daunorubicin from carminomycin in streptomyceies other than Streptomyceε peucetius 29050 and in bacterial cell extracts or by purified enzymes derived therefrom. Background of the Invention The anthracyclines of the daunorubicin group, such as doxorubicin, carminomycin and aclacinomycin, are among the most widely employed agents in antitumoral therapy [F. Arcamone, Doxorubicin, Academic Press, New York, 1981 , pp. 12-25; A. Grein, Process Biochem. 16:34 (1981 ); T. Kaneko, Chimicao p; May:1 1 (1988)]. Improved derivatives of daunorubicin and doxorubicin have been made by chemical synthesis to enhance their antitumor activity, particularly by the oral route of administration, and to combat the acute toxicity and chronic cardiotoxicity associated with the use of these drugs in the treatment of cancer [Penco, Process Biochem. 15:12 (1980); T. Kaneko, Chimicaogp/ ay:l l (1988)]. 4'- Epidoxorubicin (Epirubicin®) and 4-demethoxydaunorubicin (Idarubicin®) are examples of such analogs. These naturally occuring compounds are produced by various strains of Sireptomyces (S. peucetius, S. coeruleorubidus, S. galilaeυs, S. griseus, S. griseoruber, S. insignis, S. viridochromcgenes, S. bifυrcus and Sireptomyces sp. strain C5) and by Actiπomyces carminata. Doxorubicin is only produced by S. peucetius subsp. caesius but daunorubicin is produced by S. peucetius as well as the other Sireptomyces described above. The type strains S. peucetius subsp. caesius I MRU 3S20 (this strain is the same as ATCC 27952 and hereinafter is abbreviated to "S. peucetius 392C " ). S. peucetius ATCC 29050 ("S. peucetius 29050"), and S. peucetius subsp. caesius ATCC 27S52 ("S. peucetius 27952 " ) are publically available and are described in USA-3 590 028. S. peucetius 29050 and 27952 have been deposited at the American Type Culture Collection, Rockville, MD USA, receiving the index number ATCC 29050 and 27952. The anthracyciir.e doxorubicin (2) is made by S. peucetius 27952 from malonic acid, propionic acid, and glucose by the pathway shown in Fig. 1 of the accompanying drawings, ε- SUBSTITUTE SHEET Rhodomycinone (4), carminomycin (3) and daunorubicin (1 ) are established intermediates in this process [Grein. Advan. Appl. Microbiol. 32:203 (1987), Eckardt and Wagner, J. Basic Microbiol. 28:137 (1988)]- Two steps in this pathway involve the O-methylation of discrete intermediates: the conversion of aklanonic acid to methyl aklanonate and carminomycin (3) to daunorubicin (l ). Cell-free extracts of S. peucetius 29050, S. iπsigπis ATCC 31913. S. coerυteorυbidυs ATCC 31276 and Sireptomyces sp- C5 have been shown to catalyze the latter step in the presence of S-adenosyl-L-melhionine [Connors et al._ J. Gen. Microbiol. 136:1895 (1990)], suggesting thai all of these strains contain a specific carminomycin 4-0- methyltransferase (COMT protein). Genes for daunorubicin biosynthesis and daunorubicin resistance have been obtained from S. peucetius 29050 and S. peucetius 27952 by cloning experiments [Stutzman-Engwall and Hutchinson, Proc. Nail. Acad. Sci. USA 86:3135 (1988); Olten et a!., J. Bacteriol. 172:3427 (1990)]. These studies have shown that, when introduced into Sireptomyces lividans 1326. these cloned genes confer the ability to produce ε-rhodomycinone and to become resistant to daunorubicin and doxorubicin to this host. In subsequent work we examined whether these clones could confer the ability to convert carminomycin to daunorubicin when introduced into S. lividans. We have now isolated a 1.6 kilobase (kb) DNA segment that incorporates the carminomycin 4-O-melhyltransferase gene, which hereinafter will be abbreviated as "dnrK". Summary of the invention The present invention provides DNAs having the configuration of restriction sites shown in Fig. 2 of the accompanying drawings or a restriction fragment derived therefrom containing a gene. dnrK, coding for carminomycin 4-O-methyitransferase. For convenience, the DNA segment shown in Fig. 2 is called here "insert DNA" and is further defined by the DNA sequence shown in Fig. 3 of the accompanying drawings. The invention also provides: (1) recombinant vectors that are capable of transforming a host cell and that contain an insert DNA or a restriction fragment derived therefrom containing the dnrK gene: (2) recombinant vectors that are able to increase the number of copies of the dnrK gene and the amount of its product in a strain of Sireptomyces spp. producing daunorubicin; (3) recombinant vectors that are able to express the cnrK gene in Escherichia coli so as to enable the production of the purified carmnomycin 4-O-rτ.εihyliransferase enzyme. (4) a mϊcrobial source of carminomycin 4- O-me-hyϋransferase for the byconversion SUBSTITUTE SHEET of carminomycin into pure daunorubicin. Brief * * .*?S ricιion of tt-**? ? * = winr s Fig. 1 is a summary of the doxorubicin biosyntr.e:ic pathway. Fig. 2 is the restriction map analysis of the firs; DNA of the invention. This is an insert in recombinant plasmid DWHM902 that was constructed by insertion of a 1.6 kb SphUFvυW DNA fragment containing the carminomycin 4-O-methyltransferase (dnrK) gene, which was obtained from recombinant plasmid pWHM901 by its digestion with Sph\ and PvυW, into the Sph\/Sma\ sites of the pWHM3 plasmid. an Escheric ia ccii-Streptomyces shuttle vector [Vara et a!., J. Bacteriol. 171 :5872 (1989)]. The map shown in Fig. 2 does not necessarily provide an exhaustive listing of all restriction sites present in the DNA segment. However, the reported sites are sufficient for an unambiguous recognition of the segments. Fig. 3 is a schematic illustration of a nucleolide sequence of the dnrK DNA segment which corresponds to that encoding carminomycin 4-O-methyltransferase. This covers the reci ' on between the SpΛI and the PvυW restriction sites of pWKfv.902 and shows the coding strand in the 5' to 3 ' direction. The derived amino acid sequence of the translated open reading frame encoding carminomycin 4-O-methyltransferasε is shown below the nuclectide sequence cf the dnrK gene. £.SEQ ID NO : l , SEQ ID ' NO : 2 ) Fig. 4 is the restriction map analysis of the second DNA of the invention. This is an insert in recombinant piasmid pWHM903 that was constructed by insertion of a =1.4 k NdeUEcoR\ DNA fragment, obtained from the 5.8 kb Sen; DNA fragment of pWHM90l by site- directed utagenesis. into the Nde\ and EccP.\ sites of ..-.= ?T7-7 E. coli expression plasmid vector [Tabor and Richardson, Proc. Nail. Acac. Sci. US.-. 52:1074 (1985)]. The map s .own in Fig. 4 does not necessarily provide an exhaustive listiπc cf all restriction sites present in the DNA segment. However, the reported sites are sufficiε.".: for an unambiguous recognition of the segments. Dgte'lPd dr?cr:t **on C* ■ ■"*= : -!v = n 'io π The insert DNAs and restriction fraςrπe.-.is of the invention contain a gene [dnrK, coσ ' inc for carminomycin 4-C-rτ :e:hyltrans;erase. Fcr such a ς = ~ e to be expressed, the DNA m y carry its own transcriptiona! control sequence and. in particular, its own promoter which is cperabiv connected to the gene =.-.c which is recognised cy a hes: ceil F. polymerasε. Alternatively, the insert DNA or rεstric:ic~ fragment may be licsted to a.-ctr.er transcriptional control sεcuence in the correct fashion c: cioned into a vectcr at a restriction site appropriately located SUBSTITUTE SHEET neighboring a transcriptional control sequence in the vector. An insert DNA or restriction fragment carrying a carminomycin &-0- melhyitransferase gene may be cloned into a recombinant DNA cloning vector. Any autonomously replicating and/or integrating agent comprising a DNA molecule to which one or more additional DNA segments can be added may be used. Typically, however, the vector is a plasmid. A preferred plasmid is the high copy number plasmid pWHM3 or plJ702 [Katz et al., J. Gen. Microbiol. 129:2703 (1983)]. Other suitable plasmϊds are plJ385 [Mayeri et al., J. Bacteriol. 172:6061 (1990)]. plJ680 (Hopwood et al.. Genetic Manipulation of Sireptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985). pWHM601 [Guilfoile and Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553 (991)] or pPM927 [Smokina et al., Gene 94:52 (1990)]. Any suitable technique may be used to insert the insert DNA or restriction fragment thereof into the vector. Insertion can be achieved by ligating the DNA into a linearized veβcdr at an appropriate restriction site. For this, direct combination of sticky or blunt ends, omopoiymer tailing, or the use of a linker or adapter molecule may be employed. The recombinant vector is used to transform a suitable host cell- The host cells may be ones that are carminomycin- or daunorubicϊn-sensitive, i. e., cannot grow in the presence of a certain amount of carminomycin or daunorubicin, or that are carminomycin- or daunorubicin- resistanl. The host may be a microorganism. Strains of S. peucetius, in particular S. peucetius 29050, and other strains of Sireptomyces species that produce anthracyclines or do not produce them may therefore be transformed. Transformants of Sireptomyces strains are typically obtained by protoplast transformation- The dnrK gene may also be incorporated into other vectors and expressed in non-streptomycetes like E. coli. The COMT protein obtained by the transformed host may be employed for bioconverting carminomycin to daunorubicin. This method would allow the preparation of highly pure daunorubicin starting from a cell extract produced by a fermentation process and containing the undesired intermediate carminomycin besides the daunorubicin. The bicconversicn process can be carried out eϊtner by using directly the f ee or immobilized transformed cells or by isolating the COMT protein, which can be used in the free form, immobilized according to known techniques to resins, class, cellulose or similar substances by ionic or covalent bonds, or grafted to fibers permeable to the substrate or insolubilized by cross-linkage- The COMT protein may also be used in the raw cellular extract. The recombinant vector of the present invention may be also used to transform a suitable SUBSTITUTE SHEET host cell, which produces daunorubicin, in order to enhance the byconversion of carminomycin and to minimize the presence of said unwanted intermediate into the final cell extract. The host cells may be ones that are carminomycin, daunorubicin- or doxorubicin-resistant, i.e., can grow in the presence of any amount of carminomycin, daunorubicin or doxorubicin. Strains of S. peucetius, in particular S. peucetius 29050, and other strains of Sireptomyces species that produce anthracyclines may therefore be transformed. Transformants of Sireptomyces strains are typically obtained by protoplast transformation. Daunorubicin can be obtained by culturing a transformed strain of S. peucetius or another Sireptomyces species that does not contain a dnrK gene and recovering the daunorubicin or related anthracyclines thus-produced. The insert DNAs are obtained from the genσmic DNA of S. peucetius 29050. This strain has been deposited at the American Type Culture Collection, Rockville, MD. USA under the accession number ATCC 29050. A strain derived from S. peucetius 29050, like S. peucetius 27952, may also be used, which typically will also be able to convert carminomycin to daunorubicin. Insert DNAs may therefore be obtained by: (a) preparing a library of the genomic DNA of S. peucetius 29050 or a strain derived therefrom; (b) screening the library for clones with the ability to convert carminomycin to daunorubicin; (c) obtaining an insert DNA from a recombinani vector that forms part of the library and that has been screened as positive for the ability to convert carminomycin to daunorubicin; and (d) optionally, obtaining from the insert DNA a restriction fragment that contains a gen coding for carminomycin 4-O-methyliransferasε. The library may be prepared in step (a) by partially digesting the genomic DNA of S. peucetius 29050 or a strain derived therefrom. The restriction enzyme Mbo\ is perferably used. The DNA fragments thus obtained can be size fractionated; fragments from 3 to 5 kb in size are preferred. These fragments are iigated into a linearized vector such as pWHM3 or plJ702. Host cells are transformed with the ligation mixture. Typically, the host cells can not produce carminomycin or daunorubicin and can be carminomycin- or daunorubicin-sensitive, for example, sensitive to 10 microgram or less of carminomycin cr Caunorubicin per ml. For example. S. lividans Jl ' 623 protoplasts (Hopwood et al., Genetic Manipulation of Sireptomyces. A Laboratory Manual. John Innes Foundation, Norwich, UK, 1985) may be transformed. SUBSTITUTE SHEET In step (b). tne transformants thus obtained are screened for the ability to take up carminomycin. convert it to daunorubicin. and excrete daunorubicin- Clones able to convert carminomycin to daunorubicin are identified by chromatc raphic analysis of extracts of a culture medium containing carminomycin for the presence of daunorubicin. Such clones are isolated and recombinant vectors contained therein are extracted.- On digestion of the recombinant vectors with suitable restriction enzymes in step (c). the S. peucetius 29050 DNA inserted into each vector may be identified, sized and mapped- In thi≤ way, it may be checked that the vector contains an insert DNA of the invention. Further, two or more overlapping inserts may be isolated that are wholly or partly embraced within the DNA of the invention. These may be fused together by cleavage at a common restriction site and subsequent ligation to obtain a DNA of the invention, pared in length using appropriate restriction enzymes if necessary- Restriction fragments of an insert DNA that contains a gene coding for the COMT protein may be obtained in step (d) also by cleaving an insert DNA with an appropriate restriction enzyme. DNA of the invention may be mutated in a way that does not affect its ability to confer the abilit to convert carminomycin to daunorubicin. This can be achieved by site-directed mulagenesis for example- Such mutated DNA forms part of the invention. The DNA of the invention may also be incorporated into vectors suitable for expression of the dnrK gene in a non-streptomycete host like E. coll The following examples illustrate the invention. Materials and Methods Bacterial strains and otasrnics: E. coli strain DH5c. which is sensitive to ampicillin and εpra~v'cin, is used for s bcloning MA fragments a~.z Ξ.coli K3S / π εsel & i-iocet , J. Bactεriol. 159 ; iC-3- (1554) / is used for expr-ession of the S. Peucetius cnr gene. Ξ. coli J.-.1C5 is us for -s ir-g single strar- ec DNA for sequencing the r_X ge M ys s.ngi bu» ~ e f '?: E. coii DK5c is maintained cπ 3 agar (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd e - Cold Spring Harcor Press. Cold Spring Har ' oor, NY. 19SΞ.. When selecting for irar.s.or ants, ampicillin or aoramycin are added at concentrations of 50 liC.'ml a d 100 ς. l. respectively- E. cell JM10Ξ is maintained on M9 minimal acar medium (Sarr.orcok ei a!-. .'.icsc εr C.'cr.inc. A Labcrsicγ Manual, 2nd ed. Cold Spring Harbor Press. Cold Spring Harbor. NY. 1 Ξ9). and a colony is '-rar.sferred to L3 medium and grown overnic t at 37=C to obtain the bacteria used in tne preparation 0! single stranded DNA. H acar (Sambrook et el.. Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Press. Cold Spring Harbor, NY, 1989) is used to plate E. coli DH5α transformed with the replicative form of M13 DNA ((Yansch-Perron et al., Gene ?3:103 (1985)]. S. lividans is maintained on R2YE agar (Hopwood et al.. Genetic Manipulaiion of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) for the preparation of spores as well as for the regeneration of protoplasts. Subcloninς DNA fragments: DNA samples are digested with appropriate restriction enzymes and separated on agarose gels by standard methods (Sambrook et al.. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). Agarose slices containing DNA fragments of interest are excised from a gel and the DNA is isolated from these slices using the GENECLEAN device (Bio10l , La Jolla, CA). The isolated DNA fragments are subcloned using standard techniques (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) into E. coli and E. coli/Streptomyces shuttle vectors for biotransformation and expression experiments, respectively, and into M13 vectors [(Yansch-Perron et al., Gene 33:103 (1985)] for sequencing. DNA seouencino: After subcloning DNA fragments of interest into an M13 vector, single stranded DNA is prepared by standard techniques (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and used in sequencing. DNA sequεncε data arε obtained using a Sequenase version 2.0 sequencing kit (US Biochemicals, Cleveland, OH) according to the manufacturers suggestions. 7-Deaza dGTP is used instead of dGTP to avoid compressions. Initially, an universal primer of the M13 vector is used to obtain the sequencε of thε first 200-250 bases, then from these sequence data, an 17- mer oligoncleotidε is svnthesised using an Applied Biosystems 391 DNA synthesizer accordino'to the manufacturer's directions and used as a primer to continue DNA sequencing until the complete DNA sequence cata are obtained. Tran?form=ιion cf Streptomvces sp*? i?? snd E. ccir Competent cells of E. coli are prepared by the calcium chloride method (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and transformed by standard techniques (Sambrook et a!., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1969). S. lividans TK24 mycelium is crown in YEME medium (Hopwood et al., Genetic Manipulation of Sireptomyces. A Laboratory Manual, John Innes Foundation. Norwich. UK, 1 S5) and harvested after 48 hr. The mycelial pellet is SUBSTITUTE SHEET washed twice with 10.3% sucrose solution and used to prepare protoplasts according to the method outlined in the Hopwood manual (Hopwood et al.. Genetic Manipulation of Sireptomyces. A Laboratory Manual, John Innes Foundation, Norwich. UK, 1985). The protoplast pellet is suspended in about 300 microlitres of P buffer (Hopwood et al., Genetic Manipulation of Sireptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985) and a 50 microlitre aliquot of this suspension is used for each transformation- Protoplasts are transformed with plasmid DNA according to the small-scale transformation method of Hopwood et al. (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK. 1985). After 17 hr of regeneration on R2YE medium at 30°C, the plales are overlayed with 50 μg/ml of thiostrepton and allowed to grow at 30°C until sporulated. Bioconversion of carminomvcin to daunorubicin: S. lividans transformants harboring different plasmids are inoculated into liquid R2YE medium (Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John innes Foundation, Norwich, UK, 1985) containing 5 μg/ml of thiostrepton. After 2 days of growth at 30°C, 2.5 ml of this culture is transferred to 25 ml of Strohl medium [(Dekleva et a!., Can J. Microbiol. 31 :287 (1985) containing 20 μg/ml of thiostrepton. Cultures are grown in baffled Erienmeyer flasks on a rotary shaker at 300 rpm at 30°C for 72 hr, after which carminomycin (as a solution in water at a concentration of 10 milligrams/ml) is added to cultures to give a final concentration of 5 μg/ml. After 24 h of further incubation on the shaker, the cultures are incubated in a water bath at 60°C for 45 min after the addition of 150 milligrams/ml of oxalic acid to hydrolize the glycosidic forms of the anthracycϋne metabolites. The metabolites are extracted from the cultures with 15 ml of chloroform after adjusting the pH of cultures to 8.4-8.6. The chloroform solution is filtered through a 0-45 μm Acrociso CR filter (Gelman Sciences, Ann Arbor. Ml) and 100 microlitres of this filtrate is analyzed by HPLC on a Waters Nova-Pak C., ε cartridge (8 mm x 10 cm) with a mobile phase of methanol-water (85:15) adjusted to pH 2.5 with phosphoric acid a* a flow rate of 3 ml/min. The column output was monitored using Waters 6000 solvent delivery system, a 441 UV detector operated at 254 nm, and a 740 data module. Carminomycin and daunorubicin (10 μg/ml in methanol) were used as external standards to quantitste the amount of these metabolites isolated irom the cultures- Example I SUBSTITUTE SHEET ClOPino of *hp en rK <- - ■ - ( • p nt-cdinπ c rninomv i" ^ ■ Q-mc 'hylirP '-.'jf f gc o Several of the cosmid clones described by Stutzman-Engwall and Hutchinson [(Proc. Natl. Acad. Sci. USA 56:3135 (1989). representing approximately 96 kb of S. peυceiius 29050 genomic DNA, are transformed into S. lividans TK24 and the transformants are analyse for the byconversion of carminomycin to daunorubicin according to the method described in th materials and methods section. Cosmid clone pWHM239 [Otten et al., J. Bacteriol. 172:3427 (1990)] bioconverts 22% of added carminomycin to daunorubicin. A 1 1.2 kb EcoRI fragment from the insert in pWHM339 is subcloned into the cosmid vector pKC505 [Richardson et al.. Gene 61 :231 (1987)] to yield plasmid pWHM534. S. lividans TK24 transformed with pWHM534 shows a 25 to 60% byconversion of added carminomycin to daunorubicin. A 5.8 k Sphl fragment from pWHM534 is subcloned in the Sphl site of the high-copy number plasmid pWHM3 to yield the plasmid pWHM901 . S. lividans . transformed with pWHM901 exhibits a 5 to 85% bioconversiorj of carminomycin to daunorubicin. A 1.6 kb Sphl/PvυU fragment is cloned from pWHM90i first into the Sphl/Smal sites of pUC19 [Yansch-Perron et al.. Gene 33:103 (1985)], then the 1.6 kb DNA fragment is subcloned from the latter plasmid as an HindttVEcoFU fragment into the Hinc\\\/EcoP * \ sites of pWHM3 to yield plasmid pWHM902 (Fi 2). S. lividans transformed with pWHM902 bioconverts 100% of the carminomycin added to the culture to daunorubicin. DNA seσuenc? o' t e region containing the dnrK g Sequencing a 2.5 kb DNA segment of the 5.8 kb Sphl fragment in pWHM90l is carried out by subcioninc 0.4 kb Sphl/Xhcl , 0.7 kb Xhol/Ssll, 0.5-kb Sstl/Sall, and 0.8 kb Sai\fXho\ fragments from pWHM902 into Mi3mp18 and -mpi 9 vectors [Yansch-Pεrron et al.. Gene 33:103 (1985)] to obtain both orientations of each DNA segment. DNA sequencing of the resulting four clones is performec as described in the materials and matrices section. The resulting DNA sequence of a 1.6 kb DNA fragment containing the enr.K ger,-. , and the amino acid sequence o; the COMT protein ceduced by analysis of this DNA sequence with the CODON PREFERENCE program described by Deverεux et al. [Nucl. Acids Rεs. 12:387 (1984)]. are shown in Fig. 3. The cr.r.< open reading frame icεntified by CODONFRΞFΞRΞNCE and TRANSLAT analysis [Deveraux et a!.. Nucl. Acids Res. 12:2S7 (1 984)] ccces for the COMT protein. Example 2 BSTITUTE SHEET An approx. 1.6 kb SpΛI/rVuI! DNA fragment containing the entire dnrK " open reading frame along with some flanking sequence (Fig. 3) is subcloned into Sp ή /I and Stnal-dlgested pUC19 [Yansch-Perron 81 βl., Gene 33:103 (1985)] lc give the plasmid pWHM904 (not shown). The following two oligodeoxynucleotloe primers, corresponding to sequences on eithe side of the ctotΛ'-containlng fragment to be amplified, are synthesized with an Applied Biosystems 391 DNA synthesizer according to the manufacturer's directions: x & l B&ΠΆI ∑bs Ndθl - GGG τ r^ ~ QQ-r-Tc.r. AGGAG CAG CATAT?? AC C GCT GAA CCG ACC GTC GCG GCC CGG CCG CAG CAG AT - 3' : Printer II ■ ( SEQ ID NO: 3 ) Sphl Pst l 3' - AC CGC TAG CCT GAC GAG CTC CTC r g '- ^ft fl.t.*_ yyy CCC - 5' : Primer 12 JF ( SEQ ID NO: 4 ) The third poslton of second, third and sixth codons (Indicated by bold face letters) of the dnrK gene is changed by using primer #1 to refieci the most frequently used codon In highly expressed E. coli genes as a means to enhance Ihe expression of the dnrK gene In £ coir. ATG ACC GCT GAA CCG ACC GTC GCG GCC CGG CCG CAG CAGA: Kutated sequence (SEQ A G AC£ GCC GAA CCG ACS GTC GCG GCC CGG CCG CAG CAGA: Wild ty e sequence (SEQ These two primers are used to amplify the dnrK sequence of pWHM904 Irom nucieotldε 205 (the beginning oi the dnrK erf) to 445 cf Fig. 3 by standard melhoBs for the polymerase chain reaction with Streptomyces DNA [for example, ses Guilfoile and Hutchinson, J. Bacterlol. 174:3559 (1 SS2)]. From the amplified product, an 85 t NdeUNco\ fragment is excised and ligatec to a 1.3 kb Λ/ccl rcoRI fragment (obtained from pWHM9C2), containing the remaining σnr ' gena (Figs. 2 & 3), and Λ/tfel/Ec RI-digested pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82:1074 (1985)], which results in tne fusion cf the dnrK orf to the T7 gene 10 promoter of this E. cell expression vector. Competent ceils cf Ξ. coli DKSc are transformed with the ligatec DNA and transformants were screened for pT7-7 with dnrK. The resulting plasmid is designated ?WKM903 (Fig. 4). ^ p^cyion of '• • "? dn r K « n? \r. E. li Competent E. coli cells containing the plasmid pG?l-2 [Tabor and Richardson, Proc. SUBSTITUTE SHEET selected on LB agar (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) containing ampicillin (100 μg/mi) and kanamycin (50 μg/ml) after growth at 30°C. The procedure for preparing competent cells of E. coli containing pGPl -2 is the same as that for any other E. coli strain, except that the cultures are maintained at 30OC instead of 370C. Competent cells of E. coli containing pGPl -2 are prepared from cells grown at 30°C to a OD5 50 of 0.5 to 0.6 in LB medium containing kanamycin. It is very important to maintain strains containing DGP1 -2 at 3Q Q C for routine maintainance and pre-induction growth to avoid over expression of T7 RNA polvmerase which might otherwise result in a mutated plasmid. A single transformant harboring both pGPl -2 and pWHM903 is inoculated into 25 ml ofi 2 X YT medium (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) containing 100 μg/ml ampicillin and 50 μg/mli kanamycin and grown overnight at 30°C with vigorous agitation. The next morning cultures are heat shocked at 42°C for 30 min in a shaking water bath and then transferred back to 30OC. After 90 min further incubation, one ml of the culture is centriluged at 14,000 rpm in a microcentrifuge for 1 min, the supernatant is discarded, and the cell pellet is resuspended in 100 microliters of Laemmli buffer [Laemmli, Nature (London), 227:680 (1970)] and boiled for 5 min. The proteins contained in the boiled sample are analyzed on a 10% SDS- polyacrylamide gel using standard methods [Laemmli, Nature (London), 227:680 (1970)] by comparison with the proteins obtained from the cell extract of E. coli transformed with the pT7- 7 vector that does not contain the dnrK gene. The COMT protein migrates at M r 38,700. Example 3 Conversion of carminomycin to daunorubicin bv a cell containing the COMT protein A single E. coli transformanl harboring both pGPi -2 and pWHM903 was inoculated into 25 ml of 2 X YT medium containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grown overnight at 30°C with vigorous agiiation. The next morning cultures are heat shocked at 4 ©C for 30 min in a shaking waier bath and then transferred back to 30OC after adding 5 μg/ml of carminomycin. The cultures are allowed to grow for additional 90 min, after which the anthracycline metabolites are isolated using standard methods and analysed on HPLC. Comparison of the relative areas of the signal peaks for carminomycin and daunorubicin in the SUBSTITUTE SHEET KPLC chromatogra n indicates that 75 to 80% of the carminomycin added to the culture medium is converted to daunorubicin. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: HUTCHINSON, Charles R. MADDURI, Krishna M. TORTI, Francesca COLOMBO, Anna L. (ii) TITLE OF INVENTION: PROCESS FOR PREPARING DAUNORUBICIN (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Nikaido, Marmelstein, Murray & Oram (B) STREET: 655 Fifteenth Street N.W. Suite 330 (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: U.S.A. (F) ZIP: 20005-5701 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS fD) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 793,873 (B) FILING DATE: 18-NOV-1991 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Chin, Monica F. (B) REGISTRATION NUMBER: P-36,105 (C) REFERENCE/DOCKET NUMBER: 1615-1816CIP (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (202)638-5000 (B) TELEFAX: (202)638-4810 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1632 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 204..1271 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCATGCCGGC AACCGGGCGC CGGTTCTCCG GTGAGCAGAT CCACCTCATC CGCATCGTCG ACGGCAAGAT CCGCGATCAC CGCGACTGGC CCGACTACCT CGGCACCTAC CGCCAGCTCG GCGAGCCCTG GCCCACCCCC GAGGGCTGGC GCCCCTGACC CCCCATCACC CCGCCGACGC CACGACAGGA GCACGGACAC ACC ATG ACA GCC GAA CCG ACG GTC GCG GCC Met Thr Ala Glu Pro Thr Val Ala Ala 1 5 CGG CCG CAG CAG ATC GAC GCC CTC AGG ACC CTG ATC CGC CTC GGA AGC Arg Pro Gin Gin lie Asp Ala Leu Arg Thr Leu lie Arg Leu Gly Ser 10 15 20 25 CTG CAC ACG CCC ATG GTC GTC CGG ACG GCC GCC ACC CTG CGG CTC GTC Leu His Thr Pro Met Val Val Arg Thr Ala Ala Thr Leu Arg Leu Val 30 35 40 GAC CAC ATC CTG GCC GGG GCC CGC ACC GTG AAG GCC CTG GCG GCC AGG Asp His lie Leu Ala Gly Ala Arg Thr Val Lys Ala Leu Ala Ala Arg 45 50 55 ACA GAC ACC CGG CCG GAA GCA CTC CTG CGA CTG ATC CGC CAC CTG GTG Thr Asp Thr Arg Pro Glu Ala Leu Leu Arg Leu lie Arg His Leu Val 60 65 70 GCG ATC GGA CTG CTC GAG GAG GAC GCA CCG GGC GAG TTC GTC CCG ACC Ala lie Gly Leu Leu Glu Glu Asp Ala Pro Gly Glu Phe Val Pro Thr 75 80 85 GAG GTC GGC GAG CTG CTC GCC GAC GAC CAC CCA GCC GCG CAG CGT GCC Glu Val Gly Glu Leu Leu Ala Asp Asp His Pro Ala Ala Gin Arg Ala 90 95 100 105 TGG CAC GAC CTG ACG CAG GCC GTG GCG CGC GCC GAC ATC TCC TTC ACC Trp His Asp Leu Thr Gin Ala Val Ala Arg Ala Asp lie Ser Phe Thr 110 115 120 CGC CTC CCC GAC GCC ATC CGT ACC GGC CGC CCC ACG TAC GAG TCC ATC Arg Leu Pro Asp Ala lie Arg Thr Gly Arg Pro Thr Tyr Glu Ser lie 125 130 135 TAC GGC AAG CCG TTC TAC GAG GAC CTG GCC GGC CGC CCC GAC CTG CGC Tyr Gly Lys Pro Phe Tyr Glu Asp Leu Ala Gly Arg Pro Asp Leu Arg 140 145 150 GCG TCC TTC GAC TCG CTG CTC GCC TGC GAC CAG GAC GTC GCC TTC GAC Ala Ser Phe Asp Ser Leu Leu Ala Cys Asp Gin Asp Val Ala Phe Asp 155 160 165 GCT CCG GCC GCC GCG TAC GAC TGG ACG AAC GTC CGG CAT GTG CTC GAC Ala Pro Ala Ala Ala Tyr Asp Trp Thr Asn Val Arg His Val Leu Asp 170 175 180 185 GTG GGT GGC GGC AAG GGT GGT TTC GCC GCG GCC ATC GCG CGC CGG GCC Val Gly Gly Gly Lys Gly Gly Phe Ala Ala Ala He Ala Arg Arg Ala 190 195 200 CCG CAC GTG TCG GCC ACC GTG CTG GAG ATG GCG GGC ACC GTG GAC ACC Pro His Val Ser Ala Thr Val Leu Glu Met Ala Gly Thr Val Asp Thr 205 210 215 GCC CGC TCC TAC CTG AAG GAC GAG GGC CTC TCC GAC CGT GTC GAC GTC Ala Arg Ser Tyr Leu Lys Asp Glu Gly Leu Ser Asp Arg Val Asp Val 220 225 230 GTC GAG GGG GAC TTC TTC GAG CCG CTG CCC CGC AAG GCG GAC GCG ATC Val Glu Gly Asp Phe Phe Glu Pro Leu Pro Arg Lys-Ala Asp Ala He 235 240 245 ATC CTC TCT TTC GTC CTC CTC AAC TGG CCG GAC CAC GAC GCC GTC CGG He Leu Ser Phe Val Leu Leu Asn Trp Pro Asp His Asp Ala Val Arg 250 255 260 265 ATC CTC ACC CGC TGC GCC GAG GCC CTG GAG CCC GGC GGG CGC ATC CTG He Leu Thr Arg Cys Ala Glu Ala Leu Glu Pro Gly Gly Arg He Leu 270 275 280 ATC CAC GAG CGC GAC GAC CTC CAC GAG AAC TCG TTC AAC GAA CAG TTC He His Glu Arg Asp Asp Leu His Glu Asn Ser Phe Asn Glu Gin Phe 285 290 295 ACA GAG CTC GAT CTG CGG ATG CTG GTC TTC CTC GGC GGT GCC CTG CGC Thr Glu Leu Asp Leu Arg Met Leu Val Phe Leu Gly Gly Ala Leu Arg 300 305 310 ACC CGC GAG AAG TGG GAC GGC CTG GCC GCG TCG GCG GGC CTC GTG GTC Thr Arg Glu Lys Trp Asp Gly Leu Ala Ala Ser Ala Gly Leu Val Val 315 320 325 GAG GAG GTG CGG CAA CTG CCG TCG CCG ACC ATC CCG TAC GAC CTC TCG SUBSTITUTESHEET 16 Glu Glu Val Arg Gin Leu Pro Ser Pro Thr He Pro Tyr Asp Leu Ser 330 335 340 345 CTC CTC GTC CTT GCC CCC GCG GCC ACC GGC GCC TGACACACGA GGTACGGGAA Leu Leu Val Leu Ala Pro Ala Ala Thr Gly Ala 350 355 GGGTTCATCA GCAATGCCGA CACGCATGAT CACCAACGAT GAGGTGACCC TGTGGAGCGA 1 AGGGCTCGGC GATCCGGCCG ACGCCCCGTT GCTCCTGATC GCCGGCGGCA ACCTCTCGGC 1 CAAATCGTGG CCGGACGAGT TCGTCGAACG CCTGGTCGCG GCCGGGCACT TCGTGATCCG 1 CTACGACCAC CGGGACACCG GGCGCTCCTC CCGGTGCGAC TTCGCGCTCC ACCCCTACGG 1 CTTCGACGAG CTGGCCGCCG ACGCGCTGGC CGTCCTGGAC GGCTGGCAGG TCCGCGCCGC 1 CCATGTGGTG GGCATGTCGC TGGGCAACAC CATCGGCCAG C 1 f (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 356 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ( i) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Thr Ala Glu Pro Thr Val Ala Ala Arg Pro Gin Gin He Asp Ala 1 5 10 15 Leu Arg Thr Leu He Arg Leu Gly Ser Leu His Thr Pro Met Val Val 20 25 30 Arg Thr Ala Ala Thr Leu Arg Leu Val Asp His He Leu Ala Gly Ala 35 40 45 Arg Thr Val Lys Ala Leu Ala Ala Arg Thr Asp Thr Arg Pro Glu Ala 50 55 60 Leu Leu Arg Leu He Arg His Leu Val Ala He Gly Leu Leu Glu Glu 65 70 75 80 Asp Ala Pro Gly Glu Phe Val Pro Thr Glu Val Gly Glu Leu Leu Ala 85 90 95 sp Asp His Pro Ala Ala Gin Arg Ala Trp His Asp Leu Thr Gin Ala 100 105 110 SUBSTITUTESHEE Val Ala Arg Ala Asp He Ser Phe Thr Arg Leu Pro Asp Ala He Arg 115 120 125 Thr Gly Arg Pro Thr Tyr Glu Ser He Tyr Gly Lys Pro Phe " Tyr Glu 130 135 140 Asp Leu Ala Gly Arg Pro Asp Leu Arg Ala Ser Phe Asp Ser Leu Leu 145 150 155 160 Ala Cys Asp Gin Asp Val Ala Phe Asp Ala Pro Ala Ala Ala Tyr Asp 165 170 175 Trp Thr Asn Val Arg His Val Leu Asp Val Gly Gly Gly Lys Gly Gly 180 185 190 Phe Ala Ala Ala He Ala Arg Arg Ala Pro His Val Ser Ala Thr Val 195 200 205 Leu Glu Met Ala Gly Thr Val Asp Thr Ala Arg Ser Tyr Leu Lys Asp 210 215 220 Glu Gly Leu Ser Asp Arg Val Asp Val Val Glu Gly Asp Phe Phe Glu 225 230 235 240 Pro Leu Pro Arg Lys Ala Asp Ala He He Leu Ser Phe Val Leu Leu 245 250 255 Asn Trp Pro Asp His Asp Ala Val Arg He Leu Thr Arg Cys Ala Glu 260 265 270 Ala Leu Glu Pro Gly Gly Arg He Leu He His Glu Arg Asp Asp Leu 275 280 285 His Glu Asn Ser Phe Asn Glu Gin Phe Thr Glu Leu Asp Leu Arg Met 290 295 300 Leu Val Phe Leu Gly Gly Ala Leu Arg Thr Arg Glu Lys Trp Asp Gly 305 310 315 320 Leu Ala Ala Ser Ala Gly Leu Val Val Glu Glu Val Arg Gin Leu Pro 325 330 335 Ser Pro Thr He Pro Tyr Asp Leu Ser Leu Leu Val Leu Ala Pro Ala 340 345 350 Ala Thr Gly Ala 355 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 base pairs 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ( i) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGTCTAGAG. GATCCAGGAG CAGCATATGA CCGCTGAACC GACCGTCGCG GCCCGGCCGC AGCAGAT (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACCGCTAGCC TGACGAGCTC CTCCGTACGG ACGTCCCC (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ( i) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGACCGCTG AACCGACCGT CGCGGCCCGG CCGCAGCAGA (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTESHEET (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATGACAGCCG AACCGACGGT CGCGGCCCGG CCGCAGCAGA

Claims: CL-AI S 1. An isolated DNA sequence comprising a gene which codes for carminomycin 4-O- methyltransferse. 2. The DNA sequence of claim 1. comprising all or part of the DNA of Fig. 3. 3. A vector comprising a gene which codes for carminomycin 4-O-methyltraπsferase. 4. The vector of claim 3 which is a plasmid. 5. The plasmid of claim 4 selected from the group consisting of pWH 901. pWHM902 and pWHM903- 6. A transformed host cell comprising a vector which comprises a gene which codes for carminomycin 4-O-methyltransferase. 7. The transformed host cell of claim 6, wherein said vector is a plasmid. 8. The transformed host cell of claim 7, wherein said host cell is selected from the group consisting of E. coli and Streptomyces. 9. The transformed host cell of claim 6, wherein said vector is a plasmid selected from the group consisting of p H 901. pWH 902 and p H 903. 10. A cell extract obtained from cells which have been transformed with a vector which expresses a gene coding for carminomycin 4-O-methyltransferase. 11. A peptide exhibiting carminomycin 4-O-methyltraπsferase activity, comprising all or pa of the amino acid sequence: (SEQ ID NO: 2) 1 MTAEPTVAAR PQQIDALRTL IR GS HTPM WRTAAT RL VDHI AGART 51 VKALAARTDT RPEALLRLIR HLVAIG EE DAPGEFVPTE VGE LADDHP 101 AAQRAWHD T QAVARADISF TR PDAIRTG RPTYESIYGK PFYEDLAGRP 151 DLRASFDSL ACDQDVAFDA PAAAYDWTNV RHV DVGGGK GGFAAAIARR 201 APHVSATVLE MAGTVDTARS Y KDEG SDR VDWEGDFFE P PRKADAII 251 LSFVLLNWPD HDAVRILTRC AEA EPGGRI LIHERDDLHE NSFNEQFTE 30L DLRMLVFLGG ALRTREKWDG LAASAGLWE EVRQ PSPTI P DLS LViA 351 PAATG 12. A process for preparaing a peptide exhibiting carminomycin 4-O-methyltransferasβ activity, comprising a gene coding for carminomycin 4-O-methyltransferase activity in an expression vector, transforming suitable host ceils with the said expression vector, culturing said host cells in a proper medium, allowinα for the expression of said gene thus obtaining 13. A process according to claim 12 wherein said host cells are selected from the group consisting of E. coli and Streptomyces. 14. A process according to claim 12 wherein said vector is a plasmid. 15. A process according to claim 14 wherein said plasmid is selected from the group consisting of pWH 901. pWHM902 and pWH 903. SUBSTITUTE SHEET 16. A process for the production of daunorubicin comprising transforming suitable host cells with a vector comprising a gene which codes for carminomycin 4-O-methyltransferase, culturing said host cells in a proper medium, allowing for the expression of said gene and recovering daunorubicin. 17. A process according to claim 16 wherein said host cells are Streptomyces cells. 18. A process according to claim 16 wherein said vector is a plasmid. 19. A process according to claim 18 wherein said plasmid is selected from the group consisting of pWHM901 , pWH 902 and pWHM903. 20. A process for the conversion of carminomycin into ' daunorubicin through the use of a preparation containing a peptide exhibiting carminomycin 4-O-methyltransferase activity or of a transformed host cell of claim 6. 21. The process of claim 20 wherein said preparation essentially consists of the peptide of claim 1 1 . 22. The process of claim 20 wherein said preparation essentially consists of the cell extract of claim 10. IT- SUBSTITUTE SHEET

Inventors: COLOMBO ANNA LUISA, HUTCHINSON CHARLES RICHARD, MADDURI KRISHNA MURTHY, TORTI FRANCESCA, COLOMBO, ANNA LUISA, HUTCHINSON, CHARLES, RICHARD, MADDURI, KRISHNA, MURTHY, TORTI, FRANCESCA
Applicants: ERBA CARLO SPA, HUTCHINSON CHARLES RICHARD, FARMITALIA CARLO ERBA, S.R.L., HUTCHINSON, CHARLES, RICHARD
Application year: 1992
Main classification: C12P19
IPC codes: C12N1, C12N9, C12N15, C12P19, C12R1