MXPA97002451A - Vectors and procedure for the production of 6,12-didesoxieritromycin a, of high purity, through fermentac - Google Patents

Abstract

A method for producing 6,12-dideoxytitromycin A, of high purity, using recombinant DNA technology is described. The erythromycin-producing strain, Seccharopolyspora erythraea, which lacks the C-12 and C-6 hydroxylases of erythromycin, produces a mixture of 6,12-dideoxytitromycin A and the precursor molecule, 6-deoxyerythromycin D. To achieve the conversion of the precursor to the final product, a second copy of eryG is inserted into a non-essential region of the Sac chromosome. erythraea that results in 6,12-dideoxytitromycin A, high purity

Description

VECTORS AND PROCEDURE FOR THE PRODUCTION OF 6.12-DIDESOXIERITROMYCIN A. HIGH PURITY. THROUGH FERMENTATION This application claims the benefit of the Provisional Application of E.U.A. No. 60 / 123,456, filed on June 8, 1995.

FIELD OF THE INVENTION The present invention relates to the production of an erythromycin derivative. In particular, the present invention relates to 6,12-dideoxytitromycin A of high purity, through genetic manipulation of the production organism.

BACKGROUND OF THE INVENTION Erythromycin A is a clinically useful, broad-spectrum macrolide antibiotic produced by the gram positive bacterium, Saccharopolyspora erythraea (Sac erythraea). Intermediaries of erythromycin biosynthesis, which may be useful in the design and development of new drugs, are produced in minute amounts by Sac. erythraea and occur as mixtures with other erythromycin derivatives, complicating the chemical modifications of these compounds. It is taught in the technique, (see Donadío et al, Genetics and Molecular Biology of Industrial Microorganisms, eds CL Hershbergeir, SW Queener, and G. Hegeman, 1989, American Society for Microbiology, Washington, DC 20005) the biosynthesis of the erythromycin A through Sac. erythraea, is achieved according to the trajectory on the right hand side, proposed, shown in Figure 1. The 14-member cyclic macrol, 6-deoxyerythronolide B, is first made from thioesters propionyl and 2-methylmalonic, and is then . hydroxylated at position C-6 to form erythronolide B. Micarosa of sugars and desosamine are synthesized from glucose and are added to erythronolide B to make erythromycin D. The next steps in the proposed trajectory are (in any order) the hydroxylation of erythromycin D at the C-12 position (resulting in the formation of erythromycin C) or the methylation of the C-3 position "(resulting in the formation of erythromycin B) Subsequent to the hydroxylation of erythromycin B or methylation of erythromycin C produces erythromycin A. Current understanding of genes responsible for erythromycin biosynthesis and techniques to inactivate genes in Sac. erythraea allows direct path manipulation to produce erythromycin A precursors and derivatives By means of these methods, erythromycin A naturally occurring precursors, such as erythromycin B and erythromycin D, are easily produced. Attempts to make highly pure erythromycin A derivatives, in vivo, are not always successful, especially when alterations are made, which change substrates by enzymes that; they act in the last stages of biosynthesis. It is, in these cases, when additional genetic modifications become necessary.

COMPENDIUM OF THE INVENTION The method of the present invention includes the genetic modification of an erythromycin-producing microorganism, such that it is transformed into a strain that produces high-purity 3"-O-methylated erythromycin derivatives, in particular, a non-essential region of DNA chromosomal is genetically modified by the insertion of a second copy of eryG whose product is 3"-0-methyl transferase, and which normally converts erythromycins D and C to erythromycin B and A, respectively. A microorganism that modalizes the present invention is a novel strain of Sac. erythraea which, under culture in an aqueous medium, produces 6,12-dideoxytitromycin A, of the formula: [1] The transformation of an erythromycin-producing microorganism to a producer strain of 6,12-dideoxytitromycin A is achieved by mutagenic techniques, and in particular, by means of gene replacement by analogous recombination. Using this methodology, the eryF and eryK genes, which encode the cytochrome P-450 enzymes essential for the hydroxylation of erythromycin at positions C-6 and C-12, respectively, are replaced by integrative plasmids, which carry deletions in these genes. As a result of the replacement, the wild type genes with deleted copies, neither the C-6 position nor the C-12 are hydroxylated. As shown theoretically on the left side of Figure 1, a deletion in the eryF gene prevents the conversion of 6-deoxyerythronolide B to erythronolide B; the addition of the sugar groups then results in the formation of 6-deoxyerythromycin D. The second elimination mutation, ie in the eryK gene, prevents the hydroxylation of 6-deoxyerythromycin D to 6-deoxyerythromycin C. In this way , in the absence of a functional eryK gene, the methylation of 6-deoxyerythromycin D directly results in the formation of 6,12-dideoxytitromycin A (which can also be designated 6-deoxyerythromycin B). However, a complicating factor in the formation of a producer strain of 6,12-dideoxytitromycin A is that 6-deoxyerythromycin D serves as a poor substrate for the 3"-0-methyltransferase of erythromycin, which converts the substrate to 6,12-didescxierithromycin A. This results in a low ratio of the desired product of 6,12-dideoxythritromycin A to 6-deoxyerythromycin D, the precursor.Thus, an additional requirement for the production of 6,12-dideoxythritromycin A high purity, is the introduction of a second copy of the gene, eryG, which encodes the 3"-0-methyltransferase, to the production organism In this particular embodiment of the invention, a plasmid was constructed, which allowed a The second copy of eryG, activated by the ermE * promoter, will be inserted via homologous recombination to a non-essential region of the Sac chromosome. erythraea and remained stably in the Sac strain. erythraea.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be readily appreciated in conjunction with the accompanying drawings, in which: Figure 1 is a proposed metabolic pathway for the biosynthesis of erythromycin A (right side) and 6,12-dideoxyrithromycin A in Sac erythraea; (left side); Figure 2 is a flow chart depicting the construction of pDPE4; Figure 3 is a flow chart depicting the construction of pGM504; Figure 4 is a flow chart depicting the construction of pDPE35; Figure 5 is a schematic representation of the replacement of the gene in Sac. erythraea; Figure 6 is thin layer chromatography of fermentation products of ER720-KF; Figure 7 is thin layer chromatography of fermentation products of ER720-KFG +; Figure 8a illustrates the amounts of 6,12-dideoxytithromycin A and 6-deoxyerythromycin D produced in a strain ER720-KF of Sac. erythraea genetically produced by engineering; Figure 8b illustrates the amounts of 6,12-dideoxytithromycin A and 6-deoxyerythromycin D produced in a strain ER720-KFG + from Sac. erythraea genetically produced by engineering; Figure 9 is a flow chart depicting the construction of pKAS37; Figure 10 represents a restriction map of pKAS37; Figure 11 is a flow chart depicting the construction of pKASI37; Figure 12 depicts a restriction map of pKASI37; Figure 13 depicts a DNA sequence of individual strand structure of a DNA fragment contained within the second site region.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for the genetic modification of erythromycin-producing microorganisms, which allows them to produce highly pure erythromycin derivatives, which have -O-methylation at the 3"position of the molecule.The compounds of the present invention include: , 12-didesoxieritromicina A, which is represented by the structural formula [1] This compound was obtained by developing the genetically modified erythromycin-producing microorganism in a liquid culture, and then extracting the compound from culture medium; it was found that the compound is the dominant erythromycin derivative in the fermentation. The present invention provides a method for the preparation of erythromycin derivatives 3"-O-methylated, highly pure, which comprises transforming an erythromycin producing strain into a variant that produces the desired compound. In one embodiment of the invention, the microorganism The erythromycin producer is the bacterium, Sac.Erythraea.After genetic manipulation, the resulting transformant is not only deficient in the P-450 cytochrome enzymes encoded by eryF and eryK, but also contains an additional copy of eryG, which it encodes the erythromycin 3"-0- m 1 i1-iititransferase. It is the presence of this second copy of eryG, which allows the efficient conversion of 6-deoxyeritramycin D to 6,12-dideoxyrithromycin A, and in doing so, provides a strain of Sac. erythraea, which produces a high purity final product instead of a mixture of erythromycins, as shown in Figures 8a and 8b. The present invention also provides, as an example, a particular method for introducing the second copy of eryG to a non-essential region of the Sac chromosome. erythraea, which comprises replacing, by homologous recombination, a section of that nonessential region of the chromosome with a copy of the same region containing, embedded therein, a resistance marker of thiostrepton and a second copy of eryG, which is low the promoter control ermE *. The methods of the present invention are widely applicable to erythromycin producing microorganisms, including but not limited to, Saccharopolyspora, Streptomyces griseoplanus, Nocardia sp., Micromonospora sp., Arthrobacter sp., And Streptomyces antibioticus species. Of these, the most preferred is Sac. erythraea. Of course, the specific sequence of the second homologous site in a non-essential region of a different microorganism may vary somewhat from that shown in SEQ ID NO: 1 for Sac. erythraea, but the method to identify such a site is within the routine experience of those who practice the technique.

The hydroxylations of both C-6 and C-12 are catalyzed by cytochrome P-450 enzymes encoded by the eryF and eryK genes, respectively. It could be predicted that a Sac. erythraemia producing erythromycin, which lacks these two activities, can produce 6,12-dideoxyrythromycin A. A means to eliminate these hydroxylation reactions is through a disabling mutation of the cellular genes required for the operation of the monooxygenase system of cytochrome P-450. This can be achieved by replacing these genes with copies containing deletions, thus making the genes non-functional and non-invertible. Any plasmid designated for gene replacement can be used by homologous recombination, which interrupts the steps of hydroxylation in the biosynthesis of erythromycin. In addition, the method of the present invention is in no way limited to the use of gene replacement to produce defective roots in the hydroxylation of C-6 and C-12 of erythromycin. Other systems can be used, which break the hydroxylase systems, such as gene disruption, transposon mutagenesis or light- or chemical-induced mutagenesis, to produce the desired genetic modification of the microorganism. Such alternative methods are well known to those skilled in the art. Although various methods for inserting foreign DNA into a plasmid to form a gene replacement plasmid are known in the art, the preferred method, according to this invention, is shown schematically in Figures 2 and 3, and is demonstrated in the Examples which are presented later. In a preferred embodiment of the invention, selectable DNA plasmids are constructed, which comprise, (a) a fragment of plasmid plJ702 or plJ486, containing an origin of replication and a DNA fragment that confers resistance to the antibiotic thiostrepton (tsr) , each of which are functional in Streptomyces; (b) an origin of replication and a DNA fragment that confers resistance to the antibiotic ampicillin (amp), each of which are functional in E. coli; and (c) a DNA fragment of the Sac chromosome. erythraea containing the mutated (ie, deleted) gene of interest and at least about 1 kb of the contiguous DNA flanking both sides of the mutated gene, each of which is capable of acting as a recognition sequence for the integration of the plasmid and the subsequent excision of the plasmid from the genome. If the case of excision occurs on both sides of the deletion, opposed to those of the integration case, the wild-type gene will be replaced by the deleted one, as shown schematically in Figure 5. Example 1 and Figure 2 are examples of a plasmid constructed to create a deletion in eryK. AND? I Example 2 and Figure 3 are examples of a plasmid constructed to create a deletion in eryF. The particular genes of resistance to antibiotics and the functional origins of replication, identified above, are necessary not only because they allow the selection and replication of the desired recombinant plasmids. Other functional markers and origins of replication can also be used in the practice of the invention. Likewise, any recognition sequence can be used, which allows the recombinant plasmid to be integrated into a portion of the genome adjacent to the gene of interest and divides the other side of the gene of interest in order to replace that gene with a copy mutated In addition, the plasmid of the invention can be constructed without the use of partial genomic digestion, as in the previous examples. Rather, if the sequences of the regions flanking eryF and eryK are known, a recognition sequence can again be iterated (for example, by polymerase chain reaction) and ligated with the necessary fragments of polymerase chain reaction. origin and resistance to form the gene replacement plasmids. In Example 4, a Sac strain was genetically modified. erythraemia producing erythromycin, appears to be deficient in the C-6 and C12 hydroxylases of erythromycin. This was accomplished by first replacing the wild-type copy of eryK (which encodes hydroxylase C-12) with an deleted copy using the plasmid pDPE4, described in Example 1. As it is said from the proposed trajectory for erythromycin biosynthesis, the mutant strain produced erythromycin B, also producing at the beginning of the fermentation some erythromycin D. The eryF gene (which codes for the hydroxy lasa C-6) of this mutant strain was then replaced by a deleted copy of the gene using the plasmid pGM504, described in example 2. The expected product of this doubly deleted strain, 6, 12-dideoxyrythromycin A, was performed, but the strain also produced large amounts of 6-deoxyerythromycin D through a six-day fermentation, the dominant derivative being 6-deoxyerythromycin D, from days 1 to 6, as shown in Figure 6. In order to produce highly pure 6,12-dideoxytitromycin A, an extra copy of eryG was introduced to a non-essential region of the Sac chromosome. erythraea. The product of eryG is 3"-0-methyltransfe rasa, which normally converts erythromycins D and C to erythromycins B and A, respectively.The preferred method to construct a gene replacement plasmid for the addition of a second copy of eryG to the Sac. erythraea chromosome, is shown schematically in Figure 4 and is described in Example 3. In a preferred embodiment of the invention, a selectable DNA plasmid is constructed, which comprises, a) a fragment of the plasmid pCD1 which contains a functional origin of replication in Streptomyces and Saccharopolyspora; b) an origin of replication and a fragment of DNA that confers resistance to antibiotic ampicillin, each of which are functional in E. coli; and c) a DNA fragment from a region of the Sac chromosome. erythraea of unknown but essential function capable of acting as a recognition site for the integration and excision of the plasmid. (Hereinafter, this DNA fragment of unknown but essential function is referred to as the "second site" region, Figure 3 represents approximately 1kb of the individual chain structure DNA sequence, which is a portion of the second site region). Embedded within the "second site" region are two additional DNA fragments, one encoding 3"-O-methyltransferase operably linked to the ermE * promoter, and a second fragment of the plasmid pWHM3 (also referred to herein as pCS5). , which confers resistance to the antibiotic thiostrepton A culture of E. coli DH5a, which contains a plasmid that modalizes the invention, designated as pDPE35, has been deposited in the Agricultural Research Culture Collection, Peoria , Illinois, and has been agreed upon with accession number NRRL B-21486.As in the previous examples, the particular antibiotic resistance genes and the functional origins of replication identified above are necessary only if they allow the selection and replication of the recombinant plasmid. Other markers and origins of replication can be used, and any fragment can be used DNA, which is homologous to a non-essential region of the Sac chromosome. erythraea, as the integration / excision recognition sequences surrounding the eryG and tsr genes. Example 5 describes the use of pDPE35 in the construction of a Sac strain. erythraea, which has previously been deleted in eryK and eryF, and which now contains an extra copy of eryG. This extra copy of eryG allows the production of 6,12-dideoxytitromycin A of high purity, during a fermentation period of at least four days, as shown in Figure 7. The strain of Sac. erythraea, which has deletions in eryK and eryF, denaminated herein as strain ER720-KFG +, has been deposited with the Agricultural Research Culture Collection, Peoria, Illinois and has been registered with Accession No. NRRL 21484.

A. DEFINITIONS The following words and phrases have the meanings set forth below. The term "cytochrome P-450 monooxygenase system", as used herein, refers to a group of proteins (2 flavoproteins, one iron-sulfur protein and the C-6 or C-hydroxylase enzymes). 12", which work together to cause hydroxylation of erythromycin B or its derivatives in Sac erythraea The term" cytochrome P-450 enzymes "refers to the hydroxylase enzymes of C-6 or C-12 of the system of cytochrome P-450 monooxygenase The term "erythromycin derivative", as used herein, refers to any erythromycin-like compound having antibiotic and / or prokinetic activity.The compounds of the erythromycin type are typically characterized by have a macrolactone ring of 14 members and two sugar molecules linked to O, such as those found in erythromycins A, B, C and D. "Derivatives of erythromycin" are intended to include erythromycin-like compounds that have modifications and / or sub in the macrolactone ring and / or sugar portions, provided that they serve as a substrate for 3"-O-methyltransferase. For example, known common modifications include: 4"-deoxycerithromycin; 6-deoxyerythromycin D; 6,9- eDoxi erythromycin; 6-O-methylerythromycin; 4" -amino-6,4"-dideoxerythromycin A; 9.4" - diamino-6,9,4"-tridesoxerythromycin A; 6,9-hemicetal of 8,9-anhydro-4" -deoxycerithromycin A; 6,9-hemicketal of 8,9-anhydro-4"-deoxycerithromycin B; 6,9-hemicketal of 8,9-anhydro-4" -deoxy-3'-N-desmethyl erythromycin A; 6,9-hemicetal of 8,9-anhydro-4"-deoxy-3'-N-desmethyl-3'-N-ethyl erythromycin A; 6,9-hemicetal bromide of 8,9-anhydro-4" - deoxy-3'-N-propargylethromycin A; 6,9-hemicketal of 8,9-anhydro-4"-deoxy-3'-N-desmethyl-erythromycin B; 6,9-hemicketal of 8,9-anhydro-4" -deoxy-3'-N-desmethyl -3'-N-erythromycin B; 6,9-hemicetal bromide of 8,9-anhydro-4"-deoxy-3'-N- propargilerithromycin B; 9-de!> oxo-4", 6-dideoxy-8-epi-6,9- epoxierithromycin A; 9-deoxo-3'-N-desmethyl-4", 6-dideoxy-8-epi-6,9-epoxy-erythromycin A; 9-deoxy-3'-N-demethyl-4", 6-dideoxy- 8-epi-3'-N-ethyl-6,9-epoxy-erythromycin A; 9-deoxo-4"bromide, 6-dideoxy-8-epi-6,9-3'-N-propargyl-erythromycin A; 9-deoxo-4", 6-dideoxy-6,9-epoxy-erithromycin A; 9-des oxo-3'- N-desmethyl-4", 6-dideoxy '6,9-epoxy-erithromycin A; 9-des-oxo-3' N-desmethyl-4", 6-d idesoxy-6,9-epoxy -3'-N-ethyl-erythromycin A; and 9-deoxo-4", 6-dideoxy-6,9-epoxy-3'N-propargyl-erythromycin bromide A. The term" expression ", as used herein, refers to the combination of intracellular procedures , including transcription and translation undergone by a coding DNA molecule, such as a structural gene, to produce a polypeptide The term "homologous recombination", as used herein, refers to complementary base linkage and interlacing between structures of DNA containing identical or nearly identical sequences The term "origin of replication", as used herein, refers to a DNA sequence that controls and allows the replication and maintenance of a plasmid or other vector in a host cell.

The term "operably linked", as used herein, refers to the control exercised by the promoter upon the initiation of the transcription of a structural gene. The term "promoter", as used herein, refers to a site of recognition on a DNA sequence or group of DNA sequences that provides an expression control element for a structural gene and to which the RNA polymerase it specifically binds and initiates RNA synthesis (transcription) of that gene. The term "restriction fragment", as used herein, refers to any linear DNA generated by the action of one or more restriction enzymes. The term "structural gene" refers to a gene that is expressed to produce a polypeptide. The term "transformation", as used herein, refers to a method for introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast, in which said exogenous DNA is incorporated into a chromosome or is capable of autonomous replication. The term "vector", as used herein, refers to a DNA molecule capable of replication in a host cell and / or to which another DNA segment can be operatively linked, in order to perform segment replication. United. A plasmid is an illustrative vector.

B. BACTERIAL CEPAS, PLASMIDE VECTORS, AND MEANS OF GROWTH The erythromycin producing microorganism used to practice the following examples of the invention was Sac. erythraea ER720 (DeWitt, J.P.J. Bacteriol., 164: 969 (1985)). The host strain for the growth of plasmids derived from E. coli was DH5a from Bethesda Research Laboratories (BRL), Gaithersburg, MD. Plasmid pUC18, pUC19 and pBR322 can be obtained from BRL. Plasmid pCS5 is a multifunctional vector for the integrative transformation of Sac. erythraea. (Plasmid pCS5 has been described by Vara, and others J. Bacteriol 171 (11): 5872 (1989) and was originally designated pWHM3). Plasmids plJ702 (described by Katz et al., J. Gen. Microbiol. 129: 2703 (1983)) and plJ4070, were obtained from John Innes Institute. Plasmid? CD1 was obtained from Claude Dery, University of Sherbrook, Quebec, Canada. Restriction map analysis and partial sequencing have shown that this plasmid is related to pJV1, described by Doull, J. L. and other FEMS Microbiol. Lett. 16: 349 (1983) Sac. erythraea for the transformation of protoplastc and routine liquid culture in 50 ml of the SGGP medium (Yamamoto et al., J. Antibiot, 39: 1304 (1986)), supplemented with 10 micrograms / milliliters (μg / ml) of thiostrepton for the selection of plasmid, where appropriate.

C. REAGENTS AND GENERAL METHODS Commercially available reagents were used to make the compounds, plasmids and genetic variants of the present invention, including ampicillin, thiostrepton, restriction endonucleases (purchased from Sigma Chemical Co., St. Loius, MO), ligase T4-DNA, and calf intestine alkaline phosphatase (CIAP) (purchased from New England Biolabs, Beverly, MA). Normal molecular biology procedures were used (Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1982)) for the construction and characterization of integrative plasmids. Plasmid DNA was routinely isolated through the alkaline lysis method (Birnboim, H.C. and Doly, J., Nucleic Acids Res. 7: 1513 (1979)). Restriction fragments of 0.8-1% agarose gels were recovered with either Prep-A-Gene (BioRad, Hercules, CA) or Gene Clean II (Bio101, Vista, CA). The ligation products for each step of plasmid construction were used to transform the intermediate host, E. coli DH5a (purchased from BRL), which was cultured in the presence of ampicillin to select host cells carrying a recombinant plasmid. When appropriate, the classification for the presence of the DNA insert with X-gal was used. Plasmid DNAs were isolated from the individual transformants that developed in the liquid culture and were characterized with respect to known restriction sites. The integrative transformation of Sac protoplasts. erythraea, and routine development and sporulation, were carried out according to the procedures described by Donadío et al., Science 115: 97 (1991), Weber and Losick, Gene 68: 173 (1988), and Yamamoto and others, J Antibiot. 39: 1304 (1986). The following abbreviations are used throughout the application: a. TES: N-tris (hydroxymethyl) methyl-2-aminoethane-sulphonic acid. b. R3M: a growth medium containing 1 liter aqueous solution: 103 g of sucrose, 0.25 g of K2SO4, 4 g of yeast extract, 4 g of casamino acids, 4 g of tryptone, 22 grams of agar in 830 ml of H2O . The solution was sterilized by autoclaving. After sterilization, the following ingredients were added: 20 ml of 2.5 M MgCl 2, 20 ml of 50% agarose, 20 ml of 2.5 M of CaCl 2, 12.5 ml of 2 M of Tris-HCl, pH 7.0, 2 ml of a trace element solution (Hopwood et al., 1985, Genetic Manipulation of Streptomyces to Laboratory Manual, The John Innes Institute), 0.37 ml of 0.5 M KH2PO4, and 2.5 ml of NaOH. c. PM: a pH regulator containing 1 liter aqueous solution: 200 grams (g) of sucrose, 0.25 g of K2SO4 in 890 ml of H2O, with the addition, after sterilization, of 100 ml of 0.25 M TES, pH 7.2, 2 ml of a trace element solution (Hopwood et al., 1985, Genetic Manipulation of Streptomyces a Laboratory Manual, The John Innes Foundation), 0.08 ml of 2.5 M of CaCl2, 10 ml of 0.5 KH2PO4, and 2 ml of 2.5 M of MgCl2. d. A4Bf: a growth medium containing 1 liter of aqueous medium: 15 g of soy flour, 50 g of glucose, 5 g of NaCl, and 1 g of CaCO3. and. SCM: a growth medium containing 1 liter of aqueous medium: 20 g of soy, 15 g of soluble starch, 10.5 g of MOPS, 1.5 g of yeast extract, and 0.1 g of CaCl2. The foregoing can be better understood by reference to the following examples, which are provided as non-limiting illustrations of the practice of the present invention. Both below and throughout the specification, it is intended that the chas to literature be expressly incorporated by reference.

EXAMPLE 1 Construction of Plasmid pDPE4 PDPE4 was constructed using normal methods of recombinant DNA technology according to the schematic profile shown in Figure 2. A 2.55 kb fragment of EcoRI-PstI, containing eryK and flanking portions of ORF 19 and ORF 21, was isolated from pEVEHβ and ligated to pUC18 cut with the same enzymes to generate the plasmid pDPE1. This plasmid was then cut with Eco0109 / and two of the three generated fragments were isolated (ie, those with sizes of 0.9 and 4.3 kb). These two fragments were ligated to generate pDPE2, which contains a small deletion within the eryK gene. Then, a 2.1 kb EcoRI-Psfl fragment of pDPE2 was ligated to pCS5 cut with the same enzymes to produce pDPE3. An additional contiguous DNA sequence downstream of eryK was added by dividing a 0.765 kb fragment of Psfl containing ORF 19 from pEVEHß and ligating this to Psfl cut, pDPE3 treated with CIAP, to generate pDPE4. Orientation was confirmed by restriction analysis.

EXAMPLE 2 Construction of Plasmid pGM504 PGM504 was constructed using normal methods of recombinant DNA technology according to the schematic profile shown in Figure 3. pGM420, a promiscuous vector of Streptomyces-E, was constructed. coli, cutting pUC18 with Ssfl and ligating this plasmid to the Ssfl site of plJ702. The pUC18 polylinker is oriented close to the BglH, Sph \ and Asp718 sites of plJ702. A 5.3 kb fragment of Psfl from Sac DNA. erythraea containing eryF, flanking and near the DNA that includes part of the eryG, was cloned into the Psfl site of pGM420 to give pMW65. A 0.5 kb outside of frame removal in eryF was made by sequential partial digestions of pMW65 with Asp718 and Ssfl and then filling in the ends of adhesion with poMK and relinking to produce pGM504.

EXAMPLE 3 Construction of Plasmid pDPE35 PDPE35 was constructed using normal methods of recombinant DNA technology according to the schematic profile shown in Figure 4. A 4.5 kb fragment of EcoRI-Sa / Ti HI and of the cosmid p7A2 (Paulus et al., J. Bacteriol. : 2541 (1990)) containing eryG, was ligated to pBR322 cut with the same enzymes, to give pGM403. Then, the EcoRI-Sph fragment from pGM403 containing eryG was ligated pUC18 cut with the same enzymes to general pDPE8. The ermE * promoter (carried on an EcoRI-SamH I fragment of plJ4070) was inserted upstream of eryG towards the EcoR \ -Bgl \ \ sites of pDPE8 to create pKAS2. PKAS2 was digested to complete with EcoRI and then partially with α / ael in order to isolate the 1.5 kb fragment containing the ermE * -eryG fusion. This fragment was ligated to pUC 18 cut with EcoR I and HincW for general pKAS3. PKAS3 was digested with Sspl and Sp? L to obtain a 3.4 kb fragment; this fragment was ligated to the 0.6 kb fragment of pUC 19 cut with the same enzymes in order to add an EcoRI site downstream of eryG for general pKAS4. The ge n eryG was inserted into the "second site" region of AD N de Sac. erythraea as follows. A 1 1 kb fragment of Hind \\\ Sac chromosomal DNA. erythraea was ligated to a derivative of pBR322 for general pGM469. This fragment of Hind \\\ contains a unique Stu \ site, to which an adapter EcoR \ -Stu \ was inserted, to generate pGM473. This plasmid was digested with EcoRI and treated with CIAP. The 1.6 kb EcoRI fragment, from pKAS4 containing the ermE * -eryG fusion, was isolated and ligated to pGM473 to generate pKAS19. Then, the 14 kb fragment of Hind \\\ from pKAS19, containing the "second site" region construct, was ligated to pCDI cut with Hind \\\ and treated with CIAP to produce pKAS 20. The resistance gene of thiostrepton was placed downstream of eryG in the following manner. A 1.1 kb fragment of Bcl \, containing the tsr gene, of plasmid pCS5, was inserted into pUC19 (cut with SamHI and treated with CIAP) to generate pDPE23A. In order to insert a multiple cloning site (MCS) downstream of tsr, this plasmid was digested with EcoRI and Seal and ligated to pUC18 cut with the same enzymes to give pDPE26. Then, a 1.1 kb fragment of Xoal containing tsr could be isolated from pDPE26 and ligated to Xbal cut and treated with CIAP, pKAS20, to generate pDPE34. The removal of the second copy of tsr from pDPE34 was achieved in the following way. The 3 kb fragment of? / El-EcoRI of pCD1, containing an origin of Sac. Replication erythraea was ligated to pUC19 digested with the same enzymes to give plasmid pDPE21. This 15 kb fragment of Hind \\\ Sac DNA. erythraea, containing the promoter ermE *, eryG and tsr of pDPE34, was then ligated to the Hind site of pDPE21 to give the plasmid pDPE35.

EXAMPLE 4 Construction of the Cepa Sac. erythraea eryK. ervF (ER720-KF) An example of a 6,12-dideoxyrylromycin A producing microorganism was prepared by replacing cells, wild-type, ce erK and Sac eryF. erythraea, with deletions in these genes carried by the recombinant plasmids of Examples 1 and 2. The transformation and resolution of the integration case was carried out by the following method. Sac ER720 cells were developed. erythraea in 50 ml of SGGP medium for 3 days, at 32 ° C, and then washed in 10 ml of 10.3% sucrose. The cells were resuspended in 10 ml of pH regulator containing 1 mg / ml of lysozyme and incubated at 30 ° C for 15-30 minutes until most of the meralian fragments were converted to spherical protoplasts. The protoplasts were washed once with PM and then resuspended in 3 ml of the same pH buffer containing 10% DMSO for storage in aliquots of 200 ml at -80 ° C. The transformation was performed by rapidly dissolving an aliquot of protoplasts, centrifuging for 15 seconds in a microfuge, decanting the supernatant, and resuspending the protoplasts in the remaining PM in the tube. 10 μL of DNA solution (3 μL of pDPE4 DNA of Example 1 at approximately 1 μg / μL in 7 μL of PM buffer) was added and mixed are the protoplasts covering the tube moderately. Then, two tenths of a mL of 25% PEG 8000 in pH T buffer (Hopwood et al., 1985, Genetic Manipulation of Streptomyces A Laboratory Manual, The John Innes Institute) were added, pipetted into the solution three times and the suspension was immediately spread on a dry plate of R3M. The plate was incubated at 30 ° C for 20 hours and covered with 2 ml of water containing 100 μg / ml thiostrepton, dried briefly and incubated 4 more days at 30 ° C. To select the members, transformants were replicated in the plate on a non-selective medium of R3M (ie, without thioestreptop), allowed to sporulate and then replicated on the plate on R3M medium containing 10 μg / ml thiostrepton. '0 colonies were incubated in SGGP containing thiostrepton. Of these, 8 were developed and selected as members. The integration of plasmid DNA was confirmed by Southern hybridization, and it was found that the 8 strains, by TLC analysis, form erythromycin A. The integrants were then grown on non-selective R3M and allowed to sporulate. The spores were plated to obtain individual colonies on R3M plates, which were then classified for sensitivity to thiostrepton, indicating the loss of the plasmid sequence of the chromosome. Eight thiostrepton-sensitive colonies were selected, and two of these were conformed by Southern hybridization and through the production of erythromycins D and B to contain the deleted copy of eryK on the chromosome. The replacement of eryF with a deleted copy was performed, as described above for the elimination of eryK, except that the deleted strain eryK was used as the container pGM504 (described in Example 2). By means of Southern analysis, the integration and excision of the plasmid was verified from the Sac chromosome. erythraea, and it was found that the resulting strain, called ER720-KF, produces a mixture of 6,12-dideoxytithromycin A and 6-deoxy-erythromycin D.

EXAMPLE 5 Construction of Sac. erythraea eryK, eryF, 'second site' :: ervG + (ER720-KFG +) A preferred example of the 6,12-c idesoxierithromycin A producing microorganism of the present invention was prepared by transforming ER720-KF cells with the recombinant plasmid of Example 3 (ie, pDPE35) to construct a strain, which produces 6.12 -didesoxierithromycin A highly pure, instead of a mixture of 6,12-dideoxytithromycin A and 6-deoxyerythromycin D. The integration and excision of pDPE35 of the Sac chromosome. erythraea to leave behind a second copy of eryG activated by the promoter ermE *, was made as follows. Cell protoplasts were transformed into ER720-KF with pDPE35 as described in Example 4. In order to solve the duplication created by the integration of the plasmid into a region of homology of unknown function but not essence on the Sac chromosome. erythraea, and as a result of leaving behind eryG and the resistance marker of thiostrepton carried by the plasmid, the transformants were striated twice consecutively on plates of R3M containing thioestrepton. Those colonies that were able to develop after two passages on thiostrepton, were found by Southern analysis, which contains a second copy of eryG integrated to the region of 'seg undo s itio' of the chromosome. The strain was designated as E R720-KFG +.

EXEM PLO 6 Fermentation of ER720-KF v ER720KFG +. and Identification of compounds produced by the two strains The cups of Sac. Recombinant erythraea, produced in Examples 4 and 5, were cultured using the following fermentation procedure. Seed cultures of 600 ml of E R720-KF / ER720KFG + were grown in an A4Bf medium in two liter flasks with cotton plugs at 32 ° C, at 225 rpm for 48 and 72 hours, respectively. Inoculated, with 1.5 liters of seed stock, LH 45-liter fermenters (Incel Tech, Hayward, CA) containing 20 liters of SCM medium (with thiostrepton added at 10 μg / ml for ER720-KFG +) . Cells were grown at 32 ° C, at 250 rpm with a head pressure at 0.3515 KG / cm2, and an aeration rate of 0.7-1 volumes of 02 / culture volume / minute. Antiespumeinte was added at 0.01%, initially, and the pH was controlled at 7.0 with propionic acid and KOH. Culture samples were taken at 0, 24, 40, 48, 66, 72 and 144 hours for ER720-KF and at 24, 40, 48, 66, 72, 88 and 144 hours for ER720-KFG +. Erythromycin derivatives were isolated from the culture broth of the producing strains by the following procedure. Cells of 1.5 ml of culture were removed, by centrifugation for one minute in a microfuge. One ml of supernatant was removed to another tube and the pH was adjusted to 9.0 by the addition of 6 μl of NH OH. Half of a ml of ethyl acetate was added, the tube swirled for 10 seconds and then centrifuged for about 5 minutes to separate the phases. The organic phase was removed to another tube, and the aqueous phase was extracted with 0.5 ml of ethyl acetate. The second organic phase was combined with the first / dried in Speed Vac. The residue was taken up in 11 μl of ethyl acetate and 1 μl was placed as spots on the TLC plates. A normal curve of 6,12-dideoxyrythromycin A was also included to ensure that the amounts of compound applied to the plate were on the linear scale of the detection method. Silica gel thin layer chromatography plates (Merck 60F-254) were developed using isopropyl ether-methanol-NH 4 OH (7535: 2). The compounds were visualized by spraying the plates with anisaldehyde-sulfuric acid-ethanol (1: 1: 9). With this reagent, 6,12-dideoxythritromycin A and 6-deoxyerythromycin D appeared as blue spots and were further identified by comparing their Rf values (relationship of movement of the spot to the frontal movement of the solvent) with those of the normals (see Figures 6). and 7). The ratio of 6,12-dideoxytitromycin A to 6-deoxyerythromycin D, produced by genetically engineered strains, was analyzed by measuring the spots by TLC with a Molecular Dynamics Personal Densitometer (PD-120 Laser-Based Transmission Scanner) 100 μm resolution. Figure 8a shows that in a 6-day fermentation, while the strain lacking hydroxylases of C-6 and C-12 produced 6,12-dideoxytitromycin A, it also accumulated a large amount of the non-methylated precursor, 6-deoxyerythromycin D. However, as shown in Figure 8b, when an extra copy of the 3"-0-methyltransferase gene was added to a non-essential region of this strain, it was able to overcome the accumulation of 6-deoxy-eritromicinei D, and convert this precursor to 6, 12-dideoxythromycin A.

EXAMPLE 7 Construction of Plasmid pKAS37 PKAS37 was constructed using normal methods of recombinant DNA technology according to the schematic profile of Figure 9. The ermE * promoter from plJ4070 was inserted into the SamHI / EcoRI sites of pUC19, to give pKAS7. A polyadaptator region, including the Kpn sites a / g / ll, was removed from plJ4070 to pUC19 to give pKAS8. PKAS7 was digested with Ssp \ BamY and the ermE * promoter fragment inserted into SspMBglW was digested pKASd to give pKAS16. To remove the PvuU site, plasmid pKAS16 was digested with? / Ael / EcoO109, filled with Klenow and linked to generate pKAS17. Then, pKAS17 was digested with Sspl / H / pd? Ll and the ermE * fragment was ligated to a similarly digested plJ4070, to give pKAS18. The tsr gene was divided from pCS5 by digestion of Bcl \ and ligated to pUC19 digested with BamHl, dephosphorylated with CIAP to give pDPE23B. Then, pDP23B was digested with Ssp \ / Nde \ and the fragment ermE * was isolated from pKAS18 digested with Ssp \ / Ase \ and ligated to generate pKAS23. pKAS23 was digested with Ssfll / PvtIl and the 1.1 kb fragment of Ssfll / Smal from pCS5 were ligated to give pKAS33. pDPE35 was digested with Hind \\\ / Kpn \ and the replication fragment pCD1 was ligated to a similarly digested pGM469 to give pKAS30. p <; AS30 was digested separately with Hind W and Kpn \ with concurrent filling with Klenow to give pKAS34. pKAS34 was partially digested with Stu, dephosphorylated with CIAP and the fragment containing ermE * and the tsr gene of pKAS33 was digested with Sspl / Fspl, and ligated to generate pKAS35 (-). PDPE36 was generated through the digestion of pDPE21 with Mlu \ INde \, filling with Klenow and ligand. PKAS35 (-) was digested with EcoR \ IStu \ and the pCD1 replica of similarly digested pDPE36 were ligated for general pKAS36. pKAS36 and pKAS35 (-) were digested with Sspl / EcoRI to generate pKAS37. A detailed restriction map of this plasmid is shown in Figure 10. A culture of E. coli DH5a, which contains plasmid pKAS37, was deposited, as previously, in the Agricultural Research Culture Collection and has been under Accession No. NRRL B-21485.

EXAMPLE 8 Construction of PKASI37 Plasmid Plasmid pKASI37, an alternative modality of pKAS37, was constructed using recombinant DNA technology methods according to the schematic profile of Figure 11. To obtain the 'second site' region of pKASI37, the chromosomal DNA of ER720 or NRRL2338 of Sac. erythraea was digested with Hind \\\ and approximately 12 kb fragments of 0.7% agarose gel were isolated. This fragment combination was ligated to pUC19 (also digested with Hind \\\). Transformants were classified for plasmids carrying the second site by digestion of miniprep DNA with SamHI, Mlu \ and Sful to generate the expected fragments of 6.8, 5.6 and 2.1 kb (for Bam \), 11.3, 3.3 and 0.3 kb (for Mlu \) and 14.9 kb (for Stu). Appropriate orientation of the Hind fragment was determined by Kpnl digestion, since the Kpn site at one end of the 'second site' region must be adjacent to the Kpn site in the pUC19 polylinker. The resulting plasmid is pX1. Then, plasmid pCD1 was digested with Mlu \, treated with Klenow and digested with Kpnl. The resulting fragment was approximately 3 kb containing the replica of Sac. erythraea, ligated to pX1 digested with? / del (filled with Klenow) and Kpnl to form plasmid pX2. Then, plasmid pX2 was digested with Kpn \, treated with Klenow and religated to give plasmid pX3. PX3 was digested with Hindlll, treated with Klenow and re-ligated to give the plasmid pX4. The final steps of plasmid construction involve insertion of promoter cell ermE *, polyadapter and tsr gene. The plasmid plJ4070 was digested with Kpnl, treated with Klenow and ligated again to form the plasmid pX5. Then, two oligonucleotides were synthesized, which when strengthened will contain the following restriction sites: ßg / l-EcoRI-Kpnl-Xbal- / V / ndlll-ßg / ll-SamHI-EcoRI-Psfl (synthesized polyadaptator). This double structured chain fragment was ligated to pX5 digested with Sa HI and Psfl to give plasmid pX6. Then, the plasmid pSC5 was digested with Bell and the resulting fragment containing 1 kb of tsr was ligated to the SamH I site of pX6 to give the plasmid pX7. Next, pX7 was partially digested with EcoR I, treated with Klenow and the 1 kb fragment of DNA containing the tsr polyadapter gene synthesized with the ermE * promoter, inserted into the unique Stul site of the 'second site' region of Sac . erythraea in pX4 to form the plasmid gone pKAS I37. A detailed restriction map of plasmid pKASI37 is shown in Figure 12.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: Diane L. Stassi Gregory T. Maine David A. Post Mark Satter (ii) TITLE OF THE INVENTION: VECTORS AND PROCEDURE FOR THE PRODUCTION OF 6,12-DIDESOXIERITROMYCIN A, OF HIGH PURITY, THROUGH FERMENTATION (iii) NUMBER OF SEQUENCES: 1 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Abbott Laboratories (B) STREET: 100 Abbott Park Road ( C) CITY: Abbott Park (D) STATE: Illinois (E) COUNTRY: USA (F) POSTAL CODE: 60064-3500 (v) "COMPUTER LEGIBLE ORMA: A) TYPE OF MEDIUM: flexible disk B) COMPUTER: IBM PC compatible C) OPERATING SYSTEM: PC-DOS / MS-DOS D) PROGRAM: Patent In Relay # 1.0, Version # 1.30 (vi) CURRENT INFORMATION OF THE APPLICATION: (A) REQUEST NUMBER: (B) DATE OF PRESENTATION: C) CLASSIFICATION: (viii) EMPLOYEE / AGENT NORMATION: A) NAME: Andreas M. Danckers B) NO. REGISTRATION: 32,652 [C) NO. REFERENCE / APPORTER: 5789. US.01 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 708 / 937-9803 (B) TELEFAX: 708 / 938-2623 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 917 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: double (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: genomic DNA (iii) HYPOTHETIC: No (v) TI = OR OF FRAGMENT: internal (vi) SOURCE OF ORIGIN: Saccharoolyspora erythraea (ix) ASPECT: (A) NAME / KEY: 1 kb from the region of second site (B) LOCATION: (C) OTHER INFORMATION: non-essential function (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAGCGACCAC AGGTGGGCCC GGATGTTGCA GCCTTGGTCG GGGTAGTCGA GCGGATTCG 60 GAACAGTGCC ACGGCTGTGG TGTTCGAAGG TGGAAG CTT GAGCTGCTGG TGCCACCGGA 120 TTGCTTGCTC CAGCGAGACC GCGTTGCCGT TGACGAAGGC CAACGCGTCA AACACCGCCT 180 GGGAGTGCTC GGGTCGCAGT TTCTTCAAGT CATCGCTGAG AATCCCGGCA CCGAGCGTGA 240 TAGGCATCCT GCACCGCCCC ACACGGCGCG GAGATTGCGG TCCAGGCCCG GCAACATACC 300 AGCGCTTCGT CGAACTCGTC CGCCTCGACG TGGGCCCGCA GTTGTTCCGC GAACACTGCG 360 CAGTTCGGA CAGCTTCTGG CCCAGGGCTT GCGACAACCT TGGGTGGGGT GTGCGCGGGG 420 TTGGTGCTG ^ AGTCGTTGCG GAAACCCAGC ATCGTCAGAG CGTGGTCGAA CTGTGCTGGA 480 CTGAGGTGCT CAGACAGCAC ACGAATCCAG CTCCCTGCCG GTGTGCTGCC AGAAGGGGAC 540 CGCGAGGCCC GCGGAATCTC CGCCGGATCG CCCCGAAGCC GACCCAGCTC ACGCAACACC 600 GAATCGGTGT CCGGCCGAGG TGACCGTGTG CCCGACCCGG AGCCGGGAGC ACGCCGCGCA 660 CTGGGCCTC; TCGGTTGTGT GTGTGAGATC GTCGTTCCTC GAATTTAAGC AAGCCGGCGA 720 TGAACTTCG .: CCGGCGCGCG GACAACGTCG TCACATCACC GTCCGCCCCG ACGCCAGAAG 780 CCGAGCCAGC: CCCCGCACTG CGGCCCGAAC GGAACCTCCT CGGAAGTTAC GCCGGAGCTG 840 CCCGGTGCCG CCGTGGTCAG GAAAGCCTGC GCGTGCTGAG GGAGCCGTCC ATGTTGATAA 900 TTATTATCTC AGATGAC 917

Claims (2)

1. - A recombinant DNA vector for integrating a DNA sequence of interest to the chromosome of an erythromycin-producing host cell, said vector comprising a first DNA sequence from a non-essential region of the chromosome Saccharopclyspora erythraea, a second DNA sequence allowing the replication in a particular host cell and a third sequence of DNA encoding a selectable marker gene. 2 - The DNA vector according to claim 1, wherein said second DNA sequence is derived from plasmid pCD1. 3. The DNA vector according to claim 1, further comprising a DNA sequence encoding the ermE * promoter, wherein said ermE * promoter is operably linked to a gene of interest. 4 - The DNA vector according to claim 3, wherein said gene of interest is the eryG gene. 5. The DNA vector according to claim 1, further comprising a multiple cloning site. 6. The DNA vector according to claim 1, wherein dichD vector is the plasmid pKAS37. 7. The DNA vector according to claim 6, further comprising the eryG gene. 8. The DNA vector according to claim 1, wherein said vector is the plasmid pDPE35. 9. A method for making a derivative of erythromycin 3"-metilade ?, substantially pure, said method comprising: a) introducing an integrating recombinant vector containing the eryG gene into host cells, wherein said host cells p-oduce a mixture of said erythromycin derivative 3"-methylade? and a non-methylated erythromycin 3"derivative: b) selecting stable members for said host cells, said members having the eryG gene stably integrated to a non-essential region of the chromosome of said members, c) culturing said stable members in a medium of culture, and d) isolating said 3-methylated erythromycin derivative from the culture medium. 10. The method according to claim 9, wherein said integration recombinant DNA vector is the vector of claim 1, claim 4, claim 6 or claim 8. 11.- A method to make a 6 , 12-dideoxythromycin A, substantially pure, said method comprising: a. introducing an integrative recombinant vector containing the eryG gene into host cells, wherein said host cells produce a mixture of said 6,12-dideoxyrithromycin A and 6-deoxyeri: romicin D; b. selecting stable members for said host cells, said members having the eryG gene stably integrated to a non-essential region of the chromosome of said members; c. cultivate said stable members in a culture medium; and d. isolating said 6, 12-dídesoxieritromicina A from the culture medium. 1

2. The method according to claim 11, wherein said integrating recombinant DNA vector is the vector of claim 1, claim 4, claim 6 or claim 8. 13 - Jn method for increasing the activity of 3"-0-methyltransferase in host cells, which produces at least one substrate for said 3" -0-methyltransferase activity, said method comprising: a. introducing a recombinant integration vector containing said eryG gene into said host cells, said eryG gene encoding said activity of 3"-0-methyltransferase, and b) selecting stable integrants of said host cells, said members having the eryG gene stably integrated to a non-essential region of the chromosome of said members 14 - The method according to claim 13, wherein said integrating recombinant DNA vector is the vector of claim 1, claim 4, claim 6 or claim 8. A method for making a modified deoxyeritrichycin producing host strain, wherein said modification is an increase in the activity of 3"-0-methyltransferase, said method comprising: a. introducing an integrative recombinant vector containing the eryG gene into said host strain, said eryG gene encoding the activity of 3"-0-methyltransferase; and b. selecting stable members of said host strain, said members having the eryG gene stably integrated to a non-essential region of the chromosome of said members. 16. The method according to claim 15, wherein said integrating recombinant DNA vector is the vector of claim 1, claim 4, claim 6 or claim 8. 17.- A host strain that produces a erythromycin derivative, said host strain being modified to produce the activity of 3"-0-methyltransferase, wherein said modification is stable to the integration of the eryG gene encoding said 3" -0-methyl-ansferase activity to a non-specific region. essential chromosome of said strain.