| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 22, 1992 |
| PATENT TITLE |
Tylosin biosynthetic genes tylA, tylB and tylI |
| PATENT ABSTRACT | Provided are gene sequences encoding tylosin biosynthetic gene products. In particular, recombinant DNA vectors comprising DNA sequences encoding the tylA, tylB, tylI and tylG activities of Streptomyces fradiae are provided. Also provided are host cells transformed with the noted vectors and a method for increasing the tylosin-producing ability of a tylosin-producing organism |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 30, 1991 |
| PATENT REFERENCES CITED |
Kirby and Hopwood, 1976, J. Gen. Microbiol. 97:1-14. Baltz et al., 1982, Genentics and Biochemistry of Tylosin Production in Trends in Antibiotic Research (eds. Unezawa et al.; published by the Japan Antibiotics Research Association). Feitelson et al., 1983, Mol. Gen. Genet. 190:394-398. Hopwood et al., 1983, Trends in Biotech. 1(2):42-48. Chater and Bruton, 1983, Gene 26:67-78. Malpartida and Hopwood, 1984, Nature 309:462-464. Hopwood et al., 1985, Nature 314:642-644. De Main, 1985, Nature 314:577-578. Seno and Hutchinson, 1986, The biosynthesis of tylosin and erythromycin: model system for studies of the genetics and biochemistry of antibiotic formation, in The Bacteria, A Treatise on Structure and Function, (eds. J. R. Sokatch and L. N. Ornston), vol. IX, Antibiotic-Producing Streptomyces (vol. eds: S. W. Queener and L. E. Day), Academic Press, Inc., Orlando, FL, pp. 231-279. Stonesifer et al., 1986, Mol. Gen. Genet. 202:348-355. Birmingham et al., 1986, Mol. Gen. Genet. 204:532-539. Cox et al., J. Nat. Prod., 49 971 (1986). Fishman et al., PNAS, U.S.A., 84 8248 (1987). Fishman et al., Mar. 1986, Poster Session of the American Society of Microbiologists (ASM) meeting in Washington, D.C. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An isolated DNA sequence which comprises a gene sequence which encodes an activity selected from the group consisting of the tylA, tylB and tylI biosynthetic gene products of Streptomyces fradiae. 2. An isolated DNA sequence as claimed in claim 1 comprising a gene sequence which encodes the tylA gene product of Streptomyces fradiae. 3. An isolated DNA sequence as claimed in claim 1 comprising a gene sequence which encodes the tylB gene product of Streptomyces fradiae. 4. An isolated DNA sequence as claimed in claim 1 comprising a gene sequence which encodes the tylI gene product of Streptomyces fradiae. 5. A DNA sequence as claimed in claim 2 which comprises the .about.9.8 kb EcoRI-BglII fragment of pSET552, the .about.8.6 kb BamHI fragment of pSET555, or the .about.8.6 kb BamHI fragment of pSET556. 6. A DNA sequence as claimed in claim 2 which comprises the .about.6.2 kb EcoRI-PstI fragment of plasmid pSET552. 7. A DNA sequence as claimed in claim 3 which comprises the .about.9.8 kb EcoRI-BglII fragment of pSET552, the .about.8.6 kb BamHI fragment of pSET555, or the .about.8.6 kb BamHI fragment of pSET556. 8. A DNA sequence as claimed in claim 3 which comprises the .about.6.2 kb EcoRI-PstI fragment of plasmid pSET552. 9. A DNA sequence as claimed in claim 4 which comprises the .about.13 kb PstI fragment of pSET551, the .about.9.8 kb EcoRI-BglII fragment of pSET552, the .about.8.6 kb BamHI fragment of pSET555, or the .about.8.6 kb BamHI fragment of pSET556. 10. A DNA sequence as claimed in claim 4 which comprises the .about.1.4 kb EcoRI-PstI fragment of plasmid pSET551, pSET552, pSET555, or pSET556. 11. A recombinant DNA vector which comprises a DNA sequence as claimed in claim 1. 12. A recombinant DNA vector as claimed in claim 11 which is an expression vector. 13. A recombinant DNA vector as claimed in claim 11 which is a plasmid. 14. A plasmid as claimed in claim 13 which is selected from the group consisting of plasmids pSET551, pSET552, pSET555 and pSET556. 15. A host cell transformed with a recombinant vector as claimed in claim 11. 16. A host cell transformed with a recombinant vector as claimed in claim 12. 17. A host cell transformed with a recombinant vector as claimed in claim 13. 18. A host cell transformed with a recombinant vector as claimed in claim 14. 19. A host cell as claimed in claim 15 which is Streptomyces. 20. A host cell as claimed in claim 19 which is Streptomyces fradiae. 21. The Streptomyces fradiae as claimed in claim 20 which is S. fradiae GS14, S. fradiae GS50 or S. fradiae GS77. 22. A host cell as claimed in claim 15 which is E. coli. 23. A host cell as claimed in claim 22 which is E. coli K12 DH5.alpha.. 24. A transformed host cell which is selected from the group consisting of E. coli DH5.alpha./pSET551, E. coil DH5.alpha./pSET552, E. coli DH5.alpha./pSET555 and E. coli DH5.alpha./pSET556. 25. A method for increasing the tylosin-producing ability of a tylosin-producing microorganism, said method comprising 1) transforming with a recombinant DNA vector or portion thereof a microorganism that produces tylosin or a tylosin precursor by means of a biosynthetic pathway, said vector or portion thereof comprising a DNA sequence as claimed in claim 1 that codes for the expression of an activity that is rate limiting in said antibiotic 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 said antibiotic or antibiotic precursor. 26. A method as claimed in claim 25 in which the vector or portion thereof comprises a DNA sequence encoding the tylA gene product. 27. A method as claimed in claim 25 in which the vector or portion thereof comprises a DNA sequence encoding the tylB gene product. 28. A method as claimed in claim 25 in which the vector or portion thereof comprises a DNA sequence encoding the tylI gene product. |
| PATENT DESCRIPTION |
SUMMARY OF THE INVENTION The invention relates to novel DNA sequences that code for antibiotic biosynthetic gene products, recombinant DNA expression vectors, the products encoded by said genes, and transformed microbial host cells. The invention further comprises a novel method for increasing the antibiotic-producing ability of an antibiotic-producing organism. The method involves transforming a microbial host cell with a DNA sequence that codes for the expression of a gene product of the invention. The present invention represents a significant commercial exploitation of recombinant DNA technology in antibiotic-producing organisms such as streptomycetes. Previously, the development and exploitation of recombinant DNA technology has been limited, for the most part, to the expression of specific polypeptides in E. coli and, in some instances, mammalian cells. These advances led to the comparatively simple expression of heterologous gene products such as human insulin A and B chains, human proinsulin, human growth hormone, human protein C, human tissue plasminogen activator, bovine growth hormone, and other compounds of potential therapeutic value. In each case, heterologous gene expression is primarily independent of and does not interact with, take part in, or modulate operative biosynthetic pathways. Recombinant DNA technology now can be applied to improve selected biosynthetic pathways for the expression of increased yields of known or new antibiotics or antibiotic precursors. Most recombinant DNA technology applied to streptomycetes and other antibiotic-producing organisms has been limited to the development of cloning vectors. Early attempts include the disclosures of Reusser U.S. Pat. No. 4,332,898 and Manis et al. U.S. Pat. Nos. 4,273,875; 4,332,900; 4,338,400; and 4,340,674. Transformation of streptomycetes was not disclosed or taught in these early references. Improved vectors showing greater potential for use in antibiotic-producing organisms were disclosed by Fayerman et al. in U.S. Pat. No. 4,513,086; Nakatsukasa et al. in U.S. Pat. Nos. 4,513,085 and 4,416,994; Malin et al. in U.S. Pat. No. 4,468,462; PCT International Application WO/79/01169; Bibb et al., 1980, in Nature 284:526; Thompson et al., 1980, in Nature 286:525; Suarez et al., 1980, in Nature 286:527; Malpartida et al., 1984, in Nature 309:462; Hershberger, 1982, in Ann. Reports on Fermentation Processes, 5:101-126 (G. T. Tsao, ed., Academic Press N.Y.); Hershberger et al., 1983, in Ann. N.Y. Acad. Sci. 413:31-46; and Larson and Hershberger, 1984, in J. Bacteriol. 157:314-317. These improved vectors contain markers that are selectable in streptomycetes, can be used to transform many important Streptomyces strains, and constitute the tools required for conducting more complicated gene cloning experiments. One such experiment is reported by Hopwood et al., 1985, in Nature 314:642. Although Hopwood et al. reported the production of novel hybrid antibiotic pigments, the disclosure does not focus on increasing the antibiotic-producing ability or biosynthetic efficiency of a given host cell but instead describes the transferring of actinorhobin pigment biosynthetic genes from one Streptomyces strain to another. The previously described references provided the background for research leading to studies of the Streptomyces genome. In particular, European Patent Application, EP A 0 238 323 (Publication No. 87302318.8, published Sep. 23, 1987) discloses a small portion of the Streptomyces fradiae genome. This portion of the genome comprises a gene cluster comprising several tylosin biosynthetic genes. This latter work is described in S. E. Fishman et al., Proc. Nat'l. Acad. Sci., U.S.A., 84, 8248 (1987) and K. L. Cox, et al., J. Natural Products, 49 971 (1986). Fishman et al. describe the biosynthetic gene cluster which comprises the tylE, tylD, tylH, tylF, tylJ, tylC, tylK, tylL and tylM biosynthetic genes. Those researchers, however, were not able to define DNA sequences corresponding to the tylA, tylB, tylG and tylI biosynthetic genes. Mutations in these genes block tylactone biosynthesis (tylG), prevent the attachment or biosynthesis of all tylosin sugars (tylA) or just mycaminose (tylB), or block oxidation at the C-20 position of tylactone (tylI). These genes are responsible, therefore, for activities necessary in the early steps of biosynthesis of tylosin. See FIG. 1 in this regard. In contrast to this previous work, the present invention provides an unexpected second biosynthetic gene cluster, physically removed from the cluster described in Fishman, et al. This cluster has been shown to comprise DNA sequences which complement tylG, tylB, tylA, and tylI mutations. Thus, there are provided DNA sequences encoding four tylosin biosynthetic gene products, tylA, tylB, a tylG, and tylI. The invention also provides novel recombinant DNA expression vectors, the gene products of the noted genes, and host cells transformed with vectors comprising these genes. The present invention is particularly useful in that it allows for the commercial application of recombinant DNA technology to streptomycetes and other antibiotic-producing organisms. Because over half of the clinically important antibiotics are produced by streptomycetes, it is especially desirable to develop methods that are applicable to that industrially important group. The present invention provides such methods and allows for the cloning of genes both for increasing the antibiotic-producing ability as well as for the production of new antibiotics and antibiotic precursors in an antibiotic-producing organism. The following terms, as defined below, are used to described the invention. 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 activity, such as an enzymatic activity, or encodes a product that regulates expression of an activity, that is necessary for a reaction in the process of converting primary metabolites to antibiotic intermediates, which also can possess antibiotic activity, and then to antibiotics. Antibiotic Biosynthetic Pathway--the entire 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. ApR--the ampicillin-resistance phenotype or gene conferring same. Host Cell--an organism, including the viable protoplast thereof, that can be transformed with a recombinant DNA cloning vector. Operation of Antibiotic Biosynthetic Pathway--the expression of antibiotic biosynthetic genes and the related biochemical reactions required for the conversion of primary metabolites into antibiotics. Recombinant DNA 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, and which also can include DNA sequences necessary for the expression of the inserted additional DNA. 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, which cannot grow in the presence of a given antibiotic without the presence of a DNA segment that confers resistance to the antibiotic. 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, that changes the genotype of the recipient cell. tsrR--the thiostrepton-resistance phenotype or gene conferring same. DESCRIPTION OF THE FIGURES The plasmid and chromosomal maps depicted in the Figures are drawn approximately to scale. The spacing of restriction sites on the map is proportional to the actual spacing of the restriction sites on the vector, but actual restriction site distances may vary somewhat from calculated distances. The tylosin biosynthetic genes of the invention, although linked, are scattered across an .about.9.8 kb segment of DNA. Restriction site mapping data exists only for a few regions of the tylosin biosynthetic gene-containing DNA fragment. The maps do not necessarily provide an exhaustive listing of all the restriction sites of a given restriction enzyme. FIG. 1--The Tylosin Biosynthetic Pathway. FIG. 2--Restriction Site and Function Map of Plasmid pHJL401. FIG. 3--Restriction Site and Function Map of Plasmid pKC644. FIG. 4--Restriction Site and Function Map of Plasmid pKC668. FIG. 5--Restriction Site and Function Map of Plasmid pSET551. FIG. 6--Restriction Site and Function Map of Plasmid pSET552. FIG. 7--Restriction Site and Function Map of Plasmid pSET55. FIG. 8--Restriction Site and Function Map of Plasmid pSET556. FIG. 9--Genomic Map of S. fradiae Tylosin Biosynthetic Genes. DETAILED DESCRIPTION OF THE INVENTION The invention comprises related antibiotic biosynthetic genes, recombinant DNA cloning vectors, and antibiotic or antibiotic precursor-producing microorganisms transformed with the aforementioned genes and vectors. In particular, the invention provides the previously unknown tylA, tylB, tylG and tylI biosynthetic genes, vectors containing these genes and the polypeptide activity expressed by these genes. Further, the invention relates to the polypeptide products encoded by the individual antibiotic biosynthetic genes of the invention. The present invention also provides a method for increasing the antibiotic-producing ability of an antibiotic-producing microorganism, said method comprising 1) transforming with a recombinant DNA cloning vector or portion thereof a microorganism that produces an antibiotic or antibiotic precursor by means of an antibiotic biosynthetic pathway, said vector or portion thereof comprising an antibiotic biosynthetic gene of the invention that codes for the expression of an enzyme or other gene product that is rate limiting in said antibiotic 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 said antibiotic or antibiotic precursor. The biosynthetic genes of the invention, when inserted into a stably maintained vector in a host cell, preferably a Streptomycete host cell, can produce, upon expression, a higher level of gene product. This in turn may accelerate the steps in the tylosin biosynthetic pathway affected by the resulting activity, ultimately resulting in increased yields of the final antibiotic product. In addition, the introduction of the biosynthetic genes of the invention into other macrolide or similar antibiotic producing organisms can be used to produce novel hybrid antibiotics. For example, as noted earlier, tylB encodes an activity responsible for the biosynthesis or mycaminose. When introduced into, for example, an erythromycin producer, the gene could produce mycaminosyl derivatives of erythromycin. Tables I and II are non-exhaustive lists of macrolide, or similar antibiotic-producing organisms, in which the biosynthetic genes of the invention may be useful. TABLE I ______________________________________ Macrolide, Lincosamide, and Streptogramin Antibiotic-Producing Organisms Organism Antibiotic ______________________________________ Micromonospora rosaria rosaramicin 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 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 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 II ______________________________________ 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 demethylchlor- tetracycline rimosus oxytetracycline ______________________________________ Several Streptomyces fradiae strains are described herein that have mutant tylosin biosynthetic genes and thus make much less or ny tylosin compared to the strain from which they were derived. Table III provides a brief description of these mutant strains. TABLE III ______________________________________ Streptomyces fradiae Mutants Defective in Tylosin Biosynthesis Strain Mutant ATCC* or NRRL Designation Gene Accession No. Deposit Date ______________________________________ GS5 tylG NRRL 18415 Sept. 14, 1988 GS14 (A252.5) tylA NRRL 12188 June 13, 1980 GS50 (A252.6) tylB NRRL 12201 July 10, 1980 GS77 (A252.8) tylI, tylD ATCC 31733 Oct. 16, 1980 ______________________________________ Alternate designations *ATCC is the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, and NRRL is the Agricultural Research Culture Collection of the Northern Regional Research Laboratory, Peoria, IL 61604 These strains were deposited and are maintained in the permanent culture collections of the noted depositories in accordance with the terms of the Budapest Treaty. NRRL 12188, NRRL 12201, and ATCC 31733 are available to the public under the noted accession numbers. All restrictions on the availability of NRRL 18415 will be irrevocably removed upon issuance or publication of the present patent application or its foreign equivalents. Streptomyces fradiae ATCC 31733 and the products produced by this tylI blocked mutant are described in detail in U.S. Pat. No. 4,304,856 (Dec. 8, 1981), incorporated herein by reference. Streptomyces fradiae NRRL 12188 and the product produced by this tylA blocked mutant are described in detail in U.S. Pat. No. 4,366,247 (Dec. 28, 1982), incorporated herein by reference. Further, Streptomyces fradiae NRRL 12201 and the products produced by this tylB mutant are described in U.S. Pat. No. 4,440,857 (Apr. 3, 1984), also incorporated herein by reference. These mutant strains help to verify the presence or absence of the gene sequences of the invention. In particular, using complementation or hybridization studies familiar to those skilled in the art, one can determine whether a given gene sequence of the invention is present on a particular gene fragment. For example, transformation of a Streptomyces fradiae mutant deficient for a particular gene product with an expression vector supplying upon expression the missing polypeptide product should restore normal, or near normal, production of the final end product, for example, tylosin. By way of illustration, one can transform Streptomyces fradiae GS14 (tylA deficient, NRRL 12188) with a gene believed to comprise the tylA DNA sequence. Upon culturing the transformed organism under conditions suitable for cell growth and expression of the transformed gene, GS14 should then produce at least near normal quantities of tylosin, the final product in the biosynthetic pathway. Likewise, similar analyses can be performed using mutant strains deficient in a single tylG, tylB or tylI product if one wants to determine whether the sequence in questions encodes the tylG, tylB, or tylI products, respectively. If the sequence fails to supply the missing activity, the biosynthetic pathway will be "clocked" at the step at which the mutant fails to produce the questioned activity. In the specific case of GS77, a second mutation in the tylD gene exists. Thus, to find tylosin as the final product, the tylD product also must be supplied. The tylD gene is described in European Patent Publication, EPA 0 238 323 (Published Sep. 23, 1987). Under usual circumstances, however, if the sequence in question fails to provide the missing activity and if the host cell contains, for example, only a single mutation, for example, in the tylI gene, upon transformation with the expression vector, O-mycaminosyl-tylactone, as well as the shunt product, 4'-O-mycarosyl-O-mycaminosyltylactone, would tend to accumulate rather than the desired tylosin final product. For the purposes noted above, one skilled in the art will appreciate that the specific mutants noted can be substituted with other mutant strains. In addition, one skilled in the art is familiar with the methods for producing alternate mutant strains. Any mutagenic method, for example, treatment with ultraviolet light, x-rays, gamma rays or N-methyl-N'-nitro-N-nitrosoguanidine, is satisfactory for preparing alternate mutant strains. As long as one knows for what gene the mutant is deficient, the resulting organism can be used to determine the presence or absence of the desired gene in the manner described above. The vectors of the invention provide a means for increasing the efficiency of the tylosin biosynthetic pathway by not only providing a non-defective gene but also by increasing the copy number of the tylA, tylB, tylI and tylG biosynthetic genes in mutant or non-mutant strains and by increasing the intracellular amount of the products specified by these genes. The concentration of available tylG gene product, for example, will thus be increased, resulting in an elevated amount of the activity responsible for synthesizing tylactone, followed by the conversion of tylactone to O-mycaminosyltylactone to tylosin in the tylosin biosynthetic pathway (See FIG. 1). Similarly, the concentration of available tylA, tylB or tylI gene product can be increased, resulting in the production of elevated amounts of the polypeptide activity necessary for driving the corresponding conversion of the tylosin precursors (noted in FIG. 1). In addition, having these genes localized, one skilled in the art will appreciate that it is possible to directly manipulated the antibiotic biosynthetic genes and encoded products. Thus, one skilled in the art will be able to modify, for example, by mutation, deletion, or direct chemical modification or synthesis of the natural gene sequence so as to obtain DNA sequences encoding the same, improved or modified activity. Such modified activities, for example, may produce broader or more specific substrate specificity of the encoded activity and may allow for the production of novel antibiotics in the manner described previously. Likewise, one skilled in the art can modify the biosynthetic genes in a manner which allows for greater throughput or efficiency of the activity even though functionally the modified activity is equivalent to that encoded by the natural sequence. Further, for those activities related to regulatory functioning of the biosynthetic pathway, one skilled in the art is familiar with modification techniques which would allow for greater control over later steps in the biosynthetic pathway. Thus, one could manipulated the pathway and the products produced in any manner desired. These functionally equivalent modified or synthesized genes and gene products, therefore, are meant to be encompassed by the terms "gene", "DNA sequence", or the "polypeptide", "amino acid sequence", "activity" or "product" encoded by such genes. A schematic representation of the tylosin biosynthetic pathway is presented in FIG. 1; each arrow in FIG. 1 represents a conversion step in the biosynthesis of tylosin that is catalyzed by one or more tylosin biosynthetic gene products, as indicated by the gene name(s) located above each arrow. For example, tylG is involved in the synthesis of tylactone, tylA is involved in the attachment or biosynthesis of tylosin sugars whereas tylB is related to the attachment or synthesis, specifically, of mycaminose. Further, tylI is required for the oxidation at the C-20 position. Each genotypic designation may represent a class of genes that contribute to the same phenotype. A number of vectors are used to exemplify the present invention. These vectors comprise one or more tylosin biosynthetic genes and can be obtained from the Northern Regional Research Laboratories (NRRL), Peoria, Ill. 61604. Table IV provides a brief description of each of the plasmids containing the tylosin biosynthetic genes of the present invention. TABLE IV ______________________________________ Plasmids Comprising Tylosin Biosynthetic Genes NRRL Host/Designation Tylosin Gene(s) Accession No. Map ______________________________________ E. coli K12 G, I B-18411 FIG. 5 DH5.alpha./pSET551 E. coli K12 I, A, B B-18412 FIG. 6 DH5.alpha./pSET552 E. coli K12 G, I, A, B B-18413 FIG. 7 DH5.alpha./pSET555 E. coli K12 G, I, A, B B-18414 FIG. 8 DH5.alpha./pSET556 ______________________________________ The tylA, tylB, tylG and tylI genes provided by the present invention are located within a previously unknown tylosin biosynthetic gene cluster approximately 30 kb rightward of the previously described Streptomyces fradiae tylosin biosynthetic gene cluster (See e.g. published European Application, EP A 0 238 323 (Sep. 23, 1987) and Fishman, et al., Proc. Natl. Acad. Sci. U.S.A., 84, 8248 (1987)). This cluster is bounded on the right by a tylosin resistance gene, tlrC, and on the left by repeating sequences, RS.sub.2 and RS.sub.1. See FIG. 9 in this regard. These genes were obtained from a cosmid library of Streptomyces fradiae DNA inserted into cosmid vector pKC462A. Cosmid vector pKC462A has been transformed into host cell E. coli K12 SF-8 and deposited in the NRRL. A sample of this host containing this cosmid can be obtained from the NRRL under the accession number B-15973. Plasmid pKC668 contains a fragment of Streptomyces ambofaciens DNA which complements tylA and tylB mutant strains such as GS14 and GS50. This plasmid served as a hybridization probe to identify homologous Streptomyces fradiae DNA present on the cosmids obtained from the Streptomyces fradiae cosmid library described above. Plasmid pKC668 is generated by digesting cosmid pKC644 with restriction enzyme EcoRI, isolating the resulting .about.10 kb EcoRI restriction fragment, and ligating the fragment with EcoRI-digested plasmid pHJL401 (FIG. 2). Plasmid pHJL401 is available from the NRRL under the accession number NRRL B-18217 and is described in Larson and Hershberger, 1986, Plasmid 15:199-209. Cosmid pKC644 is deposited in E. coli K12 DK22 in the Northern Regional Research Center (NRRL), Peoria, Ill. under the accession number NRRL B-18238. The restriction map of pKC644 is shown in FIG. 3. The .about.10 kb EcoRI fragment, when ligated to digested pHJL401, results in two plasmids differing only in the orientation of the inserted DNA. These plasmids are designated pKC668A and pKC668. The orientation of the .about.10 kb EcoRI restriction fragment can be determined by restriction enzyme analysis familiar to one skilled in the art. Other fragments, including overlapping restriction fragments, from the S. fradiae cosmid library were used to determine by complementation the presence of of the tylG, tylI, tylA and tylB gene sequences. For example one cosmid vector designated AUD 8-2 contains an .about.13 mb PstI fragment which, when subcloned into pHJL401, complements tylosin mutant strains GS5 and GS77. Likewise, cosmid vector tlrC 8-6 contains an .about.8.6 BamHI fragment which when subcloned into pHJL401, is found to complement upon transformation GS5, GS14, GS50 and GS77 thereby confirming the presence of the tylG, tylA, tylB and tylI genes, respectively. Similarly, an .about.9.8 kb EcoRI-BglII fragment of plasmid tlrC 4-3.24, when inserted in pHJL401, is found to complement the tylI, tylA and tylB mutants previously described. As noted, plasmid pHJL401 (NRRL B-18217) is a shuttle vector containing a replicon specifying a moderate plasmid copy number in streptomycetes as well as being able to replicate in E. coli. Plasmid pHJL401 contains a polylinker sequence located between the HindIII and EcoRI sites in the lacZ region of the plasmid (FIG. 2). This polylinker provides unique insertion sites for HindIII, PstI, XbaI, BamHI, XmaI, SmaI, SacI and EcoRI. These sites are useful for "sticky-end" ligations for the tylosin biosynthetic gene fragments noted above. In particular, the .about.13 kb PstI fragment of AUD 8-2, when inserted into PstI digested pHJL401 produces plasmid pSET551, (NRRL B-18411), the restriction map of which is shown in FIG. 5. Likewise, insertion of the .about.9.8 kb EcoRI-BglII fragment of tlrC 4-3.24 into a EcoRI-BamHI digested pHJL401 results in plasmid pSET552 (NRRL B-18412), the restriction map of which is shown in FIG. 6. Also, the .about.8.6 kb BamHI fragment of tlrC 8-6 when inserted into BamHI digested pHJL401 results in plasmids pSET555 (NRRL B-18413) and pSET556 (NRRL B-18414), the restriction maps of which are shown in FIGS. 7 and 8, respectively. Further complementation with overlapping sequences and restriction site analyses would indicate that the tylG biosynthetic gene is comprised on an .about.4.2 kb BamHI-EcoRI fragment of pSET555, pSET551 or pSET556. In addition, the tylI gene is contained on an .about.1.4 kb EcoRI-PstI fragment of pSET555, pSET556, pSET551 or pSET552. Although the orientations of the tylA and tylB biosynthetic genes are unclear, as shown in FIG. 9, these genes can be isolated on an .about.6.2 kb EcoRI-PstI restriction fragment of pSET552. As one skilled in the art will appreciate, appropriate routine restriction enzyme treatment of the indicated vectors will produce fragments containing the biosynthetic genes of the invention. The tyl gene-containing fragments noted above can be ligated into other vectors to make other useful vectors. Such other vectors may include, for example, those vectors disclosed in U.S. Pat. Nos. 4,468,462; 4,513,086; 4,416,994; 4,503,155; and 4,513,185; and also plasmids pIJ101, pIJ350, pIJ702 (ATCC 39155), SCP2* (NRRL 15041), pOJ160 (NRRL B-18088), pHJL192, pHJL197, pHJL198, pHJL210, pHJL211, pHJL400, pHJL302, pIJ922, pIJ903, pIJ941, pIJ940, and pIJ916. These vectors replicate and are stably maintained in Stretomyces fradiae and other tylosin-producing strains and, therefore, are useful for cloning the present antibiotic biosynthetic genes. Likewise, as discussed below, if integration of the vector into the genome is desired, a variety of techniques are available. Particularly useful integrative vectors may include, for example, derivatives of .phi.C31 (Chater, et al., Gene, 26, 67 (1983); Methods of Microbiology, Ch. 4, (1981)). One such vector is phasmid pKC331 which can be obtained from E. coli K12 BE447/pKC331 (NRRL B-15828). Likewise, other integrative vectors comprise vectors such as the S. coelicolor minicircle (for example, pIJ4210) [See, for example, Lydiate, et al., Proc. of the 5th Intl. Symposium on the Genetics of Industrial Microorganisms pp. 49-56 (1986); Lydiate, et al., Mol. Gen., Genet. 203, 79 (1986)] or derivatives of pSAM2 See, for example, Pernodet, et al., Mol. Gen. Genet. 198, 35 (1984); Simonet, et al., Gene 59 137 (1987). Illustrative host strains for the vectors noted above may include, for example, S. fradiae, S. fradiae GS5, S. fradiae GS14, S. fradiae GS50, S. fradiae GS77, S. coelicolor, S. lividans, S. thermotolerans, and S. ambofaciens. Preferably integrative vectors derived from .phi.C31 or pSAM2 are transformed into host strains S. ambofaciens, S. coelicolor, S. lividans or S. fradiae. The preferred Streptomyces host strains for the pHJL401 derived vectors are S. fradiae GS5, S. fradiae GS14, S. fradiae GS50 or S. fradiae GS77. Vectors derived from the S. coelicolor minicircle are preferably transformed into host strains S. lividans, S. coelicolor, or S. fradiae. Other representative Streptomyces host strains may include, for example, S. rimosus and S. hygroscopicus. Streptomyces hygroscopicus and S. rimosus are well known, having been deposited at the American Type Culture Collection (ATCC), Rockville, Md. 20852. A number of strains of S. hygroscopicus can be obtained under the accession numbers ATCC 27438, ATCC 21449, ATCC 15484, ATCC 19040, and ATCC 15420, and s. rimosus can be obtained under the accession number ATCC 10970. Streptomyces fradiae is also an especially well known microorganism and several strains are available, on an unrestricted basis, from the Northern Regional Research Laboratory (NRRL), Peoria, Ill. 61604 and the ATCC under the accession numbers NRRL 2702, NRRL 2703, and ATCC 19609. Streptomyces ambofaciens, also well-known, is available from the ATCC under the accession numbers ATCC 15154 and ATCC 23877, or from the NRRL under the accession number NRRL 2420. Likewise, strains of S. coelicolor are available from the ATCC under the accession numbers ATCC 3355, ATCC 10147, ATCC 13405, ATCC 19832 or ATCC 21666. S. lividans is available from the ATCC under the accession number ATCC 19844. Finally, S. thermotolerans is available from the ATCC under the accession number ATCC 11416. As noted, the vectors of the present invention can increase the antibiotic-producing ability of an antibiotic-producing organism by providing higher levels, as compared to an untransformed organism, of an enzyme or other gene product or activity that is rate-limiting in an antibiotic biosynthetic pathway. However, plasmid maintenance in an antibiotic-producing host cell sometimes requires significant expenditures of the cell's energy, energy that might otherwise be used to produce antibiotic. Thus, certain microorganisms transformed with autonomously replicating vectors actually show a decrease in antibiotic-producing ability, even though the same vectors can increase the antibiotic-producing ability of other organisms. The synthesis of antibiotics is also believed to be a dispensable function in antibiotic-producing organisms, for mutants blocked in the biosynthesis of antibiotics are viable and grow as well as the antibiotic-producing parent. Wild-type strains produce a relatively small amount of antibiotic, which is apparently adequate to provide the organism with a selective advantage. The development of industrial antibiotic producing strains from natural isolates involves many cycles of mutation and selection for higher antibiotic production. Because the synthesis of antibiotics drains primary metabolites and cellular energy away from growth and maintenance functions, selection for higher antibiotic production frequency occurs at the expense of the vitality of the organism. Thus, the generation of high antibiotic-producing strains involves finely balancing the cells nutritional and energy resources between growth-maintenance functions and antibiotic production. As a consequence of this fine-tuning, high-yielding production strains tend to be extremely sensitive to factors that affect cellular physiology. For example, introduction of autonomously-replicating vectors, notably multicopy plasmids, sometimes tends to decrease the antibiotic-producing ability of an organism that normally produces antibiotics at high levels. The mechanism of this inhibition is not clear, but it is through to occur at an early step in the biosynthesis of the antibiotic, because measurable levels of antibiotic precursors do not accumulate under these conditions. In addition, autonomously replicating vectors may drain pools of precursors for DNA or RNA synthesis or, in high copy number, may titrate DNA binding proteins, such as RNA polymerase, DNA polymerase, polymerase activators, or repressors of gene expression. Another frequent limitation of autonomously replicating vectors is spontaneous loss. Spontaneous loss is especially problematical when the vector reduces growth rate, as frequently occurs. Selection for a resistance marker on the plasmid can ensure the growth of homogeneous, plasmid-containing populations but can also disrupt the physiological balance of an antibiotic fermentation. Selection for unstable plasmids operates by killing or inhibiting the bacteria that lose the plasmid and can result in a reduced growth rate. The negative effect, sometimes observed, of autonomously replicating vectors on the antibiotic-producing ability of a microorganism is greatest in high-producing strains that are delicately balanced with respect to growth-maintenance functions and antibiotic production. The problem of the negative effect of autonomous plasmid replication on high-producing strains can be overcome by methods of culturing the transformed host cell to facilitate identification of transformed cells containing integrated plasmid and, in addition, by providing vectors with features that also facilitate detection of integration. Selecting a culturing procedure that results in integration is important in improving the antibiotic-producing ability of highly selected and conventionally improved antibiotic-producing organisms. Organisms or strains that have a low antibiotic-producing ability can be improved by transformation via either integration or autonomous vector replication. As those skilled in the art of fermentation technology will appreciate, the greatest improvement in antibiotic-producing ability is shown when the present invention is applied to low antibiotic-producing strains. Therefore, if desired, integration of plasmid DNA is readily accomplished by transforming, according to standard transformation procedures, with a vector which is either segregationally unstable or which is unable to replicate in the strain, a given antibiotic-producing strain or mutant thereof, selecting or otherwise identifying the transformants, and then culturing the cells under conditions that do not require the presence of plasmid DNA sequences for the host cell to grow and replicate. After several generations under non-selective conditions, certain cells will no longer contain free plasmid DNA. By selecting for or otherwise identifying plasmid DNA sequences present in the host cell, one can identify host cells in which the plasmid DNA has integrated into the chromosomal (genomic) DNA of the cell. This culturing technique to obtain integration of vector DNA is especially useful when used in conjunction with a vector that is inherently unstable in the transformed host cell, so that culturing without selective pressure to maintain the vector generates segregants that are free of the plasmid. Bibb et al., 1980, Nature 384:526-531, described a DNA sequence needed for stable inheritance of a vector, and a variety of vectors have been constructed that lack this stability sequence. For instance, cloning vector pHJL401 (NRRL B-18217), which was used to construct the plasmids of the invention, lacks this stability sequence. As used, "unstable" refers to plasmids that are lost at high frequency by transformed cells only when those cells are cultured in the absence of selective pressure for plasmid maintenance. Normally plasmids such as pHJL401 are quite stable when selective pressure is applied to the transformed host cell. When host cells transformed with stable vectors are cultured in the absence of selective pressure, the vector is not lost with the high frequency observed with unstable vectors, and identification of integrants is made difficult by the great number of cells that still contain autonomously replicating plasmid even after growth under nonselective conditions. Selection for integrants is more fully described below. Once the vector DNA has integrated into the chromosomal DNA of the host cell, one observes the maximum increase in antibiotic-producing ability for that host cell, because inhibition by autonomously replicating plasmids no longer occurs. Integration of vectors containing cloned genes into the genome of the producing organism can be achieved in a number of ways. One way is to use a lysogenic bacteriophage or other phage vector that can integrate into the genome of the host strain. Another approach is to use a plasmid vector carrying the cloned genes and to screen for integration of the recombinant plasmid into the host genome by a single recombination event between the cloned sequence and the homologous chromosomal sequence. Integration frequency of a vector can be dramatically increased by adding DNA homologous to the genomic DNA of the host cell to the vector. As used, "integration" refers both to a single recombination event, known as Campbell-type recombination, and also to a double-crossover event, which results in exchange of genetic information between the vector and the chromosome. With double-crossover recombination, only a portion of the vector integrates into the chromosomal DNA. For example, a plasmid carrying cloned tylosin biosynthetic genes (tyl) could integrate into the Streptomyces fradiae genome by a single crossover between the tyl genes on the plasmid and the homologous tyl genes in the genome. Another option would be to put a non-tyl S. fradiae DNA sequence on the plasmid in addition to the cloned tyl genes and to screen for integration at the locus corresponding to the non-tyl sequence. The latter approach avoids the possible mutagenic effects of integration into the tyl sequences, but if double-crossover recombination is desired, the vector should comprise the antibiotic biosynthetic genes flanked by separate sequences of homologous DNA. To avoid the potentially adverse effects, however remote, of a recombinant plasmid (either autonomously replicating or integrated) on tylosin production, one can make use of the ability of Streptomyces fradiae to take up tylosin precursors from the culture medium and convert them to tylosin. Thus, one can develop specific strains of S. fradiae containing multiple copies of the present biosynthetic genes and high enzyme levels to act as converters of accumulated precursors to tylosin. These converter strains can be used in several different ways: (1) the converter strain can be co-inoculated into the fermentor with the normal production strain at a low ratio of converter:producer; (2) the converter strain can be introduced into a production fermentation culture late in the cycle to convert intermediates; (3) the converter strain can be kept in a separate "reactor", to which the fermentation production broth from the producer strain would be added; or (4) the converter strain can be immobilized on a column, and fermentation broth from the producer strain passed through. Those skilled in the art will recognize that having separate production and converting populations eliminates the adverse effects that recombinant plasmids sometimes have on antibiotic production in high antibiotic-producing strains. Separate populations also eliminate vector stability problems, because the converting strains can be grown in small vessels in which antibiotic selection or some other selection means for maintenance of the plasmid can be carefully regulated and controlled. In essence, the converting strain is a source of enzymes, and the production of these enzymes at high level can be approached in much the same way as production of proteins from recombinant plasmids in E. coli. Normally, antibiotic production is only increased when the transforming DNA comprises a gene, the expression of which enhances the activity of the rate-limiting product of the untransformed strain. Various methods for determining the rate-limiting step in the biosynthesis of an antibiotic are known in the art (Seno and Baltz, 1982, Antimicrobial Agents and Chemotherapy 21:758-763), but there is no need to identify the rate-limiting step when the entire set of antibiotic biosynthetic genes are available for introduction into the antibiotic-producing strain. If a rate-limiting enzyme is not known, the antibiotic-producing strain is transformed with the entire set of antibiotic biosynthetic genes, thus ensuring that, no matter what enzyme is rate-limiting, the transformed host cell will have higher levels of the rate-limiting enzyme than the untransformed host cell. Often, however, the rate-limiting enzyme of an antibiotic biosynthesis pathway will be known, and the genes of the invention can be used to increase the antibiotic-producing ability of the organism by transforming the organism with a vector that encodes the rate-limiting antibiotic biosynthetic enzyme. The recombinant plasmids described in the present invention each comprise one or more antibiotic biosynthetic genes. Unless part of a polycistron, an antibiotic biosynthetic gene normally comprise: (1) a promoter that directs transcription of the gene; (2) a sequence that, when transcribed into mRNA, directs translation of the transcript ("translational activating sequence"); (3) a protein-coding sequence; and (4) a transcription terminator. Each of these elements is independently useful and can, through the techniques of recombinant DNA technology, be used to form recombinant genes of great variety. As one example, the protein-coding sequence for the tylG gene can be linked to the promoter, translation-activating sequence, and transcription-terminating sequence from a non-Streptomyces fradiae gene to form a recombinant gene that functions in the host from which the non-S. fradiae sequences were isolated. Such a novel gene could be used to produce a hybrid antibiotic if introduced into an organism that produced an antibiotic or antibiotic intermediate that is not found in the tylosin pathway but which could serve as a substrate for the novel gene product. Similarly, the promoter and other regulatory elements of the tylG gene could be linked to the coding sequence of a non-tylosin antibiotic biosynthetic gene to prepare a hybrid gene that would function in S. fradiae. Thus, the individual elements of each of the antibiotic biosynthetic genes on each of the plasmids of the invention comprise an important component of the present invention. That is, the promoter, translational activating sequence, protein-encoding sequence and transcription termination sequences, of the biosynthetic genes of the invention, individually, comprise important aspects of the invention. Streptomyces fradiae strains can be cultured in a number of ways using any of several different medium. 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. S. fradiae strains are grown under aerobic culture conditions over a relative wide pH range of about 6 to 8 at temperatures ranging from about 25.degree. to 34.degree. C. The following non-limiting examples further illustrate and describe the invention. The invention is not limited in scope by reason of any of the following Examples. Sources of reagents are provided merely for convenience and in no way limit the invention. |
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