MXPA96006114A - Process for dienone macrolides - Google Patents

Abstract

The process of this invention is directed to isolating or otherwise obtaining en olefinic macrolide producing microorganism which microorganism does not contain epoxide enzyme activity. This invention also relates to a process for preparing said olefinic macrolide by fermenting a mutant microorganism lacking epoxidase activity, designated rosX herein, which mutant is obtained from the wild-type microorganism. This invention also relates to a rosX mutant of Micromonospora rosaria, and to any microorganism having the identifying characteristics thereof, said mutant also designated ATCC 55709. This invention also relates to a process to preparing repromicin, the compound of fórmula(II), (see fórmula) by mutating a wild-type microorganism capable of producing rosamicin to produce a mutant microorganism lacking epoxidase activity such that repromicin is produced by said mutant microorganism.

Description

PRQCEDIPiENTQ PflRñ HñCRQLIPQS PE PIENONñ BACKGROUND OF THE INVENTION This invention relates to a method for isolating or otherwise obtaining a microorganism producing olefinic rnacrolides, said microorganism not containing < Je e ci epoxidase. This invention also relates to a process for preparing said olefinic rnacrolide by fermentation of a mutant microorganism lacking epoxidase activity, designated rosX in the present, said mutant being obtained from the wild-type microorganism. This invention also relates to a rosX rosy lichromonospore mutant, and to a microorganism having the identification characteristics thereof, said rn? Tter also designated OTC55709. Rosapucma, the compound of formula I ) is an antibacterial rnacrolide produced by Ha chrome nos pora rosaría fermenter, flTCC 55708.

It is known that the immediate precursor to rosamycin in the biosynthesis thereof is repromycin, the compound of formula II.

(») Repromycin differs structurally from roea icine only in one way: repromycin has a double bond at position C-12 / C-13 where rosamycin has an epoxide. Repromycin can not be isolated directly from the fermentation broths produced by the wild-type flicromonospora rosaria in significant amounts because the organism has an epoxidase enzyme responsible for epoxidation of the C-12 oifen, repromycin C-13 to form the epoxide. C-12, C-13, rosamycin. The repromycin is prepared chemically by the depoxidation of rosamycin. The reaction suffers from very low yields. A high-throughput procedure for the preparation of repromycin is desirable, since repromycin is a well-known key intermediate in the synthesis of various antibiotics having the same ring structure or a similar ring structure as repromycin.

The present invention discloses that repromycin can be prepared by fermenting a rosX mutant of picro onospora rosaría, and particularly of the rosmoría rosmoría micrornonospora rosX mutant which is designated ATTCC 55709 and isolating the reromycin from the fermentation broth. A pink x-chromosome rosmor mutant does not contain enzyme activity ep > oxidase that is found in the wild type of Micromonospora rosaría. As such, said mutant can not produce the epoxidized acrylic acid rosamycin.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a process for preparing a microorganism that can produce an olefinic macrolide comprising inactivating an epoxidase activity of a wild-type microorganism producing an epoxide macrolide. In the present invention it is particularly directed to a process as described in the previous paragraph wherein said olefinic macromido is a dienone macrolide. The present invention is further directed to a process for preparing a microorganism that can produce an olefinic macrolide comprising inactivating the epoxidase activity of a wild type microorganism producing the epoxide macrolide and further comprising the isolation of a gene encoding said activity. epoxidase of said wild-type microorganism producing an epoxy rnacrolide and inactivating said gene. The present invention is particularly directed to a method of the preceding paragraph wherein said olefinic macrolide is a dienone macrolide. The present invention is also particularly directed to a method for preparing a microorganism capable of producing an olefinic macrolide comprising isolating from the wild-type microorganism corresponding to said microorganism a gene which codes for an epoxidase activity and inactivating said epoxidase activity, wherein said The gene encoding said epoxidase activity is isolated from the wild type microorganism through co-plementation. The present invention is also directed to a process for preparing an olefinic macrolide comprising fermenting in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen, a first microorganism that has been prepared by inactivating an epoxidase encoding gene present in the wild-type microorganism corresponding to said first microorganism. The present invention is further directed to a process co or described in the preceding paragraph which further comprises isolating said olefinic macrolide from the fermentation broth.

The present invention is particularly directed to a process as described in the preceding paragraph wherein said olefinic macrolide is a dienone acrylic acid. The present invention is also directed to a process for preparing repromycin, the compound of formula II above, comprising fermenting a mutant microorganism, said microorganism being able to produce repromycin and not being able to produce rosarnicin due to the absence of any activity of epoxidase, in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen to produce a fermentation broth. The present invention is further directed to a method as described in the preceding paragraph which further comprises recovering said re-glycine from the fermentation broth. The present invention is particularly directed to a method of the preceding paragraph wherein said mutant microorganism is a rosX mutant of Micromonospora rosaria. The present invention is directed very particularly to a method of the preceding paragraph wherein said mutant microorganism is the rosX mutant of Micrornonospora rosaria . said mutant being designated as flTCC 55709. The present invention is further directed to a rosX mutant of Micromonospora rosaria.

The present invention is particularly directed to the RosX Micromonospora rosarian specimen which is designated as flTCC 55709. The present invention is also particularly directed to a rosX mutant of My chromospore which has all the identifying characteristics of RTCC 55709. The present invention it is also particularly directed to a rosx mutant of Micromonos or rosaria which is capable of directing isolated amounts of repromycin. The present invention is also directed to a process for preparing repromycin, the method of example II above comprising mutating a microorganism producing rosamycin to produce a mutant microorganism, said microorganism can produce repromycin and can not produce rosamycin due to the absence of any activity of epoxidase and subsequently fermenting said mutant microorganism in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen to produce a fermentation broth. The present invention is particularly directed to a process for preparing repromycin as described in the preceding paragraph wherein said rosamycin-reproducing microorganism is My romonospora rosaría RTCC 29337 or Mirromnnospnra rosaría PTCC 55708. The present invention is further directed to a method as described in the two preceding paragraphs, further comprising isolating repromycin from the fermentation broth. The present invention is further directed to a process for preparing repromycin which comprises fermenting a mutant microorganism in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen. The present invention is particularly directed to a method as described in the previous paragraph wherein said mutant microorganism is a rosX mutant of Micro onuspora rosaria. The present invention is now directed more particularly to a method as described in the previous paragraph wherein said rosmaria Hicromonospora rosaria mutant is designated fiTCC 55709. The present invention is also directed to a method for preparing repromycin as described in any of the three previous paragraphs also comprising recovering said repromycin. The present invention is also directed to a process for preparing an olefinic macrolide comprising a) mutating a microorganism producing an epoxide macrolide to provide a mutant microorganism, said mutant microorganism being able to produce an olefinic macrolide corresponding to said epoxide macrolide; and b) fermenting said mutant microorganism in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen. Where it is used in the present, the term "wild-type microorganism" refers to a microorganism having the epoxidase activity that is absent in the corresponding microorganism. Oditionally, where the term "wild type Micromonospo a rosaría" is used herein, it refers to a rosicrucian Micromonospora culture having epoxidase activity and therefore can produce insoluble amounts of rosamycin, said oxidase activity being absent in the corresponding rosX mutant of Micromonospora rosaria.

DESCRIPTION PETflLLflPR PE Lfl INVENTION icromo ospora rosaría is a microorganism that produces the potent antibiotic rosamycin, which is the macrolide antibiotic that contains epoxide of the aforementioned formula I. Within the genome of Micromonospora rosaría exists an epoxidase gene that codes for an enzyme that converts C-12 / C-13 double bond of repromycin (the compound of formula II mentioned above to an epoxide). As such, the Micromonospora ROSN ODN is used according to the method of this invention to develop a zonda to determine the segment of RDN that codes for the enzyme epoxidase in other microorganisms producing rnacrómidos epoxi. A lyophilized sample of Micromonospora rosaría NRRL 3718 (ATCC 29337) has been deposited in the American-type culture deposit, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, under the terms of the Budapest treaty on September 5, 1995. fl east The newly deposited deposit was given the new deposit number of flTCC 55708. All restrictions on the availability to the public of the microorganism culture so deposited will be irrevocably removed under the patent insurance of this application. A Micromonospora rosaria mutant is a microorganism that produces the macromycin potent macrolide antibiotic, which is a rosamycin derivative that has an olefinic bond between the -12 and C-13 carbons rather than an epoxide. A rosx mutant of Micromonospora rosaría lacks the enzyme epoxidaea that is present in Micromonospora rosaría. Therefore, the olefinic macrolide intermediate of C-12 / C-13 can not be converted to the epoxy macrolide rosamycin by a rosX mutant of Micromonospora rosaria. As such, a Rosfru Micromonospora rosX mutant is used in accordance with the method of the invention to produce repromycin through the fermentation of said rosX methyl ester and isolating repromycin from the fermentation broth thereof in accordance with well-known methods for one skilled in the art. This isolation can be achieved by extracting said repromycin directly from fermentation broth or by filtering said fermentation broth to remove the whole cells and extracting the repromycin from the filtered product. Generally, the volume of the repromycin is recovered by extracting the filtered product. However, packed cells obtained by filtration can also be extracted to recover a small amount of repromycin. It is generally preferred to extract the repromycin from the filtered product after filtering the fermentation broth. A lyophilized sample of the rosmoría Micromonospora rosX mutant (R94-304-99 SC23) has been deposited in the American type culture tank 12301 Parklawn Drive, Rockville, Maryland, 20852, E.U.fl. under the terms of the Budapest treaty on September 5, 1995. 0 this deposited crop was given the new deposit number of ftTCC 55709. All restrictions on the availability to the public of the microorganism culture thus deposited will be irrevocably removed under the guarantee of a patent from this application. The present invention describes the preparation of repromycin, in compounds of formula II above. To prepare repromycin by the method of this invention, a rosX mutant of Micromonospora rosaria. preferably in the amount designated PTCC 55709, it is inoculated into a suitable growth medium and agitated for a sufficient period to ensure production, such as 2 to 4 days. Agitation is preferred for three days. A preferred growth medium is JDYTT, which is prepared by mixing cerelosa (10 g / 1), corn starch (5 g / 1), corn maceration solids (2.5 g / 1), NZ-amine YTT (5 g / 1), CoCla-BHaO (0.002 g / 1), P2000 (1 ml / 1) and CaCOa (3 g / 1) and adding water to achieve a final volume of one liter. NZ-amine YTT is a hydrolyzed protein preparation (casein) and can be purchased from Sheffield Products, Uoods Corner, Norwich, New York. One skilled in the art will recognize that there are many other alternative products or means that can provide sufficient nutrients to ensure growth of the microorganism. Before use, the growth medium is adjusted to pH 7.0 using a weak anhydrous solution of hydrochloric acid if the pH is basic or a weak anhydrous solution of sodium hydroxide if the pH is acidic. Using the preferred medium, the pH of the medium is acidic and therefore must be adjusted by the addition of dilute aqueous sodium hydroxide. Then the growth medium is sterilized for about 20 minutes to one hour, and preferably for 30 minutes at elevated temperatures. A preferred temperature for sterilization is about 120 ° C using the normal autoclave temperature of 121 ° C. Generally the mixture of microorganism and growth medium is agitated with a 5 cm stroke agitator, between 200 to 300 rprn at slightly elevated temperatures. A preferred temperature is 30 ° C. The term "five-centimeter stroke" refers to the horizontal displacement of the flask that is orbitally agitated by an agitator. One of ordinary skill in the art will recognize that any way of providing good agitation of the mixture is sufficient to mix the ingredients. Approximately 20% volume of a suitable cryoprotectant, such as glycerol, is added per volume of the medium of the growth medium mixture of the microorganism, and the culture is stored at -80 ° C for the short term (less than one year ) and under liquid nitrogen for long-term storage. When necessary, the frozen culture is transferred to a flask containing a sufficient amount of a suitable growth medium such as JDYTT and the culture is grown until a substantial amount of microorganism is present. It is preferred to shake the contents of the flask at 250 rpm and at 30 ° C in a 5-centimeter shaker for 3 days. However, other rp s, temperatures and times of the agitator may be satisfactory to obtain effective growth of the desired microorganism. The content of this fermentation is added to a sufficient quantity of a production medium such as RSM-β; RSM-6 is prepared by mixing corn starch (50 g / 1), cerelosa (10 g / 1), burn in PH (5 g / 1), Farmamedia (10 g / 1), MgHP0 ^ -3H20 (10 g / 1), casein hydrolyzate (2.5 g / 1), asparagine (0.5 g / 1), P2000 (1 ml / 1) and adding water to reach a final volume of one liter. One skilled in the art will recognize that there are other means of production that can be substituted by RSM-6, with similar results. When used, RSM-6 is adjusted to pH 7.0 with a dilute aqueous solution of sodium hydroxide and autoclaved for approximately 100 minutes at approximately 121 ° C immediately before use. The fermentation can be carried out in any suitable fermentation vessel such as flasks, New Brunswick fermenter containers (New Brunswick, Scientific, New Brunswick, New Jersey), tanks and the like. This fermentation is carried out at slightly elevated temperatures, generally about 30 ° C, with a stirring speed of 100-1000 rpm. Generally, the preferred stirring speed is about 450 rpm. The agitation of the fermentation vessel provides aeration to the contents thereof. Aeration can also be effected by bubbling air through the mixture. Preferably, the pH of the fermentation broth is controlled during fermentation by the addition of NaOH or HaS0", as necessary, so that the pH remains between 6.7 and 7.3. Fermentation will generally yield the highest repromycin titers between approximately 60 and 120 hours after fermentation begins. Samples containing repromycin are isolated from the fermentation mixture using methods well known to those skilled in the art. A suitable solvent for extracting the repromycin from the fermentation broth is any solvent or mixture of solvents that separates the repromycin from the desired byproducts of the fermentation and that does not react or adversely affect the re-thromycin to be isolated. A preferred solvent mixture for this extraction is a mixture of methanol and potassium diphosphate 0.1M (KHaPO). It is especially preferred that this mixture have a pH of 3.5 and that the relative amounts of methanol and KHaPO_ ". 0.1M are 35% methanol and 65% KHzPO *. This invention is also directed to the preparation of a microorganism that can produce an olefinic macrolide from a wild-type microorganism that ordinarily produces epoxy acrylics. The wild-type microorganisms that are used in the process of this invention are microorganisms that produce epoxide nacrolides and therefore have an epoxidase enzyme that is responsible for catalyzing the intracellular reaction that converts an olefinic macrolide to the corresponding epoxide macrolide. The epoxidase enzyme that catalyzes this intracellular reaction, remains unchanged, ordinarily converts all or essentially all of the intermediate diene to the epoxide. The efficiency of this intracellular process is manifested by the inability to isolate any appreciable amount of olefin from the fermentation broths of these microorganisms. In this way, to obtain these definitions, the microorganism must be prevented from converting the olefin to the epoxide. This is achieved by the process of this invention. The enzyme epoxidase that is present in the Micromonospora rpflflrj ATCC 55708 wild-type is produced by Micromonospora rosaria using presumably ordinary cellular procedures such as transcription of a DNA segment followed by translation of the RNA transcript. The DNA segment responsible for the production of the epoxidase protein by this method is isolated from wild type Micromonospora rosary DNA by methods well known to those skilled in the art, such as complementation; said DNA segment is able to complement the deficient mutant of Micromonospora rosaria ATCC 55709 epoxidase. To obtain the DNA segment responsible for the production of the epoxidase protein by complementation, the following complementation protocol is followed. The gene that directs the formation of the epoxidase responsible for the epoxidation of the C-12 olefin, C-13 of repromycin to the form C-12, C.? A rosamicin epoxide is cloned from wild-type Micromonospora rosaría using mass cloning and trans mutant complementation. The term "mass cloning" is well understood by those skilled in the art and refers to the random insertion of large numbers of different DNA restriction fragments into the plasmids. The complementation here refers to the ability of a cloned genomic fragment to direct the synthesis of the epoxidase and to produce wild-type phenotype when introduced in trans configuration into RosX-rosmarian Micromonospora rosX cells. The trans configuration refers to the intracellular presence of two epoxidase genes on two different DNA molecules. In a trans completion analysis, the mutated epoxidase gene is part of the genome or chromosome of the host culture, and the normal or wild-type epoxidase gene is located in a vector or plasmid molecule. Although trans-complementation is preferred, one of ordinary skill in the art will recognize that cis-complementation can also be used in this complementation protocol. ROSX cells of Mi romonospora rosaría are unable to produce rosamycin because the microorganism lacks the epoxidase enzyme responsible for epoxidation of the C-12 oleophoresis, C-13 of repromycin. As a consequence, the last compound accumulates in the cultivation. However, when a particular DNA fragment, which has the ability to direct the synthesis of the epoxidase enzyme, is introduced into the RosXian rosmorium of Micromonospora cells by normal transformation procedures, the component that is absent or inactivated by the mutation is restored. enzyme function epoxidase). Therefore, as a result of the complementation experiment, the mutated culture regains the ability to epoxidize the repromycin to form rosamycin and now exhibits a wild-type phenotype. This complementation test is used to identify and clone the DNA fragment carrying the epoxidase gene from a nymphosonospore rosary library. The first step in cloning the gene that governs Micromonosoora rosodia epoxidase protein formation is to build a library . This library consists of chromosomal fragments of DNA prepared from the wild type of My romonospora rosaría (for example strain ATCC 55708, described in this application). The terms "genic and genomic" are used herein as synonyms and refer to a group of DNA fragments cloned together that represent the entire genome of the wild type of Micromonospora rosaria. It is well known to one of ordinary skill in the art that Micromonospora rosaria is a gram-positive organism that has an extremely high content of G + C (about 70%) within its chromosomal DNA. Like other species of ñc? InPffl'Ce eS, it is expected that Micromonospora rosaría has a relatively large genome. This genome complexity makes the cloning of large fragments particularly useful when building a DNA library of Micromonospora rosaría. Assuming random DNA cut and optimal representation of the chromosome in the library, the number of clones to be selected is directly related to the size of the genome and inversely to the average size of the DNA inserts. Therefore, to reduce the number of clones to be selected by the epoxidase gene, it is advantageous to clone large pieces of DNA. Furthermore, it is extremely useful in minimizing the number of analyzes and manipulations required to establish a restriction map of a large chromosomal region by identifying contiguous or overlapping clones. Techniques for preparing libraries are well known to those skilled in the art. A general description of the preparation of libraries is described in Desmbtook, J., E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual", 1989, 2a. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. A full description of chromosomal library preparation of actin icetos is described in Hopwood, and others "Genetic rnanipulation of Strepto vces - A Laboratory Manual", 1985, The John Innes Foundation, Norwich, UK. The chronosomal DNA is prepared from a culture of Micromonospora rosaria ATCC 55708 developed in liquid medium as described above. The mycelium is recovered by centrifugation and genomic DNA is prepared following the protocol found in Hopwood, et al., Mentioned above. DNA pellets are resuspended in an aqueous pH regulating solution such as pH regulator TE (Tris 10 rnM-HCl, pH 8.0; EDTA 1 mM). Then the crsenoric DNA is partially digested with a restriction enzyme such as HaelTT or Sau3A. Sau3R is preferred because it recognizes 4-base sequences that produce a random collection of insert fragments. Enzymes recognizing 5-base sequences in this procedure can also be used but are less preferred since such enzymes recognize a less random collection of insert fragments than do enzymes that recognize 4-base sequences. The fragments are ligated into the unique BamHI site present in the multiple cloning site of a vector. The preferred vector is pCD25, a promiscuous cosmid vector, although it will be understood by one of ordinary skill in the art that other promiscuous vectors can be used. A full description of pCD425 and other promiscuous vectors useful for cloning into actinomycota can be found in European Patent Publication No. 0 620280. It is advantageous to clone the gene fragments into a vector that can transform both Eschepchia coli and Micromonospora rosaria. The promiscuous cosmid vector CD425 exhibits a high copy number in Escherichia coli cells and a low copy number of actin icetos cells. It is further preferred to use a vector that exhibits a low copy number or a single one per genome in Micromonospora rosaria, a preferred low copy number vector is pCD425 which has the ability to stably maintain large DNA fragments (up to 40 kb) and avoid possible effects of gene doses. The person skilled in the art will recognize that there are alternative low copy number vectors that are useful in this invention, such as pCD396, which is described in European Patent Publication No. 0 618297. Both pCD396 and pCD425 have the ColEl origin of replication. widely used (see Sambrook, 3 et al., infra) which allows replication in all strains of Escherichia coli commonly used in recombinant DNA experiments, and the SCP2 * origin of replication that allows the maintenance of a low copy number and a broad host scale in actinomycetes (see Lydiate, D. 3., Malpartida, F., and Hopwood, DA, Gene 3 223-235, 1985). These vectors also carry the tsr gene, which confers resistance to the antibiotic thiostrepton under transformation in actinomycete cells, and the amp gene that confers resistance to the antibiotic amplicillin by transformation to Escherichia coli cells. The tsr gene and the amp gene are used to select the transformed cells from the cell mixture. In addition, the promiscuous vector pCD425 is a cosmid vector having AT-rich recognition sequences for different restriction enzymes flanking the BarnHI cloning site. Since the AT-rich recognition sequences are frequently represented in the Micromonospora rosaria genome, there is a great opportunity to find at least one of these enzymes that will allow the recovery of the genomic DNA insert in GC containing the epoxidase gene as a single restriction fragment. This feature is useful for the reconstruction and in vitro map design of a large chromosomal region that contains the epoxidase gene in Micro onosora rosaría. A description of cosmic vectors in general and a list of alternative cosmid vectors is described in Sambrook and other ß? Ora. In this way, the entire genome of the wild type of the Micrornonospora rosaria culture can be represented in the form of randomly generated DNA fragments that are linked to the promiscuous vector pCD425. The genomic library constructed as described above can be used later for an identification and cloning experiment. The identification and cloning experiment refers to the identification and cloning of a particular DNA fragment carrying the epoxidase gene from a library of randomly generated DNA fragments linked to a vector. Plasmid DNA can be prepared from the genomic picroinon SP ra os rí constructed in Escherichia coli by modifying the Brimboim and Doly method described in Brimboim et al., Nucleic Acids Research, 1979, 7, 1513-1525 ,. Other methods for preparing plasmid DNA are found in Sambrook, mentioned above. The plasmid DNA preparation is subsequently used to directly transform protoplasts of Micromo ospora rosaria rQSX. The protoplasts of the picrpmPnPSPPra rosaria rosX cells are prepared following normal procedures such as those described by Hopwood and others, mentioned above. Alternatively, the plasmid DNA prepared from the genomic library Micromonospora rosaria constructed in Escherichia coli is used to transform an intermediate host cell as the TK64 Streptomvces lividians cells. In this way, a genomic DNA library of Micromonospora rosaría is obtained in an intermediate host as Streptomvces livi dians. The plasmid DNA is prepared from the host library and this plasmid DNA is used to transform protoplasts from Micromonospora rosaria following protocols similar to those described herein above. This procedure results in a better transformation efficiency of Micromonospora rosaria cells that can be achieved when plasmid DNA prepared directly from Escherichia coli cells is used directly. The protocols used for the preparation of protoplasts St rentomvces l víti ns, transformation of S ren or ines livida s and preparation of plasmid DNA of host Streptornvces lividans are well known to those skilled in the art and are found in Hopwood, and others, previously mentioned and in the publication number U0995 / 16781 of international patent application. In the transformation of rosarium jchromonospora, cell transformants capable of growing on agar plates containing the antibiotic thiostrepton (thiostrepton resistant transformants (tsr ~ R)) are first selected from agar plates containing the antibiotic thiostrepton as a selective factor. These transformants are detected by visual observation of the agar plates. Those forms that are resistant to thiostrepton are then screened for rosamycin production. In this way, the fermentation products made by the transformants are recovered by the usual way, such as by extraction. These transformants grow in a suitable liquid medium such as RSM-6, described hereinabove, and rosarnicin is extracted therefrom according to the normal methods of macrolide isolation as described herein. The obtained macrolide is characterized by HPLC. A culture exhibiting a rosamycin * phenotype is assumed to be due to the co-suspension of the rosX mutation. Representative rosamycin transformants * subsequently grow under non-selective conditions (following protoplast formation and regeneration), which allows for plasmatic loss, and the resulting thiostrepton-sensitive colonies (tsr-S) are isolated and tested for antibiotic production. These colonies have lost the ability to produce detectable amounts of rosamycin, and instead have produced repromycin. The latter is a strong indication d? that the rosamycin '*' phenotype was due to the presence of the transforming DNA and not to the reversal of the rosX mutation. The plasmid DNA of the transformed cultures of Micrornonospora rosaria is subsequently rescued by transforming competent DH5-alpha cells of Escherichia-Q with total DNA recovered from the representative rosamycin * transformants. Esoueriauia coli competent cells such as the DH5-alpha cells of Esqueriauia coli strain (available from Life Technologies, Inc., Gaithersburg, MD) are transformed with purified plasmid DNA by the vendor-recommended method. The recovered transformants resistant to ampicillin are cultured, plasically isolated and analyzed following normal procedures as described in Sambrook., and others, mentioned above and in any place as known to those skilled in the art. In addition, plasmid DNA is also prepared from the original Escherichia coli library clone. A restriction analysis carried out by comparing the electrophoretic profiles of agarose gßl of plasmid d? The original library and reisolated plasmids must be indistinguishable in all cases. This analysis shows whether the plasmids existed as the unaltered free replicons in the rosamycin * transformants. The epoxidase activity is subsequently rnapeada in the fragment of DNA clonado by means of a combination of analysis of elimination and s? Bclonación, followed by the complementation. To terminate which region of cloned DNA fragment codes for epoxidase, a series of DNA segments of this region is sclized into promiscuous vector CD425 using procedures similar to those described above. These new constructions are subsequently transformed into ATCC 55709 Micromonospora rosaría rosX cells and the new transformants are screened for the production of repromycin. A subclone is identified by its ability to re-establish rosamycin production in the rosX mutant culture. The smallest cloned DNA fragment that exhibits the ability to complement the rosX mutation is sequenced. A preferred sequencing method is the dideoxy sequencing method described in Sanger, and others; Proc. Nati Acad. Sci. USA, 1977.74.5463-5467. The sequence is then analyzed for the epoxidase coding region, using a sequence analysis program. The preferred computer equipment to carry out this sequence analysis is CodonPreference *, which is a trademark of Genetics Computer Group, Inc., Madison, Wisconsin. This computer equipment can be obtained from Genetics Computer Group, Inc., Madison, Wisconsin. When carrying out this sequence analysis, a person skilled in the art seeks the use of characteristic coding and the third position G + C pathways of actinomycete genes. Using this procedure, the open reading frame containing the complete epoxidase gene is identified. The term "open reading frame", when used herein, refers to a continuous sequence of genetic information without any sign of termination. The sequence is translatable into a protein. In particular in this invention, the open reading frame contains the sequence information necessary to direct the synthesis of the epoxidase enzyme during the translation process.

It is known to those skilled in the art that alternative approaches to the completion procedure described above can be employed to clone the gene encoding the epoxidase protein responsible for epoxidizing the C-12, C13 olefin of repromycin to form the C-epoxide. -12, C-13 d? rosamycin of the wild type Micromonospora rosaría. In particular, two alternative approaches include reverse genetics and homology testing. In reverse genetics, the enzyme epoxidase is purified using normal protein purification procedures and the N-terminal amino acid sequence of the enzyme epoxidase is determined. Subsequently, based on this amino acid sequence, a DNA sequence is deduced and used to prepare a synthetic DNA probe. This probe is subsequently used to clone the epoxidase gene from the wild-type genomic library Micromonospora rosaria using a conventional screening approach based on hybridization. Homology probing is accomplished by the derivation of synthetic DNA primers or synthetic oligonucleic DNA probes by comparing other related epoxidases available from the literature. These DNA primers or synthetic oligoeric DNA probes are subsequently used to clone the wild-type genomic DNA gene Micromonospora rosaria using either PCR or hybridization techniques or a combination of both. These and other approaches are well known to those skilled in the art and a general description of these methods can be found in? Arnbrook, et al., Mentioned above. A fragment of this DNA segment is used as a probe to determine the segment of fiDN responsible for the production of epoxidase enzyme, which catalyzes the intracellular epoxidation of olefins in other wild-type microorganisms that generally produce epoxy acrylics. It is well known to those skilled in the art how a small DNA segment of a microorganism can be used as a probe for segments that have similar sequences in other microorganisms. This procedure is described, for example, in many normal laboratory manuals, such as Current Protocols in Molecular Bioioav. Greene Publishing Associates and Uiley-Interscience, Do n Uiley to Sons, NY (1987). Using these techniques, the DNA segment encoding the epoxidase enzyme can be easily determined, which is responsible for catalyzing the intracellular epoxidation of olefinic macrolides to epoxy macrolides found in microorganisms capable of producing macrolide epoxy antibiotics. The following table contains a list of microorganisms that are capable of producing an epoxy macrolide antibiotic and the macrolide epoxy antibiotic, which is produced by the microorganism. This table is of exemplary nature and is not designed to include all microorganisms capable of producing macrolide epoxy antibiotics. One skilled in the art will readily appreciate that the method of this invention for preparing a microorganism capable of producing an olefinic macrolide is capable of being carried out without any microorganism capable of producing epoxy rnacrolides, including but not limited to the microorganisms listed in the table. 1 below.

Table 1.- 16 member ring macrolides containing epoxy groups. Source: S. Om? Ra and H. Tanaka, Macrolide Antibiotics. S. Omuara, Ed., 1984, Academic Press, Orlando, FL 32887, pp.3-36, ("Production and Anti-microbial Activity of Macrolides"). A DNA fragment encoding the epoxidase enzyme was found, using procedures well known to those skilled in the art, of a strain of a microorganism that produces an epoxydienone macrolide. To achieve this, total genomic DNA is hybridized to the probe DNA segment, which has already been isolated from pink njcrpinpnpsppr. Hybridization is achieved using methods well known to those skilled in the art. Thus, genomic DNA is generally cut into DNA fragments using restriction enzymes, such as Bam Hl, EcoRl or other well-known restriction enzymes. It is preferred that the resulting fragments range from about Ikb to about 10kb. Of course, there will generally be fragments that are smaller or larger than l-10kb depending on the sequence of the particular DNA and the particular restriction enzymes chosen. These fragments are analyzed using electrophoresis, generally using an agarose gel. After the separation has been achieved, the fragments are transferred to a nitrocellulose filter and the DNA is fused to separate the chains using a suitable temperature such as 90 ° C to 100 ° C. The Micromonospora rosario probe is made radioactive using well known techniques and is added to the filter in an aqueous solution. The temperature is lowered to approximately 45 ° to 65 ° C to refine the DNA. One skilled in the art will recognize that the optimum for refixation depends on the specific DNA sequence of the particular microorganism that is being hybridized. The probe will bind only to that DNA strand having a substantially complementary nucleotide sequence. The degree of complementarity that will be necessary for the probe to hybridize with the DNA is controlled by the temperature of the hybridization reaction. After obtaining the hybrid DNA, the filter is washed and dried and the DNA segment hybrid / probe is detected by the use of X-ray film autoradiography. If a DNA fragment is hybridized as determined by the film autoradiography X-ray, a DNA library of the strain of interest is then prepared using methods well known to those of ordinary skill in the art. This library is itself subsequently probed with the original radioactive probe of the Micromonospora rosaría to determine which colony in the library contains the gene of interest. The DNA of the colony of interest is then cut using several restriction enzymes until a DNA fragment is obtained which is small enough to conveniently sequence. Those skilled in the art will appreciate that this commonly requires the preparation of a restriction map that organizes the results of the cuts. When a sufficiently small DNA fragment containing the gene of interest is obtained, which hybridizes with the probe, the fragment is sequenced using methods well known to those skilled in the art to determine the neucleotide sequence of the ism. It is generally preferred that the DNA fragment contains only one gene with few extraneous nucleotide bases fixed to each end. However, it will occasionally be impractical to reduce the size of the DNA fragment to be sequenced to less than about 2-5kb. In such situations, the largest DNA fragment is sequenced. The DNA sequence of the gene expressing the protein that catalyzes the epoxidation is thus obtained by this method. To deactivate this DNA sequence so that it is unable to express the enzyme epoxidase for the microorganism of interest, a portion of the DNA of the gene is cut off. Generally, the amount of DNA that is removed is approximately 1 to 30 nucleotide bases in size, although more bases can be removed depending on the particular gene being deactivated and the method used to deactivate the gene. The nucleotide base cut of the gene is achieved using methods well known to those skilled in the art, such as but not limited to the use of restriction enzymes either alone or in combination with exonuclases, and the use of the chain reaction of polymerase (PCR) in combination with a pair of specific mutagenic primers that specifically direct the gene encoding epoxidase. After the cut has been achieved, if the nucleotide bases have been removed from an internal portion of the gene, then two fragments of DNA generated in this way will be ligated to form the gene that contains a deletion. This engineered defective gene is then sequenced to determine the nucleotide sequence of the defective gene. One skilled in the art will realize that a deletion will ordinarily result in deactivation of the gene's ability to express an active protein. However, rarely, a deletion can not result in a deactivation as such. In such rare circumstances, a different portion of the nucleotide sequence will be required to be deleted to deactivate the gene. Alternatively, to ensure complete inactivation of the epoxidase gene an antibiotic marker is inserted instead of the null sequence. The preferred antibiotic marker for this work is the errnE gene of SaccharoDolvsoora erv. raea (formerly Streptomvc.es ervthraeus). For a description of this marker, see Hopwood, and others suora. This marker conferred resistance to erythrocyte for the transformed cells Micromonospora rosaría. Additional useful markers that confer resistance to other antibiotics such as neomycin, hygroicin, vioicin can be used for a similar purpose. For a list of these markers, including the complete restriction map information, see D.A. Hopwood and others suora. Normal cloning procedures are used to insert the ermE marker at the site of elimination of the epoxidase gene. The marker is placed in an orientation opposite to that of the opoxidase open reading frame to avoid a possible lethality caused by the over expression of genes under current. To insert this marker to interrupt a gene of interest see International Patent Publication No. W095 / 16781. The antibiotic marker inserted into the truncated epoxidase gene will facilitate the replacement of the mutated gene in the wild-type chain allowing the monitor the success of gene replacement in the microorganism of interest as will be discussed in the following sections. The wild-type microorganism of the chain from which the gene that was deactivated was obtained, which is capable of epoxidizing the diene to form an epoxide, is then manipulated to contain the gene deactivated by genetic engineering. This is achieved in accordance with gene replacement methods well known to those skilled in the art. Generally, the gene is inserted into a DNA delivery vector such as a bacteriophage, plasmid or cosmid. It is preferred to use a plasmid to deliver the gene engineered to the microorganisms useful in the present invention by techniques well known to those skilled in the art such as transformation or conjugation. The transformation is achieved by transforming the microorganism with the plasmid, which transfers the gene engineered to the DNA of the microorganism. The transfer of the mutated gene into the chromosome of the microorganism of interest occurs when the host chromosome and the plasmid containing a region homologous to it are harvested. It is assumed that the replacement of the gene segments with altered DNA sequences in the plasmid depends on homologous recombination in vivo. Supposedly, two crosses that occur simultaneously or an individual cross that leads to integration and a subsequent resolution step where the integrated gone plase is cut, causes reciprocal exchange between the cloned and resident sequences. By using this proposal, it is possible to interrupt the open reading frame of the epoxidase enzyme coding gene. The alteration involves a chromosomal deletion or an insertion of an antibiotic marker or both. The resulting mutant chain, which lacks the epoxidase activity, is stable and can be used to generate valuable olefinic macrolides that are isolatable by fermentation. The construction of the plasmid, also called integration vector, constitutes a crucial step in the development of the chain ruled by the replacement of the gene. An important feature of the integration vector is that after the replacement of genes in the actinomycete chromosome, the vector is completely eliminated from the host cells. A number of vectors are described in Kieser, T., and Hopwood, D. A. "Methods in Enzymology" (Methods in Enzymology), 1991, vol. 204, 430-458). In the present invention, the promiscuous vector pCD262 is employed in the construction of a deficient epoxidase chain of the microorganism of interest. This vector is part of a series of versatile promiscuous vectors useful for cloning into actinomycetes and Escherichia coli. and for a variety of gene replacement applications in actinomycetes, see, e.g., European Patent Application Publication No. 0 618297. Vector pCD262 carries a replication origin controlling for a moderate high copy number in actinomycetes. When the cells of Micromonospora rosaría that have been transformed with the vector are subjected to extreme conditions, such as high temperature, sporulation, or protoplasting and subsequent regeneration, numerous colonies without plasmid can be recovered. The construction of the plasmid carrying an inactivated version of the gene encoding the epoxidase protein involves the cloning of the deactivated epoxidase gene into the integration vector. The inactivated epoxidase gene is located approximately in the middle of the genomic fragment Micromonospora rosaria. This fragment should be at least 3 kb long to facilitate the exchange of the epoxidase genes (deactivated and wild type) at high frequency. It is understood by the person skilled in the art that the fragment is limited in size by the particular promiscuous vector being used. The integration vector contains several marker genes such as amp that confers resistance to amplicin when the vector is repeating in Escherichia coli and tsr cells. which confers resistance to triostrepton when the vector is introduced by means of the transformation into Micromonospora rosaria cells. In addition, the integration vector contains the ermE marker inserted in the middle of the inactivated epoxidase gene, which confers resistance to erythromycin in the transformed Micromonospora rosaria cells. The preferred vector for these manipulations is the promiscuous vector pCD262. However, it will be understood by those skilled in the art that other integrating vectors and antibiotic markers can also be effectively used to construct the plasmid (see Kieser, t., And Hopwood, DA 1991, Genetic Manipulation of Streptomvces: Integration and replacement vectors. of gene, in two: methods in enzymology, vol 204, 430-458). At the time of transformation, colonies that have been transformed are challenged with an antibiotic that kills all microorganisms that do not contain the labeled gene used to inactivate the epoxidase open reading frame. The only remaining cells are those that have been absorbed by the integration vector. Among these will be those cells that have the gene engineered by defective genetic engineering inserted into the chromosome and no gene expressing wild type epoxidase. The transformed microorganism containing the gene engineered by defective engineering then fermented TS by using fermentation methods well known to those skilled in the art. Because the microorganism is already known to produce an epoxide-containing macrolide, a normal fermentation process will have already been established by this microorganism. The fermentation broth is analyzed for the presence of the epoxidized macrolide which is formed by the wild-type microorganism. The absence of epoxidized macrolide that the wild-type microorganism originally produced and the presence of the diene nacrolide indicates that the microorganism has been successfully made to produce only the diene macrolide precursor. The diene macrolide is isolated by using methods well known to those skilled in the art. To convert the wild-type microorganism into a microorganism that is incapable of producing an epoxide macrolide and thus produces an olefinic rnacrolide as a final and isolable by-product, the gene present in the wild-type microorganism can also be inactivated by chemical mutagenesis. at the molecular level or by other methods well known to those skilled in the art such as gene disruption. One skilled in the art will realize that any methodology involving molecular genetic applications will require identification of the gene as described above. The inactivated gene is then unable to synthesize the enzyme epoxidase in the microorganism. The antibiotic systems carried by the microorganism in this way are stopped in the olefin stage. The olefin is isolated by using the methods taught herein to isolate reromycin or other standard procedures well known to those skilled in the art. To obtain a rosx of Micromonospora rosaría ATCC 55709, a wild type Micrornonospora rosaria culture is mixed with an appropriate growth medium and agitated at 200-225 rrnp at 25 ° C-30 ° C. Those of skill in the art will understand that this method of utagénesis can be carried out in any culture or mutant of Micrornonospora rosaría that contains epoxidase activity to prepare a rosX mutant of Micromonospora rosaria. The suitable growth medium is well known to those skilled in the art. A preferred growth medium for the growth of polyproline is the YPD-6 medium. It is generally preferred to use a YPD-6 force medium. It is preferred that the cultivation of Micromonospora rosaría be a light growing crop. A light growing crop is a crop with a slight possible turbidity as compared to a full growth culture that exhibits heavy, dense growth. A medium strength YPD-6 is prepared by mixing Difco yeast extract (5 g / L), Bacto peptone (5 g / L), dextrose (2.5 g / L) MOPS regulator (5 g / L), and adding enough water to give the total volume to one liter. The pH is adjusted to a pH of 7.0 with dilute aqueous sodium hydroxide. This culture is treated with an agent that induces suitable chemical mutagenesis such as m-ta-sulphonic acid, ethyl ester (EMS) or sodium bisulfite. The concentrations of the molecular agent can be determined empirically by using the criteria established below. When EMS is used, for example, the amount of EMS is generally around 15-50 μL for a 2 mL aliquot of light growth of a culture of the microorganism. The preferred mutagenesis-inducing agent is EMS, the mutagenesis reaction is incubated at temperatures such as 15 ° C to 40 ° C for 3 to 10 hours. It is preferred that the mutagenesis reaction is carried out at 30 ° C for 4.5 to 5 hours with stirring. It is also preferred that the mutagenesis reaction flask be stirred at 30 ° C at 200-225 rrnp. The mutagenase reaction culture is diluted with a suitable medium such as fresh SCM medium and centrifuged. The supernatant is usually removed and discarded. The centrifugation cell pellets are resuspended in a fresh growth medium. It is generally preferred that this growth medium is identical to the growth medium that is used during the initial growth of the Micromonospora rosaría d? wild type. This is concentrated in solid medium to determine the number of survivors of mutagenesis. The protocol continues only if 1000 or more colonies remain. If fewer colonies are discovered, the above procedure is generally repeated and less EMS (15-20 μL) is used. If less than 50% colony death is observed, compared to a non-mutagenized control, then the above procedure can be repeated and more EMS (30 to 50 uL) will be used. Tubes containing survivors are incubated for 2-3 days until the culture is fully grown. Sterile glycerol (80% 2mL) is added to each survivor tube and each tube is placed in a freezer at -20 ° C. After 34-48 hours, the frozen culture is concentrated for future reference. To isolate the rosX meter. the individual colonies of the mutagenized p rent 1 frozen storage are grown and fermented in 2mL cultures. The products of this culture are examined after the broths are extracted with 8rnL of extraction regulator and carried out through an HPLC. The extraction regulator that is used to extract the mutant is identical to the "mobile phase" that is used in the HPLC analysis. One skilled in the art will realize d? that there are other methods of mutagenesis that are capable of producing the RosX number of Micromonospora rosaria. The utility of the macrolide olefins which are prepared by the process of this invention as antibiotics is demonstrated by testing such compounds using procedures well known to those skilled in the art such as those described in Wagrnan et al., Journal of Antibiotics, 1972. 25. 641-646. When used herein, the term "CLAR" ST refers to "high performance liquid chromatography", which is an analytical and isolation technique used by those skilled in the art. The present invention is illustrated by the following examples, but is not limited to the details thereof. P-2000 is polypropylene glycol and is purchased from George Mann & amp;; Co., Inc., 175 Terminal Road, Providence, Rhode Island, 02905. Phar amedia is a protein nutrient derived from cottonseed and is purchased from Traders Protein, The Buckeye Cellulose Corporation, P.O. Box 8407, Memphis, Tennessee, 38108. Ardarnina Ph. Is purchased from Charnplain Industries Inc., 79 State Street, Harbor Beach Michigan, 48441. Pepticase is purchased from Sheffield Chemical, Norwich, New York.

EXAMPLES Example 1 • 1- Preparation of repromycin - fermenter scale To prepare frozen lots to be used as a normal inoculum, Micromonospora rosaria was inoculated in a marked JDYTT medium (cerelosa 10 g / 1, corn starch 5 g / 1, corn solids 2.5 g / 1, amine NZ YTT g / 1, CoCla, dHaO 0.002 g / 1, P2000 1 1/1, CaCOa 3 g / 1) and stirred (250 rprn, 320 ° C, released at 5.6 c) for about 3 days. The TDYTT medium adjusted to a pH of about 7.0, was sterilized at about 121 ° C for about 30 minutes before use. After cell growth was completed, glycerol, final constitution of 20%) was added as a protective trio, and the culture was stored at about -80 ° C. To prepare the inoculum, 5 ml of the frozen culture lot was transferred to 1 liter of JDYTT medium in a 2.8 liter fernbach flask. The culture was allowed to grow for about 3 days at about 30 ° C with shaking (250 rpm, cast at 5.6 cm). The total content of the fernbach flask was transferred to 8 liters of RS-6 production medium in a 14 liter flask (New Brunswick Scientific, NEw Brunswick, NJ) with two 12.06 cm shaker blades. The composition of RSM-6 was corn starch 50 g / 1, cellulose 10 g / 1, ardamine, pH 5 g / 1, farrnamedia 10 g / 1, rngHPO, 3Ha 0 10 g / 1, casein hydrolyzate 2.5 g / 1, aspargin 0.5 g / 1, FeSO 7Ha0 0.028 g / 1 gSO *, 7Ha0 0.5 g / 1, KaHP0A, 0.75 g / 1 uSO * 5HaO 9.003 g / 1 MnCla 4Ha0 0.003 g / 1, ZnSO * 7HaI 0.0003 g / 1 , CoCla 6Ha 0.003 g / 1, P2000 1 rnl / 1. RSM-6 was adjusted to a pH of 7.0 with dilute aqueous OH NaOH and autoclaved for about 9 minutes at about 121 ° C before use. The fermentations were carried out at approximately 30 ° C, 450 rpm, 0.34 v / v / m, air, with controlled pH between 6.7 and 7.3 with NaOH / HaSO or by the addition of g / 1 of MOPS to the production medium. The repromycin titers typically reached a peak between 69 and 1166 hours. Samples were drawn to a solvent mixture 3.5 2.6.5 pH-regulating methanol from kHaP0 * to 0.5 pH) for CIAR testing (Inertsil C-8 column, Su, 250 x .6 nm (Etache Technologies, Torrance, CA), 30 ° C flow rate of 0.6 l / min ^ detected through UV at 280 n Ha0 mobile phase acetonitrile: THF: 60: 28; 12, 0.05% trifluoroacetic acid). Micromonospora rosaria R94-304-99 S23 (ATC 55709) produced 36T-398 mg / 1 of repromycin under these conditions. * 2 repromycin preparation - flask scale. The inoculum was prepared as described above or by the addition of 2 ml of a frozen culture lot to 30 rnl of JDYTT inoculum medium from a 300 ml Erlen eyer flask. The culture was allowed to grow for about 3 days at about 30 ° C with shaking (250 rpm, cast at 5.0 cm). Two inoculum rnl were transferred to approximately 30 ml of modified RSM-5 medium (corn starch g / 1 10 g / 1 Pharmarnedia, 10 g / 1 cereelly, 5.0 g / 1 ardamine pH 0.5 g / 1 aspargin FeSO * 7H »0 9,028 g / 1, gSOA, 7Hβ0 0.5 g / 1, Ka HPO * 0.75 g / 1, CuSO« 5HßIO 0.0002 g / 1, nCla 4Ha0 0.003 g / 1 Zn SO * 7HaO 0.003 g / 1 , OPS 6g / l, casein idrolisate 2.5 g / 1 and gHP04 3Ha0 10 g / 1, P2000 1 rnl / L, P2000 ml / L pH 7.0 (adjusted with dilute NaOH), autoclaved at approximately 121 ° C during approximately 20 minutes) in a 300 ml Erlenrneyer flask. The flasks were shaken for 3 to 4 days at approximately 30 ° C. The fermentation broth was extracted as described above and was subjected to ClAr test Micromonospora rosaria R94-304 99 SC23 (ATCC 55709) produced 455 mg / 1 d? repromycin under these conditions.

Example 2 Preparation of the rosx mutant of Micromonospora rosarí a freshly grown culture slightly Micromonospora rosaría YPD- was mixed with medium resi tence medium and agitated at 200-225 rpm at 28 ° C - 30 ° C. A lightly grown crop is a crop with a slight visible turbidity compared to a fully grown crop that shows dense growth, strong. A medium resistance YPD-6 was prepared by mixing Difco yeast extract (5g / l), bacto-pectone (5 g / 1) dextrose (2.5 g / 1, pH buffer of MOPS in sufficient water so the total volume of the mixture was 1 liter.The pH was adjusted to pH 7.0 with diluted aqueous sodium hydroxide.Rethanesulfonic acid, ethyl ester (EMs, 25 μl was added to this culture mixture / medium (2 ml) and the mixture of The reaction was incubated at 30 ° C for 4.5 to 5 hours with shaking at 200-225 rpm.The culture was diluted with fresh dCSM medium (8 ml), which was centrifuged and the supernatant carefully removed and discarded. Pellets of centrifugation cells were resuspended in YPD media of medium resistance (2 ml) and titrated on solid medium to determine the number of survivors of mtmatogenesis.The protocol was continued only if 1000 colonies or more remained If there were few colonies, the previous procedure was repeated and used less s EMS (15-20μl) If less than 50% of colony deaths were observed, compared to a non-mutagenized control, then the above procedure was repeated and used more. In this experiment, 25 μl produced colonies within the desired range so the procedure could be continued. Tubes containing survivors covered for 2-3 days until the culture was completely grown. Sterile glycerol (80% 2 ml) was added to the tube of surviving colonies and each tube was placed in a -20 ° C freezer after 24-48 hours, the frozen culture was titrated for future reference. To isolate the rosX meter. Isolated colonies on YPD binding medium from the frozen supply of mutagenized original culture were grown and fermented in d? 2 mi. The products of the cultures were examined after the broths were extracted with 8 ml of extraction pH reagent and were canalized by HPLC, the extraction pH regulator that was used to extract the mutant is identical to the "mobile phase". "which is used in the analysis of CLAR. Samples were extracted in a solvent mixture (3.5: 6.5 methanol: pH regulator KHaPO at 0.1M) for CIAR test (Inertsil C-8 column, Su, 250 x 4.6 mm (Metachen Technologies, Torrance, CA), 30 ° C, flow rate of 0.6 rnl / rnin, detected through UV at 2B0 nm, HaO mobile phase: acetonitrile: THF: 60: 28: 12, 0.05% trifluoroacetic acid). The repromycin has a DVmmat of 288 and typically takes time d? 50% more for the? Ir than the rosamycin, therefore the repromycin can be differentiated from the rosamycin by submitting together a sample of the product d? this example and the repromycin. The UVm_ "« for rosamycin is 242 nm. The rosmoría micromonospora rosX mutant was detected and isolated on the basis of its production of repromycin and the absence of any rosamycin.

Claims (28)

NQVEPFíP PE Lfl INVENTION CLAIMS

1. - A method for preparing a microorganism that can produce an olefinic macrolide comprising inactivating the epoxidase activity of a wild-type microorganism producing the epoxide macrolide.

2. A process according to claim 1, further characterized in that said olefinic rnacrolide is a dienone macrolide.

3.- A procedure d? according to claim 2, further characterized in that it comprises isolating a gene encoding said epoxidase activity from said wild type microorganism producing the epoxide macrolide and inactivating said gene.

4. A process according to claim 3, further characterized in that said olefinic rnacrolide is a dienone macrolide.

5. A method according to claim 3, further characterized in that said gene coding for said activity d? Epoxidase is isolated from the wild type microorganism through complementation.

6. A process for preparing an olefinic macrolide comprising fermenting a microorganism prepared according to the method of clause 3 in an aqueous nutrient medium containing assimilable sources of nitrogen to produce a fermentation broth.

7. A process according to claim 6, further characterized in that it comprises isolating said olefinic macrolide from said fermentation broth.

8. A process according to claim 1, further characterized in that said olefinic macrolide is a dienone macrolide.

9. A process for preparing repromycin comprising a) fermenting a mutant microorganism obtained in accordance with the method of clause 3 in a medium d? aqueous nutrient containing assimilable sources of carbon and nitrogen to produce a fermentation broth.

10. A method according to claim 9, further characterized in that it comprises recovering said repromycin from d? said fermentation broth.

11. A method according to claim 10, further characterized in that said microorganism is a mutant rosX Micromonospora rosaría.

12. A method according to claim 11, further characterized in that said RosX miRNA of Micromonospora mutant is designated ATC 55709.

13. A method according to claim 11, further characterized in that said mutant microorganism is the rp§X mutant. of M.ic prnpn0SPPra rosaría. said mutant also designated as ATCC 55709.

14.- A rosx micromonospora rosarnant.

15. A rosX reporter according to claim 14 which is designated ATTC 55709.

16. A rosX mutant according to claim 14, which has all the identifying characteristics of ATCC 55709.

17. - a rpsX mutant of nicrompnpsppra rs in accordance with claim 14, which can produce isolatable amounts of rβpromycin.

18. A method for preparing repromycin comprising a) producing a microorganism producing rosamycin or a nucleus of said microorganism producing rosarnicin to provide a mutant microorganism, said mutant microorganism being capable of producing repromycin; and b) fermenting said mutant microorganism into an aqueous nutrient medium containing assimilable sources of carbon and nitrogen.

19. A method according to claim 18, further characterized in that it comprises recovering said repromycin.

20. A method according to claim 18, further characterized in that said microorganism producing rosamycin is Micromonospora rosaria ATCC 29337 or ATCC 55708.

21. - A method according to claim 18, further characterized in that said rosamycin-producing microorganism is Micrornonospora rosaria ATCC 29337 or ATCC 55708.

22. A method for preparing repromycin which comprises fermenting a mutant microorganism in an aqueous nutrient medium containing sources assimilable carbon and nitrogen.

23. A method according to claim 22, further characterized in that it comprises recovering said repromycin.

24. A method according to claim 22, further characterized in that said mutant microorganism is a RosX specimen of Micrornonosoora rpsarí. A method according to claim 24, further characterized in that it comprises recovering said repromycin. 26. A method according to claim 24, further characterized in that said Micromonospora rosaria is designated as ATC 55709. 27.- A method according to claim 26, further characterized in that it comprises recovering said reprornicin. 28. A process for the preparation of an olefinic rnacrolide comprising a) mutating a microorganism producing an epoxide macrolide to provide a mutant microorganism, said mutant microorganism being capable of producing an olefinic macrolide corresponding to said epoxide macrolide; and b) fermenting said microorganism in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen. SUMMARY PE LR INVENTION The process of this invention is directed to isolate or otherwise obtain a microorganism producing olefinic rnacrolide, said microorganism not containing epoxidase enzyme activity; this invention also relates to a process for preparing said olefinic macrolide by fermenting a mutant microorganism lacking epoxidase activity, designated rosX in the present, said mutant being obtained from the wild-type microorganism; This invention also relates to a RosX Micromonospora rosX specimen: and to any microorganism having the identification characteristics thereof, said mutant also designated ATCC 55709. This invention also relates to a method for preparing reromycin, the compound of formula II . (ID by measuring a wild-type microorganism that can produce rosamycin to produce a mutant microorganism that lacks epoxidase activity such that repromycin is produced by said mutant microorganism. 3J / EA / JN / BS / mvs »ieoh * casv P96 / 681