MXPA98004995A - Genes of sugar biosynthesis associated with policet - Google Patents

Abstract

The present invention provides polynucleotides from Saccharomyces erythraea which encode enzymes involved in the biosynthesis of sugars associated with polyketide. Methods for using the polynucleotides to produce novel polyketides modified by glycosylation are also provided.

Description

GENES OF BIOSYNTHESIS OF SUGAR ASSOCIATED WITH PO ICETIDA This application claims the benefit of E U A Series No. 08 / 576,626 filed on December 21, 1995, now pending FIELD OF THE INVENTION The present invention relates to methods for directing the biosynthesis of specific polyketide analogs by genetic manipulation. In particular, sugar biosynthesis genes are manipulated to produce precise, novel macrolides modified with ghcosylation of predicted structure.

BACKGROUND OF THE INVENTION Polyketides are a large class of natural products that include many antibiotic, antifungal, anticancer, and anti-helminthic compounds such as epitromycins, amphotepins, daunorubicins, and avermectins. Their synthesis comes from an ordered condensation of acyl esters to generate carbon chains. side chain with variant length and reduction pattern that are differentially cycled and subsequently modified to give the mature polyketides. For many polychaetes, maturation includes the addition of one or more sugar residues to the cycled carbon chain. Sugar residues are frequently critical to the biological activity of the mature polyketide Streptomyces and the closely related genera of Saccharopolyspora are prodigious producers of pohcetide metabolites. Due to the commercial importance of these compounds, a great deal of effort has been spent in the study of genetics of Streptomyces C consequently, much is known about Streptomyces and there are several cloning vectors for introducing DNA into these organisms. Although many polyketides have been identified, the need remains to obtain novel modified polyketide structures by glycosylation (as defined herein) with improved properties. Current methods for obtaining such molecules include sifting biological samples and chemically modifying existing polyketides, both of which are costly and time consuming. Current screening methods are based on net properties of the molecule, ie, antibacterial, antifungal activity, etc. , and both an a priori knowledge of the structure of the molecules obtained and the predetermination of improved properties are almost impossible. The normal chemical modification of existing structures has been successfully used, but is limited by the number of types of compounds obtainable. Poor synthetic synthesis of multiple steps often limits the practice of this proposal The following modifications to sugar residues bound to pohcetides are particularly difficult or ineffective at this time changes the stereochemistry of specific hydroxyl or methyl groups, changes the oxidation state of specific hydroxyl groups, and deoxygenation of specific carbons Accordingly, there is a need to obtain molecules wherein said changes are specified and made that would represent an improvement in the technology to produce modified polyketide molecules with altered glycosylation with predicted structure. The present invention overcomes these Problems in providing the genetic sequence of sugar biosynthesis genes in the biosynthesis of sugars associated with polyketide BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a single or double chain isolated polynucleotide, typically DNA, having a nucleotide sequence comprising (a) a nucleotide sequence selected from the group consisting of (i) the sense sequence of Figure 4A (SEQ ID NO 1) from about nucleotide position 54 to about nucleotide position 1136, (n) the sense sequence of SEQ ID NO 1 from about nucleotide position 1147 to about the position of nucleotide 2412, (m) the sense sequence of SEQ ID NO 1 from about nucleotide position 2409 to about the nucleotide position 3410, (iv) the sense sequence of Figure 4B (SEQ ID NO 2) from about nucleotide position 80 to about nucleotide position 1048, (v) the sense sequence of SEQ ID NO 2 around the position of nucleotide 1048 at about nucleotide position 2295, (vi) the sense sequence of SEQ ID NO 2 from about nucleotide position 2348 to about nucleotide position 3061, (vn) the sense sequence of SEQ ID NO 2 from about nucleotide position 3214 to about nucleotide position 4677, (vm) the sense sequence of SEQ ID NO 2 from about nucleotide position 4674 to about nucleotide position 5879, (ix) the sequence of sense of SEQ ID NO 2 from about nucleotide position 5917 to about nucleotide position 7386, and (x) the sense sequence of SEQ ID NO 2 from about nucleotide position 7415 to about the position nucleotide ion 7996, (b) sequences complementary to the sequences of (a), (c) sequences that, in expression, encode a polypeptide encoded by the sequences of (a), and (d) analogous sequences that hybridize under demanding conditions to the sequences of (a) and (b) A preferred molecule is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. In another embodiment, a DNA molecule of the present invention is found in an expression vector. preferably expression vector further comprises an enhancer promoter operably linked to the polynucleotide. In a preferred embodiment, the DNA molecule in the vector is one of the preferred sequences mentioned above. In an especially preferred embodiment, the DNA molecule in the vector is the sequence of SEQ ID NO 2 from about nucleotide position 80 to about nucleotide position 1048 The present invention further provides a transforming host cell with a polynucleotide or expression vector of this invention Preferably, the host cell is a bacterial cell selected from the group consisting of Saccharopolyspora spp, Streptomyces spp and E coli. The present invention also provides methods for producing novel structures of modified polyketide with glycosylation when designing and introducing specific changes in the governing DNA synthesis and binding to sugar residues to polyketides. According to one method, the biosynthesis of specific polyketides modified by ghcosylation is achieved by genetic manipulation of a microorganism that produces polyketide comprising the steps of isolating a DNA sequence containing the sugar biosynthesis gene from those described above; identify within the DNA sequence containing the gene one or more DNA fragments responsible for the biosynthesis of a sugar associated with polyketide or its binding to the polyketide, create one or more specific changes in the fragment or DNA fragments, thus resulting in an altered DNA sequence, introducing the altered DNA sequence into a microorganism that produces polyketide to replace the original sequence with the altered DNA sequence, when translated, results in altered enzymatic activity capable of carrying out the production of the specific modified polyketide by ghcosylation, growing a culture of the microorganism which produces altered polyketide under conditions suitable for the formation of the specific polyketide modified by glycosylation, and isolating said modified specific polyketide by glycosylation of the culture. In a second method, the biosynthesis of specific polyketides modified by glycosylation is achieves by isolating a DNA sequence containing a sugar biosynthesis gene of those described above, to identify within the DNA sequence containing the gene one or more DNA fragments responsible for the biosynthesis of a sugar associated with polyketide or its attachment to the drug; inverting the strand orientation of the fragment or DNA fragments, thus resulting in an altered DNA sequence which, when transcribed, results in the production of an anti-sense mRNA, introducing the altered DNA sequence into a microorganism that produces pohcetide which has an mRNA capable of binding the anti-sense mRNA resulting in altered enzymatic activity capable of producing the specific polyketide modified by glycosylation, growing a culture of the microorganism that produces altered polyketide under conditions suitable for the formation of the polyketide modified by glycosylation, and isolate the specific polyketide modified by glycosylation In a third method, the biosynthesis of specific polyketides modified by garylation is achieved by isolating a DNA sequence containing sugar biosynthesis gene from those described above, identifying within the DNA sequence containing one or more gene fragments of DNA responsible for the biosynthesis of a sugar associated with pohcetide or its binding to the polyketide, introducing the fragment or fragments of DNA into a microorganism that produces pohcetide wherein the transcription and translation of the fragment or fragments of DNA generate a microorganism that produces altered polyketide which is capable of producing the specific polyketide modified by glycosylation; growing a culture of the polyketide-producing microorganism containing the DNA fragment or fragments under conditions suitable for the formation of the specific polyketide modified by glycosylation, and isolating the modified specific polyketide by glycosylation of the culture. Preferably, the DNA sequence containing the gene. of sugar biosynthesis of the processes described above includes genes encoding an enzymatic activity involved in the biosynthesis of L-micarosa and / or D-desosamine. More preferably, the DNA sequence containing sugar biosynthesis gene comprises the sequence of SEQ ID NO 2 from about nucleotide position 80 to about nucleotide position 1048. The present invention is especially useful for manipulating sugar biosynthesis genes from Streptomyces and Saccharopolyspora, organisms that provide more than half of clinically useful antibiotics BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A illustrates the organization of the epitomycin biosynthetic gene cluster and the genetic designations of biosynthetic genes, Figure 1B illustrates a biosynthetic scheme of abbreviated erythromycin that broadly associates biosynthetic genes with their role in ephromycin biosynthesis Seven eryB genes, eryBI - eryBVIl, are responsible for the biosynthesis of L-micarosa or its attachment to the ring of eptronohda B, and six eryC genes, eryCI - eryCVI, are responsible for the biosynthesis of D-desosamine or its attachment to 3-am? ler? tronol? da B Divided arrows indicate that the trajectory through erythromycin B is not the main natural biosynthetic pathway for eptromycin A Figure 2 illustrates the proposed scheme for the biosynthesis of L-micarosa and the eryB genes responsible for the steps Specificities Figure 3 illustrates the proposed scheme for the biosynthesis of D-desosamine and the eryC genes responsible for the specific steps. 4A (1-4) illustrates the nucleotide sequence (SEQ ID NO 1) of the sugar biosynthesis genes eryCII (coordinates 54-1136), eryCIII (coordinates 1147-2412), and eryBII (coordinates 2409-3410), with corresponding translation of the open reading frames (SEQ ID NO 3, SEQ ID N04 and SEQ ID NO.5, respectively) The normal one-letter codes for the amino acids appear below their respective nucleic acid codons as described herein . Figure 4B (1-9) illustrates the nucleotide sequence (SEQ ID NO 2) of the sugar biosynthesis genes eryBIV (coordinates 80-1048), eryCVI (coordinates 2348-3061), eryBVI (coordinates 3214-4677), eryCIV (coordinates 4674-5879), eryCV (coordinates 5917-7386), and eryBVIl (coordinates 7415-7996) with corresponding translation of the putative open reading frames (SEQ ID NO 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively). The normal one-letter codes for the amino acids appear below their respective nucleic acid codons as described herein. Figure 5A illustrates the identity of the amino acid sequence between the sugar biosynthesis enzyme encoded by the eryBIV gene of Sac erythraea (SEQ ID NO 6) and the sugar biosynthesis enzymes encoded by the ascF gene of Yersmia pseudotuberculosis [Thorson et al. others, J Bacterio! , 176 5483 (1994)], (SEQ ID NO 13), the rfbJ gene of Salmonella enterica [Jiang et al., Mol Microbio! , 5695 (1991)] (SEQ ID NO 14), the strL gene of Streptomyces griseus [Pissowotzki et al., Mol Gen Genet 241 193 (1993)] (SEQ ID NO 15) and the galE gene of Eschenchia coli [Lemaire and Hill , Nucí Acids Res 147705 (1986)] (SEQ ID NO 16) In this and all other figures where the identity of the amino acid sequence is compared, the capital letters represent consensus amino acids (identical) between species or amino acids that are conservative substitutions for consensus residues Also in each figure, the sequence identified as "consensus" is simply a convenient representation of conserved amino acids and should not be a representation of any existing polypeptide sequence. Figure 5B illustrates the identity of the amino acid sequence between the sugar biosynthesis enzyme encoded by the eryBVII gene of Sac erythraea (SEQ ID NO 12) and the sugar biosynthesis enzymes encoded by the strM gene of Streptomyces griseus [Pissowotzki et al., Mol Gen Genet 241 193 (1993)] (SEQ ID NO 17) and the rfbC gene of Salmonella enterica [Jiang et al., Mol Microbio! 5695 (1991)] (SEQ ID NO 18) and the rfbF gene from Yersima entercolitica [Zang et al., Mol Microbiol 9309 (1993)] (SEQ ID NO 19) and the ascE gene from Yersema pseudotuberculosis [Thorson et al., J Bacterium ! 176 5483 (1994)] (SEQ ID NO 20) Figure 5C illustrates the identity of the amino acid sequence between the sugar biosynthesis enzyme encoded by the eryCIV gene of Sac erythraea (SEQ ID NO 10) and the biosynthesis enzymes of sugars encoded by the eryCI gene of Sac erythraea [Dhillon et al., Mol Microbe! , 3,1405 (1989)] (SEQ ID NO 21), the ascC gene Yersima pseudotuberculosis [Weigel et al., Biochemistry, 31 2129 (1992) Thorson et al., J Am Chem Soc, 1156993 (1993), Thorson et al., J Bacterium! , 1765483 (1994)] (SEQ ID NO 22), the Streptomyces dnrj gene [Stutzman-Eng all and others, J Bactenol, 174 144 (1992)] (SEQ ID NO 23), the prgl gene of Streptomyces alboniger [Lacalle and others, EMBO J, 11,785 (1992)] (SEQ ID NO 24), and the strS gene of Streptomyces griseus [Distler et al., Gene, 115 105 (1992)] (SEQ ID NO 25) Figure 5D illustrates the identity of the amino acid sequence between the sugar biosynthesis enzymes encoded by the eryBV and eryCIII genes of Sac erythraea (SEQ ID NO 7 and SEQ ID NO 4, respectively) and the sugar biosynthesis enzyme encoded by the Streptomyces dnrS gene peucetius [Otten and others, J Bacterio! 1776688 (1995)] (SEQ ID NO 26) Figure 5E illustrates the identity of the amino acid sequence between the sugar biosynthesis enzyme encoded by the eryCVI gene of Sac erythraea (SEQ ID NO 8) and the sugar biosynthesis enzymes encoded by the srmX gene of Streptomyces ambofaciens [Geistlich et al., Mol Microbio! , 62019 (1992)] (SEQ ID NO 27, the rdmD gene from Streptomyces purpurascens [GenBank Access U10405] (SEQ ID NO 28) and the glycine methyltransferase from Rattus norvegious [Ogawa et al., Eur J Biochem 168 141 (1987) ] (SEQ ID NO 29) Figures 6A to 6D illustrate the compounds formed in conceivable manner in Examples 1-4 respectively and are representative of the compounds formed of Type I (Figure 6A), Type II (Figure 6B) and Type III (Figures 6C and 6D) Figure 7 illustrates the construction of expression plasmid pASX2 described in Example 2 For Figures 7-13 the following abbreviations amp, ampicillin resistance gene, tsr, resistance gene, thiostrepton, ROP, repressor of plasmid synthesis gene, eryB !, eryBII, eryBIII, eryBIV, eryBV, eryBVI, eryBVIl, eryCI, eryCII, eryCIII, eryCIV, eryCV, and eryCVI, the biosynthetic genes of eptromicin involved in the synthesis of micarosa or its attachment to the macrolide ring (eryB) or the s synthesis of desosamma or its fixation of the ring of macrophyte (eryC) [the thin arrows above a gene indicate its relative size and the direction of transcription], op-E coli, a replication origin of DNA that works in £ coli, in the specific examples the origin ColE1, ori-Streptomyces, a replication origin of DNA that works in Streptomyces, in the specific examples the origin pJV1 [Servin-Gonzalez et al., Microbiology, 141 2499 (1995)], p-ermE *, a modified promoter for the ephromycin resistance gene, t-fd, the transcription terminator of bacteriophage fd gene VIII, PCR, polymerase chain reaction Restriction enzyme sites have been indicated by their normal trade names (i.e. BamHl EcoRI, etc.) The abbreviations attached to the long arrows in the synthetic plasmid schemes summarize each of the steps involved in the plasmid constructions. These steps are fully described in the relevant examples. Figure 8 illustrates the construction of plasmid pASBVIII for antisense expression of eryBVIl described in Example 2 Figure 9A illustrates the construction of vehicle plasmid pK1 Figures 9B-E illustrate the construction of plasmid pKB6 carrying all eryB genes and is described in Example 3 Figure 10 illustrates the construction of the expression plasmid pX1 described in Example 3 Figure 11 illustrates the construction of plasmids pXSB6 and expression pXB6 of eryB described in Example 3 Figures 12A-B illustrate the construction of the pKC4 plasmid carrying all the eryC genes described in example 4 Figure 13 illustrates the construction of the eXC expression plasmids pXSC4 and pXC4 described in example 4 DETAILED DESCRIPTION OF THE INVENTION I. THE INVENTION The present invention provides isolated and purified polynucleotides encoding enzymes or fragments thereof responsible for the biosynthesis of sugars associated with polyketide or its binding to polyketides, vectors containing those polynucleotides, host cells transformed with those vectors, a process for making glycosylated polyketides Novelties using those polynucleotides and vectors, and isolated and purified recombinant polypeptides and polypeptide fragments thereof.

II. DEFINITIONS For the purposes of the present invention as described and claimed herein, the following terms are defined. The term "polyketide" as used herein refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer and anti-helminthic compounds. Antibiotics include, but are not limited to, anthracyclines and macrolides of different types (polyenes and avermectins as well as classical macrolides such as erythromycins).

The term "glycosylated polyketide" refers to any pohcetide that contains one or more sugar residues. The term "polyketide modified by glycosylation" refers to a polyketide having a changed pattern or configuration of glycosylation relative to that of the unmodified or native state. The particular term The term "microorganism that produces polyketide" as used herein includes any microorganism that can produce a polyketide naturally or after it is formed (i.e., genetically) Examples of actinomycetes and the polyketides they produce in a manner include, but are not limited to, those listed in Table 1 below (see Hopwood, DA and Sherman, DH, Annu Rev Genet, 2437-66 (1990) incorporated herein by reference) TABLE 1 Organism Pohcetida produced Saccharopolyspora erythraea Eptromycin Streptomyces ambofaciens Spiramycin Streptomyces avermitilis Avermectin Streptomyces fradiae Tilosma Streptomyces gpseus Candicidin, mona ct ina, gpseusin Streptomyces violaceoniger Granaticina Streptomyces thermotolerans Carbomycin Streptomyces pmosus Oxytetracycline Streptomyces peucetius Daunorubicin Streptomyces coelicolor Actinorhodina Streptomyces glaucescens Tetracenomycin Streptomyces roseofulvus Frenolicin Streptomyces cinnamonensis Monensin Streptomyces curacoi Curamycin Amycolatopsis mediterranei Rifamycin Other examples of polyketide-producing microorganisms that naturally produce polyketides include vain strands of Actinomadura, Dactylosporangium and Nocardia. The term "sugar biosynthesis genes" as used herein refers to DNA sequences from Saccharopolyspora erythraea that encode enzymes of sugar biosynthesis and DNA sequences should be included from other polyketide-producing microorganisms that are identical or analogous to those obtained from Saccharopolyspora erythraea. The term "sugar biosynthesis enzymes" as used herein refers to polypeptides that are involved in the biosynthesis and / or fixation of sugars associated with polyketide and its derivatives and intermediates The term "sugar associated with polyketide" refers to a sugar that is known to bind to pohcetides or that can be fixed to pohcetides by the processes described in present The term "sugar derivative" is referred to A sugar that is naturally associated with a polyketide, but that is altered with respect to the unmodified or native state, examples include only N-3-a-desd? met? l D-desosamine, D-mycarose, 4-keto -Lm? Carose, 4-keto-D-micarose, 3-desmet? L L-micarose and 3-desmet? L D-micarose The term "sugar intermediate" refers to an intermediate compound produced in a biosynthetic pathway of Sugar The term "eryB" as used herein refers to DNA sequences encoding enzymes specifically involved in the biosynthesis of L-micarosa deoxyazugar. The term "eryC" as used herein refers to DNA sequences that encode enzymes specifically involved in the biosynthesis of deoxyazugar D-desosamma lll. POLYUCLEOTIDES The organization of the chromosome segment Saccharopolyspora erythraea (Sac erythraea) which determines the biosynthesis of eptromicin and the corresponding genes that determine the biosynthesis of the sugars L-micarosa and D-desosamine, designated eryB and eryC, respectively, are shown in figure 1A. It is observed that several genes are require for the biosynthesis of each of the sugars and that these genes are separated from each other. It is predicted that each gene encodes an enzyme that catalyses one or several steps in the biosynthesis of L-micarosa or D-desosamina from d? phospho- 4-keto-6 thymidine deoxyglucose (TDP-glucose), these steps are delineated in Figure 2 and Figure 3 In the case of L-mycarose, (shown in Figure 2), these steps include (1) C-2"deoxygenation, (2) C-2" / C-3"reduction of enoyl, (3) C-5" epimerazion, (4) C-3"C-methylation, (5) C-4" reduction keto, and (6) Transfer to Eptronolide B For D-desosamine, shown in Figure 3, these steps comprise (1) C-473 'isomepzation, (2,3) C-3' deoxygenation and eduction, (4) C-3 'amination, (5, 6) N-3a'N-dimethylation, and transfer to micarosil eptrono da B This classification of genes (belonging to class eryB or class eryC) was determined by altering first the wild-type genes of interest in a strand that produces eptromycin (i.e., live m) to inactivate its expression. Then the eptromicin products resulting from said alterations were analyzed. The genes whose alterations caused an accumulation of eptronolide B (indicating a lack of L-mycarose, or failure to fix L-mycarose to the ephotonohda ring) were classified as eryB genes, the genes whose alterations caused an accumulation of 3-alam expensive? l eptronolide B (indicating a lack of D- desosamine, or failure to bind D-desosamine to the ring of 3-alam expensive? l eptronohda B) were classified as eryC genes Therefore, it should be noted that all such genes identified in the present as eryB or eryC are involved in the synthesis of L-micarose or D-desosamma The predicted functional activities of the polypeptides encoded by eryB and eryC will be discussed in detail below. Then, in one aspect, the present invention provides eryB and eryC polynucleotides isolated and purified from Sac erythraea encoding enzymes involved in the production of ghcosylated polyketides A polynucleotide of the present invention that encodes a sugar biosynthesis enzyme is an isolated single or double chain polynucleotide having a nucleotide sequence comprising (a) a nucleotide sequence selected from group consisting of (i) the sense sequence of Figure 4A (SEQ ID NO 1) from about nucleotide position 54 to about nucleotide position 1136, (n) the sense sequence of SEQ ID NO 1 of around nucleotide position 1147 to about nucleotide position 2412, (ni) the sense sequence of SEQ ID NO 1 from about nucleotide position 2409 to about nucleotide position 3410, (iv) the sense sequence of Figure 4B (SEQ ID NO 2) from about nucleotide position 80 to about nucleotide position 1048, ( v) the sense sequence of SEQ ID NO 2 from about nucleotide position 1048 to about nucleotide position 2295, (vi) the sense sequence of SEQ ID NO 2 from about nucleotide position 2348 to about nucleotide position 3061, (vn) the sense sequence of SEQ ID NO 2 from about the position of nucleotide 3214 to about the position of nucleotide 4677, (vm) the sequence of sense of SEQ ID NO 2 around the position from nucleotide 4674 to about the position of nucleotide 5879, (ix) the sense sequence of SEQ ID NO 2 from about the position of nucleotide 5917 to about the position of nucleotide 7386, and (x) the sequence of sense of SEQ ID NO 2 from about nucleotide position 7415 to about the nucleotide position 7996(b) sequences complementary to the sequences of (a), (c) sequences that, in expression, encode a pohpeptide encoded by the sequences of (a), and (d) analogous sequences that hybridize under demanding conditions to the sequences of (a) A preferred polynucleotide is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. The nucleotide sequence and deduced amino acid residue sequences of the sugar biosynthesis genes are set forth in Figure 4A (1-4). ) and Figure 4B (1-9) The nucleotide sequences of Figure 4A (1-4) (SEQ ID NO 1) and Figure 4B (1-9) (SEQ ID NO 2) represent full-length DNA clones of the sense strand of two distinct groups of sugar biosynthesis genes and must represent both the sense strand (shown at the top) and its complement The amino acid sequences illustrated below the sense strand correspond to pohpeptides encoded by a sequence of nucle selected from the group consisting of (i) the sense strand of SEQ ID NO 1 from about nucleotide position 54 to about nucleotide position 1136, (n) the sense sequence of SEQ ID NO 1 of around the position of nucleotide 1147 at about the position of nucleotide 2412, (m) the sense sequence of SEQ ID NO 1 around the position of nucleotide 2409 at about the position of nucleotide 3410, (iv) the sequence of sense of SEQ ID NO 2 from about the position of nucleotide 80 to about the position of nucleotide 1048, (v) the sequence of sense of SEQ ID NO 2 from about the position of nucleotide 1048 to about the position of nucleotide 2295, ( vi) the sense sequence of SEQ ID NO 2 from about nucleotide position 2348 to about nucleotide position 3061, (vn) the sense sequence of SEQ ID NO 2 around nucleotide position 321 4 at about the position of nucleotide 4677, (vni) the sense sequence of SEQ ID NO 2 from about nucleotide position 4674 to about nucleotide position 5879, (ix) the sense sequence of SEQ ID NO 2 of around the position of nucleotide 5917 at about the position of nucleotide 7386, and (x) the sequence of sense of SEQ ID NO 2 around the position of nucleotide 7415 at about the position of nucleotide 7996 The polypeptides encoded by the sequences of nucleotides of (?) - (x) above are set forth as SEQ ID NO 3-SEQ ID NO 12, respectively. The present invention also contemplates analog DNA sequences that hybridize under stringent hybridization conditions to the DNA sequences set forth above. Strict hybridization are well known in the art and define a degree of sequence identity greater than about 80-90%. The "analogue" modifier refers to those nucleotide sequences which encode analogue polypeptides (ie, in relation to a sugar biosynthesis enzyme), analogous polypeptides being those which have only conservative differences and which retain the conventional characteristics and activities of sugar biosynthesis enzymes (more further a more detailed description of analogous polypeptides) The present invention also contemplates variations and allelic mutations that occur naturally of the DNA sequences set forth above as long as said variations and mutations encode, in expression, for a sugar biosynthesis gene of This invention as set forth hereinbelow As is well known in the art, due to the degeneracy of the genetic code, there are numerous different DNA and RNA molecules that can code for the same polypeptides as those encoded by the biosynthesis genes of sugar and fragments thereof mentioned above Therefore, the present invention contemplates those other DNA and RNA molecules that in expression, encode polypeptides of SEQ ID NO 3-SEQ ID NO 11 or fragments thereof After identifying the amino acid residue sequence encoded by a sugar biosynthesis gene, and with the knowledge of all triple codons for each amino acid residue In particular, it is possible to describe all said RNA and DNA sequences encoding the DNA and RNA molecules different from those specifically described herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the Scope of this invention The 20 common amino acids and their representative abbreviations, symbols and codons are well known in the art (see for example, Molecular Biology of the Cell Second Edition B Alberts et al., Garland Publishing Inc, New York and London, 1989) As is well known in the art, codons constitute triple nucleotide sequences in mRNA molecules and as such , are characterized by base uracil (U) instead of base thymidine (T) that is present in DNA molecules A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the polypeptide encoded by For example, it can be observed from SEQ ID NO 1 that an AGC codon for sepna exists at nucleotide positions 126-128 and once again at positions 420-422 and 561-563 However, it can also be observe from the same sequences that sepA can be encoded by a TCG codon (see for example, nucleotide positions 192-194) and a TCC codon (see for example, nucleotide positions 204-206) The substitution of the Last codons for sepna with the AGC codon for sepna, or vice versa, do not substantially alter the DNA sequence of SEQ ID NO 1 and result in the production of the same polypeptide. Similarly, substitutions of the cited codons can be made with other codons equivalent in a similar manner but without departing from the scope of the present invention A polynucleotide of the present invention can also be an RNA molecule An RNA molecule contemplated by the present invention is complementary to or hybrid under strict conditions to any of the DNA sequences established above Preferred and preferred RNA molecules are mRNA molecules that encode sugar biosynthetic enzymes of this invention IV. POLYPEPTIDES In another aspect, the present invention provides polypeptides that are reasonably believed to be sugar biosynthesis enzymes. A sugar biosynthesis enzyme of the present invention is a polypeptide of about 21 kdal to about 47 kdal. As set forth in FIGS. 5E, analogs of the predicted polypeptides encoded by certain eryB and eryC genes have been identified in several species and their sequences compared to using the PRETTY routine (Genetics Computer Group (GCG) Sequence Analysis Software Package, Madison, WI) Due to the degree of identity of the amino acid sequence that exists between the polypeptides of these other sugar biosynthesis genes and the polypeptides encoded by the eryB and eryC genes, certain enzyme activities can be reasonably attributed to the eryB and eryC polypeptides. example, analogs of the polypeptide encoded by the eryBIV gene have been identified in Yersinia pseudotuberculosis , Enteric Salmonella, Streptomyces griseus and Eschenchia coll (see Figure 5A) Several analogs have been identified with 290-328 amino acid residues and are characterized by a low degree of amino acid sequence identity (eg, the identity between the enzyme biosynthesis of sugar encoded by the eryBIV gene of Sac. erythraea and the sugar biosynthesis enzyme encoded by the E. coli galE gene is 20% at the amino acid level). However, there is a conserved amino acid sequence motif, G xx G xx G (where G represents the amino acid glycine and x represents any amino acid residue) within the first 30 amino acid residues of all the analogs shown. has shown that the polypeptide encoded by the galE gene is an epimerase (whose mechanism includes ketoreduction (Bauer et al., Proteins 12 372 (1992)), the eryBIV gene product is reasonably predicted to be a ketoreductase. As set forth in the figure 5B, analogs of the sugar biosynthesis enzyme by the eryBVIl gene have been identified in Streptomyces griseus, enteric Salmonella, Yersima entercolitica and Yersinia pseudotuberculosis. Analogous spans have been identified with 183-200 amino acid residues and are characterized by a moderate degree of amino acid identity By way of example, the identity at the amino acid level between the sugar biosynthesis enzyme codi The eryBVIl gene of Sac erythraea and the sugar biosynthesis enzyme encoded by the rfbC gene of Salmonella enterica or the strM gene of Streptomyces griseus are 37% and 61%, respectively. Furthermore, a common feature of these particular polypeptides (including that one of eryBVIl), is that they are only associated with the biosynthesis of L-sugar and not with the biosynthesis of D-sugar. In this way, the gene product of eryBVIl is reasonably predicted to function as a C-5 epimerase that converts the stereochemistry of sugar from the "D" configuration to the "L" configuration As set forth in Figure 5C, the analogs of the sugar biosynthesis enzyme encoded by the eryCIV gene have been identified in Sac erythraea and Yersmia pseudotuberculosis As established in Figure 5C, the predicted amino acid sequences of the eryCI and eryCIV protein products share 34% sequence identity with each other, 27% and 25% respectively at the same time. predicted amino acid sequence encoded by Yssema pseudotuberculosis ascC We have shown that the enzyme encoded by ascC removes a hydroxyl group located at the C-3 position of L-ascaplosa (Liu and Thorson, Annu Rev Microbio! 48223 (1994)) Thus, at least one of the polypeptides encoded by eryCI or eryCIV is predicted to be an enzyme that functions in deoxygenation reactions. In addition, the enzyme encoded by the ascC gene requires the biochemical cofactor pipdoxamine, which is the same cofactor used in biochemical transamination reactions Consequently, it has been proposed that some protein analogs (such as Streptomyces peucetius dnrJ, Streptomyces alboniger prgl and Streptomyces griseus strs) that have a moderate degree of sequence similarity to the polypeptide encoded by ascC function as transaminases in amino sugar biosynthesis ( Thorson et al., J Am Chem Soc 1156993 (1993)) Since the biosynthesis of D-desosamine requires both deoxygenation and transamination, it is reasonable to predict that at least one of the polypeptides encoded by eryCI or eryCIV genes functions in transamination reactions. is set forth in Figure 5D, the predicted polypeptides encoded by eryBV and eryCIII share 43% identity at the amino acid level and as such, can be assumed to have similar activities with respect to their particular sugars However as shown in Figures 2 and 3 there are no common steps in the proposed trajectories of L-mycarose and D-desosamine biosynthesis instead of having similar sugar biosynthesis functions, these polypeptides are predicted to be nucleotidyl-sugar transferases which, (in Sac erythraea at least), function to fix L-micarose and D-desosamine to B-type and 3-ammonium Eppendolide B, respectively As set forth in Figure 5E, analogs of the polypeptide encoded by the eryCVl gene have been identified in Streptomyces ambofaciens, Streptomyces purpurascens, and Rattus norvegicus. Several analogs have been identified with 237-293 amino acid residues and characterized by a low to moderate degree of amino acid identity. For example, the identity between the pohpeptide encoded by the eryCVl gene of Sac erythraea and the glycine methyltransferase of Rattus norvegicus is 26% at the amino acid level. Furthermore, these enzymes sugar biosynthesis share a common sequence motif, LDVACGTG (SEQ ID NO 30 = amino acid positions 64-71 in the consensus sequence in Figure 5E), with m Rat glycine ethyltransferase whose biochemical function is known (Ogawa et al., Eur J Biochem 168 141 (1987)) Thus, these polypeptides are predicted to be N-methyltransferases. In another aspect, the present invention provides a C-4" Recombinant keto reductase from Sac. erythraea A C-4"Sacred erythraea ketoreductase of the present invention is a polypeptide of about 322 or less amino acid residues A C-4" recombinant ketoreductase of Sac. erythraea is that encoded by the nucleotide sequence of SEQ ID NO: 2 from about nucleotide position 80 to about nucleotide position 1048. The present invention also contemplates amino acid residue sequences that substantially duplicate the sequences set forth in the present so that those sequences show biological activity similar to the sequences described. Said contemplated sequences include those analogous sequences characterized by a minimal change in sequence or type of amino acid residue (eg, conservatively substituted sequences), which non-substantial changes do not alter the fundamental nature and biological activity of the biosynthesis enzymes of sugar mentioned before. It is well known in the art that modifications and changes in the structure of a polypeptide can be made without substantially altering the biological function of that peptide. For example, certain amino acids may be substituted with other amino acids in a given polypeptide without appreciable loss of function. Such changes, modifications, substitutions of similar amino acid residues can be made based on the relative similarity of side chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like. As detailed in the US Pat. No. 4,554,101, incorporated herein by reference, the following hydrophobicity values have been assigned to amino acid residues Arg (+ 30), Lys (+ 30), Asp (+ 30), Glu (+ 3 0), Ser (+ 03), Asn (+ 02), Gln (+ 02), Gly (0), Pro (-0 5), Thr (-04), Ala (-0 5), His (-0 5), Met (-1 3), Val (-1 5), Leu (-1 8), Tyr (-2 3), Phe ( -2 5), and Trp (-34) It is understood that an amino acid residue can be substituted with another having a similar hydrophilicity value (for example, within a value of plus or minus 2 0) and still obtain a biologically equivalent polypeptide Similarly, substitutions can be made based on the similarity in hydropathic index. Each amino acid residue has been assigned a hydropathic index based on its hydrophobicity and loading characteristics. Those values of hydropathic index are Me (+ 45). , Val (+ 42), Leu (+ 3 8), Phe (+ 2 8), Cys (+ 2 5), Met (+ 1 9), Ala (+1 8), Gly (-04), Thr ( -07), Ser (-08), Trp (-09), Tyr (-1 3), Pro (-1 6), His (-32), Glu (-35), Gln (-3 5), Asp (-35), Asn (-35), Lys (-39), and Arg (-4 5) When making a substitution based on the index hydropathic, a value is preferred within plus or minus 2 0 V. PRODUCTION OF NOVELTY GLYCOSYLLED POLYCYCLES In another aspect, the present invention comprises a general method for producing novel pohcetide structures in vivo to selectively alter, inactivate, or increase the genetic information of the organism that naturally produces a related polyketide. That is, in the present invention, novel polyketides are produced of desired structure by manipulation of the eryB and / or eryC genes followed by their introduction into various microorganisms that produce polyketide. These manipulations result in the formation of "modified glycosylation" polyketides (ie, pohcetides having a glycosylation pattern or pattern). altered with respect to its native state) For example, polyketides "modified by glycosylation" are those that have additional sugar groups fixed (where none existed before), different sugars (such as sugar intermediates) fixed instead of sugars natural or sugar-free groups (in the positions where there were sugar groups before). In the case of Type I and Type II alterations (described below), polyketides modified by glycosylation may arise through mechanisms that cause (i) the non-production of the sugar-binding enzyme (ie, the enzyme involved in the fixation of a sugar to the structure of the polyketide) or (2) the non-production of a sugar biosynthesis enzyme In the first case, the sugar will not be fixed to the pohcetide since the enzyme that works to fix the sugar will be missing In the second situation a sugar intermediate will be produced from the path of biosynthesis (depending on the enzyme that is missing) and will be fixed to the polyketide if it is recognized as a suitable substrate by the sugar-binding enzyme, alternatively, it will not be recognized and therefore not fixed In the case of Type III alterations (also described below), glycosylated modified proteins arise through the addition of additional sugars. are different (ie, not normally found in a chain that produces particular polyketide) to the polyketide It should be noted, that these postulated mechanisms are provided simply to improve understanding of the novel processes described herein, are not currently known. Real mechanisms whereby Type I, II and III alterations produce polyketides modified by ghcosylation In the first type of alteration (referred to herein as Type I alterations), eryB and / or eryC genes genetically altered in the chromosome are introduced of Sac Erythraea or another organism that produces glycosylated polyketide that also produces L-mycarose, D-desosamine, or its closely related derivatives such as mycaminosa (4-h? drox? -D-desosam? na) Genetic alteration of an eryB gene and / or eryC is such that it causes a non-functional enzyme to be synthesized. Once introduced into an appropriate chain, the altered gene replaces its wild-type correspo gene. chain causing the chain to lose the ability to produce a particular enzymatic activity involved in sugar biosynthesis. As a result, a glycosylated modified protein is produced by the mechanisms previously described for a Type I alteration in a Type I change. described herein, a specific mutation in an eryB and / or eryC gene of the Sac Erythraea chromosome is achieved by a three-step process that involves 1) specifically altering the DNA sequence of a desired sugar biosynthesis gene, 2) subcloning the altered sequence in a suitable vector capable of recombining in the chromosome of an appropriate host; and 3) introducing a vector containing the subcloned sequence into the appropriate host, so that exchange of the wild type allele with the mutated one will occur. achieved by using normal recombinant DNA techniques to perform an omission, base pair conversion, or frame change in the DNA sequence The second step, which also employs normal recombinant techniques, involves subcloning the altered sequence into a vector that does not replicate in Sac erythraea or the desired host. In the final step, the vector is introduced into a suitable host, where by the process of gene replacement, the altered allele replaces the wild-type allele All the techniques employed in a Type I change are well known to those skilled in the art Example 1 illustrates the process of gene replacement of an eryB gene As shown in example 1, the gene eryss of intent is mutated and together with adjacent sequences of DNA beyond the 5 'end and 3' end, cloned into a plasmid vector of Sac erythraea without replication The vector leading to the mutated allele and attached DNA is then introduced into the strand host by the protoplast transformation process Transformers are regenerated under selective conditions (ie, conditions that require the expression of a plasmid marker parti cular) in order to induce the recombination of the plasmid in the host cell chromosome In other words, since the plasmid does not replicate autonomously, it must reside in the chromosome to be maintained when the regenerated cells go through a single homologous recombination between one of the two segments of DNA that change the mutation in the plasmid and its homologous counterpart in the chromosome The cells then grow without selection for the marker, which induces plasmid loss from the chromosome This loss arises after the cells undergo a second recombination between the second segment of DNA that changes the mutation and its counterpart of homologous chromosome This second fact of recombination results in the loss of the plasmid sequences and the wild-type allele from the chromosome, however, the mutant allele is retained in a variation of a Type I change, the non-production of the sugar biosynthesis enzyme (or enzyme of tion) can be achieved by alternative mechanisms of promoter inactivation and / or insertion of trans-transcriptional terminator. These variations do not form the gene sequence itself, but rather regulatory mechanisms involved in the transcription of the "promoter" gene as used herein. to that region of a DNA molecule that controls the initiation of RNA transcription. Said regions are known to bind RNA polymerases (ie, the inactivation of the promoter) involves two steps of 1) identifying the promoter region of the desired gene and 2) making the promoter region inoperable by mutation As in the replacement mechanism described above, such mutations can be carried out by creating omissions in the promoter sequence or by base pair conversion. In the case where the promoter controls the transcription of a single gene, inactivation of the promoter will eliminate the expression of that particular gene. , of course, where the promoter controls the expression of an entire operon (ie, a series of genes whose expression is controlled by a single promoter), the inactivation of the promoter will effectively eliminate the expression of all genes in that operon. Similarly, the non-production of a sugar biosynthesis enzyme (or binding enzyme) can arise by inserting a transcriptional terminator beyond the 5 'end of the gene to be inactivated. A "transcriptional terminator" as used in the present is a nucleotide sequence that signals RNA polymerase to stop transcription. An example of a transcriptional terminator is a palindromic sequence capable of forming a stem-loop structure that is followed by a stretch of U residues (e.g. the trans-transcriptional terminator following gene VIII of bacteriophage fd (Beck and Zmk, Gene, 1635 (1981)) The realization of a change in the production of a sugar biosynthesis gene by this process involves 1) identifying the gene or genes of interest (in the case of an operon arrangement) to inactivate and 2) cloning a trans-sectional terminator sequence into a region of the DNA beyond the 5 'end of d? cho (s) gene (s) A Transcourse terminator will cause the polymerase involved in the transcription of RNA to stop (in or near the signaling region), thus preventing transcription of any sequence beyond the 3 'end. In this manner, changes such as promoter inactivation and trans-occlusional insertion, which directly effect the expression of sugar biosynthesis genes, should also be within the scope of the invention. In the second case (referred to herein as Type II alterations), eryB and / or eryC genes are arranged in a vector in an anti-sense orientation with respect to a promoter capable of allowing expression of the gene in Sac erythraea or Streptomyces. vector is then introduced into a microorganism that produces pohcetide As a result of this vector construction, antisense messenger RNA (mRNA) is produced which interferes with the translation of wild-type mRNA. In a manner similar to Type I manipulation, polyketides will be produced. novel products modified by glycosylation in which the normal mycarose, desosamine and / or closely related sugar residue is it missing or is replaced by an intermediary sugar change Type II, inactivation of genes is achieved. eryB and / or eryC by anti-sense expression by a two-step procedure wherein (1) a specific sugar biosynthesis gene is subcloned into an expression vector in an anti-sense orientation (ie, reverse), and (2) the anti-sense expression vector is introduced into the desired chain. The first step is achieved by using normal recombinant DNA techniques employing E. coli or Streptomyces as the host, and an expression vector (capable of replicating at any host) that can be assembled to contain a Streptomyces promoter. Streptomyces promoters can be obtained from any commercially available Streptomyces plasmid or combined Streptomyces-E coli plasmids. In step 2, the anti-sense expression vector is introduced into a suitable Streptomyces chain and the transformed cells are grown under selective conditions in order to maintain the expression plasmid in the cell As described in the example or 2, the gene for inactivation is subcloned in its reverse orientation beyond the 3 'end of a Streptomyces promoter (which is within a replication plasmid of Sac erythraea). The plasmid carrying the anti-sense gene is introduced. then in the host chain by protoplast transformation Transformers regenerate under selective conditions in order to keep the plasmid that replicates autonomously in the cells The subsequent expression of the anti-sense gene causes the production of an anti-sense messenger RNA (mRNA) ) that is complementary to the mRNA of the native allele of the selected gene Through normal nucleotide base pair interactions, the anti-sense mRNA and the native mRNA form an RNA duplex that occludes the ribosome binding site of the native mRNA This interaction prevents ribosomal translation of the native mRNA and the corresponding synthesis of the enzyme encoded by that mRNA. In this way, the enzymatic steps s specific in sugar biosynthesis corresponding to the identity of the gene expressed in the anti-sense orientation are blocked leading to the production of novel sugar intermediates which, when attached to the polyketide ring of the host microorganism, give rise to polyketides Novel glycosylation-modified novels Alternatively, the anti-sense expression vector can be constructed by using a non-replicated vector of Sac erythraea which includes changing DNA from a non-essential region of the Sac erythraea chromosome, such as the region immediately beyond the end. The eryK gene (Figure 1) This vector can then be used to stably insert the anti-sense construct into the chromosome by homologous recombination in a manner normal to that described for the construction of a Type I alteration. In the third case (referred to in FIG. the present as alterations of Type III), novel pohcetides modified by g cosilaci are produced n desired structure by providing all or subsepe of eryB and / or eryC genes in a vector replication and introduce these genes en bloc in a "different" organism producing polyketide, ie, a different one to the microorganism from which the eryB and / or eryC genes were taken. As an example, eryB and / or eryC genes of Sac erythraea can be taken and introduced into Streptomyces violaceoniger or Streptomyces venezuelae. In this case, micarosa will be synthesized. , desosamma, its biochemical intermediates and / or its closely related derivatives and will be fixed in specific positions to polyketide compounds that do not necessarily carry these, or any sugar residue Some examples of novel ghcosylated polyketides that can be produced in hosts that carry such manipulations are shown in Figure 6 In Type III changes, genes for the biosynthesis of micarosa and / or desosamine are introduced into an organism that produces polyketide other than Sac erythraea by another simple two-step procedure 1) all or a sub-section of the eryB and / or eryC genes are assembled together in a replication plasmid beyond the 3 'end of a Streptomyces promoter, and 2) the plasmid is introduced into the polyketide-producing organism Step 1 requires normal recombinant DNA manipulations employing E. coli and / or Streptomyces as the host Step 2 requires one or more plasmids outside the different Streptomyces vectors or combined vectors of available E. coli Streptomyces, one or more promoters running on Streptomyces, and a selection for the presence of the plasmid carrying chain As described in Examples 3 and 4, the eryB and / or eryC genes are sequenced subcloned in sequence. in a replicate vector beyond the 3 'end of a suitable promoter that functions in the desired host. The plasmid carrying the pooled genes is then introduced into the host chain by electroporation or transformation of protoplasts that employ selection for a marker of plasmid GENERAL METHODS MATERIALS. PLASMID. AND BACTERIAL CHAINS Restriction endonucleases, T4 DNA hase, DH5a competent coli cells, X-gal, IPTC and plasmids pUC18, pUC19, and pBR322 were purchased from Bethesda Research Laboratories (BRL), Gaithersburg, MD VentR® DNA polymerase was purchased from New England Biolabs (Beverly, MA) Plasmids pGEM®5Zf, pGEM®7Zf, and pGEM11Zf® were from Promega, Madison, Wl, plasmids plJ4070 and plJ702 were obtained from John Innes Institute, Norwich, England, and plasmids pWHM3 and pWHM4 (J Bacterium! 1989 171 5872) were obtained from CR Hutchinson, University of Wisconsin, Wl [a-32P] dCTP, Hybond ™ -N nylon membranes, and Megappme nick translation equipment were from Amersham Corp Chicago, IL SeaKem®Le agarose and SeaPlaque® agarose of low gelling temperature were from FMC Bioproducts, Rockland, ME The chains of £ coli K12 carrying plasmids pWHM3 and pWHM4 combined from £ coli-Sac erythraea (Vara et al., J Bacterio!, 171 5872 ( 1989)) and pAIX have been deposited in Agp Cultural Research Collection Collection (NRRL) 1815 N University Street, Peona, Illinois 61604, December 5, 1995, under the terms of the Budapest Treaty and shall be maintained for a period of thirty (30) years from the date of deposit. , or for five (5) years after the last request for the deposit, or during the current period of the US patent, whichever is longer The plasmids pWHM3, pWHM4 and pAIX received accession numbers NRRL B-21512, NRRL B-21513 and NRRL B-21514, respectively NRRL2338 of Sac Erythraea chain is also available from the culture collection of Agpcultural Research Service ThR of Staphylococcus aureus (resistant to thiostrepton) was obtained by plating 108 cells of S aureus in agar medium containing 10 μg / ml thiostrepton and by choosing a survivor after 48 hours of growth at 37 ° C Thiostrepton was obtained from Sigma Chemical, St Louis, MO All other chemicals and reagents were rum from normal commercial sources, unless otherwise specified. DNA MANIPULATIONS Normal conditions were used for restriction endonuclease digestion, electrophoresis with agarose gel, isolation of DNA fragments from low melting agarose gels, DNA ligation, plasmid isolation from £ coli by alkaline lysis, and transformation of coli using selection for ampicillin resistance (150 μg / ml) on LB agar plates (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview NY, 1989) The total DNA of Sac erythraea and Streptomyces (including S fradiae, S celestial, S violaceoniger, S hygroscopicus, S Venezuelae) were prepared in accordance with the described procedures (Hopwood et al., Genetic Mampulation of Streptomyces, A Laboratory Manual, John Innes Foundation, Norwich, UK (1985) ) The DNA transfer of agarose gels was done in accordance with the manufacturer's instructions AMPLIFICATION OF DNA FRAGMENTS Synthetic deoxyoligonucleotides were synthesized on an ABI Model 380A synthesizer (Applied Biosystems, Foster City, CA) following manufacturers' recommendations Amplification of DNA fragments was performed by polymerase chain reaction (PCR) using a PCR System 9600 from Perkm Elmer GeneAmp® The reactions contained 100 pmol of each primer, 1 μg of templated DNA (chromosomal DNA of Sac Erythraea NRRL2338), 2 units of VentR® DNA polymerase in 100 μl volume of PCR buffer (10mM Kcl, 10 mM (NH4) 2S04, 20 mM Tp-HCl (pH 88, at 25 ° C), 2.5 mM MgSO4, 0 1% Triton® X-100) containing dATP (200 μM), dTTP (200 μM), dCTP (250 μM) and dGTP (250 μM) The reaction mixture was subjected to 30 cycles. Each cycle consisted of a period of 35 seconds at 96 ° C and a period of 2 minutes at 72 ° C. The reaction products were visualized and purified. from low melt agarose The PCR primers described in the examples were derived of the nucleotide sequence of the eryB and eryC genes of figure 4 TRANSFORMATION PE REPLACEMENT OF GENE IN Sac. ervthraea Protoplasts of Sac erythraea chains were prepared and transformed with miniprep DNA isolated from £ coli in accordance with published procedures (Yamamoto et al., J Antibiotics, 39 1304 (1986)) Non-integrating transformers were selected, in the case of derivatives of pWHM4, by regenerating the protoplasts and overlapping with thiostrepton (final concentration 20 μg / ml) as described (Webe et al., Gene, 68 173 (1988)) Integrating transformers were selected, in the case of pWHM3 derivatives, in plates of agar containing thiostrepton (15 μg / ml) as described by Weber et al., Gene, 68 173 (1988) The loss of the ThR phenotype was verified after two rounds of non-selective growth in SGGP medium (Yamamoto et al., J. Antibiotics, 39 1304 (1986)) followed by protoplast formation and serial dilution of non-selective agar medium Plaques were plated on regenerated protoplasts in medium containing thiostrepton. Ths (sensitive to thiostrepton) at a frequency of 101 Retention of the mutant allele was established by Southern hybridization of vain Ths colonies FERMENTATION Sac erythraea or Streptomyces cells were inoculated in 100 ml SCM medium (1 5% soluble starch, 2 0% Difco Soytone, 0 15% yeast extract, 001% CaCl 2) and allowed to grow for 3 a 6 days All the culture was then inoculated into 10 liters of fresh SCM medium. The fermenter operated for a period of 4 to 7 days at 32 ° C, maintaining constant aeration and pH at 70. After completing the fermentation, the cells were removed by centrifugation at 4 ° C and the fermentation beer stays cold until used When an antibiotic selection is required to maintain a plasmid, such as pXC4 or pXB6, thiostrepton (10 μg / ml) is added to the 100 ml start culture and the fermenter of 10 liters The invention will be better understood in relation to the following examples, which should be an illustration and not a limitation of the scope of the invention Both in the following specification and in all, it is intended that citations to the literature They are expressly incorporated by reference EXAMPLE 1: CONSTRUCTION AND CHARACTERIZATION OF Sac. erythraea ERBIV THAT PRODUCES 4"-deoxy-4" -oxo-erythromycin A A Construction of Plasmid pRBIV A fragment of Pst \ -Hmdlll of 4 3 kb, which included the eryBIV gene, was isolated from plasmid pAIX5 and subclone in pUC19 digested from Pst \ -H? Nd \\\ to generate pUCBIV plasmid after the transformation and isolation of the plasmid from £ coll, the identity of pUCBIV was confirmed by digestion with Mun \ which released a fragment of 370 bp Then plasmid pUCBIV was cut with the restriction enzyme Ncol, the restriction site filled with Klenow enzyme, and the religated plasmid to generate plasmid pNCOBIV, (which now carried a frame-shifting mutation in the eryBIV gene) After transformation and isolation of the plasmid from £ coli, the identity of pNCOBIV was confirmed by digestion with? / sly HindlM that released a fragment of 1 59 kb (The? / S / l site was formed by the filling and religation of the Ncol site) Finally, pNCOBIV plasmid was digested with HindIII and SsfI and the 3 2 kb fragment leading to the eryBIV gene to lterado was isolated and ligated in pWHM3 digested with Hindlll and Sstl to generate plasmid pRBIV After transformation and isolation of the plasmid from £ coli, the identity of pRBIV was confirmed by digestion with Kpnl which released 52 kb, 44 kb fragments, and 072 kb B Construction of Sac erythraea ERBIV Protoplasts of Sac erythraea were transformed with pRBIV plasmid and selected integrating transformers as described in General Methods The resolution in the members by nonselective growth as described in General Methods produced ERBIV from Sac erythraea where the wild-type copy of the eryBIV gene was replaced with the inactive mutant copy. Gene replacement was confirmed by Southern analysis of Sac erythraea DNA digested with Ncol and Sac erythraea DNA digested with Ncol-Nsil using the? / cabbage fragment -H / ndIII of 1.58 kb isolated from the pUCBIV plasmid (coordinates 681-2214, Figure 4B) as a probe. Wild type Sac erythraea resolvents exhibit a 2 75 kb hybridizing DNA fragment when digested with Ncol or Ncol-γ / s / l, whereas Sac Erythraea chain ERBIV is characterized by hybridization to a 16 kb DNA fragment or a 2 75 kb DNA fragment when digested with Ncol or Ncol-Nsil, respectively C. Isolation, purification and properties of 4"-deoxy-4" -oxo-erythromycin A a from ERBIV de Sac. erythraea: ERBIV from Sac chain was fermented. erythraea for 4 days in the middle of SCM as described in General Methods. The fermentation broth of ERBIV de Sac. erythraea is then cooled to 4 ° C and adjusted to pH 4.0 and extracted once with methylene chloride. The aqueous layer is again adjusted to a pH of 9.0 and extracted twice with methylene chloride and the combined basic methylene chloride extracts are concentrated to a solid residue. This is digested in methanol and chromatographed on a column of Sephadex LH-20 in methanol. Fractions of bioactivity are tested against a sensitive organism, such as Staphylococcus aureus ThR, and the active fractions are combined. The combined fractions are concentrated and the residue is digested in 10 ml of the upper phase of a solvent system consisting of n-heptane, benzene, acetone, isopropanol, 0.05 M, pH 7.0 of aqueous phosphate buffer (5:10: 3: 2: 5, v / v / v / v / v), and chromatographed on an Ito Coil Planet Centrifuge in the same system. The active fractions are combined, concentrated and divided between methylene chloride and dilute ammonium hydroxide (pH 9.0). The methylene chloride layer is separated and concentrated to produce the desired product as a white foam.

EXAMPLE 2: CONSTRUCTION AND CHARACTERIZATION OF Sac.

Ervthraea ER720 (pASBVII) THAT PRODUCES 3-a-D-m? Carosil-5-ß-D-desosaminoyl-12-hydroxy-erythronolide B A Plasmid construction pASX2 (see figure 7) The 290 bp £ coRI-8a / 77HI seg leading to the ermE * promoter was isolated from plasmid plJ4070 and ligated into pWHM4 DNA digested with £ coRI-SamHI to form pASX1. of the transformation and isolation of the plasmid from £ coli, the identity of pASX1 was confirmed by digestion with ApaLI which releases frags of 39 kb, 2 5 kb, 1 2 kb, 0 5 kb, and 04 kb Two oligonucleotides from the sequences SEQ ID NO 31 (5'-GT-3 GATCCAGCGTCTGCAGGCATGCTCTA GATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAGATTTTCAA ') and SEQ ID NO 32 (5'-AGCTACGTTGAAAATCTCCAAAAAAAAA GGCTCCAAAAGGAGCCTTTAATTGTATCTAGAGCATGCCTGCAGACGC TG-3'), which correspond to strands (+) and (-) transcription terminator gene VIII of bacteriophage fd (t-fd) (Beck et al., (1978) Nucí Actds Res 54495) and including restriction enzyme sites for the enzymes Pstl, Sphl, and Xbal, and pendant ends compatible with BamHl and HindlW are synthesized and approximately 250 n g of each oligonucleotide are then mixed together in TE buffer and heated at 99 ° C for 1 minute. The solution is cooled slowly to room temperature allowing the oligonucleotides to be quenched due to auto-comple, and the quenched oligonucleotides are then ligated into pASX1 digested with SamHI-tf.ncNII to give pASX2 After transformation and isolation of the plasmid from £ coli, the identity of pASX2 is confirmed by DNA sequence of the frag £ coRI-Sa / l of 1 2 kb containing the ErmE * promoter and the bacteriophage terminator fd B Construction of plasmid pASBVIl (see figure 8) The seg of base pair DNA 598 leading to the eryBVIl gene, which comprises coordinates 7398-7996 (Figure 4B), is amplified by PCR using two oligonucleotides, SEQ ID NO 33 (5'-GATCGCATGCTC TAGAGTACG-TGAGCTGGCGGTGGCGGGC-3 ') and SEQ ID NO 34 (5'-GA TCCGGATCCGCATGCTT-CACCTGCCGGTGCTGGCGGG-3') After digestion of the purified PCR product with n BamHl-Xbal the PCR frag was ligated to pASX2 digested with ßamHI-XJbal to give pASBVIl After transformation and isolation of the plasmid from £ coli, the identity of pASBVIl is verified by DNA sequence of the insert EcoRI-Xal 880 bp C Construction of Sac ervthraea ER720 (pASBVII) Protoplasts of Sac erythraea chain ER720 were transformed with pASBVIl plasmid and transformers were selected with thiostrepton (15 μg / ml) To confirm the transformation, total DNA was isolated from ThR colonies and used to transform the coli After transformation and isolation of the plasmid from the coli, the identity of pASBVIl is verified by restriction analysis with the enzymes Pvull and Sa HI that releases a frag of 1 48 kb Those colonies of Sac erythraea that is found contain pASBVIl are designated Sac erythraea ER720 (pASBVII) D Isolation, purification and properties of 3-aDm? expensive? l-5-3-D-desosam? no? l-12-h? drox? -er? tronol? d a B from Sac ervthraea ER720 (pASBVII) Sac erythraea fer ER720 (pASBVII) for 3 days in SCM medium with selection of thiostrepton as described in General Methods The feration broth was then cooled to 4 ° C and adjusted to pH 4.0 and extracted once with methylene chloride. The aqueous solution is adjusted to a pH of 90 and extracted twice with methylene chloride and the combined extracts are concentrated to a solid residue. This is digested in methane and chromatographed on a column of Sephadex LH-20 in methanol. The fractions are tested. of bioactivity against a sensitive organism, such as Staphylococcus aureus ThR, and the active fractions are combined. The combined fractions are concentrated and the residue is digested in 10 ml of the upper phase of a solvent system consisting of n-heptane, benzene, acetone, isopropanol, 005 M, pH 70 of aqueous phosphate buffer (5 1032 5, v / v / v / v / v), and chromatographed on an Ito Coil Planet Centpfuge in the same system. They combine, concentrate and divide the acti fractions Between methylene chloride and dilute ammonium hydroxide (pH 90) The methylene chloride layer is separated and concentrated to produce the desired product as a white foam.

EXAMPLE 3: CONSTRUCTION AND CHARACTERIZATION OF Streptomvces antibioticus ATCC 11891 (pXB6l PRODUCING 3-de-oleandrosyl-3-micarosyl oleandomycin A. Construction of plasmid pKB6 (see figure 9) i) Construction of plasmid pK1: The DNA sequences of pBR322 (GenBank Access #: J01749) and pUC19 (GenBank Access #: X02514). The 805 nt DNA segment comprising coordinates 1673 to 2478 of pBR322 is amplified by PCR using two oligodeoxynucleotides, SEQ ID NO: 35 (5'-GATC ACATGTTCTTTCCTG-CGTTATCCCCTG-3 ') and SEQ ID NO: 36 (5'- GATC GGATCCATGCA-TGTCTAGAGCATCGCAGGATGCTGCTGGC-3 '). After digestion of the PCR product purified with Afllll and ßa HI the fragment is ligated into pUC19 digested with Afllll and BamHl to give the plasmid pK1. The identity of plasmid pK1, after transformation and isolation from £. coli, is verified by digestion of Pvulll that releases fragments of 0.55 kb and 2.55 kb. Plasmid pK1 contains the ROP region of pBR322 which controls the number of plasmid copies. ü) Construction of plasmid pKB1: The 2.24 kb DNA segment carrying the eryBIV and eryBV genes, comprised between the coordinates 56 and 2296 sequence presented in SEQ ID NO: 2, is amplified by PCR using two deoxyoligonucleotides, SEQ ID NO 37 (5'-GAATGCATCCTGGAAAGCGAGCAAATGCTCCGGTG-3 ') and SEQ ID NO 38 (5-'GATCTAGAGCTAGCCGGCGTGGCGGCGCGTG-3') After digestion? / S / l and Xbal the fragment is ligated into pK1 digested with? / S / ly and X £ > al to produce the plasmid pKB1, 53 kb in size The identity of the plasmid pKB1, after transformation from £. coli is verified by Kpnl digestion that releases fragments of 072 kb, 14 kb and 342 kb MI) Construction of plasmid pKB2 The DNA segment of 1 56 kb that leads to the eryBVI gene, included between coordinates 3121 and 4677 of the sequence presented in SEQ ID NO.2, is amplified by PCR using two deoxyoligonucleotides, SEQ ID NO 39 (5'-GATCGCTAGCCGTGACCGGACCCTTACAGTGAGTT G-3 ') and SEQ ID NO 40 (5'-GATCTAGACTTAAGTCATCCGGCGGTCCT GGTGTA GACGGC-3') After digestion with Nhel and Xbal the fragment is ligated into pKB1 digested with Nhel and Xbal to give the plasmid pKB2, 6.9 kb in size The identity of the plasmid pKB2, after transformation and isolation from £. coli, is confirmed by digestion of SamHI that releases fragments of 0.22 kb, 040 kb, 2.6 kb and 3.7 kb iv) Construction of plasmid pKB3: The 0.6 kb DNA segment carrying the eryBVIl gene, included between coordinates 7385 and 7987 of the sequence presented in SEQ ID NO: 2, is amplified by PCR using two deoxyoligonucleotides, SEQ ID NO 41 (α-GATCTTAAGAACCGGAGTTGCGAGTACGTGAGCT GGCG-3 ') and SEQ ID NO 42 (5'-GATCTAGACCTAGGTCACCTGCCGG TGCTGGCGGGCTC-3') After digestion with Afll and Xbal the fragment is ligated into pKB2 digested with Afll and Xbal giving plasmid pKB3, 75 kb in size The identity of the plasmid pKB3, after transformation and isolation from £ coli, is verified by digestion of Psfl that liberates fragments of 1 1 kb and 64 kb v) Construction of plasmid pKB4 The DNA segment of 1 0 kb carrying the eryBII gene, included between coordinates 2385 and 3410 of the sequence presented in SEQ ID NO 1, is amplified by PCR using two deoxiohgo nucleotides, SEQ ID NO 43 (5'-GATCCTAGGCCGCAGGAAGGAGAGAACCACG-3 ') and SEQ ID NO 44 (5'-GATCTAGATTAATCACTGCAACCAGGCTTCCGGC-3') After digestion with Avrll and Xbal the fragment is ligated into pKB3 digested with Avrll and Xbal giving plasmid pKB4 The identity of the plasmid pKB4, 85 in size, after transformation and isolation from £ colt, is verified by digestion of BglII and EcoRI that releases fragments of 041 kb, 1 6 kb, 3 1 kb and 34 kb vi) Construction of plasmid pKB5. The DNA sequence of eryBIII has been reported (Haydock et al. (1991) Mol Gen Genet 230120) The DNA segment of 1 3 kb leading to the eryBIII gene, included between coordinates 3965 and 5232 of the sequence illustrated in Haydock et al. , it is amplified by PCR using two oligonucleotides, SEQ ID NO 45 (5'-GATTAATTGGCCGCGGCGCCGC GCTC-GTTATG-3 ') and SEQ ID NO 46 (5'-GATCTAGATAATTAATCATAC GACTTCCAGTC-GGGGTAG-3') After digestion with Msel and Xbal the fragment is ligated into pKB4 digested with Msel and Xbal to give the desired plasmid pKB5, 98 in size The identity of pKB5, after transformation and isolation from £ co, is verified by digestion of Pstl which releases fragments of 1 1 kb, 2 5 kb, and 6 1 kb, visualized by gel electrophoresis vn) Construction of plasmid pKB6 The eryBI gene has been mapped (Haydock et al. (1991) Mol Gene Genet 230 120) and the DNA sequence is known in both changes of eryBI (Haydock et al. 1991) Mol Gen Genet 230 120) and GenBank Access # M11200 The 2 5 kb DNA segment leading to the eryBI gene, included between coordinates 1 1 and 36 of the map presented in Haydock et al., Is amplified by PCR using two deoxyoligonucleotides. SEQ ID NO 47 (d'GATTAATTAATGATCA-AGCTGAAAATTGTTTGCATG -3 ') and SEQ ID NO 48 (5'-G) ATCTAGACTGCCGGCT-CAGCCTTCCCAGG TTCG-3 ') After digestion with Pací and Xbal the fragment is ligated in pKB5 digested with Pací and Xal to give the plasmid pKB6, 12 3 kb in size The identity of pKB6, after transformation and isolation to from £ coli, it is verified by digestion of BamHl that releases 022 kb, 040 kb, 1 4 kb, 26 kb, 33 kb and 44 kb fragments. Plasmid pKB6 carries all the eryB, eryB-eryBVH genes, which are involved in the biosynthesis of micarosa and its binding to the pohcetide B Construction of plasmid pXSB6 (see figure 11) f The segment of? / s / l-Xsal of 92 kb of pKB6, prepared as described in example 3 (A) (v ??) above, which carries all the eryB genes is isolated and ligated into pASX2 digested with Psfl-Xal, prepared as described in example 2 (a) above, to give the plasmid pXSB6 After transformation and isolation of the plasmid a From £ coli, the identity of pXSB6, 172 kb in size, is verified by the observation Ion fragments of 041 kb, 1 9 kb, and 149 kb after digestion of £ coRI Plasmid pXSB6 carries all eryB genes in a trans-transcriptional fusion beyond the 5 'end of the ermE * promoter in a combined plasmid of E. coli ? ~ Streptomyces C Construction of plasmid pXB6 i) Construction of plasmid pN702 (see Figure 10) Two or gonucleotides of the sequences SEQ ID NO 49 (5'-GGAATTCAGA TCTATGCATTCTAGAA-3 ') and SEQ ID NO 50 (5'-) are synthesized. CGCGTTCTAGAATGCA TAGATCTGAATTCCTGCA-3 ') which include restriction enzyme sites for the enzymes £ coRI, BglII,? / S / ly and Xbal and pendant ends compatible with Psfl and Mlul Approximately 250 ng of each ohgonucleotide are then mixed together in TE buffer and they are heated at 99 ° C for one minute. After the solution is slowly cooled to room temperature allowing the ohgonucleotides to warm up due to auto-complement, the tempered oligonucleotides are ligated into plJ702 digested with Pstl-Mlu for p roducing the desired plasmid pN702 After transformation and isolation of the plasmid from Streptomyces lividans 1326, the identity of the plasmid pN702, 4 3 kb in size is verified by the observation of fragments of 0 75 kb and 36 kb after digestion of fcoRI-ßamHI or Xal-ßamHI n) Construction of plasmid pX1 (see figure 10) The 290 bp £ coRI-ßamHI segment leading to the ermE * promoter is isolated from plasmid plJ4070 and ligated into pN702 digested with coRI-BglII to give the plasmid pX1 The resulting mixture contains the desired plasmid pX1 After transformation and isolation of the plasmid from Streptomyces lividans 1326 the identity of the plasmid pX1, 46 kb in size, is verified by the observation of 1 0 kb fragments and 36 kb after digestion of? / S / l-ßa HI ni) Construction of plasmid pXB6 (see figure 11) The segment of Ns? \ - Xba \ of 92 kb of pKB6, prepared as described in example 3 ( A) (v ??) previous, that carries all The genes eryB is isolated and ligated in pX1 digested with Ns? \ - Xba \ to give the desired plasmid pXB6 After transformation and isolation of the plasmid from Streptomyces lividans 1326, the identity of plasmid pXB6, 138 in size, is verified by the observation of fragments of 041 kb, 1 9 kb and 11 5 kb after EcoRI digestion Plasmid pXB6 carries all eryB genes in a transcppional fusion to the ermE * promoter in a Streptomyces plasmid D Construction of Streptomyces antibioticus ATCC 11891 (pXB6) Approximately 500 μg of plasmid pXB6, isolated from Streptomyces lividans 1326 (pXB6), is electroporated into the oleandomycin producer Streptomyces antibioticus ATCC 11891 and some of the Th? oR colonies Results that appear on R3M-agar plates containing thiostrepton are analyzed for their plasmid content The presence of plasmid pXB6, 138 kb in size, is verified by observing fragments of 041 kb, 1 9 kb and 11 5 kb after of digestion £ coRI E Isolation, purification and properties of 3-des-oleandros? l-3-m? s? oleandomycin of Streptomyces antibioticus ATCC 11891 (pXB6) Streptomyces antibio was fermented ticus ATCC 11891 (pXB6) for 5 days in SCM medium with thiostrepton selection as described in General Methods The fermentation broth is then cooled to 4 ° C and adjusted to a pH of 40 and extracted once with methylene chloride The aqueous layer is re-adjusted to pH 9.0 and extracted twice with methylene chloride and the combined basic methylene chloride extracts are concentrated to a solid residue. This is digested in methanol and chromatographed on a Sephadex LH column. -20 in methanol Fractions of bioactivity are tested against a sensitive organism, such as Staphylococcus aureus ThR, and the active fractions are combined. The combined fractions are concentrated and the residue is digested in 10 ml of the upper phase of a solvent system which consists of n-heptane benzene, acetone, isopropanol, 005 M pH 7 0 of aqueous phosphate buffer (5 10325, v / v / v / v / v), and is chromatographed on an Ito Coil Planet Centpfuge in the same system combine, concentrate and divide the active fractions between methylene chloride and dilute ammonium hydroxide (pH 90). The methylene chloride layer is separated and concentrated to produce the desired product as a white foam.

EXAMPLE 4: CONSTRUCTION AND CHARACTERIZATION OF Streptomvces violaceoniaer NRRL 2834 (pXC4) WHICH PRODUCES 5- des-calcosyl-5-desosaminoyl lancamycin A Construction of plasmid pKC4 and intermediates (see figure 121 i) Construction of plasmid pKC1 The 24 kb DNA segment carrying the eryCII and eryCIII genes, comprised between coordinates 33 and 2413 of the sequence presented in SEQ ID NO 1, is amplified by PCR using two deoxyoltgonucleotides, SEQ ID NO 51 (5'-GAATGCATCTGGCTGGGCGGAGGGAATTCATG-3 ') and SEQ ID NO 52 (5'-GATCTAGACTTAAGTCATCGTGGTTCTCTCCTTCCTGC GGC-3') After digestion with? / s / l and Xbal the purified PCR fragment is ligated into pK1 digested with? / s / ly and X? al to give plasmid pKC1, 55 kb in size The identity of plasmid pKC1, after transformation and isolation from £ coll, is verified by digestion of £ coRI that liberates fragments of 22 kb and 33 kb II) Construction of plasmid pKC2 The 732 bp DNA segment carrying the eryCVl gene, included between coordinates 2331 and 3063 of the sequence presented in SEQ ID NO 2, is amplified by PCR using two deoxy-oligonucleotide tidos, SEQ ID NO 53 (5'-GATCCTTAAGCTCCGGAGGGAGCAGGGATC-3 ') and SEQ ID NO 54 (5'-GATCTAGACCTAGGTCATCCGCGCACACCGACG AAC-3) After digestion with Afll and Xbal the purified PCR fragment is ligated into pKC1 digested with Afll and Xbal to give the plasmid pKC2, 62 in size The identity of the plasmid pKC2, after the transformation and isolation from £ coli, is verified by digestion of Xbalcole which releases fragments of 095 kb, 2 2 kb and 3 1 kb MI) Construction of plasmid pKC3 The DNA segment of 2 7 kb carrying the genes eryCIV and eryCV, comprised between coordinates 4650 and 7386 of the sequence presented in SEQ ID NO 2, is amplified by PCR using two deoxio gonucleotides, SEQ ID NO 55 (5'-GATCCTAGGCCGTCTACACCAGGACCGCCGG-3 ') and SEQ ID NO 56 (5'-GATCTAGATTAATCACCTTCCGCGCAGGAAGCCGC-3') After digestion with Avrll and Xbal the purified PCR fragment is ligated into pKC2 digested with Avrll and Xbal to produce Plasmid pKC3, 90 in size The identid ad of plasmid pKC3, after transformation and isolation from £ coli, is verified by digestion of Sphl releasing 40 kb and 5 0 kb fragments iv) Construction of plasmid pKC4 The DNA sequence of the eryCI genes has been determined (GenBank Access # X15541). The 1 1 kb DNA segment carrying the eryCI gene, comprised between coordinates 38 and 1161 of the sequence indicated above, is amplified by PCR using two deoxyoligonucleotides, SEQ ID NO 57 (5'-GATCTTAAGCCGCCACTCGAACGGACACTCG-3 ') and SEQ ID NO 58 (5'-GATCTAGATCAAGCCCCAGCCTTGAGGG-3 ') After digestion with Msel and Xbal the fragment is ligated into pKC3 digested with Msel and Xbal to give the plasmid pKC4, 10 1 kb in size The identity of the plasmid pKC4, after the transformation and isolation from £ coli, is verified by Kpnl digestion that releases fragments of 0 15 kb, 0.31 kb, 4 1 kb and 5.5 kb The plasmid pKC4 carries all the genes eryC, eryCI-eryCVI, which are involved in biosynthesis of desosamine and its binding to polyketide B. Construction of plasmid pXSC4 (see figure 13) The segment of Nsi \ -Xba \ 6.9 of pKC4 carrying all the genes of eryC is isolated and ligated in pASX2 digested with Pstl-Xbal , prepared as described in example 2 (A), to give the desired plasmid pXSC4, 14.9 kb in size, where all the eryC genes are transcriptionally linked beyond the 5 'end of the ermE * promoter in a combined plasmid of £. coli-Streptomyces. The identity of plasmid pXSC4, after transformation and isolation from £ coli, is verified by the observation of fragments of 0.29 kb, 2 2 kb and 12 4 kb after the digestion of £ coRI C Construction of plasmid pXC4 (see Figure 13): The Nsil-Xbal segment 69 of pKC4 carrying all the eryC genes is isolated and ligated in pX1 digested with Nsil-Xbal, prepared as described in Example 3 (C) (??), to give the desired plasmid pX1SC4, 11 5 kb in size, where all the eryC genes are trans-transcriptionally linked beyond the 5 'end of the ermE * promoter in a Streptomyces plasmid The identity of the plasmid pXC4, after transformation and isolation from Streptomyces lividans 1326, it is verified by the observation of fragments of 0.29 kb, 22 kb and 90 kb after digestion of the coRI. D Construction of Streptomyces violaceoniaer NRRL 2834 (pXC4) Approximately 500 μg of plasmid pXC4, isolated from Streptomyces lividans 1326 (pXC4), is electroporated into the lancamycin producer Streptomyces violaceoniger NRRL 2834 and some of the resulting ThioR colonies that appear on the plates of R3M-agar containing thiostrepton are analyzed for their plasmid content The presence of plasmid pXC4 is verified by the observation of fragments of 0.29 kb, 2.2 kb and 9.1 kb in size after EcoRI digestion of plasmid E. Isolation, purification and properties of 5-des-desosaminoyl lancamycin S. violaceoniger NRRL 2834 (pXC4) was fermented for 5 days in SCM medium with thiostrepton selection as described in General Methods The fermentation broth is then cooled to 4 ° C and adjusted to pH of 40 and extracted once with methylene chloride. The aqueous layer is again adjusted to a pH of 90 and extracted twice with methylene chloride and the Combined methylene chloride extracts are concentrated to a solid residue. This is digested in methanol and chromatographed on a column of Sephadex LH-20 in methanol. The bioactivity fractions are tested against a sensitive organism, such as Staphyloccocus aureus ThR, and combined. active fractions The combined fractions are concentrated and the residue is digested in 10 ml of the upper phase of a solvent system consisting of n-heptane, benzene, acetone, isopropanol, 005 M, pH 7.0 of aqueous phosphate buffer ( 5 10 32 5, v / v / v / v / v), and chromatographed on an Ito Coil Planet Centpfuge in the same system. The active fractions are combined, concentrated and divided between methylene chloride and dilute ammonium hydroxide (pH 9.0). The methylene chloride layer is separated and concentrated to produce the desired product as a white foam. Although the present invention is illustrated in the examples listed above in terms of preferred embodiments, these examples should not be construed as limiting the scope of the invention. The above illustrations serve to describe the principles and methodologies involved in the creation of the types of alterations. Genetics that can be introduced in Sac. erythraea and / or other Streptomyces that result in the synthesis of novel products of polyketide modified by glycosylation Although a single alteration of Type I, leading to the production of for example 4"-desox? -4" -oxo-er? throm? c A, it is specified herein that it is obvious to those skilled in the art that other Type I changes in the eryB and / or eryC genes can be introduced leading to novel structures of modified polyketide by costing. Examples of additional alterations of the Type I that lead to useful novel compounds include, but are not limited to, mutations in the eryBVIl gene that conceivably leads to 3-aDm? Expensive? L-5-ß-D-desosam? No? L-12-h? drox? -er? tronol? da B and mutations in the eryCVl gene that lead in a conceivable way to N-3a'-des-d? met? l eptromicin A In addition, it is obvious that Type I alterations in two or more genes eryB and / or eryC can be combined leading to novel structures of polycheid modified by ghcosil Examples of combinations of two Type I alterations that lead to useful compounds include, but are not limited to, mutations in the eryBIV and eryBVI1 genes that conceivably lead to 3-aD-4"-desox? -4" -oxo- m? 5-ß-D-desosam? no? l-12-h? drox? -er? tronol? da B, mutations in the eryBIV and eryCVl genes that conceivably lead to 4"-desox? -4"-oxo- (N-3a'-des-d? Met? L) -er? Trom? C? Na A, and mutations in the eryBIV, eryBVIl and eryCVl genes that lead conceivably to 3-aD-4"-desox? -4" -oxo-m? expensive? l-5-ß-D- (N-3a'- des-d? met? l) -desosa-m? no? l-12-h? drox? -er? tronol? da B All mutations of Type I or combinations of two or more mutations of Type I in the eryBII genes eryBIV, eryBV, eryBVI, eryBVIl, eryCII, eryCIII, eryCIV, eryCV or eryCVl, the strands of Sac erythraea carrying such mutations or combinations of mutations, and the corresponding pohcetides produced from said strands, therefore, are within range of the present invention Although the Type II mutation in the present was constructed with the eryBVII gene in a self-replicating plasmid, it is obvious that other eryB genes and eryC genes can be expressed in an anti-sense orientation leading to novel structures Modified Polypeptide by Glycosylation Examples of additional Type II alterations leading to useful compounds include, but are not limited to ex anti-sense pressure of the eryBIV gene which conceivably leads to 4"-desox? -4" -oxo-eptrom? c? na A and antisense expression of the eryCVl gene which leads conceivably to N-3a'-des In addition, it will occur to those skilled in the art that promoters other than the ermE * promoter, for example the melC promoter of plJ702, will be suitable for anti-sense expression, and that many vectors of Self-replication in addition to pWHM4 will work to carry the anti-sense alteration It will also occur for those of skill in the art that a self-replicating vector is not required for this invention and that the anti-sense alteration can be introduced directly into the chromosome by using the same principles used to construct a Type I gene alteration An example of a Type II alteration that is introduced directly into the chromosome is the eryBVII antisense alteration described in Example 2, where s DNA segments immediately beyond the 3 'end of the eryK gene are used to change the ending of e / 777 £ -eryß \ /// - phage fd that it groups into a pWHM3 vector, and this vector is integrated into and then resolves from the chromosome leaving the terminator of ermE * -eryB V / -phago fd clustering stably incorporated in its nonessential region of the Sac erythraea chromosome that leads conceivably to the production of expensive 3-aDm? l-5-ß- D-desosam? No? L-12-h? Drox? -er? Tronohda B All Type II mutations in the genes eryBII, eryBIV, eryBV, eryBVI, eryBVIl, eryCII, eryCIII, eryCIV, eryCV or eryCVl carried in a self-replicating plasmids or integrated in a non-essential region of the chromosome, the strands of Sac erythraea carrying said mutations, and the corresponding polyketides produced from said strands, therefore, are within the scope of the present invention. Type III alterations, which lead to the production of 5-decalcings? l-5- In the case of L. lancamycin in Streptomyces violaceoniger and 3-de-olearyl-3-mers expensive oleandomycin in Streptomyces antibioticus, are specified here, it is obvious that Type III alterations can be introduced in any microorganism that Produces polyketide leading to novel polyketides modified by glycosylation It will also occur to those skilled in the art that the eryB and eryC genes can be cotransformed into a microorganism that produces polyketide or clustered together into a single vector that is introduced into a microorganism that produces polyketide. Example of a change in Type III that uses the eryB and eryC genes together is its introduction in Streptomyces violaceoniger that conceivably leads to 3-des- (4"-0-acet? larcanos? l) -3-m? l-5-de-chalcos? l-5-desosam? no? lancamycin Although the Type III alterations specified herein have indicated a specific genetic order of the eryB or eryC genes, it will occur for those who are In the art, many different genetic arrangements of the eryB or eryC genes will produce similar results. It will also occur for those skilled in the art that certain arrangements of the eryB or eryC genes that lack one or more of the eryB and / or eryC genes will lead to the production of novel glycosylated polyketides where the intermediates in the biosynthesis of micarosa and / or desosamine, respectively, such as those delineated in Figures 2 and 3, are fixed to the polyketide. An example of a Type III alteration in which only a sub-domain of the eryB and / or eryC genes is used is the introduction of a derivative of pXC4. lacking the eryCVl gene, removed by digestion of plasmid pXC4 with Aflll and Avrll followed by treatment with the Klenow fragment of DNA polymerase I and religation, in Streptomyces violaceoniger leading to the production of 5-decalcings? l- (N- 3a'-des-dimethyl desosaminoyl) lancamycin. It will also occur for those who are skilled in the art that promoters other than ermE or ermE *, such as the melC promoter of plasmid plJ702, and vectors other than pWHM4 or plJ702 can also be used in the construction of a Type III alteration, and these vanishing ones, of course, are considered within the scope of the invention. Finally, it will also occur for those of skill in the art that a self-replicating vector is not required for this invention and that an assembly of sugar biosynthesis genes can be enter directly into the chromosome of a heterologous host by using the same principles used to construct an alteration of Type I gene once a non-essential region of the heterologous host chromosome has been identified. Alternatively, plasmids or bacteriophages that undergo site-specific recombination with host genes can also be used to introduce eryB and eryC genes into a host to perform Type III alterations. All Type III alterations that use one or more of the eryBII genes, eryBIV, eryBV, eryBV !, eryBVIl, eryCII, eryCIII, eryCIV, eryCV or eryCVl, the polyketide producing strands carrying such alterations, and the corresponding polyketides produced from said strands, therefore, are included within the scope of the present invention.

In addition, it is also possible to create combinations of Type I and Type II alterations such as some mutations of eryB and / or eryC of Type I are introduced directly into the chromosome of Sac erythraea in the appropriate place while other genes eryB and / or eryC are activated by Type II alterations when using a self-replicating or integrating vector. For example, a combination of a Type I alteration, such as a mutation in eryBIV, and a Type II alteration, such as transformation with pASBVIll, will lead conceivably to the production of 3-aD-4"-desox? -4" -oxo-m? expensive? l-5-ß-D-desosam? no? l-12-h? drox? -er? tronol ? da B All combinations of two or more alterations of Type I and Type II, strands of Sac erythraea carrying such alterations, and glycosylated polyketides produced from said strands are included within the scope of the present invention as an extension of the examples recorded with the eryB and / or eryC genes, it is possible to apply r the method described herein to heterologous sugar biosynthesis genes that are similar to the eryB and / or eryC genes The construction of strands carrying heterologous sugar biosynthesis genes that lead to the production of novel glycosylated polyketides requires (i) clone the sugar biosynthesis genes from any other actinomycete that produces glycosylated polyketide, (n) determine the nucleotide sequence of the cloned gene, (ni) cut and assemble the cloned genes into vectors suitable for Type I, Type II alterations , or Type III, and (iv) transform microorganisms that produce pohcetide and screen the novel compound. In this way, any sugar biosynthesis gene associated with polyketide can be accurately cut from the genome of a microorganism that produces gaseous and altered pohcetide g or disposed with other sugar biosynthesis genes and then introduced into the same or different microorganism that produces polyketide to create a novel ghcosylated pohcetide of predicted structure In this way, for example a Type I or Type II alteration of a heterologous gene that is similar to an eryB and / or eryC gene, as can be found in the homolog eryBVIl for the synthesis of L-oleandrosa in Streptomyces antibioticus, to result in the production of 3-de-L-oleandros? l-3-D-oleandros? l oleandomycin, is included within the scope of the present invention. Similarly, a Type III assembly of the genes for the synthesis of a sugar other than micarosa or desosamine, as can be found in the genes for the synthesis of angolosamine in Streptomyces eurythermus, and its transformation into Sac erythraea to result in the synthesis of des-desosamino? l-5-angolosam? no? l-eptromycin A, it is included within the scope of the present invention It will occur to those skilled in the art that the genetic manipulations of Type I, Type II and Type III described The present and the microorganisms that produce polyketide, where they are introduced, are by no means exclusive Therefore, the choice of a suitable host and the choice of a Type I or Type III alteration is based solely on the ratio of the desired novel glycosylated polyketide with a natural counterpart Thus, Type I, Type II, and Type III alterations can be constructed in any microorganism that produces pohcetide using endogenous or exogenous sugar biosynthesis genes. Thus, all mutations of the type I, Type II and Type III or various combinations thereof constructed in any microorganism that produces polyketide in accordance with the principles described herein, and the respective polyketides produced from said strands, are included within the scope of the present invention. Examples of glycosylated polyketides that can be altered when creating Type I, Type II, or Type III changes n the production of microorganisms include, but are not limited to macrolide antibiotics such as eptromycin, tylosin, spiramycin, etc., aromatic polyketides such as daunorubicin and doxorubicin, etc., polyenes such as candicidin, amphotecines, etc., and other complex pohcetides such as avermectin While the novel derivatives or modifications of ephromycin described herein have been specified as derivatives of A, such as 4"-deoxy? -4" -oxo-er? throm? c? na A, those skilled in the art understand that the wild-type strand of Sac erythraea produces a family of compounds of eptromycin, including epigomycin A, eptromycin B, epromycin C, and epromycin D Thus, the modified strands of Sac erythraea such as ERBIV strand, for example, would be expected to produce the corresponding members of the family of 4"-desox? -4" -oxo-eptrom? c? na including 4"-desox? -4" -oxo-eptrom? c? na A, 4"-desox? -4" -oxo-eptrom? c? na B, 4"-desox? -4"-oxo-er? Trom? C? Na C and 4" -desox? -4 '-oxo-eptromicin D Similarly, all other modified strands of Sac erythraea producing novel glycosylated epitromycin derivatives would be expected to produce the AB, C and D forms of such derivatives. For example, the modified strands of Sac erythraea that produce 6-deoxyric? er? trom? c? na, 6.12 -d? desox? eptrom? c? na and 6,7-anh? droer? trom? na? would be expected to produce novel glycosylation-modified pohcetides from further modification of a Type I, II or III change in a gene of sugar biosynthesis Therefore, all family members of each of the novel ephromycins described herein or produced by these methods are included within the scope of the present invention. Variations and modifications of the methods to obtain the desired plasmids , hosts for cloning and choices of eryB and eryC vectors and genes and modifying, others other than those described herein, will occur for those who are experts in the art. For example, although the use of plasmids has been described WHM3, pWHM4, and plJ702, other vectors may be employed where all or part of said plasmids are replaced by other DNA segments that function in a similar manner, such as replacing the pUC19 component of pWHM3 and pWHM4 with pBR322, available from BRL, or employ different segments of the replicon pl J 01 in pWHM3 and plJ702, or the replicon pJV1 in pWHM4, respectively, or employ selectable markers other than resistance to thiostrepton or ampicillin There are only a few of a long list of possible examples of which all are included Within the scope of the present invention Similarly, the segments of the eryB and eryC that have been specified herein to generate the vain alterations of Type I, Type II and Type III can be readily replaced by other segments of different length than encode the same functions, whether produced by PCR amplification of genomic DNA or from an isolated clone, or by isolating restriction fragments Sac erythraea In the same way, it is possible to create Type I mutations functionally equivalent to those described by altering through omission, insertion or site-directed mutagenesis different portions of the corresponding genes It is also possible to create equivalent Type II mutations functionally to those described herein by employing larger or smaller portions of the corresponding genes, and it is possible to create Type III mutations by using larger or smaller segments of the corresponding genes in the same or different linear order described in the present additional modifications include changes in the restriction sites used for cloning or in the general methodologies described above. All said changes are included in the scope of the present invention. It will also occur for those who are skilled in the art that there are different methods available to ferment Sac. . erythraea and other microorganisms that produce polyketide and to extract the novel pohcetides specified herein, and all such methods are also included within the scope of this invention. It will also be apparent that many modifications and variations of the invention as set forth herein are possible without departing from the spirit of the scope thereof, and that, therefore, such limitations are imposed only as indicated by the appended claims.

Claims (21)

1 - . 1 - An isolated single or double-stranded polynucleotide having a nucleotide sequence comprising (a) a nucleotide sequence selected from the group consisting of (i) the sense sequence of SEQ ID NO 1 of about the position from nucleotide 54 to about the nucleotide position 1136, (n) the sense sequence of SEQ ID NO 1 from about the position of nucleotide 1147 to about the nucleotide position 2412, (ni) the sense sequence of SEQ ID NO 1 from about nucleotide position 2409 to about nucleotide position 3410, (iv) the sense sequence of SEQ ID NO 2 from about the position of nucleotide 80 to about the nucleotide position 1048, (v) the sequence of sense of SEQ ID NO 2 from about nucleotide position 1048 to about nucleotide position 2295, (vi) the sense sequence of SEQ ID NO 2 from about nucleotide position 2348 to about the position of nucleotide 3061; (vn) the sense sequence of SEQ ID NO 2 from about nucleotide position 3214 to about nucleotide position 4677, (vin) the sense sequence of SEQ ID NO 2 from about nucleotide position 4674 to about the position of nucleotide 5879, (ix) the sense sequence of SEQ ID NO 2 from about nucleotide position 5917 to about nucleotide position 7386, and (x) the sense sequence of SEQ ID NO 2 of about the position of nucleotide 7415 at about the position of nucleotide 7996, (b) sequences complementary to the sequences of (a), (c) sequences which, in expression, encode a polypeptide encoded by the sequences of (a), and (d) ) analogous sequences that hybridize under demanding conditions to the sequences of (a)

2 - The polynucleotide according to claim 1, which is a DNA molecule or an RNA molecule

3 - The polynucleotide according to claim ication 2, wherein the nucleotide sequence is the nucleotide sequence of (a) selected from the group consisting of (i) the sense sequence of SEQ ID NO 1 from about nucleotide position 54 to about the position of nucleotide 1136, (n) the sense sequence of SEQ ID NO 1 from about nucleotide position 1147 to about nucleotide position 2412, (ni) the sense sequence of SEQ ID NO: 2 around the nucleotide position 2348 at about nucleotide position 3061, (iv) the sense sequence of SEQ ID NO 2 from about nucleotide position 4674 to about nucleotide position 5879, and (v) the sense sequence of SEQ ID NO 2 from about the position of nucleotide 5917 to about the position of nucleotide 7386.

4 - The polynucleotide according to claim 2, wherein the nucleotide sequence is the nucleotide sequence of (a) sele cited from the group consisting of (i) the sense sequence of SEQ ID NO 1 from about the position of nucleotide 2409 to about the nucleotide position 3410, (n) the sense sequence of SEQ ID NO 2 around from the position of nucleotide 80 to approximately the nucleotide position 1048, (MI) the sense sequence of SEQ ID NO 2 from about the position of nucleotide 1048 to about the position of nucleotide 2295, (iv) the sequence of sense of SEQ ID NO 2 from about the position of nucleotide 3214 to about the position of nucleotide 4677, and (v) the sequence of sense of SEQ ID NO 2 around the position of nucleotide 7415 to about the position of nucleotide 7996

5 - The polynucleotide according to claim 2, wherein the nucleotide sequence is the nucleotide sequence of (a) having the sense sequence of SEQ ID NO 2 from about the position of nucleotide 80 to about The nucleotide position 1048

6 - A vector comprising the DNA molecule according to claim 2 - The vector according to claim 6, which further comprises an enhancer-promoter operably linked to the polynucleotide 8 - The vector according to claim 6, wherein the polynucleotide has the nucleotide sequence according to claim 5 - A host cell transformed with the vector in accordance with claim 6 or claim 7 or claim 8 - The transformed host cell according to claim 9, is a bacterial cell 11 - The transformed host cell according to claim 10, wherein the bacterial cell is selected from from the group consisting of Streptomyces and £ coli 12 - A method for directing the biosynthesis of specific polyketides modified by glycosylation by genetic manipulation of a microorganism that produces polyketide, said method comprising the steps of (1) isolating a DNA sequence containing sugar biosynthesis gene in accordance with the law indication 1, (2) identify within said DNA sequence containing gene one or more DNA fragments responsible for the biosynthesis of a sugar associated with pohcetide or its binding to the polyketide, (3) create one or more specific changes in said fragment or fragments of DNA, thus resulting in an altered DNA sequence, (4) introducing said altered DNA sequence into a microorganism that produces polyketide to replace the original sequence, said altered DNA sequence, when translated, resulting in enzymatic activity altered capable of carrying out the production of said specific polyketide modified by g cosilation, (5) growing a culture of said microorganism that produces altered pohcetide under conditions suitable for the formation of said specific polyketide modified by glycosylation, (6) isolating said specific modified polyketide by glycosylation of said culture 13 - The method according to claim 12, wherein said specific change of said DNA fragment or fragments results in the inactivation of at least one enzymatic activity involved in the biosynthesis of a sugar associated with polyketide or in its attachment to a polyketide 14 - The method according to claim 13, wherein said sugar associated with polyketide is L-mycarose 15 - The method according to claim 13, wherein said sugar associated with polyketide is D-desosamine 16 - A method for directing the biosynthesis of specific polyketides modified by ghcosylation of a microorganism that produces polyketide, said method comprising the steps of (1) isolating a DNA sequence containing a biosynthesis gene from sugar according to claim 1, (2) identify within said DNA sequence containing g in one or more DNA fragments responsible for the biosynthesis of a sugar associated with pohcetide or its binding to the polyketide, (3) reversing the strand orientation of said fragment or DNA fragments, thus resulting in an altered DNA sequence which, when transcribed, it results in the production of an anti-sense mRNA, (4) introducing said altered DNA sequence into a microorganism that produces polyketide having an mRNA capable of binding said anti-sense mRNA to produce a microorganism that produces altered polyketide capable of producing said specific polyketide modified by glycosylation, (5) growing a culture of said microorganism that produces altered polyketide under conditions suitable for the formation of said specific polyketide modified by glycosylation, (6) isolating said modified specific polyketide by glycosylation of said culture. 1

7 - A method for directing the biosynthesis of specific polyketides modified by glycosylation by genetic manipulation of a microorganism that produces polyketide, said method comprising the steps of (1) isolating a DNA sequence containing sugar biosynthesis gene in accordance with claim 1; (2) identifying within said DNA sequence containing gene one or more DNA fragments responsible for the biosynthesis of a sugar associated with pohcetide or its binding to the polyketide; (3) introducing said fragment or fragments of DNA into a different microorganism to produce a microorganism that produces altered polyketide capable of producing said specific polyketide modified by glycosylation, (4) growing a culture of said microorganism that produces polyketide containing said fragment or DNA fragments under conditions suitable for the formation of said specific polyketide modified by glycosylation, and (6) isolating said specific modified polyketide by glycosylation of said culture 1

8 - The method according to claim 13 or claim 16 or claim 17, wherein said fragment of DNA comprises one or more genes encoding an enzymatic activity involved in the biosynthesis of L-micarosa or in its attachment to a polyketide 1

9 - The method according to claim 13 or claim 16 or claim 17, wherein said fragment of DNA comprises one or more genes that encode an enzymatic activity involved in the biosynthesis of D-desosamine or in its attachment to a pohcetide 20 - The method according to claim 13 or claim 16 or claim 17, wherein said fragment of DNA is the sequence according to claim 8 - An isolated polypeptide having an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of the sense sequence of SEQ ID NO 1 around the position of nucleotide 54 to about the nucleotide position 1136, the sense sequence of SEQ ID NO 1 from about the position of nucleotide 1147 to about the position of nucleotide 2412, the sense sequence of SEQ ID NO 1 around the position of nucleotide 2409 to about the position of nucleotide 3410 , the sense sequence of SEQ ID NO 2 from about nucleotide position 80 to about nucleotide position 1048, the sense sequence of SEQ ID NO 2 from about nucleotide position 1048 to about nucleotide position 2295 , the sense sequence of SEQ ID NO 2 from about nucleotide position 2348 to about nucleotide position 3061, the sense sequence of SEQ ID NO 2 from about nucleotide position 3214 to about nucleotide position 4677 , the sense sequence of SEQ ID NO 2 around the position of nucleotide 4674 at approximately the position of nucleotide 5879, the sequence of sense of SEQ ID NO 2 from about nucleotide position 5917 to about nucleotide position 7386, and the sense sequence of SEQ ID NO 2 from about nucleotide position 7415 to about nucleotide position 7996