| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | June 19, 1990 |
| PATENT TITLE |
Method of isolating antibiotic biosynthetic genes |
| PATENT ABSTRACT | The present invention is a method for isolating antibiotic biosynthetic genes. To practice the method, an antibiotic resistance-conferring DNA segment is labelled and used as a probe to find, via DNA hydridization, homologous DNA in a genetic library which comprises chromosomal and plasmid DNA of an antibiotic-producing organism. Individual vectors of the genetic library which hybridize to the antibiotic resistance-conferring gene, and which comprise .about.1-45 kb of contiguous DNA from the antibiotic-producing organism, which also comprise an antibiotic biosynthetic gene. The present method is exemplified by using the erythromycin resistance-conferring gene of Streptomyces erythreus to clone the erythromycin biosynthetic pathway from the same organism. The erythromycin biosynthetic pathway isolated with the present method synthesizes erythromycin when introduced into S. lividans TK23. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 7, 1985 |
| PATENT REFERENCES CITED |
Thompson et al; Nature 286: 525, (1980. Isreali-Reches et al; Mol. Gen. Genet. 194: 362, (1984). Grunstein et al; Proc. Natl. Acad. Sci. USA 72: 3961, (1975). Murakami et al; Chem. Abstr. 100: 1505p, (1984), of J. Antibiotic, 36(10), 1305, (1983). Hopwood; in Biochemistry and Genetic Regulation of Commercially Important Antibiotics, 1983, Vining (ed.), Addison-Wesley Publishing Co., Reading, MA, pp. 1-23. Chater, 1984, Microbial Development, Streptomyces Differentiation, R. Locisk and L. Shapiro, eds., Cold Spring Harbor Laboratories, 1984, pp. 89-115. Kirby et al., 1975, Nature 254 (5497):265. Kirby and Hopgood, 1977, J. Gen. Microbiol. 98:239. Gil and Hopwood, 1983, Gene 25:119. Rhodes et al., 1984, Biochem. Soc. Trans. 12:586. Feitelson and Hopwood, 1983, Mol. Gen. Genet. 190:394. Jones and Hopwood, 1984, J. Biol. Chem. 259(22):14151. Malpartida and Hopwood, 1984, Nature 309:462. Murakami et al., 1984, Abstract from the Annual Meeting of the Society of Fermentation Technology of Japan. McGraw-Hill Newswatch, Oct. 7, 1985, at 3. Murakami et al., Mol. Gen. Genet. (1986), 205:42-50. Stanzak et al., Biotechnology (1986), 4:229-232. |
| PATENT CLAIMS |
We claim: 1. A method for identifying and isolating an antibiotic biosynthetic gene linked to an antibiotic resistance-conferring gene, said method comprising identifying an antibiotic biosynthetic gene-containing DNA sequence, said DNA sequence not previously isolated, by hybridizing a labelled antibiotic resistance conferring-gene to said antibiotic biosynthetic gene-containing DNA and isolating said DNA thus identified. 2. The method of claim 1, which comprises: (1) isolating chromosomal and plasmid DNA from an antibiotic-producing organism; (2) preparing a genetic library of said chromosomal and plasmid DNA, such that the recombinant vectors of said library comprise a fragment of said DNA of a size ranging from .about.1-45 kb; (3) hybridizing a labelled antibiotic resistance conferring gene to said library; (4) identifying which recombinant vectors of said library hybridize to said antibiotic resistance-conferring gene; (5) isolating said hybridizing vectors from said library. 3. The method of claim 1, wherein said DNA comprises the genes which encode the enzymes comprising an antibiotic biosynthetic pathway. 4. The method of claim 1, wherein said DNA is isolated from a species of a genus selected from the group of genera consisting of Actinomadura, Actinoplanes, Bacillus, Cephalosporium, Corynebacterium, Dactylosporangium, Micromonospora, Nocardia, Penicillium, Saccharopolyspora, Streptomyces, and Streptoverticillium. 5. The method of claim 4, wherein said genus is Streptomyces and said species is selected from the group consisting of albireticuli, ambofaciens, antibioticus, aureofaciens, cattleya, chartreusis, cinnamonensis, clavuligerus, erythreus, fradiae, hygroscopicus, kanamyceticus, lavendulae, macrosporeus, narbonensis, olivaceus, rimosus, tenebrarius, thermotolerans, toyocoensis, and virginiae. 6. The method of claim 5, wherein said species is erythreus. 7. The method of claim 5, wherein said species is fradiae. 8. The method of claim 1, wherein said antibiotic resistance-conferring gene confers resistance to an antibiotic selected from the group consisting of antibiotics apramycin, .beta.-lactams, erythromycin, hygromycin, kanamycin, methelenomycin, neomycin, spiramycin, tobramycin, thiostrepton, tylosin, and viomycin. 9. The method of claim 1, wherein said antibiotic resistance-conferring gene encodes an enzymatic activity selected from the group consisting of acetyltransferase activity, .beta.-lactamase activity, phosphotransferase activity, and rRNA methyltransferase activity. 10. The method of claim 8, wherein said antibiotic resistance-conferring gene confers resistance to erythromycin. 11. The method of claim 8, wherein said antibiotic resistance-conferring gene confers resistance to tylosin. 12. The method of claim 9, wherein said antibiotic resistance-conferring gene encodes rRNA methyltransferase activity. 13. The .about.34 kb Bg1II fragment of plasmid pKC488. 14. A Streptomyces host cell transformed with plasmid pKC488. 15. The host cell of claim 14 that is Streptomyces lividans/pKC488. 16. A DNA sequence which encodes an enzyme in the erythromycin biosynthetic pathway, said DNA sequence being comprised on plasmid pK488. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
SUMMARY OF THE INVENTION The present invention is a method for isolating antibiotic biosynthetic genes. The present method is most useful when used to isolate antibiotic biosynthetic pathways, which are the entire set of antibiotic biosynthetic genes necessary for the biosynthesis of a particular antibiotic. Antibiotic biosynthetic genes can be used to enhance antibiotic production, to produce novel antibiotics, and to isolate other antibiotic biosynthetic genes. The present method is especially useful in isolating antibiotic biosynthetic genes from Streptomyces, organisms which provide over one-half of the clinically significant antibiotics. Because of the resulting commercial significance of Streptomyces, much time and effort has been expended in the study of Streptomyces genetics and the development of Streptomyces cloning vectors and transformation systems. Consequently, much is now known about Streptomyces, and several cloning vectors exist transforming the organism. However, the methods of recombinant DNA technology have not been readily applied to Streptomyces because of the difficulty of isolation of antibiotic biosynthetic genes. In contrast, the present invention provides a novel method that will have great impact on the production of antibiotics by fermentation. The present method makes use of the linkage of antibiotic resistance-conferring genes with antibiotic biosynthetic genes. Linkage describes the distance between two or more genes. Two genes are closely linked when the two genes are close together on the same chromosome or are present on the same plasmid. The linkage between antibiotic biosynthetic and resistance-conferring genes can be utilized to isolate antibiotic biosynthetic genes for purposes of improving antibiotic-producing organisms or producing novel antibiotics. Prior to the present invention, antibiotic biosynthetic genes were difficult to isolate. Prior art methods required that a diverse set of mutants be constructed from the antibiotic-producing organisms from which antibiotic biosynthetic genes were to be isolated. The mutants, called "blocked mutants," had to be defective for an antibiotic biosynthetic gene. These mutants were difficult to isolate and characterize. Once the mutants were obtained, they were transformed with cloned DNA from genetic libraries, and the transformants were analyzed to determine if the "block" had been complemented by the transforming DNA. The method was time consuming, because each transformant had to be screened for enzyme activity or antibiotic production, as there were no other methods to tell which recombinant vectors of the genetic library had DNA segments that comprised an antibiotic biosynthetic gene. The present invention is a method for using antibiotic resistance-conferring genes to identify segments of DNA that comprise antibiotic biosynthetic genes. Many species that produce antibiotics also produce enzymes that catalyze reactions that confer resistance to those antibiotics. Antibiotic resistance-conferring genes can now be isolated by gene cloning. For instance, cloning DNA from a Streptomyces strain that produces a given antibiotic into a Streptomyces strain normally sensitive to that antibiotic can yield transformants which contain the antibiotic resistance-conferring gene. The clone containing the antibiotic resistance-conferring gene can be isolated by selection for resistance to the antibiotic. One embodiment of the present method consists of isolating antibiotic resistance-conferring genes on large segments of DNA, so that the antibiotic biosynthetic genes linked to the resistance-conferring genes are also cloned. However, not all antibiotic biosynthetic genes from antibiotic-producing organisms can be isolated by the above-described method. Nevertheless, the linkage between antibiotic resistance-conferring genes and antibiotic biosynthetic genes can still be used to isolate antibiotic biosynthetic genes in the majority of cases. Because of the structural similarity that exists between members of a given class of antibiotics, the antibiotic resistance-conferring genes for a given class of antibiotics will often have similar DNA sequence and encode similar enzymatic activity. Because of this similar DNA sequence, also called homology, one antibiotic resistance-conferring gene for an antibiotic of a given class can be used to identify DNA segments comprising another antibiotic resistance-conferring gene for that same class. If the DNA segments are of the proper size, antibiotic biosynthetic genes linked to the resistance-conferring gene will also be identified. The DNA segments can be of any reasonable size, allowing the isolation of a single antibiotic biosynthetic gene with a relatively small segment or the isolation of an entire antibiotic biosynthetic pathway with a relatively large segment. The antibiotic biosynthetic gene-comprising DNA segments can be cloned onto any of the available cloning vectors for a particular organism. Inserting the biosynthetic genes into multi-copy vectors, such as plasmids, and introducing those vectors into the hosts from which the genes were isolated can result in increased antibiotic production. Introducing a vector which comprises an entire antibiotic biosynthetic pathway into hosts which produce no antibiotic, but which recognize the transcriptional and translational control signals of the pathway genes, results in the production of the antibiotic in the transformed host. Introducing the same vector into hosts which produce an antibiotic different from the one produced by the host from which the biosynthetic genes were isolated can result in the production of a novel antibiotic. For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below. AmR - the apramycin resistance-conferring gene. Antibiotic - a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell. Antibiotic Biosynthetic Gene - a DNA segment that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics. Antibiotic Biosynthetic Pathway - the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. Antibiotic-Producing Organism - any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic. Antibiotic Resistance-Conferring Gene - a DNA segment that encodes an enzymatic or other activity which confers resistance to an antibiotic. ApR - the ampicillin resistance-conferring gene. Bifunctional Cloning Shuttle Vector - a recombinant DNA cloning vector which can replicate and/or integrate into organisms of two different genera. Cloning - the process of incorporating a segment of DNA into a recombinant DNA cloning vector and transforming a host cell with the recombinant DNA. cos - the lambda cohesive end sequence. Cosmid - a recombinant DNA cloning vector which not only can replicate in a host cell in the same manner as a plasmid but also can be packaged into phage heads. EryR - the erythromycin resistance-conferring gene. Genetic Library - a set of recombinant DNA cloning vectors into which segments of DNA, comprising substantially all of the DNA of a particular organism, have been cloned. Hybridization - the process of annealing two single-stranded DNA molecules to form a double-stranded DNA molecule, which may or may not be completely base-paired. NmR - the neomycin resistance-conferring gene. ori - a plasmid origin of replication. Recombinant DNA Cloning Vector - any autonomously replicating or integrating agent, including, but not limited to, plasmids, comprising a DNA molecule to which one or more additional DNA molecules can be or have been added. Restriction Fragment - any linear DNA molecule generated by the action of one or more restriction enzymes. rRNA - ribosomal ribonucleic acid. Sensitive Host Cell - a host cell that cannot grow in the presence of a given antibiotic without a DNA segment that confers resistance thereto. Str ori - a Streptomyces plasmid origin of replication. Transductant - a recipient host cell that has undergone transformation by recombinant phage infection. Transformant - a recipient host cell that has undergone transformation. Transformation - the introduction of DNA into a recipient host cell that changes the genotype and results in a change in the recipient cell. BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Restriction Site and Function Map of Plasmid pKC462A. FIG. 2. Restriction Site and Function Map of Plasmid pKC488. DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for isolating antibiotic biosynthetic genes, said method comprising: (1) isolating chromosomal and plasmid DNA from an antibiotic-producing organism; (2) preparing a genetic library of said chromosomal and plasmid DNA, such that each recombinant vector of said library comprises a fragment of said DNA of a size ranging from .about.1-45 kb; (3) hybridizing an antibiotic resistance-conferring gene to said library; (4) identifying which recombinant DNA cloning vectors of said library hybridize to said antibiotic resistance-conferring gene; and (5) isolating said hybridizing vectors from said library. Each of the five steps of the present method are described more fully below. Step one of the present method requires the isolation of chromosomal and plasmid DNA from an antibiotic-producing organism. A variety of methods for isolating Streptomyces chromosomal and plasmid DNA are well known in the art, and one such method is presented in Example 1, below. The method of Example 1 is generally applicable to all antibiotic-producing organisms, although some minor modifications, such as incubation time or reaction volume, may be necessary to optimize the procedure for a particular organism. A variety of antibiotic-producing organisms and the antibiotics which those organisms produce are presented in Tables I-IX below. The present method is applicable to, but not limited to, all of the organisms listed in the Tables. TABLE I ______________________________________ Aminocyclitol Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Bacillus various species various aminocyclitols Micromonospora various species gentamycins Saccharopolyspora various species various aminocyclitols Streptomyces albogriseolus neomycins albus var. metamycinus metamycin aquacanus N-methyl hygromycin B atrofaciens hygromycins bikiniensis streptomycin bluensis var. bluensis bluensomycin canus ribosyl paromamine catenulae catenulin chrestomyceticus aminosidine crystallinus hygromycin A erythrochromogenes streptomycin var. narutoensis eurocidicus A16316-C fradiae hybrimycins and neomycin fradiae var. italicus aminosidine galbus streptomycin griseus streptomycin griseoflavus MA 1267 hofuensis seldomycin complex hygroscopicus hygromycins, leucanicidin, and hygrolidin hygroscopicus forma glebomycin glebosus hygroscopicus var. validamycins limoneus hygroscopicus var. spectinomycin sagamiensis kanamyceticus kanamycin A and B Streptomyces kasuqaensis kasugamycins kasugaspinus kasugamycins lavendulae neomycin lividus lividomycins mashuensis streptomycin microsporeus SF-767 netropsis LL-AM31 noboritoensis hygromycins olivaceus streptomycin olivoreticuli var. destomycin A cellulophilus poolensis streptomycin rameus streptomycin ribosidificus SF733 rimofaciens destomycin A rimosus forma paromomycins and paromomycinus catenulin spectabilis spectinomycin tenebrarius tobramycin and apramycin Streptoverticillium flavopersicus spectinomycin ______________________________________ TABLE II ______________________________________ Ansamycin Antibiotic-Producing Organism Organism Antibiotic ______________________________________ Micromonospora various species various ansamycins Nocardia mediterranei rifamycin Streptomyces collinus ansatrienes and napthomycins diastochromogenes ansatrienes and napthomycins galbus subsp. griseosporeus napthomycin B hygroscopicus herbimycin hygroscopicus var. geldanus geldamycin var. nova nigellus 21-hydroxy-25-demethyl 25-methylthioproto- streptovaricin rishiriensis mycotrienes sp. E/784 actamycin and mycotriene sp. E88 mycotrienes spectabilis streptovaricins tolypophorous tolypomycin ______________________________________ TABLE III ______________________________________ Anthracycline and Quinone Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Streptomyces caespitosus mitomycins A, B, and C coelicolor actinorhodin coeruleorubidicus daunomycin cyaneus ditrisarubicin flavogriseus cyanocycline A galilaeus aclacinomycin A, auramycins, and sulfurmycins lusitanus napthyridinomycin peuceticus daunomycin and adriamycin violochromoqenes arugomycin ______________________________________ TABLE IV ______________________________________ .beta.-Lactam Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Cephalosporium various species various .beta.-lactams Nocardia lactamadurans cephamycin C Penicillium various species various .beta.-lactams Streptomyces antibioticus clavulanic acid argenteolus asparenomycin A, MM 4550, and MM 13902 cattleya thienamycin chartreusis SF 1623 and cephamycin A and B cinnamonensis cephamycin A and B clavuligerus PA-32413-I, cephamycin C, A16886A, clavulanic acid, and other clavams fimbriatus cephamycin A and B flavovirens MM 4550 and MM 13902 flavus MM 4550 and MM 13902 fulvoviridis MM 4550 and MM 13902 griseus cephamycin A and B halstedi cephamycin A and B heteromorphus C2081X and cephamycin A and B hygroscopicus deacetoxycephalosporin C lipmanii penicillin N, 7-methoxyceph alosporin C, A16884, MM 4550, and MM 13902 olivaceus (MM 17880) epithienamycin F, MM 4550, and MM 13902 panayensis C2081X and cephamycin A and B pluracidomyceticus pluracidomycin A rochei cephamycin A and B sioyaensis MM 4550 and MM 13902 sp. OA-6129 OA-6129A sp. KC-6643 carpetimycin A tokunomensis asparenomycin A viridochromogenes cephamycin A and B wadayamensis WS-3442-D ______________________________________ TABLE V ______________________________________ Macrolide, Lincosamide, and Streptogramin Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Micromonospora rosaria rosaramicin Streptomyces albireticuli carbomycin albogriseolus mikonomycin albus albomycetin albus var. coilmyceticus coleimycin ambofaciens spiramycin and foromacidin D antibioticus oleandomycin avermitilis avermectins bikiniensis chalcomycin bruneogriseus albocycline caelestis M188 and celesticetin cinerochromogenes cineromycin B cirratus cirramycin deltae deltamycins djakartensis niddamycin erythreus erythromycins eurocidicus methymycin eurythermus angolamycin fasciculus amaromycin felleus argomycin and picromycin fimbriatus amaromycin flavochromogenes amaromycin and shincomycins fradiae tylosin fungicidicus NA-181 fungicidicus var. espinomycins espinomyceticus furdicidicus mydecamycin goshikiensis bandamycin griseofaciens PA133A and B griseoflavus acumycin griseofuscus bundlin griseolus griseomycin griseospiralis relomycin griseus borrelidin Streptomyces griseus ssp. sulphurus bafilomycins halstedi carbomycin and leucanicidin hygroscopicus tylosin hygroscopicus subsp. aureolacrimosus milbemycins kitastoensis leucomycin A.sub.3 and josamycin lavendulae aldgamycin lincolnensis lincomycin loidensis vernamycin A and B macrosporeus carbomycin maizeus ingramycin mycarofaciens acetyl-leukomycin, and espinomycin narbonensis josamycin and narbomycin narbonensis var. leucomycin A josamyceticus and josamycin olivochromogenes oleandomycin platensis platenomycin rimosus tylosin and neutramycin rochei lankacidin and borrelidin rochei var. T2636 volubilis roseochromogenes albocyline roseocitreus albocycline spinichromoenes var. kujimycins suragaoensis tendae carbomycin thermotolerans carbomycin venezuelae methymycins violaceoniger lankacidins and lankamycin ______________________________________ TABLE VI ______________________________________ Miscellaneous Antibiotic-Producing Streptomyces Antibiotic Type Streptomyces Species Antibiotic ______________________________________ amino acid sp. cycloserine analogues cyclopentane ring- coelicolor methylenomycin A containing erythrochromogenes sarkomycin violaceoruber methylenomycin A nitro-containing venezuelae chloramphenicol polyenes griseus candicidin nodosus amphotericin B noursei nystatin tetracyclines aureofaciens tetracycline, chlor- tetracycline, demethyltetra- cycline, and demethylchlortetra cycline rimosus oxytetracycline ______________________________________ TABLE VII ______________________________________ Nucleoside Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Corynebacterium michiganese pv. rathayi tunicamycin analogues Nocardia candidus pyrazofurin Streptomyces antibioticus ara-A chartreusis tunicamycin griseoflavus var. streptoviridans thuringiensis griseolus sinefungin lysosuperificus tunicamycin ______________________________________ TABLE VIII ______________________________________ Peptide Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Actinoplanes missouriensis actaplanin teichomyceticus teicoplanin Bacillus various species bacitracin, polymixin, and colistin Nocardia candidus A-35512 and avoparcin lurida ristocetin orientalis vancomycin Streptomyces antibioticus actinomycin aureus thiostrepton canus amphomycin eburosporeus LL-AM374 haranomachiensis vancomycin pristinaespiralis pristinamycin roseosporus lipopeptides, such as A21978C toyocaensis A47934 virginiae A1030 ______________________________________ TABLE IX ______________________________________ Polyether Antibiotic-Producing Organism Organism Antibiotic ______________________________________ Actinomadura various species various polyethers Dactylosporangium various species various polyethers Nocardia various species various polyethers Streptomyces albus A204, A28695A and B, and salinomycin aureofaciens narasin cacaoi var. lysocellin asoensis chartreusis A23187 cinnamonensis monensin conglobatus ionomycin eurocidicus var. laidlomycin asterocidicus flaveolus CP38936 gallinarius RP 30504 griseus grisorixin hygroscopicus A218, emericid, DE3936, A120A, A28695A and B, etheromycin, and dianemycin lasaliensis lasalocid longwoodensis lysocellin mutabilis S-1173a ribosidificus lonomycin violaceoniger nigericin Streptoverticillium various species various polyethers ______________________________________ After isolating the chromosomal and plasmid DNA from a particular antibiotic-producing organism, the present method requires the construction of a genetic library of the prepared DNA. There are many methods of preparing genetic libraries, and so long as certain size constraints described herein are met, any of those methods can be used in the present method. A preferred method of constructing a Streptomyces genetic library has been disclosed and claimed in U.S. patent application Ser. No. 06/655,178, filed 9/27/84, Docket No. X-6422 and the continuation-in-part application of Ser. No. 655,178, which is U.S. patent application Ser. No. 06/742,172, filed 6/7/85, Docket No. X-6422A. The aforementioned method is preferred, because very large segments of DNA, .about.30-45 kb, can be incorporated into the cosmid cloning vectors disclosed and claimed in the aforementioned application. Using this preferred method increases the likelihood that an entire antibiotic biosynthetic pathway will be cloned. This preferred method is illustrated in Example 2 below. The various methods of constructing genetic libraries primarily differ with respect to the restriction enzyme and/or molecular linkers used to prepare the chromosomal and plasmid DNA for insertion into the recombinant DNA cloning vector used to construct the library. Ideally, several libraries are constructed from each antibiotic-producing species, with each library constructed with chromosomal and plasmid DNA prepared for insertion into the recombinant DNA cloning vector with a different restriction enzyme and/or molecular linker. Preparing a variety of libraries in this manner will minimize the risk of cutting, and thereby possibly destroying, a particular antibiotic biosynthetic gene during the construction of the library. Genetic libraries also differ on the average size of the chromosomal and plasmid DNA inserted into the recombinant DNA cloning vector used to construct the library. As stated above, larger inserts are desired so that entire biosynthetic pathways can be cloned. One method of controlling the size of a DNA insert is to carefully monitor digestion of large DNA fragments. As a general rule, the longer the digestion time, the smaller the resulting digestion products, until complete digestion is obtained. DNA can also be fragmented by a variety of mechanical, as opposed to enzymatic, techniques. Mechanical methods usually require that the DNA be modified by the addition of DNA linkers to the DNA or by treatment to make the DNA blunt-ended after the fragmentation process. The construction of a genetic library is also governed by the choice of the recombinant DNA cloning vector used to construct the library. The preferred recombinant DNA cloning vector of the present invention is able to replicate in both E. coli and Streptomyces. Such a bifunctional cloning vector, also called a "shuttle vector," allows manipulation of the antibiotic biosynthetic gene-containing DNA in E. coli, an organism eminently suited for the manipulation of DNA, and introduction of the antibiotic biosynthetic gene-containing DNA into Streptomyces without construction of numerous intermediate recombinant DNA cloning vectors. Preferred recombinant DNA cloning vectors for the construction of genetic libraries for use in the method of the present invention are disclosed in the aforementioned U.S. patent application Ser. No. 655,178 and the continuation-in-part application of Ser. No. 655,178, Ser. No. 06/742,172, Docket No. X-6422A these vectors are pKC420, pKC427, pKC428, pKC448, pKC462, pKC462A and pKC467. However, the present method is not limited to the use of a particular recombinant DNA cloning vector. A great number of cloning vectors exist that are suitable for the purposes of the present invention, such as, for example, E. coli-Streptomyces shuttle vectors disclosed in U.S. Pat. Nos. 4,416,994; 4,343,906; 4,477,571; 4,362,816; and 4,340,674. Nor is the present invention limited to the use of bifunctional vectors or to bifunctional vectors of E. coli and Streptomyces, because bifunctional vectors between E. coli and an antibiotic-producing organism such as Bacillus, Cephalosporium, or Penicillium, for example, would be equally useful. The present method can also be executed using mono-functional vectors, such as, for example, plasmid pBR322, the Charon phages, phage .PHI.C3l, plasmid SCP2, plasmid SCP2*, plasmid pIJ702, and derivatives of these vectors. After the genetic library is constructed, segments of DNA known to confer antibiotic resistance are first labelled and then brought into contact with the genetic library under conditions that allow complementary strands of DNA to anneal. This annealing process, or hybridization, is an important step of the present invention and a variety of important factors are critical to successful execution of this step. Because the resistance-conferring gene functions as a probe that seeks out and identifies segments of chromosomal and/or plasmid DNA containing antibiotic biosynthetic genes, the selection of a particular antibiotic resistance-conferring DNA segment is an important factor in the present method. In an especially preferred embodiment of the present invention, the resistance-conferring gene used in the present method confers resistance to the antibiotic produced by the organism from which the chromosomal or plasmid DNA has been isolated. For example, acetyltransferases, adenylyltransferases, phosphotransferases, and methyltransferases can confer resistance to the aminocyclitol antibiotics. Thus, using a DNA segment that encodes an acetyltransferase, adenylyltransferase, phosphotransferase, or methyltransferase in the present method to identify aminocyclitol biosynthetic gene-containing DNA segments is a preferred embodiment of the present invention. Not all aminocyclitol-producing organisms will contain the same type of aminocyclitol resistance-conferring gene. Thus, it may be necessary to use a variety of resistance-conferring genes before successfully isolating an aminocyclitol biosynthetic gene-containing DNA fragment. Macrolide, lincosamide, and streptogramin antibiotic-producing organisms often contain antibiotic resistance-conferring genes such as methyltransferases. In fact, most rRNA methyltransferases confer resistance to a wide variety of macrolide, lincosamide, and streptogramin antibiotics and are therefore called "MLS resistance determinants." Therefore, a preferred embodiment of the present invention comprises the use of a DNA segment encoding a methyltransferase to identify macrolide, lincosamide, and streptogramin biosynthetic gene-containing DNA segments. It is especially important to note that the present method provides a means to isolate antibiotic biosynthetic gene from organisms which have been only poorly characterized genetically. Because antibiotic resistance genes from different species often show homology, and indeed, some scientists believe many organisms possess antibiotic resistance-conferring genes through the action of trans-poson-like elements (Israeli-Reches et al., 1984, Mol. Gen. Genet. 194:362), the present method can be used to isolate antibiotic biosynthetic genes from a species of a genus that is distinct from the genus from which the antibiotic resistance-conferring gene was isolated. No resistance-conferring genes are known to exist for some of the antibiotics listed in the preceding Tables. However, the present invention is not limited to a particular antibiotic resistance-conferring gene. Instead, the present method describes how to use any antibiotic resistance-conferring gene to identify antibiotic biosynthetic genes. Therefore, even should antibiotic resistance-conferring genes be discovered after the filing date of the present invention, the present invention comprises the use of those novel resistance-conferring genes in the present method. Furthermore, the present method can be used to isolate antibiotic biosynthetic genes from organisms which produce antibiotics for which no resistance-conferring gene is known. In such an embodiment of the present invention, the chromosomal and plasmid DNA of the organism is hybridized to resistance-conferring genes specific for antibiotics other than those the organism is known to produce. Some organisms possess silent or cryptic antibiotic biosynthetic genes (Jones et al., 1984, J. Biol. Chem. 259: 14158). These silent genes could not only be linked to the organism's active biosynthetic genes, but also could possess a resistance-conferring gene, specific for the antibiotic that the silent pathway could produce. Furthermore, the unknown resistance-conferring gene for the antibiotic the organism does produce could be homologous to a known resistance-conferring gene. In either case, the present invention can be used to isolate antibiotic biosynthetic genes from organisms that produce antibiotics for which no resistance-conferring gene is known. A great many resistance genes are known. The following table, Table X, presents many, but not all, of the known resistance-conferring genes and provides citations to the references that disclose the genes. Depository accession numbers in Table X refer to either the strain or the plasmid from which the resistance-conferring gene can be isolated. The ATCC is the American Type Culture Collection, located in Rockville, Md. 20852. The NRRL is the Northern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, located in Peoria, Ill. 61604. __________________________________________________________________________ ANTIBIOTIC RESISTANCE-CONFERRING GENES Resistance- Organism Plasmid Depository Conferring Comprising Comprising Accession Reference to Article Describing Gene Gene Gene Number the Resistance-Conferring __________________________________________________________________________ Gene hygromycin B: E. coli JR225 pKC203 ATCC 31912 Kaster et al., 1983, Nucleic Acids phosphotransferase Research 11(19): 6895 kanamycin and neomycin: E. coli/Tn5 pKC7 ATCC 37084 Beck et al., 1982, Gene 19:327 phosphotransferase neomycin: Streptomyces PIJ1 and ATCC 10745 Thompson et al., 1980, Nature 286:525 acetyltransferase fradiae PIJ4 Thompson et al., 1982, Gene 20:51 thiostrepton: rRNA Streptomyces pIJ5 and ATCC 14921 Thompson et al., 1980, Nature 286:525 methylase azureus pIJ6 Thompson et al., 1982, Gene 20:51 kanamycin: Streptomyces pIJ2 and ATCC 10745 Thompson et al., 1980, Nature 286:525 phosphotransferase fradiae pIJ3 Thompson et al., 1982, Gene 20:51 viomycin: Streptomyces pIJ36 No deposit Thompson et al., 1982, Gene 20:51 phosphotransferase vinaceus erythromycin: Streptomyces pIJ43 ATCC 39156 Thompson et al., 1982, Gene 20:51 rRNA methyltrans- erythreus ferase apramycin: E. coli. pKC309 NRRL B-15827 U.S. Pat. application Ser. No. acetyltransferase 06/655,180, filed 09/27/84 tylosin: rRNA Streptomyces pSVB2 NRRL 15880 U.S. Pat. Application Ser. No. methyltransferase fradiae 06/653,975, filed 09/25/84 spiramycin: rRNA Streptomyces pNAS105 NRRL B-15919 U.S. Pat application Ser. No. methyltransferase ambofaciens 06/685,677, filed 12/24/84 methylenomycin Streptomyces pSCP111, No deposit Bibb et al., 1980, Nature 284: 526 coelicolor 19332 pSLP111, and pSLP112 penP: Bacillus pTB2 No deposit Neugebauer et al., 1981, Nucl. Acids .beta.-lactamase licheniformis Res. 9:2577 Gray and Chang, 1981, J. Bacteriol. 145:422 erythromycin: Bacillus pBD90 No deposit Gryczan et al., 1984, Mol. Gen. ermD, a rRNA licheniformis Genet. 194:349 methyltransferase erythromycin: Staphylococcus pEI94 No deposit Shivakumar and Dubnau, 1981, Nucl. ermC, a rRNA aureus methyltransferase __________________________________________________________________________ As stated above, the present invention is not limited to a particular set of antibiotic resistance-conferring genes. Methods of isolating antibiotic resistance-conferring genes are well known in the art. One such method is described in Thompson et al., 1980, Nature 286:525. Methods for isolating antibiotic resistance-conferring genes, such as the method of Thompson et al., usually involve cloning DNA from an antibiotic-producing organism and transforming the cloned DNA into a second organism that is sensitive to the antibiotic the first organism produces. Transformants of the second organism that are resistant to the antibiotic are presumed to comprise an antibiotic resistance-conferring gene. Although the above method is primarily an effective way to clone antibiotic resistance-conferring genes, the method can be adapted, by keeping the cloned sequences large enough so that genes flanking the antibiotic resistance-conferring gene are also cloned on the same DNA segment as the antibiotic resistance-conferring gene to clone antibiotic biosynthetic genes. Therefore, the present method also comprises isolating an antibiotic resistance-conferring gene of an organism in order to obtain the antibiotic biosynthetic genes linked or adjacent to that antibiotic resistance-conferring gene. When using a resistance-conferring gene, such as one of the genes listed in the preceding Table, in the present method, it is desirable to label only the protein-coding sequence, or some subfragment thereof, of the gene to use for identification of an antibiotic biosynthetic gene-comprising DNA segment. Of course, not all of the antibiotic resistance-conferring genes in the preceding Table have been sequenced, so the exact boundaries of the protein-coding sequence of those genes are unknown. However, it is still possible to eliminate any extraneous DNA that is not necessary to confer resistance before using the antibiotic resistance-conferring gene in the present method. The purpose of using only the protein-coding sequence of the antibiotic resistance-conferring gene in the present method is to prevent hybridization of DNA sequences adjacent to the antibiotic resistance-conferring gene from hybridizing with homologous sequences present in the chromosomal or plasmid DNA from which antibiotic biosynthetic genes are to be isolated. Because these flanking sequences might not be specific to antibiotic resistance-conferring genes or antibiotic biosynthetic genes, the presence of these sequences could lead to false positives if the sequences were present during the hybridization step of the present method. However, if the vector on which the antibiotic resistance-conferring gene is present is not homologous to the chromosonal or plasmid DNA from which the antibiotic biosynthetic genes are to be isolated, the presence of those vector sequences will not impair the successful operation of the present method. In order to identify any antibiotic resistance-conferring DNA that hybridizes with the chromosomal or plasmid DNA of an antibiotic-producing organism, the antibiotic resistance-conferring DNA must be labelled. Usually, this labelling of the DNA is done through techniques which cause radioactive molecules, such as radioactive phosphorous (.sup.32 P) or deoxyribonucleotides comprising .sup.32 P, to be incorporated into the DNA. One such method of labelling DNA through the use of radioactive deoxyribonucleotides is presented in Example 3. Another method of labelling DNA involves biotinylation of the DNA; this method is described and marketed by Bethesda Research Laboratories (Gaithersburg, Md. 20877) and works well in the method of the present invention. The present invention is not limited to a particular method of labelling the antibiotic resistance-conferring gene. After the antibiotic resistance-conferring gene has been labelled, the resistance-conferring gene is brought into contact with the chromosomal or plasmid DNA from the antibiotic-producing organism under conditions which allow single strands of the antibiotic resistance-conferring gene DNA to anneal with homologous regions of the single-stranded chromosomal or plasmid DNA of the antibiotic producing organism. This annealing process is called hybridization; the process is described in more detail in Example 4. After the hybridization reaction, the unannealed, labelled DNA is washed from the system. Various parameters of the wash, also described in Example 4, can be adjusted to set the degree of homology required for a labelled DNA to remain annealed. In the method of the present invention, the washes are controlled such that the labelled DNA must be at least 50% homologous to the chromosomal and plasmid DNA of the antibiotic-producing organism before it will not be washed out of the system. The labelled DNA which remains in the system is bound to the chromosomal and plasmid DNA of the antibiotic producing organism which, in turn, is bound to a substrate, usually nitrocellulose. The substrate-bound DNA is produced by culturing the organisms comprising the aforementioned genetic library on a material such as nitrocellulose; lysing the organisms under conditions that denature the DNA of the organisms which becomes bound to the nitrocellulose in single-stranded form; and, finally, treating the nitrocellulose-bound DNA to conditions, such as prolonged heat, that cause the DNA to become covalently bound to the nitrocellulose. In this manner, the DNA of the antibiotic-producing organism becomes bound to a substrate in a single-stranded form such that hybridization with the antibiotic resistance-conferring DNA can readily occur. Other systems for preparing the DNA of the antibiotic-producing organism for hybridization involve a material, such as nylon-based membranes or Whatman chromatography paper, to which the DNA is bound by becoming intertwined with the fibers of which the material is made. Before or during hybridization, however, the DNA must be denatured into single strands, so that the antibiotic resistance-conferring DNA can hybridize with it. The actual system used for immobilizing and denaturing the DNA of the antibiotic-producing organism in no way limits the method of the present invention. A variety of techniques exist or are possible for binding the DNA of a genetic library to a substrate in such a manner that hybridization with a labelled antibiotic resistance-conferring DNA segment can take place so that specific vectors of the genetic library that hybridize can be identified. The method of the present invention is not limited to any particular method or set of methods for accomplishing the hybridization step. The system used for immobilizing and denaturing the DNA of the antibiotic-producing organism must allow for subsequent identification of those recombinant vectors in the genetic library which comprise sequences to which the antibiotic resistance-conferring DNA hybridizes. Usually, this goal is accomplished by culturing the hosts comprising the recombinant vectors of the genetic library on a master plate, which is specially marked or shaped. Replicas of this master plate set of cultures are then made, preferably on the material which will be used to bind the DNA comprised in the genetic library. The replicas are marked such that cultures on the replica plate can be identified with the cultures on the master plate from which they arose; the markings on, or shape of, the master plate are for aiding this purpose. The replica plates are then used for the purposes of the present invention. The master plate is preserved, with its cultures intact and viable, so that after recombinant vectors of the genetic library which comprise antibiotic biosynthetic genes are identified, the organisms comprising those recombinant vectors can be isolated and cultured. Recombinant vectors comprising antibiotic biosynthetic genes are identified in accordance with method used to label the antibiotic resistance-conferring DNA. If a method employing the radioactive labelling of the DNA was used, identification will proceed by measuring the radioactivity preserved on the material which binds the genetic library DNA after the hybridization and washing steps. Radioactivity can be conveniently measured by exposing X-ray film to the material bound to the genetic library DNA (the "filters") after the hybridization and washing steps. Other methods, such as those employing scintillation or geiger counters, can also be employed in the present method. When the biotinylation method is used, the filters are exposed to dyes which allow vectors that hybridized to the antibiotic resistance-conferring gene to be identified by colorimetric means. Once a recombinant vector of a genetic library from an antibiotic-producing organism has been shown to comprise sequences which are homologous to and hybridize with DNA sequences from an antibiotic resistance-conferring gene, it then becomes necessary to identify the antibiotic biosynthetic genes which are linked to the antibiotic resistance-conferring homologous sequences and which are also present on the plasmid. A variety of methods exist for identifying the presence and type of antibiotic biosynthetic genes on a vector; the methods are briefly summarized below. The presence of an antibiotic biosynthetic pathway on a recombinant DNA cloning vector can be determined by introducing the recombinant vector into hosts which do not naturally produce an antibiotic but which recognize the control signals of the pathway genes and replication functions of the recombinant vector. Presence of an antibiotic pathway, or at least a regulator of a cryptic pathway, on the recombinant vector is indicated by the production of an antibiotic by the transformed organism, because the untransformed organism produces no antibiotic. Alternatively, the pathway-comprising recombinant vector could be introduced into an organism that does produce an antibiotic, and presence of a novel antibiotic pathway, or of at least antibiotic biosynthetic genes, would be indicated by the production of an antibiotic not produced by the untransformed organism. The presence of antibiotic biosynthetic genes on a recombinant DNA cloning vector can also be identified by the transformation of antibiotic blocked mutants to wild-type state with the recombinant vector. Furthermore, the presence and type of antibiotic biosynthetic genes can also be determined by enzyme assays, such as those described by Seno and Baltz, 1981, Antimicrobial Agents and Chemotherapy 20(3):370. The Example section, below, is presented to illustrate the method of the present invention. The Examples illustrate the use of the erythromycin resistance-conferring gene, which is thought to encode a ribosomal RNA methyltransferase, of plasmid pIJ43 to identify erythromycin biosynthetic genes in accordance with the method of the present invention. Not only did the erythromycin resistance-conferring gene hybridize, in a specific manner, to the chromosomal and plasmid DNA of several erythromycin or other antibiotic-producing organisms, but also the erythromycin resistance-conferring gene was used in the method of the present invention to identify a recombinant vector shown to encode the antibiotic pathway for erythromycin which drives expression of erythromycins in a host that when untransformed produces no useful antibiotic. Thus, the Examples illustrate the utility of the present invention and the general applicability of the present invention to antibiotic-producing organisms. |
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