| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | March 2, 1993 |
| PATENT TITLE |
Use of the site-specific integrating function of phage .phi.C31 |
| PATENT ABSTRACT | The present invention provides a method for transforming an actinomycete with an integrating vector which has the advantages of high transformation rates into a broad host range, site-specific integration, and stable maintenance without antibiotic selection. Also provided are methods for the increased production of antibiotics and for the production of hybrid antibiotics |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 12, 1989 |
| PATENT REFERENCES CITED |
Balakrishnan and Backman, Gene 67:97-103 (1988). Pernodet et al., Mol. Gen. Genet. 198:35-41 (1984). Kuhstoss et al., J. Bact. 171:16-23 (1989). Rodicio et al., Gene 34:283-292 (1985). Suarez and Chater, Nature 286:527-529 (1980). Chater et al., Gene 15:249-256 (1981). Ow and Ausubel, J. Bact. 155:704-713 (1983). Boccard et al., Mol. Gen. Genet. 212:432-439 (1988). Omer and Cohen, Mol. Gen. Genet. 196:429-438 (1984). Omer and Cohen, J. Bact. 166:999-1006 (1986). Ramaswamy et al. Gene 67:97 (1988). Harris et al. Gene 22:167 (1983). Epp et al. Biol. Actinomycetes '88 (82-85) Abstract 1988. Pouwels et al., Cloning Vectors, Elsevier Sci. Pub. (1985). Charles et al. Gene 19(1):21 (1982). Jones et al. Abstr. Annu. Meet. Am. Soc. Microbiol. (84:101) (1984 Abstract). Cox et al. Lilly Research Labs J Nat Prod. (49, 6, 971-80). 1986 Abstract. Blatz et al. Lilly Res. Lab. (32, 6, Meet., 55-64) 1986. Abstract. |
| PATENT CLAIMS |
We claim: 1. A method for directing integration of a plasmid into a streptomycete genome which comprise the step of introducing into said streptomycete a plasmid comprising a DNA sequence, such DNA sequence containing site-specific integrating functions of phage .phi.C31 subject to the limitation that the plasmid not be capable of directing plaque formation. 2. The method of claim 1, wherein said DNA sequence comprising the site-specific integrating functions of phage .phi.C31 is __________________________________________________________________________ GAC GTC CCG AAG GCG TGG CGC GGC TTC CCC GTG CCG GAG CAA TCG CCC TGG GTG GGT TAC ACG ACG CCC CTC TAT GGC CCG TAC TGA CGG ACA CAC CGA AGC CCC GGC GGC AAC CCT CAG CGG ATG CCC CGG GGC TTC ACG TTT TCC CAG GTC AGA AGC GGT TTT CGG GAG TAG TGC CCC AAC TGG GGT AAC CTT TGA GTT CTC TCA GTT GGG GGC GTA GGG TCG CCG ACA TGA CAC AAG GGG TTG TGA CCG GGG TGG ACA CGT ACG CGG GTG CTT ACG ACC GTC AGT CGC GCG AGC GCG AGA ATT CGA GCG CAG CAA GCC CAG CGA CAC AGC GTA GCG CCA ACG AAG ACA AGG CGG CCG ACC TTC AGC GCG AAG TCG AGC GCG ACG GGG GCC GGT TCA GGT TCG TCG GGC ATT TCA GCG AAG CGC CGG GCA CGT CGG CGT TCG GGA CGG CGG AGC GCC CGG AGT TCG AAC GCA TCC TGA ACG AAT GCC GCG CCG GGC GGC TCA ACA TGA TCA TTG TCT ATG ACG TGT CGC GCT TCT CGC GCC TGA AGG TCA TGG ACG CGA TTC CGA TTG TCT CGG AAT TGC TCG CCC TGG GCG TGA CGA TTG TTT CCA CTC AGG AAG GCG TCT TCC GGC AGG GAA ACG TCA TGG ACC TGA TTC ACC TGA TTA TGC GGC TCG ACG CGT CGC ACA AAG AAT CTT CGC TGA AGT CGG CGA AGA TTC TCG ACA CGA AGA ACC TTC AGC GCG AAT TGG GCG GGT ACG TCG GCG GGA AGG CGC CTT ACG GCT TCG AGC TTG TTT CGG AGA CGA AGG AGA TCA CGC GCA ACG GCC GAA TGG TCA ATG TCG TCA TCA ACA AGC TTG CGC ACT CGA CCA CTC CCC TTA CCG GAC CCT TCG AGT TCG AGC CCG ACG TAA TCC GGT GGT GGT GGC GTG AGA TCA AGA CGC ACA AAC ACC TTC CCT TCA AGC CGG GCA GTC AAG CCG CCA TTC ACC CGG GCA GCA TCA CGG GGC TTT GTA AGC GCA TGG ACG CTG ACG CCG TGC CGA CCC GGG GCG AGA CGA TTG GGA AGA AGA CCG CTT CAA GCG CCT GGG ACC CGG CAA CCG TTA TGC GAA TCC TTC GGG ACC CGC GTA TTG CGG GCT TCG CCG CTG AGG TGA TCT ACA AGA AGA AGC CGG ACG GCA CGC CGA CCA CGA AGA TTG AGG GTT ACC GCA TTC AGC GCG ACC CGA TCA CGC TCC GGC CGG TCG AGC TTG ATT GCG GAC CGA TCA TCG AGC CCG CTG AGT GGT ATG AGC TTC AGG CGT GGT TGG ACG GCA GGG GGC GCG GCA AGG GGC TTT CCC GGG GGC AAG CCA TTC TGT CCG CCA TGG ACA AGC TGT ACT GCG AGT GTG GCG CCG TCA TGA CTT CGA AGC GCG GGG AAG AAT CGA TCA AGG ACT CTT ACC GCT GCC GTC GCC GGA AGG TGG TCG ACC CGT CCG CAC CTG GGC AGC ACG AAG GCA CGT GCA ACG TCA GCA TGG CGG CAC TCG ACA AGT TCG TTG CGG AAC GCA TCT TCA ACA AGA TCA GGC ACG CCG AAG GCG ACG AAG AGA CGT TGG CGC TTC TGT GGG AAG CCG CCC GAC GCT TCG GCA AGC TCA CTG AGG CGC CTG AGA AGA GCG GCG AAC GGG CGA ACC TTG TTG CGG AGC GCG CCG ACG CCC TGA ACG CCC TTG AAG AGC TGT ACG AAG ACC GCG CGG CAG GCG CGT ACG ACG GAC CCG TTG GCA GGA AGC ACT TCC GGA AGC AAC AGG CAG CGC TGA CGC TCC GGC AGC AAG GGG CGG AAG AGC GGC TTG CCG AAC TTG AAG CCG CCG AAG CCC CGA AGC TTC CCC TTG ACC AAT GGT TCC CCG AAG ACG CCG ACG CTG ACC CGA CCG GCC CTA AGT CGT GGT GGG GGC GCG CGT CAG TAG ACG ACA AGC GCG TGT TCG TCG GGC TCT TCG TAG ACA AGA TCG TTG TCA CGA AGT CGA CTA CGG GCA GGG GGC AGG GAA CGC CCA TCG AGA AGC GCG CTT CGA TCA CGT GGG CGA AGC CGC CGA CCG ACG ACG ACG AAG ACG ACG CCC AGG ACG GCA CGG AAG ACG TAG CGG CGT AGC GAG ACA CCC GGG AAG CCT __________________________________________________________________________ wherein A is a deoxyadenyl residue, G is a deoxyguanyl residue, C is a deoxycytidyl residue, and T is a thymidyl residue. 3. The method of claim 1 wherein said plasmid further comprises an E. coli origin of replication. 4. The method of claim 1 wherein said plasmid further comprises an antibiotic resistance gene. 5. The method of claim 1 wherein said plasmid further comprises a multiple cloning site. 6. The method of claim 3 wherein said origin of replication is derived from a pUC plasmid. 7. The method of claim 4 wherein said antibiotic resistance gene confers resistance to apramycin. 8. The method of claim 1 wherein said plasmid is pKC796. 9. The method of claim 1 wherein said plasmid further comprises an antibiotic biosynthetic gene. 10. The method of claim 9 wherein said plasmid comprises a tylosin biosynthetic gene. 11. The method of claim 9 wherein said plasmid comprises a carbomycin biosynthetic gene. 12. The method of claim 10 wherein said plasmid is pSKC50. 13. The method of claim 10 wherein said plasmid is pSKC51. 14. The method of claim 11 wherein said plasmid is pOJ242. 15. The method of claim 11 wherein said plasmid is pOJ243. 16. A plasmid selected from the group consisting of pKC796, pOJ242, pOJ243, pSKC50, and pSKC51. 17. The plasmid of claim 16 that is pKC796. 18. The plasmid of claim 16 that is pOJ242. 19. The plasmid of claim 16 that is pOJ243. 20. The plasmid of claim 16 that is pSKC50. 21. The plasmid of claim 16 that is pSKC51. 22. An streptomycete transformed with a plasmid comprising the site-specific integrating functions of phage .phi.C31 subject to the limitation that the plasmid not be capable of directing plaque formation. 23. The streptomycete of claim 22 transformed with a plasmid selected from the group consisting of pKC796, pOJ242, pOJ243, pSKC50, and pSKC51. 24. The streptomycete of claim 23 transformed with plasmid pKC796. 25. The streptomycete of claim 23 transformed with plasmid pOJ242. 26. The streptomycete of claim 23 transformed with plasmid pOJ243. 27. The streptomycete of claim 23 transformed with plasmid pSKC50. 28. The streptomycete of claim 23 transformed with plasmid pSKC51 |
| PATENT DESCRIPTION |
SUMMARY OF THE INVENTION The in vivo amplification of genes coding for proteins involved in the biosynthesis of antibiotics can serve to increase production of that antibiotic. In actinomycetes, the usual route to this amplification has been via autonomously replicating plasmids. For reviews of Streptomyces cloning systems, see Hopwood et al., in 153 Methods in Enzymology 116 (1987) and Hopwood et al., in 9 The Bacteria 159 (1986). The present invention provides methods for increasing a given gene dosage and for adding heterologous genes that lead to the formation of new products such as hybrid antibiotics. The procedures of the present invention have many advantages over methods involving autonomously replicating plasmids. Plasmids comprising the site-specific integrating function of phage .phi.C31 can be used to permanently integrate copies of the gene of choice into the chromosome of many different hosts. The vectors can transform these hosts at a very high efficiency. Because some of the vectors do not have actinomycete origins of replication, the plasmids cannot exist as autonomously replicating vectors in actinomycete hosts. The plasmids only exist in their integrated form in these hosts. The integrated form is extremely stable which allows the gene copies to be maintained without antibiotic selective pressure. The result is highly beneficial in terms of cost, efficiency, and stability of the fermentation process. The integrating vectors can be used to integrate genes which increase the yield of known products or generate novel products, such as hybrid antibiotics or other novel secondary metabolites. The vector can also be used to integrate antibiotic resistance genes into strains in order to carry out bioconversions with compounds to which the strain is normally sensitive. The resulting transformed hosts and methods of making the antibiotics are within the scope of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention represents a significant advance in the introduction and maintenance of cloned genes in the antibiotic producing streptomycetes and related organisms. The invention is based on the use of an .about.2 kb fragment of actinophage .phi.C31 (Chater et al., Gene 19:21-32 (1982). A vector which comprises this fragment, plasmid pKC796, is available from the NRRL (Northern Regional Research Laboratories, Peoria, Illinois 61604) under the accession number B-18477. The plasmid has been deposited in accordance with the terms of the Budapest Treaty. See FIG. 1 for a restriction map of pKC796. For purposes of the present invention as disclosed and claimed herein, the following terms are as defined below. Antibiotic--a substance produced by a microorganism that, either naturally or with limited chemical modification, inhibits the growth of or kills another microorganism or eukaryotic cell. Antibiotic Biosynthetic Gene--a DNA segment that encodes an enzymatic activity or encodes a product that regulates expression of an enzymatic activity that is necessary for an enzymatic reaction in the process of converting primary metabolites to antibiotic intermediates, which can also possess antibiotic activity, and then to antibiotics. Antibiotic Biosynthetic Pathway--the set of antibiotic biosynthetic genes and biochemical reactions necessary for the process of converting primary metabolites to antibiotic intermediates and then to antibiotics. Antibiotic-Producing Microorganism--any organism, including, but not limited to Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, that either produces an antibiotic or contains genes that, if expressed, would produce an antibiotic. Antibiotic Resistance-Conferring Gene--a DNA segment that encodes an activity that confers resistance to an antibiotic. AmR--the apramycin-resistant phenotype or gene conferring same. ApR--the ampicillin-resistant phenotype or gene conferring same. attP--the attachment site of phage .phi.C31 for integration into the host chromosome. cos site--the lambda cohesive end sequence. Host Cell--an organism, including the viable protoplast thereof, that can be transformed with a recombinant DNA cloning vector. Hybrid Antibiotic--an antibiotic produced when a heterologous antibiotic biosynthetic gene is introduced into an antibiotic producing microorganism, said antibiotic biosynthetic gene encoding an enzyme that is capable of modifying the antibiotic produced by the original host cell. Integrating Vector--a vector which, when transformed into a host cell, does not autonomously replicate within the host cell but rather integrates into the host chromosome by recombination. ori--as used in the Figures herein, an E. coli origin of replication. Recombinant DNA Cloning Vector--any selectable and autonomously replicating or chromosomally integrating agent, including but not limited to plasmids and phages, comprising a DNA molecule to which additional DNA can be or has been added. rep--as used in the Figures herein, a Streptomyces plasmid origin of replication. Restriction Fragment--any linear DNA generated by the action of one or more restriction enzymes. Sensitive Host Cell--a host cell, including the viable protoplast thereof, that cannot grow in the presence of a given antibiotic without a DNA segment that confers resistance thereto. Site-specific integration--the process of integration by a vector into the host chromosome that utilizes specific bacterial (attB) and plasmid or phage (attP) attachment sites and specific recombinational systems (int) coded by plasmid or phage. Transformant--a recipient host cell, including the viable protoplast thereof, that has undergone transformation. Transformation--the introduction of DNA into a recipient host cell, including the viable protoplast thereof, and subsequent maintenance of said DNA that results in a change in the genotype of the recipient cell. tsr--the thiostrepton-resistant phenotype or gene conferring same. DESCRIPTION OF THE FIGURES The restriction site and function maps presented in the Figures are approximate representations of the recombinant DNA vector discussed herein. The spacing of restriction sites on the map is proportional to the actual spacing of the restriction sites on the vector, but observed restriction site distances may vary somewhat from calculated map distances. The maps do not necessarily provide an exhaustive listing of all the cut sites of a given restriction enzyme; therefore, there may be more restriction sites of a given type on the vector than actually shown on the map. FIG. 1 - Restriction Site and Function Map of Plasmid pKC796. FIG. 2 - Restriction Site and Function Map of Plasmid pOJ171. FIG. 3 - Restriction Site and Function Map of Plasmid pOJ242. FIG. 4 - Restriction Site and Function Map of Plasmid pOJ243. FIG. 5 - Restriction Site and Function Map of Plasmid pHJL280. FIG. 6 - Restriction Site and Function Map of Plasmid pSKC50. FIG. 7 - Restriction Site and Function Map of Plasmid pSKC51. The present invention provides plasmid vectors which comprise the site-specific integrating function of the actinomycete phage .phi.C31. The DNA sequence of this region, presented below, was unknown prior to the present invention. Only one strand of the sequence is shown, reading in the 5'.fwdarw.3'0 direction. ##STR1## wherein A is a deoxyadenyl residue, G is a deoxyguanyl residue, C is a deoxycytidyl residue, T is a thymidyl residue, and X is a deoxyadenyl, deoxycytidyl, deoxyguanyl or thymidyl residue. The DNA sequence comprising all the elements of DNA required for site-specific integration is bounded by the AatII restriction site (underlined) beginning at position 279 and the underlined thymidyl nucleotide at position 2369 of the above sequence. Plasmids comprising this sequence transform actinomycetes at extremely high rates. The plasmids are superior to phage cloning vectors comprising this sequence (Suarez, J.E. and Chater, K.F., Nature 286:527-529 (1980)) in that there is less limitation on the amount of DNA which can be cloned into the plasmid. The cloning capacity of phage vectors is limited to the amount of DNA which can be packaged. Those skilled in the art will readily recognize that the variety of vectors which can be created that comprise this fragment is virtually limitless. The only absolute requirement is that the plasmid comprise an origin of replication which functions in the host cell in which constructions are made, such as E. coli or Bacillus. No actinomycete origin of replication is required. In fact, a preferred plasmid comprising the .phi.C31 fragment comprises no actinomycete origin of replication. Other features, such as an antibiotic resistance gene, a multiple cloning site and cos site are useful but not required. A description of the generation and uses of cosmid shuttle vectors can be found in Rao et al., 1987, in Methods in Enzymology, 153:166-198 (R.Wu and L. Grossman, eds. Academic Press, NY). In short, any plasmid which comprises the .phi.C31 fragment and which does not direct the formation of phage plaques is within the scope of this invention. A preferred embodiment of the present invention is plasmid pKC796 (see FIG. 1). The plasmid has an E. coli origin of replication derived from the pUC plasmids (available from Bethesda Research Laboratories, Inc., P.O. Box 577, Gaithersburg, MD 20760) which facilitates plasmid construction. Because plasmid construction in E. coli is so simple and quick compared to constructions carried out in Streptomyces, it is advantageous that all the initial steps can be carried out in E. coli. Only the final product is then transformed into Streptomyces for use in antibiotic production. The vector has no Streptomyces origin of replication, which is a great advantage in an integrating vector. Multiple origins of replication in a chromosome can lead to instability of the construction. Experiments have shown that transformation rates for vectors with a Streptomyces origin of replication and an integrating function are far lower than transformation rates for vectors with either function alone. This result may be due to the instability problem. Plasmid pKC796 also comprises the attachment site (attP) of Streptomyces phage .phi.C31. Chater et al., Gene 19: 21-32 (1982). The site is on an .about.4 kb ClaI-KpnI fragment derived from .phi.C31. The protein(s) recognizing the attP and attB site direct site-specific integration into the chromosome. Once integrated, the construction is extraordinarily stable with virtually no reversion to the natural state. The site-specific nature of the integration facilitates analysis of the integrants. Plasmid pKC796 also comprises the apramycin resistance gene, which is conveniently selectable in both E. coli and Streptomyces. The apramycin selection during the transformation process in Streptomyces ensures that integration has occurred due to the fact that there is no Streptomyces origin of replication on the vector. The apramycin selection can then be removed without fear of loss of the desired phenotype, as the integrated DNA is stable. The vector comprises a multiple cloning site within the lac.alpha.(.beta.-galactosidase) gene. See FIG. 1 for the available cloning sites. An E. coli transformant carrying the pKC796 plasmid with an insert is easily detected as a white colony (as opposed to blue) when grown on media containing Xgal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and IPTG (isopropyl-.beta.-D-thiogalactoside). One of the most important assets of the present invention is the vectors' ability to transform many actinomycete strains at very high rates. The following table shows the results of some representative transformations with plasmid pKC796. TABLE I ______________________________________ Approximate Transformation Strain Frequency (per .mu.g DNA) ______________________________________ S. ambofaciens .gtoreq.10.sup.6 S. griseofuscus .gtoreq.10.sup.6 S. lividans .gtoreq.10.sup.6 S. lipmanii .gtoreq.10.sup.4 S. fradiae .gtoreq.10.sup.3 S. thermotolerans .gtoreq.10.sup.3 Amycolatopsis orientalis .gtoreq.10.sup.2 ______________________________________ The following tables provide a non-exhaustive list of antibiotic-producing microorganisms to which the present invention may be applied. The invention, in some instances, may also be used to generate increased amounts of products or novel products other than antibiotics. TABLE II ______________________________________ 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 neomycins 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 kasugaensis 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 III ______________________________________ Ansamycin Antibiotic-Producing Organisms 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 mycotrienes sp. E88 mycotrienes spectabilis streptovaricins tolypophorous tolypomycin ______________________________________ TABLE IV ______________________________________ 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 violochromogenes arugomycin ______________________________________ TABLE V ______________________________________ .beta.-Lactam Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Nocardia lactamadurans cephamycin C uniformis nocardicin Streptomyces antibioticus clavulanic acid argenteolus asparenomycin A, MM 4550, and MM 13902 cattleya thienamycin chartreusis SF 1623 and cephamycin A and B Streptomyces cinnamonensis cephamycin A and B clavuligerus PA-32413-I, cephamycin C, A16886A, penicillins cephalosporins, 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 and carpetimycin A and B halstedi cephamycin A and B heteromorphus C2081X and cephamycin A and B hygroscopicus deacetoxycephalosporin C lipmanii cephamycin, penicillin N, 7-methoxycephalosporin C, A16884, MM4550, MM13902 olivaceus epithienamycin F, MM 4550, and MM 13902 panayensis C2081X and cephamycin A and B rochei cephamycin A and B sioyaensis MM 4550 and MM 13902 sp. OA-6129 OA-6129A sp. KC-6643 carpetimycin A viridochromogenes cephamycin A and B wadayamensis WS-3442-D ______________________________________ TABLE VI ______________________________________ Macrolide, Lincosamide, and Streptogramin Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Micromonospora rosaria rosaramicin Saccharopolyspora erythraea erythromycins Streptomyces albireticuli carbomycin albogriseolus mikonomycin albus albomycetin albus var. coleimycin coilmyceticus 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 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 griseus ssp. sulphurus bafilomycins halstedi carbomycin and leucanicidin hygroscopicus tylosin hygroscopicus subsp. milbemycins aureolacrimosus 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.sub.3 josamyceticus and josamycin olivochromogenes oleandomycin platensis platenomycin rimosus tylosin and neutramycin rochei lankacidin and borrelidin rochei var. T2636 volubilis roseochromogenes albocycline roseocitreus albocycline spinichromogenes var. kujimycins suragaoensis tendae carbomycin thermotolerans carbomycin venezuelae methymycins violaceoniger lankacidins and lankamycin ______________________________________ TABLE VII ______________________________________ Miscellaneous Antibiotic-Producing Streptomyces Antibiotic Type Streptomyces Species Antibiotic ______________________________________ amino acid sp. cycloserine analogues cyclopentane ring- coelicolor methylenomycin A containing erythrochromogenes sarkomycin kasugaensis aureothricin and thiolutin 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 VIII ______________________________________ Nucleoside Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Corynebacterium michiganese pv. rathayi tunicamycin analogues Nocardia candidus pyrazofurin Streptomyces antibioticus ara-A chartreusis tunicamycin griseoflavus var. thuringiensis streptoviridans griseolus sinefungin lysosuperificus tunicamycin ______________________________________ TABLE IX ______________________________________ Peptide Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Actinoplanes missouriensis actaplanin teichomyceticus teicoplanin 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 A41030 ______________________________________ TABLE X ______________________________________ Polyether Antibiotic-Producing Organism Organism Antibiotic ______________________________________ Actinomadura various species various polyethers oligosporus A80190 Dactylosporangium various species various polyethers Nocardia various species various polyethers Streptomyces albus A204, A28695A and B, and salinomycin aureofaciens narasin bobili A80438 cacaoi var. asoensis lysocellin chartreusis A23187 cinnamonensis monensin conglobatus ionomycin eurocidicus var. asterocidicus laidlomycin flaveolus CP38936 gallinarius RP 30504 griseus grisorixin hygroscopicus A218, emericid, DE3936, A120A, A28695A and B, etheromycin, and dianemycin lasaliensis lasalocid longwoodensis lysocellin mutabilis S-11743a pactum A80438 ribosidificus lonomycin violaceoniger nigericin Streptoverticillium various species polyethers ______________________________________ If it is desired to create integrating vectors with features other than those of pKC796, the .phi.C31 fragment may be obtained from plasmid pKC796, deposited with the NRRL under accession number NRRL B-18477 by a combination of site-specific mutagenesis and restriction enzyme digestion. First, the plasmid may be site-specifically mutagenized in accordance with the method of Adelman et al., DNA 2:183-193 (1983), herein incorporated by reference, using the synthetic DNA fragment TGTTAGGCGCTGAGACGGGCCCACAGCGGGCTTCCTGGGGC. Incorporating this fragment into the plasmid generates the restriction site ApaI at the 3' end of the fragment. The fragment can then be isolated as a AatII-ApaI restriction fragment. In some contexts it may be necessary to insert a promoter functional in actinomycetes at the AatII site. Such promoters are well-known to one skilled in the art. One skilled in the art may then insert the fragment as is into the plasmid of their choice or may adapt the ends with oligonucleotide linkers to insert the fragment at a desired restriction site. One skilled in the art will also recognize that a synthetic DNA fragment comprising any restriction site may be inserted into the plasmid using site-specific mutagenesis and the fragment subsequently isolated by digestion with that restriction enzyme. The integrating vector of the present invention has great utility in many aspects of Streptomyces research and commercialization of Streptomyces fermentation products. Preferred uses are to integrate into the host chromosome extra copies of homologous genes or new copies of heterologous genes. The use is not limited to genes involved in antibiotic production or resistance. For example, genes involved in amino acid production could be integrated into an auxotrophic strain, which would enable the growth of the strain on media not supplemented with the particular amino acid. The salient feature of all such experiments is the subsequent maintenance of the genes without antibiotic selection. One preferred use is exemplified by the integration of the carE gene (described and claimed in U.S. Patent Application No. 07/194,672 filed May 13, 1988) of Streptomyces thermotolerans into the Streptomyces ambofaciens genome. The S. thermotolerans carE gene encodes a 4"-O-isovaleryl acylase which attaches the isovaleryl group of isovaleryl coenzyme A to a mycarose sugar residue of the macrolide antibiotic carbomycin. S. ambofaciens produces the macrolide antibiotic spiramycin and a variety of other spiramycin-related compounds that contain a mycarose residue with a 4"-OH group. S. ambofaciens does not produce a 4"-O-isovaleryl acylase activity. The carE gene can be isolated from plasmid pOJ171, which can be obtained from the NRRL in E. coli K12 SF8 under the accession number NRRL B-18169. A restriction site and function map of pOJ171 is presented in FIG. 2. The .about.2.4 kb BamHI restriction fragment isolated from plasmid pOJ171 is inserted into BamHI-digested pKC796 (NRRL B-18477). The fragment can be inserted in both orientations, yielding plasmids pOJ242 and pOJ243 (see FIGS. 3 and 4, respectively). Both plasmids and the control vector pKC796 were transformed into an S. ambofaciens strain such as NRRL 2420. Transformants were initially selected with the antibiotic apramycin. The plasmids necessarily integrate into the chromosome because no Streptomyces origins of replication are present on the vectors. Due to the stable integration, no subsequent maintenance with apramycin is required. The presence of the carE gene in the S. ambofaciens chromosome causes the strain to produce isovaleryl spiramycin. The S. ambofaciens strains which have integrated the parent pKC796 vector continue to produce spiramycin. This method of producing isovaleryl spiramycin has two major advantages over methods involving the introduction of the carE gene on an autonomously replicating plasmid. First, the transformants can be grown without antibiotic selection, thus decreasing cost and increasing efficiency. The second advantage is that the integrated transformants produce a greater amount of isovaleryl spiramycin than replicating plasmids carrying the same gene because replicating plasmids seem to depress antibiotic production. These advantages apply generally when the integrating vector is used with any gene and are not limited to situations where the carE gene is utilized. The present invention thus provides a method for producing hybrid antibiotics, said method comprising 1) transforming a microorganism that produces an antibiotic with a plasmid vector comprising a DNA sequence which comprises the site-specific integrating functions of phage .phi.C31, said vector also comprising an antibiotic biosynthetic gene that codes for an enzyme or other gene product not previously expressed in said microorganism and that convert said antibiotic to an antibiotic not previously produced by said microorganism and 2) culturing said microorganism transformed with said vector under conditions suitable for producing the hybrid antibiotic. Another preferred use of the integrating vector is to increase the production of an antibiotic by integrating the antibiotic biosynthetic gene(s) into the host chromosome via the vector. When production of the antibiotic is increased, it may also be valuable to integrate extra copies of the respective antibiotic resistance gene(s) to avoid inhibition by the antibiotic. The production of many fermentation products such as tylosin, monensin, and narasin may be improved by this method. Integration of cloned genes using the integrating vector has numerous advantages. Stable maintenance of cloned genes in streptomycete fermentations (which involve many cell generations) in the absence of a selective agent has been a significant problem which is overcome by the present invention. For example, in tylosin production, stable maintenance of the cloned tylF gene enhances the conversion of macrocin to tylosin, providing larger quantities of tylosin. In addition to its stability, the integrative vector has a lesser inhibitory effect on antibiotic production than autonomous plasmids. The use of the integrating vector to stably maintain cloned genes in order to increase antibiotic production is exemplified herein by using the vector and cloned tylosin biosynthetic genes to increase the production of tylosin. See Examples 3-5. Tylosin production strains such as Streptomyces fradiae T1405 accumulate the tylosin precursors demethylmacrocin and macrocin. Fermentation cultures of the strain with extra copies of the tylE and tylF genes integrated into the chromosome produce increased amounts of the tylF-encoded macrocin O-methyltransferase and tylE-encoded demethylmacrocin O-methyltransferase. The result is increased conversion of demethylmacrocin to macrocin and macrocin to tylosin and thus, a greater yield of the desired end product tylosin. The tylE and tylF genes along with other tylosin biosynthetic genes were described in European Patent Publication Ser. No. 0238323, published Sep. 23, 1987. The BamHI fragment comprising the tylF gene is derived from plasmid pHJL280 (available from the NRRL in E. coli K12 HB101 as NRRL B-18043; see FIG. 5). The fragment is then inserted into BamHI-digested pKC796. The resulting plasmids (differing by the orientation of the inserted BamHI fragment) are called pSKC50 and pSKC51. When transformed into the S. fradiae production strain T1405, the integrated vector causes tylosin production to increase to 136% as compared to the production levels of the T1405 control of 100%. The stability of the transformants is demonstrated by the fact that virtually 100% of the colony forming units retain the apramycin resistance phenotype provided by the vector after at least three passages in the absence of selection. The ability to transform tylosin production strains is a feature not found with other site-specific integrative vectors tested. The experiment was attempted with two other Streptomyces site-specific integrating vectors lacking a Streptomyces origin of replication: pIJ4210, which is the Streptomyces coelicolor minicircle cloned into the E. coli plasmid pBR325 (Lydiate et al., Mol. Gen. Genet. 203:79-88 (1986)) and plasmids (Kuhstoss et al., J. Bact. 171:16-23 (1989)) derived from the integrative S. ambofaciens plasmid pSAM2 (Pernodet et al., Mol. Gen. Genet. 198:35-41 (1984)). These plasmids could not be stably introduced into tylosin production strains of S. fradiae. Stable maintenance without antibiotic selection and very high transformation frequencies are the key advantages of an integrating plasmid vector comprising the site-specific integration functions of .phi.C31. The vector comprising cloned tylosin biosynthetic genes can also be used to increase the tylosin production of S. fradiae mutant strains such as GS15 (tylF mutant; NRRL 18058) and GS16 (tylE mutant; available from the American Type Culture Collection, Rockville, MD 20852 under accession number ATCC 31664). The aforementioned plasmids pSKC50 and pSKC51 comprise the tylE and tylF genes. These plasmids can be transformed into the S. fradiae mutant strains. The resulting transformants will produce increased yields of tylosin and be stably maintained. Thus, an important aspect of the present invention is to provide a method for increasing the antibiotic-producing or antibiotic precursor-producing ability of an antibiotic-producing or antibiotic precursor-producing microorganism, said method comprising 1) transforming a microorganism that produces an antibiotic or an antibiotic precursor by means of an antibiotic biosynthetic pathway with an integrating vector comprising a DNA sequence which comprises the site-specific integrating functions of phage .phi.C31, said vector also comprising an antibiotic biosynthetic gene that codes for an enzyme or other gene product that is rate-limiting in said biosynthetic pathway; and 2) culturing said microorganism transformed with said vector under conditions suitable for cell growth, expression of said antibiotic biosynthetic gene, and production of antibiotic or an antibiotic precursor, subject to the limitation that said antibiotic biosynthetic gene selected in step (1) provides for an increase in the antibiotic-producing or antibiotic precursor-producing ability of said microorganism. Illustrative tylosin biosynthetic genes that can be used for purposes of the present invention include, for example, the tylA, tylB, tylC, tylD, tylE, tylF, tylG, tylH, tylI, tylJ, tylK, tylL, and tylM, genes. Of this group, the tylF gene is preferred, because the macrocin O-methyltransferase enzyme encoded thereby appears to be rate-limiting in the tylosin biosynthetic pathway of most tylosin-producing strains. Macrocin accumulates to unacceptable levels under conditions of optimum fermentation of Streptomyces fradiae because of the rate-limiting step catalyzed by the tylF gene product. The tylF enzyme catalyzes the conversion of macrocin to tylosin. Overproduction of the tylF gene product, macrocin O-methyltransferase, results in the more efficient operation of the tylosin biosynthetic pathway as indicated by increased antibiotic yield and lower cost of fermentation. Streptomyces strains can be cultured in a number of ways using any of several different media. Carbohydrate sources that are preferred in a culture medium include, for example, molasses, glucose, dextran, and glycerol, and nitrogen sources include, for example, soy flour, amino acid mixtures, and peptones. Nutrient inorganic salts are also incorporated into the medium and include the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate, and like ions. As is necessary for the growth and development of other microorganisms, essential trace elements are also added. Such trace elements are commonly supplied as impurities incidental to the addition of other constituents of the medium. Streptomyces strains are grown under aerobic culture conditions over a relatively wide pH range of about 6 to 8 at temperatures ranging from about 25.degree. to 37.degree. C. At temperatures of about 34.degree.-37.degree. C., antibiotic production may cease. |
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