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Product USA. E. No. 1

PATENT NUMBER This data is not available for free
PATENT GRANT DATE July 14, 1987
PATENT TITLE Method of selecting recombinant DNA-containing streptomyces

PATENT ABSTRACT A novel method of selecting Streptomyces recombinant DNA-containing host cells and vectors useful in the method are described. The vectors confer tylosin resistance to sensitive Streptomyces host cells and thus provide a convenient method of selecting Streptomyces transformants. The novel tylosin resistance-conferring gene described can be isolated on an .about.2.6 kb KpnI restriction fragment from plasmid pSVB2. Plasmid pSVB2 can be isolated from Streptomyces lividans TK23/pSVB2 (NRRL 15880
PATENT INVENTORS This data is not available for free
PATENT ASSIGNEE This data is not available for free
PATENT FILE DATE September 25, 1984
PATENT REFERENCES CITED Katz, et al., Journal of General Microbiology (1983) 129:2703.
Benveniste and Davies, 1973, Proceedings of the National Academy of Sciences, USA 70(8):2276.
Thompson et al., 1980, Nature 286:525.
Thompson et al., 1982, Gene 20:51.
PATENT CLAIMS We claim:

1. A method for selecting a recombinant DNA-containing Streptomyces host cell, said method comprising:

(a) transforming a tylosin-sensitive, restrictionless Streptomyces host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Streptomyces host cell, said vector comprising a DNA sequence that confers resistance to tylosin, and

(b) culturing said transformed cell under growth conditions suitable for selection for tylosin resistance,

subject to the limitation that said host cell is susceptible to transformation, cell division and culture.

2. The method of claim 1 wherein the recombinant DNA cloning vector is a plasmid.

3. The method of claim 1 wherein the recombinant DNA cloning vector is a phage.

4. The method of claim 2 wherein the plasmid is selected from the group consisting of pSVB2, pSVB12, pSVB23, pSVB16, pSVB18,pSVB20 and pSVB22.

5. The method of claim 2 wherein the plasmid is pSVB2.

6. The method of claim 2 wherein the plasmid is pSVB12.

7. The method of claim 2 wherein the plasmid is pSVB23.

8. The method of claim 2 wherein the plasmid is pSVB16.

9. The method of claim 2 wherein the plasmid is pSVB18.

10. The method of claim 2 wherein the plasmid is pSVB20.

11. The method of claim 2 wherein the plasmid is pSVB22.

12. The method of claim 3 wherein the phage is selected from the group consisting of pSVB3310 and pSVB3311.

13. The method of claim 1 wherein the transformed Streptomyces host cell is selected from the group consisting of Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces griseofuscus, Streptomyces lividans, Streptomyces cinnamonensis and Streptomyces toyocaenisis.

14. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB2.

15. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB12.

16. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB23.

17. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB16.

18. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB18.

19. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB20.

20. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB22.

21. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB2.

22. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB12.

23. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB23.

24. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB16.

25. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB18.

26. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB20.

27. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB22.

28. A recombinant DNA cloning vector capable of autonomous replication or integration in a Streptomyces host cell, said vector comprising a DNA sequence that confers resistance to tylosin in a tylosin-sensitive Streptomyces host cell.

29. A vector claim 28 which is selected from the group consisting of plasmids pSVB2, pSVB12, pSVB23, pSVB16, pSVB18, pSVB20 and pSVB22; and phages pSVB3310 and pSVB3311.

30. The vector of claim 29 that is plasmid pSVB2.

31. The vector of claim 29 that is plasmid pSVB12.

32. The vector of claim 29 that is plasmid pSVB23.

33. The vector of claim 29 that is plasmid pSVB16.

34. The vector of claim 29 that is plasmid pSVB18.

35. The vector of claim 29 that is plasmid pSVB20.

36. The vector of claim 29 that is plasmid pSVB22.

37. The vector of claim 29 that is plasmid pSVB3310.

38. A Streptomyces host cell transformed by a vector of claim 28, said Streptomyces host cell being tylosin-sensitive when not transformed with said vector.

39. The Streptomyces host cell of claim 38 transformed by a vector from the group consisting of plasmids pSVB2, L pSVB12, pSVB23, pSVB16, pSVB18, pSVB20 and pSVB22; and phage pSVB3310 and pSVB3311.

40. The transformed Streptomyces host cell of claim 39 that is Streptomyces ambofaciens.

41. The transformed Streptomyces host cell of claim 39 that is Streptomyces lividans.

42. The transformant of claim 40 that is Streptomyces ambofaciens/pSVB2.

43. The transformant of claim 41 that is Streptomyces lividans/pSVB3310.

44. A method for selecting a recombinant DNA-containing Nocardia host cell, said method comprising:

(a) transforming a tylosin-sensitive, restrictionless Nocardia host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Nocardia host cell, said vector comprising a DNA sequence that confers resistance to tylosin, and

(b) culturing said transformed cell under growth conditions suitable for selection for tylosin resistance,

subject to the limitation that said host cell is susceptible to transformation, cell division and culture.

45. The method of claim 44 wherein the recombinant DNA cloning vector is a plasmid.

46. A Nocardia host cell transformed by a recombinant DNA cloning vector capable of autonomous replication or integration in said Nocardia host cell, said vector comprising a DNA sequence that confers resistance to tylosin in a tylosin-sensitive Nocardia host cell, said Nocardia host cell being tylosin-sensitive when not transformed with said vector.
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PATENT DESCRIPTION SUMMARY OF THE INVENTION

The present invention is a method for selecting a recombinant DNA-containing Streptomyces host cell. The invention further comprises recombinant DNA cloning vectors and transformants useful in executing the method.

The present method provides tylosin resistance-conferring cloning vectors for use in Streptomyces. The development and exploitation of recombinant DNA technology in Streptomyces is dependent upon the availability of selectable genetic markers on suitable cloning vectors. This development has been somewhat retarded by the low number of selectable markers presently available for use in Streptomyces. The present invention is useful and especially important in that it expands the number of selectable markers suitable for such use.

The vectors of the present method are particularly useful because they are small, versatile and can be transformed and selected in any Streptomyces cell that is sensitive to tylosin. Streptomyces provides over half of the clinically important antibiotics and thus is a commercially significant group. The present invention provides new and useful cloning systems and vectors for this industrially important group and allows for the cloning of genes both for increasing the yields of known antibiotics, as well as for the production of new antibiotics and antibiotic derivatives.

The present invention further provides a method of selecting Streptomyces transformants from a background of untransformed cells. The method allows one to add non-selectable DNA to the present vectors, transform Streptomyces with the modified vectors and select tylosin-resistant transformants containing this otherwise non-selectable DNA. Since transformation is a very low frequency event, such a functional test is a practical necessity for determining which cell(s), of among the millions of cells, has acquired the transforming DNA.

For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.

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 segments can be or have been added.

Transformation--the introduction of DNA into a recipient host cell that changes the genotype and results in a change in the recipient cell.

Transfectant--a recipient host cell that has undergone transformation by phage DNA.

Transformant--a recipient host cell that has undergone transformation.

Sensitive Host Cell--a host cell that cannot grow in the presence of a given antibiotic without a DNA segment that provides resistance thereto.

Restriction Fragment--any linear DNA molecule generated by the action of one or more restriction enzymes.

Phasmid--a recombinant DNA vector that may act as a phage or as a plasmid.

Ap.sup.R --the ampicillin-resistant phenotype

tsr.sup.R --the thiostrepton-resistant phenotype

tyl.sup.R --the tylosin-resistant phenotype

Tc.sup.R --the tetracycline-resistant phenotype

mel--the tyrosinase gene

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for selecting a recombinant DNA-containing Streptomyces host cell, said method comprising:

(1) transforming a tylosin-sensitive, restrictionless Streptomyces host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Streptomyces host cell, said vector comprising a DNA sequence that confers resistance to tylosin, and

(2) culturing said transformed cell under conditions suitable for selection for tylosin resistance, subject to the limitation that said host cell is susceptible to transformation, cell division and culture. The present invention further comprises the vectors and transformants used to practice the aforementioned method.

The present method for selecting Streptomyces transformants by the tylosin-resistant phenotype is best exemplified by transforming Streptomyces lividans TK23 with plasmid pSVB2 and selecting the resulting transformants on tylosin-containing media. Plasmid pSVB2 comprises a novel tylosin resistance-conferring gene that was isolated from Streptomyces fradiae and cloned into plasmid pIJ702. Plasmid pSVB2 can be obtained from Streptomyces lividans TK23/pSVB2, a strain deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Agricultural Research Service, 1815 North University Street, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL 15880. A restriction site and function map of plasmid pSVB2 is presented in FIG. 1 of the accompanying drawings.

As shown in FIG. 1, the .about.2.6 kb KpnI restriction fragment of plasmid pSVB2 contains the entire tylosin resistance-conferring gene of the present invention. Knowing the location of the tylosin resistance-conferring gene allows for construction of other cloning vectors also useful in the present method. Thus, plasmids pSVB12 and pSVB23 were constructed by inserting the .about.2.6 kb tylosin resistance-conferring KpnI restriction fragment into the KpnI site of plasmid pIJ702. The two resultant plasmids, pSVB12 and pSVB23, differ only with respect to the orientation of the inserted fragment. The plasmid pIJ702 starting material can be obtained from Streptomyces lividans/pIJ702, a strain deposited and made part of the permanent stock culture collection of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, under the accession number ATCC 39155. Restriction site and function maps of plasmids pIJ702, pSVB12 and pSVB23 are respectively presented in FIGS. 2, 3 and 4 of the accompanying drawings.

Plasmids pSVB16 and pSVB18 were constructed by inserting the .about.3.8 kb PstI restriction fragment of plasmid pSVB2 into the PstI site of plasmid pIJ702. Again, two plasmids resulted because of the two possible orientations of the inserted fragment. Plasmids pSVB16 and pSVB18 are useful in the present method, because the inserted fragment contains the .about.2.6 kb KpnI tylosin resistance-conferring restriction fragment of plasmid pSVB2. Restriction site and function maps of plasmids pSVB16 and pSVB18 are respectively presented in FIGS. 5 and 6 of the accompanying drawings.

Additional illustrative plasmids were constructed by inserting the .about.2.9 kb BamHI-BglII restriction fragment of plasmid pSVB2 into the BglII site of plasmid pIJ702, thus inactivating the tyrosinase gene present in plasmid pIJ702. Although the resultant plasmids, designated as pSVB20 and pSVB22, differ with respect to the orientation of the inserted fragment, both confer tylosin resistance to tylosin-sensitive Streptomyces host cells. Restriction site and function maps of plasmids pSVB20 and pSVB22 are respectively presented in FIGS. 7 and 8 of the accompanying drawings.

Restriction fragments used to construct vectors illustrative of the present invention can be conventionally modified to facilitate ligation. For example, molecular linkers can be provided to a particular tylosin resistance gene-containing restriction fragment or to DNA comprising replication or integration functions. Thus, specific sites for subsequent ligation can be constructed conveniently. In addition, the various tylosin resistance gene-containing restriction fragments, origin of replication or integration sequences can be modified by adding, eliminating or substituting certain nucleotides to alter characteristics and to provide a variety of restriction sites for ligation of DNA. Those skilled in the art understand nucleotide chemistry and the genetic code and thus which nucleotides are interchangeable and which DNA modifications are desirable for a specific purpose. It is also noteworthy that the .about.2.6 kb KpnI tylosin resistance gene-containing restriction fragment is not limited to a particular position on a cloning vector, as long as the critical vector-controlled functions are not disrupted. Those skilled in the art understand or can readily determine which sites on a vector are advantageous for the ligation or insertion of a particular tylosin resistance gene-containing restriction fragment.

Although the above-described vectors comprise the Streptomyces replicon derived from plasmid pIJ702, a variety of known Streptomyces replicons can be used to construct similar vectors. Table 1 is an illustrative, but not comprehensive, listing of Streptomyces plasmids from which additional functional Streptomyces replicons can be obtained. Those skilled in the art recognize that all or part of the plasmids may be used to construct vectors exemplifying the present invention so long as the replicon function is not disrupted. the plasmid-containing host and depository accession number are also listed in Table 1.


TABLE 1
______________________________________
Streptomyces Plasmids
Accession
Plasmid Host Number
______________________________________
SCP2 Streptomyces coelicolor A3 (2)
NRRL* 15042
SCP2* Streptomyces coelicolor M110
NRRL 15041
pEL7 Streptomyces ambofaciens/pEL7
NRRL 12523
pUC6 Streptomyces espinosus
NRRL 11439
pUC3 Streptomyces 3022A NRRL 11441
SLP1 Streptomyces lividans
NCIB** 11417
pNM100 Streptomyces virginiae
NRRL 15156
pEL103 Streptomyces granuloruber
NRRL 12549
A39912.13/pEL103
______________________________________
*Agricultural Research Culture Collection (NRRL), 1815 North University
Street, Peoria, Illinois 61604, United States of America
**National Collection of Industrial Bacteria (NCIB), Torry Research
Station, Post Office Box 31, 135 Abbey Road, Aberdeen AB98DG, Scotland,
United Kingdom



Phage .phi.C31 is a well-known Streptomyces phage that is an excellent source of starting material for constructing integrative tylosin resistance-conferring vectors that further exemplify the present invention. A derivative of phage .phi.C31, phasmid pKC331, is especially preferred for constructing such integrating vectors and can be obtained from E. coli K12 BE447/pKC331, a strain deposited and made part of the permanent stock culture collection of the aforementioned Northern Regional Research Laboratory under the accession number NRRL B-15828. Ligation of the .about.37 kb PstI restriction fragment of phasmid pKC331 to the .about.3.8 kb tylosin resistance-conferring PstI restriction fragment of plasmid pSVB2 results in the derivative phages pSVB3310 and pSVB3311. These phages are integrative vectors which confer tylosin resistance to Streptomyces and thus further exemplify the present invention. Restriction site and function maps of phages pSVB3310 and pSVB3311 are respectively presented in FIGS. 9 and 10 of the accompanying drawings.

The vectors of the present invention comprise a Streptomyces replicon and a tylosin resistance-conferring restriction fragment. Because amplification and manipulation of plasmids is done faster and more efficiently in E. coli than in Streptomyces, it is convenient to add DNA sequences that also allow for replication in E. coli. Thus, the additions of functional replicon-containing and antibiotic resistance-conferring restriction fragments from E. coli plasmids such as, for example, pBR322, pACYC184, pBR325, pBR328 and the like are highly advantageous and add to the general utility of the present illustrative vectors.

The vectors used in the present method confer tylosin resistance to tylosin-sensitive Streptomyces or related host cells. Although 10 .mu.g/ml of tylosin is generally toxic to tylosin-sensitive Streptomyces, vectors of the present invention confer resistance to levels approaching 10 mg/ml of tylosin. The preferred tylosin concentration for purposes of selection, however, is about 50 .mu.g/ml for Streptomyces griseofuscus and 500 .mu.g/ml for S. lividans. The preferred tylosin concentration for purposes of selection for other Streptomyces species is readily determined by procedures well-known in the art. While all embodiments of the present invention are useful, some of the recombinant DNA cloning vectors and transformants are preferred. Accordingly, preferred vectors and transformants are listed in Table 2.


TABLE 2
______________________________________
Preferred Vectors and Transformants
Vector Transformant
______________________________________
pSVB2 Streptomyces lividans
pSVB2 Streptomyces griseofuscus
pSVB12 Streptomyces lividans
pSVB12 Streptomyces griseofuscus
pSVB23 Streptomyces lividans
pSVB23 Streptomyces griseofuscus
pSVB16 Streptomyces lividans
pSVB16 Streptomyces griseofuscus
pSVB18 Streptomyces lividans
pSVB18 Streptomyces griseofuscus
pSVB20 Streptomyces lividans
pSVB20 Streptomyces griseofuscus
pSVB22 Streptomyces lividans
pSVB22 Streptomyces griseofuscus
______________________________________



The method and recombinant DNA cloning vectors of the present invention are not limited for use in a single species or strain of Streptomyces. To the contrary, the method and the vectors are broadly applicable and can be used with tylosin-sensitive host cells of many Streptomyces taxa, particularly restrictionless strains of economically important taxa that produce antibiotics such as aminoglycoside, macrolide, .beta.-lactam, polyether and glycopeptide antibiotics. Such restrictionless strains are readily selected and isolated from Streptomyces taxa by conventional procedures well-known in the art (Lomovskaya et al., 1980, Microbiological Reviews 44:206). Host cells of restrictionless strains lack restriction enzymes and, therefore, do not cut or degrade plasmid DNA upon transformation. For purposes of the present application, host cells containing restriction enzymes that do not cut any of the restriction sites of the present vectors are also considered restrictionless.

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa that produce aminoglycoside antibiotics, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: S. kanamyceticus (kanamycins), S. chrestomyceticus (aminosidine), S. griseoflavus (antibiotic MA 1267), S. microsporeus (antibiotic SF-767), S, ribosidificus (antibiotic SF733), S. flavopersicus (spectinomycin), S. spectabilis (actinospectacin), S. rimosus forma paromomycinus (paromomycins, catenulin), S fradiae var. italicus (aminosidine), S. bluensis var. bluensis (bluensomycin), S. catenulae (catenulin), S. olivoreticuli var. cellulophilus (destomycin A), S. lavendulae (neomycin), S. albogriseolus (neomycins), S. tenebrarius (tobramycin, apramycin), S. albus var. metamycinus (metamycin), S. hygroscopicus var. sagamiensis (spectinomycin), S. bikiniensis (streptomycin), S. griseus (streptomycin), S. erythrochromogenes var. narutoensis (streptomycin), S. poolensis (streptomycin), S. galbus (streptomycin), S. rameus (streptomycin), S. olivaceus (streptomycin), S. mashuensis (streptomycin), S. hygroscopicus var. limoneus (validamycins), S. rimofaciens (destomycins), S. hygroscopicus forma glebosus (glebomycin), S. fradiae (hybrimycins neomycins), S. eurocidicus (antibiotic A16316-C), S. aquacanus (N-methyl hygromycin B), S. crystallinus (hygromycin A), S. noboritoensis (hygromycin), S. hygroscopicus (hygromycins), S. atrofaciens (hygromycin), S. kasugaspinus (kasugamycins), S. kasugaensis (kasugamycins), S. netropsis (antibiotic LL-AM31), S. lividus (lividomycins), S. hofuensis (seldomycin complex) and S. canus (ribosyl paromamine).

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa that produce macrolide antibiotics, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: S. caelestis (antibiotic M188), S. platensis (platenomycin), S. rochei var. volubilis (antibiotic T2636), S. venezuelae (methymycins), S. griseofuscus (bundlin), S. narbonensis (josamycin, narbomycin), S. fungicidicus (antibiotic NA-181), S. griseofaciens (antibiotic PA133A, B), S. roseocitreus (albocycline), S. bruneogriseus (albocycline), S, roseochromogenes (albocycline), S. cinerochromogenes (cineromycin B), S. albus (albomycetin), S. felleus (argomycin, picromycin), S. rochei (lankacidin, borrelidin), S. violaceoniger (lankacidin), S. griseus (borrelidin), S. maizeus (ingramycin), S. albus var. coilmyceticus (coleimycin), S. mycarofaciens (acetyl-leukomycin, espinomycin) S. griseospiralis (relomycin), S. lavendulae (aldgamycin), S. rimosus (neutramycin), S. deltae (deltamycins), S. fungicidicus var. espinomyceticus (espinomycins), S. furdicidicus (mydecamycin), S. ambofaciens (spiramycin, foromacidin D), S. eurocidicus (methymycin), S. griseolus (griseomycin), S. flavochromogenes (amaromycin, shincomycins), S. fimbriatus (amaromycin), S. fasciculus (amaromycin), S. erythreus (erythromycins), S. antibioticus (oleandomycin), S. olivochromogenes (oleandomycin), S. spinichromogenes var. suragaoensis (kujimycins), S. kitasatoensis (leucomycin), S. narbonensis var. josamyceticus (leucomycin A3, josamycin), S. albogriseolus (mikonomycin), S. bikiniensis (chalcomycin), S. cirratus (cirramycin), S. djakartensis (niddamycin), S. eurythermus (angolamycin), S. goshikiensis (bandamycin), S. griseoflavus (acumycin), S. halstedii (carbomycin), S. tendae (carbomycin), S. macrosporeus (carbomycin), S. thermotolerans (carbomycin), and S. albireticuli (carbomycin).

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa that produce .beta.-lactam antibiotics, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: S. lipmanii (A16884, MM4550, MM13902), S. clavuligerus (A16886B, clavulanic acid), S. lactamdurans (cephamycin C), S. griseus (cephamycin A, B), S. hygroscopicus (deacetoxycephalosporin C), S. wadayamensis (WS-3442-D), S. chartreusis (SF 1623), S. heteromorphus and S. panayensis (C2081X); S. cinnamonensis, S. fimbriatus, S. halstedii, S. rochei and S. viridochromogenes (cephamycins A, B); S. cattleya (thienamycin); and S. olivaceus, S. flavovirens, S. flavus, S. fulvoviridis, S. argenteolus and S. sioyaensis (MM 4550 and MM 13902).

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa that produce polyether antibiotics, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: S. albus (A204, A28695A and B, salinomycin), S. hygroscopicus (A218, emericid, DE3936), A120A, A28695A and B, etheromycin, dianemycin), S. griseus (grisorixin), S. conglobatus (ionomycin), S. eurocidicus var. asterocidicus (laidlomycin), S. lasaliensis (lasalocid), S. ribosidificus (lonomycin), S. cacaoi var. asoensis (lysocellin), S. cinnamonensis (monensin), S. aureofaciens (narasin), S. gallinarius (RP 30504), S. longwoodensis (lysocellin), S. flaveolus (CP38936), S. mutabilis (S-11743a) and S. violaceoniger (nigericin).

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa or related genera such as, for example, Nocardia that produce glycopeptide antibiotics, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: Nocardia orientalis and S. haranomachiensis (vancomycin); Nocardia candidus (A-35512, avoparcin), S. eburosporeus (LL-AM 374), S. virginiae (A41030) and S. toyocaensis (A47934).

Preferred host cells of restrictionless strains of tylosin-sensitive Streptomyces taxa, and in which the present method is especially useful and the present vectors can be transformed, include restrictionless cells of, for example: S. coelicolor, S. granuloruber, S. roseosporus, S. lividans, S. tenebrarius, S. acrimycins, S. glaucescens, S. parvilin, S. pristinaespiralis, S. violaceoruber, S. vinaceus, S. espinosus, S. azureus, S. griseofuscus, S. fradiae, S. ambofaciens and S. toyocaensis.

The method and recombinant DNA cloning vectors of the present invention have broad utility and help fill the need for suitable cloning vehicles for use in Streptomyces and related organisms. Moreover, the ability of the present vectors to confer tylosin resistance provides a functional means for selecting transformants. This is important because of the practical necessity for determining and selecting the particular cells that have acquired vector DNA.

Additional DNA segments, that lack functional tests for their presence, can also be inserted onto the present vectors, and then transformants containing the non-selectable DNA can be isolated by tylosin selection. Such non-selectable DNA segments can be inserted at any site, except within regions necessary for plasmid function and replication or within the tylosin resistance-conferring gene, and include, but are not limited to, genes that specify antibiotic modification enzymes and regulatory genes of all types.

More particularly, a non-selectable DNA segment that comprises a gene is inserted on a plasmid such as, for example, plasmid pSVB2 at the central ClaI restriction site of the thiostrepton resistance gene. Such an insertion inactivates the thiostrepton resistance gene and thus allows for the easy identification of transformants containing the recombinant plasmid. This is done by first selecting for tylosin resistance and, secondarily, identifying those tylosin-resistant transformants tht are not resistant to thiostrepton. Therefore, the ability to select for tylosin resistance in Streptomyces and related cells allows for the efficient isolation of the extremely rare cells that contain the particular non-selectable DNA of interest.

The functional test for tylosin resistance, as described herein above, is also used to locate DNA segments that act as control elements and direct expression of an individual antibiotic resistance-conferring gene. Such segments, including, but not limited to, promoters, attenuators, repressor and inducer binding-sites, ribosomal binding-sites and the like, are used to control the expression of other genes in cells of Streptomyces and related organisms.

The tylosin resistance-conferring vectors of the present invention are also useful for ensuring that linked DNA elements are stably maintained in host cells over many generations. These genes or DNA fragments, covalently linked to the tylosin resistance-conferring restriction fragment and propagated in Streptomyces, are maintained by exposing the transformants to levels of tylosin that are toxic to non-transformed cells. Therefore, transformants that lose the vector, and consequently any covalently linked DNA, cannot grow and are eliminated from the culture. Thus, the vectors of the present invention can stabilize and maintain any DNA sequence of interest.

The method, cloning vectors and transformants of the present invention provide for the cloning of genes to improve yields of various products that are currently produced in Streptomyces and related cells. Examples of such products include, but are not limited to, Streptomycin, Cephalosporins, Actaplanin, Apramycin, Narasin, Monensin, Tobramycin, Erythromycin and the like. The present invention also provides selectable vectors that are useful for cloning, characterizing and reconstructing DNA sequences that code: for commercially important proteins such as, for example, human insulin, human proinsulin, glucagon, interferon and the like; for enzymatic functions in metabolic pathways leading to commercially important processes and compounds; or for control elements that improve gene expression. These desired DNA sequences also include, but are not limited to, DNA that codes for enzymes that catalyze synthesis of derivatized antibiotics such as, for example, Streptomycin, Cephalosporin, Apramycin, Actaplanin, Narasin, Tobramycin, Monensin and Erythromycin derivatives, or for enzymes that mediate and increase bioproduction of antibiotics or other products. The capability for inserting and stabilizing such DNA segments thus allows for increasing the yield and availability of antibiotics that are produced by Streptomyces and related organisms.

Streptomyces can be cultured in a number of ways using any of several different media. Carbohydrate sources which are preferred in a culture medium include, for example, molasses, glucose, dextrin and glycerol. Nitrogen sources include, for example, soy flour, amino acid mixtures and peptones. Nutrient inorganic salts are also incorporated and include the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate and the 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 is grown under aerobic culture conditions over a relatively wide pH range of about 5 to 9 at temperatures ranging from about 15.degree. to 40.degree. C. For plasmid stability and maintenance, it is desirable to start with a culture medium at a pH of about 7.2 and maintain a culture temperature of about 30.degree. C.

The following examples further illustrate and detail the invention disclosed herein. Both an explanation of and the actual procedures for constructing the invention are described where appropriate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the restriction site and function map of plasmid pSVB2.

FIG. 2 shows the restriction site and function map of plasmid pIJ702.

FIG. 3 shows the restriction site and function map of plasmid pSVB12.

FIG. 4 shows the restriction site and function map of plasmid pSVB23.

FIG. 5 shows the restriction site and function map of plasmid pSVB16.

FIG. 6 shows the restriction site and function map of plasmid pSVB18.

FIG. 7 shows the restriction site and function map of plasmid pSVB20.

FIG. 8 shows the restriction site and function map of plasmid pSVB22.

FIG. 9 shows the restriction site and function map of phage pSVB3310.

FIG. 10 shows the restriction site and function map of phage pSVB3311
PATENT EXAMPLES available on request
PATENT PHOTOCOPY available on request

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