EA003905B1 - Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins - Google Patents

Abstract

1. An isolated polynucleotide molecule comprising the complete aveC open-reading frame (ORF) of Streptomyces avermitilis or a substantial portion thereof, comprising at least about 70% of the complete aveC encoding functionally equevalent AveC gene product, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the Streptomyces avermitilis chromosome, and which ORF has the nucleotide sequence of FIGURE 1 (SEQ ID NO:1). 2. The isolated polynucleotide molecule of claim 1, further comprising nucleotide sequences that naturally flank the aveC gene in situ in S avermitilis. 3. An isolated polynucleotide molecule, comprising a nucleotide sequence that is homologous to the Streptomyces avarmitilis AveC gene product-encoding nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof, comprising at least about 70% of the complete aveC which encodes functionally equevalent AveC gene product. 4. An isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID ID:3 or a substantial portion thereof, or a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO:3. 5. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1),under highly stringent conditions. 6. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence of SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of SEQ ID NO:3, under highly stringent conditions. 7. The oligonucleotide molecule of claim 5, which is complementary to the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) 8. The oligonucleotide molecule of claim 5, which is complementary to the nucleotide sequence of SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence that is the complement of the nucleotide sequence of SEQ ID NO:3. 9. A recombinant vector comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof; and a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO: 1) or substantial portion thereof. 10. The recombinant vector of claim 9, further comprising a nucleotide sequence encoding one or more regulatory elements, which regulatory element encoding nucleotide sequence is in operative association with the nucleotide sequence of the polynucleotide molecule of the vector of claim 9. 11. The recombinant vector of claim 10, further comprising a nucleotide sequence encoding a selectable marker. 12. The recombinant vector of claim 11, which is plasmid pSE186 (ATCG 209604). 13. A host cell comprising the recombinant vector of claim 11. 14. The host cell of claim 13, which is Stratomyces avermitilis. 15. A recombinant vector comprising polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of the nucleotide Sequence of SEQ ID NO:3 or a substantial portion thereof; a nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO:3; and a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO:4. 16. The recombinant vector of claim 15, further comprising a nucleotide sequence encoding one or more regulatory elements, which regulatory element encoding nucleotide sequence is in operative association with the nucleotide sequence of the polynucleotide molecule of the vector of claim 15. 17. The recombinant vector of claim 16, further comprising a nucleotide sequence encoding a selectable marker. 18. A host cell comprising the recombinant vector of claim 17. 19. The host cell of claim 18, which is Streptomyces hygroscopicus. 20. An isolated S. avermitilis AveC gene product having the amino acid sequence encoded by the S. avermitilis AveC gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of SEQ ID NO:2 or substantial portion thereof, comprising at least about 70% of the complete aveC which is a functionally equevalent AveC gene product, in which optionally one or more amino acid residue is conservatively replaced with another amino acid residue, wherein such a replacement brings to a functionally equevalent gene product. 21. An isolated S. hygroscopicus AveC homolog gene product having the amino acid sequence of SEQ ID NO:4. 22. A method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector according to any one of claims 9 to 11 under conditions conducive to the production of the recombinant AveC gene product and recovering the AveC gene product from the cell culture. 23. A method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector according to any one of claims 15 to 17 under conditions conducive to the production of the recombinant AveC gene product and recovering the AveC gene product from the cell culture. 24. A polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ IS NO:1) or a degenerate variant thereof, but which nucleotide sequence further comprises one or more mutations, so that cells of S, avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a reduced cyclohexyl B2:cyclohexyl B1 ratio of avermectins when fermented in the presence of cyclohexanecarboxylic acid than is produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele. 25. The polynucleotide molecule of claim 24, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is less than 1.6:1. 26. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.94:1. 27. The polynucleotide molecule of claim 26, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid substitution at residue 139 of the AveC gene product from alanine to threonine. 28. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.88:1. 29. The polynucleotide molecule of claim 28, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes an amino acid substitution at residue 138 of the AveC gene product from serine to threonine. 30. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.84:1. 31. The polynucleotide molecule of claim 30, wherein the mutation to the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1) encodes a first amino acid substitution at residua 138 of the AveC gene product from serine to threonine, and a second amino and substitution at residue 139 of the AveC gene product from alanine to threonine. 32. A polynucleotide molecule comprising a strong promoter in operative association with the S. avermitilis aveC ORF of FIGURE 1 (SEQ ID NO:1). 33. The polynucleotide molecule of claim 32, wherein the strong promoter is the ermE promoter from Saccharopolyspora erythraea. 34. A polynucleotide molecule comprising a nucleotide sequence encoding the AveC gene product-encoding ORF of FIGURE 1 (SEQ ID NO:1) that has been inactivated by insertion into said nucleotide sequence of a heterologous nucleotide sequence. 35. A polynucleotide molecule comprising an aveC allele that has been inactivated by deleting a 640 by Psf/Sph1 fragment from the aveC ORF of FIGURE 1 (SEQ ID NO: 1). 36. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a frameshift mutation into the aveC ORF of FIGURE 1 (SEQ ID NO:1). 37. The polynucleotide molecule of claim 36, in which the frameshift mutation was introduced by adding two G nucleotides after the C nucleotide at position 471 of the aveC ORF of FIGURE 1 (SEQ ID NO:1). 38. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a termination codon at the nucleotide position that encodes amino acid 116 of the S. avermitilis AveC gene product encoded by the aveC ORF of FIGURE 1 (SEQ ID NO:1). 39. A polynucleotide molecule comprising an aveC allele that has been inactivated by introducing a first mutation at amino acid position 256 of the AveC gene product that changes a glycine to an aspartate, and a second mutation at position 275 of the AveC gene product that changes a tyrosine to a histidine. 40. A recombinant vector comprising the polynucleotide molecule of any of claims 24 to 39. 41. A recombinant vector comprising the polynucleotide molecule of claim 34, which vector is pSE180 (ATCC 209605). 42. A host Streptomyces cell comprising the recombinant vector of claim 40. 43. A strain of S. avermitilis comprising cells expressing a mutated aveC allele which results in the production by the cells of avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele. 44. The strain of claim 43, wherein the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio of less than 1.6:1. 45. The strain of claim 43, wherein the cells produce cyclohexyl B2:cyclohexyl B1 avermectins in a ratio

Description

The present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health. More specifically, the present invention relates to polynucleotide molecules containing nucleotide sequences encoding the AueC gene product, which can be used to modulate the ratio of avermectins of classes 2: 1, obtained by fermenting cultures of human tissue, as well as compositions and methods for screening such polynucleotide molecules. In addition, the present invention relates to vectors, transformed host cells, and new mutant strains of 8.Aunty1Sh, in which the AueC gene is mutated in such a way as to modulate the ratio of produced avermectins of classes 2: 1.

2. Background of the invention

2.1. Avermectins

Types of Herbalis produce a wide variety of secondary metabolites, including avermectins, which contain a series of eight related hexacid macrocyclic lactones with strong anthelmintic and insecticidal activity. These eight distinct but closely related compounds are called A1a, A1b, A2a, A2b, B2a, B2b, B2a, and B2b. The series of compounds “a” refers to natural avermectin, where the substituent in position C25 is (8) -butyl, and the series “b” refers to those compounds where the substituent in position C25 is isopropyl. The designations "A" and "B" refer to avermectins, where the substituent at position C5 is methoxy or hydroxy, respectively. The number “1” refers to avermectins, where a double bond is present at position C22,23, and the number “2” refers to avermectins having hydrogen at position C22 and hydroxy at position C23. Among the related avermectins, type B1 avermectin is recognized as having the most effective antiparasitic and pesticidal action, and as a result, it is the most commercially suitable avermectin.

Avermectins and their production by means of aerobic fermentation of the 8.Aurety1Sh strains are described in US Patents 4,310,519 and 4,429,042. The natural avermectins biosynthesis is believed to be initiated endogenously with the CoA-thioether analogs of isobutyric acid and (8) - (+) - 2 methyl butyric acid.

The combination of both the improvement of the strains through nonspecific mutagenesis and the use of exogenously added fatty acids led to the efficient production of analogues of avermectin. Mutants 8. auyuti1Sh, which are deficient in dehydrogenase 2-oxo acids with a branched chain (Lb-deficient mutants), can produce avermectins only when added to the fermentation of fatty acids. Screening and isolation of mutants deficient in branched chain dehydrogenase activity (for example, 8.AUETY1Sh ATCC 53567) are described in European Patent (EP) 276103. Fermentation of such mutants in the presence of exogenously added fatty acids leads to the production of only four avermectins corresponding to the fatty acid used. Thus, the addition during the fermentation of 8. auettic1 (ATCC 53567) (8) - (+) - 2 methyl butyric acid leads to the production of the natural avermectins A1 a, A2a, B2a and B2a; during the fermentation, the addition of isobutyric acid leads to the production of natural avermectins A1b, A2b, B1b and B2b; and the addition of cyclopentanecarboxylic acid during fermentation leads to the production of four new cyclopentyl avermectins A1, A2, B1 and B2.

When adding other fatty acids, new avermectins are produced. With the help of screening, more than 800 potential precursors have identified more than 60 other new avermectins (see, for example, EnCon et al., 1991, 1. LpNIoR 44: 357-365; and Wapch et al., 1994, Wow. 8os. Syesh 147: 16-26). In addition, the mutants 8. auetyySh, lacking in activity of 5-O-methyltransferase, produce, in essence, only B-similar avermectins. As a result, the mutants 8.out1Ιίΐίχ. lacking both the branched-chain 2-oxo-acid dehydrogenase activity and the 5-O-methyltransferase activity, only B-avermectins are produced, corresponding to the fatty acid, which is used for addition during fermentation. Thus, the addition of (8) - (+) - 2-methylbutyric acid to such double mutants leads to the production of only natural avermectins B1a and B2a, while the addition of isobutyric acid or cyclopentanecarboxylic acid leads to the production of natural avermectins B1b and B2b or new cyclopentylvermectins B1 and B2, respectively. Adding double mutants of cyclohexanecarboxylic acid to the strain is the preferred method of obtaining a commercially important new avermectin, cyclohexylavermectin B1 (doramectin). The isolation and characterization of such double mutants, for example 8. Chevron (ATCC 53692), are described in EP 276,103.

2.2. Genes involved in avermectin biosynthesis

It has been found that in many cases the genes involved in the production of secondary metabolites and the genes encoding a specific antibiotic are associated on the chromosome together. Such is, for example, the case of a polyketide synthase 81gerUtuseh (PK8) gene cluster (see Normiów apbéttap, 1990, App. Reu.

Sepek 24: 37-66). Thus, one way to clone genes in the biosynthetic pathway was to isolate the drug resistance gene, and then test the adjacent chromosome regions for other genes related to the biosynthesis of this particular antibiotic. Another way of cloning genes involved in the biosynthesis of important metabolites was complementation of mutants. For example, parts of a library of DNA fragments from an organism capable of producing a specific metabolite are introduced into a non-producing mutant, and transformants are screened for the production of this metabolite. In addition, for the identification and cloning of genes in biosynthetic pathways, library hybridization was used using probes of origin from other types of Gertusus 5.

It was found that the genes involved in the biosynthesis of avermectin (ayu genes), like the genes required for the biosynthesis of other secondary metabolites of Gertusus 5 (for example PK8), are associated on the chromosome. A number of AUE genes have been successfully cloned using vectors to complement the 3-auger mutants with blocked avermectin biosynthesis. The cloning of such genes is described in US Pat. No. 5,252,474. In addition, 1keba e! A1., 1995, 1. ΑηίίЫо1. 48: 532-534, describes the localization of the chromosome region that causes the dehydration stage C22,23 (aeC) for the WatH1 Z.AegtTbb fragment of 4.82 kb in length, as well as mutations in the ACC gene. which result in a single component producer B2a. Since ivermectin, a strong anthelmintic compound, can be chemically obtained from avermectin B2a, such a single-component producer of avermectin B2a is considered particularly useful for the industrial production of ivermectin.

Identification of mutations in the AueC gene that minimize the complexity of producing avermectin, such as, for example, mutations that reduce the B2: B1 ratio of avermectins, should simplify the production and purification of commercially important avermectins.

3. Summary of the Invention

The present invention proposes an isolated polynucleotide molecule containing a full-size open reading frame (ORF) of ACE 8. avayetch or its substantial part, and the following full-size ORF is missing in the isolated polynucleotide molecule, which is localized in the forward direction from the ORS aeC Шиη Shi to the chromosome 8.a . The isolated polynucleotide molecule of the present invention preferably contains a nucleotide sequence that is the same as the sequence of the plasmid p8E186 (ATCC 209604), which encodes the product of the AueC gene. 1 (8ЕО Ш Ν: 1), or its essential part.

The present invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the sequence of the plasmid p8E186 (ATCC 209604), which encodes the product of the AueC gene. 1 (8ЕО Ш NO: 1), or its essential part.

Further, the present invention provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence encoded by the plasmid p8E186 (ATCC 209604) sequence, or the amino acid sequence shown in FIG. 1 (8ЕО Ш NO: 2), or its essential part.

The present invention further provides an isolated polynucleotide molecule containing a nucleotide sequence encoding the product of the homolog of the AueC gene. In a preferred embodiment, the isolated polynucleotide molecule contains a nucleotide sequence encoding the product of the AueC gene homolog from Z. Goodtoxorki, wherein this gene homolog product contains the amino acid sequence 8ЕО Ш NO: 4 or a substantial part of it. In a preferred embodiment, the isolated polynucleotide molecule of the present invention, which encodes the product of the homologue of the AueC gene 8.Hyudgo5sor1ci8, contains the nucleotide sequence EO W NO: 3 or a substantial part of it.

Further, the present invention provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the nucleotide sequence of 8.11 F5 5core 5 8 EO W NO: 3. Further, the present invention provides a polynucleotide molecule containing a nucleotide sequence that encodes a polypeptide that is homologous to the product of the homologue of the AueC gene Z. iudtoxorku having the amino acid sequence 8ЕО Ш ^: 4.

The present invention further provides oligonucleotides that hybridize with a polynucleotide molecule having the nucleotide sequence shown in FIG. 1 (8ЕО Ш NO: 1) or 8ЕО Ш NO: 3, or with a polynucleotide molecule having a nucleotide sequence that represents the complement of the nucleotide sequence shown in FIG. 1 (EE GO ^: 1) or EE GO ^: 3.

Further, the present invention provides recombinant cloning vectors and expression vectors that are useful in cloning or expressing a polynucleotide of the present invention, including polynucleotide molecules containing the ORES S. e. In a non-limiting embodiment, the present invention provides the plasmid p8E186 (ATCC 209604), which contains the full-length ORF of the AueC gene Z.Augteysch. Further, in the present invention proposed transformed host cells containing a polynucleotide molecule or a recombinant vector according to the invention, as well as new strains or cell lines derived from them.

Further, in the present invention, a recombinantly expressed product of the AueC gene or the product of the homolog of the AueC gene, which is substantially purified or isolated, or a substantial portion thereof, as well as its homologues, is proposed. Further, the present invention provides a method for producing a recombinant AuEC gene product, in which a host cell transformed with a recombinant expression vector is cultured, said recombinant expression vector containing a polynucleotide molecule having a nucleotide sequence encoding the AueC gene product or the product of the AUeC gene homologue Auroa iso iso object. the molecule is in operative association with one or more regulatory elements that control the expression of this olinukleotidnoy molecule in the host cell, under conditions conducive for production of the recombinant gene product or product avec avec homolog gene, and isolating the gene product avec or homolog gene product from the cell culture avec.

The present invention further provides a polynucleotide molecule containing a nucleotide sequence that is otherwise the same as the sequence of the plasmid p8E186 (ATCC

209604), which encodes the product of the AueC gene of 8.Aegtey, or the nucleotide sequence of the ORF, AeSAAAAA, as shown in FIG. 1 (8EO GO N0: 1), but which additionally contains one or more mutations, so that cells of the strain Z.Aegtyysch ATCC 53692, in which the wild-type alleS allele is inactivated and which express a polynucleotide molecule containing the mutated nucleotide sequence, produce another the ratio or number of avermectins, which is produced by the cells of strain 8.AntiSSATCC 53692, which instead express only the wild type allele allele. According to the present invention, such polynucleotide molecules can be used to produce new 8.AE strains15, which exhibit a detectable change in the production of avermectins compared to the same strain, which instead expresses only the wild-type allele allele. In a preferred embodiment, such polynucleotide molecules are useful for producing new 8.AE strains15 that produce avermectins in a reduced ratio of 2: 1 classes compared to the ratio of the same strain, which instead expresses only the wild-type allele allele. In the following preferred embodiment, such polynucleotide molecules are useful for producing new Z.AugesPi strains that produce elevated levels of avermectins compared with the same strain that instead expresses only the wild type AeC allele. In yet another preferred embodiment, such polynucleotide molecules are useful for producing new 8.apps1111 strains. in which the AueS gene is inactivated.

In the present invention, methods are proposed for identifying ORS aeC Z. aegtychk mutations capable of changing the ratio and / or amount of avermectins produced. In a preferred embodiment, the present invention provides a method for identifying ORS aeC mutations capable of changing the ratio of 2: 1 classes of avermectins produced, in which: (a) the ratio of 2: 1 classes of avermectins produced by the cells of strain Z.AegteyPy in which the native AeC allele is determined the polynucleotide molecule containing the nucleotide sequence encoding the product of the mutated AueC gene is inactivated and in which the polynucleotide molecule is inserted and expressed; (b) determine the ratio of 2: 1 classes of avermectins produced by cells of the same Z.AegtyShk strain, as in stage (a), but which instead express only the AUC allele having the ORF nucleotide sequence shown in FIG. 1 (8EO GO NO: 1), or a nucleotide sequence that is homologous to this; and (c) compare the ratio of the 2: 1 classes of avermectins produced by the H. aegtyy cells of stage (a) with the ratio of the classes of 2: 1 avermectins produced by the cells of the 8. aaergetic stage (b); so, if the ratio of 2: 1 classes of avermectins produced by the H. aegtyy cells of stage (a) is different from the ratio of 2: 1 classes of avermectins produced by the cells of the z.auetteyyy stage (b), then the ORS aus mutation of mutations 2: 1 avermectins, identified. In a preferred embodiment, this mutation reduces the avermectin 2: 1 class ratio.

In the following preferred embodiment, the present invention proposes a method for identifying ORF aeC mutations or genetic constructs containing ORC aeC that can change the amount of avermectins produced, in which: inactivated and into which a polynucleotide molecule is introduced and expressed, containing a nucleotide sequence encoding a product of the mutated AueC gene, or containing a gene a tic construct comprising a nucleotide sequence encoding the product of the AueC gene; (b) Determine the amount of avermectins produced by cells of the same 8.AeGT strain, as in stage (a), but which instead express only the AluC allele having the ORF nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ N0: 1), or a nucleotide sequence that is homologous to this; and (c) compare the amount of avermectins produced by the H.Aegteis cells of stage (a) with the amount of avermectins produced by the cells of aaustectus stage3 (b); so, if the number of avermectins produced by the cells of H.Aegtes Stage (a) differs from the number of avermectins produced by the cells of H.Augerschase Stage (b), the ORS mutation or genetic construct capable of changing the number of avermectins is identified. In a preferred embodiment, this mutation increases the amount of avermectins produced.

Further, in the present invention, recombinant vectors are proposed that are useful in creating new 8.aegty1115 strains having altered avermectin production. For example, the present invention proposes vectors that can be used to deliver any of the polynucleotide molecules containing the mutated nucleotide sequences of the present invention to the AeC gene of the Z.AegtyS chromosome either for insertion into it, or to replace it or OC by means of homologous recombination. According to the present invention, however, a polynucleotide molecule containing the mutated nucleotide sequence of the present invention, proposed here, can also function to modulate the biosynthesis of avermectin when inserted into the C.Aegteschz chromosome at a site other than the Aus gene, or while maintaining an episomal state in cells 8. august Thus, the present invention also proposed vectors comprising a polynucleotide molecule containing the mutated nucleotide sequence of the present invention, and these vectors can be used to embed this polynucleotide molecule into a site on the chromosome 8. Acorta, other than the CayeC gene, or maintain episomal state. In a preferred embodiment, the present invention proposes gene replacement vectors that can be used to embed a mutated AluC allele into the Z.Aegtyus chromosome to form new cell strains that produce avermectins in a reduced ratio of 2: 1 classes compared to cells of the same strain, which instead Only the wild-type AeC allele express this.

Further, the present invention proposes methods for creating new 8.Aegteysh strains containing cells that express the mutated AeC allele and that produce altered ratios and / or amounts of avermectins compared to cells of the same 8.Aegty1113 strain, which instead express only the Allee Wild wild allele type In a preferred embodiment, the present invention proposes a method for creating new strains of C.Aegreths containing cells that express the mutated AeC allele and which produce an altered ratio of 2: 1 avermectins compared to cells of the same strain 8.AegtySHz, which instead express only the allele wild type Aureus, in which cells of the 8.Aegteysh strain are transformed with a vector that carries the mutated AueC allele encoding a gene product that changes the ratio of 2: 1 classes of avermectins, produced by cells of the 8.Aegteysh strain expressing this mutated allele compared to cells of the same strain, which instead express only the wild type Allele C allele, and select transformed cells that produce avermectins in a modified class ratio of 2: 1 compared to the class ratio 2: 1, produced by cells of the strain, which instead express the wild type AeC allele. In a preferred embodiment, the ratio of 2: 1 classes of avermectins produced is reduced in the cells of the novel strain.

In the following preferred embodiment, the present invention proposes a method for creating new strains of C.Aegteshz containing cells that produce altered amounts of avermectin, in which the cells of the strain of C.Aegteshz are transformed with a vector that carries a mutated AeC allele or a genetic construct that contains the Aeus allele whose expression leads to an altered amount of avermectins produced by cells of the 8.Aegteysh strain expressing this mutated AeC allele or genetic construct, along with compared to cells of the same strain, which instead express only the wild type Aureas allele, and select transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by the strain cells, which instead express only the wild type Allele C allele. In a preferred embodiment, the amount of avermectins produced is increased in the cells of the new strain.

In the following preferred embodiment, the present invention proposes a method for creating new strains of H. Swannieus, whose cells contain an inactivated aeC allele, in which cells of the Aiuyu strain 188, which express the wild type AeC allele, are transformed with a vector that inactivates the AeC allele and select transformed cells in which the allele is inactivated.

Further, in the present invention, new strains of Z.Auettyshk are proposed, containing cells that have been transformed with any of the polynucleotide molecules or vectors containing the mutated nucleotide sequence of the present invention. In a preferred embodiment, the present invention proposes new strains of 8.Auetstey containing cells that express a mutated AluC allele instead of a wild-type AluC allele or in addition to a wild-type AuleC allele, and the cells of a new strain produce avermectins in a modified ratio of 2: 1 classes compared to with cells of the same strain, which instead express only the wild-type AeC allele. In a more preferred embodiment, the cells of the new strain produce avermectins in a reduced ratio of 2: 1 classes compared to cells of the same strain, which instead express only the wild type allele allele. Such new strains are useful in large-scale production of commercially suitable avermectins, such as doramectin.

In a further preferred embodiment, the present invention proposes new strains of Z.AuetteyIsch containing cells that express the mutated aeuC allele or a genetic construct containing the aeuC allele instead of the wild-type aeC allele or in addition to the wild-type aeus allele, which leads to the production of these cells the change in the amount of avermectins compared with the amount of avermectins produced by cells of the same strain, which instead express only the wild type allele allele. In a preferred embodiment, the new cells produce an increased amount of avermectins.

In the following preferred embodiment, the present invention proposes new strains of Z.AuettiSchS containing cells in which the AueC gene is inactivated. Such strains are useful for both the different spectrum of avermectins that they produce compared to the wild-type strain and for complementation screening assays, as described here, to determine whether directed or non-specific mutagenesis of the AeU gene is affected by the production of avermectin.

Further, the present invention provides a method for producing avermectins, wherein cells of the Z.AuntuyPsch strain, which express a mutated AluC allele, which encodes a gene product that changes the ratio of 2: 1 classes of avermectins produced by the Z.ouetteCy strain cells, expressing this mutated AusaC allele, compared to cells of the same strain, which do not express the mutated AeC allele, but instead express only the wild type AeC allele, are cultured in culture media under conditions that make it possible to produce or induce avermectin production of them and secrete said avermectins from the culture. In a preferred embodiment, the ratio of 2: 1 classes of avermectins produced by cells expressing this mutation is reduced. This method provides increased efficiency in the production of commercially important avermectins, such as doramectin.

Further, the present invention proposes a method of obtaining avermectins, wherein cells of the strain Z. Auretica, which express a mutated AluC allele or a genetic construct containing the AeC allele, which leads to the production of an altered amount of avermectins produced by the Z. Waile Tyrate strain, expressing this mutated allele. or a genetic construct compared to cells of the same strain that do not express the mutated AeC allele or genetic construct, but instead essiruyut only the wild-type allele avec cultured in culture media under conditions that allow production or induce avermectin production of them, and secrete said avermectins from the culture. In a preferred embodiment, the amount of avermectins produced by cells expressing this mutation or genetic construct is increased.

Further, the present invention proposes a new composition of avermectins produced by the strain Z.AuyettyIsch expressing the mutated AuC allele of the present invention, and the avermectins are produced in a reduced ratio of 2: 1 classes compared to the classes of 2: 1 avermectins produced by cells of the same strain Z. Auntestech, which do not express a mutated AluC allele, but instead express only the wild-type AeC allele. This new composition of avermectins may be present as produced in the fermentation culture liquid or it can be collected from this liquid, and it can also be partially or substantially purified from it.

4. Brief description of graphic materials

FIG. 1. The DNA sequence (8EO GO ΝΘ: 1), containing the ORS of S. Cayettyus, and the deduced amino acid sequence (8EO GO N0: 2).

FIG. 2. Plasmid vector p8E186 (ATCC 209604), containing the full-length ORF of the AueC gene 8. start out.

FIG. 3. The gene replacement vector p8E180 (ATSS 209605), containing the gene egTE 8ass. The Shetaa is built into the ODP Aeus Z. Auettyk.

FIG. 4. Restrictive map of the WatH1 gene cluster of avermectin-polyketide synthase from Z.AuettyiIschI with five identified overlapping cosmid clones (i.e. p8E65, p8E66, p8E67, p8E68, p8E69). The ratio of p8E118 and p8E119 is also indicated.

FIG. 5. HPLC analysis of fermentation products produced by the strains of Z.Auettyk. Quantitative determination of the peaks was carried out by comparison with standard amounts of cyclohexyl B1. The retention time of cyclohexyl B2 was 7.4-7.7 min; the retention time of cyclohexyl B1 was 11.912.3 minutes. FIG. 5A. Strain Z. auettyShk 8E180-11 with inactivated ORS aeus. FIG. 5 B. Strain 8.auetty11 8E180-11, transformed R8E186 (ATSS 209604). FIG. 5B. Strain 8. ouetyty11 8E180-11, transformed p8E187. FIG. 5G. Strain Z. auettyShz 8E180-11, transformed p8E188.

FIG. 6. Comparison of the deduced amino acid sequences encoded by the LFS aueC Z. Auetetyk (8EO GO N0: 2), the partial ORS of the homologue of aeU from Z. dpzeostotodepez (8E0 GO N0: 5) and the ORS of the homologue AeES from Z. Hudgoksoryik (8E0 GO) ). Valine residue in bold is the estimated starting site for protein. Conservative residues are shown in capital letters for homology in all three sequences and lower case letters for homology in 2 of 3 sequences. Amino acid sequences contain approximately 50% sequence identity.

FIG. 7. Hybrid plasmid construct containing the fragment Bg-A1 / Crp1 with a length of 564 bp. from the homologue of the AueS gene Z. Iudgoxoryushch, embedded in the site Bg-A1 / Crp1 in the LFS Aeus 8. go back 11 st.

5. Detailed Description of the Invention

The present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences that encode the product of the AueC gene from 81 AUCT5, to the design of new 8.auretych strains, which can be used to screen the products of the mutated AueC gene for their effect on the production of avermectins, as well as the discovery that some products of the mutated AueC gene can reduce the B2: B1 ratio of avermectins produced by Z.AuettetyShz. In the following sections, the invention is described by way of example for a polynucleotide molecule having either a nucleotide sequence that is the same as the sequence encoding the product of the AueC gene 8, Strand, the plasmid p8E186 (ATCC 209604), or the nucleotide sequence of the ORF of FIG. 1 (8E0 GO NO: 1), and for polynucleotide molecules that have mutated nucleotide sequences derived from them. However, the principles outlined in the present invention can be applied in a similar way to other polynucleotide molecules, including the homologues of the AeC gene from other types of Gertus 5. including, for example, Z.iudgozsorksh and Z.dgéosygotodepez, among others.

5.1. Polynucleotide molecules that encode the product of the AueC gene Z. auetstiShz

The present invention proposes an isolated polynucleotide molecule containing a full-sized ORS S. Z. Auettyushz or a substantial part thereof, and the following full-sized ORF is missing in this isolated polynucleotide molecule, which is localized in the forward direction from the ORS aeus η § in chromosome 8. auetus

The isolated polynucleotide molecule of the present invention preferably contains a nucleotide sequence that is the same as the sequence of the plasmid p8E186 (ATCC 209604), which encodes the product of the AueC gene Z.Auettyyk, or which is the same as the ORF nucleotide sequence shown in FIG. 1 (8E0 GO NO: 1), or its essential part. As used herein, a substantial portion of the isolated polynucleotide molecule containing the nucleotide sequence encoding the AueC gene product 8.AegtySh§ means an isolated polynucleotide molecule containing at least about 70% of the sequence of the full-length 0PC aeueC shown in FIG. 1 (8E0 GO NO: 1), and which encodes a functionally equivalent product of the AueC gene. In this regard, the functionally equivalent product of the AueC gene is defined as the product of the gene, which, when expressed in the Z.AuettyShs ATCC 53692 strain, in which the native AUS allele is inactivated, results in the production of essentially the same ratio and amount of avermectins as produced by B. Auetusch ATSC 53692, which instead expresses only the functionally active allele of the wild-type AeC, native to the B. Awetch strain ATSS 53692.

In addition to the ORF auC nucleotide sequence, an isolated polynucleotide molecule of the present invention may additionally contain nucleotide sequences that naturally flank the AueC ίη Kyi gene in B.auette, such as those flanking nucleotide sequences shown in FIG. 1 (Beau ΝΘ: 1).

The terms polynucleotide molecule, polynucleotide sequence, coding sequence, open reading frame and ORF are used to refer to both DNA and RNA molecules, which can be either single-stranded or double-stranded and which can be transcribed and translated (DNA) or translated (RNA ) into the product of the AueC gene or, as described below, into the product of the homologue of the AueC gene or into the polypeptide that is homologous to the product of the AueC gene or the product of the homologue of the AueC gene, in a suitable system e Xpress host cell when placed under the control of appropriate regulatory elements. The coding sequence may include prokaryotic sequences, cDNA sequences, genomic DNA sequences, as well as chemically synthesized DNA and RNA sequences, but is not limited to them.

The nucleotide sequence shown in FIG. 1 (BEO ΙΌ ΝΘ: 1), contains four different GTR codons at the p.o. 42, 174, 177 and 180. As described in section 9 below, to help determine which of these codons can function in ORF aeC as starting sites for protein expression, multiple deletions of the 5 'ORC aeueC region have been constructed (Fig. 1; Beau ΝΘ: 1). The deletion of the first OTO site in p. 42 did not eliminate the activity of AueC. An additional deletion of all the GRT codons simultaneously in the b.p. 174, 177 and 180 eliminated the activity of AueC, which indicates that this region is necessary for protein expression. Thus, the present invention encompasses the OPC of a varying length AUs, which initiate translation in any of the GRT sites located in p. 174, 177 or 180, as shown in FIG. 1 (BEO ΙΌ ΝΘ: 1), and the corresponding polypeptides for each.

Further, the present invention provides a polynucleotide molecule having a nucleotide sequence that is homologous to the sequence of the rBE186 plasmid (ATCC 209604), which encodes the product of the AueC gene B. Bauettet, or the nucleotide sequence of the ORC aeC shown in FIG. 1 (BEO ΙΌ ΝΘ: 1), or its essential part. When the term homologous is used in relation to a polynucleotide molecule that is homologous to the sequence that encodes the product of the AueC B.auettycc gene, it means a polynucleotide molecule having a nucleotide sequence that: (a) encodes the same product of the AueC gene as the sequence of the plasmid rE186 ( ATCC 209604), which encodes the product of the AueC B.Aoutes family of the gene, or encodes the same product of the AUES gene, as the nucleotide sequence of the AESC ORF, shown in FIG. 1 (BEO ΙΌ ΝΘ: 1), but includes one or more silent substitutions in the nucleotide sequence in accordance with the degeneracy of the genetic code; or (b) hybridizes with a complement of a polynucleotide molecule that has a nucleotide sequence that encodes the amino acid sequence encoded by the plasmid sequence rBE186 (ATCC 209604), which encodes the product of the AueC gene, or which encodes the amino acid sequence shown in FIG. 1 (BEO ΙΌ ΝΘ: 2), under conditions of moderate severity, i.e., hybridization with filter associated with DNA in 0.5 M NaΗPΘ ₄ 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C and washing in 0.2хББС (Standard saline solution is 0.15 M No. C1, 0.015 M sodium citrate, pH 7.0) / 0.1% SDS at 42 ° C (see Aikie1 e! a1. (ebk.), 1989, SitheyrHyoiOeί Molecule Vuidu, Wo1. 1, Ogep Rinkepd Akkoslaek, Shs., Apb boNp \ UPeu & Bopk, Shs., Νο \ ν Watk, p. 2.10.3), and encodes a functionally equivalent product of the AueC gene, as defined above. In a preferred embodiment, this homologous polynucleotide molecule hybridizes with the complement of the nucleotide sequence of the plasmid rBE186 (ATCC 209604), which encodes the product of the AueC gene, or with the complement of the nucleotide sequence shown in FIG. 1 (BEO ΙΌ ΝΘ: 1), or its essential part, under very strict conditions, i.e., hybridization with filter associated with DNA in 0.5 M NaΗPΗ ₄ 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° С and washing in 0.1 x BS / 0.1% SDS at 68 ° C (see Aikie1 et al., 1989, above), and encodes a functionally equivalent product of the AueC gene, as defined above.

The activity of the AueC gene product and its potential functional equivalents can be determined by HPLC analysis of fermentation products, as described in the examples below. Polynucleotide molecules with nucleotide sequences that encode functional equivalents of the product of the AueC B. auett gene include the naturally occurring AeC genes present in other strains of B. OwettySc, homologues of the AueC genes present in other types of 81ger (compartments, and mutated alleles Spice, either found in nature or designed.

The present invention further provides a polynucleotide molecule containing a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence encoded by the plasmid p8E186 (ATCC 209604) that encodes the product of the AueE gene 8.aeg1111, or the amino acid sequence shown in FIG. . 1 (8EP ΝΘ: 2), or its essential part. As used herein, a substantial portion of the amino acid sequence shown in FIG. 1 (8EP ΝΘ: 2), means a polypeptide containing at least about 70% of the amino acid sequence shown in FIG. 1 (8EP ΝΘ: 2), and which constitutes a functionally equivalent product of the AueC gene, as defined above.

As used herein, the term homologous to amino acid sequences that are homologous to the amino acid sequence of the AueC gene product from 8.AEG, refers to a polypeptide encoded by the sequence of the plasmid p8E186 (ATCC 209604), which encodes the product of the AueC gene, or that has the amino acid sequence shown in FIG. . 1 (8EP ΝΘ: 2), but in which one or more amino acid residues are conservatively replaced by another amino acid residues, and this conservative replacement leads to a functionally equivalent gene product, as defined above. Conservative amino acid substitutions are known in the art. The rules for the creation of such substitutions include those which, among other things, are described by Jacob, .Ό., 1987,; · ιΕ Βίοтб. Cake Roipb., ^ Akidiu, IS, Wo1. 5, 8ir. 3. More specifically, conservative amino acid substitutions are those that typically occur within a family of amino acids that are related in acidity, polarity, or bulk in their side chains. Genetically encoded amino acids are generally divided into four groups: (1) acidic = aspartate, glutamate; (2) basic = 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. Phenylalanine, tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or more substitution within any particular group, for example, leucine for isoleucine or valine, or aspartate for glutamate, or threonine for serine, or any other amino acid residue for a structurally related amino acid residue, for example, an amino acid residue with similar acidity, polarity, bulk or with a similarity in some combination of them, will usually have a negligible effect on the function of this polypeptide.

The present invention further provides an isolated polynucleotide molecule containing a nucleotide sequence encoding the product of the homolog of the AueC gene. As used herein, the product of the AueC gene homolog is defined as a gene product having at least 50% amino acid sequence identity with the AueC Z.Aegtyshc gene product containing the amino acid sequence encoded by the plasmid p8E186 sequence (ATCC 209604), which encodes the AueC gene product, or the amino acid sequence the sequence shown in FIG. 1 (8EP ΝΟ: 2). In a non-limiting embodiment, the homologue product of the AueC gene from 8.budgo5sor1s5 (described in EP 0 98423; deposition of RECM BP-1901) contains the amino acid sequence 8 Ep ΙΌ ΝΘ: 4 or a substantial part thereof. A substantial part of the amino acid sequence 8ER ΙΌ ΝΟ: 4 means a polypeptide containing at least about 70% of the amino acid sequence 8 Ep ΙΌ ΝΟ: 4, and which is a functionally equivalent product of the homologue of the AueC gene. A functionally equivalent product of the AueC gene homolog is defined as a gene product that, when expressed in the strain Z.budgorkorkie RECM ΒΡ-1901, in which the native allele of the AueC homolog is inactivated, produces the same ratio and amount of milbemycins as produced by the strain Z.budgorkorkik REKM ΒΡ1901, expressing instead only the functionally active allele of the wild-type homologue of aeUs, native to the strain Z.budgorkorkik REKM-1901. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention, which encodes the product of the homologue of the AueC gene Z.Budgokorkik, contains the nucleotide sequence 8 Ep ΙΌ: 3 or a substantial part thereof. In this regard, a substantial part of an isolated polynucleotide molecule containing the nucleotide sequence 8epp ΝΘ ΝΘ: 3 means an isolated polynucleotide molecule containing at least 70% of the nucleotide sequence 8epp ΙΌ ΝΘ: 3, which encodes the functionally equivalent product of the homologue of the AueC gene, as determined directly above.

Further, in the present invention, a polynucleotide molecule is proposed that contains a nucleotide sequence that is homologous to the nucleotide sequence of 8.Hydroxoric 8EO ΙΌ N0: 3. The term is homologous when used in relation to a polynucleotide molecule containing a nucleotide sequence that is homologous to a sequence that encodes the product of the homologue of the AueC gene. 8.Hydroxoric 8E0 ΙΌ NO: 3, means a polynucleotide molecule having a nucleotide sequence that: (a) encodes the same product gene, as the nucleotide sequence 8E0 ΙΌ N0: 3 or its essential part, but includes one or more than one silent substitution in this nucleotide sequence in accordance with dance with the degeneracy of the genetic code; or (b) hybridizes with a complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence 8E0 ΙΌ NO: 4, under conditions of moderate stringency, that is, hybridization with 7% sodium dodecyl sulfate (SDS) associated with a DNA filter in 0.5 M NaΗΡO4 , 1 mM EDTA at 65 ° C and washed in 0.2x88C / 0.1% SDS at 42 ° C (see Aikie1 e1 a1. Above), and encodes a functionally equivalent product of the AueC homologue as defined above. In a preferred embodiment, this homologous polynucleotide molecule hybridizes with the complement of the nucleotide sequence 8E0 ΙΌ NO: 3, which encodes the product of the homologue of the AueC gene, or its essential part under very strict conditions, i.e., hybridization with the filter associated with the 0.5 M NaHΡO ₄ DNA in 7% Sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C and washed in 0.1x88 C / 0.1% SDS at 68 ° C (see Aikie1 e1 a1. above), and encodes a functionally equivalent product of the homolog of the AueC gene, as defined above.

Further, the present invention provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the product of the homologue of the AueC gene 8.yudgoxoric. The term homologous, as used herein in relation to polypeptides that are homologous to the product of the homologue of the gene AueC 8E0 ΙΌ NO: 4 out of 8. hudgoxor1, means a polypeptide having the amino acid sequence 8E0 ΙΌ NO: 4, but in which one or more than one amino acid residue is conservative replaced by another amino acid residue, and this conservative replacement leads to a functionally equivalent product of the homologue of the AueC gene, as defined above.

The present invention further provides oligonucleotides that hybridize with a polynucleotide molecule having the nucleotide sequence shown in FIG. 1 (8E0 ΙΌ NO: 1) or 8E0 ΙΌ NO: 3, or with a polynucleotide molecule having a nucleotide sequence that represents the complement of the nucleotide sequence shown in FIG. 1 (8E0 ΙΌ NO: 1) or 8E0 ΙΌ NO: 3. The length of such oligonucleotides is at least about 10 nucleotides, and preferably from about 15 to about 30 nucleotides, and they hybridize with one of the above polynucleotide molecules under very strict conditions, i.e., washing in 6x88C / 0.5% sodium pyrophosphate at ~ 37 ° С for oligonucleotides of ~ 14 bases, at ~ 48 ° С for oligonucleotides of ~ 17 bases, at ~ 55 ° С for oligonucleotides of ~ 20 bases, and at ~ 60 ° С for oligonucleotides of ~ 23 bases. In a preferred embodiment, these oligonucleotides are complementary parts of one of the aforementioned polynucleotide molecules. These oligonucleotides are useful for a variety of purposes, including encoding or acting as antisense molecules, useful in regulating genes, or as primers in amplifying polynucleotide molecules that encode aeC or a homolog of aeus.

Additional homologues of the AueC genes can be identified in other species or strains of Türkus using the polynucleotide molecules or oligonucleotides described here, in conjunction with known techniques. For example, an oligonucleotide molecule containing a part of the nucleotide sequence of 8.Aeg1111 shown in FIG. 1 (8E0 ΙΌ NO: 1), or a part of the nucleotide sequence 8.hudgoxor1ic 8E0 ΙΌ NO: 3, you can enter a detectable radioactive tag and use it to screen a genomic library constructed from DNA originating from the organism of interest. The severity of the conditions of hybridization is chosen on the basis of the relation of the organism in question, in this example, 8.Aegtyyk or 8.Yudgoksor1sik, to the organism of interest. The requirements for various stringency conditions are known to those skilled in the art, and such conditions, as expected, will vary depending on the particular organisms from which the library and labeled sequences are isolated. The length of such oligonucleotides is preferably at least about 15 nucleotides, and they include, for example, those described in the examples below. Amplification of homologues can be performed using these and other oligonucleotides using standard techniques such as the polymerase chain reaction (PCR), although other amplification techniques known in the art, such as ligase chain reaction, can also be used.

Clones identified as containing nucleotide sequences of aUs homologs can be tested for their ability to encode the functionally active product of the Lués homolog gene. For this purpose, these clones can be sequenced to identify a suitable reading frame, as well as initiation and termination signals. Alternatively or additionally, the cloned DNA sequence can be inserted into a suitable expression vector, i.e., into a vector that contains the necessary elements for transcription and translation of the inserted protein coding sequence. Any of a variety of vector / host systems can be used as described below, including bacterial systems such as plasmid, bacteriophage or cosmid expression vectors, but not limited to. Suitable host cells transformed with such vectors containing the potential coding sequences of the AeC homolog can then be analyzed for AeC-type activity using methods such as HPLC analysis of fermentation products, as described, for example, in section 7 below.

The preparation and manipulation of the polynucleotide molecules described here are within the competence of specialists in this field, and can be carried out according to recombinant techniques described, for example, in MashaOk e! A1., 1989, The Council of Directors. A bogaogu Mapia1, Co1b Srppd Nagyog bAoga! Ogu Ryukyu So 16 Srppd Nagog, ΝΥ; Aikie1 e! A1., 1989, Siggep! Rgo! Oso1k ίη Monumenti Vy1oda, Szhepe Riukydd Akkostilakek Ubayu 1n1si8s1epse,; Wait a minute! a1., 1989, Molesciente: A La Logogu Mapia1, 2b eb., Co1b Srppd Nagyog Balaigogu Reggk, S1b Srppd Nagog, ΝΥ; PTTK e! a1. (ebk), 1995, RAC Z1ga! edilek, Asabetk Rgekk, 1ps., Zap Ekdo; and Yegysy (EB), 1992, RAC Tes11. OhGogb Ikegkyu Rgekk, Νο \ ν Wagk, all of these publications are incorporated herein by reference. Polynucleotide clones encoding products of the AueC gene and products of the homologue of the AueC gene can be identified using any method known in the art, including, but not limited to, the methods outlined in section 7 below. Screening of genomic DNA libraries for sequences encoding aeUs and the homologue of aeUs can be performed using techniques such as the methods outlined in the Vep op Aub 1977, Zyppé 196: 180, for bacteriophage libraries and in Hypopathic Nodpekk, 1975, Progos . No! 1 Asab. ZSK I8A, 72: 3961-3965, for plasmid libraries. Polynucleotide molecules having nucleotide sequences that are known to include the ORC aeC, as is present, for example, in plasmid p8E186 (ATCC 209604) or plasmid p8E119 (described in section 7 below), can be used as probes in these screening experiments. Alternatively, oligonucleotide probes can be synthesized that correspond to nucleotide sequences derived from partial or complete amino acid sequences of the purified product of the homologue of the AueC gene.

5.2. Recombinant Systems

5.2.1. Cloning vectors and expression vectors

Further, in the present invention, recombinant cloning vectors and expression vectors are proposed that are useful in cloning or expressing the polynucleotide molecules of the present invention, containing, for example, ORC 8.AEHT1111K or ORF of any homologs of aeUEs. In a non-limiting embodiment, the present invention provides the plasmid p8E186 (ATCC 209604), which contains the full-length ORF of the aeC gene Z.autecsch.

All subsequent descrip- tion regarding the ORF of the AueC from the H.Auschtus or a polynucleotide molecule containing the ORS of the AeS from the Z.Aegtyshk or its part, or the product of the AueS gene, also refers to the aeus homologs and products of the AueC homologs, if not found. details or in context.

A number of different vectors have been developed for specific use in the Trigger Box, including, among others, phage, multi-copy plasmids, low-copy plasmids and Shuttle vectors E.soy-Strainer Trim, and any of them can be used to implement the present invention. A variety of drug resistance genes have also been cloned from the germs, and some of these genes have been inserted into vectors as selective markers. Examples of currently used vectors for the Germouths are presented, among other things, in Neapolis, 1980, Arriebe Vusiet. Vu! Es. 16: 169-190.

Recombinant vectors of the present invention, in particular, expression vectors, are preferably designed so that the coding sequence for the polynucleotide molecule of the invention is in operative communication with one or more regulatory elements necessary for the transcription and translation of this coding sequence to produce the polypeptide. As used herein, the term regulatory element includes nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators, and other elements known in the art that serve to direct and / or regulate the expression of polynucleotide coding sequences, but is not limited to . In addition, the coding sequence used here is in operative connection with one or more regulatory elements, where these regulatory elements efficiently regulate and ensure the transcription of the coding sequence or the translation of its mRNA, or both.

Typical plasmid vectors that can be designed to contain the polynucleotide molecule of the present invention include, among many others, ccc-Bccc1, ccc2.1 (1yyyy), ρΟΕΜ3Ζ £ (Regeda), and the shuttle vector r№HM3 (Var f! a1., 1989, 1. You !. 171: 58725881).

Methods for constructing recombinant vectors containing specific coding sequences in operative association with suitable regulatory elements are known in the art and can be used to implement the present invention. These methods include ίη νίίτο recombinant techniques, synthetic techniques and genetic recombination ίη νίνο. See, for example, the techniques described in Masha! To e! A1., 1989, above; Liebero! A1., 1989, above; 8th eto! A1., 1989, above; Ιηηίδ e! A1., 1995, above; and His111. 1992, above.

The regulatory elements of these vectors can vary in their strength and specificity. Depending on the vector / host system used, any of a number of suitable transcriptional and translational elements may be used. Non-limiting examples of transcriptional regulatory sites or promoters for bacteria include the β-dac promoter T7 promoter, the TAC promoter, left and right λ promoters, promoters! Gr and 1ac, merged promoters! Gr-1as, and more specifically for the 8th terminus §, its promoters, te1C and! ΊρΑ and so on. In the specific embodiment described in section 11 below, an expression vector was created that contained an ORC aeU cloned under the strong constitutive égE promoter from the 8th corenator of horns estaea. This vector was transformed by Z.Auggshiz, and subsequent analysis by HPLC of fermentation products showed an increased titer of avermectins produced compared to their production by the same strain, which instead expressed the wild type allele.

The expression vectors of the fusion proteins can be used to express a protein fused to the product of the AueC gene. This purified fusion protein can be used to generate antisera against the AUEC gene product, to study the biochemical properties of the AUES gene product, to construct AUES fusion proteins with various biochemical activities, or to help identify or purify the expressed AUES gene product. Possible expression vectors of fusion proteins include vectors in which sequences are inserted that encode fusions for β-galactosidase and GRE, fusions to malto-binding proteins, fusions to glutathione-8-transferase, and fusions to polyhistidine (portions of the carrier), but are not limited to by them. In an alternative embodiment, the product of the AueC gene or its part can be merged with the product of the homologue of the AueC gene or its part, originating from another species or strain of ternary, such as, for example, Z. yudtoxoruik or 8.d ^ backdose ^ this site. In the specific embodiment described in section 12 below and depicted in FIG. 7, a chimeric plasmid was constructed that contains the ORF region of the H.C. homologue Z. Iudgoxoruic, 564 bp long, replacing the homologous section of the CCСA.AUSETtyk SNA, 564 bp. Such hybrid vectors can transform 8. aegtCPCC cells and test them in order to determine their effect, for example, on the ratio of 2: 1 classes of avermectins produced.

AueC fusion proteins can be designed to contain a site useful for purification. For example, AeC-malto-binding protein fusions can be purified using an amylose resin; fusion proteins AueC-glutathione-8-transferase can be purified using glutathione-agarose granules; and the fusion of AueC-polyhistidine can be cleaned using a divalent nickel resin. Alternatively, antibodies against the carrier protein or peptide can be used to purify the fusion protein using affinity chromatography. For example, a nucleotide sequence encoding a target epitope of a monoclonal antibody can be genetically engineered to be inserted into an expression vector in operative association with regulatory elements and positioned so that the expressed epitope is fused with Aurespolypeptide. For example, the nucleotide sequence encoding the EBAO ™ epitope tag (Iη! E ^ ηaί ^ οηa1 B ^ ο! Uy η1 1 ^ e8 1ps.), Which is a hydrophilic marker protein, can be inserted into the expression vector at the point corresponding to for example, the carboxyl terminus of an AueC polypeptide. The expressed EbAO ™ epitope-AEP polypeptide fusion product can then be detected and affinity purified using commercially available anti-EEAO ™ antibodies.

An expression vector encoding an AueC-fused protein can also be designed to contain polylinker sequences that encode cleavage sites for specific proteases, so that the expressed AueC polypeptide can be isolated from the carrier region or fusion partner by processing with a specific protease. For example, a fusion protein vector may include DNA sequences encoding, among others, thrombin or factor Xa cleavage sites.

The signal sequence in the opposite direction and in reading frame with the OCR aeC can be inserted genetically into the expression vector using known methods to direct the transport and secretion of the expressed gene product. Non-limiting examples of signal sequences include, among others, signal sequences from α-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors.

To facilitate the selection of host cells transformed or transfected with cloning vectors or expression vectors of the present invention, this vector can be designed so that it additionally contains the coding sequence for the product of the genereporter or other selective marker. Such a coding sequence is preferably in operative communication with a regulatory element encoding sequences as described above. Gene reporters that are useful in the invention are known in the art and include those that encode, among others, green fluorescent protein, luciferase, x1E, and tyrosinase. Nucleotide sequences encoding selective markers are known in the art and include those that encode gene products that confer resistance to antibiotics or antimetabolites, or that satisfy auxotrophic needs. Examples of such sequences, among many others, include those that encode resistance to erythromycin, thiostrepton, or kanamycin.

5.2.2. Transformation of host cells

Further, in the present invention proposed transformed host cells containing a polynucleotide molecule or a recombinant vector according to the invention, as well as new strains or cell lines derived from them. The host cell useful in practicing the invention is preferably tertustec cells, although other prokaryotic cells or eukaryotic cells can also be used. Such transformed host cells usually include microorganisms, such as bacteria, transformed with bacteriophage recombinant DNA, plasmid vector DNA or cosmid vector DNA, or yeast transformed with recombinant vector, among other things, among other things, among other things.

The polynucleotide molecules of the present invention are designed to function in h1n1seuse cells, but they can also transform other bacterial or eukaryotic cells, for example, for cloning or expression purposes. It is usually possible to use an E.co11 strain, such as, for example, the ΌΗ5α strain, which can be obtained from Leshepsap Toure Cnite Soyesjoi (ATCC), Koskushe, ΜΌ, I8A (no. 31343 catalog) and from commercial sources (81stademy). Preferred eukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be used effectively.

The recombinant expression vector of the invention preferably transforms or transfects one or more host cells of a substantially homogeneous cell culture. This expression vector is usually introduced into host cells in accordance with known techniques, such as, for example, transformation of protoplasts, calcium phosphate precipitation, treatment with calcium chloride, microinjection, electroporation, transfection by contact with a recombinant virus, mediated by liposomes, transfection, DEAE diethylaminoethyl) -dextran transfection, transduction, conjugation or micro bombardment bombardment. The selection of transformants can be performed using standard techniques, such as the selection of cells expressing a selective marker, for example, resistance to an antibiotic associated with a recombinant vector, as described above.

Once the expression vector has been introduced into the host cell, the integration and maintenance of the AuC coding sequence either in the chromosome of the host cell or in the episomal state can be confirmed using standard techniques, for example, using Southern hybridization analysis, restriction analysis, PCR analysis, including reverse transcriptase PCR (τί-PCR), or by immunoassay to detect the expected gene product. Cell host containing and / or expressing the recombinant auC coding sequence can be identified using any of at least four conventional approaches that are well known in the art, including: (1) DNA-DNA hybridization, DNA-RNA or RNA-antisense RNA ; (2) detecting the presence of functions of the “marker” gene; (3) an estimate of the level of transcription, which is measured by the expression of Aus-specific mRNA transcripts in the host cell; and (4) detecting the presence of a mature polypeptide product, which is measured, for example, by immunological analysis or by the presence of the biological activity of AueC (for example, the production of specific ratios and amounts of avermectins indicative of LuéS activity, for example, in H.AughttyShz host cells) .

5.2.3. Expression and characterization of the product of the recombinant gene Lues

As soon as the coding sequence of the AueC is stably introduced into a suitable host cell, this transformed host cell is cloned multiply, and the resulting cells can be grown under conditions leading to maximum production of the LuéS gene product. Such conditions typically include growing cells to high density. Where the expression vector contains an inducible promoter, suitable induction conditions are used, such as, for example, temperature shift, nutrient depletion, the addition of spontaneous inducers (for example, carbohydrate analogues, such as isopropyl-P-E-thiogalactopyranoside (IPTG)), accumulation an excess of metabolic by-products and the like, as is necessary for the induction of expression.

Where the expressed LueC gene product is stored inside the host cell, these cells are harvested and lysed, and this product is isolated and purified from the lysate under extraction conditions known in the art that minimize protein degradation, such as, for example, at 4 ° C or in the presence of protease inhibitors, or under those and other conditions. Where the expressed product of the LuéS gene is secreted from host cells, a depleted nutrient medium can simply be collected and the product can be isolated from it.

The expressed LueC gene product can be isolated or substantially purified from cell lysates or culture medium, as appropriate, using standard methods, including any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density gradient centrifugation and affinity chromatography, but not limited to them. Where the expressed product of the LuéS gene exhibits biological activity, the increased purity of the preparation can be monitored at each stage of the purification procedure by using a suitable assay. Shows the expressed product of the LuéS gene or does not exhibit biological activity, it can be detected on the basis of, for example, size or reactivity with an antibody that is otherwise specific to LuéS, or by the presence of a fusion tag.

Further, in the present invention, a recombinantly expressed product of the LuES Z.Auyuteshz gene is proposed, which contains the amino acid sequence encoded by the plasmid p8E186 (ATCC 209604) sequence, which encodes the product of the AueC gene, or the amino acid sequence shown in FIG. 1 (8ЕО ΙΌ ΝΘ: 2), or its essential part, as well as its homologues.

Further, in the present invention, a recombinantly expressed product of the homologue of the AueC gene 8.Hyudo5sor1ci5 is proposed, which contains the amino acid sequence 8ЕО ΙΌ ΝΘ: 4 or its essential part, as well as its homologues.

The present invention further provides a method for producing an AueC gene product, in which a host cell transformed with a recombinant expression vector is cultured, said vector comprising a polynucleotide molecule having a nucleotide sequence encoding an AueC gene product, and the polynucleotide molecule is in operative communication with one or more than one regulatory element that controls the expression of this polynucleotide molecule in the host cell under the conditions leading to the production the product of the recombinant AuC gene, and the AuC gene product is isolated from the cell culture.

The recombinantly expressed product of the AueC gene Z.ameccine is useful for a variety of purposes, including screening for compounds that alter the function of the AueC gene product and therefore modulate the biosynthesis of avermectins, as well as for the production of antibodies directed against the AueC gene product.

As soon as the product of the AEEC gene of sufficient purity is obtained, it can be characterized using standard methods, including SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), gel chromatography, amino acid sequence analysis, biological activity when producing the corresponding products in the biosynthesis pathway avermectins and so on. For example, the amino acid sequence of the AueC gene product can be determined using standard peptide sequencing techniques. The AueC gene product can be further characterized using a hydrophilicity analysis (see, for example, NorrHapHoooy. 1981, Proc. NI. Asai. 8cE of A 78: 3824) or similar software algorithms for identifying hydrophobic and hydrophilic sites in the AueC gene product. . Structural analysis can be performed to identify areas of the AueC gene product that adopt specific secondary structures. For mapping and studying the interaction sites between the AueC gene product and its substrate, one can use biophysical methods, such as X-ray crystallography (Eidkbot, 1974, Vyuyet. Εχρ. ΒίοΙ. 11: 7-13), computer simulation (Meijsk apb 2оlt (ebk), 1986 , ίη: Sittep! Sottisseyopk ίη Method of research Соοίοβν. So1b 8rjpd Nagog Labota1ot, So1b 8rjpd NatЬot, ΝΥ) and nuclear magnetic resonance (NMR). The information obtained from these studies can be used to select new sites for mutation in the ORC of aeus to facilitate the development of new 8.aoutey1k strains with more desirable avermectin production characteristics.

5.3. Construction and use of mutants AueC

The present invention further provides a polynucleotide molecule containing a nucleotide sequence, which in other respects is the same as the ρ8 плаз186 plasmid sequence (ATCC

209604), which encodes the product of the AueC gene of 8.AEHT115, or the nucleotide sequence of the ORF AUS 8, of the nucleus, as shown in FIG. 1 (8ΕΟ GO NO: 1), but which additionally contains one or more mutations, so that the cells of the strain 8.years of ATCC 53692, in which the wild-type CayeS allele is inactivated, and which express a polynucleotide molecule containing the mutated nucleotide sequence, produce another the ratio or number of avermectins, which is produced by the cells of the strain 8.outset ATCC 53692, which instead express only the wild type allele allele.

According to the present invention, such polynucleotide molecules can be used to produce new 8.Aegty111 strains, which exhibit a detectable change in the production of avermectins compared to the same strain, which instead expresses only the wild-type allele allele. In a preferred embodiment, such polynucleotide molecules are useful for producing new 8.Avc111 strains that produce avermectins in a reduced ratio of 2: 1 classes compared to the same strain, which instead expresses only the wild-type allele allele. In a further preferred embodiment, such polynucleotide molecules are useful for producing new 8.Avc strains that produce elevated levels of avermectins compared to the same strain that instead expresses only the wild-type allele allele. In a further preferred embodiment, such polynucleotide molecules are useful for producing new 8.A strains in which the AueC gene is inactivated.

Mutations in the AUs coding sequence include any mutations that introduce amino acid deletions, additions or substitutions to the AUES gene product or that shorten the AUES gene product, or any combination thereof, and that give the desired result. For example, in the present invention, polynucleotide molecules containing the nucleotide sequence of the plasmid p8E186 (ATCC 209604), encoding the AueC gene product, or the ORF nucleotide sequence of the AUSC 8, as shown in FIG. 1 (8ΕΟ GO NO: 1), but which additionally contain one or more mutations that encodes the replacement of an amino acid residue with another amino acid residue at selected positions in the AueC gene product. In some non-limiting embodiments, examples of which are given below, such substitutions can be made at any of the positions of amino acids 55, 138, 139 or 230, or in some combination thereof.

Mutations in the AUEC coding sequence are performed by any of a number of known methods, including the use of error-prone PCR or cassette mutagenesis. For example, directional site-directed mutagenesis using oligonucleotides can be used to change the sequence of the aluC ORF in a specific way, such as, for example, introducing one or more than one restriction site or termination codon into specific sites within the ausc OPC sequence. You can also use methods such as those described in US Pat. No. 5,605,793, which involve random fragmentation, repetitive mutagenesis cycles, and nucleotide shift, to create large polynucleotide libraries with nucleotide sequences encoding Aurea mutations.

Directed mutations can be useful, in particular, where they serve to change one or more of the conservative amino acid residues in the AUeC gene product. For example, a comparison of the deduced amino acid sequences of the AueC gene products and the products of the AueC gene homologues from 8.Aegteyshk (8ΕΟ GO NO: 2), Z. dykeostogodetepepek (8ΕΟ GO NO: 5) and 8. Nudgooxorik (8ΕΟ GO NO: 4) as presented in fig. 6 indicates the sites of significant conservativeness of amino acid residues between these species. Directed mutagenesis, which leads to a substitution in one or more of these conservative amino acid residues, may be particularly effective in obtaining new mutant strains that exhibit the desired changes in the production of avermectins.

Nonspecific mutagenesis can also be useful, and it can be accomplished by exposing Z.AuschCTP cells under ultraviolet irradiation or X-rays, or with chemical mutagens such as N-methyl-M'-nitrosoguanidine, methanesulfonic acid ethyl ester, nitrous acid, or nitrous acid and primers . See, for example, Li5iBc1. 1989, above, as a review of mutagenesis techniques.

As soon as mutated polynucleotide molecules are obtained, they are screened to determine if they can modulate the biosynthesis of avermectins in Z.auettet. In a preferred embodiment, a polynucleotide molecule having a mutated nucleotide sequence is tested by complementing a strain Z.auettytSh, wherein the avec gene has been inactivated, for aueSnegativnogo (avec ^-) genetic background. With a non-limiting method, this mutated polynucleotide molecule is inserted at complementary ends into an expression plasmid in operative association with one or more regulatory elements, and this plasmid preferably also contains one or more than one drug resistance gene, which allows selection of transformed cells. This vector ^is then used to transform aUeC host cells using known techniques, and the transformed cells are selected and cultured in suitable fermentation media under conditions that allow the production of avermectins or induce it. The fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell. Examples of several vectors carrying mutated polynucleotide molecules capable of reducing the B2: B1 ratio of avermectins, including p8E188, p8E199 and p8E231, are given in section 8.3 below.

The present invention proposes methods for identifying ORS Aurea 8.15 mutations that can alter the ratio and / or amount of avermectins produced. In a preferred embodiment, the present invention provides a method for identifying ORS aeC mutations capable of changing the ratio of 2: 1 classes of avermectins produced, in which: (a) the ratio of 2: 1 classes of avermectins produced by the cells of strain Z. Xenus, in which the aeus allele is inactivated, and into which the polynucleotide molecule is inserted and expressed, containing the nucleotide sequence encoding the product of the mutated AueC gene; (b) determine the ratio of 2: 1 classes of avermectins produced by cells of the same strain of Z. Carets, as in stage (a), but which instead express only the AU allele having the ORF nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ ЫО: 1), or a nucleotide sequence that is homologous to this; and (c) compare the ratio of the 2: 1 classes of avermectins produced by the Z.Auntitus cells of stage (a) with the ratio of the 2: 1 classes of avermectins produced by the cells of 8.Austesia stage (b); so, if the ratio of the 2: 1 classes of avermectins produced by the Z.AirculaSHT cells (a) differs from the ratio of the 2: 1 classes of avermectins produced by the Z.AustaVEySHB cells (b), then the ORS mutation of the mutation 2: 1 avermectins, identified. In a preferred embodiment, this mutation reduces the avermectin 2: 1 class ratio.

In the following preferred embodiment, the present invention proposes a method for identifying ORF aeC mutations or genetic constructs containing ORC aeC that can change the amount of avermectins produced, in which: (a) determine the number of avermectins produced by cells of strain 8-ayuttyych in which the native allele of aeus inactivated, and in which a polynucleotide molecule is inserted and expressed, containing a nucleotide sequence encoding a product of the mutated AueC gene, or containing a gene cally construct comprising a nucleotide sequence encoding a gene product avec; (b) Determine the amount of avermectins produced by cells of the same Z.AuettetyCh strain, as in stage (a), but which instead express only the AeC allele having the ORF nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ N0: 1), or a nucleotide sequence that is homologous to this; and (c) compare the amount of avermectins produced by the 3A. cells of stage (a) with the amount of avermectins produced by the cells of the 8th stage (b); so, if the number of avermectins produced by the cells of the Z.Abettius stage (a) differs from the number of avermectins produced by the cells of the 8th unit class 18 (b), the ORS mutation or genetic construct capable of changing the amount of avermectins is identified. In a preferred embodiment, this mutation increases the amount of avermectins produced.

Any of the aforementioned methods for identifying mutations are performed using fermentation culture media to which cyclohexanecarboxylic acid is preferably added, although other suitable fatty acid precursors, such as any of the fatty acid precursors listed in Table 1, can also be used. one.

Once the mutated polynucleotide molecule, which modulates the production of avermectins in the desired direction, is identified, the localization of the mutation in this nucleotide sequence can be determined. For example, a polynucleotide molecule having a nucleotide sequence encoding a mutated product of the AueC gene can be isolated by PCR and subjected to DNA sequencing using known methods. The mutation (mutations) responsible for the change in the production of avermectins can be determined by comparing the DNA sequence of the mutated AuleC allele with the DNA sequence of the AeC allele of the wild type. In specific, though non-limiting, embodiments of the present invention, the products of the AueC 8 gene are known to be 1115, containing either single amino acid substitutions in any of the residues 55 (855E), 138 (8138T), 139 (A139T) or 230 (6230Ό), or double substitution in provisions 138 (8138T) and 139 (A139T), led to changes in the function of the AueC gene product, so that the ratio of 2: 1 classes of avermectins produced was changed (see section 8 below). Accordingly, polynucleotide molecules having nucleotide sequences that encode the products of the mutated AueC 8.Aegtyy15 gene, containing amino acid substitutions in one or more of the amino acid residues 55, 138, 139, or 230, or any combination thereof, are included in the scope of the present invention.

Further, in the present invention, compositions are proposed for the creation of new 8.Aegtey strains, the cells of which contain the mutated AeC allele, which leads to a change in the production of avermectins. For example, the present invention proposes recombinant vectors that can be used to deliver any of the polynucleotide molecules containing the mutated nucleotide sequences of the present invention to the AeC gene site of the 8.AeHTCP | x chromosome either for insertion into it, or to replace the AeC or her parts by homologous recombination. According to the present invention, however, a polynucleotide molecule containing the mutated nucleotide sequence of the present invention, proposed here, can also function to modulate the biosynthesis of avermectins when inserted into the chromosome 8. aegTCHx at a site other than the aeC gene, or while maintaining episomal status in cells 8. aegtTsP | x. Thus, the present invention also proposed vectors containing a polynucleotide molecule containing the mutated nucleotide sequence of the present invention, and these vectors can be used to embed this polynucleotide molecule into a site on chromosome 8.ae1111 | 11x, other than the AueC gene, or maintain episomal state.

In a preferred embodiment, the present invention proposes gene replacement vectors that can be used to embed a mutated AluC allele into the cells of the 8.AEHT11x strain with the formation of new 8.AEUHT11 | 115 strains, whose cells produce avermectins in a modified ratio of 2: 1 classes compared to cells the same strain, which instead express only the wild-type AluC allele. In a preferred embodiment, the ratio of 2: 1 classes of avermectins produced by these cells is reduced. Such gene replacement vectors can be constructed using mutated polynucleotide molecules present in the expression vectors proposed here, such as p8E188, p8E199 and p8E231, and examples of these expression vectors are given in section 8.3 below.

In a further preferred embodiment, the present invention proposes vectors that can be used to embed a mutated AuC allele into the cells of the 8. aegtCP | x strain to form new strains from cells that produce altered amounts of avermectins compared to cells of the same strain, which instead express only allele ayus wild type. In a preferred embodiment, the amount of avermectins produced by these cells is increased. In a specific, though non-limiting, embodiment, such a vector additionally contains a strong promoter, as is well known in the art, such as, for example, the strong constitutive ЕгтЕ promoter from the basalisorus uterus, which is located in the opposite direction from the OPC aeues or is in operative communication with it. Such a vector can be the plasmid p8E189 described in Example 11 below, or it can be constructed using the mutated AUeC allele of the plasmid p8E189.

In a further preferred embodiment, the present invention proposes gene replacement vectors which are useful for inactivating the aeC gene in strain 8. aegtTCP | x wild type. In a non-limiting embodiment, such gene replacement vectors can be constructed using a mutated polynucleotide molecule present in the plasmid p8E180 (ATPC 209605), an example of which is given in section 8.1 below (Fig. 3). Further, the present invention proposes gene replacement vectors that contain a polynucleotide molecule that contains nucleotide sequences or consists of nucleotide sequences that naturally flank the AeC генη δ вιι gene in the chromosome H. augustisch, including, for example, those flanking nucleotide sequences that are shown on FIG. 1 (8EO GO NO: 1), and these vectors can be used for the deletion of the LFS aeus 8. auger.

Further, the present invention proposes methods for creating new 8.Aegtey1118 strains containing cells that express the mutated AeC allele and that produce an altered ratio and / or number of avermectins compared to cells of the same Strainus strain, which instead express only the Allee Wild wild allele type In a preferred embodiment, the present invention proposes a method for creating new strains of C.Aegteysch containing cells that express the mutated AeC allele and which produce an altered ratio of 2: 1 avermectins compared to cells of the same strain 8.Aegty1115, which instead express only the allele wild type AeuS, in which cells of the strain Z. Aegtyisch are transformed with a vector that carries the mutated AeC allele, which encodes a gene product that changes the ratio of 2: 1 classes of avermectins, produced by the cells of the strain Z.AegtyYasch expressing this mutated AuC allele compared to cells of the same strain, which instead express only the wild type AeC allele, and select transformed cells that produce avermectins in a modified ratio of 2: 1 classes compared to the ratio 2: 1 classes produced by the strain cells, which instead express only the wild-type AluC allele. In a preferred embodiment, the altered class ratio of 2: 1 avermectins is reduced.

In the following preferred embodiment, the present invention proposes a method for creating new strains of C. aegtes containing cells that produce altered amounts of avermectin, in which the cells of the strain of C. aegis are transformed with a vector that carries the mutated AeC allele or a genetic construct containing the Aeus allele whose expression leads to a change in the number of avermectins produced by the cells of the strain Z.auerrgyP expressing this mutated allele of aeS or a genetic construct, cf. vneny to cells of the same strain that instead express only the wild-type allele avec and selected transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only the wild-type allele avec. In a preferred embodiment, the amount of avermectins produced in transformed cells is increased.

In the following preferred embodiment, the present invention proposes a method for creating new 8.Aegteyi strains whose cells contain an inactivated CpA allele, in which cells of the Aegte1115 strain, which express the wild-type CpeS allele, are transformed with a vector that inactivates the AueC allele and select transformed cells in which the allele is inactivated. In a preferred, though non-limiting, embodiment, cells of the strain 8.AAlHTC | 115 are transformed with a gene substitution vector that carries the AUC allele, which is inactivated by mutation or substitution of a portion of this AUC allele with the sequence of the heterologous gene, and selects the transformed cells in which the native AUS allele of these cells is replaced inactivated alleleS. Inactivation of the AueC allele can be determined using HPLC analysis of fermentation products, as described below. In a specific, though non-limiting, embodiment, as described in section 8.1 below, the AeC allele is inactivated by inserting the HergE gene from 8 of the Massage pathway into the ORF of the AeC.

Further, in the present invention, new strains of Z.Augtyysch are proposed, containing cells that have been transformed by any of the polynucleotide molecules or vectors of the present invention. In the preferred embodiment, the present invention proposes new strains of Z.Augteysch containing cells that express the mutated AuC allele instead of the wild-type AeC allele or in addition to the wild-type AeC allele compared to with a 2: 1 class ratio of avermectins produced by cells of the same strain, which instead express only the wild type allele allele. In a preferred embodiment, this altered 2: 1 class ratio produced by these new cells is reduced. Such new strains are useful in large-scale production of commercially desirable avermectins, such as doramectin.

The most important task of the screening assays described here is the identification of mutated alleles of the AueC gene, the expression of which in cells of the WAA cell changes and, more specifically, reduces the class 2: 1 ratio of avermectins produced. In a preferred embodiment, the B2: B1 ratio of avermectins produced by the cells of the new strain Z.Aegtybshch of the present invention expressing the mutated AeC allele, which reduces the ratio of classes

2: 1 produced avermectins, is below about 1.6: 1 to about 0: 1; in a more preferred embodiment, the ratio is between about 1: 1 and about 0: 1; and in the most preferred embodiment, the ratio is between about 0.84: 1 and about 0: 1. In the specific embodiment described below, the new cells of the present invention produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of less than 1.6: 1. In another specific embodiment, described below, the novel cells of the present invention produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.94: 1. In the following another specific embodiment, described below, the novel cells of the present invention produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.88: 1. In the following another specific embodiment, described below, the novel cells of the present invention produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.84: 1.

In a further preferred embodiment, the present invention proposes new 8.AEHTIC strains containing cells that express the mutated AluC allele or a genetic construct containing the AluC allele, instead of the AyC allele of the wild type or in addition to the AluC allele of the wild type, where these cells of this new strain produce a modified number of avermectins compared to cells of the same strain, which instead express only the wild type allele allele. In a preferred embodiment, this new strain produces an increased amount of avermectins. In a non-limiting embodiment, the genetic construct additionally contains a strong promoter, such as the strong constitutive promoter egTe from the eighth side of the route, which is located in the opposite direction from the LFS or in operative connection with it.

In the following preferred embodiment in the present invention proposed new strains 8.aegteyshk containing cells in which the allele of aeS is inactivated. Such strains are useful for both the different spectrum of avermectins that they produce compared to the wild-type strain, and in complementation screening assays, as described here, to determine whether directed or non-specific mutagenesis of the AeU gene is affected by the production of avermectins. In a specific embodiment, described below, the host cell 8.Aegtysch was genetically engineered to contain an inactivated AeC gene. For example, the strain 8E180-11, described in the examples below, was obtained using the plasmid replacement of the p8E180 genes (ATCC 209605) (Fig. 3), which was designed to inactivate the auC gene 8.aegty111 by inserting the resistance gene egEE into the coding region of the AeC.

Further, in the present invention, recombinantly expressed mutated AueC 8Aegty111k gene products encoded by any of the aforementioned polynucleotide molecules of the invention, and methods for their preparation, are proposed.

Further, the present invention provides a method for producing avermectins, wherein cells of the strain 8. augers that express a mutated Caye allele that encodes a gene product that changes the ratio of the 2: 1 classes of avermectins produced by the cells of the strain 8.aegteysc expressing this mutated AeleC allele compared to cells of the same strain, which instead express only the wild-type AluC allele, are cultured in culture media under conditions that allow production or induction t avermectin production of them, and secrete said avermectins from the culture. In a preferred embodiment, the ratio of the 2: 1 classes of avermectins produced in this culture by cells expressing this mutated AeC allele is reduced. This method provides increased efficiency in the production of commercially significant avermectins, such as doramectin.

Further, in the present invention, a method for producing avermectins is proposed, wherein cells of the strain 8.AeGT that express a mutated AeC allele or a genetic construct containing the AeC allele, which leads to the production of an altered amount of avermectins produced by the cells of the 8.AEG strain that express this mutated allele. or a genetic construct compared to cells of the same strain that do not express the mutated AeC allele or genetic construct, but instead They express only the wild-type AuC allele, cultured in culture media under conditions that allow or induce the production of avermectins from them, and isolate these avermectins from this culture. In a preferred embodiment, the amount of avermectins produced in culture by cells expressing this mutated AluC allele or genetic construct is increased.

Further, the present invention proposes a new composition of avermectins produced by the 8.AEGT strain expressing the mutated AUeC allele, which encodes the gene product, which reduces the ratio of 2: 1 classes of avermectins produced by the 8.AEGTSCH strain cells expressing this mutated AUSE alleleC, compared with cells of the same strain, which instead express only the wild type AeC allele, and these avermectins in the new composition are produced in a reduced ratio of 2: 1 classes compared to Ocean class 2: 1 avermectins produced by cells of the same strain Z.auettyShk that instead express only the wild-type allele avec. This new composition of avermectins can either be present as produced in the depleted fermentation culture liquid, or it can be collected from this liquid. This new avermectin composition can be partially or substantially purified from this culture fluid using well-known biochemical purification techniques, such as ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, and so on.

5.4. Use of avermectins

Avermectins are highly active antiparasitic agents that have particular benefits as anthelmintic agents, ectoparasiticides, insecticides, and acaricides. Avermectin compounds obtained according to the methods of the present invention are useful for any of these purposes. For example, avermectin compounds prepared according to the present invention are useful for treating various diseases or conditions in humans, in particular, where these diseases or conditions are caused by parasitic infections, as is known in the art. See, for example, 1kaba aib otita, 1997, syt. Keu. 97 (7): 2591-2609. More specifically, the avermectin compounds prepared according to the present invention are effective in treating a number of diseases or conditions caused by endoparasites, such as parasitic nematodes, which can infect humans, domestic animals, pigs, sheep, poultry, horses, or cattle.

More specifically, the avermectin compounds obtained according to the present invention are effective against nematodes that infect humans as well as those that infect various species of animals. Such nematodes include gastrointestinal parasites such as Aisu1okYuta, Iesa1og, Aksayk, 81toidu1o1bek, TisYie11a, SarShapa, TpsNipk, Ei1etoshk, IboGbapa and parasites which are found in the blood or other tissues or organs, such as worms filaria and extracted intestinal states 81toidopicus and Tpscie11a.

The avermectin compounds produced according to the present invention are also useful in treating ectoparasitic infections including, e.g., infestation of arthropods in mammals and birds, you rank ticks, mites, lice, fleas, padalnymi flies, biting insects, or migrating dipterous larvae that can to hit cattle and horses, among other things.

The avermectin compounds prepared according to the present invention are also useful as insecticides against household pests, such as, for example, cockroach, clothes moth, leather, and houseflies, as well as insect pests of harvested grain and agricultural plants, among which these pests include themselves, among others, spider mites, aphids, caterpillars and orthoptera, such as locusts.

Animals that can be treated with avermectin compounds prepared according to the present invention include sheep, cattle, horses, deer, goats, pigs, birds including poultry, as well as dogs and cats.

The avermectin compound prepared according to the present invention is administered in a preparation in accordance with the specific intended use, the specific type of host animal being treated and the parasite or insect involved. For use as a parasiticide, the avermectin compound prepared according to the present invention can be administered orally in the form of a capsule, bolus, tablet or liquid medication, or it can alternatively be administered as an infusion or by injection or as an implant. Such drugs are prepared in a conventional manner in accordance with standard veterinary practice. Thus, capsules, boluses or tablets can be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier, further comprising a disintegrant and / or binding agent, such as starch, lactose, talc, magnesium stearate, and so on. The liquid medication can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc. Injectable preparations can be prepared in the form of a sterile solution that may contain other substances, such as, for example, a sufficient amount of salts and / or glucose to make this solution isotonic with blood.

Such preparations will vary with respect to the weight of the active compound depending on the patient or animal species of the host to be treated, the severity and type of infection, as well as the body weight of the host. As a rule, for oral administration, a dose of the active compound from about 0.001 to 10 mg per kg of body weight of the patient or animal, which is given as a single dose or divided into treatment courses over a period of 1 to 5 days, will be satisfactory. However, there may be cases where higher or lower dosage ranges are indicated, as determined, for example, by a doctor or veterinarian on the basis of clinical symptoms.

Alternatively, the avermectin compound prepared according to the present invention can be administered in combination with the animal feed, and for this purpose it is possible to prepare a concentrated food supplement or a pre-prepared mixture for mixing with the usual animal feed.

For use as an insecticide and for treating agricultural pests, the avermectin compound prepared according to the present invention can be used in the form of a sprayed solution, pulverulent preparation, emulsion, and the like in accordance with standard agricultural practice.

6. Example: Fermentation of $ 1 Toner Aeurens and Analysis of Avermectins B2: B1

Strains lacking both activities, branched-chain 2-oxo-acid dehydrogenases and 5-O-methyltransferase, do not produce avermectins if no fatty acids are added to the fermentation medium. This example demonstrates that in such mutants a wide range of B2: B1 ratios of avermectins can be obtained, when biosynthesis is initiated in the presence of various fatty acids.

6.1. Materials and methods

The Zeru Yutuşeş auetus Š ATCC 53692 was stored at -70 ° C in the form of a full broth prepared in a seed medium consisting of: starch (Yaabekh, Latda ·; · ιΙίοη; · ι1) - 20 g, ReagtateFa (Ttabet Rto1esh, Metréi, ΤΝΙί) ) - 15 g, Agbatte pH (hr 15% 15 pps) - 5 g, calcium carbonate - 1 g. The final volume was adjusted to 1 l with tap water, the pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C for 25 min .

ml of thawed suspension of the above preparation was used to inoculate a flask containing 50 ml of the same medium. After 48 h of incubation at 28 ° C on a rotary shaker at 180 rpm, 2 ml of broth was used to inoculate a flask containing 50 ml of production nutrient medium consisting of: starch - 80 g, calcium carbonate - 7 g, REagtateb1a - 5 g , potassium hydrogen phosphate - 1 g, magnesium sulfate - 1 g, glutamic acid - 0.6 g, iron (II) sulfate heptahydrate - 0.01 g, zinc sulfate - 0.001 g, manganese (II) sulfate - 0.001 g. Final volume it was adjusted to 1 liter with tap water, the pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C for 25 minutes.

Various carboxylic acid substrates (see Table 1) were dissolved in methanol and added to the fermentation broth 24 hours after inoculation to obtain a final concentration of 0.2 g / l. This fermentation broth was incubated for 14 days at 28 ° C, then this broth was centrifuged (2500 rpm for 2 min) and the supernatant was discarded. The mycelial residue was extracted with acetone (15 ml), then dichloromethane (30 ml), and the organic phase was separated, filtered, then evaporated to dryness. This residue was dissolved in methanol (1 ml) and analyzed by HPLC using a liquid chromatograph He \ 1e11Raskagb 1090A, equipped with a scanning detector device with a diode array at 240 nm. The column used was a Wectap IETacrete C-18, 5 µm, 4.6 mm x 25 cm column, which was maintained at 40 ° C. 25 μl of the above methanol solution was added to the column. Elution was performed with a linear gradient of methanol-water from 80:20 to 95: 5 over 40 minutes at 0.85 ml / min. To calibrate the detector signal, two standard concentrations of cyclohexyl B1 were used and the area under the curves for avermectins B2 and B1 was measured.

6.2. results

The retention time observed for avermectins B2 and B1, and the ratio of 2: 1 are presented in table. one.

Table 1

Retention time in HPLC, min Ratio Substrate AT 2 IN 1 B2: B1 4-tetrahydropyrancarboxylic acid 8.1 14.5 0.25 Isobutyric acid 10.8 18.9 0.5 3-Furancarboxylic acid 7,6 14.6 0.62 8 - (+) - 2-methyl butyric acid 12.8 21.6 1.0 Cyclohexanecarboxylic acid 16.9 26.0 1.6 3-Thiophencarboxylic acid 8,8 16,0 1.8 Cyclopentane carboxylic acid 14.2 23.0 2.0 3-Trifluoromethylbutyric acid 10.9 18,8 3.9 2-Methylpentanoic acid 14.5 24.9 4.2 Cycloheptancarboxylic acid 18.6 29.0 15.0

The data presented in table. 1, demonstrate an extremely wide range of avermectin B2: B1 ratios, indicating a significant difference in the results of the dehydrative conversion of class 2 compounds to class 1 compounds depending on the nature of the starting unit of the added fatty acid side chain. This indicates that changes in the B2: B1 ratios as a result of changes in the AueC protein can be specific for specific substrates. Therefore, screening for mutants exhibiting changes in the B2: B1 ratio obtained with a particular substrate must be carried out in the presence of this particular substrate. In the subsequent examples described below, cyclohexanecarboxylic acid was used as a screening substrate. However, this substrate was used solely to give an example of the possible, and it is not intended to limit the applicability of the present invention.

Ί. Example: Isolation of an Auec Gene

This example describes the isolation and characterization of a portion of the BDerYutusek AeuretcD chromosome, which encodes the product of the AueC gene. As demonstrated below, the aueC gene was identified as being capable of modifying the ratio of avermectins produced (B2: B1) to cyclohexyl-B2 and cyclohexyl-B1.

7.1. Materials and methods

7.1.1. Growing BDJetusek for DNA extraction

For the cultivation of Broker, the following method was performed. Single colonies of B.Aegtyyshk ATSS 31272 (single colony isolate No. 2 isolate) were isolated on URE-6 1/2 concentration, containing: IgGso yeast extract -5 g, bactopeptone E | Gso - 5 g, dextrose - 2.5 g, MORB - 5 g, baktoagar IGGo - 15 g. The final volume was adjusted to 1 liter with distilled ^ Θ, the pH was adjusted to 7.0, and the medium was autoclaved at 121 ° C for 25 minutes.

Mycelium grown in the above medium was used to inoculate 10 ml of TBV medium (tryptic Iso Tsoy broth (Ijso TgurDs Bow Vgo111) - 30 g, in 1 liter of distilled H ₂ O, autoclaved at 121 ° С for 25 min) in tube 25 mm x 150 mm, which was held with shaking (300 rpm) at 28 ° C for 48-72 h.

7.1.2. Isolation of chromosomal DNA from BDerUtusek

Aliquots of samples (0.25 ml or 0.5 ml) of mycelia grown as described above were placed in 1.5 ml microcentrifuge tubes, and the cells were concentrated by centrifugation at 12000 x d for 60 s. The supernatant was discarded, and the cells were resuspended in 0.25 ml of TBE buffer (20 ml of 1.5 M sucrose, 2.5 ml of 1 M Tris-HC1, pH 8.0, 2.5 ml of 1 M EDTA, pH 8.0 and 75 ml of distilled H ₂ O) containing 2 mg / ml lysozyme. Samples were incubated at 37 ° C for 20 min with shaking, loaded into an automated AOUTOep 540 ™ nucleic acid extraction device ([BelagaDeraBeraDop Buk1etk, No.DSC, MA) and genomic DNA was isolated using cycle 159 (equipment by software) according to instructions manufacturer.

Alternatively, 5 ml of mycelia were placed in a 17 mm x 100 mm tube, the cells were concentrated by centrifugation at 3000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 1 ml of TBE buffer, concentrated by centrifugation at 3000 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in 1 ml of TBE buffer containing 2 mg / ml of lysozyme and incubated at 37 ° C with shaking for 30-60 minutes. After incubation, 0.5 ml of 10% sodium dodecyl sulfate (SDS) was added, and the cells were incubated at 37 ° C until complete lysis was observed. This lysate was incubated at 65 ° C for 10 min, cooled to room temperature, divided into two 1.5 ml Eppendorf tubes, and extracted with 1x 0.5 ml of a phenol / chloroform mixture (50% phenol, previously equilibrated with 0.5 M Tris pH 8.0; 50% chloroform). The aqueous phase was collected and extracted from 2 to 5x with chloroform: isoamyl alcohol (24: 1). DNA was precipitated by adding 1/10 volume of 3 M sodium acetate, pH 4.8, incubating the mixture on ice for 10 minutes, centrifuging the mixture at 15,000 rpm at 5 ° C for 10 minutes and removing the supernatant in a clean tube to which 1 volume of isopropanol was added. The supernatant plus isopropanol mixture was then incubated on ice for 20 minutes, centrifuged at 15,000 rpm for 20 minutes at 5 ° C, the supernatant was removed, and the DNA was washed with 1x 70% ethanol. After the precipitate had dried, the DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

7.1.3. Isolation of plasmid DNA from BDerUtusek

An aliquot sample (1.0 ml) of mycelium was placed in 1.5 ml microcentrifuge tubes, and the cells were concentrated by centrifugation at 12000 x d for 60 s. The supernatant was discarded, and the cells were resuspended in 1.0 ml of 10.3% sucrose, concentrated by centrifugation at 12000 x d for 60 s, and the supernatant was discarded. The cells were then resuspended in 0.25 ml of TBE buffer containing 2 mg / ml of lysozyme, incubated at 37 ° C for 20 minutes with shaking, and loaded into the Ai1O-Oep 540 ™ nucleic acid extraction device. Plasmid DNA was isolated using loop 106 (hardware software) according to the manufacturer's instructions.

Alternatively, 1.5 ml of mycelia were placed in 1.5 ml microcentrifuge tubes, and the cells were concentrated by centrifugation at 12000 x d for 60 s. The supernatant was discarded, the cells were resuspended in 1.0 ml of 10.3% sucrose, concentrated by centrifugation at 12000 x d for 60 s, and the supernatant was discarded. The cells were resuspended in 0.5 ml of TBE buffer containing 2 mg / ml of lysozyme and incubated at 37 ° C for 1530 minutes. After incubation, 0.25 ml of alkaline SDS was added (0.3 N No. 1OH, 2% SDS), and the cells were incubated at 55 ° C for 15-30 minutes or until the solution became clear. Sodium acetate (0.1 ml, 3 M, pH 4.8) was added to the DNA solution, and then it was incubated on ice for 10 minutes. DNA samples were centrifuged at 14,000 rpm for 10 minutes at 5 ° C. The supernatant was removed to a clean tube, 0.2 ml of phenol / chloroform (50% phenol: 50% chloroform) was added and gently stirred. The DNA solution was centrifuged at 14,000 rpm for 10 minutes at 5 ° C, and the upper layer was removed in a clean Eppendorf tube. Isopropanol (0.75 ml) was added, and the solution was gently stirred, and then incubated at room temperature for 20 minutes. The DNA solution was centrifuged at 14000 rpm for 15 minutes at 5 ° C, the supernatant was removed, and the DNA residue was washed with 70% ethanol, dried and resuspended in TE buffer.

7.1.4. Isolation of plasmid DNA from E.co11

A single transformed colony of E.co11 was inoculated in 5 ml of Luria-Bertani (LB) medium (bacterotrypton -10 g, bactro-yeast extract - 5 g and №-1С1 - 10 g in 1 l of distilled H ₂ O, pH 7.0, autoclaved at 121 ° C for 25 min and 100 μg / ml ampicillin was added). This culture was incubated overnight and a 1 ml aliquot sample was placed in a 1.5 ml microcentrifuge tube. Culture samples were loaded into an automated Anlocep 540 ™ nucleic acid isolator and plasmid DNA was isolated using cycle 3 (hardware software) according to the manufacturer's instructions.

7.1.5. Production and transformation of protoplasts Z.AegtbPb

Single colonies of Z.AegtbPI were isolated at ΥΡΌ-6 1/2 concentrations. Mycelium was used to inoculate 10 ml of T8B medium in a 25 mm x 150 mm tube, which was then incubated with shaking (300 rpm) at 28 ° C for 48 hours. One milliliter of mycelia was used to inoculate 50 ml of UEME medium. UEME medium contains per liter: yeast extract E | Gso - 3 g; bactopepton E | Gso - 5 g; malt extract E | Gso - 3 g; sucrose - 300 g. After autoclaving at 121 ° C for 25 min, the following was added: 2.5 M MdCl ₂ 6H ₂ O (autoclaved separately at 121 ° C for 25 min) - 2 ml and glycine (20%) (sterilized by filtration) - 25 ml.

Mycelium was grown at 30 ° C for 48-72 h and collected by centrifugation in a 50 ml centrifuge tube (EA1SOP) at 3000 rpm for 20 minutes. The supernatant was discarded, and the mycelium was resuspended in buffer P, which contains: sucrose - 205 g; K ₂ 8O ₄ - 0.25 g; MdC1 ₂ -6H ₂ O - 2.02 g; H ₂ O - 600 ml; K ₂ PO ₄ (0.5%) - 10 ml; trace element solution * - 20 ml; CaCl ₂ -2H ₂ O (3.68%) -100 ml; and ME8 buffer (1.0 M, pH 6.5) —10 ml. (* The solution of trace elements contains per liter: Ζηί'1 ₂ - 40 mg; GeС1 ₃ -6Н ₂ О - 200 mg; CС1 ₂ -2Н ₂ О - 10 mg; МпС1 ₂ -4Н ₂ О - 10 mg; №1 ₂ В ₄ O - 10H ₂ O - 10 mg; (NH ₄ ) ₆ Mo7O ₂₄ · 4H ₂ O - 10 mg). The pH was adjusted to 6.5, the final volume was adjusted to 1 L, and the medium was filtered hot through a 0.45 micron filter.

Mycelium was pelleted by centrifugation at 3000 rpm for 20 minutes, the supernatant was discarded, and the mycelium was resuspended in 20 ml of buffer P containing 2 mg / ml of lysozyme. Mycelium was incubated at 35 ° C for 15 min with shaking and checked under a microscope to determine the degree of formation of protoplasts. When protoplast formation was completed, these protoplasts were centrifuged at 8000 rpm for 10 minutes. The supernatant was removed, and the protoplasts were resuspended in 10 ml of buffer P. These protoplasts were centrifuged at 8000 rpm for 10 min, the supernatant was removed, the protoplasts were resuspended in 2 ml of buffer P, and approximately 1 x10 ⁹ protoplasts were distributed into cryogenic test tubes with a volume of 2 , 0 ml (№1depe).

The tube containing 1x10 ⁹ protoplasts was centrifuged at 8000 rpm for 10 minutes, the supernatant was removed, and the protoplasts were resuspended in 0.1 ml of buffer P. To the protoplasts, 2 to 5 μg of transforming DNA was added, followed by the immediate addition of 0.5 ml of working buffer T. The basis of buffer T contains: PEG-1000 (81dta) - 25 g; sucrose - 2.5 g; H2O - 83 ml. The pH was adjusted to 8.8 with 1N No. UN (filter sterilized), and the basis of buffer T was sterilized by filtration and stored at 4 ° C. The working buffer T prepared on the day of consumption was 8.3 ml from the basis of buffer T; K2PO4 (4 mM) - 1.0 ml; CaCl ₂ .2H ₂ O (5 M) - 0.2 ml and TE8 (1 M, pH 8) - 0.5 ml. Each component of working buffer T was individually sterilized by filtration.

Within 20 s of the addition of T buffer, 1.0 ml of buffer R was also added to the protoplasts and the protoplasts were centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded and protoplasts were resuspended in 0.1 ml of buffer P. Then protoplasts were sown on KM14 medium, which contains: sucrose - 205 g; K ₂ 8O ₄ - 0.25 g; MdCl ₂ -6H ₂ O - 10.12 g; glucose 10 g; EGso casamic acids - 0.1 g; yeast extract E | Gso - 5 g; EGso oat agar 3 g; Baktoagar E | Gso - 22 g; distilled H2O - 800 ml. This solution was autoclaved at 121 ° C for 25 minutes. After autoclaving, sterile stock solutions of the following substances were added: K ₂ PO ₄ (0.5%) - 10 ml; SaS1 ₂ -2H ₂ O (5 M) - 5 ml; L-proline (20%) -15 ml; buffer ME8 (1.0 M, pH 6.5) - 10 ml; trace element solution (the same as above) - 2 ml; Cycloheximide stock solution (25 mg / ml) - 40 ml and 1 n. No. UN - 2 ml. The BM14 medium was divided into 25 ml aliquots per plate, and the plates were dried for 24 hours before use.

Protoplasts were incubated at 95% humidity at 30 ° C for 20-24 hours. To select thiostrepton-resistant transformants, 1 ml of coating buffer containing 125 μg per milliliter of thiostrepton was evenly distributed on the surface of KM14 regeneration plates. The coating buffer contains 100 ml: sucrose - 10.3 g; a solution of trace elements (the same as above) - 0.2 ml and MEZ (1.0 M, pH 6.5) - 1 ml. The protoplasts were incubated at 95% humidity at 30 ° C for 7-14 days, until the thiostrepton-resistant colonies (THOy ^g ) became visible.

7.1.6. Transformation of the Zkgerkotusez Shiapz Protoplasts

З.11У1йзз ТК64 (provided by YoNp 1Pezıncılıkı, №г \\ '1с11, и.К.) in some cases used for transformations. Methods and compositions for the cultivation, production of protoplasts and the transformation of 8.1Myapz are described in Norteoi et al., 1985, by Sepéls Mashriyopooperneni Zergerotusez. A LABOTAKOTA Mapia1. Salary, EoipiLop, No. d \\ 'lc11, and K.K., and they were carried out as described in this publication. Plasmid DNA from transformants 8.1Myapz were isolated as described in section 7.1.3 above.

7.1.7. Fermentation analysis of strains of Z. auettyShz

Mycelium Z. auettices, grown on URE-6 1/2 concentration for 4-7 days, were inoculated into 2.5 x 15 cm tubes containing 8 ml of the preferred fermentation medium and two 5 mm glass granules. The preferential medium contains soluble starch (either small boiled starch, or KOZO, 1arai Sogp Skaty So., Shdoua) - 20 g / l; Ryattatefa 15 g / l; Ajatshe pH - 5 g / l (Syatr1at 1y., Siyop, N1); Caso ₃ - 2 g / l; 2x bc £ a (bc £ a refers to branched chain fatty acids) containing a final concentration in this medium of 50 · 10 ^-3 2 - (+/-) - methyl butyric acid, 60 · 10 ^-3 isobutyric acid and 20 · 10 ^-3 isovaleric acid. The pH was adjusted to 7.2 and the medium was autoclaved at 121 ° C for 25 minutes.

This tube was shaken at an angle of 17 ° at 215 rpm at 29 ° C for 3 days. A 2 ml aliquot of this seed culture was used to inoculate a 300 ml Erlenmeyer flask containing 25 ml of production nutrient medium that contains starch (either small boiled starch or COZO) - 160 g / l; Mypzou (Atsyet Iashek M1y1apy, Yesakit, 1b) -10 g / l; Ajatshe pH -10 g / l; K ₂ NRA ₄ - 2 g / l; MDO ₄ 4H ₂ O - 2 g / l; EZO ₄ ^ 7H ₂ About 0.02 g / l; MPS1 ₂ - 0.002 g / l; 2pZO ₄ ^ 7H ₂ O - 0.002 g / l; Caso ₃ - 14 g / l; 2 x bc £ a (as above) and cyclohexanecarboxylic acid (CGC) (dissolved in the form of a 20% solution at pH 7.0) - 800–10 ^–3 . The pH was adjusted to 6.9 and the medium was autoclaved at 121 ° C for 25 minutes.

After inoculation, this flask was incubated at 29 ° C for 12 days with shaking at 200 rpm. After incubation, a 2 ml sample was taken from this flask, diluted with 8 ml of methanol, stirred, and the mixture was centrifuged at 1250 x d for 10 min to precipitate the debris. The supernatant was then analyzed by HPLC using a Wectap and Lazazriege SEZ column (25 cm x 4.6 mm ID), at a flow rate of 0.75 ml / min and detected by absorption at 240 nm. The mobile phase was 86 / 8.9 / 5.1 methanol / water / acetonitrile.

7.1.8. RKZ isolation of genes Z.amettshnz

A cosmid library of chromosomal DNA, Z.Awegt1kShz (ATCC 31272, ZS-2), was obtained and hybridized with the ketosynthase probe (CG) obtained from the fragment of the polyketidesynthase gene (RKZ) of SassLatorocellar etuktetaa. A detailed description of the acquisition of cosmid libraries can be found in Zatdock ek a1., 1989, see above. A detailed description of the acquisition of chromosomal DNA libraries Zkgerkotusez presented in Norteoy Ek A1, 1985, see above. Cosmid clones containing areas that hybridize with ketosintase were identified by hybridization with the fragment She1 / Eco47111 with a length of 2.7 kb. from REH26 (courtesy of Ότ. R. Leighau, Satpaid, IC). Approximately 5 ng of the ReX26 were subjected to enzymatic hydrolysis using She1 and Eco 47S. This reaction mixture was applied on 0.8% agarose gel Zeap1ac | ie STS (EMC VERTISC, Roscapien, ME). Fragment / She1 / Eso47Sh 2.7 kb long were cut out of the gel after electrophoresis, and DNA was isolated from the gel using CEbase ™ from Erkektte Teslo1odlez using the fast protocol. Fragment She1 / Eso47111 length 2.7 kb were labeled [a- ³² P] ISTP (deoxycytidine 5'-triphosphate, tetra (triethylammonium) salt, [alpha- ³² P] -) (ZHSIyropk, Vozkop, MA), using the nick translation system from VC (VC bober Teslo1, 1ps ., SacLegie, MI), following the supplier’s instructions. A typical reaction was performed in a volume of 0.05 ml. After adding 5 μl of the Buffer Zcor buffer, labeled DNA was separated from unincorporated nucleotides using an Oshsk Zrsh ™ Sephadex C-25 column (Voeppdet MappLash), following the supplier’s instructions.

Screened for hybridization of colonies of approximately 1,800 cosmid clones. Identified ten clones that showed a strict hybridization with a KZ-probe Zass.etukLtaaa. E. coli colonies containing cosmid DNA were grown in LB liquid medium and cosmid DNA from each culture was isolated in an automated AikoCep 540 ™ nucleic acid isolator using cycle 3 (hardware by software) according to the manufacturer's instructions. Restriction endonuclease mapping and Southern blot hybridization analyzes revealed that five of these clones contain overlapping chromosomal regions. The restriction genomic map of Z. Waett1kShz WatN1 from these five cosmids (i.e., pZE65, pZE66, pZE67, pZE68, pZE69) was constructed by analyzing overlapping cosmids and hybridizations (Fig. 4).

7.1.9. Identification of DNA that modulates the ratio of avermectins B2: B1, and identification of ΘRS aU

To test subcloned fragments originating from the cosmid clone p8E66 for their ability to modulate the avermectin B2: B1 ratios in AueC mutants, the following methods were used. p8E66 (5 μg) was subjected to enzymatic hydrolysis of 8aL and CatΗI. This reaction mixture was applied on a 0.8% agarose gel 8eP1as. SRT (PMC Β ^ οΡ ^ Disc, Koscapman, ME), fragment 8acI / ΒаtI with a length of 2.9 kb. were cut out of the gel after electrophoresis, and DNA was isolated from the gel using CEENAK ™ from Eplsebbe Tesbolodilc using the fast protocol. Approximately 5 μg of the shuttle vector pHHM3 (Ouage et al., 1989, I. Β ί ί ο1. 171: 58725881) were subjected to enzymatic hydrolysis of 8aL and 8atHK. and 0.5 μg of hydrolyzed pUNM3 were mixed together and incubated overnight with 1 unit. ligases (№ \ ν Епд1апб ЕщЕЕк, Шс., Βеνе ^ 1у, МА) at 15 ° С in a total volume of 20 μl according to the instructions of the supplier. After incubation, 5 μl of this ligase mixture was incubated at 70 ° C for 10 minutes, cooled to room temperature, and used to transform competent E. so EN5A (ΒΡΕ) cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of an 8acI / 8atHI insert with a length of 2.9 kb. confirmed by restriction analysis. This plasmid was designated p8E119.

The protoplasts of strain 8.aebt1b11k 11008C38 (strain of the company ΡΓί / eg) were obtained and transformed with p8E119, as described in section 7.1.5 above. Strain 1100-8C38 is a mutant that produces significantly more avermectin form of cyclohexyl-B2 compared to avermectin form of cyclohexyl-B1 when adding cyclohexanecarboxylic acid (B2: B1 approximately 30: 1). p8E119, used to transform 8.Aegtbcc protoplasts, was isolated either from the E.sub CM2163 strain (obtained from Eg. VE. Asbta η Си, Cigaute E.sob Seppets 81os Septegg, Valée Ishueg8Yu), from the E.sob EmsIm1ImI strain, from the E.sub Emi1mI strain, from the E1ImImIm1ImIeg8Yu, from the E.sob E.Emb.Emb.E.V. , or from strain 8.11U1bapk TK64. Thiostrepton-resistant transformants of strain 11008C38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of the strain 1100-8C38 8. aue1bcc containing p8E119 produced an altered ratio of cyclohexyl B2: cyclohexyl B1 avermectins of about 3.7: 1 (Table 2).

Having established that p8E119 is capable of modulating the proportions of avermectins B2: B1 in mutant AueC, the insert DNA was sequenced. Approximately 10 µg of p8E119 were isolated using plasmid DNA extraction kit (P1adep,

The Cell, CA), following the manufacturer's instructions, and sequenced using the automated DNA sequencer АΒI 373А (Regkt Eteg, Rock Sag Sbu, CA). Sequence data was collected and edited using the Sepebs Sotroshog (CAS, Mab1cop, \ νΙ) programs. The DNA sequence and ORS of the aeC are shown in FIG. 1 (8EP ΝΘ: 1).

A new plasmid, designated p8E118, was constructed as follows. Approximately 5 µg of p8E66 were subjected to enzymatic hydrolysis of 8p1H and НatHI. This reaction mixture was applied on a 0.8% agarose gel 8eP1ac. | Ee STS (EMC Β ^ οΡ ^ οisk), fragment 8rbI / НatНI with a length of 2.8 kb. were cut out of the gel after electrophoresis, and DNA was isolated from the gel using SEQac ™ (Erkebbe Tesbolodik) using the fast protocol. Approximately 5 μg of the shuttle vector pVH чел3 were subjected to enzymatic hydrolysis of 8pY and KatHI. Approximately 0.5 μg insert length 2.8 Kb. and 0.5 µg of hydrolyzed pVHΜ3 were mixed together and incubated overnight with 1 unit. ligases (№ \ ν Епд1апб ЕАбк) at 15 ° С in a total volume of 20 µl according to the instructions of the supplier. After incubation, 5 μl of this ligase mixture was incubated at 70 ° C for 10 minutes, cooled to room temperature, and used to transform competent E. so ELEN5 cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of an insert was 2.8 kb in length. 8rbI / KatNI was confirmed by restriction analysis. This plasmid was designated p8E118. The DNA of the inserts in p8E118 and p8E119 overlaps by about 838 nucleotides (Fig. 4).

The protoplasts of strain 8.aebt1bbk 11008C38 transformed p8E118, as described above. Thiostrepton-resistant transformants of strain 1100-8C38 were isolated and analyzed by HPLC analysis of fermentation products. Transformants of strain 11008C38 8. aaegts containing r8E118 did not change the ratio of cyclohexyl B2: avermectin cyclohexyl-B1 avermectin compared with strain 1100-8 (Table 2).

7.1.10. PCR amplification of the AueC gene from chromosomal DNA 8. AEG

A fragment of ~ 1.2 kb in length, containing the ORS aUeC, was isolated from chromosomal DNA of 8.AETP using PCR amplification using primers designed on the basis of the AueC nucleotide sequence obtained above. PCR primers were provided by Sepokuk ^ oΐesbpo1od ^ ek, Shs. (Techak). The right-handed primer was 5'-TACACAAACCASSASASAS-3 '(8ER ΝΘ: 6), and the left-sided primer was the 5'-SATSATSSSSAASCASS-3' (8EO ΙΌ ΝΘ: 7). The PCR reaction was performed with Heer Uep! ™ polymerase (No. \ ν

In the buffer supplied by the manufacturer, in the presence of 300 μM dNTP, 10% glycerol, 200 pmol of each primer, 0.1 μg of the matrix and 2.5 units. the enzyme in a final volume of 100 μl, using a Regcte1Te Ce! thermal cycler. The temperature profile of the first cycle was 95 ° C for 5 min (denaturation stage), 60 ° C for 2 min (annealing stage) and 72 ° C for 2 min (elongation stage). The subsequent 24 cycles had a similar temperature profile, except that the denaturation stage was shortened to 45 s, and the annealing stage was shortened to 1 min.

The PCR product was subjected to electrophoresis in 1% agarose gel and a single ~ 1.2 kb DNA band was detected. This DNA was purified from the gel and ligated with 25 ng of the linearized vector pCB-B1A1 (Chipudap) with blunt ends at 1:10 the molar ratio of the vector to the insert, following the manufacturer's instructions. This ligase mixture was used to transform the competent cells of E.soi 0n 8yo! ™ Chipugolep), following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of an insert was ~ 1.2 kbp. confirmed by restriction analysis. This plasmid was designated p8E179.

DNA inserts from p8E179 were isolated by enzymatic hydrolysis of WatH1 / XBa1, separated by electrophoresis, purified from the gel and ligated with the shuttle vector p \ UNM3, which was also subjected to enzymatic hydrolysis of WatH1 / XBa1, with a total DNA concentration of 1 μg in 1: 5 molar ratio of vector to insert. This ligase mixture was used to transform E. soy EN5a competent cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of an insert was ~ 1.2 kbp. confirmed by restriction analysis. This plasmid, which was designated as p8E186 (Fig. 2, ATCC 209604), was transformed with E. Soi ΌΜ1 and plasmid DNA was isolated from ampicillin-resistant transformants.

7.2. results

It was identified that the 8cass1 / WatH1 fragment is 2.9 kb in length. from p8E119 during the transformation by it of the strain 8.Aegteuse 11008С38 significantly changes the ratio of production of avermectins B2: B1. Normally, the strain 8.aegtyyshz 1100-8C38 has a B2: B1 ratio of about 30: 1, but when transformed with a vector containing the 8ac1 / WatN1 fragment of 2.9 kb in length, the ratio of avermectins B2: B1 decreased to about 3, 7: 1. Post-fermentation analysis of cultures of transformants confirmed the presence of transforming DNA.

This fragment is 2.9 kb in length. p8E119 was sequenced and an ORF of ~ 1.2 kbp was identified. (Fig. 1) (8EC ΙΌ NO: 1), which includes the Pz11 / 8рй1 fragment, which was previously mutated elsewhere to produce only B2 products (1kaba e1 A1., 1995, above). Comparison of this ORF or its corresponding isolated polypeptide with known databases (SepEMB, 8 ^ 188ΡΚΌΤ) did not show any strong homology with known DNA or protein sequences.

In tab. 2 shows the fermentation analysis of the strain 8. auetts Shz 1100-8C38, transformed with various plasmids.

table 2

Strain 8.augtyys (transforming plasmid) The number of tested transformants The average ratio of B2: B1 1100-8С38 (none) 9 30.66 1100-8С38 (р№НМ3) 21 31.3 1100-8C38 (r8E119) 12 3.7 1100-8C38 (r8E118) 12 30.4 1100-8C38 (r8E185) 14 27.9

8. Example: Mutant Construction

Aueas 8. Awesome

This example describes the construction of several different mutants of AueC 8. auetty using the compositions and methods described above. A general description of the methods for introducing mutations into a gene in 8 (the hers gene is presented by Klezeberg and Norden, 1991, Me.I. Ethos. 204: 430-458. A more detailed description is given by Λο / ηί е1 а1., 1988, 1. APBIOE XH1 (2): 226-233, and ΐΐΐζ Ешп-Едд ^ а11 е1 а1., 1992, I. Vastegu1. 174 (1): 144-154. These references are hereby incorporated in their entirety by reference.

8.1. Inactivation of the AueS gene 8.Aegteyzyz

AueC mutants containing inactivated AueC genes were constructed using several methods, as described in detail below.

In the first method, a 6pbp / 6pb fragment of 640 bp, internal to the AueC gene in p8E119 (plasmid described in section 7.1.9 above), was replaced by the EgEE (Erythromycin resistance) gene from 8ass. This gene was isolated from rP4026 (provided by Leuppienis 1pl111i (e, lcd, cc, and c.; See also Bilber et al., 1985, Sepa 41: 357-368) by hydrolysis with restriction enzymes / ΙΙ and EsocB, followed by electrophoresis and gel-purified. This fragment, ~ 1.7 kb in length, was ligated into pCEM72 £ (Rgheda), which was subjected to the enzymatic hydrolysis of WatH1 and EcoCb, and this ligand mixture transformed competent E cells xenoy EN5a, following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of an insert of ~ 1.7 tp length was confirmed by restriction analysis, and this plasmid was designated p8E27.

p8E118 (described in section 7.1.9 above) was subjected to enzymatic hydrolysis of 8pY and WatH1, the hydrolyzate was subjected to electrophoresis, and the insert of 8pX / 8atH1 ~ 2.8 kb in length. purified from gel. p8E119 was subjected to enzymatic hydrolysis of P3 (I and EcoC1, the hydrolyzate was subjected to electrophoresis, and an RS / EchoK insert of -1.5 kb was purified from the gel. The shuttle vector p \ UNM3 was subjected to enzymatic hydrolysis of WatH1 and EcoC1. P8E27 was subjected to enzyme hydrolysis of WatH1 and EcoC1. P3 hydrolysis (I and 8pY, the hydrolyzate was subjected to electrophoresis, and the Pg! 1 / 8pY insert with a length of ~ 1.7 kb was purified from gel. All four fragments (i.e., ~ 2.8 kb). , ~ 1.5 kbp, ~ 7.2 kbp, ~ 1.7 kbp) were ligated together when ligated in 4 directions. With this ligase mixture, competent cells were transformed E.soy OH5a , following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated p8E180 (Fig. 3; ATCC 209605).

p8E180 was transformed with 8.11) 3631'15 TK64 and identified transformed colonies for resistance to thiostrepton and erythromycin. p8E180 was isolated from 8.11U16ZP5 and used to transform 8.Aegty11k protoplasts. Four thiostrepton-resistant transformant 8.Aegt1! 11k were identified, and protoplasts were obtained and sown under non-selective conditions on KM14 medium. After these protoplasts regenerated, single colonies were screened for resistance to erythromycin and lack of resistance to thiostrepton, indicating chromosomal integration of the inactivated aeC gene and loss of free replicon. Identified one transformant Egttyo and identified it as strain 8E 180-11. From strain 8E180-11, total chromosomal DNA was isolated, hydrolyzed with restriction enzymes 8atH1, NSbN, RZI or 8pY, separated by electrophoresis on a 0.8% agarose gel, transferred to nylon membranes and hybridized with an Egte probe. These analyzes showed that the chromosomal integration of the resistance gene of egte and the concomitant deletion of the FD1 / 8pY fragment of length 640 bp. occurred due to a double crossover event. HPLC analysis of the fermentation products of strain 8E180-11 showed that normal avermectins were no longer produced (Fig. 5A).

In the second method for inactivating the AueC gene, the HerEE gene is 1.7 kb in length. were removed from the chromosome of the strain 8E180-11 8.augte1y1k, leaving 640 p. the Pg! 1 / 8pY deletion in the AeC gene. The gene replacement plasmid was designed as follows: p8E180 was subjected to partial enzymatic hydrolysis of XBa1, and a fragment of ~ 11.4 kb in length. purified from gel. In this moose ~ 11.4 kb. there is no resistance gene egt 1.7 kb in length. Then this DNA was ligated and transformed into E.soy IN5a cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as p8E184, was transformed into E.soi ΌΜ1 and plasmid DNA was isolated from ampicillin-resistant transformants. This plasmid was used to transform the protoplasts of the strain 8E180-11 8. august. Protoplasts were obtained from thiostrept-resistant transformants of strain 8E180-11 and were seeded with single colonies on KM14. After these protoplasts regenerated, single colonies were screened for the absence of both resistances, erythromycin and thiostrepton, indicating chromosomal integration of the inactivated aeC gene and the loss of a free replicon containing the egT gene. Identified one transformant Egz’tyo and identified it as strain 8Е184-1-13. The fermentation analysis of 8E184-1-13 showed that normal avermectins were not produced and that 8E184-1-13 had the same fermentation profile as 8E180-11.

In the third method, in order to inactivate the aueC gene, the chromosomal aeueC gene was introduced to shift the reading frame by adding two O (guanine) after C (cytosine) at position 471 using PCR, thereby creating the B2pE1 site. The presence of this gene-engineered site BgpE1 was useful in detecting a gene replacement event. PCR primers were designed to introduce a mutation with a shift of the frame into the Aus gene, and were provided by Oxy5y5 B ^ ο! Ёйηο1од ^ е8, 1пс. The right-handed primer was 5'06TTCCS66AT6CC6TTSTS0-3 '(8ЕО ΙΌ N0: 8), and the left-directed primer was 5'-AASTSSOOTSOASTSSSTTS-3' (8E0 ΙΌ N0: 9). PCR conditions were as described in section 7.1.10 above. The PCR product 666 bp subjected to enzymatic hydrolysis 8pY with the receipt of two fragments 278 p. and 388 bp respectively. Fragment length 388 p. purified from gel.

The gene replacement plasmid was designed as follows: the shuttle vector p \ UNM3 was subjected to enzymatic hydrolysis of EcoC1 and 8ATH1. p8E119 was subjected to enzymatic hydrolysis of WatH1 and 8pH, the hydrolyzate was subjected to electrophoresis, and a fragment of ~ 840 bp in length. purified from gel. p8E119 was subjected to enzymatic hydrolysis of EcoC1 and Hti !, the hydrolyzate was separated by electrophoresis, and a fragment of length ~ 1.7 kb. purified from gel. All four fragments (i.e., ~ 7.2 kb, ~ 840 bp, ~ 1.7 kb, and 388 bp) were ligated together when ligated in 4 directions . With this ligase mixture, competent cells of E.soy EN5a were transformed. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequencing. This plasmid, which was designated as pZE185, was transformed into E.soy EM1 and plasmid DNA was isolated from ampicillin resistant transformants. This plasmid was used to transform the protoplasts of strain 1100-ЗС38 З. аегтйШк. Thiostrepton-resistant transformants of strain 1100-ZS38 were isolated and analyzed by HPLC analysis of fermentation products. rZE185 during the transformation of strain 1100-ZS38 Z.auegtyysh did not significantly change the ratio of avermectins B2: B1 (Table 2).

rZE185 was used to transform the protoplasts Z.Aegtyyshk to create a mutation with a shift of the frame in the chromosomal aeus gene. Protoplasts were obtained from thiostrept-resistant transformants and sown by single colonies on KM14. After these protoplasts regenerated, single colonies were screened for the absence of thiostrepton resistance. Chromosomal DNA was isolated from thiostreptone-sensitive colonies and screened by PCR for a frame-shifted mutation integrated into the chromosome. PCR primers were designed based on the nucleotide sequence of the AeC gene and were provided by Sepokuk B1o131, 1ps. (Techak). The right-handed PCR primer was 5'-SSAASSATASSC SASTAS-3 '(ZEZ ΙΌ N0: 10), and the left-handed PCR primer was: 5'SAASSCSSSSSSSATAS-3' (ZGE) N0: 11), and the PCR conditions were as described in section 7.1.10 above. The resulting PCR product was 543 p. O., and during enzymatic hydrolysis of VkrE1, three fragments were observed: 368 bp, 96 p. and 79 bp, which indicated the chromosomal integration of the inactivated AeC gene and the loss of free replicon.

Fermentation analysis of Z.AegtyShk mutants containing a mutation with a frame shift in the AueC gene showed that normal avermectins were no longer produced and that these mutants had the same HPLC-fermentation profile as ZE180-11 and ZE184-1-13. One transformant, Tsho ^k , was identified and identified as strain ZE185-5a.

In addition, a mutation was obtained in the AueC gene, which replaces C with A (adenine) at nucleotide position 520, which leads to the replacement of the codon encoding tryptophan (V) at position 116 with a termination codon. Strain Z. aulegty with this mutation does not produce normal avermectins and has the same fermentation profile as strains ЗЕ180-11, ЗЕ184-1-13 and ЗЕ185-5а.

In addition, mutations were obtained in the AuC gene, which replace both (1) C with A at nucleotide position 970, which replaces the amino acid at position 275 from glycine (C) to aspartate (), and (2) T (thymidine) to C at the position of nucleotide 996, which replaces the amino acid at position 275 from tyrosine (Υ) to histidine (H). Strain Z. aegtyyshk with these mutations (C256E / U275N) does not produce normal avermectins and has the same fermentation profile as strains ЗЕ180-11, ЗЕ184-1-13 and ЗЕ185-5а.

Strains Z.Aegtyyshk, mutant for inactivation of the AueS gene, ЗЕ180-11, ЗЕ184-1-13, ЗЕ185-5а and others proposed here, provide screening tools for assessing the effect of other mutations in the AeU gene. rZE186, which contains a copy of the wild-type AUeC gene, was transformed with E.sub.EI and plasmid DNA was isolated from ampicillin-resistant transformants. This pZE186 DNA was used to transform the protoplasts of the ZE180-11 strain Z.AegtyShk. ZE180-11 isolated transformants of strain resistant to thiostrepton was determined presence of erythromycin resistance and the transformants TCO ^g Egsh ^g were analyzed by HPLC analysis of fermentation products. The presence of the functionally active AueC gene in the trans position was able to restore the normal production of avermectins in strain ME180-11 (Fig. 5B).

8.2. Analysis of mutations in the AueC gene that alter the ratios of B2: B1 classes

As described above, the ZE180-11 strain of Z.Aegtyshk containing the inactive aUeC gene was complemented with the help of a transformation with a plasmid containing the functionally active aUeC gene (pZE186). Strain ZE180-11 was also used as a host strain to characterize other mutations in the aeC gene, as described below.

Chromosomal DNA was isolated from strain 1100-ZS38 and used as a template for PCR amplification of the AueC gene. LFS length 1.2 T. p. were isolated by PCR amplification using primers designed on the basis of the nucleus sequence of aeC. The right-handed primer was ZEC ΙΌ NO: 6, and the left-handed primer was ZEC Ц NO: 7 (see section 7.1.10 above). The PCR and subcloning conditions were as described in section 7.1.10. Sequencing of 1.2 kb ORS DNA shows a mutation in the AuC gene, which replaces C with T at the position of nucleotide 337, which replaces the amino acid at position 55 of serine (3) with phenylalanine (B). The AueC gene, containing the Z55B mutation, was subcloned into pVHM3 to produce a plasmid, which was designated as rZE187 and which was used to transform the protoplasts of strain 8E180-11 8. augercine. Isolated thiostrepton resistant transformants of strain 8E180-11 determined presence of erythromycin resistance and transformants TYo ^g Egt ^g were analyzed by HPLC analysis of fermentation products. The presence of the AueC gene encoding a substitution at amino acid residue 55 (855P) was able to restore normal production of avermectins in strain 8E180-11 (Fig. 5B); however, the cyclohexyl-B2: cyclohexyl-B1 ratio was about 26: 1 compared with the 8E180-11 strain transformed with p8E186, which had a B2: B1 ratio of about 1.6: 1 (Table 3), which indicated that a single mutation (855E) modulates the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1.

Identified another mutation in the aUc gene, which replaces 6 with A at nucleotide position 862, which replaces the amino acid at position 230 from glycine (6) with aspartate (Ό). Strain 8.aegtyyk having this mutation (6230Ό), produces avermectins in a ratio of B2: B1 of about 30: 1.

8.3. Mutations that reduce the ratio of B2: B1

Several mutations that reduce the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 were constructed as follows.

A mutation has been identified in the AueC gene, which replaces 6 with A at nucleotide position 588, which replaces the amino acid at position 139 from alanine (A) with threonine (T). The AueC gene, containing the A139T mutation, was subcloned into RANM3 to obtain a plasmid, which was designated as p8E188 and which was used to transform the protoplasts of strain 8E180-11 8. aegteic. Thiostrepton-resistant transformants of strain 8E180-11 were isolated, the presence of erythromycin resistance was determined, and the TYo ^g transformers Egt ^{g were} analyzed using HPLC analysis of fermentation products. The presence of a mutated AuC gene encoding a substitution at amino acid residue 139 (A139T) was able to restore the production of avermectins in strain 8E180-11 (Fig. 5G); however, the B2: B1 ratio was about 0.94: 1, indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1. This result was unexpected, since the published results, as well as the results on the mutations described above, showed only either inactivation of the AueC gene or increased production of avermectin form B2 relative to the B1 form (Table 3).

Since the A139T mutation changed the B2: B1 ratios in a more favorable direction for B1, a mutation was constructed that encodes threonine instead of serine at amino acid position 138. Thus, p8E186 was subjected to enzymatic hydrolysis of EcoC1 and cloned into p6EM32E (Rgothed), which was hydrolyzed to EcoC1. This plasmid, which was designated p8E186a, was subjected to the enzymatic hydrolysis of Ara1 and Krp1, the DNA fragments were separated on an agarose gel, and two fragments ~ 3.8 kb. and ~ 0.4 kb purified from this gel. DNA insert ~ 1.2 kb in length from p8E186 used as a PCR template for introducing a single base substitution at nucleotide position 585. PCR primers were designed to introduce a mutation at nucleotide position 585, and were provided with Vocampo 11c, 1u. (Techak). The right-handed primer was 5'66666С666ССС666Т6С66А66С66АААТ6ССССТ66С6АС6-3 '(8 ЕС) GO N0: 12); and the left-handed primer was 5'-66ААСС6АСС6ССТ6АТАСА-3 '(8 ЕС) GO N0: 13). The PCR reaction was carried out using the AbgashSade 6C genomic PCR kit (Cipopicum Baciochek, Paolo AIO, SA) in the buffer supplied by the manufacturer, in the presence of 200 μM dNTP, 200 pmol of each primer, 50 ng template DNA, 1.0 M 6C- ME and 1 unit. K1PTAC polymerase mixture in a final volume of 50 μl. The temperature profile of the first cycle was 94 ° C for 1 min, followed by 25 cycles of 94 ° C for 30 s and 68 ° C for 2 min, and 1 cycle at 68 ° C for 3 min. PCR product 295 bp were subjected to enzymatic hydrolysis of Ara1 and Krp1 with the release of a fragment of 254 bp in length, which was separated by electrophoresis and purified from the gel. All three fragments (~ 3.8 kb, ~ 0.4 kb, and 254 bp) were ligated together when ligated in 3 directions. This ligase mixture transformed the competent cells of E.soi BN5a. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated p8E198.

p8E198 was subjected to enzymatic hydrolysis of EcoC1 and cloned into RANM3, which was enzymatically hydrolyzed by EcoC1, and E. oxy BN5a cells were transformed. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequencing. This plasmid DNA was transformed into E. soy BM1 cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as p8E199, was used to transform the protoplasts of strain 8E180-11 8. auger. Thiostrep-resistant transformants of strain 8E180-11 were isolated, the presence of erythromycin resistance was determined, and the TYo ^g transformer Egt ^{g were} analyzed using HPLC analysis of fermentation products. The presence of a mutated AuC gene encoding a substitution at amino acid residue 138 (8138T) was able to restore normal production of avermectins in strain 8E180-11; however, the B2: B1 ratio was 0.88: 1, indicating that this mutation reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (Table 3). This B2: B1 ratio is even lower than the 0.94: 1 ratio observed with the A139T mutation, produced by transforming the 8E180-11 p8E188 strain, as described above.

Another mutation was designed to introduce threonine at both positions of amino acids 138 and 139. DNA insertion of ~ 1.2 kb in length. from r8E186 was used as a PCR template. PCR primers were designed to introduce mutations at nucleotide positions 585 and 588, and were provided by Sepokhukh Vyuyypo1od8, 1pc. (Techak). The right-handed primer was 5'66666С666ССС666Т6С66А66С66АААТ6Ц6СТ66С6АС6АСС-3 '(8ER GO N0: 14), and the left-directed primer was 5'-66ААТСЦАС66СТТСАСС-3' (8 ЕS) Г N0-GO606 (8 EU) ГО N0 The PCR reaction was performed using the conditions described directly above in this section. PCR product 449 bp were subjected to enzymatic hydrolysis of Ara1 and Krp1 with the release of a fragment of 254 bp in length, which was separated by electrophoresis and purified from the gel. P8E186a was subjected to the enzymatic hydrolysis of Ara1 and Krp1, the DNA fragments were separated on an agarose gel, and two fragments ~ 3.8 kb. and ~ 0.4 kb purified from this gel. All three fragments (~ 3.8 kb, ~ 0.4 kb, and 254 bp) were ligated together when ligated in 3 directions, and this ligase mixture transformed the competent E cells .soy ΌΗ5α. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid was designated p8E230.

p8E230 was subjected to enzymatic hydrolysis of EcoB1, cloned into p ^ HM3, which was enzymatically hydrolyzed with EcoP1, and E. oi 5α cells were transformed. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequencing. E. soy ΌΜ1 was transformed with this plasmid DNA, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis. This plasmid, which was designated as p8E231, was used to transform the protoplasts of strain 8E180-11 8. aegun | 5. Thiostrepton-resistant 8E180-11 transformants were isolated, the presence of erythromycin resistance was determined, and the transformants Ty1o ^g Ett ^{g were} analyzed using fermentation. The presence of the double-mutated AuC gene encoding 8138T / A139T was able to restore normal production of avermectins in strain 8E18011; however, the B2: B1 ratio was 0.84: 1, indicating that this mutation further reduces the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1 (Table 3), compared with the reductions resulting from the transformation of strain 8E180 -11 p8E188 or p8E199, as described above.

Table 3

Strain 8.ahegsh118 (transforming plasmid) The number of protetir. transformants Relative conc. AT 2 Relative conc. IN 1 The average ratio of B2: B1 8E180-11 (no) thirty 0 0 0 8E180-11 (p ^ NME) thirty 0 0 0 8E180-11 (r8E186) 26 222 140 1.59 8E180-11 (r8E187) 12 283 eleven 26.3 8E180-11 (r8E188) 24 193 206 0.94 8E180-11 (r8E199) 18 155 171 0.88 8E180-11 (r8E231) 6 259 309 0.84

These results were the first to demonstrate specific mutations in the aeC gene, which lead to the production of elevated levels of more commercially desirable class 1 avermectins relative to class 2 avermectins.

9. Example: Construction of 5 'deletion mutants

As explained in section 5.1 above, the nucleotide sequence of the 81e1 rounds is shown in FIG. 1 (8EC GO NO: 1), contains four different SRT codons in the positions of the p. 42, 174, 177 and 180, which are potential launch sites. This section describes the construction of multiple deletions of the 5 'portion of the OCR AUES (Fig. 1; 8EC GO N0: 1) to help determine which of these codons can function as start sites in the ODP AU for the expression of the protein.

Fragments of the AueC gene, delegated in various ways at the 5 'end, were isolated from chromosomal 8.AurethI5 DNA using PCR amplification. PCR primers were designed on the basis of the aeC DNA sequence and provided by Sepokhukh Vyutseipo, 1 ps. The right-handed primers were 5'-AASSCATS6A6CC6 CTC-3 '(8 EC) GO N0: 16) (1Ό1); 5'-TS66SST6S SAASSAAS-3 '(8 EC) GO N0: 17) (Ό1Ε2); 5'CCAAC6AAC6T6TA6TA6-3 '(8 EC) GO N0: 18) (Ό1Ρ3) and 5'-TSSASSCSTASSTSTTSASS-3' (8ΕΟ ΙΌ N0: 19) (9Ό2Ρ2). Left-handed primers were 5'SATSATSSSTSAASSA-3 '(8NO ΙΌ ^: 20); 5'-SATSATSSSTSAASSASSA-3 '(8ΕΟ ^: 21) and 5'-ASSACTSTSTSTSSSTSSA-3' (8Ε0 ΙΌ NO: 22). The PCR reaction was performed as described in section 8.3 above.

PCR products were separated by electrophoresis in 1% agarose gel and individual bands of ~ 1.0 kb were found. and ~ 1.1 kb These PCR products were gel purified and ligated with 25 ng of the linearized vector pCK2.1 (1 pp) at 1:10 molar vector insertion, following the manufacturer's instructions. These ligase mixtures were used to transform competent B.so11 Ope8yo1 ™ cells (1-year-old), following the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequencing. These plasmids were designated as ρ8Ε190 (obtained with primer Ό1Ρ1), ρ8Ε191 (obtained with primer Ό1Ρ2), ρ8Ε192 (obtained with primer 1Ρ3) and ρ8Ε193 (obtained with primer 2Ρ2).

Each of the DNA inserts was subjected to enzymatic hydrolysis of WatH1 / XBa1, separated by electrophoresis, purified from the gel, and ligated separately with the shuttle vector ρΑΗΜ3, which was enzymatically hydrolyzed with WatH1 / XBa1, in a total DNA concentration of 1 μg in 1: 5 molar ratio vector. These ligase mixtures were used to transform competent B.soH α5α cells. Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the insert was confirmed by restriction analysis. These plasmids are designated as ρ8Ε194 (Ό1Ρ1), r8E195 (Ό1Ρ2), ρ8Ε196 (Ό1Ρ3) and ρ8Ε197 (Ό2Ρ2), each separately transformed strain B.soN ΌΜ1, from transformants resistant to ampicillin, plasmid DNA was isolated and the presence of the correct inserts confirmed by restriction analysis. This DNA was used to transform the protoplasts of strain 8-180-11 8.Aegty111k. Isolated thiostrepton resistant transformants of strain 8Ε180-11, determined by the presence of erythromycin resistance and the transformants TNyuEli ¹ 'analyzed by HPLC analysis of fermentation products to determine which sites are necessary for STS avec expression. The results indicate that the CTC codon at position 42 can be removed without affecting the expression of AeuC, whereas each of ρ8Ε194, ρ8Ε195 and ρ8Ε196, each of which lacks a CTC site at position 42, but each of which contains all three CTC sites in positions 174, 177, and 180, was capable of restoring normal production of avermectins during transformation 8-180-11. Normal production of avermectins was not restored when the strain 8-180-11 transformed ρ8Ε197, in which all four CTS sites are absent (Table 4).

Table 4

Strain 8.ahegsh118 (transforming plasmid) The number of protetir. transformants Relative conc. AT 2 Relative conc. IN 1 The average ratio of B2: B1 8-180-11 (none) 6 0 0 0 8Ε180-11 (ρ№ΗΜ3) 6 0 0 0 8Ε180-11 (ρ8Ε186) 6 241 152 1.58 8Ε180-11 (ρ8Ε194) 6 35 15 2.43 8Ε180-11 (ρ8Ε195) 6 74 38 1.97 8Ε180-11 (ρ8Ε196) 6 328 208 1.58 8Ε180-11 (ρ8Ε197) 12 0 0 0

10. Example: Cloning AusC Homologues from 8.Budgo “Sor1sik and 8gpheosKgotcech”. The present invention makes it possible to identify and clone homologues of AusC genes from other avermectin and milbemycin-producing species of 8ΐgrΐΐussek. For example, a cosmid library of genomic DNA 8.Wod ^ οксoρ ^ nik (ΡΕΚΜ BP-1901) was hybridized with an AeC probe of 1.2 kb in length. of 8.aνe ^ sh ^ ί ^ 1 ^ κ described above. Identified several cosmid clones that showed strict hybridization. Chromosomal DNA was isolated from these cosmids and a Krp1 fragment of 4.9 kb in length was identified, which hybridized with the AeC probe. This DNA was sequenced and the ORF (8Ε0 ΙΌ N0: 3) was identified, which has significant homology with the ORS AueC 8.аνе ^ sh ^ ί ^ 1 ^ κ. The amino acid sequence (8Ε0 ΙΌ N0: 4), derived from the ORF of the homologue Aus 8.Lud ^ οκсοрρкик, is shown in FIG. 6

In addition, the cosmid library of genomic DNA of 8.dipkeal 11gopepeck was hybridized with an AeC probe of 1.2 kb in length. from 8.11 ^ 011111115 described above. Identified several cosmid clones that showed strict hybridization. Chromosomal DNA was isolated from these cosmids and a RSh fragment of 5.4 kbp was identified, which hybridized with the AeC probe. This DNA was sequenced and a partial ORS of the aeC homologue was identified, which has significant homology with the ayC 8 ORS. The derived partial amino acid sequence (8Ε0 ΙΌ N0: 5) is shown in FIG. 6

DNA sequence and amino acid sequence analysis of AueC homologues from 8.1 · ·ΐΌ βΐΌ5Μρίαΐ5 and 8.д ^ ^ еееοοο ^ ^ ^ ^ш οοее указывает indicates that these sites share significant homology (~ 50% sequence identity at the amino acid level) with each other and with OCR aeus 8. auigtyy and an AeeC gene product (Fig. 6).

11. Example: Constructing a plasmid with the Aurec gene under the EEE promoter

ODP AueS length 1.2 T. p. from r8E186 was subcloned into p8E34, which is a shuttle vector ρ ^ ΗΜ3, having a 300E bp promoter, embedded as a fragment of Kri1 / VatH1 in Kri1 / VatN1 site p \ UNM3 (see \\\ Uagb! a1., 1986, Mo1. Sei. SeieE 203: 468-478). p8E186 was subjected to the enzymatic hydrolysis of VatH1 and Nshbsh, the hydrolyzate was separated by electrophoresis, and a fragment of 1.2 kb in length. was isolated from an agarose gel and ligated with p8E34, which was hydrolyzed with WatH1 and NShbsH. This ligase mixture transformed the competent cells of E. col11 EN5a according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of an insertion of 1.2 kb was used. confirmed by restriction analysis. This plasmid, which was designated as p8E189, was transformed to E.co11 1, and plasmid DNA was isolated from ampicillin-resistant transformants. r8E189 transformed the protoplasts of strain 1100-8C38 8. augt111k. Thiostrepton-resistant transformants of strain 1100-8C38 were isolated and analyzed by HPLC analysis of fermentation products.

Transformants of strain 1100-8C38 8. auger containing P8E189 had altered ratios of cyclohexyl-B2: avermectin cyclohexyl-B1 produced by avermectin and cyclohexyl-B1 (approximately 3: 1) compared to strain 11008С38 (approximately 34: 1), and the total production of avermectins increased by about 2.4 times compared with the strain 1100-8C38 transformed with p8E119 (Table 5).

r8E189 also transformed the protoplasts of the wild-type strain 8.aegteyshk. Thiostrepton-resistant transformants were isolated and analyzed by HPLC analysis of fermentation products. The total production of avermectins by the wild-type strain of aaaumteum transformed with p8E189 increased by about 2.2 times compared with the wild-type strain of anaurethyne transformed by p8E119 (Table 5).

Table 5

Strain 8.al ^- ergSh (transforming plasmid) The number of protetir. transformants Relative [B2] Relative [B1] Relates total avastins The average ratio of B2: B1 1100-8С38 6 155 4.8 176 33.9 1100-8C38 (r8E119) 9 239 50.3 357 4.7 1100-8C38 (r8E189) sixteen 546 166 849 3.3 Wild type 6 59 42 113 1.41 Wild type (r8E119) 6 248 151 481 1.64 Wild type (r8E189) five 545 345 1071 1.58

12. Example: Chimeric plasmid containing sequences as from ORF aeU

8. AegtNSHk, and from the homologue of AueS

8.Budgo "sor1sik

A hybrid plasmid, designated as p8E350, which contains a portion of the aueC 8.yudgoxor1Ci8 homologue, 564 bp in length, replacing the homologous portion of the CES aeC 8.AEG of 564 bp (Fig. 7), constructed as follows. p8E350 was constructed using the restriction site BkaA1, which is conserved in both sequences (AeC position 225), and the K1A restriction site, which is present in the AueC gene 8.Auscious (AeC position 810). Site Kri1 8.yudgo8sor1si8 introduced into DNA via PCR using primer pravonapravlenny 5'STTSASSTSTASSTSTTSS-3 '(8EO GO N0: 23) and the leftward primer 5'SAASTSSTASSASTSSSS-3' (8EO GO N0: 24) (provided Seiokuk Vyu1es1sho1od1e8) using the PCR conditions described in section 7.1.10 above. The PCR product was enzymatically hydrolyzed with VkaA1 and Kri1, the fragments were separated by electrophoresis in 1% agarose gel, and the fragment with VkaA1 / Kri1 564 bp in length. isolated from this gel. p8E179 (described in section 7.1.10 above) were subjected to enzymatic hydrolysis of Kri1 and NSbH, the fragments were separated by electrophoresis in 1% agarose gel, and a fragment of ~ 4.5 kb. isolated from this gel. p8E179 was subjected to enzymatic hydrolysis of NSbn and VkaA1, the fragments were separated by electrophoresis in 1% agarose gel, and the fragment VkaA1 / H1b111 with a length of ~ 0.2 kb. isolated from this gel. Fragment NshbSh / Kry length ~ 4.5 kb, Fragment VkaA1 / NshbSh length ~ 0.2 kb and fragment VkaA1 / Kri1 length 564 p. of 8.yudgoxoric were ligated together when ligated in 3 directions, and this ligase mixture was used to transform competent E.co 11 EN5a cells. Plasmid DNA was isolated from ampicillin-resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using Kri1 and AuaE. \ UNM3, which was enzymatically hydrolyzed Nshb and XHa1. This ligase mixture transformed competent E. coli OH5a cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using NShP and AuaE. This plasmid DNA was transformed with E. coli 11 ΌΜ1, from ampicillin resistant transformants plasmid DNA was isolated, and the presence of the correct insert was confirmed by restriction analysis and DNA sequencing. This plasmid was designated as p8E30 and was used to transform the protoplasts of strain 8E180-11 8. aegtyy1 Thioostrepton-resistant transformants of strain 8E180-11 were isolated, the presence of erythromycin resistance was determined, and the transformers TYO'Egt ¹ 'were analyzed by HPLC analysis of the products of the enzyme. The results show that those transformants that contain the hybrid plasmid 8.AegtSH5 / 8.hudgo5sor1si8 have an average B2: B1 ratio of about 109: 1 (Table 6).

Table 6

Strain 8.al ^- itsSh118 (transforming plasmid) The number of protetir. transformants Relative conc. AT 2 Relative conc. IN 1 The average ratio of B2: B1 8E180-11 (no) eight 0 0 0 8E180-11 (p ^ NME) eight 0 0 0 8E180-11 (r8E350) sixteen 233 2 109

Deposition of biological materials

The following biological materials were deposited with the help of Atepsai Touré SIYige SoPesOop (ATSS) on 12301 Raq1a ^ and Elouy, Vosku111e, ΜΌ, 20852 I8A on January 29, 1998, and they were assigned the following catalog numbers:

Plasmid

Plasmid p8E180

Plasmid p8E186 catalog number

209605

209604

All patents, patent applications and publications cited above are fully incorporated herein by reference.

The present invention is not limited in scope to the specific embodiments described herein, which are intended as individual illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of this invention. In fact, various modifications of the invention, other than those shown and described herein, will be apparent to those skilled in the art from the foregoing description and accompanying graphic materials. Such modifications are intended to be introduced within the scope of the appended claims.

Claims (54)

CLAIM 1. An isolated polynucleotide molecule containing a full-size open reading frame (OPC) auyC 8! Germeteus iguyu or part of it containing at least about 70% of the full-sized OPC auyC that encodes a functionally equivalent product of the auyC gene, and in the selected polynucleotide molecule the following full-sized OPC is absent, which is located in the forward direction from the OPC ayeC ίη 8Yi in the chromosome 8! gerus uterus§ aueptum, and wherein the OPC has the nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ НО: 1). 2. The selected polynucleotide molecule according to claim 1, additionally containing nucleotide sequences that in nature flank the auyC gene 3. An isolated polynucleotide molecule containing a nucleotide sequence that is homologous to the nucleotide sequence of the aufc OPC encoding the product of Aufc 81er1otus5 auigtSh gene shown in FIG. 1 (8EO ΙΌ ЫО: 1), or a part thereof containing at least about 70% of the indicated nucleotide sequence, which encodes a functionally equivalent product of the AuueC gene. 4. The selected polynucleotide molecule containing the nucleotide sequence 8EO ΙΌ NО: 3 or its essential part, or the nucleotide sequence that is homologous to the nucleotide sequence 8ЕО ΙΌ НО: 3. 5. An oligonucleotide molecule that hybridizes to a polynucleotide molecule having the nucleotide sequence shown in FIG. 1 (8EO ΙΌ WO: 1), or with a polynucleotide molecule having a nucleotide sequence, which is a complement of the nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ НО: 1), in very strict conditions. 6. An oligonucleotide molecule that hybridizes with a polynucleotide molecule having the 8ЕО ΙΌ ОО: 3 nucleotide sequence, or with a polynucleotide molecule having the nucleotide sequence, which is a complement of the 8ЕО ΙΌ НО: 3 nucleotide sequence, under very strict conditions. 7. The oligonucleotide molecule according to claim 5, which is a complementary nucleotide sequence shown in FIG. 1 (8EO ΙΌ WO: 1), or a polynucleotide molecule having a nucleotide sequence, which is a complement of the nucleotide sequence shown in FIG. 1 (8ЕО ΙΌ НО: 1). 8. The oligonucleotide molecule according to claim 5, which is a complementary nucleotide sequence of 8EO ΙΌ NО: 3 or a polynucleotide molecule having a nucleotide sequence that is a complement of the nucleotide sequence of 8EO ΙΌ ЫО: 3. 9. A recombinant vector containing a polynucleotide molecule containing a nucleotide sequence selected from the group consisting of the nucleotide sequence of the OEC auP shown in FIG. 1 (8ЕО ΙΌ НО: 1), or its essential part; and the nucleotide sequence, which is homologous to the OPC encoding the product of the AueeC gene B. auegte Sch3 shown in FIG. 1 (8ETS GO NO: 1), or its essential part. 10. The recombinant vector according to claim 9, additionally containing a nucleotide sequence encoding one or more than one regulatory element, the nucleotide sequence encoding a regulatory element being operatively associated with the nucleotide sequence of the polynucleotide molecule of the vector according to claim 9. 11. The recombinant vector of claim 10, further comprising a nucleotide sequence encoding a selectable marker. 12. The recombinant vector according to claim 11, which is a plasmid p8E186 (ATCC 209604). 13. A host cell containing the recombinant vector according to claim 11. 14. The host cell according to item 13, which is 81 15. A recombinant vector containing a polynucleotide molecule containing a nucleotide sequence selected from the group consisting of the nucleotide sequence of 8EC GO N0: 3 or an essential part thereof; a nucleotide sequence that is homologous to the nucleotide sequence of 8EC GO N0: 3; and a nucleotide sequence that encodes a polypeptide having the amino acid sequence of 8EC GO N0: 4. 16. The recombinant vector of claim 15, further comprising a nucleotide sequence encoding one or more than one regulatory element, wherein the nucleotide sequence encoding a regulatory element is operatively associated with the nucleotide sequence of the polynucleotide vector molecule of claim 15. 17. The recombinant vector according to clause 16, further comprising a nucleotide sequence encoding a selectable marker. 18. A host cell containing the recombinant vector according to 17. 19. The host cell according to claim 18, which is 81 geriotusu 5 Eudoxoxoic. 20. The selected AuueC 8.augt1Sh15 gene product having the amino acid sequence encoded by the nucleotide sequence of the plasmid p8E186 (ATCC 209604) encoding the AuueC 8.augt1Sh15 gene product, or the amino acid sequence 8EC EC GO N0: 2 or a part thereof containing at least about 70 % of this amino acid sequence, which is a functionally equivalent product of the AueC gene, in which, possibly, one or more of the amino acid residue is conservatively replaced by another amino acid residue, and such Replacing results in a functionally equivalent gene product. 21. The isolated product of the homologue of the AueC gene 8.Eudgo5cor1SI5, having the amino acid sequence of 8EC GO GO N0: 4. 22. A method of obtaining a product of the recombinant AueC gene, in which the host cell transformed with the recombinant vector according to any one of claims 9-11 is cultured under conditions leading to the production of the product of the recombinant AueC gene, and the product of the AueC gene is isolated from the cell culture. 23. A method of obtaining a product of a homologue of a recombinant AueC gene, in which a host cell transformed with a recombinant vector according to any one of claims 15-17 is cultured under conditions leading to the production of a product of a homologue of a recombinant AueC gene, and the product of the AuueC gene homologue is isolated from the cell culture . 24. A polynucleotide molecule containing the nucleotide sequence, which is the sequence of the plasmid p8E186 (ATCC 209604), encoding the product of the AuyeC gene Z. auigtshSh, or the nucleotide sequence 0RS auuC Z. auigtshSh, as shown in FIG. 1 (8th EC GO NO: 1), or a degenerate variant thereof, containing one or more than one mutation, such that cells of the Z. augetESh ATCC 53692 strain in which the wild-type ayeC allele are inactivated and which express a polynucleotide molecule containing a mutated nucleotide sequence, when fermented in the presence of cyclohexanecarboxylic acid, a reduced ratio of avermectins cyclohexyl B2: cyclohexyl B1 is produced, which is produced by cells of the Z. augenius ATCC 53692 strain, which instead express only the wild ipa. 25. The polynucleotide molecule of claim 24, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is less than 1.6: 1. 26. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.94: 1. 27. The polynucleotide molecule of Claim 26, wherein the mutation in 0PC auC Z. auegtEShk shown in FIG. 1 (8ETS GO NO: 1), encodes the amino acid substitution at residue 139 of the AueC gene product from alanine to threonine. 28. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.88: 1. 29. The polynucleotide molecule according to claim 28, wherein the mutation in 0PC ayeC Z. auegtEShk shown in FIG. 1 (8EC GO NO: 1), encodes the amino acid substitution at residue 138 of the AueC gene product from serine to threonine. 30. The polynucleotide molecule of claim 25, wherein the reduced ratio of cyclohexyl B2: cyclohexyl B1 is about 0.84: 1. 31. The polynucleotide molecule of claim 30, wherein the mutation in the OPC ayeC 8.auegpSh1118 shown in FIG. 1 (8ETS ΙΌ NO: 1), encodes the first amino acid substitution at residue 138 of the AueC gene product from serine to threonine and the second amino acid substitution at residue 139 of the AueC gene product from alanine to threonine. 32. A polynucleotide molecule containing a strong promoter in operative association with the OPC ayeC 8.auegpSh1118 shown in FIG. 1 (8ES) ΙΌ ^: 1). 33. The polynucleotide molecule according to claim 32, wherein the strong promoter is an ectE promoter from an 8assyagoro1ukrog eeguidea. 34. A polynucleotide molecule containing the nucleotide sequence encoding the OPC shown in FIG. 1 (8ETS ΙΌ NO: 1) encoding the AueC gene product, which is inactivated by inserting a heterologous nucleotide sequence into the specified nucleotide sequence. 35. A polynucleotide molecule containing ayeC allele that is inactivated by deletion of a 640 bp PkP / 8pY fragment. from the Oyu SEC presented in FIG. 1 (8ES) ΙΌ ^: 1). 36. A polynucleotide molecule containing an auyC allele that is inactivated by introducing a frame-shift mutation in the auyC OPC of FIG. 1 (8EC ΙΌ NO: 1). 37. The polynucleotide molecule of claim 36, wherein the frame-shift mutation is introduced by adding two nucleotides 6 (guanine) after nucleotide C (cytosine) at the position of nucleotide 471 OPC ayeC shown in FIG. 1 (8ЕЕ) ΙΌ ^: 1). 38. A polynucleotide molecule containing an auyC allele that is inactivated by introducing a termination codon into the position of a nucleotide that encodes amino acid 116 of the product of AuyC 8.aνе ^ ш ^ ΐ ^ 1 ^ κ encoded by OPC auyC shown in FIG. 1 (8ЕЕ) ΙΌ ^: 1). 39. A polynucleotide molecule containing an ayeC allele that is inactivated by introducing the first mutation at amino acid position 256 of the AueC gene product, which replaces glycine with aspartate, and the second mutation at position 275 of the AueC gene product, which replaces tyrosine with histidine. 40. Recombinant vector containing a polynucleotide molecule according to any one of paragraphs.24-39. 41. A recombinant vector containing a polynucleotide molecule according to claim 34, wherein the vector is p8E180 (ATCC 209605). 42. A host cell of 8ΐ ^ epococcus, containing the recombinant vector of claim 40. 43. Strain 8.auegpShSh8, containing cells expressing the mutated auyC allele, which leads to the production of avermectins by the cells in a reduced ratio of classes 2: 1 compared to cells of the same strain, which instead express only the wild-type ayeC allele. 44. The strain according to item 43, where the cells produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of less than 1.6: 1. 45. The strain of claim 43, wherein the cells produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.94: 1. 46. The strain according to item 43, where the cells produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.88: 1. 47. The strain according to item 43, where the cells produce avermectins cyclohexyl B2: cyclohexyl B1 in a ratio of about 0.84: 1. 48. Strain 8.auegpShSh8, containing cells in which the ayeC gene is inactivated. 49. A method for identifying ayec OPC mutations capable of decreasing a 2: 1 class ratio of avermectins produced, wherein (a) a 2: 1 class ratio of avermectins produced by cells of the 8. aueught1118 strain of claim 48, into which the polynucleotide molecule is introduced and expressed, is determined containing a nucleotide sequence encoding a product of a mutated AuEx gene; (b) determine the ratio of the 2: 1 classes of avermectins produced by cells of the same strain 8.auegtSh118, as in step (a), which do not express the mutated auyC allele, but instead express only the auyC allele having the OPC nucleotide sequence shown in FIG. . 1 (8EC ΙΌ NO: 1), or a nucleotide sequence that is homologous to this sequence; and (c) comparing the ratio of the 2: 1 classes of avermectins produced by stage 8.aueught1118 cells of the stage (a) with the ratio of the 2: 1 classes of avermectins produced by the stage 8.baugett118 cells of stage (b); so if the ratio of the 2: 1 classes of avermectins produced by the cells of 8.auegtSh118 of stage (a) is lower than the ratio of the 2: 1 classes of avermectins produced by the cells 8.AuegpSH1118 stage (b), the mutation of the OPC AuP mutation capable of reducing the ratio of the 2: 1 classes of avermectins is identified. 50. The method of creating strain 8. auegpSh1118 according to item 43, wherein the cells of strain 8. auegpSh1118 are transformed with a vector that carries a mutated auuC allele encoding a gene product that reduces the ratio of the 2: 1 classes of avermectins produced by cells of strain 8. auegtSh118 expressing mutated auyC allele, in comparison with cells of the same strain, which instead express only wild-type auyC allele, and transformed cells that produce avermectins in a reduced ratio of classes 2: 1 compared to the corresponding HAND class 2: 1, produced by cells of the strain that instead express the wild-type allele avec. 51. The method of creating a B. aeughtyShk strain according to claim 48, wherein the cells of the B. aeughtyShk strain are transformed with a vector that inactivates the auh allele, and transformed cells in which the auh allele are inactivated are selected. 52. The method of claim 51, wherein the vector is pBE180 (ATCC 209605). 53. A method of producing avermectins, in which the cells of the strain B. auegtyShk according to item 43 are cultured in culture media under conditions that enable production or induce the production of avermectins from them and isolate these avermectins from the culture. 54. The composition of avermectins produced by cells of the B. aeughty Schk strain according to claim 43, wherein the avermectins are produced in a reduced ratio of classes 2: 1 compared to the ratio of the 2: 1 classes of avermectins produced by cells of the same B. aeughty Schk strain that do not express a mutated allele aeuC, but instead, only the wild-type auCe allele is expressed.