MXPA99008462A - Streptomyces avermitilis regulatory genes for avermedtium production of avermecti - Google Patents

Abstract

The present invention relates to compositions and methods for producing avermectins and is primarily in the field of animal health. The present invention relates to the identification and characterization of two new genes, herecited as the aveR1 and aveR2 genes, which are involved in regulating the expression of polyketide synthase (PKS) of avermectins and the synthesis of avermectins in Streptomyces avermitilis. The present invention is based on the discovery that the deactivation of these genes results in an increase in the amount of avermectins produced by S. Avermitil

Description

REGULATORY GENES OF STREPTOMYCES AVERMITILIS FOR INCREASED PRODUCTION OF AVERMECTINES 1. FIELD OF THE INVENTION The present invention relates to compositions and methods for producing avermectins, and is found primarily in the field of animal exit. More particularly, the present invention relates to the identification and characterization of two new genes, cited herein as the aveR1 and aveR2 genes, which are involved in the regulation of the expression of avermectin-polyketide synthase (PKS) and the biosynthesis of avermectins in Streptomyces avermitilis. The present invention is based on the discovery that deactivation of these genes results in an increase in the amount of an avermectin produced by S. Avermitilis. 2. BACKGROUND OF THE INVENTION Streptomyces species produce a wide variety of secondary metabolites, including avermectins, which comprise a series of eight related, sixteen-member macrocyclic lactones that have potent anthelmintic and insecticidal activity. The eight distinct but closely related compounds are cited as Ala, Alb, A2a, A2b, B1a, B1b, B2a and B2c. The "a" series of compounds refers to natural avermectin, wherein the substituent at the C25 position is (S) -sec-butyl, and the "b" series refers to the compounds in which the substituent on the C25 position is isopropyl. The designations "A" and "B" refer to avermectins in which the substituent on C5 is methoxy and hydroxy, respectively. The number "1" refers to avermectins in which a double bond is present at the C22, 23 position, and the number "2" refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position. Among the related related avermectins, type B1 of avermectin is recognized as having the most effective antiparasitic and pesticidal activity, and therefore it is the most commercially desirable avermectin. The avermectins and their production by aerobic fermentation of strains of S. Avermitilis are described, inter alia, in United States of America patents 4,310,519 and 4,429,042.

The genes of avermectin (bird) like many genes involved in the production of secondary metabolites and other Streptomyces antibiotics, are grouped together in the bacterial chromosome. The bird gene cluster for avermectin biosynthesis encompasses a 95 kb genomic fragment of DNA, which includes the DNA encoding avermectin-polyketide synthase (PKS) (MacNeil et al., 1992, Gene 115: 119-125).

The regulation of antibiotic biosynthesis in Streptomyces is possibly optimally characterized in the Streptomyces coelicolor species. Four antibiotics produced by S. Coelicolor include actinorrodine (Act), undecylprodigiosin (Red), calcium dependent antibiotic (CDA), and methyleneomycin (Mmy). Each of these antibiotics is encoded by a different grouping of gene different genetically. Genes have been identified that are linked to either the Act gene cluster or the Red gene cluster, which encode products that specifically regulate the expression of Act's biosynthetic gene cluster or Red's biosynthetic gene cluster. respectively. A number of 1oci have also been identified that contain genes that globally regulate more than one of the biosynthetic gene clusters of antibiotics. For example, it has been shown that mutations in two independent 1oci, absA and absB, block the synthesis of all four antibiotics in S. Coelicolor (Brian et al, 1996, J. Bact, 178: 3221-3231). The absA locus has been cloned and characterized, and it has been shown that its gene products are involved in a signal transduction pathway that normally acts as a global negative regulator of the synthesis of antibiotics in S. coelicolor (Brian et al., 1996, previous).

The patent of the U.S.A. 5,707,839 granted to Denoya and the US patent. 5,728,851 granted to Denoya et al. Refer to DNA sequences encoding complexes of alpha- (branched chain ketoacid) -Dehydrogenase Streptomyces and methods to enhance the production of new avermectins.

The understanding of the mechanism by which expression of type I polyketide-synthase is regulated in S. Avermectilis will allow the genetic manipulation of bird genes to increase the production of avermectins. 3. BRIEF DESCRIPTION OF THE INVENTION The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of the aveR1 gene from S. Avermitilis. In a preferred embodiment, the aveR1 gene product comprises the amino acid sequence of SEQ ID NO: 2. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR1 ORF of S. Avermitilis, as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317. In another additional non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO: 1.

The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of the ORF of aveR1 of S. Avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 2.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR1-related polynucleotide molecules. In a preferred embodiment, the substantial portion of the aveR1-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of an aveR1 gene product of S. Avermitilis or a aveR1-related homologous polypeptide of the present invention. In a specific but non-limiting embodiment, the present invention provides a polynucleotide molecule that consists of a nucleotide sequence encoding a peptide fragment consisting of a subsequence of the amino acid sequence of SEQ ID NO: 2.

The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 ORF of S. Avermitilis in situ. Said flanking sequences can be selected from the nucleotide sequence of SEQ ID NO: 1 from about nt 1 to about nt 1111 and from about nt 2.318 to about nt 5.045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to the nucleotide sequences that naturally flank the aveR1 ORF of S. Avermitilis in situ. Each flanking sequence, or a homologous thereof, in the isolated polynucleotide molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences which naturally flank the aveR1 ORF of S. Avermitilis in situ, or which are homologous to said sequences of nucleotides, and further comprising one of the aforementioned aveR1-related nucleotide sequences of the present invention, such as, for example, the nucleotide sequence of the aveR1 ORF of S. Avermitilis as shown in SEQ ID. NO: 1 from about nt 1112 to about nt 2.317 or a substantial portion of it.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of the aveR2 gene from S. Avermitilis. In a preferred embodiment, the aveR2 gene product comprises the amino acid sequence of SEQ ID NO: 4. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR2 ORF of S. Avermitilis as shown in SEQ ID NO: 3 (note: SEQ ID NO: 3 is identical to SEQ ID NO: 3). NO: 1) from about nt 2.314 to about nt 3.021. In another additional non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO: 3.

The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of the aveR2 ORF of S. Avermitilis as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3,021.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 4.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR2 related polynucleotide molecules of the present invention. In a preferred embodiment, the substantial portion of the aveR2-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of the aveR2 gene product of S. Avermitilis or a aveR2-related homologous polypeptide of the present invention. In a specific but non-limiting embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a subsequence of the amino acid sequence of SEQ ID NO: 4.

The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR2 ORF of S. Avermitilis in situ. Said flanking sequences can be selected from the nucleotide sequence of SEQ ID NO: 3 from about nt 1 to about nt 2.313, and from about nt 3.022 to about nt 5.045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous with the nucleotide sequences that naturally flank the aveR2 ORF of S. Avermitilis in situ. Each flanking sequence in the isolated polynucleotide molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR2 ORF of S. Avermitilis in situ, or that are homologous with said sequences of nucleotides, and further comprising one or more of the above-mentioned aveR2-related nucleotide sequences of the present invention, such as, for example, the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO: 3 from about e! nt 2.314 to about nt 3.021 or a substantial portion of it.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding both products of the aveR1 and aveR2 genes from S. Avermitilis. In a preferred embodiment, the products of the aveR1 and aveR2 genes comprise the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. In a non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of the ORF of aveR1 of S. Avermitilis as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317 and the ORF of aveR2 of S Avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021. In another additional non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 1 from about nt 1.112 to about nt 3.021. In another additional non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 1.

The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of both ORFs of aveR1 and aveR2 of S. Avermitilis. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of the ORG of aveR1 of S. Avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and the ORF of aveR2 of S. Avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a first polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 2 and a second polypeptide having a second sequence of amino acids that is homologous with the amino acid sequence of SEQ ID NO: 4.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the above-mentioned polynucleotide molecules, comprising a nucleotide sequence that encodes both the aveR1 and aveR2 gene products from S. Avermitilis or any of the aforementioned polynucleotide molecules, which are homologous thereto. In a specific but non-limiting embodiment, the substantial portion of the polynucleotide molecule consists of the nucleotide sequence of the aveR1 ORF as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317. In another specific but non-limiting embodiment, the substantial portion of the polynucleotide molecule consists of the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3.021.

The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 and aveR2 ORFs of S. amitmitilis in situ. Said flanking sequences can be selected from the nucleotide sequence of SEQ ID NO: 1 from about nt 1 to about nt 1111, and from about nt 3.022 to about nt 5.045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to the nucleotide sequence that naturally flank the ORRs of aveR1 and aveR2 of S-avermitilis in situ. Each flanking sequence, or a homologous thereof, in the isolated polynucleotide molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR1 and aveR2 ORF's of S. Avermitilis in situ, or which are homologous with said nucleotide sequences, and further comprising one of the aforementioned nucleotide sequences of the present invention, encoding either or both of the products of the aveR1 and aveR2 genes from S. avermitilis such as, for example, example the nucleotide sequence of the ORF of aveRIde S. Avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and the nucleotide sequence of the aveR2 ORF of S. Avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021, and substantial portions of them.

The present invention further provides oligonucleotide molecules that are useful as primers for amplifying any of the aforementioned polynucleotide molecules of the present invention, or portions of them, or that can be used to encode or act as anti-sense molecules useful in the regulation of the expression of bird genes and in the production of avermectins. The present invention further provides compositions and methods for cloning and expressing any of the polynucleotide molecules or oligonucleotide molecules of the present invention, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and new strains or lines cell phones derived from them. In a non-limiting embodiment, the present invention provides a recombinant expression vector comprising a polynucleotide molecule of the present invention in operative association with one or more regulatory elements that are necessary for the expression of the polynucleotide molecule. In a specific but non-limiting embodiment, the present invention provides plasmid SE201 (ATCC 203182), which comprises the complete ORF's of both aveR1 and aveR2 genes of S. avermitilis. Other plasmids are described below. The present invention further provides substantially purified or isolated mpolypeptide, encoded by a polynucleotide molecule of the present invention. In a specific but non-limiting embodiment, the polypeptide is a product of the aveR1 gene comprising the sequence comprising the amino acid sequence of SEQ ID NO: 2. In another specific but non-limiting embodiment, the polypeptide is a product of the aveR2 gene comprising the amino acid sequence of SEQ ID NO: 4. The present invention also provides substantially purified or isolated polypeptides that are homologous with any of the products of the aveR1 or aveR2 genes of the present invention. The present invention further provides peptide fragments substantially purified or isolated from the products of the aveR1 or aveR2 genes, or homologous polypeptides of the present invention. The present invention further provides a method of preparing a product of the aveR1 gene, a product of the aveR2 gene, a homologous polypeptide or a peptide fragment of the present invention, substantially purified or isolated, comprising culturing a host cell transformed or transfected with a vector of recombinant expression of the present invention under conditions that lead to the expression of the particular encoded gene product, polypeptides or peptide fragment, and recover the expressed gene product, the polypeptide or the peptide fragment, from the cell culture. The present invention further provides compositions and methods for genetically modifying the cells of a Streptomyces species or strain, including artificial genetic structures, such as, eg, gene replacement vectors. As provided by the present invention, the cells of a Streptomyces species or strain are genetically modified to produce an amount of avermectins that is detectably different from the amount of avermectins produced from the same species or epa that have not been modified in this way. . In a preferred embodiment, the cells of a Streptomyces species or strain are genetically modified to produce a detectably increased amount of avermectins, compared to the amount of avermectins produced by cells of the same species or strain that has not been modified as such. In another additional preferred embodiment, the species of Streptomyces is S. avermitilis. According to the present invention, said genetic modification preferably comprises mutating any one of the genes, the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes, wherein said mutation results in a detectable increase in the amount of avermectins produced by cells of a strain of S. avermitilis that are carriers of the mutation in the aveR1 or aveR2 genes, or in both aveR1 and aveR2 genes, compared with cells of the same strain that are not carriers of the genes, the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes, can be carried out using classical mutagenic techniques, including exposure to a chemical mutagen or radiation, or using an artificial genetic structure provided by the present invention, such such as, eg, a gene replacement vector, to mutate to the aveR1 gene or the aveR2 gene or to both aveR1 and aveR2 genes, eg, by adding, deleting or substituting nucleotides, or by entering a frame shift, or introducing a different or heterologous nucleotide sequence into the aveR1 gene or the aveR2 gene, or deleting a portion or all of any of the genes, the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes, or replacing a portion or the totality of any of the genes, the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes, by a different or heterologous nucleotide sequence, or by a combination of such mutations. The present invention further provides a method for identifying a mutation of an aveR1 gene or an aveR2 gene, or both aveR1 and aveR2 genes, in a strain or strain of Streptomyces, whose mutation is capable of detectably increasing the amount of avermectins produced by cells of the species or strain of Streptomyces that are carriers of the gene mutation, in comparison with cells of the same species or strain of Streptomyces that are not carriers of the gene mutation, comprising: (a) measuring the amount of avermectins produced by cells of the particular Streptomyces species or strain; (b) introducing a mutation in the aveR1 gene or in the aveR2 gene or in both aveR1 and aveR2 genes, of cells of the species or strain; and (c) comparing the amount of avermectins produced by the cells that are carriers of the gene mutation that has occurred in step (b) with the amount of avermectins produced by the cells of operation (a) that are not carriers of gene mutation; such that if the amount of avermectins produced by the cells that are carriers of the gene mutation that has occurred in step (b) is detectably greater than the amount of avermectins produced by the cells of operation (a) that they are not carriers of the gene mutation, then a mutation of the aveR1 or aveR2 gene or both aveR1 and aveR2 genes has been identified, capable of detectably increasing the amount of avermectins. In a preferred embodiment, the Streptomyces species is S. avermitilis. The present invention further provides a method for preparing preceding genetically modified cells of a particular species or strain of Streptomyces, which modified cells produce a detectably increased amount of avermectins compared to unmodified cells of the species or strain, which comprises mutating the aveR1 gene or the aveR2 gene or both aveR1 and aveR2 genes, in cells of the Streptomyces species or strain, and selecting the cells that produce a detectably increased amount of avermectins as a result of the mutation compared with cells of the same species or Streptomyces epa. that are not carriers of the gene mutation. In a preferred embodiment, the Streptomyces species is S. avermitilis. In a specific but non-limiting embodiment, described later in section 6.9.1, both aveR1 and aveR2 genes of S. avermitilis were mutated by replacing a portion of ORF of each gene per heterologous gene, resulting in S. avermitilis cells producing a detectably increased amount of avermectins compared to cells from the same S. avermitilis strain in which the aveR1 and aveR2 genes have not been mutated in this way . In another specific but non-limiting embodiment, described later in section 6.9.2, the aveR2 gene of S. avermitilis was mutated by introducing a heterologous gene into the aveR2 ORF, resulting in S. avermitilis cells that produce a detectably amount increased avermectins compared to cells from the same strain of S. avermitilis in which the aveR2 gene has not been mutated in this manner. The present invention further provides new strains of Streptomyces, whose cells produce a detectably increased amount of avermectins as a result of one or more mutations in the aveR1 gene or in the aveR2 gene, or in both aveR1 and aveR2 genes, compared to cells of the Same strain of Streptomyces that are not carriers of the gene mutation. In a preferred embodiment, the Streptomyces strain is from the species S. avermitilis. The new strains of the present invention are useful in the large scale production of avermectins such as commercially desirable doramectin. The present invention further provides a method for increasing the amount of avermectins produced by Streptomyces cultures, which comprises culturing cells of a particular species or strain of Streptomyces, which cells comprise a mutation in the aveR1 gene or in the aveR2 gene, or in both genes aveR1 and aveR2, and whose gene mutation serves to detectably increase the amount of avermectins produced by cells of the species or strain of Streptomyces that are carriers of the gene mutation compared with cells of the same species or strain that are not carriers of the mutation of genes, in culture media and in conditions that allow or induce the production of avermectins from them, and recover the avermectins from the culture. In a preferred embodiment, the species Streptomyces is S. avermitilis. This procedure is useful to increase the production efficiency of avermectins. The present invention further provides antibodies directed against a product of the aveR1 gene, a product of the aveR2 gene, a homologous polypeptide or a peptide fragment of the present invention. 4. BRIEF DESCRIPTION OF THE INVENTION Figure 1. A. Comparison of deduced amino acid sequences that are encoded by the S. coelicolor histidine kinase homolog (absAI) and the aveR1 gene of S. avermitilis indicates an identity between sequences of about 32%. B. The comparison of the deduced amino acid sequences that are encoded by the S. coelicolor response regulator homolog (absA2) and in the aveR2 gel of S. avermitilis indicates an identity between sequences of approximately 45%. The highly conserved amino acids are in bold type. Figure 2. A. Plasmid vector pSE201 (ATCC 203182) containing the ORF's of aveR1 and aveR2. B. Plasmid vector pSE210 containing the ORFs of aveR1 and aveR2. Figure 3. A. Gene replacement vector pSE214 containing the ermE gene, which has replaced a portion of the ORFs of aveR1 and aveR2. B. Vector gene replacement pSE216 containing the ermE gene introduced in the ORF of aveR2. Figure 4. BamHI restriction map of the avermectin-polyketide-synthase gene cluster from S. avermitilis which has identified five overlapping cosmid clones (ie, pSE65, pSE66, pSE67, pSE68, pSE69).

. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences encoding the products of the aveR1 and aveR2 genes from S. avermitilis, and to the discovery that the mutation of these genes can modulate the amount of avermectins produced. As an example, the invention in the following sections for a polynucleotide molecule comprising the nucleotide sequence of the ORF of aveR1, as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317, or as is present in the plasmid pSE201 (ATCC 203182), and for a polynucleotide molecule comprising the nucleotide sequence of the ORF of aveR2, as shown in SEQ ID NO: 3 (note: SEQ ID NO: 3 is identical to SEQ ID NO: 1) from about nt 2.314 to about nt 3.021, or as it is present in plasmid pSE201 (ATCC 203182), and for polynucleotide molecules comprising mutated sequences of nucleotides that are derived from them. It is intended that the references herein made to the nucleotide sequences shown is SEQ ID NOS: 1 and 3, and to substantial portions thereof, also refer to the corresponding sequencies of nucleotides and substantial portions thereof, respectively, that are present in plasmid pSE201 (ATCC 203182), unless otherwise indicated. In addition, it is intended that the references herein made to the amino acid sequences shown in SEQ ID NOS: 2 and 4, and to peptide fragments thereof, also refer to the corresponding amino acid sequences and peptide fragments thereof, respectively, encoded by the corresponding nucleotide sequences encoding AveR1 and AveR2, which are present in the plasmid pSE201 (ATCC 203182), unless otherwise indicated. . 1 Polynucleotide Molecules As used in the present context, it is intended that the terms "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "Open reading frame (ORF)," and other similar ones refer to both DNA and RNA molecules, which may be single-stranded or double-stranded. A coding sequence or an ORF may include, but is not limited to, prokaryotic sequences, cDNA sequences, genomic DNA sequences, and chemically synthesized DNA and RNA sequences. The production and manipulation of the polynucleotide molecules and oligonucleotide molecules that are considered here are within the skill in the art and can be carried out according to recombinant techniques described, inter alia, in those of Maniatis et al 1989 Molecular Cloning: A Laboratory Manual, Cold Apring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloning: A Laboratorv Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Innis and collaborators (editing coordinators), 1995, PCR Strategies, Academic Press, Inc., San Diego; Eriich (editing coordinator), PCR Technology, Oxford University Press, New York; and Hopwood et al., 1985, Genetic Manipulation od Streptomvces, A Laboratorv Manual, Kjohn Innes Foundation, Norwich, United Kingdom (R.U.9, all of which are incorporated herein by reference. . 1.1. Polynucleotide molecules related to aveR1 The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of the aveR1 gene from S. avermitilis. In a preferred embodiment, the aveR1 gene product comprises the amino acid sequence of SEQ ID NO: 2. In a non-limiting embodiment, the isolated polynucleotide molecule of the ORF of aveR1 of S. avermitilis as shown in SEQ ID NO.1 from about nt 1112 to about nt 2.317. In another additional non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the polynucleotide sequence of SEQ ID NO: 1. The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of the ORF of aveR1 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317. The term "homologous", when used in this respect, means a polynucleotide molecule comprising a nucleotide sequence: (a) encoding the same polypeptide as SEQ ID NO: 1 from about nt 1.112 to about nt 2.317 , but which includes one or more silent changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) hybridizing with the complement of a polynucleotide molecule comprising the amino acid sequence of SEQ ID NO: 2 under moderately stringent conditions, i.e., hybridization to a DNA attached to a filter in 0.5 M NaHP04, sodium dodecyl sulfate (SDS) at 7% 1 mM EDTA at 65 ° C, and washed in 0.2xSSC / 0.1% SDS at 42 ° C (see Ausubel et al. (editing coordinators), 1989, Current Protocols in Molecular Biology, volume I, Green Publishing Associates, Inc. and John Wiley &Sons, Inc., New York, on page 2.10.3), and is useful for practicing the invention. In a preferred embodiment, the homologous polynucleotide molecule hybridizes with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 under highly stringent conditions, i.e. hybridization to a DNA attached to a filter in NaHPO40.5 M, 7% SDS 1 mM EDTA at 65 ° C, and washed in 0.1xSSC / 0.1% SDS at 68 ° C (see Ausubel et al., 1989, above), and is useful for practicing the invention. In another further preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and is useful for practicing the invention. As used in the present context, a polynucleotide molecule related to aveR1 is "useful for practicing the invention" when the polynucleotide molecule can be used to introduce mutations in ORF of S. avermitilis aveR1 by site-directed mutagenesis, such as by homologous recombination, or to amplify a polynucleotide molecule comprising the nucleotide sequence of the aveR1 ORF of S. avermitilis, using classical amplification techniques. Said homologous polynucleotide molecules can include the aveR1 genes existing in nature, present in other species of Streptomyces, except S. coelicolor, or in other strains of S. avermitilis, as well as alleles of mutated aveR1, either existing in nature, synthesized chemically or treated by genetic technology. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 2. As used in the present context to refer to polypeptides having amino acid sequences that are homologous to the amino acid sequence of a product of the aveR1 gene from S. avermitilis, the term "homologous" means a polypeptide comprising the amino acid sequence of this have been replaced conservatively by a different amino acid residue, in which the resulting polypeptide is useful for practicing the invention. Conservative amino acid substitutions are well known in the art, the rules for producing such substitutions include those described by Dayhof, M.D., 1978, Nat. Biomed. Res. Found., Washington, D.C., volume 5, supplement 3, among other citations. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in terms of the acidity, polarity or bulkiness of their side chains. Genetically encoded amino acids are generally divided into four groups: (1) those of an acid character = aspartate, glutamate; (2) those of a basic nature = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The amino acids phenylalanine, tryptophan and tyrosine are also classified together as aromatic amino acids. One or more replacements within any particular group, eg, of a leucine by an isoleucine or valine, or of an aspartate by a glutamate, or of a threonine by a serine, or of any other amino acid residue by a residue of structurally related amino acid, eg, an amino acid residue with similar characteristics of acidity, polarity, bulkiness, or similarity in some combination thereof, will generally have an insignificant effect on the function of the polypeptide. As used in the present context, an aveR1-related polypeptide is "useful for practicing the invention" when the polypeptide can be used to incite antibodies against an aveR1 gene product from S. avermitilis, or to screen for compounds that modulate the activity of AveR1 or the production of avermectins in Streptomyces.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the above-mentioned aveR1-related polynucleotide molecules of the present invention. As used in the present context, a "substantial portion" of a polynucleotide molecule related to aveR1 means a polynucleotide molecule that consists of less than the complete coding sequence of a product of the aveR1 gene of S. avermitilis, or a polypeptide homologue related to aveR1, of the present invention, but constituting at least about 20%, and more preferably at least about 30%, of said nucleotide sequence, and that it is useful to practice the invention, as the utility is defined above for polynucleotide molecules related to aveR1. In a non-limiting embodiment, the substantial portion of the aveR1-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of an aveR1 gene product of S. avermitilis or a aveR1-related homologous polypeptide of the present invention. A "peptide fragment" of a aveR1-related polypeptide is referred to a polypeptide that consists of a sub-sequence of the amino acid sequence of a full-length aveR1 gene product or a homologous polypeptide, whose sub-sequence has a longer length short that the product of the full-length aveR1 gene or the homologous peptide, and whose sub-sequence is useful for practicing the invention, as the utility is defined above for aveR1-related polypeptides. In a preferred embodiment, the present invention provides a polynucleotide molecule that consists of a nucleotide sequence encoding a peptide fragment that consists of a sub-sequence of the amino acid sequence of SEQ ID NO: 2. The peptide fragments of the invention preferably have a length of at least about 15 amino acid residues. The aveR1-related polynucleotide molecules described herein can be used to express the aveR1 gene product, to prepare new strains of Streptomyces in which the aveR1 gene has been mutated, and to identify aveR1 homologous genes in other species or bacterial strains using known techniques. Therefore, the present inventor further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of a homologous gene aveR1. As used in the present context, a "product of a homologous gene aveR1" is defined as a gene product encoded by a homologous gene aveR1 which, in turn, is defined as a gene from a different species of Streptomyces or from the genus Saccharopolyspora is closely related and is recognized by experts in this field of technology as a homologue of the aveR1 gene based on S. avermitilis at a degree of identity between nucleotide sequences greater than approximately 80%, or containing pro residues from active sites that are typically found in histidine kinase components of two-component signaling systems. For example, comparisons of homologies of aveR1 with two-component bacterial systems from the Nar / Deg subgroups show a 100% conservation of the histidine residue (H) which is the site of self-phosphorylation, and the residue of asparagine (N) which is required for the autokinases activity. Methods for identifying clones of polynucleotides containing aveR1 homologous genes are known in the art industry.

For example, a polynucleotide molecule comprising a portion of the ORF of aveR1 of S. avermitilis can be detectably labeled and used to screen a genomic library constructed from DNA derived from the organism of interest. The stringency of the hybridization conditions can be selected based on the relationship of the reference organism, in this example S. avermitilis, with the organism of interest. The requirements for different stringency conditions are well known to those with experience in the technology sector, and said conditions will vary predictably depending on the specific organisms from which the library is derived and the sequences marked. Genomic DNA libraries can be screened for sequences encoding aveR1 homologous genes using the techniques discussed, inter alia, in Benton and Davis, 1977, Science 196: 180, for bacteriophage libraries, and in Grunstein and Hogness, 1975 , Proc. Nati Acad. Sci. USA, 72: 3961-3965, for plasmid libraries, the publications of which are incorporated herein by reference. Polynucleotide molecules having nucleotide sequences that are known to include the aveR1 ORF, as shown in SEQ ID NO: 1, or the oligonucleotide molecules that represent portions of those, can be used as probes in these experiments Alternatively, polypeptides can synthesize oligonucleotide probes that correspond to nucleotide sequences deduced from the amino acid sequence of the purified product of the aveR1 gene. Clones identified as containing sequences encoding aveR1 homologous genes can be tested for an appropriate biological function. For example, clones can be subjected to sequence analysis in order to identify an appropriate reading frame, as well as initiation and termination signals. Then, the cloned DNA sequence can be introduced into an appropriate expression vector which is then transformed into the cells of a strain of S. avermilitis that have been made aveR1 to test them for complementation. Then the transformed S avermitilis host cells can be analyzed for avermectin production using methods such as pro HPLC analysis of fermentation products, as described, eg in Section 6.6, below. The present invention further provides an isolated polynucleotide molecule comprising one or more other nucleotide sequences that naturally flank the averRI ORF of S. avermitilis in situ. Such flanking sequences can be selected from the nucleotide sequence of SEQ ID NO: 1 from about nt 1 to about nt 1111, and from about nt 2.318 to about nt 5.045. The present inventor further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous with nucleotide sequences that naturally flank the aveR1 ORF of S. avermitilis in situ. As used in the present context, a nucleotide sequence is homologous with a nucleotide sequence that naturally flanks the ORF of aveR1 of S. avermitilis in situ when the homologous nucleotide sequence hybridizes with the complement of the flanking nucleotide sequence naturally to the ORF of aveR1 of s. avermitilis in situ under moderately stringent conditions, ie hybridization to a DNA bound to a filter in 0.5 M NaHP0, 7% SDS AL, 1 mM EDTA at 65 ° C, and washing in 0.2xSSC / 0.1% SDS at 42 ° C (see Ausubel et al., 1989, supra), and is useful for practicing the invention, as defined above for the polynucleotide molecules related to aveRL Each flanking sequence, or homologous thereof, in the The polynucleotide isolated molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences, which naturally flank the aveR1 ORF of S. avermitilis in situ, or which are homologous with said nucleotide sequences, and which further comprise one of the aforementioned aveR1 related nucleotide sequences of the present invention, such as for example, the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1. 112 to approximately nt 2.317, or a substantial portion of it. . 1.2. Polynucleotide Molecules Related to aveR2 The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of the aveR2 gene from S. avermitilis. In a preferred embodiment, the aveR2 gene product comprises the amino acid sequence of SEQ ID NO: 4. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3.021. In another additional non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO: 3. The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3.021 The term "homologous" when used in this regard, means a polynucleotide molecule comprising a nucleotide sequence: (a) encoding the same polypeptide as SEQ ID NO: 3 from about nt 2.314 to about nt 3.021, but which includes one or more silent changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) hybridizing with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 under moderately stringent conditions, i.e., hybridization to a bound DNA to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washing in 0.2xSSC / 0.1% SDS at 42 ° C (Ausubel et al., 1989, supra) and is useful for practicing the invention. In a preferred embodiment, the homologous polynucleotide molecule hybridizes with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 under highly stringent conditions, i.e. hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washing in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al. 1989, above) and is useful for practicing the invention. In still another preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 from about nt 2.314 to about nt 3.021, and is useful for practicing the invention.

As used in the present context, a polynucleotide molecule related to aveR2 is "useful for practicing the invention" when the polynucleotide molecule can be used to introduce mutations in the ΑR2 ORF by site-directed mutagenesis, such as by homologous recombination, or to amplify a polynucleotide molecule comprising the nucleotide sequence of the aveR2 ORF of S. avermitilis, using classical amplification techniques. Said homologous polynucleotide molecules may include aveR2 genes existing in nature, present in other Streptomyces species, except S. coelicolor, or in other strains of S. avermitilis, as well as in alleles of mutated aveR2, either existing in nature, chemically synthesized or treated by genetic engineering.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 4. As used in the present context to refer to polypeptides having amino acid sequences that are homologous with the amino acid sequence of a product of the aveR2 gene from S. avermitilis, the term "homologous" means a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, but in which one or more amino acid residues thereof have been substituted conservatively by a different amino acid residue, such as previously defined conservative amino acid substitutions, in which the resulting polypeptide is useful for practicing the invention.

As used in the present context, an aveR2-related polypeptide is "useful for practicing the invention" when the polypeptide can be used to incite antibodies against an aveR2 gene product from S. avermitilis, or to screen for compounds that modulate the activity of aveR2 or the production of avermectins in Streptomyces.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR2 related polynucleotide molecules of the present invention. As used in the present context, a "substantial portion" of a polypeptide molecule related to aveR2 means a polynucleotide molecule that consists of less than the complete coding sequence of a product of the aveR2 gene of S. avermitilis or of a prolypeptide homologue related to aveR2 of the present invention, but constituting at least about 25% and more preferably at least about 30% of said nucleotide sequence, and which is useful for practicing the invention, as defined above for utility for molecules of polynucleotides related to aveR2.

In a non-limiting embodiment, the substantial portion of the aveR2-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of a aveR2 product of S. avermitilis or a aveR2-related homologous polypeptide of the present invention, a "Poptidic fragment" of a aveR2-related polypeptide refers to a polypeptide that consists of a sub-sequence of the amino acid sequence of a full length aveR2 gene product or a homologous polypeptide, whose sub-sequence has a shorter length than that of the full-length aveR2 gene product or the homologous polypeptide, and whose sub-sequence is useful for practicing the invention, as the utility is defined above for aveR2-related polypeptides. In a preferred embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a sub-sequence of the amino acid sequence of SEQ ID NO: 4. The peptide fragments of the invention preferably have a length of at least about 15 amino acid residues. The polynucleotide molecules related to aveR2, which are described herein, can be used to express the aveR2 gene product, to prepare new strains of Streptomyces in which the aveR2 gene has been mutated, and also to identify aveR2 homologous genes in other species or bacterial strains, using known techniques. Therefore, the present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a product of a homolog gene aveR2. As used in the present context, a "product of a homolog gene aveR2" is defined as a gene product encoded by a homolog gene aveR2 which, in turn, is defined as a gene from a different species of Streptomyces, or of the closely related Saccharo-polyspora genus, and which is recognized by those having experience in the technology sector as a homolog of the aveR2 gene of S. avermitilis based on S. avermitilis at a degree of identity between nucleotide sequences greater than about 80%, or for containing residues of conserved active sites that are typically found in response regulatory components of two-component signaling systems. For example, the homology comparisons of aveR2 with two-component eubacterial systems from the Nar / Deg subgroup show a 100% conservation of two aspartate residues (D), one of which is the phosphorylation site, and one residue of preserved lysine (K).

Methods for identifying clones of polynucleotides containing aveR2 homologous genes are known in the art industry. for example, a polynucleotide molecule comprising a portion of the ORF of aveR2 of S. avermitilis can be detectably labeled and used to screen a genomic library constructed from DNs derived from the organism of interest. The stringency of the hybridization conditions can be selected based on the relationship of the reference organism, in this example S. avermitilis, with the organism of interest. Genomic DNA libraries can be screened for coding sequences of aveR2 homologous genes using the techniques cited in Section 5.1.1. Polynucleotide molecules having nucleotide sequences that are known to include the aveR2 ORF, as shown in SEQ ID NO: 3, or oligonucleotide molecules that represent portions thereof, can be used as probes in these experiments. scrutiny. Alternatively, oligonucleotide probes corresponding to nucleotide sequences deduced from the amino acid sequence of the purified product of the aveR2 gene can be synthesized.

Clones identified as containing encoding sequences of homologous aveR2 genes can be tested for an appropriate biological function. For example, clones can be subjected to sequence analysis in order to identify an appropriate reading frame, as well as initiation and termination signals. Then, the cloned DNA sequence can be introduced into an appropriate expression vector which is then transformed into the cells of a strain of S. avermitilis that have been made aveR2 ~ to test them for complementation. Then the transformed S. avermitilis host cells can be analyzed for avermectin production using methods such as HPLC analysis of fermentation products, as described, eg in Section 6.6. later. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR2 ORF of S. avermitilis in situ. Said flaking sequences can be selected from the nucleotide sequence of SEQ ID NO: 3 from about nt 1 to about nt 2.313, and from about nt 3.022 to about nt 5.045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous with nucleotide sequences that naturally flank the aveR2 ORF of S. avermitilis in situ. As used in the present context, a nucleotide sequence is homologous with a nucleotide sequence that naturally flanks the aveR2 ORF of S. avermitilis in situ when the nucleotide homologous sequence hybridizes with the complement of the flanking nucleotide sequence naturally to the OFRF of aveR2 of S. avermitilis in situ under moderately stringent conditions, i.e. hybridization to a DNA attached to a 0.5 M NaHP04 filter, 7% SDS m, 1 mM EDTA at 65 ° C, and washing in 0.2xSSc / 0.1% SDS at 42 ° C (see Ausubel et al., 1989, above), and is useful for practicing the invention, as defines the utility above for polynucleotide molecules related to aveR2. Each flanking sequence, or homologous thereof, in the isolated polynucleotide molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences which naturally flank the aveR2 ORF of S. avermitilis in situ, or which are homologous with said sequences of nucleotides, and further comprising one of the nucleotide sequences related to aveR2 mentioned above of the present invention, such as p. ex. the nucleotide sequence of the ORF of aveR2 as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3.021, or a substantial portion thereof. . 1.3 Polynucleotide molecules related to aveR1 / aveR2 The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding both products of the aveR1 and aveR2 genes from S. avermitilis (hereinafter referred to as "aveR1 / aveR2"). In a preferred embodiment, the products of the aveR1 and aveR2 genes comprise the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. In a non-limiting embodiment, the aveR1 / aveR2 polynucleotide isolated molecule comprises the nucleotide sequence of the aveR1 ORF of S. avermitilistal as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317, and the ORF of aveR2 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021. In another additional non-limiting embodiment, the aveR1 / aveR2 polynucleotide isolated molecule comprises the nucleotide sequence of SEQ ID N: 1 from about nt 1.112 to about nt 3.021. In another additional non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID N: 1 shows a partial overlap between the ORF's of aveR1 and aveR2 however, the present invention also includes aveR1 / aveR2 polynucleotide molecules in which the ORFs for aveR1 and aveR2 do not overlap.

The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising a nucleotide sequence of the ORF of aveR1 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about 31 nt 2.317, and the ORF of aveR2 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021. The term "homologue" when used in this regard means a polynucleotide molecule comprising a nucleotide sequence: (a) encoding the same polypeptides as the ORF's of aveR1 and aveR2 as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and from about nt 2.314 to about nt 3.021, respectively, but including one or more dumb changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) hybridizing with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a second polypeptide comprising the amino acid sequence of SEQ. ID NO: 4, under moderately stringent conditions, ie hybridization with a DNA attached to a filter in 0.5 m NaHP04, 7% SDS AL, 1 M EDTA at 65 ° C, and washing in 0.2xSSC / 0.1% SDS at 42 ° C (Ausubel et al., 1989, supra), and is useful for practicing the invention. In a preferred embodiment, the homologous polynucleotide molecule hybridizes with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 4, under highly stringent conditions, ie, hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 M EDTA at 65 ° C, and washing in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above), and is useful for practicing the invention. In a further preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule comprising the nucleotide sequence of the aveR1 and aveR2 ORFs of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317 and from about nt 2. 314 to about nt 3.021, respectively, and is useful for practicing the invention.

As used in the present context, a polynucleotide molecule related to aveR1 / aveR2 is "useful for practicing the invention" when the polynucleotide molecule can be used to introduce mutations into either the aveR1 ORF or the aveR2 ORF , or in both ORFs of aveR1 and aveR2, of S. avermitilis, by site-directed mutagenesis, such as by homologous recombination, or to amplify a polynucleotide molecule comprising the nucleotide sequence of either the aveR1 or ORF ORF of aveR1, or of both ORF's of aveR1 and aveR2, of S. avermitilis, using classical amplification techniques. Said homologous polynucleotide molecules can include aveR1 / aveR2 genes existing in nature, present in other Streptomyces species, except. S. coelicolor, or in other strains of S. avermitilis, as well as alleles of aveR1 or aveR2 mutated, either existing in nature, chemically synthesized or treated by genetic engineering.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding first and second polypeptides having amino acid sequence that are homologous to the amino acid sequences of SEQ ID NOS: 2 and 4, respectively, as used in the present context to refer to polypeptides having amino acid sequences that are homologous with the amino acid sequences of the products of the aveR1 and aveR2 genes from S. avermitilis, the term "homologue" means polypeptides comprising the amino acid sequences of SEQ ID NOS: 2 and 4, respectively, but in which one or more amino acid residues thereof have been conservatively substituted with a different amino acid residue, as defined above, conservative amino acid substitutions, in which the resulting polypeptides are useful for practicing the invention.

The present invention further provides an isolated polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR1 / aveR2 related polynucleotide molecules of the present invention.

As used in the present context, a "substantial portion" of a polynucleotide molecule related to aveR1 / aveR2 means a polynucleotide molecule that consists of less than the complete coding sequence required to encode both products of the aveR1 and aveR2 genes from S. avermiti is, or homologous polypeptides related to aveR1 and aveR2 of the present invention, but constituting at least 10%, and more preferably at least about 20%, of said nucleotide sequence related to aveR1 and aveR2. In a preferred embodiment, the substantial portion of the polynucleotide molecule related to aveR1 and aveR2 consists of a nucleotide sequence encoding either the aveR1 product of S. avermitilis or the aveR2 product of S. avermitilis of the present invention. , or polypeptides homologous thereto. In a specific but non-limiting embodiment, the substantial portion of the aveR1 / aveR2-related polynucleotide molecule consists of the nucleotide sequence of the aveR1 ORF as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317 . In another specific but non-limiting embodiment, the substantial portion of the aveR1 / aveR2-related polynucleotide molecule consists of the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO: 3 from about nt 2.314 to about nt 3.021 .

The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 / aveR2 ORFs of S. avermitilis in situ. Said flanking sequences may be selected from the nucleotide sequences of SEQ ID NO: 1 from about nt 1 to about nt 1.111, and from about nt 3,022 to about nt 5,045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous with nucleotide sequences naturally flanking or ORF's of aveR1 / aveR2 of S. avermitilis in situ.

As used in the present context, a nucleotide sequence is homologous to a nucleotide sequence that naturally flanks the aveR1 / aveR2 ORFs of S. avermitilis in situ when the nucleotide homologous sequence hybridizes with the nucleotide sequence complement which naturally flanks the ORRs of aveR1 / aveR2 of S. avermitilis in situ under moderately stringent conditions, ie hybridization to DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 M EDTA at 65 ° C , and washed in 0.2xSSC / 0.1% SDS at 42 ° C (see Ausubel et al., 1989, above), and is useful for practicing the invention, as defined above for the aveR1-related polynucleotide molecule / aveR2. Each flanking sequence, or homologous thereof, in the isolated polynucleotide molecule of the present invention preferably has a length of at least about 200 nt. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more aforementioned nucleotide sequences that naturally flank the aveR1 / aveR2 S. avermitilis ORFs in situ, or that are homologous with said sequences of nucleotides, and further comprising one of the nucleotide sequences related to aveR1 / aveR2 mentioned above of the present invention such as for example a nucleotide sequence encoding any one or both ORF's of aveR1 and aveR2 of S. avermitilis as shows in SEQ ID NO: 1 from about nt 1112 to about nt 3.021. . 2. OLIGONUCLEOTIDE MOLECULES The present invention further provides oligonucleotide molecules that hybridize with any of the aforementioned polynucleotide molecules of the present invention, or hybridize to a polynucleotide molecule having a nucleotide sequence that is the complement of any of the above-mentioned polynucleotide molecules of the present invention. Said oligonucleotide molecules preferably have a length of at least about 10 nucleotides, but may be extended up to the length of any subsequence of any of the aforementioned polynucleotide molecules of the present invention, and may hybridize with one or more of the molecules of polynucleotides mentioned above under moderate or highly stringent conditions. For shorter oligonucleotide molecules, one example of highly stringent conditions includes washing in 6xSSC / 0.5% sodium pyrophosphate at about 37 ° C for ~ 14 base oligos, at about 48 ° C for oligos of ~ 17 bases, at about 55 ° C for oligos of ~ 20 bases and at approximately 60 ° C for oligos of ~ 23 bases. For longer oligonucleotide molecules (ie, greater than about 100 nts), examples of Section 5.1 above for homologous polynucleotide molecules. Hybridization conditions can be adjusted appropriately as is known in the technology sector, depending on the particular oligonucleotide molecules that are used. In a preferred embodiment, an oligonucleotide molecule of the present invention hybridizes under highly stringent conditions with a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 1, or with a polynucleotide molecule consisting of a sequence of nucleotides which is the complement of the nucleotide sequence of SEQ ID NO: 1. The oligonucleotide molecules of the present invention are useful for a variety of purposes, including that of primers for amplifying a polynucleotide molecule encoding aveR1 or aveR2 gene products, or as anti-sense molecules useful for regulating bird genes and the biosynthesis of avermectins. in Streptomyces. The amplification can be carried out using oligonucleotide molecules appropriately in conjunction with classical techniques, such as the polymerase chain reaction (PCR), although other amplification techniques that are known in the field of technology can also be used, for example, the ligase chain reaction. For example, for a PCR, a mixture comprising appropriately designed primers, a template comprising the nucleotide sequence to be amplified, and appropriate enzymes and buffers for PCR, is prepared and treated according to classical protocols to amplify a specific sequence of oligonucleotides related to aveR1 or aveR2, of the template. . 3. SYSTEMS OF RECOMBINANT EXPRESSION . 3.1. Expression Vectors The present invention further provides recombinant cloning vectors and recombinant expression vectors, comprising a polynucleotide molecule of the present invention, which vectors are useful for cloning or expressing said polynucleotide molecules, including polynucleotide molecules comprising either the ORF of aveR1 or ORF of aveR2, or both ORF's of aveR1 and aveR2, of S. avermitilis. In a non-limiting embodiment, the present invention provides the plasmid pSE201 (ATCC 203182), which comprises the entire ORF of aveR1 and the entire ORF of aveR2 of S. avermitilis. The following description is intended to be applied to all of the polynucleotide molecules and polypeptides of the present invention mentioned above, inclusive or both of the aveR1 and aveR2 ORFs from S. avermitilis and their gene products, and all of the molecules of homologous polynucleotides, of the homologous polynucleotides, of substantial portions of said polynucleotide molecules, and of peptide fragments of said gene products and polypeptides, as defined above, unless otherwise indicated. A variety of different vectors have been developed for specific use in Streptomyces, including phage, high copy number plasmids, plasmids with low copy number, suicide plasmids, temperature sensitive plasmids, and shuttle vectors between £.

Coli-Streptomyces, among others, and any of them can be used to practice the present invention. A number of clones and drug resistance have also been cloned from Streptomyces, and several of these genes have been incorporated into vectors as selectable markers. Examples of vectors currently known for use in Streptomyces are presented, inter alia, in Hutchinson, 1980, Applied Biochem. Biotech 16: 169-190. The recombinant vectors of the present invention, particularly the expression vectors, are preferably constructed such that the coding sequence for the polynucleotide molecule of the present invention is in operative association with one or more regulatory elements that are necessary for transcription and translation. of coding sequence in order to produce a polypeptide. As used in the present context, the term "regulatory element" includes, but is not limited to, nucleotide sequences encoding inducible and non-inducible promoters, enhancers, operators and other elements in the field of technology, which serve to direct and / or regulate the expression of sequences encoding polynucleotides. Also, as used in the present context, the coding sequence is in "operative association" with one or more regulatory elements in which the regulatory elements regulate efficiently and allow the transcription of the coding sequence or the transduction of its mRNA. , or both at the same time. Typical plasmid vectors that can be engineered to contain a polynucleotide molecule of the present invention include pCR-Blunt, pCR2.1 (Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3 (Vara et al., 1989. J. Bact. 171: 5872-5881), among many others. The regulatory elements of these vectors may vary in strength and their specificities. Depending on the host and vector system used, any one of a number of appropriate transcription and translation elements can be used.

Non-limiting examples of transcriptional regulatory regions or promoters for bacteria include the β-gal promoter, the T7 promoter, the TAC promoter, the? Promoters. left and right, the trp and lac promoters, the trp-lac fusion promoters and, more specifically, Streptomyces, the promoters ermE, melC and tipA, etc. Methods for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements are well known in the art and any of these can be used to practice the present invention. These methods include in vitro recombination techniques, synthesis techniques and in vivo genetic recombination. See, for example, the techniques described in the citations of Maniatis et al., 1989, above; Ausubel and collaborators, 1989, previous; Sambrook et al., 1989, above; Innis et al., 1995, above, Eriich, 1992, supra, and Hopwood et al., 1985, supra. Fusion protein expression vectors can be used to express a fusion protein with a product of the aveR1 or aveR2 gene. The purified fusion protein can be used to incite antisera against the aveR1 or aveR2 gene product, in order to study the biochemical properties of the aveR1 or aveR2 gene product, to genetically engineer aveR1 or aveR2 fusion proteins with different biochemical activities, or to assist in the identification or purification of the expressed aveR1 or aveR2 gene product, in recombinant expression systems. Possible fusion protein expression vectors include, but are not limited to, vectors that incorporate sequences encoding trpE ß-galactosidase fusions, maltose binding protein fusions, glutathione-S-transferase fusions, and polyhistidine fusions ( carrier regions). The AveR1 and AveR2 fusion proteins can be engineered to comprise a region useful for purification.

For example, fusions of AveR1 and AveR2-proteinase binding to maltose can be purified using an amylose resin; AveR1 or AveR2-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose globules; and AveR1 or AveR2-polyhistidine fusions can be purified using a divalent nickel resin. Alternatively, antibodies against the protein or a carrier peptide can be used for purification by affinity chromatography of the fusion protein. For example, a nucleotide sequence encoding the target epitope of a monoclonal antibody can be genetically engineered into the expression vector in operative association with the regulatory elements and can be positioned such that the expressed epitope is fused to the polypeptide AveR1 or AveR2. For example, a nucleotide sequence encoding the FLAG® epitope tag (International Biotechnologies Inc.), which is a hydrophilic label peptide, can be introduced pro classical techniques into the expression vector at a site corresponding to the amino terminus or carboxyl of the AveR1 or AveR2 polypeptide. The AveR1 or AveR2 polypeptide fusion product and the FLAG® epitope can then be detected and affinity purified using commercially available anti-FLAG® antibodies. The expression vector encoding the AveR1 fusion protein or AveR2 can also be engineered such that it contains polylinker sequences encoding specific protease cleavage sites such that the expressed AveR1 or AveR2 polypeptide can be detached from the carrier or fusion partner region by treatment with a specific proteinase. For example, the fusion protein vector can include DNA sequences encoding dissociation sites by thrombin or factor Xa, among others. A signal sequence located above and in reading frame with the ORR of AveR1 or AveR2 can be genetically engineered into the expression vector by known methods for directing the trafficking and secretion of the expressed gene product. Non-limiting examples of signal sequences include those from factor a, immunoglobulins, outer membrane proteins, penicillinase and T cell receptors, among others. To help make the selection of host cells transformed or transfected with cloning or expression vectors of the present invention, the vector can be engineered so that it further comprises a coding sequence for a reporter gene product or another selectable marker. Said coding sequence is preferably in operative association with the coding sequences of regulatory elements, as described above. Reporter genes that may be useful in the invention are well known in the art sector and include those encoding green fluorescent protein, luciferase, xy1E and tyrosinase, among others. Nucleotide sequences encoding selectable markers are well known in the art and include those encoding gene products that confer resistance to antibiotics or anti-metabolites, or that provide an auxotrophic requirement. Examples of such sequences include that encoding resistance to erythromycin, thiostrepton or kanamycin, among many others. . 3.2. Host cells The present invention further provides transformed host cells comprising a polynucleotide molecule or a recombinant vector of the invention, and new strains or cell lines derived therefrom. Host cells useful in the practice of the invention are preferably Streptomyces cells, although they can also use other prokaryotic cells or eukaryotic cells. Said transformed host cells typically include, but are limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA vectors, plasmid DNA or cosmid DNA or a yeast transformed with recombinant vectors, among others. Bacterial cells are generally preferred as host cells. It should be understood that the polynucleotide molecules of the present invention are intended to function in Streptomyces cells, but can also be transformed into other bacterial or eucharistic cells, for example for purposes of cloning or expression. An E. coli strain, such as, for example strain DH5a, which is available either from the American Type Culture Collection (ATCC), Rockville, MD, USA, may typically be used. (access number 31343) or commercial sources (Stratagene). Preferred eukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be used efficiently. The recombinant expression vector of the inventors preferably transformed or transfected into one or more host cells of a substantially homogeneous cell culture. The expression vector is generally introduced into the host cells according to known techniques such as, for example by transformation with protoplasts, calcium phosphate precipitation, treatment with calcium chloride, microinjection, electroporation, transfection by contact with a recombinant virus, liposome-mediated transfection, transfection in DEAE-dextran, transduction, conjugation or bomber with microprojectiles. The selection of transformants can be carried out by a conventional method, such as the selection of cells that express a selectable marker, for example, of antibiotic resistance, associated with the recombinant vector, as described above. Once the expression vector has been introduced into the host cell, the integration and maintenance of the coding sequence related to aveR1, aveR2 / aveR2, either on the chromosome of the host cell or episomally, can be confirmed by techniques classical, for example, by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), or by immunological analysis to detect the expected gene product. Host cells that contain and / or express the recombinant coding sequence related to aveR1, aveR2 or aveR1 / aveR2, can be identified by any one of at least four general approaches that are well known in the art sector, including: (i) DNA hybridization with DNA, DNA with RNA or RNA with antisense RNA; (ii) detection of the presence of "marker" gene functions; (ii) determining the level of transcription as measured by the expression of mRNA transcripts specific for aveR1 or aveR2 in the host cell; and (iv) detecting the presence of a mature polypeptide product as measured, for example by immunoassay or by the presence of biological activity of aveR1 or aveR2. . 3.3. Expression and characterization of a aveR1 or recombinant aveR1 gene product Once the coding sequence related to aveR1, aveR2 or aveR1 / aveR2 has been stably introduced into an appropriate host cell, the transformed host cell is clonally propagated, and the resulting cells are grown under conditions that lead to maximum production of the gene products related to aveR1 and / or aveR2, such conditions typically include growing cells to a high density. When the expression vector comprises an inducible promoter, suitable induction conditions, such as, for example, temperature shift, nutrient depletion, addition of free inducers (e.g., carbohydrate-like compounds, such as isopropyl-β -D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, is used as necessary to induce expression. When the gene product related to aveR1 and / or aveR2, which has been expressed, is retained within the host cells, the cells are harvested and used, and the product is isolated and purified from the lysed material under extraction conditions that are known in the field of technology in order to minimize the degradation of proteins, such as for example at 4 ° C, or in the presence of protease inhibitors, or both at the same time. When the expressed gene product of aveR1 and / or aveR2 is secreted from the host cells, the spent nutrient medium can simply be collected and the product can be isolated therefrom. The expressed gene product related to aveR1 and / or aveR2 can be isolated or purified substantially from cellular materials used or from a culture medium, as appropriate, using classical methods, including, but not limited to, any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. When the gene products related to aveR1 and / or aveR2, which have been expressed, exhibit biological activity, the increasing purity of the preparation can be monitored in each of the operations of the purification process by use of an appropriate analysis. if the expressed gene products related to aveR1 and / or aveR2 exhibit biological non-activity on the basis, for example, of the size in the reactivated with an antibody that is otherwise specific for aveR1 or aveR2, or by the presence of a tag of fusion. The present invention therefore provides a substantially purified or isolated polypeptide, which is encoded by a polynucleotide molecule of the present invention. In a specific but non-limiting embodiment, the polypeptide is a product of the aveR1 gene from S. avermitilis, which comprises the amino acid sequence of SEQ ID NO: 2. In another specific but non-limiting embodiment, the polypeptide is a product of the gene aveR2 from S. avermitilis, comprising the mainoacid sequence of SEQ ID NO: 4. The present invention further provides substantially purified or isolated polypeptides, which are homologous with any of the products of the aveR1 or aveR2 genes or homologous polypeptides of the present invention. The substantially purified or isolated polypeptides of the present invention are useful for a variety of purposes, such as, for example, screening for compounds that alter the function of the aveR1 or aveR2 gene product, thereby modulating the avermectin biosynthesis, and for incite antibodies directed against products of the aveR1 or aveR2 genes.

As used in the present context, a polypeptide is "substantially purified" when the polypeptide constitutes the majority by weight of the material in a particular preparation. Also, as used in the present context, a polypeptide is "isolated" when the polypeptide constitutes at least about 90% by weight of material in a particular preparation. The present invention further provides a method of preparing a aveR1 gene product, a product of the aveR2 gene, a homologous polypeptide or a peptide fragment of the present invention, substantially purified or isolated, which comprises culturing a host cell transformed or transfected with a vector of recombinant expression, said recombinant expression vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding the aveR1 gene product, the aveR2 gene product, the homologous polypeptide or the peptide fragment, respectively, wherein the nucleotide sequence is in operative association with one or more regulatory elements, under conditions that lead to the expression of the particular gene product, polypeptide or peptide fragment, and recovering the expressed gene product, polypeptide or peptide fragment, from the cell culture in a substantially purified form or isolated Once the aveR1 or aveR2 gene product has been obtained with sufficient purity, it can be characterized by classical methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, biological activity by producing appropriate products in the pathway of avermectin biosynthesis, etc. For example, the amino acid sequence of the aveR1 or aveR2 gene product can be determined using classical peptide sequencing techniques. The product of the aveR1 or aveR2 gene can be further characterized using hydrophilicity analysis (see, for example, Hopp and Woods, 1981, Proc. Nati.

Acad. Sci. USA 78: 3824) or analog software software algorithms, to identify hydrophobic and hydrophilic regions of the aveR1 or aveR2 gene product. A structural analysis can be carried out to identify regions of the aveR1 or aveR2 gene product that adopt specific secondary structures. Biophysical methods can be used, such as X-ray crystallography (Engstrom, 1974, Biochem Exp. Biol. 11: 7-13), modeling with ordering (Fletterick and Zoller (editing coordinators), 1986, in: Current Communications ín Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and nuclear magnetic resonance (NMR), in order to map and study the interaction sites between the aveR1 and aveR2 gene products and their substrates. The information obtained from these studies can be used to select new sites for mutation in the ORFs of aveR1 or aveR2 in order to help develop new strains of Streptomyces of avermectins. . 4 construction of aveR1 and aveR2 mutants The present invention further provides compositions and methods for genetically modifying the cells of a species or strain of Streptomyces, including artificial genetic structures such as gene replacement vectors. In a preferred embodiment, the cells of a Streptomyces species or strain with genetically modified to produce an amount of avermectins that is detectably different from the amount of avermectins produced by cells of the same species or strain, which have not been modified in this manner . In a more preferred embodiment, the cells of a strain of S. avermitilis are genetically modified to produce a detectably increased amount of avermectins as compared to the amount of avermectins produced by cells of the same strain that have not been modified in this manner. According to the present invention, said genetic modification preferably comprises mutating either the aveR1 gene or the aveR2 gene or both aveR1 and aveR2 genes, when said muting results in a detectable increase in the amount of avermectins produced by cells of a strain of S. avermitilis that are carriers of the gene mutation compared with cells of the same strain but that are not carriers of the gene mutation.

In accordance with the present invention, mutations can be introduced either into the aveR1 gene or into the aveR2 gene or into both aveR1 and aveR2 genes using any techniques currently known or to be developed in the future. For example, random mutagenesis can be carried out using classical mutagenic techniques, which include exposing Streptomyces cells to ultraviolet radiation or X-rays, or chemical mutagens such as N-methyl-N1-nitroso-guanidine, ethyl methano- sulfonate, nitrous acid or nitrogenous ostazas, and then screened for cells that exhibit detectably increased production of avermectins as the result of one or more mutations in the aveR1 and / or aveR2 genes. See, for example, Ausubel, 1989, supra, for a compilation of mutagenesis techniques.

Alternatively, mutations in the aveR1 and aveR2 genes or both aveR1 and aveR2 genes can be carried out in a site-directed manner using any of a variety of known recombination methods, including error-prone PCR, or mutagenesis. in cassete. For example, a site-directed mutagenesis, which can be used either to specifically alter the ORF of aveR1 or aveR2 or flanking sequences, or both ORF'S of aveR1 and aveR2 or flanking sequences, such that they are specifically introduced (n) one or more mutations in these genes. In addition, the methods described in U.S. Pat. 5,605,793, which comprise fragmentation of nucleotides, can be used for general large libraries of polynucleotide molecules having nucleotide sequences encoding mutations of aveR1 and / or aveR2.

Mutations in the aveR1 or aveR2 genes or in both aveR1 or aveR2 genes, which are useful in practicing the invention, include addition, deletion or substitution, or some combination thereof, of one or more nucleotides either in the aveR1 gene or in the aveR2 gene or in both aveR1 and aveR2 genes, or in flanking regulatory regions, and that produce the desired result, i.e., a detectable increase in the quantity of avermectins produced by cells of a strain of Streptomyces that are carriers of the mutation of genes compared with cells of the same species or Streptomyces pepa that are not carriers of the gene mutation. Such mutations can serve to introduce one or more new restriction sites, termination strands or frame shifts, into either or both of the ORF sequences, or in the flanking regulatory regions involved in gene transcription. Other useful mutations include those that introduce a different or heterologous sequence of nucleotides into either or both of the aveR1 and aveRRI genes; or which suppress all or a portion of either or both of the aveR1 and aveR2 genes, or which replace all or a portion of either or both of the aveR1 and aveR2 genes by a different or heterologous sequence of nucleotides; and whose mutations produce the desired result, ie a detectable increase in the quantity of avermectins produced by cells of a species or strains of Streptomyces that are carriers of the gene mutation compared with cells of the same species or strain of Streptomyces that are not carriers of the gene mutation compared to cells of the same species or strain of Streptomyces that are not carriers of the gene mutation.

Site-directed mutations are useful, particularly when they serve to alter one or more conservative amino acid residues in one of the aveR1 or aveR2 gene products or in both products of the aveR1 genes | and aveR2. For example, a comparison of the amino acid deduced sequences of the products of the aveR1 and aveR2 genes from S. avermitilis with products of analogous genes from S. coelicor, as presented in FIGURES 1A and 1B, indicates sites of significant conservation of amino acid residues between these species. Site-directed mutagenesis, which suppresses or non-conservatively replaces one or more of these conserved amino acid residues, may be particularly effective in producing novel mutant strains that exhibit desirable alterations in the production of avermectins.

In a preferred embodiment, one or more mutations are introduced by homologous recombination into either the aveR1 or the aveR2 gene, or both aveR1 and aveR2 genes, using an artificial genetic structure provided by the present invention, such as, for example, a gene replacement vector. The artificial genetic structure may comprise the complete ORF of aveR1 or a polynucleotide molecule homologous thereto, or a substantial portion thereof; or the complete ORF of aveR2 or a polynucleotide molecule homologous thereto, or a substantial portion thereof; or the two complete ORF's of aveR1 and aveR2 or a polynucleotide molecule homologous thereto, or a substantial portion thereof; or nucleotide sequences that naturally flank the ORR of aveR! or to the ORF of aveR2 or both ORF's of AveR1 and aveR2 of S. Avermitilis in situ; or a combination of them; and whose artificial genetic structure can be used to introduce a mutation into either the aveR1 gene or the aveR2 gene or both aveR1 and aveR2 genes, whose mutation results in a detectable increase in the amount of avermectins produced by cells of a species or strain of Streptomyces that are carriers of said gene mutation compared with cells of the same species or strain that are not carriers of the gene mutation. In a specific but non-limiting embodiment, an artificial genetic structure intended to be used in the practice of the present invention is a plasmid comprising a polynucleotide molecule which in turn comprises a nucleotide sequence which is otherwise the same as the nucleotide sequence. of any of the ORFs of aveR1 or aveR2 or of both ORFs of aveR1 and aveR2, or a substantial portion thereof, originating from S. Avermitilis, but also comprising one or more mutations, namely one or more deletions, insertions, substitutions, or a combination thereof, of nucleotides, whose plasmid can be used to transform Streptomyces cells, and thereby introduce the mutation in the aveR1 gene, in the aveR2 gene, or in both aveR1 and aveR2 genes, in such a way that break or otherwise alter the activity or biological function of either the aveR1 gene or the aveR2 gene or both aveR1 and aveR2 genes, respectively, or break or alter otherwise the activity of the biological function of the aveR1 gene product, the aveR2 gene product or both aveR1 and aveR2 gene products, respectively, such that the amount of avermectins produced by cells of a species or strain from Streptomyces that are carriers of said gene mutation is detectably increased compared to cells of the same species or strain that are not carriers of the gene mutation. Said plasmid further preferably comprises a selectable marker. Once it has been transformed into the host cells of Streptomyces, the polynucleotide molecule of the artificial genetic structure is specifically targeted to a target by homologous recombination with the aveR1 gene or with the aveR2 gene or with both aveR1 and aveR2 genes, I either replace the aveR1 gene or a portion of it, or the aveR2 gene or a portion of it, or both aveR1 and aveR2 genes or a portion of these, or insert inserts in the aveR1 gene or the aveR2 gene. As a result of the recombination event, either the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes, of the host cell, or the gene products encoded thereby, are partially or completely incapacitated. Transformed cells are selected, preferably by taking advantage of the presence of a selected marker in the artificial genetic structure, and screened by classical techniques, such as those described in Section 6.6 below, for cells that produce an increased amount detectably of avermectins compared to cells of the same strain that have not been transformed in this way. In a specific but non-limiting embodiment given as an example in Section 6.9.1 below, a gene replacement vector was used to break both aveR1 and aveR2 genes by replacing a portion of the ORF of each gene with a heterologous nucleotide sequence (ermE ). In another specific but not limiting embodiment, given as an example in Section 6.9.2 below, a gene replacement vector was used to break the aveR2 gene by introducing a heterologous nucleotide sequence (ermE) into the ORR of aveR2. Each of these gene replacement vectors was transformed separately into the cells of a strain of S. Avermitilis and integrated into the chromosome by homologous recombination. The analysis of the fermentation of each of these new transformants of Streptomyces indicated a significant increase in the amount of avermectins produced by cells that are carriers of these gene mutations compared to cells of the same strain that are not carriers of any of these gene mutations. The present invention further provides a method for identifying a mutation of an aveR1 gene or a aveR2 gene, or both aveR1 and aveR2 genes, in a Streptomyces species or strain, whose gene mutation is capable of detectably increasing the amount of avermectins produced by cells of the species or strain of Streptomyces that are carriers of the gene mutation compared to cells of the same species or strain of Streptomyces that are not carriers of the gene mutation, which comprises: (a) measuring the amount of avermectins produced by cells of a particular Streptomyces species or strain; (b) introducing a mutation in the averRI gene or in the aveR2 gene, or in both aveR1 and aveR2 genes, of cells of the Streptomyces species or strain from operation (a); and (c) comparing the amount of avermectins produced by the cells that are carriers of the mutation of genes that have been produced in step (b) with the amount of avermectins produced by the cells from operation (a) that are not carriers of the gene mutation; such that if the amount of avermectins produced by the cells that are carriers of the gene mutation that have occurred in step (b) is detectably greater than the amount of avermectins produced by the cells from the operation (a) that are not carriers of the gene mutation, then a mutation of the aveR1 or aveR2 gene has been identified, capable of detectably increasing the amount of avermectins produced. In a preferred embodiment, the Streptomyces species is S. Avermitilis. The present invention further provides a method for preparing genetically modified cells from a species or strain of Streptomyces, which modified cells produce a detectably increased amount of avermectins compared to unmodified cells of the same species or strain, which comprises mutating the aveR1 gene or the aveR2 gene, or both aveR1 and aveR2 genes of Streptomyces, in cells of the Streptomyces species or strain, and select the mutated cells that produce a detectably increased amount of avermectins compared to cells of the same species or strain of Streptomyces that do not they are carriers of the gene mutation. In a preferred embodiment, the Streptomyces species is S. Avermitilis. The present invention also provides new strains of Streptomyces, whose cells produce a detectably increased amount of avermectins as a result of one or more mutations in the aveR1 gene or in the aveR2 gene or in both aveR1 and aveR2 genes compared to cells of the same species or strain of Streptomyces that are not carriers of the mutation of genes. In a preferred embodiment, the Streptomyces species is S. Avermitilis. The new strains of the present invention are useful in the large scale production of avermectins, such as commercially desirable doramectin. The present invention further provides a method for producing an increased amount of avermectins produced by Streptomyces cultures, which comprises culturing cells from a Streptomyces species or strain, which cells comprise a mutation of the aveR1 gene or the aveR2 gene or both aveR1 genes and aveR2; whose gene mutation serves to detectably increase the amount of avermectins produced by cells of the species or strain of Streptomyces that are carriers of the gene mutation compared with cells of the same species or strain that are not carriers of the gene mutation, in culture media and in conditions that allow or induce the production of avermectins from them, and recover the avermectins from the culture. In a preferred embodiment, the Streptomyces species is S. avermitilis. This procedure is useful to increase the production efficiency of avermectin . 5. Anti-sense oligonucleotides and ribozymes Oligonucleotide sequences including anti-sense oligonucleotides, phosphorothioates and ribozymes that function to bind, degrade and / or inhibit the translation of aveR1 or aveR2 mRNA are also within the scope of the present invention. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, act by directly blocking the translation of mRNA by binding to a targeted mRNA and preventing the translation of proteins. For example, anti-sense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding an AveR1 or AveR2 polypeptide, e.g. eg, by conventional phosphodiester techniques. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific dissociation of RNA. The mechanism of action of ribozymes involves a specific hybridization for a sequence of the ribozyme molecule with a complementary target RNA, followed by endonucleolytic dissociation. Molecules of ribozymes with a hammerhead motif, engineered, that specifically and efficiently catalyze the endonucleolytic cleavage of aveR1 or aveR2 mRNA sequences are also within the scope of the present invention. Dissociation sites with specific ribozymes within any potential RNA target are initially identified by screening the target molecule for cleavage sites with ribozymes including the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences comprising between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site, can be evaluated for predicted structural features such as a secondary structure which may render it inappropriate to the oligonucleotide sequence. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g. eg, protection analysis with ribonucleases. Both the anti-sense oligonucleotides and the ribozymes of the present invention can be prepared by known methods. These include techniques for chemical synthesis such as, for example, by chemical synthesis of phosphoramidite in solid phase. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Said DNA sequences can be incorporated into a wide variety of vectors that incorporate appropriate RNA polymerase promoters, such as the T7 or SP6 polymerase promoters. Various modifications can be introduced into the oligonucleotides of the present invention as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking ribonucleotide or deoxyribonucleotide sequences at the 5 'and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl instead of phosphodiesterase within the framework of the oligonucleotides. . 6. Antibodies The present invention further provides polyclonal and monoclonal antibodies that bind to a product of the aveR1 gene, a product of the aveR2 gene, or a homologous polypeptide, or a peptide fragment of the present invention. Said antibodies can be used as affinity reagents, with which a product of the aveR1 or native aveR2 gene is purified, or the activity or biological function of the aveR1 or aveR2 gene products is analyzed. Antibodies can be raised against any of the aveR1 or aveR2 related polypeptides of the present invention. Various host animals can be used, including, but not limited to, cows, horses, rabbits, goats, lambs and mice, according to known methods to produce specific anti-AveR1 or anti-AveR2 antibodies. Various adjuvants known in the art can be used to enhance the production of antibodies. Polyclonal antibodies can be obtained from immunized animals and assayed for anti-AveR1 or anti-AveR2 specificity using classical techniques. Alternatively, monoclonal antibodies can be prepared for an AveR1 or AveR2 polypeptide using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hydridome technique originally described by Kohler and Milstein (Nature, 1975, 495-497); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Nati, Acad. Sci. USA 80: 2026-2030); and the EBV hybridoma technique (Colé et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). Alternatively, the techniques described for the production of single chain antibodies (see, eg, US 4,946,778) can be adapted to produce single chain antibodies specific for AveR1 or AveR2. These publications are incorporated herein by reference. Antibody fragments that contain specific binding sites for an AveR1 or AveR2 polypeptide are also encompassed within the present invention, and can be generated by known techniques. Such fragments include, but are not limited to, F (ab ') 2 fragments that can be generated by digestion with pepsin from an intact antibody molecule, and Fab fragment that can be regenerated by reducing the disulfide bridges of the F fragments ( ab ') 2. Alternatively, Fab expression libraries can be constructed (Huse et al. 1989, Science 246: 1275-1281) to allow rapid identification of Fab fragments having the desired specificity for the polypeptide AveR1 or AveR2. Techniques for the production of monoclonal antibodies and fragments of such antibodies are well known in the field of technology, and are further described, inter alia, in those of Harlow and Lane, 1988, Antibodies; A. Laboratorv Manual, Cold Spring Harbor Laboratory; and in J. W. Goding, 1986, Monoclonal Antibodies: Principies and Practice, Academic Press, London. All the aforementioned publications are incorporated herein by reference. . 7. Uses of the avermectins The avermectins are highly active antiparasitic agents which have particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides, and the avermectins prepared using the compositions and methods of the present invention are useful for any of these purposes. The avermectins prepared according to the present invention are useful for treating various diseases or conditions in humans, particularly when these diseases or conditions are caused by parasitic infections, as is known in the field of technology. See, p. ex. Ikeda and Omura, 1997, Chem. Rev. 97 (7): 2591-2609. The avermectins prepared according to the present invention are effective for treating a variety of conditions caused by endoparasites, including, for example, helminthiasis, which is most often caused by a group of parasitic nematodes, and which can cause a disease in humans, and serious economic losses in pigs, lambs, poultry, horses and cattle, as well as affecting the health of domestic animals. Therefore, the avermectins prepared according to the present invention are effective against nematodes that affect humans, as well as those that affect various species of animals, including, for example, Dirofilaria in dogs, and various parasites that infect animals. humans, including gastrointestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides, Trinchinella, Capillaria, Trichuris, Enterobius, and parasites that are found in the blood or other tissues or organs, such as filarial worms and the intestinal states of Strongyloides extracts and Trichinella. The avermectins prepared according to the present invention are also useful for treating ectoparasitic infections including, eg, infestations by arthropods of mammals and birds, caused by ticks, mites, lice, fleas, blowfly, biting insects, or larvae of migratory diptera that can affect cattle and horses, among other animals.

The avermectins prepared according to the present invention are also useful as insecticides against domestic pests such as, for example, those of cockroaches, fabric moths, carpet beetles and houseflies, among others, as well as grain insect pests. stored and agricultural plants, whose pests include spinning mites, attics, caterpillars and orthoptera such as locusts, among others. The animals that can be treated with the avermectins of the present invention include lambs, cattle, horses, deer, goats, pigs, poultry including poultry, as well as dogs and cats. The avermectins prepared according to the present invention are administered in a formulation that is appropriate for the specific intended use, the particular species of host animal being treated, and the parasite or insect involved. To be used as a parasiticide, an avermectin of the present invention can be administered orally in the form of a capsule, bolus, tablet or liquid purgative or, alternatively, it can be administered as a formulation for pouring, or by injection, or as an implant . Such formulations are prepared in a conventional manner in accordance with classical veterinary practice. In this way capsules can be prepared, boluses or tablets by mixing the active ingredient with a finely divided diluent or vehicle, additionally containing a disintegrating agent and / or binder such as starch, lactose, talc, magnesium stearate, etc. A laxative drink formulation can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc. The injectable formulations can be prepared in the form of a sterile solution which can contain other substances such as, for example, salts and / or glucose in sufficient quantity to make the solution isotonic in blood. Such formulations will vary in relation to the weight of active compound depending on the patient, or the species of host animal to be treated, the severity and type of infection, and the body weight of the host. Generally, for oral administration, a dose of active compound of about 0.001 to 10 mg per kg of body weight of the patient or animal, administered as a single dose or in divided doses over a period of 1 to 5 days, will be satisfactory. However, there may be damages in which margins of higher or lower dosages are indicated, as determined, for example, by a doctor or veterinarian, based on clinical symptoms. As an alternative, an avermectin prepared according to the present invention can be administered in combination with an animal feed, and for this purpose an additive can be prepared to concentrated feed or a premix in order to mix it with the normal animal feed. . To be used as an insectidic, and to treat agricultural pests, the compounds of the present invention can be applied as sprays, sprays, fine powders, emulsions and the like, in accordance with classical agricultural practice.

The following examples are illustrative only, and are not intended to limit the scope of the present invention. 6. EXAMPLES: ISOLATION OF THE GENES aveR1 AND aveR2 This example describes the isolation and characterization of two new genes of S. Avermitilis that encode products of the aveR1 and aveR2 genes and that are involved in the regulation of avermectin biosynthesis. 6. 1. Growth of s. Avermitilis for DNA isolation Single colonies of S. Avermitilis ATCC 31272 (isolated material No. 2 from a single colony) were isolated in YPD force medium A containing: Difco Yeast Extract - 5 g; Bacto-peptone Difco - 5 g; Dextrose - 2.5 g; MOPS - 5 g; Difco Bacto-agar - 15 g. The final volume was adjusted to 1 liter with dH20, the pH was adjusted to 7.0, and the medium was autoclaved at 121 ° C for 25 min. The mycelia that grew in the previous medium were used to inoculate 10 ml of a TSB medium (Difco Tryptic Soy Calcium in 1 liter of dH20, autoclaved at 121 ° C for 25 min) in a tube of 25 mm x 150 mm which was maintained with agitation (at 300 rpm) at 28 ° C for 48-72 hours. 6. 2. Isolation of chromosomal DNA from S. avermitilis Aliquots (0.25 ml or 0.5 ml) of mycelia that had grown as described above were placed in microfilm centrifuge tubes. 1.5 ml capacity and the cells were concentrated by centrifugation at 12,000 x g for 60 s (seconds). The excess material was discarded and the cells were resuspended in 0.25 ml of a TSE plug (20 ml of 1.5 m sucrose, 2.5 ml of IM Tris HCl, pH 8.0 2.5 ml of EDTA IM, pH 8.0, and 75 ml of dH20) containing 2mg / ml lysozyme. The samples were incubated at 37 ° C for 20 min with shaking, loaded in an automatic AutoGen 540"nucleic acid isolation instrument (Integrated Separation Systems, Natick, MA) and the genomic DNA was isolated using the Cycle 159 (software logic program of equipment) according to the manufacturer's instructions Alternatively, 5 ml of mycelia were placed in a 17 mm x 100 mm tube, the cells were concentrated by centrifugation at 3,000 rpm for 5 min, and the supernatant was removed. cells were resuspended in 1 ml of TSE buffer, concentrated by centrifugation at 3,000 rpm for 5 min and the excess material was removed.The cells were resuspended in 1 ml of TSE buffer containing 2 mg / ml lysozyme and incubated at 37 ° C with shaking for 30-50 min After incubation, 0.5 ml of 10% SDS was added and the cells were incubated at 37 ° C until the lysis was complete. Sado was incubated at 65 ° C for 10 min, cooled to room temperature (RT), divided into two Eppendorf tubes of 1.5 ml capacity and extracted 1 time with 0.5 ml of a mixture of phenol and chloroform (with 50% phenol, previously balanced with Tris 0.5 M, ph 8.0; and 50% chloroform). The aqueous phase was removed and extracted 2-5 times with a mixture of chloroform and isoamyl alcohol (24: 1). The DNA was precipitated by adding 1/10 volumes of 3m sodium acetate, pH 4.8, incubating the mixture on ice for 10 min, centrifuging the mixture to ,000 rpm at 5 ° C for 10 min and removing the supernatant material to a clean tube to which a volume of sodium propane had been added. The mixture was then incubated on ice for 20 min, centrifuged at 15,000 rpm for 20 min at 5 ° C, the supernatant was removed and the DNA pellet washed 1 time with 70% ethanol. After the sediment was dry, the DNA was resuspended in TE buffer (10mM Tris, 1mM EDTA, pH 8.0). 6. 3. Isolation of plasmid DNA from S. avermitilis An aliquot (1.0 ml) of mycelia was placed in 1.5 ml microcentrifuge tubes and the cells were concentrated by centrifugation at 12,000 xg for 60 sec. . The excess material was discarded, the cells were resuspended once again., 0 ml of 10.3% sucrose and concentrated by centrifugation at 12,000 x g for 60 s, and the supernatant was discarded. The cells were then resuspended in 0.25 ml of a TSE buffer containing 2 mg / ml lysozyme, incubated at 37 ° C for 20 min with shaking, and loaded into the AutoGen automatic nucleic acid isolation instrument. 540. The plasmid DNA was isolated using the Cycle 106 (software software program of the equipment) according to the manufacturer's instructions. Alternatively, 1.5 ml of mycelia were placed in 1.5-micron centrifuge tubes and the cells were concentrated by centrifugation at 12,000 x g for 60 s. The sobrnadante material was discarded, the cells were resuspended in 1.0 ml of sucrose .3% and concentrated by centrifugation at 12,000 x g for 60 s, and the supernatant was discarded. The cells were resuspended in 0.5 ml of TSE buffer containing 2 mg / ml lysozyme and incubated at 37 ° C for 15-30 min. After incubation, 0.25 ml of alkaline SDS (0.3 N NaOH, 2% SDS) was added, and the cells were incubated at 55 ° C for 15-30 min or until the solution was clear. Sodium acetate duel (0.1 ml, 3 m, pH 4, (8) was added to the DNA solution, which was incubated on ice for 10 min.The DNA samples were centrifuged at 14,000 rpm for 10 min at 5 °. C. The excess material was removed to a clean tube, and 0.2 ml of a mixture of phenol and chloroform (50% phenol: 50% chloroform) were added and mixed gently.The DNA solution was centrifuged at 14,000. rpm for 10 min at 5 ° C and the upper layer was retifed to a clean Eppendorf tube.Sopropanol (0.75 ml) was added and the solution mixed gently and then incubated at room temperature for 20 min. DNA was centrifuged at 14,000 rpm for 15 min at 5 ° C, the supernatant was removed, the DNA pellet was washed with 70% ethanol, dried and the DNA resuspended in a TE buffer. 6. 4. Isolation of plasmid DNA from E. coli A single colony of transformed E. coli was inoculated into 5 ml of Luria-Bertani medium (LB) (Bacto-tryptone-10 g; yeast extract Bacto-5 g; and NaCl - 10 g in 1 liter of dH20 (distilled), pH 7.0, autoclaved at 121 ° C for 25 min) supplemented with 100 * g / ml of ampicillin. The culture was incubated overnight and a 1 ml aliquot was placed in a 1 ml microcentrifuge tube. The culture samples were loaded into the AutoGen 540 automatic nucleic acid isolation instrument and the plasmid DNA was isolated using the Cycle 3 (software software program of equipment) according to the manufacturer's instructions. 6. 5. Preparation and transformation of protoplasts of S. Avermitilis Unique colonies of S. Avermitilis were isolated in YPD-6 of force Vz. The mycelia were used to inoculate 10 ml of TSB medium in a 25 mm x 150 mm rubol which was then incubated with shaking (300 rpm) at 28 ° C for 48 h. 1 ml of mycelium was used to inoculate 50 ml of the YEME medium. The YEME medium contains per liter: Difco Yeast Extract - 3 g; Bacto-peptone Difco - 5 g; Malt Extract Difco - 3 g; and sucrose - 300 g. After autoclaving at 121 ° C for 25 min, the following was added: 2.5 M MgCl2.6H20 (autoclaved separately at 121 ° C for 25 min) - 2 ml; and glycine (20%) (sterilized and filtered) - 25 ml. The mycelia were grown at 30 ° C for 48-72 h and harvested by centrifugation in a 50 ml capacity centrifuge tube (Falcon) at 3,000 rpm for 20 min. The supernatant was discarded and the mycelia were resuspended in buffer P containing: sucrose - 205 g; K2 S04, - 0.25 g; MgCl2. 6H20 - 2.02 g; H20 - 600 ml; K2P04 (0.5%) - 10 ml; trace element solution (oligoelements) * - 20 ml; CaCl2 2H20 (3.68% - 100 ml; MES buffer (1.0 M, pH 6.5) - 10 ml (* The trace element solution contains per liter: znCI2 - 40 mg, FeCI -6H20 - 200 mg; CuCl2-2H20 - 10 mg; MnCl2-4H20 - 10 mg; NaB4O7-10H2O - mg; (NH4) 6Mo 024-4H20- 10 mg). The pH was adjusted to 6.5 and the final volume was adjusted to 1 liter, and the medium was filtered hot through a 0.45 micron filter. The mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was discarded and the mycelia were resuspended in 20 ml of a P buffer containing 2 mg / ml lysozin. The mycelia were incubated at 35 ° C for 15 min with shaking, and were checked under a microscope to determine the extent of protoplast formation. When the protoplast formation was complete, they were resuspended in 10 ml of the P buffer. The protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts were resuspended in 2 ml of buffer P, and approximately 1 x 109 protoplasts were distributed in cryogenic vials of 2.0 ml capacity (Nalgene). A vial containing 1 x 109 protoplasts was centrifuged for 10 min at 8,000 rpm, the supernatant was removed, and the protoplasts were resuspended in 0.1 ml of buffer P. They were added to the protoplasts from 2 to 5 * g of DNA in transformation, immediately followed by the addition of 0.5 ml of the T buffer in work. The base of the T buffer contains: PEG 1000 (Sigma) - 25 g; sucrose - 2.5 g; and H20-83 ml. The pH was adjusted to 8.8 with 1 N NaOH (sterilized and filtered), and the base of the T buffer was filter sterilized and stored at 4 ° C. The working T buffer, produced on the same day it was used, contains the base of the T-8.3 ml buffer; K2P0 (4mM) - 10 ml; CaCI2 2H20 (5M) - 0.2 ml; and TES (1 M, pH 8) - 0.5 ml. Each component of the buffer T at work was sterilized in filter as well as stored at 4 ° C. Within 20 s of the addition of the T buffer to the protoplasts, 1.0 ml of the p-buffer was also added and the protoplasts were centrifuged at 8,000 rhoduate for 19 min. The supernatant was discarded and the protoplasts resuspended in 0.1 ml of buffer P. Then the protoplasts were seeded on Rm14 media containing: sucrose-205 g; K2S04 - 0.25 g; MgCl 2 - 6 h 20 - 10,12 g, glucose - 10 g, Casamino - Difco Acids - 0,1 g; Difco Yeast Extract - 5 g; Digco Oatmeal Agar - 3 G; Bacto Agar Difco - 22 g; and H20 - 800 m. The solution was treated in an autoclave at 121 ° C for 25 min. After autoclaving, sterile stock materials of the following were added: K2P04 (0.5%) - 10 ml; CaCl2 2 H20 (5 M) - 5 ml; L-proline (20%) - 15 ml; MES buffer (1.0 M, pH 6.5) - 10 ml; trace element solution (the same as before) - 2 ml; cycloheximide stock (25 mg / ml) -40 ml and 1 N-2 ml NaOH. 25 ml aliquots of Rm14 medium were placed per plate and the plates were dried for 24 h before use. The protoplasts were incubated with a humidity of 95% at 30 ° C for 20-24 h. To select the erythromycin-resistant transformants, 1 ml of cover buffer plus 125 g of erythromycin was spread evenly (to give a final erythromycin concentration of 5 g / ml), spread evenly over the RM14 regeneration plates. The coating buffer contains per 100 ml, sucrose - 10.3 g; trace element solution (the same as before) - 0.2 ml; and MES (1 M, pH 6.5) - 1 ml. The protoplasts were incubated with a humidity of 95% at 30 ° C for 7.14 days until erythromycin-resistant colonies (Ermr) were visible. 6. 6. Analysis of the Fermentation of Strains of S. Avermitilis Myelios of S avermitilis that had grown on YPD-6 of force Í during 4-7 days were inoculated in tubes of 1 x 6 inches that contained 8 ml of preformed medium and two globules soluble (or else diluted boiled starch or KOSO, Japan Com Starch Co., Nagoya) - 20 g / ml; Pharmamedia (Trader's Protein, Memphis, TN) - 15 g / l; Ardamine pH (Champlain Inc. Clifton, NJ) - 5 g / l; CaCO3 - 2 g / l; 2x befa ("befa" refers to branched chain fatty acids)) containing a final concentration in the medium of 50 ppm of 2- (±) - methyl-butyric acid, 60 ppm of butyric acid and 20 ppm of isovaleric acid . The pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C for 25 min.

The tube was shaken at an angle of 17 ° to 215 rpm at 29 ° C for 3 days. A 2 ml aliquot of the seed culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml of a production medium containing: starch (either diluted boiled starch or KOSO) - 160 g / l; Nutrisoy (Archer Daniels Midland, Decatur, IL) - 10 g / l; Ardamine pH - 10 g / l; K2P04 - 2 g / l; MgSO4 4H20 - 2 g / l; FeS047H20 - 0.02 g / l, MnCl2 - 0.002 g / l; ZnS047H20 - 0.002 g / l; CaCO3 - 14 g / l; and 2x befa (as above). The pH was adjusted to 6.9 and the medium was autoclaved at 121 ° C for 25 min. After inoculation, the flask was incubated at 29 ° C for 12 days with shaking at 200 rpm. After incubation, a 2 ml sample was removed from the flask, diluted with 8 ml of methanol, mixed and the mixture was centrifuged at 1250 x g for 10 min to pellet the residues. The supernatant was then analyzed by high performance liquid chromatography (HPLC) using a Beckman Ultrasphere ODS column (25 cm x 4.6 mm ID) with a flow rate of 0.75 ml / min and with detection by measurements of the absorbance at 240 nm. The mobile phase was a 86 / 8.9 / 5.1 mixture of methanol, water and acetonitrile. 6. 7 Identification and isolation of PKS genes from S. avermitilis A cosmid library of S. avermitilis (ATCC 31272) of chromosomal DNA was prepared and hybridized with a ketosynthase (KS) probe produced from a fragment of the polyketide gene. synthase (PKS) of Saccharopolyspora erythraea. A detailed description of the preparation of cosmid libraries can be found in the citation of Sambrook et al., 1989, supra. A detailed description of the preparation of Streptomyces chromosomal DNA libraries is presented in the citation of Hopwood et al., 1985, supra. Cosmid clones containing regions that hybridized with ketosynthase were identified by hybridization with a 2.7 kb Ndel / Eco47lll fragment from pEX26 (kindly supplied by Dr. P. Leadley, Cambridge, R.U.). Approximately 5 μg of pEX26 was digested using the restriction endonucleases Ndel and Eco47lll. The reaction mixture was loaded on a 0.8% SeaGlaque® GTG agarose gel (FMC BíoProducts, Rockland, ME). The 2.7 kb Ndel / Eco47lll fragment was excised from the gel after electrophoresis and the DNA was recovered from the gel using GELase® (Epicenter Technologies) using the Fast Protocol. The 2.7 kb Ndel / Eco47lll fragment was labeled with [-32P] dCTP ([a-32P] -deoxycytidine-5'-triphosphate, tetra- (triethylammonium) salt) (NEN-Dupont, Boston, MA) using the BRL Notching Translation System (BRL Life Technologies, Inc., Gaithersburg, MD) following the supplier's instructions. A typical reaction was performed in a volume of 0.05 ml. After addition of a Stop buffer of 5 μl, the labeled DNA was separated from unincorporated nucleotides using a column with G-25 Sephadex Quick Spin ™ Column (Boehringer Mannheim) following the supplier's instructions.

Approximately 1,800 cosmid clones were screened by colony hybridization. Ten clones were identified that hybridized strongly with the KS Sacc probe. erythraea. The colonies of E. coli containing cosmid DNA were grown in the liquid medium LB and cosmid DNA was isolated from each culture in the automatic AutoGen 540R nucleic acid isolation instrument using the Cycle 3 (computer software system) according to the manufacturer's instructions. The mapping with restriction endonucleases and the Southern blot hybridization analysis revealed that five of the clones contained overlapping chromosomal regions. A restriction map with Genomic BamHI of S. avermitilis from the five cosmids (ie, pSE65, pSE66, pSE67, pSE68, pSE69) was constructed by overlapping cosmid analyzes and hybridizations (FIGURE 4). 6. 8 Identification of ORF's of Regulatory Genes The following methods were used to subclone fragments derived from the pSE68 clone. PSE68 (5 μg) was digested with Xbal and EcoRl. The reaction mixture was loaded on a 0.8% SeaPlaque® GTG agarose gel (FMC BioProducts), a fragment of Xbal / EcoRI of -19 kb was excised from the gel after electrophoresis and the DNA was recovered from the gel. of the gel using GELase® (Epicenter Technologies) using the Fast Protocol. Approximately 5 μg of pGEM7Zf (+) (Promega) were digested with Xbal and EcoRl. Approximately 0.5 μg of the EcoRI / Xbal fragment of 19 kb and 0.5 μg of the digested pGEM7Zf (+) were mixed together and incubated overnight with 1 unit of ligase (New England Biolabs, Inc., Beverly, MA) at 15 ° C in a total volume of 20 μl, according to the supplier's instructions. After incubation, 5 μl of the ligation mixture was incubated at 70 ° C for 10 min, cooled to room temperature and used to transform competent E. coli DH5a cells (BRL) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the Xbal / EcoRI insert of -19 kb was confirmed by restriction analysis. This plasmid was designated pSE200. The pSE200 was further modified by digestion with Econucleasa III using the Erase-a-Base system (Promega) following the manufacturer's instructions. 5 μg of pSE200 was digested with Clal. Clal generates 5 'overhangs that were protected with respect to digestion with Exonuclease to be filled with alpha-phosphorothioate-deoxyribonucleotides according to the manufacturer's instructions. Then the pSE200 was digested with EcoRI and aliquots were digested with S1 nuclease for variable time periods ranging from 30 seconds to 12 minutes. Samples of pSE200 treated with S1 nuclease were ligated overnight and transformed into competent cells of E. coli HB101 (BRL) following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants and the size of the insert DNA was determined by restriction enzyme analysis.

An isolated material containing a 5.9 kb insert was identified, which was designated pSE201 (FIGURE 2A), and deposited with the ATCC (Accession No. 203182). A second material containing an insert was identified - 8.8 kb and this isolated material was designated as pSE210 (FIGURE 2B). Approximately 10 μg of pSE201 was isolated using a plasmid DNA isolation kit (Qiagen, Valencia, CA) following the manufacturer's instructions. This DNA was sequenced using an automatic DNA sequencer 373A (Perkin, Elmer, Foster City, CA). The sequence data was collected and edited using the programs of the Genetic Computer Group (GCG, Madison, Wl). The DNA sequences of the ORF's of the regulatory genes, aveR1 and aveR1, are presented as identical in both SEQ ID NO: 1 and SEQ ID NO: 3. The ORF of aveR1 is from nt 1.112 to nt 2.317 of SEQ ID NO: 1. The ORF of aveR2 is from nt 2.314 to nt 3.021 of SEQ ID NO: 1. A comparison of the deduced amino acid sequence of the The ORF of aveR1 of S. avermitilis shows a 32% identity with the product of the absAI gene deduced from S. coelicolor (FIGURE 1A) (Brian et al., 1996, J. Bacteriology 178: 3221-3231). A comparison of the deduced amino acid sequence of the aveR2 ORF of S. avermitilis shows a 45% identity with the product of the absA2 gene deduced from S. coelicolor (FIGURE 1 B). 6. 9 Construction and Use of Gene Replacement Vectors A general description of the techniques for introducing mutations into Streptomyces genes is provided by Kieser and Hopwood, 1991, Meth. Enzym. 204: 430-458. A more detailed description is provided by Anzai et al., 1998, J. Antibiot. XLI (2): 226-233; Stutzman-Engwall et al., 1992, J. Bacteriol. 174 (1): 144-154; and Oh and Chater. 1997, J. Bact. 179: 122-127. These publications are incorporated herein by reference. 6. 9.1. Deactivation of both genes aveR1 and aveR2 Both genes aveR1 and aveR2 were deactivated by replacing a fragment of BglII / Stul of 988 bp (base pairs) in pSE210 (FIGURE 2B) with the erythromycin resistance gene (ermE) from Saccharopolyspora erythraea, as follows. Approximately 5 μg of pSE210 were digested with Eglll and Stul to release a -10.8 kb fragment. The BglII end was filled in by incubating the DNA with a final concentration of 100 μM of the dNTPs in 1x Klenow buffer and 1 U of the Klenow enzyme (Boehringer Mannheim) for 30 min at 37 ° C. The -10.8 kb fragment was purified from an agarose gel incubated in 1x alkaline phosphatase buffer and 1 U alkaline phosphatase for 1 h at 50 ° C to dephosphorylate the ends. After incubation, the dephosphorylated fragment was purified by extraction with phenol and chloroform as described in Section 6.3 and resuspended in the TE buffer. The ermE gene was isolated from plJ4026 (provided by the John Innes Institute, Norwich, R.U .; see also Bibb et al., 1985, Gene 41: 357-368) by digestion with BglII, and the BglII ends were filled in by incubating the ermE fragment with a final concentration of 100 μM of the dNTPs in 1x Klenow buffer and 1U of Klenow enzyme for 30 min at 37 ° C. The - 1.7 kb ermE fragment was purified from an agarose gel and 0.5 μg thereof was mixed with 0.5 μg of the 10.8 kb fragment and ligated.

The ligation mixture was used to transform competent E. coli HB101 (BRL) cells following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the ermE insert was confirmed by restriction analysis. This plasmid, which was designated pSE214 (FIGURE 3A), was transformed into E. coli DM1 (BRL), and the plasmid DNA was isolated from ampicillin resistant transformants. The introduction of the ermE gene into the 988 bp BglII / Stul site removes 752 bp from the aveR1 gene and 232 bp from the aveR2 gene. PSE214, which will not replicate autonomously in S. avermitilis, was used as a gene replacement vector for gene integrator replacement in the following manner. 8 μl of pSE214 DNA (1 μg) was denatured by adding 2 μl of 1 M NaOH, incubating for 10 min at 37 ° C, then rapidly cooling on ice, followed by the addition of 2 μl of HCl and 1 M, according to with the procedure of Oh and Chater, 1987, earlier. The protoplasts of S. avermitilis were transformed with the denatured pSE214. The transformants were regenerated under selective conditions that required the expression of the erythromycin gene in such a way as to induce an integrative recombination with plasmid genes on the chromosome of host cells. Since the plasmid does not replicate autonomously, the erythromycin resistant transformants must have the plasmid sequences integrated in the chromosome or by a single homologous recombination event between one of the two DNA segments flanking the ermE gene and its homologous counterpart in the chromosome, which will result in the integration of the entire pSE214 vector, or a double crossing in which a second recombination event is performed between the second DNA segment that flanks the mutation and its homologous counterpart in the chromosome , which will result in the exchange of aveR1 / aveR2 (deactivated) sequences from the plasmid on the chromosome and the concomitant loss of the wild-type allele and vector sequences from the chromosome. The transformants resistant to erythromycin were isolated and screened by PCR in relation to the presence of the vector framework. All transformants lacked the vector lattice, suggesting that a double crossing event had occurred and the chromosomal aveR2 sequences had been replaced by the deactivated aveR2 sequence. The transformants resistant to erythromycin were analyzed by HPLC analysis of the fermentation products. S. avermitilis strains containing an inactivated aveR2 gene yielded an average of a multiple of 3.1 times the total avermectins of the control strain (Table 1).

TABLE 1 DEPOSIT OF BIOLOGICAL MATERIALS The following biological material was deposited in the American Type Culture Collection (ATCC) at 12301 Parkiawn Drive, Rockville, MD, 20852, USA, on September 5, 1998, and was assigned the following access numbers: Plasmid Plasmid access No. pSE201 203182 All patents, patent applications and publications referred to above are incorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specific embodiments described herein which are intended to be unique illustrations of individual aspects of the invention, and that functionally equivalent methods and components are within the scope of the invention. Of course, various modifications of the invention, in addition to those shown and described in the present context, will be apparent to those skilled in the art from the following description and the accompanying drawings. It is intended that said modifications fall within the scope of the appended claims.

LIST OF SEQUENCES < 110 > Pfizer Products Inc. < 120 > REGULATORY GENES OF STREPTOMYCES AVERMITILIS FOR INCREASED AVERMECTICINE PRODUCTION. < 130 > PC-9944A < 140 > < 141 > < 150 > 60 / 100,134 < 151 > 1998-09-14 < 160 > 6 < 170 > PatentlnVer.2.0 < 210 > 1 < 211 > 5045 < 212 > DNA < 213 > Streptomyces avermitilis < 220 > < 221 > CDS < 222 > (1112) .. (2317) < 223 > aveR1 ORF < 400 > 1 cgagttgctg gtcatcggcg tactcctccc ggcgactccg cccggtactc gaccgcggca 60 gcggtcagcc gcatgaacgc ctcttcgaga gacacggtct tccgcgtcac ctcgtgcacc 120 gtcacggcgt gcgccgcgac gagttcgccg atccgctccg cggcggcggc caccttcagg 180 ctgccgtcgg agcagtcggt gaccgtgatt cccgcgccga ccagcacgtc gcgcagccgc 240 cgcggttccg gagtgcgcac ccgcaccccc acgtccgtgt acctgtcgat gaactcgctc 300 atgctggtgt ccgcgaggag ccgaccgcgg ccgatgatga cgaggtggtc cacggtgagc 360 gccgcctcgc tcatcagatg gctggacacg aagacggtgc gtccctgtgc cgccaggtcc 420 cgcatgaggt gccgcagcca caggacgcct tccgggtcga gcccgttgac cggctcgtcg 480 agcaccagga cggcggggtc gccgagcagc gccgcggcga ttcccagccg ctgactcatg 540 • cccagcgaga acgtccccgc ccgccggcgc acggcgctcc gcaggcccgc cagctcgatc 600 acctcacgga cgcggcgggg cgggatccgg ttgctgcggg ccagccagcg caagtggttc 660 agcgcggttc ggccggggtg caccgccctg gcgtcgagca gtgcccccac cgtccgcagc 720 gggtcgcgga gccgctggta gggcgcgccg tcgatgcgta cctcgccggc cgtcgggcgg 780 tccaggccca gcatcatgcg catcgcggtg gacttcccgg cgccgttggg gccaaggaat 840 ccggtcaccc gaccggtccg tacctggaag gtaagaccct ccacaacggt ggtggtcccg 900 tagcgcttgg tcaggtccgt gacttcgatc atgccggtga tggtccgtga cgacaggctc 960 ccgccgcgtc ccgctcgggg ctgactgccc cttctccacc cccggttgga gaatgaccgc 020 cacccgcggc cgcgcatcag gctgcaggag gagcggcttt gaccaccgct ggacggaggc 080 ggagcggcgt acgcctggat atggtcgagc g gtg cat gca ggt acc gcg gtg 132 Val His Ala Gly Thr Ala Val 1 5 gac ccc gac gac cat ccg ate ctg gcc cgg cga ctg age cgg cgc cga 1180 Asp Pro Asp Asp His Pro lie Leu Wing Arg Arg Leu Being Arg Arg Arg 10 15 20 etc gcc gtc gtc gtg etc gta tc gcc tac gc gc gcg ctg 122? Leu lie Ala Leu Asp Gly Val Leu Val Phe Ala Tyr Ala Cys Ala Leu 25 30 35 ctg tcg acc ggg ccg here ggc ate tcg tcg tcg tcc gcg ccg ccg etc 1276 Leu Ser Thr Gly Pro Thr Gly lie Ser Ser Ser Ser Ala Pro Pro Leu 40 45 50 55 ccg gcc ccg gtg ccg tgg gag cgg etc gtg etc gcc gcg gcc gcc act 1324 Pro Pro Wing Val Pro Trp Glu Arg Leu Val Leu lie Wing Wing Thr 60 65__ 70 gcg ect gtc gcc gta cgg cgg ate tgg ccg ttg ecc gtg ttc gcg gtc 1372 Wing Pro Val Wing Val Arg Arg lie Trp Pro Leu Pro Val Phe Wing Val 75 80 85 gtg ctg gcg gtg acc gcc gtg gcc gtc gtg cgg gac gcg gcg tgg gac 1420 Val Leu Ala Val Thr Ala Val Ala Val Val Arg Asp Ala Ala Trp Asp 90 95 100 ccg ttc ctg tcg gcg gcg ttc gcc etc tac acc gtc gcc gtc acg gtg 1468 Pro Phe Leu Ser Ala Ala Phe Ala Leu Tyr Thr Val Ala Val Thr Val 105 110 115 ecc tcg cgc falls tgg tgg cag cgc tgg tta ecc ggc ctg gcg ate gct 1516 Pro Ser Arg His Trp Trp Gln Arg Trp Leu Pro Gly Leu Ala lie Wing 120 125 130 135 ttg ctg acc gtg gcc gcc ctt gcc ggc gcg gcg cgt gcg gag gcc 1564 leu leu thr val wing gly leu wing gly wing wing arg wing gly wing 140 145 150 ttc tgg tgg cgc ggc age ecc ggt ctg ctg ctg etc ggc ttc gcc gca 1612 Phe Trp Trp Arg Gly Ser Pro Gly Leu Leu Leu Leu Gly Phe Ala Wing 155 160 165 ctg etc ggc gcc tgg caa ctg gga cgc gcc gcg cgg cag agg cgc gca 1660 Leu Leu Gly Wing Trp Gln Leu Gly Arg Wing Wing Arg Gln Arg Arg Wing 170 175 180 tcc gcc gtc cgg gcg gcc gag cag etc gca cag cgg gcc gtc acg gag 1708 Phe Wing Val Arg Wing Wing Glu Gln Leu Wing Gln Arg Wing Val Thr Glu 185 190 195 gaa cgc ctg cgg ata gcc cgc gaa ctg cat gac gtc gtc acg falls age 1756 Glu Arg Leu Arg lie Wing Arg Glu Leu His Asp Val Val Thr His Ser 200 205 210 215 atg ggc ctg ate gcg gtc aag gtc ggc gtc gcc aac falls gtg ttg falls 1804 Met Gly Leu lie Ala Val Lys Val Gly Val Ala Asn His Val Leu His 220 225 230 ate agg ccg cag gag gcg tac gac gcg etc cag gtc ate gaa cgc acg 1852 lie Arg Pro Gln Glu Ala Tyr Asp Ala Leu Gln Val lie Glu Arg Thr 235 240 245 age cgc acc gcg ctg aac gac atg cgc cgg atg etc ggt gtg ctg cgt 1900 Being Arg Thr Ala Leu Asn Asp Met Arg Arg Met Leu Gly Val Leu Arg 250 255 260 acg tcc gag ggt gag cgg cag tea gcg gct etc ggc ccg ctg ect ggc 1948 Thr Ser Glu Gly Glu Arg Gln Ser Wing Ala Leu Gly Pro Leu Pro Gly 265 270 275 gcc ctt gct etc ect gac etc gtc ggg cag gcc ggc gcg cag ctg act 1996 Ala Leu Ala Leu Pro Asp Leu Val Gly Gln Ala Gly Ala Gln Leu Thr 280 285 290 295 atg cgc ggt gtc gag agt ctg ecc gac gga gtc gcg ctg gcc gtc tac 2044 Met Arg Gly Val Glu Ser Leu Pro Asp Gly Val Ala Leu Ala Val Tyr 300 305 310 cgg ate gtg cag gag gcg etc acc aat gtc gcc aag drops gcc ggc ccg 2092 Arg lie Val Gln Glu Ala Leu Thr Asn Val Ala Lys His Ala Gly Pro 315 320 325 gag gcc cgc tgc cgg gtg gcg gtc gat gcg aac ggc drops ggc gtc cgg 2140 Glu Wing Arg Cys Arg Val Wing Val Asp Wing Asn Gly His Gly Val Arg 330 335 340 etc gag ata gac gac gac gga ggc gac cgg age ecc etc gcg ccg aag 2188 Leu Glu lie Thr Asp Asp Gly Gly Asp Arg Ser Pro Leu Ala Pro Lys 345 350 355 ecc ggc ggc falls gga ate gtc ggc atg cgc gaa cgc gtc gcc ctg tac 2236 Pro Gly Gly His Gly lie Val Gly Met Arg Glu Arg Val Ala Leu Tyr 360 365 370 375 99c ggc ac ttc gcc gcc gga cg ggc ceg gag ggc ggc ttc gcg gta 2284 Gly Gly Thr Phe Wing Wing Gly Pro Arg Pro Glu Gly Gly Phe Wing Val 380 385 390 falls gcg tcc ctg ccg tac gag gag aac here tga cccggcccgc cgatccgccc2337 His Ala Ser Leu Pro Tyr Glu Glu Asn Thr 395 400 ggtgccccgg tccgggtcct catcgccgac gaccaggcgc tgctgcgcgg cagcctgcgg2397 gtgctcgtcg acaccgagcc cggcctggtg gccacgtcgg aggcggcgac cggcacggag2457 gcggtgcggc ttgcccggca ggatccgccg gacgtggtcc tgatggacgt gcggatgccc2517 gaaatggatg gcatcgaggc gacccggcag atctgcggtt cccccgagac cgcggacgtc2577 aaagtgctga tcctgacgat gttcgacctg gacgagtacg tctacgccgc gctgcgggcc2637 ggtgccagcg gcttcctgct gaaggacacg ccgcccagcg agttgctcgc ggcggtacgg2697 gtcatcgccg ccggcgaggc gctgctggca ccggccgtga cgcggcgcct gatcgcggag2757 ttcgtccacc gcccggagcc ctcgcgaccg ctgcgtcgca ccctggacgg cgtgaccgag2817 cgcgaacgtg aagtcctcac cctcatcgcc tgcggcctgt ccaacaccga gatcgccgag2877 cggctgtatc tcggcattgc caccgtgaag acccacgtca gccacctgct caccaagctc2937 gccacccgcg atcgcgctca gttggtgatc gtcgcgtacg agagcggcct ggtcacggtg2997 gcgcgaccac cgatcggttc ctgaggggcg ccggcgcaca cggtgcacgg cctgggcggg3057 gccgttcaga atggatcacc cgggtacacg aggcgcagtt cgtcgacatg gctcatgagg3117 tactcaccgg ggcactgggt ggatgccggg gcccgggact gcttcttgcg cggctggtgg3177 ccccagacgc tgctgatgcc gaagcggacg gccaggacgt ccacgaggac gtcgagtgtt3237 gtgagttget tgggcgtegg gtggtegtag cgtgcccact ggttctgcca gcgcggtccg3297 aagtcgccgg tgagcacgat gccgagattg ccggcgttga agagtteage gtgtgagccc3357 tegatgeega gtggccgccc ctcgtagatc gtcccggcgc cgtcgatgat gtagtggtaa3417 ccgatgtcgg ccttgtcgtc cgcgaagtgc gcccgctgga tcgtgcgcgg gccctcatgc3477 gtgtacgtga cggggtcggc cgagtggtgg atggtgatcc agcggtagac ggaggccagg3537 ggccggttct cgctgagcgg tacgggactg ccgcggtagg gcggtggcgc gagggggccg3597 gaggcggect cgtggaaatc ccaggtacgc ageggggggt cgatctgcgg cggggccgcc3657 ccccaggtgg cgcggccgac gacggacacg gtcagcggcc ctcgcggtgt catggcccac3717 aactcgtagt cgccgctcg c cggatgcagg aagcgcgact cgtcccagca ggcggcgacg3777 gggccgtgcg cggtgtcgac cggccgggtg ccgcaggacg tcagcctcag gggagtccgg3837 tgcgggcgct gccccgagac cggcgcgttg aaccggccga tgtcggtgat cacggtggtg3897 cggagttccg acaggtcgta gccgtcgcgg gggcattcga gggagcgcgg cggcggttcc3957 acgaccctga gcgccgcatc gcaccggggg cagacgagaa cgagcacctc gcgggcgacc4017 agctccgtcg tcgtaccggg cgggagccgg tggtggcggg gcagatcgag tggcgtgcgg4077 ccgggccgca gttcggtcac gggcacgggg tcggtggctt cggcggcggg tgccagctcg4137 tggtcggcgc aggcgaccgt ccaggcacgc gtcccggcgt cgggaaccat gagggtgccc4197 agcgcgtccg tcgtggccgc gatcccggaa tgccgtcctc ccgatggcgg gatgagccgt4257 acggtgaatc cggggatcgg gctgccgtcg cggcgcagga tcaccagggc cgtgtccgac4317 ggtggtgaga gttcggcggc cagccccgcc tcgacgaagt gcagcaagcg gtgtgtcagt4377 tgcagtacct cgggagagtc cggcgcgagc atggcctcgg cacggctgcg cacgctctcg4437 aacgcgccgc cgagtgcgaa gcgcaggaag tcgacggcga acgcgacgat ctccccggcg4497 acgaagccga ccgcggcgtc ggcgaagcga ggcgggccga agccaggtgc cagggggagc4557 gccggcgctc cggcactggt cctggtggcg gcgacga acg cggtgeaaeg ccggtccacg4617 gcgccgtcgt agtactcacg cagctgcgcc gccagcgagc ggtgcgggtc gaaggactcg4677 ccgaggttca ccccgtcgat gtcgcccagc agccgcggcg tcgaagcgtg gcgggcgacc4737 cagtggtcca gcgaccgacc gcggtccgcg gccggcaccc cgggcgcgtg gcgggcgcgg4797 acgtacgcgg cgagggcgcg cccgaggtca ccgctccagg tgagggcgag atccgctcga4857 ggggccgggt ccagggggcc gggcgtctgc cggtcggccc cgtcgatgcc ggccagcacc4917 tgcgccaggt cgagccgctc gaagccgtgc tgcacccgca gcagcgcggc cagccgggcg4977 gcccggcggg gcagctccca ggacgagccc ggcgtctggt cgtacggggg gatgttccgc5037 cggttctg 5045 < 210 > 2 < 211 > 401 < 212 > PRT < 213 > Streptomyces avermitilis < 400 > 2 Val His Wing Gly Thr Wing Val Asp Pro Asp Asp His Pro lie Leu Wing 1 5 10 15 Arg Arg Leu Ser Arg Arg Arg Leu lie Wing Leu Asp Gly Val Leu Val 20 25 30 Phe Ala Tyr Ala Cys Ala Leu Leu Ser Thr Gly Pro Thr Gly lie Ser 35 40 45 Being Ser Wing Pro Pro Leu Pro Wing Pro Val Pro Tr Glu Arg Leu 50 55 60 Val Leu lie Ala Ala Ala Thr Ala Pro Val Ala Ala Ar Arg Arg He Trp 65 70 75 80 Pro Leu Pro Val Phe Ala Val Val Leu Ala Val Thr Ala Val Ala Ala 85 90 95 Val Arg Asp Ala Ala Trp Asp Pro Phe Leu Ser Ala Ala Phe Ala Leu 100 105 110 Tyr Thr Val Wing Val Thr Val Pro Ser Arg His Trp Trp Gln Arg Trp 115 120 125 Leu Pro Gly Leu Wing He Wing Leu Leu Thr Val Wing Gly Leu Wing Gly 130 135 140 Ala Ala Arg Ala Gly Glu Ala Phe Trp Trp Arg Gly Ser Pro Gly Leu 145 150 155 160 Leu Leu Leu Gly Phe Ala Ala Leu Leu Gly Ala Trp Gln Leu Gly Arg 165 170 175 Ala Ala Arg Gln Arg Arg Ala Phe Ala Val Arg Ala Ala Gllu Gln Leu 180 185 190 Ala Gln Arg Ala Val Thr Glu Glu Arg Leu-Arg He Ala Arg Glu Leu 195 200 205 His Asp Val Val Thr His Ser Met Gly Leu He Wing Val Lys Val Gly 210 215 220 Val Ala Asn His Val Leu His He Arg Pro Gln Glu Ala Tyr Asp Ala 225 230 235 240 Leu Gln Val lie Glu Arg Thr Ser Arg Thr Ala Leu Asn Asp Met Arg 245 250 255 Arg Met Leu Gly Val Leu Arg Thr Ser Glu Gly Glu Arg Gln Ser Wing 260 265 270 Wing Leu Gly Pro Leu Pro Gly Wing Leu Wing Leu Pro Asp Leu Val Gly 275 280 285 Gln Ala Gly Ala Gln Leu Thr Met Arg Gly Val Glu Ser Leu Pro Asp 290 295 300 Gly Val Ala Leu Ala Val Tyr Arg He Val Gln Glu Ala Leu Thr Asn 305 310 315 320 Val Ala Lys His Ala Gly Pro Glu Ala Arg Cys Arg Val Ala Ala Asp 325 330 335 Wing Asn Gly His Gly Val Arg Leu Glu He Thr Asp Asp Gly Gly Asp 340 345 350 Arg Ser Pro Leu Pro Pro Lys Pro Gly Gly His Gly He Val Gly Met 355 360 365 Arg Glu Arg Val Wing Leu Tyr Gly Gly Thr Phe Wing Wing Gly Pro Arg 370 375 380 Pro Glu Gly Gly Phe Ala Val His Ala Ser Leu Pro Tyr Glu Glu Asn 385 390 395 400 Thr < 210 > 3 < 21 1 > 5045 < 212 > DNA < 213 > Streptomyces avermitilis < 220 > < 221 > CDS < 222 > (2314) .. (3021) < 223 > aveR2 ORF < 400 > 3 cgagttgctg gtcatcggcg tactcctccc ggcgactccg cccggtactc gaccgcggca 60 gcggtcagcc gcatgaacgc ctcttcgaga gacacggtct tccgcgtcac ctcgtgcacc 120 gtcacggcgt gcgccgcgac gagttcgccg atccgctccg cggcggcggc caccttcagg 180 ctgccgtcgg agcagtcggt gaccgtgatt cccgcgccga ccagcacgtc gcgcagccgc 240 cgcggttccg gagtgcgcac ccgcaccccc acgtccgtgt acctgtcgat gaactcgctc 300 atgctggtgt ccgcgaggag ccgaccgcgg ccgatgatga cgaggtggtc cacggtgagc 360 gccgcctcgc tcatcagatg gctggacacg aagacggtgc gtccctgtgc cgccaggtcc 420 cgcatgaggt gccgcagcca caggacgcct tccgggtcga gcccgttgac cggctcgtcg 480 agcaccagga cggcggggtc gccgagcagc gccgcggcga ttcccagccg ctgactcatg 540 cccagcgaga acgtccccgc ccgccggcgc acggcgctcc gcaggcccgc cagctcgatc 600 acctcacgga cgcggcgggg cgggatccgg ttgctgcggg ccagccagcg caagtggttc 660 agcgcggttc ggccggggtg caccgccctg gcgtcgagca gtgcccccac cgtccgcagc 720 gggtcgcgga gccgctggta gggcgcgccg tcgatgcgta cctcgccggc cgtcgggcgg 780 tccaggccca gcatcatgcg catcgcggtg gacttcccgg cgccgttggg gccaaggaat 840 ccggtcaccc gaccggtccg tacctggaag gtaagaccct ccacaacggt ggtggtcccg 900 tagcgcttgg tcaggtccgt gactt? GATC tggtccgtga atgccggtga cgacaggctc 960 ccgccgcgtc ccgctcgggg ctgactgccc cttctccacc cccggttgga gaatgaccgc 020 cacccgcggc cgcgcatcag gctgcaggag gagcggcttt gaccaccgct ggacggaggc 080 ggagcggcgt acgcctggat atggtcgagc ggtgcatgca ggtaccgcgg tggaccccga 140 cgaccatccg atcctggccc ggcgactgag ccggcgccga ctcatcgccc tggacggcgt 200 gctcgtattc gcctacgcat gcgcgctgct gtcgaccggg ccgacaggca tctcgtcgtc 260 gtccgcgccg ccgctcccgg ccccggtgcc gtgggagcgg ctcgtgctca tcgccgcggc 320 cactgcgcct gtcgccgtac ggcggatctg gccgttgccc gtgttcgcgg tcgtgctggc 380 ggtgaccgcc gtggccgtcg tgcgggacgc ggcgtgggac ccgttcctgt cggcggcgtt 440 cgccctctac accgtcgccg tcacggtgcc ctcgcgccac tggtggcaac gctggttacc 500 cggcctggcg atcgctttgc tgaccgtggc cggccttgcc ggagcagcgc gtgccggcga 560 ggccttctgg tggcgcggca gccccggtct gctgctgctc ggcttcgccg cactgctcgg 620 cgcctggcaa ctgggacgcg ccgcgcggca gaggcgcgca ttcgccgtcc gggcggccga 680 gcagctcgca caacgggccg tcacggagga acgcctgcgg atagcccgcg aactgcatga 740 cgtcgtcacg cacagcatgg gcctgatcgc ggtcaaggtc ggcgtcgcca accacgtgtt 800 gcacatcagg ccgcaggagg cgtacgacgc gctccaggtc atcgaacgca cgagccgcac 860 cgcgctgaac gacatgcgcc ggatgctcgg tgtgctgcgt acgtccgagg gtgagcggca 920 gtcagcggct ctcggcccgc tgcctggcgc ccttgctctc cctgacctcg tcgggcaggc 980 cggcgcgcag ctgactatgc gcggtgtcga gagtctgccc gacggagtcg cgctggccgt 040 ctaccggatc gtgcaggagg cgctcaccaa tgtcgccaag cacgccggcc cggaggcscg 100 ctgccgggtg gcggtcgatg cgaacggcca cggcgtccgg ctcgagataa ccgacgacgg 160 aggcgaccgg agccccctcg cgccgaagcc cggcggccac ggaatcgtcg gcatgcgcga 220 acgcgtcgcc ctgtacggcg gcaccttcgc cgccggaccg cgtccagagg gcggcttcgc 280 ggtacacgcg tccctgccgt acgaggagaa falls atg acc cgg ecc gcc gat ccg 334 Met Thr Arg Pro Wing Asp Pro 1 5 ecc ggt gcc ccg gtc cgg gtc etc gcc gc gac gac cag gcg ctg ctg 2382 Pro Gly Ala Pro Val Arg Val Leu He Wing Asp Asp Gln Ala Leu Leu 10 15 20 cgc ggc age ctg cgg gtg etc gtc gac acc gag ecc ggc ctg gtg gcc 430 Arg Gly Ser Leu Arg Val Leu Val Asp Thr Glu Pro Gly Leu Val Wing 25 30 35 acg tcg gag gcg gcg acc ggc acg gag gcg gtg cgg ctt gcc cgg cag 478 Thr Ser Glu Wing Wing Thr Gly Thr Glu Wing Val Arg Leu Wing Arg Gln 40 45 50 55 gat ccg ccg gac gtg gtc ctg atg gac gtg cgg atg ecc gaa atg gat 2526 Asp Pro Pro Asp Val Val Leu Met Asp Val Arg Met Pro Glu Met Asp 60 65 70 ggc ate gag gcg acc cgg cag ate tgc ggt tcc ecc gag acc gcg gac 2574 Gly He Glu Wing Thr Arg Gln He Cys Gly Ser Pro Glu Thr Wing Asp 75 80 85 gtc aaa gtg ctg ate ctg acg atg ttc gac ctg gac gag tac gtc tac 2622 Val Lys Val Leu He Leu Thr Met Phe Asp Leu Asp Glu Tyr Val Tyr 90 95 100 9CC gc9 ctg cgg cgg ggg ggt gcc gcc gcc ctg cg aag gcg acg cgc 2670 Ala Ala Leu Arg Ala Gly Ala Be Gly Phe Leu Leu Lys Asp Thr Pro 105 110 115 ecc age gag ttg etc gcg gcg gta cg gc gc gcc gcc gcc gag gcg 2718 Pro Ser Glu Leu Leu Ala Ala Val Arg Val He Ala Ala Ala Gly Glu Ala 120 125 130 135 ctg ctg gca ccg gcc gtg acg cgg cgc ctg ate gcg gag ttc gtc falls 2766 Leu Leu Ala Pro Ala Val Thr Arg Arg Leu He Ala Glu Phe Val His 140 145 150 cgc ccg gag ecc tcg cga ccg ctg cgt cgc acc ctg gac ggc gtg acc 2814 Arg Pro Glu Pro Ser Arg Pro Leu Arg Arg Thr Leu Asp Gly Val Thr 155 160 165 gag cgc gaa cgt gaa gtc etc acc etc gcc tgc ggc ctg tcc aac aac 2862 Glu Arg Glu Arg Glu Val Leu Thr Leu He Wing Cys Gly Leu Ser Asn 170 175 180 acc gag ate gcc gag cgg ctg tat etc ggc att gcc acc gtg aag acc 2910 Thr Glu He Wing Glu Arg Leu Tyr Leu Gly He Wing Thr Val Lys Thr 185 190 195 falls gtc age falls ctg etc acc aag etc gcc acc cgc gat cgc gct cag 2958 His Val Ser His Leu Leu Thr Lys Leu Wing Thr Arg Asp Arg Wing Gln 200 205 210 215 ttg gtg ate gtc gcg gcg tac gag age ggc ctg gtc acg gtg gcg cga cea 3006 Leu Val He Val Wing Ala Tyr Glu Ser Gly Leu Val Thr Val Wing Arg Pro 220 225 230 ccg ate ggt tcc tga ggggcgccgg cgcacacggt gcacggcctg ggcggggccg 3061 Pro He Gly Ser 235 • ttcagaatgg atcacccggg tacacgagge gcagttcgtc gacatggctc atgaggtact3121 caccggggca ctgggtggat gccggggccc gggactgctt cttgcgcggc tggtggcccc3181 agaegetget gatgccgaag cggacggcca ggacgtccac gaggacgtcg agtgttgtga3241 gttgcttggg cgtcgggtgg tcgtagcgtg cccactggtt ctgccagcgc ggtccgaagt3301 cgccggtgag cacgatgccg agattgccgg cgttgaagag ttcagcgtgt gagccctcga3361 tgccgagtgg ccgcccctcg tagatcgtcc cggcgccgtc gatgatgtag tggtaaccga3421 tgtcggcctt gtcgtccgcg aagtgcgccc gctggatcgt gcgcgggccc tcatgcgtgt3481 acgtgacggg gtcggccgag tggtggatgg tgatccagcg gtagaeggag gccaggggcc3541 ggttctcgct gagcggtacg ggactgccgc ggtagggcgg tggcgcgagg gggccggagg3601 cggcctcgtg gaaatcccag gtacgcagcg gggggtcgat ctgcggcggg gccgcccccc3661 aggtggegeg gccgacgacg gacacggtca gcggccctcg cggtgtcatg gcccacaact3721 cgtagtcgcc gctcgccgga tgcaggaagc gcgactcgtc ccagcaggcg gcgacggggc3781 cgtgcgcggt gtcgaccggc cgggtgccgc aggacgtcag cctcagggga gtccggtgcg3841 ggcgctgccc cgagaccggc gcgttgaacc ggccgatgtc ggtgatcacg gtggtgcgga3901 gttccgacag gtcgtagccg tcgcgggggc attcgaggga gcgcggcggc ggttccacga3961 ccctgagcgc cgcatcgcac cgggggcaga cgagaacgag cacctcgcgg gcgaccagct4021 ccgtcgtcgt accgggcggg agccggtggt ggcggggcag atcgagtggc gtgcggccgg4081 gccgcagttc ggtcacgggc acggggtcgg tggcttcggc ggcgggtgcc agctcgtggt4141 cggcgcaggc gaccgtccag gcacgcgtcc cggcgtcggg aaccatgagg gtgcccagcg4201 cgtccgtcgt ggccgcgatc ccggaatgcc gtcctcccga tggcgggatg agccgtacgg4261 tgaatccggg gatcgggctg ccgtcgcggc gcaggatcac cagggccgtg tccgacggtg4321 gtgagagtte ggcggccagc cccgcctcga cgaagtgcag caagcggtgt gtcagttgca4381 gtacctcggg agagtccggc gegageatgg cctcggcacg gctgcgcacg ctctcgaacg4441 cgccgccgag tgcgaagcgc aggaagtcga cggcgaacgc gacgatctcc ccggcgacga4501 agccgaccgc ggcgtcggcg aagcgaggcg ggccgaagcc aggtgccagg gggagcgccg4561 gcgctccggc actggtcctg gtggcggcga cgaacgcggt gcaacgccgg tccacggcgc4621 cgtcgtagta ctcacgcagc tgcgccgcca gcgagcggtg cgggtcgaag gactcgccga4681 ggttcacccc gtcgatgtcg cccagcagcc gcggcgtcga agcgtggcgg gcgacccagt4741 ggtccagcga ccgaccgcgg tccgcggccg gcaccccggg cgcgtggcgg gcgcggacgt4801 acgcggcgag ggcgcgcccg aggtcaccgc tccaggtgag ggcgagatcc gctcgagggg4861 ccgggtccag ggggccgggc gtctgccggt cggccccgtc gatgccggcc agcacctgcg4921 ccaggtcgag ccgctcgaag ccgtgctgca cccgcagcag cgcggccagc cgggcggccc4981 ggcggggcag ctcccaggac gagcccggcg tctggtcgta cggggggatg ttccgccggt5041 tctg 5045 < 210 > 4 < 211 > 235 < 212 > PRT < 213 > Streptomyces avermitilis < 400 > 4 Met Thr Arg Pro Wing Asp Pro Pro Gly Wing Pro Val Arg Val Leu He 10 15 Wing Asp Asp Gln Wing Leu Leu Arg Gly Ser Leu Arg Val Leu Val Asp 20 25 30 Thr Glu Pro Gly Leu Val Wing Thr Ser Glu Wing Wing Thr Gly Thr Glu 35 40 45 Wing Val Arg Leu Wing Arg Gln Asp Pro Pro Asp Val Val Leu Met Asp 50 55 60 Val Arg Met Pro Glu Met Asp Gly He Glu Wing Thr Arg Gln He Cys 65 70 75 80 Gly Ser Pro Glu Thr Wing Asp Val Lys Val Leu He Leu Thr Met Phe 85 90 95 Asp Leu Asp Glu Tyr Val Tyr Ala Ala Leu Arg Ala Gly Ala Ser Gly 100 105 110 Phe Leu Leu Lys Asp Thr Pro Pro Ser Glu Leu Leu Ala Wing Val Arg 115 120 125 Val He Wing Wing Gly Glu Wing Leu Leu Wing Pro Wing Val Thr Arg Arg 130 135 140 Leu He Wing Glu Phe Val His Arg Pro Glu Pro Being Arg Pro Leu Arg 145 150 155 160 Arg Thr Leu Asp Gly Val Thr Glu Arg Glu Arg Glu Val Leu Thr Leu 165 170 175 He Wing Cys Gly Leu Being Asn Thr Glu He Wing Glu Arg Leu Tyr Leu 180 185 190 Gly He Ala Thr Val Lys Thr His Val Ser His Leu Leu Thr Lys Leu 195 200 205 Ala Thr Arg Asp Arg Ala Gln Leu Val He Val Ala Tyr Glu Ser Gly 210 215 220 Leu Val Thr Val Ala Arg Pro Pro He Gly Ser 225 230 235 < 210 > 5 < 21 1 > 394 < 212 > PRT - < 213 > Streptomyces coelicolor < 400 > 5 Met His Arg Trp Gln Wing Val Arg Arg Arg He Glu Ser Leu Val Arg 1 5 10 15 Val Leu Gly Ser Glu Arg Pro Phe Thr Arg Arg Wing Asp Leu Val Leu 20 25 30 Leu Leu Val Leu Leu Val Pro Be Wing Phe Wing Thr Gly Thr Leu Glu 35 40 45 Thr Ala Pro Val Wing Trp Leu Thr Wing Cys Leu Leu He Wing Wing Wing 50 55 60 Val Val Val Gln Arg Thr Ala Pro Leu Leu Ser Leu Leu Leu Ala Ala 65 70 75 80 Leu Leu Thr Leu Phe Tyr Pro Trp Phe Gly Wing Asn Leu Trp Pro Ser 85 90 95 Met Ala Thr Val Val Leu Ser Cys Leu Ala Gly Arg Arg Leu Thr Arg 100. 105 110 Leu Trp Pro Wing His Leu Val Phe Leu Cys Val Wing Wing Wing Gly Leu 115 120 125 Leu Leu Val Ala Thr Val Gly Gln Gly Lys Asp Trp Leu Ser Leu Leu 130 135 140 Met Thr Glu Phe Val Wing Cys Val Leu Pro Trp Trp Wing Gly Asn Trp 145 150 155 160 Trp Ser Gln Arg Thr Ala Leu Thr His Wing Gly Trp Glu His Wing Glu 165 170 175 Gln Leu Glu Trp Arg Gln Arg Tyr He Wing Asp Gln Wing Arg Met Lys 180 185 190 Glu Arg Ala Arg He Ala Gln Asp He His Asp Ser Leu Gly His Glu 195 200 205 Leu Ser Val Met Ala Leu Leu Ala Gly Gly Leu Glu Leu Ala Pro Gly 210 215 220 Leu Ser Asp Pro His Arg Glu Ser Val Gly Gln Leu Arg Glu Arg Cys 225 230 235 240 Thr Met Ala Thr Glu Arg Leu His Glu Val He Gly Leu Leu Arg Glu 245 250 255 Asp Pro Asn Pro Ser Leu Thr Pro Wing Asp Glu Ser Val Wing Gln Leu 260 265 270 Val Arg Arg Phe Gln Arg Ser Gly Thr Pro Val Arg Phe Gln Glu Asp 275 280 285 Gly Ala Arg Asp Arg Pro Gly Thr Pro Leu Leu Ser Asp Leu Ala Ala 290 295 300 Tyr Arg Val Val Gln Glu Ala Leu Thr Asn Ala Ala Lys His Ala Pro 305 310 315 320 Gly Ala Pro He Asp Val Arg Val Thr His Thr Ala Asp Glu Thr Val 325 330 335 Val Ser Val Val Asn Glu Arg Pro Glu Arg Gly Gly Ser Val Pro Wing 340 345 350 Wing Gly Ser Gly Ser Gly Leu He Gly Leu Asp Glu Arg Val Arg Leu 355 360 365 Wing Gly Gly Thr Leu Arg Thr Gly Pro Arg Wing Gly Gly Phe Glu Val 370 375 380 Tyr Ala Arg Leu Pro Arg Gly Ala Ser Ser 385 390 < 210 > 6 < 211 > 222 < 212 > PRT < 213 > Streptomyces coelicolor < 400 > 6 Met He Arg Val Leu Leu Wing Asp Asp Glu Thr He He Arg Wing Gly 10 15 Val Arg Ser He Leu Thr Thr Glu Pro Gly He Glu Val Val Ala Glu 20 25 30 Wing Ser Asp Gly Arg Glu Wing Val Glu Leu Wing Arg Lys His Arg Pro 35 40 45 Asp Val Ala Leu Leu Asp He Arg Met Pro Glu Met Asp Gly Leu Thr 50 55 60 Wing Wing Gly Glu Met Arg Thr Thr Asn Pro Asp Thr Wing Val Val Val 65 70 75 80 Leu Thr Thr Phe Gly Glu Asp Arg Tyr He Glu Arg Ala Leu Asp Gln 85 90 95 Gly Val Ala Gly Phe Leu Leu Lys Ala Ser Asp Pro Arg Asp Leu He 100 105 110 Ser Gly Val Arg Ala Val Ala Ser Gly Gly Ser Cys Leu Ser Pro Leu 115 120 125 Val Ala Arg Arg Leu Met Thr Glu Leu Arg Arg Ala Pro Ser Pro Arg 130 135 140 Ser Glu Val Ser Gly Glu Arg Thr Thr Leu Leu Thr Lys Arg Glu Gln 145 150 155 160 Glu Val Leu Gly Met Leu Gly Wing Gly Leu Ser Asn Wing Glu He Wing 165 170 175 Gln Arg Leu His Leu Val Glu Gly Thr He Lys Thr Tyr Val Ser Wing 180 185 190 He Phe Thr Gln Leu Glu Val Arg Asn Arg Val Gln Ala Wing He He 195 200 205 Wing Tyr Glu Wing Gly Leu Val Lys Asp Wing Asp Leu Asn Arg 210 215 220

Claims (1)

NOVELTY OF THE INVENTION CLAIMS

1- An isolated polynucleotide molecule, comprising a nucleotide sequence encoding a product of the aveR1 gene from S. avermitilis. 2. The isolated polynucleotide molecule of claim 1, wherein the aveRI gene product comprises the amino acid sequence of SEQ ID NO. 2. The isolated polynucleotide molecule of claim 2, comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 1.112 to about nt 2.317. 4. The isolated polynucleotide molecule of claim 3, comprising the nucleotide sequence of SEQ ID NO: 1. 5. An isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 1112 to about nt 2.317. 6. The isolated polynucleotide molecule of claim 5, which hybridizes under moderately stringent conditions to the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 , and that is useful to practice the invention. 7. - The isolated polynucleotide molecule of claim 6, which hybridizes under highly stringent conditions with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. The isolated polynucleotide molecule of claim 7, which hybridizes under highly stringent conditions with the complement of a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 1112 to about nt 2.317. 9. An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 2. 10. An isolated polynucleotide molecule, consisting of in a nucleotide sequence that is a substantial portion of a polynucleotide molecule of claims 1, 2, 3, 4, 5, 6, 7, 8, or 9. 11- An isolated polynucleotide molecule, comprising a sequence nucleotide, comprising a nucleotide sequence that naturally flanks the ORF of aveR1 of S. avermitilis in situ, selected from the nucleotide sequence of SEQ ID NO: 1 from about nt 1 to about nt 1111 and from about nt 2.318 to about nt 5.045, or a nucleotide sequence that is homologous to said flanking sequence. 12. An isolated polynucleotide molecule, comprising a nucleotide sequence that encodes a product of the aveR2 gene from S. avermitilis. 13. The isolated polynucleotide molecule of claim 12, wherein the aveR2 gene product comprises the amino acid sequence of SEQ ID NO: 4. 14. The isolated polynucleotide molecule of claim 13, comprising the sequence of nucleotides of SEQ ID NO: 3 from about nt 2.314 to about nt 3.021. 15. The isolated polynucleotide molecule of claim 14, comprising the nucleotide sequence of SEQ ID NO: 3. 16.- An isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of SEQ. ID NO: 3 from about nt 2.314 to about nt 3.021. 17. The isolated polynucleotide molecule of claim 16, which hybridizes under moderately stringent conditions to the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 , and it is useful to practice the invention. 18. The isolated polynucleotide molecule of claim 17, which hybridizes under highly stringent conditions to the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ. ID NO: 4. 19. The isolated polynucleotide molecule of claim 18, which hybridizes under highly stringent conditions with the complement of a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 from about nt 2.314 until approximately nt 3.021. 20. An isolated polynucleotide molecule, comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 4. 21. An isolated polynucleotide molecule, which consists of a nucleotide sequence that is a substantial portion of a polynucleotide molecule of claims 12, 13, 14, 15, 16, 17, 18, 19 or 20. 22. An isolated polynucleotide molecule, comprising a sequence of nucleotides that naturally flank the ORF of bird R.sub.2 of S. avermitilis in situ, selected from the nucleotide sequence of SEQ ID NO: 3 from about nt 1 to about nt 2.313 and from about nt 3.022 to about nt 5.045, or a nucleotide sequence that is homologous with said flanking sequence. 23. - An isolated polynucleotide molecule, comprising a nucleotide sequence that encodes products of the aveR1 and aveR2 genes from S. avermitilis. 24. The isolated polynucleotide molecule of claim 23, wherein the aveRI gene product comprises the amino acid sequence of SEQ ID NO: 2, and the aveR2 gene product comprises the amino acid sequence of SEQ ID NO: 4 25. The isolated polynucleotide molecule of claim 24, comprising the nucleotide sequence of the ORF and aveR1 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 2.317, and ORF of aveR2 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021. 26. The isolated polynucleotide molecule of claim 25, comprising the nucleotide sequence as shown in SEQ ID NO: 1 from about nt 1.112 to about nt 3.021. 27. The isolated polynucleotide molecule of claim 26, comprising the nucleotide sequence of SEQ ID NO: 1. 28.- An isolated polynucleotide molecule that is homologous with a polynucleotide molecule comprising the nucleotide sequence of the polynucleotide. ORF of aveRI of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and the ORF of aveR2 of S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 until approximately nt 3.021. 29. The isolated polynucleotide molecule of claim 28, which hybridizes under moderately stringent conditions to the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 4, and which is useful for practicing the invention. 30. The isolated polynucleotide molecule of claim 29, which hybridizes under highly stringent conditions with the complement of a polynucleotide molecule comprising a nucleotide sequence encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 4. 31.- The isolated polynucleotide molecule of claim 30, which hybridizes under highly stringent conditions with the complement of a polynucleotide molecule comprising the sequence nucleotides of the aveRI ORF of S. avermitilis as shown in SEQ ID NO: 1 from about nt 1112 to about nt 2.317, and the aveR2 ORF of S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to approximately nt 3.021. 32. - An isolated polynucleotide molecule, comprising a nucleotide sequence encoding a first polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 2 and a second polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 4. 33.- An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of a polynucleotide molecule of claims 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. 34.- An isolated polynucleotide molecule comprising a nucleotide sequence that naturally flanks the ORRs of aveR1 and aveR2 of S. avermitilis in situ, selected from the nucleotide sequence of SEQ ID NO: 1 from about nt 1 to approximately nt 1111, and from approximately nt 3.022 to approximately nt 5.045, or a nucleotide sequence that is homologous with said flanking sequence. 35.- An oligonucleotide molecule that hybridizes under highly stringent conditions with a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 1, or with a polynucleotide molecule consisting of a nucleotide sequence that is the complement of the nucleotide sequence of SEQ ID NO: 1. 36.- A recombinant vector, comprising: (a) a polynucleotide molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or (b) a polynucleotide molecule that is homologous with the polynucleotide molecule from (a); or (c) a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide having an amino acid sequence that is homologous with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or (d) a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the polynucleotide molecules from (a), (b) or (c). 37.- The recombinant vector of claim 36, wherein the polynucleotide molecule is in association with one or more regulatory elements. 38.- The recombinant vector of claim 36, further comprising a nucleotide sequence encoding a selectable marker or a reporter gene product. 39.- the recombinant vector of claim 36, comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 1.112 to about nt 2.317. 40.- the recombinant vector of claim 36, comprising the nucleotide sequence of SEQ I NO: 3 from about nt 2.314 to about nt 3.021. 41.- the recombinant vector of claim 36, comprising the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in DSEQ ID NO: 1 from about nt 1112 to about nt 2.317, and the aver2 ORF S. avermitilis as shown in SEQ ID NO: 1 from about nt 2.314 to about nt 3.021. 42.- The recombinant vector of claim 41, which is the plasmid pSE201 (ATCC 203182). 43.- A transformed host cell comprising the recombinant vector of claim 36. 44.- A substantially purified or isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a homologous polypeptide or peptide fragment thereof. 45.- A method of preparing a substantially purified or isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a homologous polypeptide or peptide fragment thereof, which comprises culturing the transformed host cell of claim 43, under conditions that lead to the expression of the polypeptide or peptide fragment, and recovering the polypeptide or peptide fragment expressed from the cell culture in a substantially purified or isolated form. 46. A polynucleotide molecule: (a) comprising a nucleotide sequence that encodes a product of the aveR1 gene or a product of the aveR2 gene from Streptomyces avermitilis; or (b) which is homologous to the polynucleotide molecule from (a); or (c) which consists of a sequence of nucleotides that a substantial portion of the polynucleotide molecule from (a) or (b); or (d) comprising a nucleotide sequence that naturally flanks the ORF of aveR1 or the ORF of aveR2 of S. avermitilis in situ; whose polynucleotide molecule further comprises at least one mutation that results in a detectable increase in the amount of avermectins produced by cells from a strain of S. avermitilis that are carriers of the mutation in the aveR1 gene or in the aveR2 gene , or in both genes aveR1 and aveR2, compared with cells of the same strain that are not carriers of the gene mutation. 47. The polynucleotide molecule of claim 46, comprising the full ORF of S. avermitilis aveR or a polynucleotide molecule homologous thereto, or a substantial portion thereof, or the complete ORF of aveR2 of S. avermitilis or a polynucleotide molecule homologous thereto, or a substantial portion thereof; or the complete ORF's of aveR1 and aveR2 of S. avermitilis or a polynucleotide molecule homologous thereto, or a substantial portion thereof; or a nucleotide sequence which naturally flanks the ORF of aveR1 of S. avermitilis in situ, or which naturally flanks the ORF of aveR2 of S. avermitilis in situ, or which naturally flanks both ORFs of aveR1 and aveR2 of S. avermitilis in situ; whose polynucleotide molecule is useful for mutating either the aveRI gene or the aveR2 gene or both the aveR1 and aveR2 genes of S. avermitilis, such that the amount of avermectins produced by cells of a strain of S. avermitilis that are carriers of said mutation in the aveR1 gene or in the aveR2 gene or in both aveR1 and aveR2 genes, it is detectably increased in comparison with cells of the same strain that are not carriers of the gene mutation. 48. An artificial genetic structure useful for introducing a mutation into either the aveR1 gene or the aveR2 gene or both aveR1 and aveR2 genes of S. avermitilis, which comprises the polynucleotide molecule of claim 46 or 47. 49. - The artificial genetic structure of claim 48, which can mutate the aveR1 gene or the aveR2 gene of S. avermitilis, by deleting a portion of the a gene? eR1 or the aveR2 gene, respectively, by a different or heterologous sequence of nucleotides. 50.- The artificial genetic structure of claim 49, which can mutate to both aveR1 and aveR2 genes of S. avermitilis, by deleting a portion of the bird genes R1 and aveR2, or by replacing a portion of the aveR1 and aveR2 genes with a sequence different or heterologous nucleotides. 51.- The artificial genetic structure of claim 48, which can be mutated to the aveR1 gene or to the aveR2 gene of S. avermitilis by introducing a different or heterologous sequence of nucleotides in the aveRI gene or in the aveR2 gene, respectively. 52.- A method to identify a mutation of a aveR gene? or of a aveR2 gene or both aveR1 and aveR2 genes, in a Streptomyces species or strain, whose gene mutation is capable of detectably increasing the amount of avermectins produced by cells of the Streptomyces species or strain that are carriers of the mutation of genes compared with cells of the same species or strain of Streptomyces that are not carriers of the gene mutation, comprising: (a) measuring the amount of avermectins produced by cells of a Streptomyces species or strain; (b) introducing a mutation in the aveR1 gene or in the aveR2 gene, or in both aveRI and aveR2 genes, of cells of the species or strain of Streptomyces from operation (a); and (c) comparing the amount of avermectins produced by the cells carrying the gene mutation as produced in step (b) with the amount of avermectins produced by the cells of operation (a) that are not carriers of the gene mutation; such that if the amount of avermectins produced by the cells carrying the gene mutation as occurred in step (b) is detectably greater than the amount of avermectins produced by the cells of operation (a) that are not carriers of the gene mutation, then a mutation of the aveR1 or aveR2 gene or of both aveR1 and aveR2 genes has been identified, capable of detectably increasing the amount of avermectins produced. 53. The method of claim 52, wherein the Streptomyces species is S. avermitilis. 54.- A method of preparing genetically modified cells from a Streptomyces species or strain, whose modified cells produce a detectably increased amount of avermectins, compared to unmodified cells of the Streptomyces species or strain, which comprises mutating the aveRI gene or the aveR2 gene or both aveR1 and aveR2 genes, in cells of the Streptomyces species or strain and select the mutated cells that produce a detectably increased amount of avermectins compared to cells of the same species or strain of Streptomyces that are not carriers of the mutation of genes. 55.- The method of claim 54 wherein the Streptomyces species is S. avermitilis. 56. The method of claim 54 wherein the mutation is carried out by deleting a portion of the aveRI gene or the aveR2 gene, or by replacing a portion of the aveR1 gene or the aveR2 gene with a different or heterologous sequence of nucleotides. The method of claim 54, wherein the mutation is carried out by deleting a portion of both aveR1 and aveR2 genes, or replacing a portion of both aveR1 and aveR2 genes with a different or heterologous sequence of nucleotides. 58.- the method of claim 54 wherein the mutation is carried out by introducing a different or heterologous sequence of nucleotides into the aveR ^ gene or into the aveR2 gene. 59.- A strain of Streptomyces, whose cells produce a detectably increased amount of avermectins as a result of one or more mutations in the aveR1 gene or in the aveR2 gene or in both aveR1 and aveR2 genes, compared with cells of the same species or strain from Streptomyces that are not carriers of the gene mutation. 60. - The strain of claim 59, whose species is S. avermitilis. 61.- A process for producing an increased amount of avermectins produced by Streptomyces cultures, which comprises culturing cells of a Streptomyces species or strain, whose cells comprise a mutation in the aveR1 gene or in the aveR2 gene, or in both aveRI genes and aveR2, whose gene mutation serves to detectably increase the amount of avermectins produced by cells of the species or strain of Streptomyces that are carriers of the gene mutation compared to cells of the same species or strains that are not carriers of the mutation of genes, in culture media and in conditions that allow or induce the production of avermectins from them, and recover the avermectins from the culture. 62.- The method of claim 61 wherein the Streptomyces species is S. avermitilis. 63. An antibody directed against a product of the aveR1 gene, a product of the aveR2 gene, a homologous polypeptide or a peptide fragment of the present invention.