Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms

SUMMARY OF THE INVENTION
The present invention is 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 that is rate-limiting in an antibiotic biosynthetic pathway. The invention further comprises related DNA sequences that code for antibiotic biosynthetic gene products, recombinant DNA expression vectors, and transformed microbial host cells.
The present invention represents an early and significant commercial exploitation of recombinant DNA technology in antibiotic-producing organisms such as streptomycetes. Heretofore, the development and exploitation of recombinant DNA technology has been limited, for the most part, to the expression of specific poly-peptides 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 several other compounds of potential value. In each case, heterologous gene expression is more or less independent and does not interact with, take part in, or modulate operative biosynthetic pathways. Recombinant DNA technology can now be applied to improve selected biosynthetic pathways for the expression of more and increased yields of antibiotics or antimicrobial 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, New York); 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 was recently 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 actinorhodin pigment biosynthetic genes from one Streptomyces strain to another.
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.
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 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-resistant phenotype or gene conferring same.
Host Cell--an organism, including the viable protoplast thereof, that can be transformed with a recombinant DNA cloning vector.
NmR--the neomycin-resistant phenotype or gene conferring same.
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 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.
Transcriptional Activating Sequence--any DNA sequence that directs, promotes, or provides for the transcription of DNA into a mRNA transcript.
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.
Translational Activating Sequence--any DNA sequence that provides for the translation of a mRNA transcript into a peptide or polypeptide.
tsr--the thiostrepton-resistant phenotype or gene conferring same.

DESCRIPTION OF THE FIGURES
The plasmid and chromosomal maps depicted in the Figures are drawn to scale. However, the tylosin biosynthetic genes, although linked, are scattered across a large segment of DNA. Therefore, detailed restriction site mapping data exists only for small regions of the large tylosin biosynthetic gene-containing DNA fragment. The maps do not necessarily provide an exhaustive listing of all the cut sites of a given restriction enzyme. The location of individual genes, represented by line segments on the maps, was determined by deletion mapping and thus only approximates the exact location of a given gene.
FIG. 1--The Tylosin Biosynthetic Pathway.
FIG. 2--Restriction Site and Function Map of Plasmid pHJL280.
FIG. 3--Restriction Site and Function Map of Plasmid pHJL284.
FIG. 4--Restriction Site and Function Map of Plasmid pHJL309.
FIG. 5--Restriction Site and Function Map of Plasmid pHJL311.
FIG. 6--Restriction Site and Function Map of Plasmid pHJL315.
FIG. 7--Chromosomal Organization of the Tylosin Biosynthetic Genes.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for increasing the antibiotic-producing ability of an antibiotic-producing microorganism, said method comprising
1) transforming a microorganism that produces an antibiotic or antibiotic precursor by means of an antibiotic biosynthetic pathway with a recombinant DNA cloning vector or portion thereof, said vector or portion thereof comprising an antibiotic biosynthetic gene 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, subject to the limitation that said culturing procedure selected in step (2) provides for an increase in the antibiotic-producing ability of said microorganism.
The invention further comprises related antibiotic biosynthetic genes, recombinant DNA cloning vectors, and antibiotic or antibiotic precursor-producing microorganisms transformed with the aforementioned genes and vectors. The method of the present invention is widely applicable to all antibiotic-producing organisms. The following tables provide a non-exhaustive list of antibiotic producing organisms to which the present invention is applicable.
TABLE I______________________________________Aminocyclitol Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Bacillus various aminocyclitolsvarious speciesMicromonospora gentamycinsvarious speciesSaccharopolyspora various aminocyclitolsvarious speciesStreptomycesalbogriseolus neomycinsalbus var. metamycinus metamycinaquacanus N-methyl hygromycin Batrofaciens hygromycinsbikiniensis streptomycinbluensis var. bluensis bluensomycincanus ribosyl paromaminecatenulae catenulinchrestomyceticus aminosidinecrystallinus hygromycin Aerythrochromogenes streptomycinvar. narutoensiseurocidicus A16316-Cfradiae hybrimycins and neomycinsfradiae var. italicus aminosidineStreptomycesgalbus streptomycingriseus streptomycingriseoflavus MA 1267hofuensis seldomycin complexhygroscopicus hygromycins, leucanicidin, and hygrolidinhygroscopicus forma glebomycinglebosushygroscopicus var. validamycinslimoneushygroscopicus var. spectinomycinsagamiensiskanamyceticus kanamycin A and Bkasugaensis kasugamycinskasugaspinus kasugamycinslavendulae neomycinlividus lividomycinsmashuensis streptomycinmicrosporeus SF-767netropsis LL-AM31noboritoensis hygromycinsolivaceus streptomycinolivoreticuli var. destomycin Acellulophiluspoolensis streptomycinrameus streptomycinribosidificus SF733rimofaciens destomycin Arimosus forma paromomycins andparomomycinus catenulinspectabilis spectinomycintenebrarius tobramycin and apramycinStreptoverticillium spectinomycinflavopersicus______________________________________
TABLE II______________________________________Ansamycin Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Micromonospore various ansamycinsvarious speciesNocardia rifamycinmediterraneiStreptomycescollinus ansatrienes and napthomycinsdiastochromogenes ansatrienes and napthomycinsgalbus subsp. griseosporeus napthomycin Bhygroscopicus herbimycinhygroscopicus var. geldanus geldamycinvar. novanigellus 21-hydroxy-25-demethyl 25-methylthioproto- streptovaricinrishiriensis mycotrienessp. E/784 actamycin and mycotrienessp. E88 mycotrienesspectabilis streptovaricinstolypophorous tolypomycin______________________________________
TABLE III______________________________________Anthracycline and Quinone Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Streptomycescaespitosus mitomycins A, B, and Ccoelicolor actinorhodincoeruleorubidicus daunomycincyaneus ditrisarubicinflavogriseus cyanocycline Agalilaeus aclacinomycin A, auramycins, and sulfurmycinslusitanus napthyridinomycinpeuceticus daunomycin and adriamycinviolochromogenes arugomycin______________________________________
TABLE IV______________________________________.beta.-Lactam Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Agrobacterium various .beta.-lactamsCephalosporium penicillins andacremonium cephalosporinsChromobacterium various .beta.-lactamsGluconobacter various .beta.-lactamsNocardialactamadurans cephamycin Cuniformis nocardicinPenicillium various penicillins andchrysogenum other .beta.-lactamsSerratia various .beta.-lactamsantibioticus clavulanic acidargenteolus asparenomycin A, MM 4550, and MM 13902cattleya thienamycinchartreusis SF 1623 and cephamycin A and BStreptomycescinnamonensis cephamycin A and Bclavuligerus PA-32413-I, cephamycin C, A16886A, penicillins cephalosporins, clavulanic acid, and other clavamsfimbriatus cephamycin A and Bflavovirens MM 4550 and MM 13902flavus MM 4550 and MM 13902fulvoviridis MM 4550 and MM 13902griseus cephamycin A and B and carpetimycin A and Bhalstedi cephamycin A and Bheteromorphus C2081X and cephamycin A and Bhygroscopicus deacetoxycephalosporin Clipmanii cephamycin, penicillin N, 7-methoxycephalosporin C, A16884, MM4550, MM13902olivaceus epithienamycin F, MM 4550, and MM 13902panayensis C2081X and cephamycin A and Brochei cephamycin A and Bsioyaensis MM 4550 and MM 13902sp. OA-6129 OA-6192Asp. KC-6643 carpetimycin Aviridochromogenes cephamycin A and Bwadayamensis WS-3442-D______________________________________
TABLE V______________________________________Macrolide, Lincosamide, and StreptograminAntibiotic-Producing OrganismsOrganism Antibiotic______________________________________Micromonospore rosaramicinrosariaStreptomycesalbireticuli carbomycinalbogriseolus mikonomycinalbus albomycetinalbus var. coleimycincoilmyceticusambofaciens spiramycin and foromacidin Dantibioticus oleandomycinavermitilis avermectinsbikiniensis chalcomycinbruneogriseus albocyclinecaelestis M188 and celesticetincinerochromogenes cineromycin Bcirratus cirramycindeltae deltamycinsdjakartensis niddamycinerythreus erythromycinseurocidicus methymycineurythermus angolamycinfasciculus amaromycinStreptomycesfelleus argomycin and picromycinfimbriatus amaromycinflavochromogenes amaromycin and shincomycinsfradiae tylosinfungicidicus NA-181fungicidicus var. espinomycinsespinomyceticusfurdicidicus mydecamycingoshikiensis bandamycingriseofaciens PA133A and Bgriseoflavus acumycingriseofuscus bundlingriseolus griseomycingriseospiralis relomycingriseus borrelidingriseus ssp. sulphurus bafilomycinshalstedi carbomycin and leucanicidinhygroscopicus tylosinhygroscopicus subsp. milbemycinsaureolacrimosuskitastoensis leucomycin A.sub.3 and josamycinlavendulae aldgamycinlincolnensis lincomycinloidensis vernamycin A and Bmacrosporeus carbomycinmaizeus ingramycinmycarofaciens acetyl-leukomycin, and espinomycinnarbonensis josamycin and narbomycinnarbonensis var. leucomycin A.sub.3josamyceticus and josamycinolivochromogenes oleandomycinplatensis platenomycinrimosus tylosin and neutramycinrochei lankacidin and borrelidinrochei var. T2636volubilisroseochromogenes albocyclineroseocitreus albocyclinespinichromogenes var. kujimycinssuragaoensistendae carbomycinthermotolerans carbomycinvenezuelae methymycinsviolaceoniger lankacidins and lankamycin______________________________________
TABLE VI______________________________________Miscellaneous Antibiotic-Producing StreptomycesAntibiotic Type Streptomyces Species Antibiotic______________________________________amino acid sp. cycloserineanaloguescyclopentane ring- coelicolor methylenomycin Acontaining erythrochromogenes sarkomycin kasugaensis aureothricin and thiolutin violaceoruber methylenomycin Anitro-containing venezuelae chloramphenicolpolyenes griseus candicidin nodosus amphotericin B noursei nystatintetracyclines aureofaciens tetracycline, chlor- tetracycline, demethyltetra- cycline, and demethylchlortetra- cycline rimosus oxytetracycline______________________________________
TABLE VII______________________________________Nucleoside Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Corynebacterium tunicamycin analoguesmichiganese pv. rathayiNocardia pyrazofurincandidusStreptomycesantibioticus ara-Achartreusis tunicamycingriseoflavus var. streptoviridansthuringiensisgriseolus sinefunginlysosuperificus tunicamycin______________________________________
TABLE VIII______________________________________Peptide Antibiotic-Producing OrganismsOrganism Antibiotic______________________________________Actinoplanesmissouriensis actaplaninteichomyceticus teicoplaninBacillus bacitracin, polymixin,various species and colistinNocardiacandidus A-35512 and avoparcinlurida ristocetinorientalis vancomycinStreptomycesantibioticus actinomycinaureus thiostreptoncanus amphomycineburosporeus LL-AM374haranomachiensis vancomycinpristinaespiralis pristinamycinroseosporus lipopeptides, such as A21978Ctoyocaensis A47934virginiae A41030______________________________________
TABLE IX______________________________________Polyether Antibiotic-Producing OrganismOrganism Antibiotic______________________________________Actinomaduravarious species various polyethersoligosporus A80190Dactylosporangium various polyethersvarious speciesNocardia various polyethersvarious speciesStreptomycesalbus A204, A28695A and B, and salinomycinaureofaciens narasinbobili A80438cacaoi var. lysocellinasoensischartreusis A23187cinnamonensis monensinconglobatus ionomycineurocidicus var. laidlomycinasterocidicusflaveolus CP38936gallinarius RP 30504griseus grisorixinhygroscopicus A218, emericid, DE3936, A120A, A28695A and B, etheromycin, and dianemycinlasaliensis lasalocidlongwoodensis lysocellinmutabilis S-11743apactum A80438ribosidificus lonomycinviolaceoniger nigericinStreptoverticillium polyethersvarious species______________________________________
The present invention is best exemplified by transforming antibiotic-producing microorganisms with genes that code for enzymes that catalyze chemical reactions governing the conversion of primary metabolites into antibiotics. One such enzyme, macrocin O-methyl-transferase, catalyzes the final step in the biosynthesis of tylosin. Transforming tylosin-producing microorganisms with a macrocin O-methyltransferase-encoding gene, designated herein as tylF, results in an improved tylosin biosynthetic pathway as indicated by increased levels of the tylF gene product in the transformed cells. Accordingly, a further object of the present invention is to provide a method for increasing the tylosin-producing or tylosin precursor-producing ability of a tylosin-producing or tylosin precursor-producing microorganism, such method comprising
1) transforming a microorganism that produces tylosin or a tylosin precursor by means of an antibiotic biosynthetic pathway with a recombinant DNA cloning vector or portion thereof, said vector or portion thereof comprising a tylosin 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 tylosin or a tylosin precursor, subject to the limitation that said culturing procedure selected in step (2) provides for an increase in the tylosin-producing or tylosin precursor-producing ability of said microorganism.
The method of the present invention utilizes antibiotic biosynthetic genes to increase the antibiotic-producing ability of an organism. A small number of antibiotic biosynthetic genes have been cloned, characterized, and described in the relevant literature. Methods for isolating antibiotic biosynthetic genes have been developed, but one especially preferred method is described in Baltz et al., U.S. patent application Ser. No. 742,349, (U.S. Pat. No. 4,935,340) filed Jun. 7, 1985, attorney docket number X-6640, which is incorporated herein by reference. The tylosin antibiotic biosynthetic genes described herein as a specific exemplification of the present method were initially isolated from a .lambda. library constructed in substantial accordance with the procedure described in Fishman et al., 1985, J. Bacteriology 161:199-206.
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. Each genotypic designation may represent a class of genes that contribute to the same phenotype. A number of expression 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 X provides a brief description of each of the plasmids used to exemplify the method of the present invention.
TABLE X______________________________________Plasmids Comprising Tylosin Biosynthetic Genes Tylosin NRRLHost/Designation Gene(s) Accession No. Map______________________________________E. coli K12 HB101/pHJL280 D, E, F, H, J B-18043 FIG. 2E. coli K12 HB101/pHJL284 C, F, J B-18044 FIG. 3E. coli K12 HB101/pHJL309 L, M B-18045 FIG. 4E. coli K12 HB101/pHJL311 C, F, J, K, H B-18046 FIG. 5E. coli K12 JM109/pHJL315 D, E, F, H, J B-18047 FIG. 6______________________________________
A number of Streptomyces fradiae strains are described herein that have mutant tylosin biosynthetic genes and thus make much less tylosin than the strain from which they were derived. Table XI provides a brief description of these mutant strains.
TABLE XI______________________________________Streptomyces fradiae Mutants Defective inTylosin BiosynthesisStrain Mutant ATCC* or NRRLDesignation Gene Accession No.______________________________________GS15 tylF NRRL 18058GS16 tylE ATCC 31664GS28 tylF NRRL 18059GS33 tylLGS48 tylD NRRL 12170GS52 tylC NRRL 18060GS62 tylMGS76 tylD tylH NRRL 12171GS85 tylKGS88 tylJ______________________________________ *ATCC is the American Type Culture Collection, Rockville, MD 20852, and NRRL is the Northern Regional Research Laboratory, Peoria, IL 61604.
Plasmids pHJL280, pHJL284, and pHJL315 were used to transform Streptomyces fradiae GS15 and Streptomyces fradiae GS28. The GS15 and GS28 strains were prepared from S. fradiae C4 by nitrosoguanidine mutagenesis. S. fradiae C4 was derived from S. fradiae T59235 (ATCC 19609) by mutagenesis. The GS15 strain makes almost no tylosin, and the GS28 strain makes low levels of tylosin, as compared with the C4 strain. The decreased or nonexistent tylosin-producing ability of the GS15 and GS28 strains is believed to result from mutations affecting the tylF gene, which encodes macrocin O-methyltransferase (MOMT). The MOMT enzyme, which is required for the final conversion of macrocin to tylosin in the tylosin biosynthetic pathway, is frequently present in reaction rate-limiting amounts in tylosin-producing strains. Plasmids pHJL280, pHJL284, and pHJL315 remove this reaction limitation by providing a means for increasing both the copy number of the tylF biosynthetic gene and also the concentration of macrocin O-methyltransferase available for tylosin biosynthesis. Accordingly, fermentation of S. fradiae GS15L pHJL280, S. fradiae GS15/pHJL284, S. fradiae GS15/pHJL315, S. fradiae GS28/pHJL284, S. fradiae GS28/pHJL280, and S. fradiae GS28/pHJL315 for 72 hours results in about a 2-fold to a 6-fold increase in the production of macrocin O-methyltransferase over that produced in the C4 strain and a 120-fold increase over that produced in the GS28 strain.
Plasmid pHJL280 was also used to transform: (1) Streptomyces fradiae GS16; (2) S. fradiae GS48; (3) S. fradiae GS76; and (4) S. fradiae GS88 which produce tylosin at levels below the detection limit and were derived by mutagenesis of the C4 strain. Untransformed strains GS16, GS48, GS76, and GS88 respectively produce a defective enzyme or a rate-limiting amount of (1) the tylE, demethylmacrocin O-methyltransferase, enzyme; (2) the tylD enzyme, which is required for addition or biosynthesis of 6-deoxy-D-allose; (3) the tylH enzyme, which is required for oxidation of the C-23 methyl position of tylactone; and (4) the tylJ enzyme. Untransformed strains GS16, GS48, GS76, and GS88 respectively tend to accumulate demethylmacrocin, demycinosyl tylosin, 23-deoxydemycinosyl tylosin, and demycinosyl tylosin rather than the desired tylosin antibiotic compound.
Plasmid pHJL280 provides 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 tylD, tylE, tylH, and tylJ biosynthetic genes and by increasing the intracellular amount of the products specified by these genes. The concentration of available tylE gene product is thus increased, resulting in an elevated amount of enzyme capable of driving the conversion of demethylmacrocin to macrocin to tylosin in the tylosin biosynthetic pathway. Similarly, the concentration of available tylD, tylH, and tylJ gene products is also increased, resulting in the production of elevated amounts of the enzymes capable of driving the 6-deoxy-D-allose addition and C-23 oxidation of tylosin precursors. Fermentation of Streptomyces fradiae GS16/pHJL280, S. fradiae GS48/pHJL280, S. fradiae GS76/pHJL280, and S. fradiae GS88/pHJL280 for 144-168 hours results in yields of tylosin that are significantly increased over that of the untransformed, low-tylosin-producing, mutant strains. Such transformed strains have higher enzyme levels of the particular enzymes encoded on plasmid pHJL280 than the parent C4 strain and thus further exemplify the present invention. Plasmid pHJL280 can be used to improve the tylosin-producing ability of any organism in which the tylD, tylE, tylF, tylH, or tylJ gene products (or any combination thereof) are present in rate-limiting amounts for tylosin biosynthesis.
Plasmid pHJL284 was also used to transform Streptomyces fradiae GS52, a low tylosin-producing, mutant strain derived from the C4 strain that produces reaction-limiting amounts of an enzyme required for the biosynthesis or addition of mycarose to de-O-methyllactenocin. Thus, the tylosin biosynthetic pathway of Streptomyces fradiae GS52 tends to produce desmycosin rather than the desired tylosin antibiotic compound. Plasmid pHJL284 provides a means for improving the synthetic efficiency of this pathway by providing a non-defective biosynthetic gene and by increasing the copy number of the tylC biosynthetic gene. The concentration of available tylC gene product in the transformed strain is thus increased, resulting in the elevated production of enzyme capable of driving the desired addition reaction. Accordingly, fermentation of Streptomyces fradiae GS52/pHJL284 for 144-168 hours results in a level of tylosin production that is significantly increased over that of the untransformed mutant strain and results in higher tylC enzyme levels than those in the parent C4 strain. Plasmid pHJL284 was also used in the present method to improve the tylosin-producing ability of Streptomyces fradiae GS88, a tylJ mutant, and thus can also be used in the present method to improve the tylosin-producing ability of any organism in which the tylC, tylF, or tylJ gene products (or any combination thereof) are present in rate-limiting amounts for tylosin biosynthesis.
Plasmid pHJL309 contains the tylL and tylM biosynthetic genes and so was used in the present method to improve the tylosin-producing ability of Streptomyces fradiae GS33, a tylL mutant, and GS62, a tylM mutant. Plasmid pHJL309 can also be used in the present method to improve the tylosin-producing ability of any organism in which the tylL or tylM gene products (or both) are present in rate-limiting amounts for tylosin biosynthesis.
Plasmid pHJL311 contains the tylC, tylF, tylH, tylJ, and tylK biosynthetic genes and so was used in the present method to improve the tylosin-producing ability of Streptomyces fradiae GS52, a tylC mutant; GS88, a tylJ mutant; GS15 and GS28, both of which are tylF mutants; and GS85, a tylK mutant. Plasmid pHJL311 can also be used in the present method to improve the tylosin-producing ability of any organism in which the tylC, tylF, tylH, tylJ, or tylK gene products (or any combination thereof) are present in rate-limiting amounts for tylosin biosynthesis.
Plasmid pHJL315 contains the tylD, tylE, tylF, tylH, and tylJ biosynthetic genes and so was used in the present method to improve the tylosin-producing ability of Streptomyces fradiae GS48, a tylD mutant; GS88, a tulJ mutant; GS16, a tylE mutant; GS76, a tylD, tylH double mutant; and GS15 and GS28, both of which are tylF mutants. Plasmid pHJL315 can also be used in the present method to improve the tylosin-producing ability of any organism in which the tylD, tylE, tylF, tylH, or tylJ gene products (or any combination thereof) are present in rate-limiting amounts for tylosin biosynthesis.
The results described above demonstrate that the vectors of the present invention can increase the antibiotic-producing ability of an antibiotic-producing organism by providing higher enzyme or other gene product levels, as compared to an untransformed organism, of an enzyme or other gene product 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. Not wishing the present invention to be bound or limited in any way by theory, this apparent anomaly can be explained by the fact that antibiotics are produced from primary metabolites, such as acetate, propionate, malonyl-CoA, methylmalonyl-CoA, and glucose, by the action of specific enzymes. These enzymes are usually not present during the rapid growth phase of an organism and so do not rob the growing cell of needed compounds. As growth becomes limited by nutritional conditions, antibiotic biosynthetic genes are believed to be activated, causing the synthesis of enzymes that redirect the flow of certain primary metabolites into antibiotic products.
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, it is believed that selection for higher antibiotic production frequently 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 thought 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 fine physiological balance (already mentioned) 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 present invention overcomes this heretofore unrecognized problem of the negative effect of autonomous plasmid replication on high-producing strains by providing 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.
Integration of plasmid DNA is readily accomplished by transforming a given antibiotic-producing strain or mutant thereof according to standard transformation procedures, 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, so 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, describe 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 vectors pHJL210 and pHJL401, which were used to construct the plasmids of the present invention, lack this stability sequence. Plasmid pHJL210 is disclosed in U.S. patent application Ser. No. 639,566, filed Aug. 10, 1984, attorney docket No. X-6256, now issued as U.S. Pat. No. 4,753,886 (Jun. 28, 1988), incorporated herein by reference. Plasmid pHJL401 is disclosed in U.S. patent application Ser. No. 841,920, U.S. Pat. No. 4,898,828 filed Mar. 20, 1986, attorney docket No. X-6786A, which is a continuation-in-part of Ser. No. 763,172, filed Aug. 7, 1985, attorney docket No. X-6786. As used herein, "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, for plasmids such as pHJL210 and 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 plasmid 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 herein, 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. In one fermentation of a tylosin-producing strain that had been transformed with plasmid pHJL280 and cultured to obtain integrants, only a subpopulation (.about.18%) of the cells were thiostrepton resistant, which indicates the presence of plasmid pHJL280 sequences. However, this subpopulation contained multiple copies of the genes for two rate-limiting enzymes, demethylmacrocin-O-methyltransferase (DMOMT) and macrocin-O-methyltransferase (MOMT), and consequently elevated (about 9 fold) levels of the two enzymes, and was able to convert all of the normally accumulated demethylmacrocin and macrocin to tylosin (see Table XV).
Thus, one can develop specific strains of S. fradiae containing multiple copies of rate-limiting 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.
Of course, antibiotic production is only increased by the method of the present invention when the transforming DNA comprises a gene that encodes the rate-limiting enzyme 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 method of the present 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.
For instance, the GS15 strain, which produces no readily detectable tylosin (the level of tylosin produced by these cells is below the detection limits for the assay used to determine tylosin levels) and the GS28 strain, which produces very low levels of tylosin, contain tylF mutations, so that it is a relatively simple matter to identify the rate-limiting step in tylosin biosynthesis in these mutant strains. The strain from which the GS15 and GS28 strains were derived, designated Streptomyces fradiae C4, produces high levels of tylosin and accumulates relatively large amounts of macrocin, the immediate precursor of tylosin on which the tylF gene product acts to form tylosin. Other S. fradiae strains that produce even more tylosin than the C4 strain accumulate even more macrocin than the C4 strain. These observations indicate that the tylF gene product is present in rate-limiting amounts for the biosynthesis of tylosin in high tylosin-producing strains. Transformation of these macrocin-accumulating strains with a vector comprising the tylF gene followed by isolation of those transformants that only contain integrated copies of the vector yields transformants that produce more tylosin than the untransformed cells. The increase in tylosin production observed in these transformants is related to the amount of macrocin that accumulates in the untransformed cells. It will be apparent to those skilled in the art that the transformants produced by the foregoing procedure might still contain rate-limiting amounts of the tylF gene product, in which case a further increase of the tylF copy number would further increase tylosin yield, or the transformed strains might now contain rate-limiting amounts of yet another antibiotic biosynthetic enzyme, the level of which could be made non-rate-limiting by the method of the present invention.
The present invention provides both a method and recombinant DNA cloning vectors for increasing the production of an antibiotic by manipulation of antibiotic biosynthetic pathways. An illustrative antibiotic biosynthetic pathway involves the biosynthesis of tylosin, a complex macrolide produced by strains of Streptomyces fradiae, Streptomyces rimosus, and Streptomyces hygroscopicus. Tylosin is composed of a 16-member branched lactone (tylonolide) to which three sugars (mycarose, mycaminose, and mycinose) are attached. The lactone is derived from two acetates, five propionates, and a butyrate by condensation of a propionyl-S-coenzyme A molecule with two malonyl-S-coenzyme A molecules, four methylmalonyl-S-coenzyme A molecules, and an ethylmalonyl-S-coenzyme A molecule by a scheme believed analogous to that involved in fatty acid biosynthesis. Lactone forma-tion, sugar biosynthesis/attachment, and the conversion of resultant intermediate compounds to tylosin are catalyzed by a series of gene-encoded enzymes. Cloning genes that code for such enzymes allows for modifying and improving the operational efficiency of the tylosin biosynthetic pathway and thus is illustrative of the present invention.
Illustrative tylosin biosynthetic genes that can be used for purposes of the present invention include, for example, the tylC, tylD, tylE, tylF, tylH, 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 steps catalyzed by the tylF gene product. The tylF enzyme catalyzes the conversion of macrocin to tylosin, as depicted in FIG. 1 of the accompanying drawings. Over production 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.
Those skilled in the art will recognize that the present invention is not limited to the use of plasmids pHJL280, pHJL284, pHJL309, pHJL311, or pHJL315. The antibiotic biosynthetic genes contained in the aforementioned vectors can be excised in whole or in part and ligated into any number of different recombinant DNA cloning vectors. For instance, digestion of plasmid pHJL280 with restriction enzymes BamHI and BglII yields five Bam HI-BamHI fragments with sizes of .about.10.3 kb, .about.6.54 kb, .about.2.3 kb, .about.1.7 kb, and .about.1.0 kb; two Bam HI-BglII fragments with sizes of .about.2.9 kb and 2.0 kb; and one BglII-BglII fragment .about.0.2 kb in size. The .about.2.9 kb BamHI-BglII fragment of plasmid pHJL280 contains the tylF gene. Digestion of plasmid pHJL280 with restriction enzymes BglII and EcoRI generates four fragments: an .about.11.24 kb EcoRI-EcoRI fragment; an .about.11.5 kb BglII-EcoRI fragment; an .about.4.0 kb EcoRI-BglII fragment, and an .about.0.2 kb BglII-BglII fragment. The .about.4.0 kb EcoRI-BglII fragment of plasmid pHJL280 contains the tylE gene.
Digestion of plasmid pHJL28 4 with restriction enzymes BamHI and EcoRI generates three BamHI-BamHI fragments with sizes of .about.9.7 kb, .about.2.3 kb, and .about.1.0 kb; and four EcoRI-BamHI fragments with sizes of .about.6.24 kb, .about.4.3 kb, .about.2.3 kb, and .about.1.1 kb. The .about.2.3 kb BamHI-EcoRI fragment of plasmid pHJL284 contains the tylF gene. Digestion of plasmid pHJL284 with restriction enzyme EcoRI generates two fragments with sizes of .about.16.4 kb and .about.10.54 kb; the .about.16.4 kb fragment contains the tylF, tylC, and tylJ genes. The .about.1.7 kb EcoRI-Bam HI restriction fragment of plasmid pHJL311 comprises the tylK gene. The .about.18.5 kb EcoRI restriction fragment, as well as the .about.8.3 kb Bam HI-KpnI restriction fragment, of plasmid pHJL309 contains the tylL and tylM genes.
Any of the aforementioned tyl gene-containing fragments can be ligated into other vectors to make vectors useful in the present method. Such other vectors 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), pHJL192, pHJL197, pHJL198, pHJL210, pHJL211, pHJL400, pHJL401, pHJL302, pIJ922, pIJ903, pIJ941, pIJ940, and pIJ916. These vectors replicate in Streptomyces fradiae and other tylosin-producing strains and are thus useful for cloning the present antibiotic biosynthetic genes. The "unstable" vectors described above are preferred when integration of the vector is desired.
A number of tylosin biosynthetic gene-containing vectors have been constructed. Table XII provides a brief description of these plasmids, their construction, and the genes they complement. The tyl gene-containing restriction fragments are identified by their position on the chromosomal map provided in FIG. 7 of the accompanying drawings.
TABLE XII__________________________________________________________________________Tylosin Expressiono VectorsDerivation tyl-containingVector Cloning Vector restriction fragment Complementation__________________________________________________________________________pSKC16 BamHI-digested pHJL401 BamHI(20.0)- BamHI(25.1) E, D, H, F, and JpSKC13 BamHI-digested pHJL401 BglII(16.0)- BglII(22.2) E and DpSFH62 BamHI-digested pHJL401 BglII(16.0)- BglII(22.0) E and DpSFH61 * EcoRI- HindIII-digested KpnI(18.0)- KpnI(21.7) E pHJL40-1POJ190 BamHI- KpnI-digested BamHI(40.0)- KpnI(48.3) L and M pOJ160**pOJ163 BamHI-digested pOJ160 BamHI(40.0)- BamHI(51.0) L and M__________________________________________________________________________ *The 3.7 KpnI fragment was first cloned into the KpnI site of plasmid pUC19 to yield plasmid pSPH60 which was used as a source of the tylEcontaining EcoRI HindIII fragment. **Plasmid pOJ160 is available from the NRRL in E. coli K12 JM109 under th accession number NRRL B18088
Illustrative Streptomyces strains that can be used for purposes of the present invention include, for example, S. fradiae, S. fradiae GS52, S. fradiae GS48, S. fradiae GS16, S. fradiae GS28, S. fradiae GS15, S. fradiae GS76, 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. Of the Streptomyces taxa, S. fradiae GS16, S. fradiae GS15, and S. fradiae GS28 are preferred, especially for transformation with plasmid pHJL280. 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 respective accession numbers NRRL 2702, NRRL 2703, and ATCC 19609.
The recombinant plasmids described in the present invention each comprise one or more antibiotic biosynthetic genes. Unless part of a polycistron, each antibiotic biosynthetic gene normally comprises: (1) a promoter (transcriptional activating sequence) that directs transcription of the gene; (2) a sequence that, when transcribed into mRNA, directs translation of the transcript; (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 tylF gene can be linked to the transcriptional activating sequence, 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 that would serve as a substrate for the novel gene product. Similarly, the promoter and other regulatory elements of the tylF 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 described herein comprise an important component of the present invention.
For example, sequence data for the cluster comprised of the tylE, tylD, tylH, tylF, tylJ, as shown below, provides the direction of transcription and translation of each of these genes, possible transcription start sites ("TS"), possible translation-activating sequences ("TAS"), possible transcription end sequences ("TE") and possible translation start sites ("S"). Subscripts associated with these designations indicate for which gene the particular regulatory elements refer. For example, TS.sub.E refers to the transcription start site for the tylE gene. Because the tylE, tylD, tylH, and tylJ genes are transcribed from the complementary strand, both DNA strands of the entire tylEDHFJ cluster, and the projected corresponding amino acid sequence for the proposed coding sequences are provided. Nucleotides designated "N" or "S" indicate that the particular base is not clearly defined. Thus, the sequence for the tylEDHFJ cluster is the following: ##STR1## wherein A is a deoxyadenyl residue; G is a deoxyguanyl residue; C is a deoxycytidyl residue; T is a thymidyl residue; ALA is alanine, RAG is arginine, ASN is asparagine, ASP is aspartic acid, CYS is cysteine, GLN is glutamine, GLU is glutamic acid, GLY is glycine, HIS is histidine, ILE is isoleucine, LEU is leucine, LYS is lysine, MET is methionine, PHE is phenylalanine, PRO is proline, SER is serine, THR is theonine, TRP is tryptophan, TYR is tyrosine, and VAL is valine.
The arrows on the DNA and amino acid sequence map data above indicate the direction of transcription for each of the designated genes. In particular, one skilled in the art will appreciate that the tylE, tylD, tylH and tylJ gene products are encoded on one DNA strand. The tylF gene, however, is transcribed from the complementary strand. Initiation of translation in streptomycetes occurs at GTG codons as well as ATG codons. In the present case, initiation of translation of the tylH, tylF and tylJ genes most likely occurs at a GTG codon. The tylE gene probably starts at an ATG codon whereas the tylD gene may initiate at either an ATG or GTG.
Furthermore, transcription mapping shows that the transcript for the tylE gene overlaps the transcript of the tylD and tylH genes. Also, the tylH transcript overlaps the transcript of the tylF gene. Asterisks on the map indicate inverted repeat sequence which may be able to form stem-loop structures.
Also noted on this map are possible translational activating sequences for several of the genes. Also, the transcription termination site for the tylD gene appears to be located only one base pair from the gene appears to be located only one base pair from the tylE translation start codon.
Streptomyces fradiae 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, an 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 37.degree. C.
The following examples further illustrate and describe the invention disclosed herein. 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. Both an explanation of and the actual procedures for constructing the invention are described where appropriate.
EXAMPLE 1
Isolation of Plasmid pHJL280
A. Culture of E. coli K12 HB101L pHJL280
Lyophils of E. coli K12 HB101/pHJL280 can be B-18043. The lyophilized cells are streaked onto L-agar plates (L agar contains 10 g of Bacto Tryptone, 5 g of Bacto Yeast Extract, 10 g of NaCl, 2 g of glucose, and 15 g of agar per liter) containing 50 .mu.g/ml ampicillin to obtain a single-colony isolate of E. coli K12 HB101pHJL280. One such colony was used to inoculate 100 ml of L broth (L broth is L agar without the agar), which was then incubated aerobically at 37.degree. C. overnight (about 16 hours). The following morning, the cells were harvested by centrifugation at 10,000.times.g for 10 minutes. The .about.1 g of cells obtained by this procedure are used to prepare plasmid pHJL280 DNA in substantial accordance with the procedure described below.
B. Plasmid Isolation
The cell pellet obtained in Example 1A was resuspended in 10 ml of a solution composed of 25% sucrose and 50 mM Tris-HCl at a pH=8.0. About 1 ml of a 10 mg/ml solution of lysozyme in 50 mM Tris-HCl at a pH=8.0 was incubated on ice for 5 minutes. About 4 ml of 0.25M EDTA, pH=8.0, were then added to the cell suspension, and incubation on ice was continued for another 5 minutes. About 16 ml of lysis solution (lysis solution contains 0.4% deoxycholate; 1% Brij58 (Sigma Chemical Co., P.O. Box 14508, St. Louis, Mo. 63178); 0.05M Tris-HCl, pH=8.0; and 0.0625M EDTA) were added to the lysozyme-treated cells, and the resulting mixture was incubated at 37.degree. C. for 15 minutes.
The cell lysate was cleared by centrifugation at 48,000.times.g for 25 minutes. The supernatant was decanted into a separate tube, to which was added 0.1 volume of 3.0M NaOAc at a pH=8.0 and 0.64 volume of isopropyl alcohol. The DNA precipitate was collected by centrifugation at 20,000.times.g for 10 minutes and then redissolved in 0.1 volume of TE buffer (10 mM Tris-HCl, pH=7.8, and 1 mM EDTA). The solution of DNA was incubated at 65.degree. C. for 30 minutes and then purified by equilibrium-density-gradient ultracentrifugation in CsCl and propidium diiodide. The plasmid pHJL280 DNA obtained by this procedure was dissolved in TE buffer at a concentration of about 1 .mu.g/.mu.l. A restriction site map of plasmid pHJL280 is presented in FIG. 2 of the accompanying drawings.
EXAMPLE 2
Isolation of Plasmids pHJL284, pHJL309, pHJL311, and pHJL315
Lyophils of the E. coli strains harboring plasmids pHJL284, pHJL309, pHJL311, and pHJL315 can be listed in Table X. The desired plasmids are each obtained and purified from the lyophilized cells in substantial accordance with the teaching of Example 1. Restriction site maps of the plasmids are presented in FIGS. 2-6 of the accompanying drawings.
EXAMPLE 3
Construction of Streptomyces fradiae GS28/pHJL280
A culture of Streptomyces fradiae GS 28 was inoculated into 20 ml of trypticase-soya broth (TSB) and incubated in a water-bath incubator at 29.degree. C. at 260 rpm overnight (about 16 hours). The culture was homogenized using a homogenizing vessel (Thomas Scientific, Swedesboro, N.J.) and a T-Line laboratory stirrer and then fragmented using a Sonifier Cell Disruptor (Heat Systems Ultrasonics, Inc.) for 7 seconds at 76 Watts. Four ml of the homogenized, fragmented culture were inoculated into 20 ml of TSB (BBL) containing 0.3% weight by volume glycine, and the culture was again incubated overnight at 29.degree. C. The following morning, the culture was homogenized and recultured as described above. After this third overnight incubation, the culture was homogenized, collected, and then washed twice with P media (P media was prepared by adding 103 g of sucrose to 0.25 g of K.sub.2 SO.sub.4 and 2.03 g of MgCl.sub.2 -6H.sub.2 O and then adding deionized water to a final volume of 700 ml. The mixture was then sterilized, and to each 70 ml of solution, about 10 ml each of 0.05 g KH.sub.2 PO.sub.4 /100 ml of deionized water; 2.78 g CaCl.sub.2 /100 ml of deionized water; and 0.25M TES (2-(�tris-(hydroxymethyl)methyl!amino)ethanesulfonic acid)) at a pH=7.2 were added to form the desired P media.
The cell pellet was resuspended in 15 ml of P media containing 1 mg/ml lysozyme (Calbiochem, La Jolla, Calif. 92037) and then incubated at room temperature for about one-and-one-half hours to form protoplasts. The protoplasts were gently collected by centrifugation, washed twice with P media, resuspended in 2 ml of P media, and incubated on ice until use. About 1 .mu.g of plasmid pHJL280 DNA was added to about 50 .mu.l of 2 mg/ml heparin sulfate (Sigma) and incubated on ice for about 10 minutes. Much less plasmid DAN, about 5-100 nanograms, can be used to transform Streptomyces fradiae if prepared from a S. fradiae host. The procedure for isolating Streptomyces plasmid DNA is described in Hopwood et al., 1985, Genetic Manipulation of Streptomyces: A Laboratory Manual (John Innes foundation, Norwich, England). The DNA/heparin solution was first added to about 200 .mu.l of protoplasts, and about 0.9 ml of a solution composed of 55% PEG 1000 (Sigma) in P medium was then added to the DNA/protoplast mixture, and the resulting mixture was gently mixed at room temperature.
The mixture was plated in varying aliquots onto R2 plates using 4 ml of soft-R2-agar overlays. R2 plates contain 30 ml of R2 media and have been dried at 37.degree. C. for about 4 days. R2 media is prepared by adding 103 g sucrose, 0.25 g K.sub.2 SO.sub.4, 2 ml of trace element solution, 10.12 g MgCl.sub.2 -6H.sub.2 O, 10.0 g of glucose, 2.0 g of L-asparagine, 0.1 g of Casamino acids, and 22 g of agar to 700 ml of water; sterilizing the resulting solution; and finally, adding 100 ml of each of the following solutions: 0.05 g KH.sub.2 PO.sub.4 /100 ml of deionized water; 2.22 g CaCl.sub.2 /100 ml of deionized water; and 0.25M TES, pH=7.2. The pH of the final solution is adjusted to equal 7.2. Trace element solution contains 40 mg ZnCl.sub.2, 200 mg FeCl.sub.3 -6H.sub.2 O, 10 mg CuCl.sub.2 -2H.sub.2 O, 10 mg MnCl.sub.2 -4H.sub.2 O, 10 mg Na.sub.2 B.sub.4 O.sub.7 -10H.sub.2 O, and 10 mg (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O per liter. The soft-R2-agar overlays are prepared by adding 51.5 g of sucrose, 5.06 g MgCl.sub.2 -6H.sub.2 O, 1.11 g CaCl.sub.2, 50 ml of 0.25M TES at a pH=7.2, and 2.05 g agar to enough deionized water to achieve a final volume of 500 ml. The mixture is steamed to melt the agar, decanted into 4 ml aliquots, and autoclaved prior to use. After the transformed protoplasts had been plated, the plates were incubated at 29.degree. C. for 24 hours, and then, 4 ml of soft-R2 agar containing 25 .mu.l of 50 mg/ml thiostrepton (E. R. Squibb, Princeton, N.J. 08540) were spread over the protoplasts. Incubation of the plates at 29.degree. C. was continued until regeneration was complete, usually a period of about 7-14 days, to select for the desired S. fradiae GS28/pHJL280 strain
The Streptomyces fradiae GS28/pGJL280 strain was cultured and produced macrocin O-methyltransferase and tylosin at levels above that produced in the untransformed S. fradiae GS28 strain. Macrocin O-methyltransferase activity was assayed and determined in substantial accordance with the teaching of Yeh et al., 1984, Journal of Chromatography 288:157-165. Comparison of the macrocin O-methyltransferase activities in the transformed, GS28/pHJL280, and parental, GS28, strains showed a 60-to-100-fold increase of enzyme and 14-to-18-fold increase of tylosin production in the transformed strain. Tylosin production was assayed and determined in substantial accordance with the teaching of Baltz and Seno, 1981, Antimicrobial Agents and Chemotherapy 20:214-225; and Kennedy, J. H., 1983, Journal of Chromatography 281:288-292.
EXAMPLE 4
Construction of Streptomyces fradiae GS15/pHJL280
The desired strain was constructed in substantial accordance with the teaching of Example 3 except that Streptomyces fradiae GS15, rather than S. fradiae GS28, was used. The desired strain was cultured for 72 hours and produced macrocin O-methyltransferase and tylosin at levels above that produced in the un-transformed S. fradiae GS15 strain, which produces no readily detectable tylosin.
EXAMPLE 5
Construction of Streptomyces fradiae GS15, pHJL284
The desired strain was constructed in substantial accordance with the teaching of Example 4 except that plasmid pHJL284, rather than plasmid pHJL280, was used. The desired strain was cultured and produced macrocin O-methyltransferase and tylosin at levels above that produced in the untransformed S. fradiae GS15 strain.
EXAMPLE 6
Construction of Streptomyces fradiae GS16/pHJL280
The desired strain was constructed in substantial accordance with the teaching of Example 3 except that Streptomyces fradiae GS16, rather than S. fradiae GS28, was used. The desired strain was cultured and produced the tylE gene product, demethylmacrocin O-methyltransferase, and tylosin at levels above that produced in the untransformed strain. The demethylmacrocin O-methyltransferase activity and tylosin production respectively are assayed and determined in substantial accordance with the above-referenced procedures, except that demethylmacrocin is substituted for macrocin as substrate.
EXAMPLE 7
Construction of Streptomyces fradiae GS76/pHJL280
The desired strain was constructed in substantial accordance with the teaching of Example 3 except that Streptomyces fradiae GS76, rather than S. fradiae GS28, was used. The desired strain was cultured and produced the tylD and tylH gene products and tylosin at levels above that produced in the untransformed strain.
EXAMPLE 8
Construction of Streptomyces fradiae GS48/pHJL280
The desired strain was constructed in substantial accordance with the teaching of Example 3 except that Streptomyces fradiae GS48, rather than S. fradiae GS28, was used. The desired strain was cultured and produced the tylD gene product and tylosin at levels above that produced in the untransformed strain.
EXAMPLE 9
Construction of Streptomyces fradiae GS52/pHJL284
The desired strain was constructed in substantial accordance with the teaching of Example 3 except that Streptomyces fradiae GS52 and plasmid pHJL284, rather than S. fradiae GS28 and plasmid pHJL280, were used. The desired strain was cultured and produced the tylC gene product and tylosin at levels above that produced in the untransformed strain.
EXAMPLE 10
Specific Activity of Rate-Limiting Enzymes and Increased Tylosin Production Using the Present Method
The following Tables demonstrate the effectiveness of the present method. All transformants listed in the Tables were obtained in substantial accordance with the procedure of Example 3. The results indicated in Tables XIII and XIV were obtained from strains cultured in fermentation media (Baltz and Seno, 1981, Antimicrobial Agents and Chemotherapy 20:214-225) that also contained 20 .mu.g/ml thiostrepton if the strain being cultured harbored a plasmid. Note that the transformed strains listed in Tables XIII and XIV are low tylosin-producing, or produce amounts of tylosin that are not readily detectable, and were cultured in the presence of selective pressure (thiostrepton) for plasmid maintenance as an autonomously replicating vector.
TABLE XIII__________________________________________________________________________Specific Activity of the tylF Gene Product, Macrocin O-methyltransferase (MOMT) Transforming MOMT Specific ActivityStrain Plasmid 2 days.sup.1 3 days 4 days 6 days__________________________________________________________________________Run 1 GS15 pHJL210.sup.4 0 0 0 0 GS15 pHJL280 1.14 1.93 NT NT C4.sup.2 None Not tested (NT) 0.35 0.16 NTRun 2 GS15 pHJL210 0 0 0 0 GS15 pHJL280 4.2 3.2 2.2 1.8 C4 None 0.8 1.0 0.9 0.9 T1405.sup.3 None 0.9 1.2 1.5 1.4Run 3 GS15 pHJL210 0 0 NT NT GS15 pHJL289.sup.5 0.5 0.3 NT NT GS15 pHJL311 NT 0.4 NT NT GS15 pHJL315 0.1 0.4 NT NT C4 None 0.2 0.2 NT NTRun 4 GS28 None 0 0.01 0.03 NT GS28 pHJL210 0 0 0 NT GS28 pHJL280 0.8 0.7 1.0 NT GS28 pHJL284 0.9 1.2 0.9 NT C4 None 0.2 0.6 0.5 NT__________________________________________________________________________ .sup.1 days in fermentation. .sup.2 the strain from which GS15 and GS28 were derived. .sup.3 a strain derived from C4. .sup.4 the cloning vector into which the tylgenes were inserted to obtai plasmids pHJL280 and pHJL284. .sup.5 pHJL289 is constructed by ligating the .about.2.3 kb, tylFcontaining, EcoRI BamHI restriction fragment of pHJL284 into pHJL401
TABLE XIV______________________________________Specific Activity of the tylE Gene Product, Demethylmacrocin O-methyltransferase (DMOMT) Transforming DMBMT Specific ActivityStrain Plasmed 2 days* 3 days 4 days______________________________________GS16 pHJL210 0 0 0GS16 pHJL280 1.8 3.7 4.0GS16 pHJL280 3.8 1.7 3.0GS16 pHJL284 1.3 1.6 2.2C4 pHJL210 0.7 1.3 1.5C4 pHJL210 0.2 1.1 1.9C4 None 0.4 1.5 1.0______________________________________
The results in Table XV were obtained from transformants of high tylosin-producing strains that were cultured post-transformation to obtain integrants, transformants in which all or part of the plasmid DNA has integrated into the genome of the host cell. Two methods were used to obtain the integrants. In the first method, transformants are passaged onto selective (contains thiostrepton) and nonselective plates and incubated about 16 hours at 29.degree. C. to obtain single colonies. The single colonies on the nonselective plates that were thiostrepton-resistant on the selective plate are repassaged several times in the same manner until a single colony was found to be relatively stable without selection. In the second method for obtaining integrants, the transformants were nonselectively passaged several times by transferring spores from the surface of the plate using a cotton swab. After several passages, the colonies are grown in non-selective, liquid media (TSB), homogenized, fragmented by sonication, diluted, and plated on selective and nonselective media to identify relatively stable integrants. Other methods of obtaining integrants are apparent to those skilled in the art, and the present method is not limited to a particular method of obtaining integrants.
Relatively stable integrants were used to inoculate vegetative medium (complex vegetative medium contains, per liter, 10 g of corn steep liquor, 5 g of yeast extract, 5 g of soybean grits, 3 g of calcium carbonate, and 4.5 g of crude soybean oil, and the pH is adjusted to 7.8 with NaOH. TSB is also a suitable vegetative media) without thiostrepton (no selective pressure), and the vegetative culture was used to inoculate (10% inoculum) the fermentation medium, which also lacked thiostrepton. Fermentations were run at 260 rpm at 29.degree. C. for seven days. The total macrolide content of the fermentation broth was measured by extraction with methanol:CHCl.sub.3, reading the absorbance at 290 nm, and comparing to a standard curve. Tylosin factors were identified by spotting the fermentation broth onto silica-gel-TLC plates and developing the plates with a solvent system of 95:5 ethylacetate:diethylamine. The concentration of individual macrolide components was the total A.sub.290 times the percentage of each component as determined by HPLC.
TABLE XV__________________________________________________________________________ % Thiostrepton Transforming DMOMT MOMTStrain Resistant Plasmed Specific Activity Specific Activity Tylosin*__________________________________________________________________________C4 0 None 0.59 0.14 1C4 9.5 pHJL280 Not tested (NT) NT 1.10C4 5.9 pHJL280 NT NT 0.97T1405 0 None 1.0 0.17 1.14T1405 50 pHJL280 0.91 0.26 1.52T1405 8.7 pHJL280 NT UT 1.21T1405 11 pHJL280 NT NT 1.07T1405 6.4 PHJL280 NT UT 1.21T1405 18 pHJL280 2.5 0.43 1.60T1405 4.6 pHJL280 NT KT 0.98T1405 16 pHJL280 NT NT 1.07T1405 12 pHJL280 NT NT 1.10T1405 18 pHJL280 NT NT 1.28T1405 25 pHJL280 NT NT 1.28T1405 56 pHJL280 0.82 0.22 1.45__________________________________________________________________________ *Relative to C4 strain

	Number	Date
Parent	198672	Feb 1994
Parent	107232	Jul 1993
Parent	742222	Aug 1991
Parent	351350	May 1989

	Number	Date
Parent	18237	Feb 1987
Parent	890670	Jul 1986
Parent	842330	Mar 1986

Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms

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